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Sample records for characterization mitochondrial localization

  1. Molecular characterization of Saudi local chicken strains using mitochondrial DNA markers.

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    Yacoub, H A; Ramadan, H A I; Baeshen, Nabih A; Sadek, Mahmoud Abdel; Abou Alsoud, M E

    2015-08-01

    The current study was carried out to investigate and estimate the genetic diversity of native breeds based on cytochrome b (cyt-b) gene of mitochondrial DNA information. The obtained sequences of cyt-b gene segment have TAA as a stop codon at 488 position with no insertions or deletion in all individuals of both native chicken strains. The blast results showed that no variation was found among individuals within both native chicken strains, but when a comparison was established among them and other species of genus Gallus the variation is exploring, additionally many mutant sites were detected as single nucleotide polymorphisms (SNPs) in different sites. The phylogenetic trees exhibited three different groups. The results revealed that the native chicken strains were closely related to the cluster of Gallus gallus and subspecies of Gallus, suggesting that they may be separated from the same origin. According to this result and previously studies, the native chicken strains are genetically closer to Gallus gallus and it could be successfully distinguished from the other wild types of Gallus chicken based on cyt-b gene information. We recommended that the governmental concerns for native chicken strain should be enhanced to screen its genetic structure for large scale in the Kingdom of Saudi Arabia.

  2. cDNA cloning and characterization of mouse nifS-like protein, m-Nfs1: mitochondrial localization of eukaryotic NifS-like proteins.

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    Nakai, Y; Yoshihara, Y; Hayashi, H; Kagamiyama, H

    1998-08-14

    We have isolated a mouse cDNA which shows significant sequence similarity to the yeast nifS-like gene (y-NFS1), and termed it m-Nfs1. The deduced protein sequence (459 amino acids long) has several characteristic features common to those of bacterial NifS proteins, but distinct from them by its amino-terminal extension which contains a typical mitochondrial targeting presequence. m-Nfs1 was found to be a soluble 47-kDa protein in the matrix fraction of mouse liver mitochondria. The m-Nfs1 gene was ubiquitously expressed in most tissues, suggesting its housekeeping function in vivo. We also found that the gamma-NFS1 protein was localized in the mitochondrial matrix in yeast cells. These results suggest that both eukaryotic NifS-like proteins may play some roles in mitochondrial functions.

  3. RECQL4 localizes to mitochondria and preserves mitochondrial DNA integrity

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    Croteau, Deborah L; Rossi, Marie L; Canugovi, Chandrika

    2012-01-01

    RECQL4 is associated with Rothmund-Thomson Syndrome (RTS), a rare autosomal recessive disorder characterized by premature aging, genomic instability, and cancer predisposition. RECQL4 is a member of the RecQ helicase family, and has many similarities to WRN protein, which is also implicated...... in premature aging. There is no information about whether any of the RecQ helicases play roles in mitochondrial biogenesis, which is strongly implicated in the aging process. Here, we used microscopy to visualize RECQL4 in mitochondria. Fractionation of human and mouse cells also showed that RECQL4 was present...... in mitochondria. Q-PCR amplification of mitochondrial DNA demonstrated that mtDNA damage accumulated in RECQL4-deficient cells. Microarray analysis suggested that mitochondrial bioenergetic pathways might be affected in RTS. Measurements of mitochondrial bioenergetics showed a reduction in the mitochondrial...

  4. Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction.

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    Alecu, Irina; Tedeschi, Andrea; Behler, Natascha; Wunderling, Klaus; Lamberz, Christian; Lauterbach, Mario A R; Gaebler, Anne; Ernst, Daniela; Van Veldhoven, Paul P; Al-Amoudi, Ashraf; Latz, Eicke; Othman, Alaa; Kuerschner, Lars; Hornemann, Thorsten; Bradke, Frank; Thiele, Christoph; Penno, Anke

    2017-01-01

    1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.

  5. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

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    Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

  6. Mitochondrial fusion and ERK activity regulate steroidogenic acute regulatory protein localization in mitochondria.

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    Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J; Poderoso, Cecilia

    2014-01-01

    The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR.

  7. Sepsis-induced cardiac mitochondrial dysfunction involves altered mitochondrial-localization of tyrosine kinase Src and tyrosine phosphatase SHP2.

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    Qun S Zang

    Full Text Available Our previous research demonstrated that sepsis produces mitochondrial dysfunction with increased mitochondrial oxidative stress in the heart. The present study investigated the role of mitochondria-localized signaling molecules, tyrosine kinase Src and tyrosine phosphatase SHP2, in sepsis-induced cardiac mitochondrial dysfunction using a rat pneumonia-related sepsis model. SD rats were given an intratracheal injection of Streptococcus pneumoniae, 4×10(6 CFU per rat, (or vehicle for shams; heart tissues were then harvested and subcellular fractions were prepared. By Western blot, we detected a gradual and significant decrease in Src and an increase in SHP2 in cardiac mitochondria within 24 hours post-inoculation. Furthermore, at 24 hours post-inoculation, sepsis caused a near 70% reduction in tyrosine phosphorylation of all cardiac mitochondrial proteins. Decreased tyrosine phosphorylation of certain mitochondrial structural proteins (porin, cyclophilin D and cytochrome C and functional proteins (complex II subunit 30kD and complex I subunit NDUFB8 were evident in the hearts of septic rats. In vitro, pre-treatment of mitochondrial fractions with recombinant active Src kinase elevated OXPHOS complex I and II-III activity, whereas the effect of SHP2 phosphatase was opposite. Neither Src nor SHP2 affected complex IV and V activity under the same conditions. By immunoprecipitation, we showed that Src and SHP2 consistently interacted with complex I and III in the heart, suggesting that complex I and III contain putative substrates of Src and SHP2. In addition, in vitro treatment of mitochondrial fractions with active Src suppressed sepsis-associated mtROS production and protected aconitase activity, an indirect marker of mitochondrial oxidative stress. On the contrary, active SHP2 phosphatase overproduced mtROS and deactivated aconitase under the same in vitro conditions. In conclusion, our data suggest that changes in mitochondria-localized

  8. Dual localized mitochondrial and nuclear proteins as gene expression regulators in plants?

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    Philippe eGiegé

    2012-09-01

    Full Text Available Mitochondria heavily depend on the coordinated expression of both mitochondrial and nuclear genomes because some of their most significant activities are held by multi-subunit complexes composed of both mitochondrial and nuclear encoded proteins. Thus, precise communication and signaling pathways are believed to exist between the two compartments. Proteins dual localized to both mitochondria and the nucleus make excellent candidates for a potential involvement in the envisaged communication. Here, we review the identified instances of dual localized nucleo-mitochondrial proteins with an emphasis on plant proteins and discuss their functions, which are seemingly mostly related to gene expression regulation. We discuss whether dual localization could be achieved by dual targeting and / or by re-localization and try to apprehend the signals required for the respective processes. Finally, we propose that in some instances, dual localized mitochondrial and nuclear proteins might act as retrograde signaling molecules for mitochondrial biogenesis.

  9. The mitochondrial genome of the fission yeast Schizosaccharomyces pombe : 5. Characterization of mitochondrial deletion mutants.

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    Ahne, F; Merlos-Lange, A M; Lang, B F; Wolf, K

    1984-09-01

    The three mutator strains ana (r)-8, ana (r)-14, and diu (r)-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe.

  10. A comprehensive characterization of mitochondrial DNA mutations in glioblastoma multiforme.

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    Vidone, Michele; Clima, Rosanna; Santorsola, Mariangela; Calabrese, Claudia; Girolimetti, Giulia; Kurelac, Ivana; Amato, Laura Benedetta; Iommarini, Luisa; Trevisan, Elisa; Leone, Marco; Soffietti, Riccardo; Morra, Isabella; Faccani, Giuliano; Attimonelli, Marcella; Porcelli, Anna Maria; Gasparre, Giuseppe

    2015-06-01

    Glioblastoma multiforme (GBM) is the most malignant brain cancer in adults, with a poor prognosis, whose molecular stratification still represents a challenge in pathology and clinics. On the other hand, mitochondrial DNA (mtDNA) mutations have been found in most tumors as modifiers of the bioenergetics state, albeit in GBM a characterization of the mtDNA status is lacking to date. Here, a characterization of the burden of mtDNA mutations in GBM samples was performed. First, investigation of tumor-specific vs. non tumor-specific mutations was carried out with the MToolBox bioinformatics pipeline by analyzing 45 matched tumor/blood samples, from whole genome or whole exome sequencing datasets obtained from The Cancer Genome Atlas (TCGA) consortium. Additionally, the entire mtDNA sequence was obtained in a dataset of 104 fresh-frozen GBM samples. Mitochondrial mutations with potential pathogenic interest were prioritized based on heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. A preliminary biochemical analysis of the activity of mitochondrial respiratory complexes was also performed on fresh-frozen GBM samples. Although a high number of mutations was detected, we report that the large majority of them does not pass the prioritization filters. Therefore, a relatively limited burden of pathogenic mutations is indeed carried by GBM, which did not appear to determine a general impairment of the respiratory chain. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

  11. Human 2'-phosphodiesterase localizes to the mitochondrial matrix with a putative function in mitochondrial RNA turnover

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    Poulsen, Jesper Buchhave; Andersen, Kasper Røjkjær; Kjær, Karina Hansen

    2011-01-01

    . Interestingly, 2′-PDE shares both functionally and structurally characteristics with the CCR4-type exonuclease–endonuclease–phosphatase family of deadenylases. Here we show that 2′-PDE locates to the mitochondrial matrix of human cells, and comprise an active 3′–5′ exoribonuclease exhibiting a preference...... a role in the cellular immune system, may also function in mitochondrial RNA turnover....

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

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    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

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

  13. Identification and characterization of the mitochondrial targeting sequence and mechanism in human citrate synthase.

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    Cheng, Tsung-Lin; Liao, Ching-Chun; Tsai, Wen-Hui; Lin, Chin-Chih; Yeh, Chin-Wei; Teng, Chiao-Fang; Chang, Wen-Tsan

    2009-08-01

    Citrate synthase (CS), the first and rate-limiting enzyme of the tricarboxylic acid (TCA) cycle, plays a decisive role in regulating energy generation of mitochondrial respiration. Most mitochondrial proteins are synthesized in the cytoplasm as preproteins with an amino (N)-terminal mitochondrial targeting sequence (MTS) that directs mitochondria-specific sorting of the preprotein. However, the MTS and targeting mechanism of the human CS protein are not fully characterized. The human CS gene is a single nuclear gene which transcribes into two mRNA variants, isoform a (CSa) and b (CSb), by alternative splicing of exon 2. CSa encodes 466 amino acids, including a putative N-terminal MTS, while CSb expresses 400 residues with a shorter N terminus, lacking the MTS. Our results indicated that CSa is localized in the mitochondria and the N-terminal 27 amino acids, including a well-conserved RXY downward arrow (S/A) motif (the RHAS sequence), can efficiently target the enhanced green fluorescent protein (EGFP) into the mitochondria. Furthermore, site-directed mutagenesis analysis of the conserved basic amino acids and serine/threonine residues revealed that the R9 residue is essential but all serine/threonine residues are dispensable in the mitochondrial targeting function. Moreover, RNA interference (RNAi)-mediated gene silencing of the preprotein import receptors, including TOM20, TOM22, and TOM70, showed that all three preprotein import receptors are required for transporting CSa into the mitochondria. In conclusion, we have experimentally identified the mitochondrial targeting sequence of human CSa and elucidated its targeting mechanism. These results provide an important basis for the study of mitochondrial dysfunction due to aberrant CSa trafficking.

  14. An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

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    Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

    2016-04-19

    Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins.

  15. Mitochondrial localization of fission yeast manganese superoxide dismutase is required for its lysine acetylation and for cellular stress resistance and respiratory growth

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    Takahashi, Hidekazu, E-mail: hidetakahashi@riken.jp [Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Suzuki, Takehiro [Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 (Japan); Shirai, Atsuko; Matsuyama, Akihisa [Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Dohmae, Naoshi [Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 (Japan); Yoshida, Minoru, E-mail: yoshidam@riken.jp [Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 (Japan)

    2011-03-04

    Research highlights: {yields} Fission yeast manganese superoxide dismutase (MnSOD) is acetylated. {yields} The mitochondrial targeting sequence (MTS) is required for the acetylation of MnSOD. {yields} The MTS is not crucial for MnSOD activity, but is important for respiratory growth. {yields} Posttranslational regulation of MnSOD differs between budding and fission yeast. -- Abstract: Manganese-dependent superoxide dismutase (MnSOD) is localized in the mitochondria and is important for oxidative stress resistance. Although transcriptional regulation of MnSOD has been relatively well studied, much less is known about the protein's posttranslational regulation. In budding yeast, MnSOD is activated after mitochondrial import by manganese ion incorporation. Here we characterize posttranslational modification of MnSOD in the fission yeast Schizosaccharomyces pombe. Fission yeast MnSOD is acetylated at the 25th lysine residue. This acetylation was diminished by deletion of N-terminal mitochondrial targeting sequence, suggesting that MnSOD is acetylated after import into mitochondria. Mitochondrial localization of MnSOD is not essential for the enzyme activity, but is crucial for oxidative stress resistance and growth under respiratory conditions of fission yeast. These results suggest that, unlike the situation in budding yeast, S. pombe MnSOD is already active even before mitochondrial localization; nonetheless, mitochondrial localization is critical to allow the cell to cope with reactive oxygen species generated inside or outside of mitochondria.

  16. Drosophila clueless is highly expressed in larval neuroblasts, affects mitochondrial localization and suppresses mitochondrial oxidative damage.

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    Aditya Sen

    Full Text Available Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu's specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu's role in mitochondrial function is not critical during larval development, but is important for pupae and adults.

  17. Mitochondrial localization of the low level p53 protein in proliferative cells

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    Ferecatu, Ioana; Bergeaud, Marie; Rodriguez-Enfedaque, Aida; Le Floch, Nathalie [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Oliver, Lisa [INSERM U601, Universite de Nantes, Faculte de Medecine, Nantes Cedex (France); Rincheval, Vincent; Renaud, Flore [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Vallette, Francois M. [INSERM U601, Universite de Nantes, Faculte de Medecine, Nantes Cedex (France); Mignotte, Bernard [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Vayssiere, Jean-Luc, E-mail: jean-luc.vayssiere@uvsq.fr [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France)

    2009-10-02

    p53 protein plays a central role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis through transcription-dependent and -independent mechanisms. Emerging publications suggest that following stress, a fraction of p53 translocates to mitochondria to induce cytochrome c release and apoptosis. However, the localization of p53 under unstressed conditions remains largely unexplored. Here we show that p53 is localized at mitochondria in absence of apoptotic stimuli, when cells are proliferating, localization observed in various cell types (rodent and human). This is also supported by acellular assays in which p53 bind strongly to mitochondria isolated from rat liver. Furthermore, the mitochondria subfractionation study and the alkaline treatment of the mitochondrial p53 revealed that the majority of mitochondrial p53 is present in the membranous compartments. Finally, we identified VDAC, a protein of the mitochondrial outer-membrane, as a putative partner of p53 in unstressed/proliferative cells.

  18. Human REV3 DNA Polymerase Zeta Localizes to Mitochondria and Protects the Mitochondrial Genome.

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    Singh, Bhupendra; Li, Xiurong; Owens, Kjerstin M; Vanniarajan, Ayyasamy; Liang, Ping; Singh, Keshav K

    2015-01-01

    To date, mitochondrial DNA polymerase γ (POLG) is the only polymerase known to be present in mammalian mitochondria. A dogma in the mitochondria field is that there is no other polymerase present in the mitochondria of mammalian cells. Here we demonstrate localization of REV3 DNA polymerase in the mammalian mitochondria. We demonstrate localization of REV3 in the mitochondria of mammalian tissue as well as cell lines. REV3 associates with POLG and mitochondrial DNA and protects the mitochondrial genome from DNA damage. Inactivation of Rev3 leads to reduced mitochondrial membrane potential, reduced OXPHOS activity, and increased glucose consumption. Conversely, inhibition of the OXPHOS increases expression of Rev3. Rev3 expression is increased in human primary breast tumors and breast cancer cell lines. Inactivation of Rev3 decreases cell migration and invasion, and localization of Rev3 in mitochondria increases survival and the invasive potential of cancer cells. Taken together, we demonstrate that REV3 functions in mammalian mitochondria and that mitochondrial REV3 is associated with the tumorigenic potential of cells.

  19. Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

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    Anand, Sanjeev K; Gaba, Amit; Singh, Jaswant; Tikoo, Suresh K

    2014-02-01

    Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

  20. Characterization of mitochondrial populations during stem cell differentiation.

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    Kerscher, Petra; Bussie, Blakely S; DeSimone, Katherine M; Dunn, David A; Lipke, Elizabeth A

    2015-01-01

    Mitochondrial dynamics play an important role in numerous physiological and pathophysiological phenomena in the developing and adult human heart. Alterations in structural aspects of cellular mitochondrial composition as a function of changes in physiology can easily be visualized using fluorescence microscopy. Commonly, mitochondrial location, number, and morphology are reported qualitatively due to the lack of automated and user-friendly computer-based analysis tools. Mitochondrial Quantification using MATLAB (MQM) is a computer-based tool to quantitatively assess these parameters by analyzing fluorescently labeled mitochondria within the cell; in particular, MQM provides numerical information on the number, area, and location of mitochondria within a cell in a time-efficient, automated, and unbiased way. This chapter describes the use of MQM's capabilities to quantify mitochondrial changes during human pluripotent stem cell (hPSC) differentiation into spontaneously contracting cardiomyocytes (SC-CMs), which follows physiological pathways of human heart development.

  1. Yeast mitochondrial biogenesis: a role for the PUF RNA-binding protein Puf3p in mRNA localization.

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    Yann Saint-Georges

    Full Text Available The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators. Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings

  2. Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene.

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    Schneider, Mary D; Bains, Anupinder K; Rajendra, T K; Dominski, Zbigniew; Matera, A Gregory; Simmonds, Andrew J

    2010-11-01

    MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.

  3. Characterization of mitochondrial function in cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.

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    Atlante, Anna; Favia, Maria; Bobba, Antonella; Guerra, Lorenzo; Casavola, Valeria; Reshkin, Stephan Joel

    2016-06-01

    Evidence supporting the occurrence of oxidative stress in Cystic Fibrosis (CF) is well established and the literature suggests that oxidative stress is inseparably linked to mitochondrial dysfunction. Here, we have characterized mitochondrial function, in particular as it regards the steps of oxidative phosphorylation and ROS production, in airway cells either homozygous for the F508del-CFTR allele or stably expressing wt-CFTR. We find that oxygen consumption, ΔΨ generation, adenine nucleotide translocator-dependent ADP/ATP exchange and both mitochondrial Complex I and IV activities are impaired in CF cells, while both mitochondrial ROS production and membrane lipid peroxidation increase. Importantly, treatment of CF cells with the small molecules VX-809 and 4,6,4'-trimethylangelicin, which act as "correctors" for F508del CFTR by rescuing the F508del CFTR-dependent chloride secretion, while having no effect per sè on mitochondrial function in wt-CFTR cells, significantly improved all the above mitochondrial parameters towards values found in the airway cells expressing wt-CFTR. This novel study on mitochondrial bioenergetics provides a springboard for future research to further understand the molecular mechanisms responsible for the involvement of mitochondria in CF and identify the proteins primarily responsible for the F508del-CFTR-dependent mitochondrial impairment and thus reveal potential novel targets for CF therapy.

  4. A critical analysis of the combined usage of protein localization prediction methods: Increasing the number of independent data sets can reduce the accuracy of predicted mitochondrial localization

    Science.gov (United States)

    Lythgow, Kieren T.; Hudson, Gavin; Andras, Peter; Chinnery, Patrick F.

    2011-01-01

    In the absence of a comprehensive experimentally derived mitochondrial proteome, several bioinformatic approaches have been developed to aid the identification of novel mitochondrial disease genes within mapped nuclear genetic loci. Often, many classifiers are combined to increase the sensitivity and specificity of the predictions. Here we show that the greatest sensitivity and specificity are obtained by using a combination of seven carefully selected classifiers. We also show that increasing the number of independent prediction methods can paradoxically decrease the accuracy of predicting mitochondrial localization. This approach will help to accelerate the identification of new mitochondrial disease genes by providing a principled way for the selection for combination of appropriate prediction methods of mitochondrial localization of proteins. PMID:21195798

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

    Directory of Open Access Journals (Sweden)

    Seong-Woon Yu

    2009-10-01

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

  6. 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.

  7. Mitochondrial functional changes characterization in young and senescent human adipose derived MSCs

    Directory of Open Access Journals (Sweden)

    Bernd Robert Stab II

    2016-12-01

    Full Text Available Mitochondria are highly dynamic organelles that in response to the cell’s bio-energetic state continuously undergo structural remodeling fission and fusion processes. This mitochondrial dynamic activity has been implicated in cell cycle, autophagy and age-related diseases. Adult tissue-derived mesenchymal stromal/stem cells present a therapeutic potential. However, to obtain an adequate mesenchymal stromal/stem cell number for clinical use, extensive in vitro expansion is required. Unfortunately, these cells undergo replicative senescence rapidly by mechanisms that are not well understood. Senescence has been associated with metabolic changes in the oxidative state of the cell, a process that has been also linked to mitochondrial fission and fusion events, suggesting an association between mitochondrial dynamic and senescence. In the present work, we studied the mitochondrial structural remodeling process of mesenchymal stromal/stem cells isolated from adipose tissue in vitro to determine if mitochondrial phenotypic changes are associated with mesenchymal stromal/stem cell senescence. For this purpose, mitochondrial dynamics and oxidative state of stromal/stem cell were compared between young and old cells. With increased cell passage, we observed a significant change in cell morphology that is associated with an increase in β-galactosidase activity. In addition, old cells (population doubling seven also showed increased mitochondrial mass, augmented superoxide production, and decreased mitochondrial membrane potential. These changes in morphology were related to slightly levels increases in mitochondrial fusion proteins, Mitofusion 1 (MFN1 and Dynamin-realted GTPase (OPA1. Collectively, our results showed that adipose tissue-derived MSCs at population doubling seven develop a senescent phenotype that is characterized by metabolic cell changes that can lead to mitochondrial fusion.

  8. Bioinspired sensory systems for local flow characterization

    Science.gov (United States)

    Colvert, Brendan; Chen, Kevin; Kanso, Eva

    2016-11-01

    Empirical evidence suggests that many aquatic organisms sense differential hydrodynamic signals.This sensory information is decoded to extract relevant flow properties. This task is challenging because it relies on local and partial measurements, whereas classical flow characterization methods depend on an external observer to reconstruct global flow fields. Here, we introduce a mathematical model in which a bioinspired sensory array measuring differences in local flow velocities characterizes the flow type and intensity. We linearize the flow field around the sensory array and express the velocity gradient tensor in terms of frame-independent parameters. We develop decoding algorithms that allow the sensory system to characterize the local flow and discuss the conditions under which this is possible. We apply this framework to the canonical problem of a circular cylinder in uniform flow, finding excellent agreement between sensed and actual properties. Our results imply that combining suitable velocity sensors with physics-based methods for decoding sensory measurements leads to a powerful approach for understanding and developing underwater sensory systems.

  9. Characterization of mitochondrial haplogroups in a large population-based sample from the United States.

    Science.gov (United States)

    Mitchell, Sabrina L; Goodloe, Robert; Brown-Gentry, Kristin; Pendergrass, Sarah A; Murdock, Deborah G; Crawford, Dana C

    2014-07-01

    Mitochondrial DNA (mtDNA) haplogroups are valuable for investigations in forensic science, molecular anthropology, and human genetics. In this study, we developed a custom panel of 61 mtDNA markers for high-throughput classification of European, African, and Native American/Asian mitochondrial haplogroup lineages. Using these mtDNA markers, we constructed a mitochondrial haplogroup classification tree and classified 18,832 participants from the National Health and Nutrition Examination Surveys (NHANES). To our knowledge, this is the largest study to date characterizing mitochondrial haplogroups in a population-based sample from the United States, and the first study characterizing mitochondrial haplogroup distributions in self-identified Mexican Americans separately from Hispanic Americans of other descent. We observed clear differences in the distribution of maternal genetic ancestry consistent with proposed admixture models for these subpopulations, underscoring the genetic heterogeneity of the United States Hispanic population. The mitochondrial haplogroup distributions in the other self-identified racial/ethnic groups within NHANES were largely comparable to previous studies. Mitochondrial haplogroup classification was highly concordant with self-identified race/ethnicity (SIRE) in non-Hispanic whites (94.8 %), but was considerably lower in admixed populations including non-Hispanic blacks (88.3 %), Mexican Americans (81.8 %), and other Hispanics (61.6 %), suggesting SIRE does not accurately reflect maternal genetic ancestry, particularly in populations with greater proportions of admixture. Thus, it is important to consider inconsistencies between SIRE and genetic ancestry when performing genetic association studies. The mitochondrial haplogroup data that we have generated, coupled with the epidemiologic variables in NHANES, is a valuable resource for future studies investigating the contribution of mtDNA variation to human health and disease.

  10. Cannabinoid CB1 Receptors Are Localized in Striated Muscle Mitochondria and Regulate Mitochondrial Respiration

    Directory of Open Access Journals (Sweden)

    Juan Mendizabal-Zubiaga

    2016-10-01

    Full Text Available The cannabinoid type 1 (CB1 receptor is widely distributed in the brain and peripheral organs where it regulates cellular functions and metabolism. In the brain, CB1 is mainly localized on presynaptic axon terminals but is also found on mitochondria (mtCB1, where it regulates cellular respiration and energy production. Likewise, CB1 is localized on muscle mitochondria, but very little is known about it. The aim of this study was to further investigate in detail the distribution and functional role of mtCB1 in three different striated muscles. Immunoelectron microscopy for CB1 was used in skeletal muscles (gastrocnemius and rectus abdominis and myocardium from wild-type and CB1-KO mice. Functional assessments were performed in mitochondria purified from the heart of the mice and the mitochondrial oxygen consumption upon application of different acute delta-9-tetrahidrocannabinol (Δ9-THC concentrations (100 nM or 200 nM was monitored. About 26% of the mitochondrial profiles in gastrocnemius, 22% in the rectus abdominis and 17% in the myocardium expressed CB1. Furthermore, the proportion of mtCB1 versus total CB1 immunoparticles was about 60% in the gastrocnemius, 55% in the rectus abdominis and 78% in the myocardium. Importantly, the CB1 immunolabeling pattern disappeared in muscles of CB1-KO mice. Functionally, acute 100 nM or 200 nM THC treatment specifically decreased mitochondria coupled respiration between 12% and 15% in wild-type isolated mitochondria of myocardial muscles but no significant difference was noticed between THC treated and vehicle in mitochondria isolated from CB1-KO heart. Furthermore, gene expression of key enzymes involved in pyruvate synthesis, tricarboxylic acid (TCA cycle and mitochondrial respiratory chain was evaluated in the striated muscle of CB1-WT and CB1-KO. CB1-KO showed an increase in the gene expression of Eno3, Pkm2, and Pdha1, suggesting an increased production of pyruvate. In contrast, no significant

  11. Cannabinoid CB1 Receptors Are Localized in Striated Muscle Mitochondria and Regulate Mitochondrial Respiration

    Science.gov (United States)

    Mendizabal-Zubiaga, Juan; Melser, Su; Bénard, Giovanni; Ramos, Almudena; Reguero, Leire; Arrabal, Sergio; Elezgarai, Izaskun; Gerrikagoitia, Inmaculada; Suarez, Juan; Rodríguez De Fonseca, Fernando; Puente, Nagore; Marsicano, Giovanni; Grandes, Pedro

    2016-01-01

    The cannabinoid type 1 (CB1) receptor is widely distributed in the brain and peripheral organs where it regulates cellular functions and metabolism. In the brain, CB1 is mainly localized on presynaptic axon terminals but is also found on mitochondria (mtCB1), where it regulates cellular respiration and energy production. Likewise, CB1 is localized on muscle mitochondria, but very little is known about it. The aim of this study was to further investigate in detail the distribution and functional role of mtCB1 in three different striated muscles. Immunoelectron microscopy for CB1 was used in skeletal muscles (gastrocnemius and rectus abdominis) and myocardium from wild-type and CB1-KO mice. Functional assessments were performed in mitochondria purified from the heart of the mice and the mitochondrial oxygen consumption upon application of different acute delta-9-tetrahydrocannabinol (Δ9-THC) concentrations (100 nM or 200 nM) was monitored. About 26% of the mitochondrial profiles in gastrocnemius, 22% in the rectus abdominis and 17% in the myocardium expressed CB1. Furthermore, the proportion of mtCB1 versus total CB1 immunoparticles was about 60% in the gastrocnemius, 55% in the rectus abdominis and 78% in the myocardium. Importantly, the CB1 immunolabeling pattern disappeared in muscles of CB1-KO mice. Functionally, acute 100 nM or 200 nM THC treatment specifically decreased mitochondria coupled respiration between 12 and 15% in wild-type isolated mitochondria of myocardial muscles but no significant difference was noticed between THC treated and vehicle in mitochondria isolated from CB1-KO heart. Furthermore, gene expression of key enzymes involved in pyruvate synthesis, tricarboxylic acid (TCA) cycle and mitochondrial respiratory chain was evaluated in the striated muscle of CB1-WT and CB1-KO. CB1-KO showed an increase in the gene expression of Eno3, Pkm2, and Pdha1, suggesting an increased production of pyruvate. In contrast, no significant difference was

  12. Functional localization of two poly(ADP-ribose)-degrading enzymes to the mitochondrial matrix.

    Science.gov (United States)

    Niere, Marc; Kernstock, Stefan; Koch-Nolte, Friedrich; Ziegler, Mathias

    2008-01-01

    Recent discoveries of NAD-mediated regulatory processes in mitochondria have documented important roles of this compartmentalized nucleotide pool in addition to energy transduction. Moreover, mitochondria respond to excessive nuclear NAD consumption arising from DNA damage-induced poly-ADP-ribosylation because poly(ADP-ribose) (PAR) can trigger the release of apoptosis-inducing factor from the organelles. To functionally assess mitochondrial NAD metabolism, we overexpressed the catalytic domain of nuclear PAR polymerase 1 (PARP1) and targeted it to the matrix, which resulted in the constitutive presence of PAR within the organelles. As a result, stably transfected HEK293 cells exhibited a decrease in NAD content and typical features of respiratory deficiency. Remarkably, inhibiting PARP activity revealed PAR degradation within mitochondria. Two enzymes, PAR glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3), are known to cleave PAR. Both full-length ARH3 and a PARG isoform, which arises from alternative splicing, localized to the mitochondrial matrix. This conclusion was based on the direct demonstration of their PAR-degrading activity within mitochondria of living cells. The visualization of catalytic activity establishes a new approach to identify submitochondrial localization of proteins involved in the metabolism of NAD derivatives. In addition, targeted PARP expression may serve as a compartment-specific "knock-down" of the NAD content which is readily detectable by PAR formation.

  13. Genetic characterization of Phytophthora nicotianae by the analysis of polymorphic regions of the mitochondrial DNA.

    Science.gov (United States)

    A new method based on the analysis of mitochondrial intergenic regions characterized by intraspecific variation in DNA sequences was developed and applied to the study of the plant pathogen Phytophthora nicotianae. Two regions flanked by genes trny and rns and trnw and cox2 were identified by compa...

  14. Molecular cloning of rat acss3 and characterization of mammalian propionyl-CoA synthetase in the liver mitochondrial matrix.

    Science.gov (United States)

    Yoshimura, Yukihiro; Araki, Aya; Maruta, Hitomi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-12-21

    Among the three acyl-CoA synthetase short-chain family members (ACSS), ACSS3 is poorly characterized. To characterize ACSS3, we performed molecular cloning and protein expression of rat acss3 and determined its intracellular localization, tissue distribution, and substrate specificity. Transient expression of rat ACSS3 in HeLa cells resulted in a 10-fold increase of acetyl-CoA synthetase activity compared with that in control cells. The acss3 transcripts are expressed in a wide range of tissues, with the highest levels observed in liver tissue followed by kidney tissue. Subcellular fractionation using liver tissue showed that ACSS3 is localized into the mitochondrial matrix. Among the short-chain fatty acids examined, recombinant ACSS3, purified from Escherichia coli cells transformed with the plasmid containing rat acss3, preferentially utilized propionate with a KM value of 0.19 mM. Knockdown of acss3 in HepG2 cells resulted in a significant decrease of ACSS3 expression level and propionyl-CoA synthetase activity in cell lysates. Levels of ACSS3 in the liver and the activity of propionyl-CoA synthetase in the mitochondria were significantly increased by fasting. These results suggested that ACSS3 is a liver mitochondrial matrix enzyme with high affinity to propionic acid, and its expression level is upregulated under ketogenic conditions.

  15. Characterization of nucleotide misincorporation patterns in the iceman's mitochondrial DNA.

    Directory of Open Access Journals (Sweden)

    Cristina Olivieri

    Full Text Available BACKGROUND: The degradation of DNA represents one of the main issues in the genetic analysis of archeological specimens. In the recent years, a particular kind of post-mortem DNA modification giving rise to nucleotide misincorporation ("miscoding lesions" has been the object of extensive investigations. METHODOLOGY/PRINCIPAL FINDINGS: To improve our knowledge regarding the nature and incidence of ancient DNA nucleotide misincorporations, we have utilized 6,859 (629,975 bp mitochondrial (mt DNA sequences obtained from the 5,350-5,100-years-old, freeze-desiccated human mummy popularly known as the Tyrolean Iceman or Otzi. To generate the sequences, we have applied a mixed PCR/pyrosequencing procedure allowing one to obtain a particularly high sequence coverage. As a control, we have produced further 8,982 (805,155 bp mtDNA sequences from a contemporary specimen using the same system and starting from the same template copy number of the ancient sample. From the analysis of the nucleotide misincorporation rate in ancient, modern, and putative contaminant sequences, we observed that the rate of misincorporation is significantly lower in modern and putative contaminant sequence datasets than in ancient sequences. In contrast, type 2 transitions represent the vast majority (85% of the observed nucleotide misincorporations in ancient sequences. CONCLUSIONS/SIGNIFICANCE: This study provides a further contribution to the knowledge of nucleotide misincorporation patterns in DNA sequences obtained from freeze-preserved archeological specimens. In the Iceman system, ancient sequences can be clearly distinguished from contaminants on the basis of nucleotide misincorporation rates. This observation confirms a previous identification of the ancient mummy sequences made on a purely phylogenetical basis. The present investigation provides further indication that the majority of ancient DNA damage is reflected by type 2 (cytosine

  16. Mitochondrial localized STAT3 is involved in NGF induced neurite outgrowth.

    Directory of Open Access Journals (Sweden)

    Lihan Zhou

    Full Text Available BACKGROUND: Signal transducer and activator of transcription 3 (STAT3 plays critical roles in neural development and is increasingly recognized as a major mediator of injury response in the nervous system. Cytokines and growth factors are known to phosphorylate STAT3 at tyrosine(705 with or without the concomitant phosphorylation at serine(727, resulting in the nuclear localization of STAT3 and subsequent transcriptional activation of genes. Recent evidence suggests that STAT3 may control cell function via alternative mechanisms independent of its transcriptional activity. Currently, the involvement of STAT3 mono-phosphorylated at residue serine(727 (P-Ser-STAT3 in neurite outgrowth and the underlying mechanism is largely unknown. PRINCIPAL FINDINGS: In this study, we investigated the role of nerve growth factor (NGF induced P-Ser-STAT3 in mediating neurite outgrowth. NGF induced the phosphorylation of residue serine(727 but not tyrosine(705 of STAT3 in PC12 and primary cortical neuronal cells. In PC12 cells, serine but not tyrosine dominant negative mutant of STAT3 was found to impair NGF induced neurite outgrowth. Unexpectedly, NGF induced P-Ser-STAT3 was localized to the mitochondria but not in the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NGF induced neurite outgrowth and the production of reactive oxygen species (ROS. CONCLUSION: Taken together, the findings herein demonstrated a hitherto unrecognized novel transcription independent mechanism whereby the mitochondria localized P-Ser-STAT3 is involved in NGF induced neurite outgrowth.

  17. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    NARCIS (Netherlands)

    Matthews, G.D.; Gur, N.; Koopman, W.J.H.; Pines, O.; Vardimon, L.

    2010-01-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS

  18. Mitochondrial localization of the mevalonate pathway enzyme 3-Hydroxy-3-methyl-glutaryl-CoA reductase in the Trypanosomatidae

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Montalvetti, Andrea; Flores, Carmen-Lisset

    2004-01-01

    3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eu...... that the parasite enzyme exhibits a mitochondrial localization, suggesting a conservation between the targeting signals of both organisms....

  19. Monomer DJ-1 and its N-terminal sequence are necessary for mitochondrial localization of DJ-1 mutants.

    Directory of Open Access Journals (Sweden)

    Chinatsu Maita

    Full Text Available DJ-1 is a novel oncogene and also a causative gene for familial Parkinson's disease (park7. DJ-1 has multiple functions that include transcriptional regulation, anti-oxidative reaction and chaperone and mitochondrial regulation. Mitochondrial dysfunction is observed in DJ-1-knockout mice and fry, and mitochondrial DJ-1 is more protective against oxidative stress-induced cell death. Although translocation of DJ-1 into mitochondria is enhanced by oxidative stress that leads to oxidation of cysteine 106 (C106 of DJ-1, the characteristics of mitochondrial DJ-1 and the mechanism by which DJ-1 is translocated into mitochondria are poorly understood. In this study, immunostaining, co-immunoprecipitation, cell fractionation and pull-down experiments showed that mutants of glutamine 18 (E18 DJ-1 are localized in mitochondria and do not make homodimers. Likewise, DJ-1 with mutations of two cysteines located in the dimer interface, C46S and C53A, and pathogenic mutants, M26I and L166P DJ-1, were found to be localized in mitochondria and not to make homodimers. Mutant DJ-1 harboring both E18A and C106S, in which C106 is not oxidized, was also localized in mitochondria, indicating that oxidation of C106 is important but not essential for mitochondrial localization of DJ-1. It should be noted that E18A DJ-1 was translocated from mitochondria to the cytoplasm when mitochondrial membrane potential was reduced by treatment of cells with CCCP, an uncoupler of the oxidative phosphorylation system in mitochondria. Furthermore, deletion or substitution of the N-terminal 12 amino acids in DJ-1 resulted in re-localization of E18A, M26I and L166P DJ-1 from mitochondria into the cytoplasm. These findings suggest that a monomer and the N-terminal 12 amino acids are necessary for mitochondrial localization of DJ-1 mutants and that conformation change induced by C106 oxidation or by E18 mutation leads to translocation of DJ-1 into mitochondria.

  20. Monomer DJ-1 and Its N-Terminal Sequence Are Necessary for Mitochondrial Localization of DJ-1 Mutants

    Science.gov (United States)

    Maita, Chinatsu; Maita, Hiroshi; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2013-01-01

    DJ-1 is a novel oncogene and also a causative gene for familial Parkinson’s disease (park7). DJ-1 has multiple functions that include transcriptional regulation, anti-oxidative reaction and chaperone and mitochondrial regulation. Mitochondrial dysfunction is observed in DJ-1-knockout mice and fry, and mitochondrial DJ-1 is more protective against oxidative stress-induced cell death. Although translocation of DJ-1 into mitochondria is enhanced by oxidative stress that leads to oxidation of cysteine 106 (C106) of DJ-1, the characteristics of mitochondrial DJ-1 and the mechanism by which DJ-1 is translocated into mitochondria are poorly understood. In this study, immunostaining, co-immunoprecipitation, cell fractionation and pull-down experiments showed that mutants of glutamine 18 (E18) DJ-1 are localized in mitochondria and do not make homodimers. Likewise, DJ-1 with mutations of two cysteines located in the dimer interface, C46S and C53A, and pathogenic mutants, M26I and L166P DJ-1, were found to be localized in mitochondria and not to make homodimers. Mutant DJ-1 harboring both E18A and C106S, in which C106 is not oxidized, was also localized in mitochondria, indicating that oxidation of C106 is important but not essential for mitochondrial localization of DJ-1. It should be noted that E18A DJ-1 was translocated from mitochondria to the cytoplasm when mitochondrial membrane potential was reduced by treatment of cells with CCCP, an uncoupler of the oxidative phosphorylation system in mitochondria. Furthermore, deletion or substitution of the N-terminal 12 amino acids in DJ-1 resulted in re-localization of E18A, M26I and L166P DJ-1 from mitochondria into the cytoplasm. These findings suggest that a monomer and the N-terminal 12 amino acids are necessary for mitochondrial localization of DJ-1 mutants and that conformation change induced by C106 oxidation or by E18 mutation leads to translocation of DJ-1 into mitochondria. PMID:23326576

  1. Identification and characterization of a mitochondrial unfolded protein response transcription factor ATFS-1 in Litopenaeus vannamei.

    Science.gov (United States)

    Chen, Yong-Gui; Yue, Hai-Tao; Zhang, Ze-Zhi; Yuan, Feng-Hua; Bi, Hai-Tao; Yuan, Kai; Weng, Shao-Ping; He, Jian-Guo; Chen, Yi-Hong

    2016-07-01

    A mitochondrial specific stress response termed mitochondrial unfolded protein response (UPR(mt)) is activated in responding to disturbance of protein homeostasis in mitochondria. The activating transcription factor associated with stress-1 (designated as ATFS-1) is the key regulator of UPR(mt). To investigating the roles of ATFS-1 (LvATFS-1) in Litopenaeus vannamei mitochondrial stress remission and immunity, it's full length cDNA was cloned. The open reading frame of LvATFS-1 was 1, 557 bp in length, deducing to a 268 amino acids protein. LvATFS-1 was highly expressed in muscle, hemocytes and eyestalk. Subcellular location assays showed that N-terminal of LvATFS-1 contained a mitochondrial targeting sequence, which could directed the fused EGFP located to mitochondria. And the C-terminal of LvATFS-1, which had a nuclear localization signal, expressed in nucleus. The in vitro experiments verified that LvATFS-1 could reduced the level of intracellular reactive oxygen species (ROS). And results of real-time RT-PCR indicated that LvATFS-1 might scavenge excess ROS via ROS-eliminating genes regulation. Reporter gene assays showed that LvATFS-1 could upregulated the expression of the antimicrobial peptide genes in Drosophila Schneider 2 cells. Results of real-time RT-PCR showed that Vibrio alginolyticus or white spot syndrome virus (WSSV) infection induced the expression of LvATFS-1. And knocked-down LvATFS-1 by RNAi resulted in a higher cumulative mortality of L. vannamei upon V. alginolyticus or WSSV infection. These results suggested that LvATFS-1 not only rolled in mitochondrial specific stress responding, but also important for L. vannamei immunologic defence.

  2. Chromosomal localization of mitochondrial transcription factor A (TCF6), single-stranded DNA-binding protein (SSBP), and endonuclease G (ENDOG), three human housekeeping genes involving in mitochondrial biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Tiranti, V.; Rossi, G.; DiDonato, S. [Istituto Nazionale Neurologico, Carlo Besta (Italy)] [and others

    1995-01-20

    By using a PCR-based screening of a somatic cell hybrid panel and FISH, we have assigned the loci of mitochondrial single-stranded DNA-binding protein (SSBP), mitochondrial transcription factor A (TCF6), and mitochondrial endonuclease G (ENDOG) genes to human chromosomes 7q34, 10q21, and 9q34.1, respectively. The products of these three genes are involved in fundamental aspects of mitochondrial biogenesis, such as replication and transcription of the mitochondrial genome. The chromosomal localization of these genes is important to testing whether the corresponding proteins may play a role in the etiopathogenesis of human disorders associated with qualitative or quantitative abnormalities of mitochondrial DNA. 20 refs., 1 fig., 2 tabs.

  3. Protein structure search and local structure characterization

    Directory of Open Access Journals (Sweden)

    Ku Shih-Yen

    2008-08-01

    Full Text Available Abstract Background Structural similarities among proteins can provide valuable insight into their functional mechanisms and relationships. As the number of available three-dimensional (3D protein structures increases, a greater variety of studies can be conducted with increasing efficiency, among which is the design of protein structural alphabets. Structural alphabets allow us to characterize local structures of proteins and describe the global folding structure of a protein using a one-dimensional (1D sequence. Thus, 1D sequences can be used to identify structural similarities among proteins using standard sequence alignment tools such as BLAST or FASTA. Results We used self-organizing maps in combination with a minimum spanning tree algorithm to determine the optimum size of a structural alphabet and applied the k-means algorithm to group protein fragnts into clusters. The centroids of these clusters defined the structural alphabet. We also developed a flexible matrix training system to build a substitution matrix (TRISUM-169 for our alphabet. Based on FASTA and using TRISUM-169 as the substitution matrix, we developed the SA-FAST alignment tool. We compared the performance of SA-FAST with that of various search tools in database-scale search tasks and found that SA-FAST was highly competitive in all tests conducted. Further, we evaluated the performance of our structural alphabet in recognizing specific structural domains of EGF and EGF-like proteins. Our method successfully recovered more EGF sub-domains using our structural alphabet than when using other structural alphabets. SA-FAST can be found at http://140.113.166.178/safast/. Conclusion The goal of this project was two-fold. First, we wanted to introduce a modular design pipeline to those who have been working with structural alphabets. Secondly, we wanted to open the door to researchers who have done substantial work in biological sequences but have yet to enter the field of protein

  4. Mitochondrial Localized Stat3 Promotes Breast Cancer Growth via Phosphorylation of Serine 727*

    Science.gov (United States)

    Zhang, Qifang; Raje, Vidisha; Yakovlev, Vasily A.; Yacoub, Adly; Szczepanek, Karol; Meier, Jeremy; Derecka, Marta; Chen, Qun; Hu, Ying; Sisler, Jennifer; Hamed, Hossein; Lesnefsky, Edward J.; Valerie, Kristoffer; Dent, Paul; Larner, Andrew C.

    2013-01-01

    Signal transducer and activator of transcription 3 (Stat3) is a key mediator in the development of many cancers. For 20 years, it has been assumed that Stat3 mediates its biological activities as a nuclear localized transcription factor activated by many cytokines. However, recent studies from this laboratory and others indicate that Stat3 has an independent function in the mitochondria (mitoStat3) where it controls the activity of the electron transport chain (ETC) and mediates Ras-induced transformation of mouse embryo fibroblasts. The actions of mitoStat3 in controlling respiration and Ras transformation are mediated by the phosphorylation state of serine 727. To address the role of mitoStat3 in the pathogenesis of cells that are transformed, we used 4T1 breast cancer cells, which form tumors that metastasize in immunocompetent mice. Substitution of Ser-727 for an alanine or aspartate in Stat3 that has a mitochondrial localization sequence, MLS-Stat3, has profound effects on tumor growth, complex I activity of the ETC, and accumulation of reactive oxygen species (ROS). Cells expressing MLS-Stat3(S727A) display slower tumor growth, decreased complex I activity of the ETC, and increased ROS accumulation under hypoxia compared with cells expressing MLS-Stat3. In contrast, cells expressing MLS-Stat3(S727D) show enhanced tumor growth and complex I activity and decreased production of ROS. These results highlight the importance of serine 727 of mitoStat3 in breast cancer and suggest a novel role for mitoStat3 in regulation of ROS concentrations through its action on the ETC. PMID:24019511

  5. Mitochondrial genome variations and functional characterization in Han Chinese families with schizophrenia.

    Science.gov (United States)

    Bi, Rui; Tang, Jinsong; Zhang, Wen; Li, Xiao; Chen, Shi-Yi; Yu, Dandan; Chen, Xiaogang; Yao, Yong-Gang

    2016-03-01

    The relationship between mitochondrial DNA (mtDNA) variants and schizophrenia has been strongly debated. To test whether mtDNA variants are involved in schizophrenia in Han Chinese patients, we sequenced the entire mitochondrial genomes of probands from 11 families with a family history and maternal inheritance pattern of schizophrenia. Besides the haplogroup-specific variants, we found 11 nonsynonymous private variants, one rRNA variant, and one tRNA variant in 5 of 11 probands. Among the nonsynonymous private variants, mutations m.15395 A>G and m.8536 A>G were predicted to be deleterious after web-based searches and in silico program affiliated analysis. Functional characterization further supported the potential pathogenicity of the two variants m.15395 A>G and m.8536 A>G to cause mitochondrial dysfunction at the cellular level. Our results showed that mtDNA variants were actively involved in schizophrenia in some families with maternal inheritance of this disease.

  6. Isolation, expression, and chromosomal localization of the human mitochondrial capsule selenoprotein gene (MCSP)

    Energy Technology Data Exchange (ETDEWEB)

    Aho, Hanne; Schwemmer, M.; Tessmann, D.; Murphy, D. [Institut fuer Humangenetik der Universitaet, Goettingen (Germany)] [and others

    1996-03-01

    The mitochondrial capsule selenoprotein (MCS) (HGMW-approved symbol MCSP) is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. We describe here the isolation of a cDNA, the exon-intron organization, the expression, and the chromosomal localization of the human MCS gene. Nucleotide sequence analysis of the human and mouse MCS cDNAs reveals that the 5{prime}- and 3{prime}-untranslated sequences are more conserved (71%) than the coding sequences (59%). The open reading frame encodes a 116-amino-acid protein and lacks the UGA codons, which have been reported to encode the selenocysteines in the N-terminal of the deduced mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein (39%). The most striking homology lies in the dicysteine motifs. Northern and Southern zooblot analyses reveal that the MCS gene in human, baboon, and bovine is more conserved than its counterparts in mouse and rat. The single intron in the human MCS gene is approximately 6 kb and interrupts the 5{prime}-untranslated region at a position equivalent to that in the mouse and rat genes. Northern blot and in situ hybridization experiments demonstrate that the expression of the human MCS gene is restricted to haploid spermatids. The human gene was assigned to q21 of chromosome 1. 30 refs., 9 figs.

  7. Characterization of human mitochondrial ferritin promoter: identification of transcription factors and evidences of epigenetic control

    Science.gov (United States)

    Guaraldo, Michela; Santambrogio, Paolo; Rovelli, Elisabetta; di Savino, Augusta; Saglio, Giuseppe; Cittaro, Davide; Roetto, Antonella; Levi, Sonia

    2016-09-01

    Mitochondrial ferritin (FtMt) is an iron storage protein belonging to the ferritin family but, unlike the cytosolic ferritin, it has an iron-unrelated restricted tissue expression. FtMt appears to be preferentially expressed in cell types characterized by high metabolic activity and oxygen consumption, suggesting a role in protecting mitochondria from iron-dependent oxidative damage. The human gene (FTMT) is intronless and its promoter region has not been described yet. To analyze the regulatory mechanisms controlling FTMT expression, we characterized the 5‧ flanking region upstream the transcriptional starting site of FTMT by in silico enquiry of sequences conservation, DNA deletion analysis, and ChIP assay. The data revealed a minimal promoter region and identified the presence of SP1, CREB and YY1 as positive regulators, and GATA2, FoxA1 and C/EBPβ as inhibitors of the transcriptional regulation. Furthermore, the FTMT transcription is increased by acetylating and de-methylating agent treatments in K562 and HeLa cells. These treatments up-regulate FtMt expression even in fibroblasts derived from a Friedreich ataxia patient, where it might exert a beneficial effect against mitochondrial oxidative damage. The expression of FTMT appears regulated by a complex mechanism involving epigenetic events and interplay between transcription factors.

  8. Characterization of Thin Films Using Local Magneometer

    CERN Document Server

    Katzan N.

    2016-01-01

    SIS nanocomposite (Superconductor/Insulator/Superconductor) could improve the efficiency of bulk Nb accelerating cavities as proposed in 2006 by A. Gurevich [1]. The SRF multilayers concept takes advantage of the enhancement of HC1 of thin layers with thickness d~. The use of thin layers makes it easier to prevent avalanche penetration of vortices in case of local defects that could promote early penetration. The external field is not fully attenuated in such configuration, so several layers are necessary in order to screen the external field down to values below Nb HC1, decoupled from each other with a dielectric layer. Many deposition techniques exist that can allow the deposition of such multilayers but a few of them are adapted for accelerating cavities shapes. Moreover we do not know yet how the predicted properties evolve in realistic deposition conditions. It seems reasonable to start the optimization of such structure on samples. Two parameters need to be measured to predict their behavior in condi...

  9. Molecular cloning, functional characterization, and subcellular localization of soybean nodule dihydrolipoamide reductase.

    Science.gov (United States)

    Moran, Jose F; Sun, Zhaohui; Sarath, Gautam; Arredondo-Peter, Raúl; James, Euan K; Becana, Manuel; Klucas, Robert V

    2002-01-01

    Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH.

  10. Mutations of C19orf12, coding for a transmembrane glycine zipper containing mitochondrial protein, cause mis-localization of the protein, inability to respond to oxidative stress and increased mitochondrial Ca²⁺.

    Science.gov (United States)

    Venco, Paola; Bonora, Massimo; Giorgi, Carlotta; Papaleo, Elena; Iuso, Arcangela; Prokisch, Holger; Pinton, Paolo; Tiranti, Valeria

    2015-01-01

    Mutations in C19orf12 have been identified in patients affected by Neurodegeneration with Brain Iron Accumulation (NBIA), a clinical entity characterized by iron accumulation in the basal ganglia. By using western blot analysis with specific antibody and confocal studies, we showed that wild-type C19orf12 protein was not exclusively present in mitochondria, but also in the Endoplasmic Reticulum (ER) and MAM (Mitochondria Associated Membrane), while mutant C19orf12 variants presented a different localization. Moreover, after induction of oxidative stress, a GFP-tagged C19orf12 wild-type protein was able to relocate to the cytosol. On the contrary, mutant isoforms were not able to respond to oxidative stress. High mitochondrial calcium concentration and increased H2O2 induced apoptosis were found in fibroblasts derived from one patient as compared to controls. C19orf12 protein is a 17 kDa mitochondrial membrane-associated protein whose function is still unknown. Our in silico investigation suggests that, the glycine zipper motifs of C19orf12 form helical regions spanning the membrane. The N- and C-terminal regions with respect to the transmembrane portion, on the contrary, are predicted to rearrange in a structural domain, which is homologs to the N-terminal regulatory domain of the magnesium transporter MgtE, suggesting that C19orf12 may act as a regulatory protein for human MgtE transporters. The mutations here described affect respectively one glycine residue of the glycine zipper motifs, which are involved in dimerization of transmembrane helices and predicted to impair the correct localization of the protein into the membranes, and one residue present in the regulatory domain, which is important for protein-protein interaction.

  11. Mutations of C19orf12, coding for a transmembrane glycine zipper containing mitochondrial protein, cause mis-localization of the protein, inability to respond to oxidative stress and increased mitochondrial Ca2+

    Science.gov (United States)

    Venco, Paola; Bonora, Massimo; Giorgi, Carlotta; Papaleo, Elena; Iuso, Arcangela; Prokisch, Holger; Pinton, Paolo; Tiranti, Valeria

    2015-01-01

    Mutations in C19orf12 have been identified in patients affected by Neurodegeneration with Brain Iron Accumulation (NBIA), a clinical entity characterized by iron accumulation in the basal ganglia. By using western blot analysis with specific antibody and confocal studies, we showed that wild-type C19orf12 protein was not exclusively present in mitochondria, but also in the Endoplasmic Reticulum (ER) and MAM (Mitochondria Associated Membrane), while mutant C19orf12 variants presented a different localization. Moreover, after induction of oxidative stress, a GFP-tagged C19orf12 wild-type protein was able to relocate to the cytosol. On the contrary, mutant isoforms were not able to respond to oxidative stress. High mitochondrial calcium concentration and increased H2O2 induced apoptosis were found in fibroblasts derived from one patient as compared to controls. C19orf12 protein is a 17 kDa mitochondrial membrane-associated protein whose function is still unknown. Our in silico investigation suggests that, the glycine zipper motifs of C19orf12 form helical regions spanning the membrane. The N- and C-terminal regions with respect to the transmembrane portion, on the contrary, are predicted to rearrange in a structural domain, which is homologs to the N-terminal regulatory domain of the magnesium transporter MgtE, suggesting that C19orf12 may act as a regulatory protein for human MgtE transporters. The mutations here described affect respectively one glycine residue of the glycine zipper motifs, which are involved in dimerization of transmembrane helices and predicted to impair the correct localization of the protein into the membranes, and one residue present in the regulatory domain, which is important for protein-protein interaction. PMID:26136767

  12. Characterization of the complete mitochondrial genome of flower-breeding Drosophila incompta (Diptera, Drosophilidae).

    Science.gov (United States)

    De Ré, F C; Wallau, G L; Robe, L J; Loreto, E L S

    2014-12-01

    Drosophila incompta belongs to the flavopilosa group of Drosophila, and has a restricted ecology, being adapted to flowers of Cestrum as feeding and oviposition sites. We sequenced, assembled, and characterized the complete mitochondrial genome (mtDNA) of D. incompta. In addition, we performed phylogenomic and polymorphism analyses to assess evolutionary diversification of this species. Our results suggest that this genome is syntenic with the other published mtDNA of Drosophila. This molecule contains 15,641 bp and encompasses two rRNA, 22 tRNA and 13 protein-coding genes. Regarding nucleotide composition, we found a high A-T bias (76.6 %). The recovered phylogenies indicate D. incompta in the virilis-repleta radiation, as sister to the virilis or repleta groups. The most interesting result is the high degree of polymorphism found throughout the D. incompta mitogenome, revealing pronounced intrapopulational variation. Furthermore, intraspecific nucleotide diversity levels varied between different regions of the genome, thus allowing the use of different mitochondrial molecular markers for analysis of population structure of this species.

  13. Mitochondrial DNA COI characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) from Paraguay and Uruguay.

    Science.gov (United States)

    Arnemann, J A; James, W J; Walsh, T K; Guedes, J V C; Smagghe, G; Castiglioni, E; Tay, W T

    2016-04-07

    Since its detection in Brazil in 2013, the Old World cotton bollworm Helicoverpa armigera has been reported in Argentina, Paraguay, and Bolivia. Here we present evidence extending the South American range of H. armigera to Uruguay, using polymerase chain reaction and sequencing of the partial mitochondrial DNA (mtDNA) cytochrome oxidase I region. Molecular characterization of this gene region from individuals from Paraguay also supports previous morphological identification of H. armigera in Paraguay. Shared mtDNA haplotypes in H. armigera from Brazil, Uruguay, and Paraguay were identified. Additional surveying of populations in this region will be imperative to better monitor and understand factors that are underpinning its presence and successful adaptation in these South American regions. We discuss our findings with respect to the development of resistance pest management strategies of this invasive insect pest in a predominantly monoculture soybean crop landscape in the Southern Cone region.

  14. Cloning, characterization, and expression of Cytochrome b (Cytb)-a key mitochondrial gene from Prorocentrum donghaiense

    Institute of Scientific and Technical Information of China (English)

    ZHAO Liyuan; MI Tiezhu; ZHEN Yu; YU Zhigang

    2012-01-01

    Mitochondrial cytochrome b (Cytb),one of the few proteins encoded by the mitochondrial DNA,plays an important role in transferring electrons.As a mitochondrial gene,it has been widely used for phylogenetic analysis.Previously,a 949-bp fragment of the coding gene and mRNA editing were characterized from Prorocentrum donghaiense,which might prove useful for resolving P.donghaiense from closely related species.However,the full-length coding region has not been characterized.In this study,we used rapid amplification of cDNA ends (RACE) to obtain full-length,1124 bp cDNA.Cytb transcript contained a standard initiation codon ATG,but did not have a recognizable stop codon.Homology comparison showed that the P.donghaiense Cytb had a high sequence identity to Cytb sequences from other dinoflagellate species.Phylogenetic analysis placed Cytb from P.donghaiense in the clade of dinofiagellates and it clustered together strongly with that from P.minimum.Based on the full-length sequence,we inferred 32 editing events at different positions,accounting for 2.93% of the Cytb gent.34.4% (11) of the changes were A to G,25% (8) were T to C,and 25% (8) were C to U,with smaller proportions of G to C and G to A edits (9.4% (3) and 6.2% (2),respectively).The expression level of the Cytb transcript was quantified by real-time PCR with a TaqMan probe at different times during the whole growth phase.The average Cytb transcript was present at 39.27±7.46 copies of cDNA per cell during the whole growth cycle,and the expression of Cytb was relatively stable over the different phases.These results deepen our understanding of the structure and characteristics of Cytb in P.donghaiense,and confirmed that Cytb in P.donghaiense is a candidate reference gene for studying the expression of other genes.

  15. Localized Retroprocessing as a Model of Intron Loss in the Plant Mitochondrial Genome.

    Science.gov (United States)

    Cuenca, Argelia; Ross, T Gregory; Graham, Sean W; Barrett, Craig F; Davis, Jerrold I; Seberg, Ole; Petersen, Gitte

    2016-08-03

    Loss of introns in plant mitochondrial genes is commonly explained by retroprocessing. Under this model, an mRNA is reverse transcribed and integrated back into the genome, simultaneously affecting the contents of introns and edited sites. To evaluate the extent to which retroprocessing explains intron loss, we analyzed patterns of intron content and predicted RNA editing for whole mitochondrial genomes of 30 species in the monocot order Alismatales. In this group, we found an unusually high degree of variation in the intron content, even expanding the hitherto known variation among angiosperms. Some species have lost some two-third of the cis-spliced introns. We found a strong correlation between intron content and editing frequency, and detected 27 events in which intron loss is consistent with the presence of nucleotides in an edited state, supporting retroprocessing. However, we also detected seven cases of intron loss not readily being explained by retroprocession. Our analyses are also not consistent with the entire length of a fully processed cDNA copy being integrated into the genome, but instead indicate that retroprocessing usually occurs for only part of the gene. In some cases, several rounds of retroprocessing may explain intron loss in genes completely devoid of introns. A number of taxa retroprocessing seem to be very common and a possibly ongoing process. It affects the entire mitochondrial genome.

  16. Mitochondrial localization of the threonine peptidase PfHslV, a ClpQ ortholog in Plasmodium falciparum.

    Science.gov (United States)

    Tschan, Serena; Kreidenweiss, Andrea; Stierhof, York-Dieter; Sessler, Nicole; Fendel, Rolf; Mordmüller, Benjamin

    2010-11-01

    Plasmodium falciparum belongs to a group of eukaryotes expressing an ortholog of the prokaryotic T1-threonine peptidase, heat shock locus V (HslV). Bacterial HslV is a particularly well studied protease, due to its structural and biochemical similarity to the eukaryotic proteasome. Plasmodium falciparum HslV (PfHslV) is expressed in schizonts and merozoites of the asexual blood stage. Strong sequence conservation between plasmodial species, absence of HslV homologs in the human genome, and availability of specific inhibitors led us to explore its function and potential use as a drug target. In a first step, we investigated localization of PfHslV, using a bioinformatics approach and a transgenic P. falciparum line expressing a PfHslV-enhanced yellow fluorescent protein (EYFP) fusion protein from the endogenous pfhslV locus. PfHslV-EYFP was found in the mitochondrial matrix under fluorescence and immunoelectron microscopy. Endogenous, non-modified PfHslV was present in purified mitochondria and interference with mitochondrial membrane potential by drug treatment led to impairment of PfHslV processing. Import of heterologous EYFP into the plasmodial mitochondrion is mediated by the N-terminal 37 amino acids of PfHslV. PfHslV's targeting sequence is also functional in human cells, demonstrating strong conservation of mitochondrial targeting in eukaryotes. In conclusion, our data shows that PfHslV is located to the plasmodial mitochondrion and presumably has vital function within this organelle which makes it an attractive target for interventions.

  17. Characterization of mitochondrial DNA in various Candida species: isolation, restriction endonuclease analysis, size, and base composition.

    Science.gov (United States)

    Su, C S; Meyer, S A

    1991-01-01

    A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed. Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C. albicans, C. kefyr, C. lusitaniae, C. maltosa, C. parapsilosis, C. shehatae, and C. tropicalis) and Lodderomyces elongisporus revealed different cleavage patterns that appeared to be specific for the species. Few common restriction fragments were evident. The genome sizes of the mitochondrial DNAs ranged from 26.4 to 51.4 kilobase pairs, and the guanine-plus-cytosine contents ranged from 20.7 to 36.8 mol%. There was no correlation between the base compositions of nuclear and mitochondrial DNAs. Eight isolates of C. parapsilosis, including the type culture, and an ascosporogenous strain of L. elongisporus, which was once proposed as the teleomorph of C. parapsilosis, had similar mitochondrial DNA molecular sizes (30.2 and 28.8 kilobase pairs); however, restriction endonuclease patterns of these organisms were distinct. These data provide additional support for discrimination of these two species. The results of our experiments demonstrate that mitochondrial DNA analyses may provide useful criteria for the differentiation of yeast species.

  18. Characterization of the Complete Mitochondrial Genome Sequence of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidae from China

    Directory of Open Access Journals (Sweden)

    Guo-Hua Liu, Chun Li, Jia-Yuan Li, Dong-Hui Zhou, Rong-Chuan Xiong, Rui-Qing Lin, Feng-Cai Zou, Xing-Quan Zhu

    2012-01-01

    Full Text Available Sparganosis, caused by the plerocercoid larvae of members of the genus Spirometra, can cause significant public health problem and considerable economic losses. In the present study, the complete mitochondrial DNA (mtDNA sequence of Spirometra erinaceieuropaei from China was determined, characterized and compared with that of S. erinaceieuropaei from Japan. The gene arrangement in the mt genome sequences of S. erinaceieuropaei from China and Japan is identical. The identity of the mt genomes was 99.1% between S. erinaceieuropaei from China and Japan, and the complete mtDNA sequence of S. erinaceieuropaei from China is slightly shorter (2 bp than that from Japan. Phylogenetic analysis of S. erinaceieuropaei with other representative cestodes using two different computational algorithms [Bayesian inference (BI and maximum likelihood (ML] based on concatenated amino acid sequences of 12 protein-coding genes, revealed that S. erinaceieuropaei is closely related to Diphyllobothrium spp., supporting classification based on morphological features. The present study determined the complete mtDNA sequences of S. erinaceieuropaei from China that provides novel genetic markers for studying the population genetics and molecular epidemiology of S. erinaceieuropaei in humans and animals.

  19. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV

    Energy Technology Data Exchange (ETDEWEB)

    Han, Q.; Robinson, H.; Cai, T.; Tagle, D. A.; Li, J.

    2011-10-01

    Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  20. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV.

    Science.gov (United States)

    Han, Qian; Robinson, Howard; Cai, Tao; Tagle, Danilo A; Li, Jianyong

    2011-10-01

    Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  1. Characterizing Dust Attenuation in Local Star Forming Galaxies

    Science.gov (United States)

    Battisti, Andrew; Calzetti, Daniela; Chary, Ranga-Ram

    2017-01-01

    The dust attenuation for a sample of ~10000 local (z ≤ 0.1) star forming galaxies is constrained as a function of their physical properties. We utilize aperture-matched multi-wavelength data from the UV-to-NIR, available from the Galaxy Evolution Explorer, the Sloan Digital Sky Survey, the United Kingdom Infrared Telescope, and the Two Micron All-Sky Survey, to ensure that regions of comparable size in each galaxy are being analyzed. We characterize the dust attenuation through the slope of the UV flux density and the Balmer decrement (Hα/Hβ). The observed relationship between these quantities is similar to the local starburst relation and is not seen to vary strongly with galactic properties. We derive the total attenuation curve over the range 1250 Å < λ < 28500 Å and find that a single attenuation curve is effective for characterizing the majority of galaxies in our sample. This attenuation curve is slightly lower in the far-UV than local starburst galaxies, by roughly 15%, but appears similar at longer wavelengths and has a normalization of RV = 3.7±0.4 (V-band). This indicates that a single attenuation curve is reasonable for wide application in the local Universe.

  2. Cannabinoid CB1 Receptors Are Localized in Striated Muscle Mitochondria and Regulate Mitochondrial Respiration

    OpenAIRE

    Mendizabal-Zubiaga, Juan; Melser, Su; Bénard, Giovanni; Ramos, Almudena; Reguero, Leire; Arrabal, Sergio; Elezgarai, Izaskun; Gerrikagoitia, Inmaculada; Suarez, Juan; Rodríguez de Fonseca, Fernando; Puente, Nagore; Marsicano, Giovanni; Grandes, Pedro

    2016-01-01

    The cannabinoid type 1 (CB1) receptor is widely distributed in the brain and peripheral organs where it regulates cellular functions and metabolism. In the brain, CB1 is mainly localized on presynaptic axon terminals but is also found on mitochondria (mtCB1), where it regulates cellular respiration and energy production. Likewise, CB1 is localized on muscle mitochondria, but very little is known about it. The aim of this study was to further investigate in detail the distribution and function...

  3. 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.

  4. Characterization of 67 mitochondrial tRNA gene rearrangements in the Hymenoptera suggests that mitochondrial tRNA gene position is selectively neutral.

    Science.gov (United States)

    Dowton, Mark; Cameron, Stephen L; Dowavic, Jessica I; Austin, Andy D; Whiting, Michael F

    2009-07-01

    We present entire sequences of two hymenopteran mitochondrial genomes and the major portion of three others. We combined these data with nine previously sequenced hymenopteran mitochondrial genomes. This allowed us to infer and analyze the evolution of the 67 mitochondrial gene rearrangements so far found in this order. All of these involve tRNA genes, whereas four also involve larger (protein-coding or ribosomal RNA) genes. We find that the vast majority of mitochondrial gene rearrangements are independently derived. A maximum of four of these rearrangements represent shared, derived organizations, whereas three are convergently derived. The remaining mitochondrial gene rearrangements represent new mitochondrial genome organizations. These data are consistent with the proposal that there are an enormous number of alternative mitochondrial genome organizations possible and that mitochondrial genome organization is, for the most part, selectively neutral. Nevertheless, some mitochondrial genes appear less mobile than others. Genes close to the noncoding region are generally more mobile but only marginally so. Some mitochondrial genes rearrange in a pattern consistent with the duplication/random loss model, but more mitochondrial genes move in a pattern inconsistent with this model. An increased rate of mitochondrial gene rearrangement is not tightly associated with the evolution of parasitism. Although parasitic lineages tend to have more mitochondrial gene rearrangements than nonparasitic lineages, there are exceptions (e.g., Orussus and Schlettererius). It is likely that only a small proportion of the total number of mitochondrial gene rearrangements that have occurred during the evolution of the Hymenoptera have been sampled in the present study.

  5. Characterization of PHB1 and its role in mitochondrial maturation and yolk platelet degradation during development of Artemia embryos.

    Directory of Open Access Journals (Sweden)

    Xiang Ye

    Full Text Available BACKGROUND: To cope with harsh environments, crustaceans such as Artemia produce diapause gastrula embryos (cysts with suppressed metabolism. Metabolism and development resume during post-diapause development, but the mechanism behind these cellular events remains largely unknown. PRINCIPAL FINDING: Our study investigated the role of prohibitin 1 (PHB1 in metabolic reinitiation during post-diapause development. We found that PHB1 was developmentally regulated via changes in phosphorylation status and localization. Results from RNA interference experiments demonstrated PHB1 to be critical for mitochondrial maturation and yolk degradation during development. In addition, PHB1 was present in yolk platelets, and it underwent ubiquitin-mediated degradation during the proteolysis of yolk protein. CONCLUSIONS/SIGNIFICANCE: PHB1 has an indispensable role in coordinating mitochondrial maturation and yolk platelet degradation during development in Artemia. This novel function of PHB1 provides new clues to comprehend the roles of PHB1 in metabolism and development.

  6. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity.

    Directory of Open Access Journals (Sweden)

    Alberto Cruz-Bermúdez

    Full Text Available The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids. Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

  7. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity.

    Science.gov (United States)

    Cruz-Bermúdez, Alberto; Vicente-Blanco, Ramiro J; Hernández-Sierra, Rosana; Montero, Mayte; Alvarez, Javier; González Manrique, Mar; Blázquez, Alberto; Martín, Miguel Angel; Ayuso, Carmen; Garesse, Rafael; Fernández-Moreno, Miguel A

    2016-01-01

    The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

  8. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity

    Science.gov (United States)

    Cruz-Bermúdez, Alberto; Vicente-Blanco, Ramiro J.; Hernández-Sierra, Rosana; Montero, Mayte; Alvarez, Javier; González Manrique, Mar; Blázquez, Alberto; Martín, Miguel Angel; Ayuso, Carmen; Garesse, Rafael; Fernández-Moreno, Miguel A.

    2016-01-01

    The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations. PMID:26784702

  9. Localization and functional characterization of the human NKCC2 isoforms

    DEFF Research Database (Denmark)

    Carota, I; Theilig, F; Oppermann, M;

    2010-01-01

    AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2...... isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were...... performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant...

  10. Characterizing genetic diversity of contemporary pacific chickens using mitochondrial DNA analyses.

    Directory of Open Access Journals (Sweden)

    Kelsey Needham Dancause

    Full Text Available BACKGROUND: Mitochondrial DNA (mtDNA hypervariable region (HVR sequences of prehistoric Polynesian chicken samples reflect dispersal of two haplogroups--D and E--by the settlers of the Pacific. The distribution of these chicken haplogroups has been used as an indicator of human movement. Recent analyses suggested similarities between prehistoric Pacific and South American chicken samples, perhaps reflecting prehistoric Polynesian introduction of the chicken into South America. These analyses have been heavily debated. The current distribution of the D and E lineages among contemporary chicken populations in the Western Pacific is unclear, but might ultimately help to inform debates about the movements of humans that carried them. OBJECTIVES: We sought to characterize contemporary mtDNA diversity among chickens in two of the earliest settled archipelagos of Remote Oceania, the Marianas and Vanuatu. METHODS: We generated HVR sequences for 43 chickens from four islands in Vanuatu, and for 5 chickens from Guam in the Marianas. RESULTS: Forty samples from Vanuatu and three from Guam were assigned to haplogroup D, supporting this as a Pacific chicken haplogroup that persists in the Western Pacific. Two haplogroup E lineages were observed in Guam and two in Vanuatu. Of the E lineages in Vanuatu, one was identical to prehistoric Vanuatu and Polynesian samples and the other differed by one polymorphism. Contrary to our expectations, we observed few globally distributed domesticate lineages not associated with Pacific chicken dispersal. This might suggest less European introgression of chickens into Vanuatu than expected. If so, the E lineages might represent lineages maintained from ancient Pacific chicken introductions. The Vanuatu sample might thus provide an opportunity to distinguish between maintained ancestral Pacific chicken lineages and replacement by global domesticates through genomic analyses, which could resolve questions of contemporary

  11. Characteristics of mitochondrial calpains.

    Science.gov (United States)

    Ozaki, Taku; Tomita, Hiroshi; Tamai, Makoto; Ishiguro, Sei-Ichi

    2007-09-01

    Calpains are considered to be cytoplasmic enzymes, although several studies have shown that calpain-like protease activities also exist in mitochondria. We partially purified mitochondrial calpain from swine liver mitochondria and characterized. Only one type of mitochondrial calpain was detected by the column chromatographies. The mitochondrial calpain was stained with anti-mu-calpain and calpain small subunit antibodies. The susceptibility of mitochondrial calpain to calpain inhibitors and the optimum pH differ from those of cytosolic mu- and m-calpains. The Ca(2+)-dependency of mitochondrial calpain was similar to that of cytosolic mu-calpain. Therefore, we named the protease mitochondrial mu-like calpain. In zymogram analysis, two types of caseinolytic enzymes existed in mitochondria and showed different mobilities from cytosolic mu- and m-calpains. The upper major band was stained with anti-mu-calpain and calpain small subunit antibodies (mitochondrial calpain I, mitochondrial mu-like calpain). The lower band was stained only with anti-calpain small subunit antibody (mitochondrial calpain II, unknown mitochondrial calpain). Calpastatin was not detected in mitochondrial compartments. The mitochondrial calpain processed apoptosis-inducing factor (AIF) to truncated AIF (tAIF), releasing tAIF into the intermembrane space. These results indicate that mitochondrial calpain, which differs from mu- and m-calpains, seems to be a ubiquitous calpain and may play a role in mitochondrial apoptotic signalling.

  12. Accuracy characterization and measurement point planning for workpiece localization

    Institute of Scientific and Technical Information of China (English)

    ZHU Limin; LUO Honggen; DING Han

    2004-01-01

    This paper addresses the problem of accuracy characterization and measurement point planning for 3-D workpiece localization in the presence of part surface errors and measurement errors. Two frame-invariant functions of the infinitesimal rigid body displacement are defined to quantify the localization accuracy required by manufacturing processes. Then, two kinds of frame-invariant indices are derived to characterize the sensitivities of the accuracy measures to the sampling errors at the measurement points. With a dense set of discrete points on the workpiece datum surfaces pre-defined as candidates for measurement, planning of probing points for accurate recovery of part location is modeled as a combinatorial problem focusing on minimizing the accuracy sensitivity index. Based on an interchange rule, a greedy algorithm is developed to efficiently find a near-optimal solution. It is also shown that if the number of the measurement points is sufficiently large, there is no need to optimize their positions. Example confirms the validity of the presented indices and algorithm.

  13. Characterization of the complete mitochondrial genome of the king pigeon (Columba livia breed king).

    Science.gov (United States)

    Zhang, Rui-Hua; He, Wen-Xiao; Xu, Tong

    2015-06-01

    The king pigeon is a breed of pigeon developed over many years of selective breeding primarily as a utility breed. In the present work, we report the complete mitochondrial genome sequence of king pigeon for the first time. The total length of the mitogenome was 17,221 bp with the base composition of 30.14% for A, 24.05% for T, 31.82% for C, and 13.99% for G and an A-T (54.22 %)-rich feature was detected. It harbored 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one non-coding control region (D-loop region). The arrangement of all genes was identical to the typical mitochondrial genomes of pigeon. The complete mitochondrial genome sequence of king pigeon would serve as an important data set of the germplasm resources for further study.

  14. Complete mitochondrial DNA sequence of the endangered fish (Bahaba taipingensis): Mitogenome characterization and phylogenetic implications.

    Science.gov (United States)

    Zhao, Linlin; Gao, Tianxiang; Lu, Weihua

    2015-01-01

    To understand the systematic status of Bahaba taipingensis within Sciaenidae, the complete mitochondrial genome (mitogenome) sequence of Chinese bahaba has recently been determined by long PCR and primer walking methods. The complete mitochondrial genome is 16500 bp in length and contains 37 mitochondrial genes (13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes) as well as a control region (CR) as other bony fishes. Within the control region, we identified the extended termination associated sequence domain (ETAS), the central conserved sequence block domain (CSB-D, SCB-E and CSB-F) and the conserved sequence block domain (CSB-1, CSB-2 and CSB-3). Phylogenetic analyses revealed that Bahaba taipingensis is more closely related to Pseudosciaeniae than Argyrosominae and Sciaeninae. Additionally, Bahaba taipingensis is the sister taxon of Miichthys miiuy, and those two are sister to Collichthys plus Larimichthys.

  15. Microsatellite loci and the complete mitochondrial DNA sequence characterized through next generation sequencing and de novo genome assembly for the critically endangered orange-bellied parrot, Neophema chrysogaster.

    Science.gov (United States)

    Miller, Adam D; Good, Robert T; Coleman, Rhys A; Lancaster, Melanie L; Weeks, Andrew R

    2013-01-01

    A suite of polymorphic microsatellite markers and the complete mitochondrial genome sequence was developed by next generation sequencing (NGS) for the critically endangered orange-bellied parrot, Neophema chrysogaster. A total of 14 polymorphic loci were identified and characterized using DNA extractions representing 40 individuals from Melaleuca, Tasmania, sampled in 2002. We observed moderate genetic variation across most loci (mean number of alleles per locus = 2.79; mean expected heterozygosity = 0.53) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. De novo and reference-based genome assemblies performed using MIRA were used to assemble the N. chrysogaster mitochondrial genome sequence with mean coverage of 116-fold (range 89 to 142-fold). The mitochondrial genome consists of 18,034 base pairs, and a typical metazoan mitochondrial gene content consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a single large non-coding region (control region). The arrangement of mitochondrial genes is also typical of Avian taxa. The annotation of the mitochondrial genome and the characterization of 14 microsatellite markers provide a valuable resource for future genetic monitoring of wild and captive N. chrysogaster populations. As found previously, NGS provides a rapid, low cost and reliable method for polymorphic nuclear genetic marker development and determining complete mitochondrial genome sequences when only a fraction of a genome is sequenced.

  16. Fluence- and time-dependant lysosomal and mitochondrial damage induced by LS11 PDT characterized with light scattering

    Science.gov (United States)

    Wilson, Jeremy D.; Foster, Thomas H.

    2007-02-01

    Light scattering from cells originates from sub-cellular organelles. Our measurements of angularly resolved light scattering have demonstrated that at 633 nm, the dominant scattering centers within EMT6 cells are mitochondria and lysosomes. To assess their specific contributions, we have used photodynamic therapy (PDT) to induce organelle-specific perturbations within intact cells. We have developed a coated sphere scattering model for mitochondrial swelling in response to ALA- and Pc 4-PDT, and in the case of Pc 4-PDT we have used this model to map the scattering responses into clonogenic cell survival. More recently, we demonstrated the ability to measure the size, scattering contribution, and refractive index of lysosomes within cells by exploiting the localization and high extinction of the photosensitizer LS11 and an absorbing sphere scattering model. Here we report on time- and fluence-dependant scattering measurements from cells treated with LS11-PDT. LS11-PDT causes rapid lysosomal disruption, as quantified by uptake of acridine orange, and can induce downstream effects including release of mitochondrial cytochrome c preceding the loss of mitochondrial membrane potential (Reiners et al., Cell Death Differ. 9:934, 2002). Using scattering and these various methods of analysis, we observed that the induction of lysosomal morphology changes requires a fluence significantly higher than that reported for cell killing. At lower fluences, we observe that at 1 h after irradiation there is significant mitochondrial swelling, consistent with the onset of cytochrome c-induced cell death, while the morphology of lysosomes remains unchanged. We also expand on the ideas of lysosomal staining to demonstrate the sensitivity of scattering measurements at different wavelengths to different organelle populations.

  17. Mitochondrial DNA evidence indicates the local origin of domestic pigs in the upstream region of the Yangtze River.

    Directory of Open Access Journals (Sweden)

    Long Jin

    Full Text Available Previous studies have indicated two main domestic pig dispersal routes in East Asia: one is from the Mekong region, through the upstream region of the Yangtze River (URYZ to the middle and upstream regions of the Yellow River, the other is from the middle and downstream regions of the Yangtze River to the downstream region of the Yellow River, and then to northeast China. The URYZ was regarded as a passageway of the former dispersal route; however, this assumption remains to be further investigated. We therefore analyzed the hypervariable segements of mitochondrial DNA from 513 individual pigs mainly from Sichuan and the Tibet highlands and 1,394 publicly available sequences from domestic pigs and wild boars across Asia. From the phylogenetic tree, most of the samples fell into a mixed group that was difficult to distinguish by breed or geography. The total network analysis showed that the URYZ pigs possessed a dominant position in haplogroup A and domestic pigs shared the same core haplotype with the local wild boars, suggesting that pigs in group A were most likely derived from the URYZ pool. In addition, a region-wise network analysis determined that URYZ contains 42 haplotypes of which 22 are unique indicating the high diversity in this region. In conclusion, our findings confirmed that pigs from the URYZ were domesticated in situ.

  18. Defect localization, characterization and reliability assessment in emerging photovoltaic devices.

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Benjamin Bing-Yeh [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Cruz-Campa, Jose Luis [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Haase, Gad S. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Tangyunyong, Paiboon [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Colr, Edward Isaac [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Okandan, Murat [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Nielson, Gregory N. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2014-04-01

    Microsystems-enabled photovoltaics (MEPV) can potentially meet increasing demands for light-weight, portable, photovoltaic solutions with high power density and efficiency. The study in this report examines failure analysis techniques to perform defect localization and evaluate MEPV modules. CMOS failure analysis techniques, including electroluminescence, light-induced voltage alteration, thermally-induced voltage alteration, optical beam induced current, and Seabeck effect imaging were successfully adapted to characterize MEPV modules. The relative advantages of each approach are reported. In addition, the effects of exposure to reverse bias and light stress are explored. MEPV was found to have good resistance to both kinds of stressors. The results form a basis for further development of failure analysis techniques for MEPVs of different materials systems or multijunction MEPVs. The incorporation of additional stress factors could be used to develop a reliability model to generate lifetime predictions for MEPVs as well as uncover opportunities for future design improvements.

  19. Characterizing structural transitions using localized free energy landscape analysis.

    Directory of Open Access Journals (Sweden)

    Nilesh K Banavali

    Full Text Available BACKGROUND: Structural changes in molecules are frequently observed during biological processes like replication, transcription and translation. These structural changes can usually be traced to specific distortions in the backbones of the macromolecules involved. Quantitative energetic characterization of such distortions can greatly advance the atomic-level understanding of the dynamic character of these biological processes. METHODOLOGY/PRINCIPAL FINDINGS: Molecular dynamics simulations combined with a variation of the Weighted Histogram Analysis Method for potential of mean force determination are applied to characterize localized structural changes for the test case of cytosine (underlined base flipping in a GTCAGCGCATGG DNA duplex. Free energy landscapes for backbone torsion and sugar pucker degrees of freedom in the DNA are used to understand their behavior in response to the base flipping perturbation. By simplifying the base flipping structural change into a two-state model, a free energy difference of upto 14 kcal/mol can be attributed to the flipped state relative to the stacked Watson-Crick base paired state. This two-state classification allows precise evaluation of the effect of base flipping on local backbone degrees of freedom. CONCLUSIONS/SIGNIFICANCE: The calculated free energy landscapes of individual backbone and sugar degrees of freedom expectedly show the greatest change in the vicinity of the flipping base itself, but specific delocalized effects can be discerned upto four nucleotide positions away in both 5' and 3' directions. Free energy landscape analysis thus provides a quantitative method to pinpoint the determinants of structural change on the atomic scale and also delineate the extent of propagation of the perturbation along the molecule. In addition to nucleic acids, this methodology is anticipated to be useful for studying conformational changes in all macromolecules, including carbohydrates, lipids, and proteins.

  20. Over-expressed copper/zinc superoxide dismutase localizes to mitochondria in neurons inhibiting the angiotensin II-mediated increase in mitochondrial superoxide

    Directory of Open Access Journals (Sweden)

    Shumin Li

    2014-01-01

    Full Text Available Angiotensin II (AngII is the main effector peptide of the renin–angiotensin system (RAS, and contributes to the pathogenesis of cardiovascular disease by exerting its effects on an array of different cell types, including central neurons. AngII intra-neuronal signaling is mediated, at least in part, by reactive oxygen species, particularly superoxide (O2·−. Recently, it has been discovered that mitochondria are a major subcellular source of AngII-induced O2·−. We have previously reported that over-expression of manganese superoxide dismutase (MnSOD, a mitochondrial matrix-localized O2·− scavenging enzyme, inhibits AngII intra-neuronal signaling. Interestingly, over-expression of copper/zinc superoxide dismutase (CuZnSOD, which is believed to be primarily localized to the cytoplasm, similarly inhibits AngII intra-neuronal signaling and provides protection against AngII-mediated neurogenic hypertension. Herein, we tested the hypothesis that CuZnSOD over-expression in central neurons localizes to mitochondria and inhibits AngII intra-neuronal signaling by scavenging mitochondrial O2·−. Using a neuronal cell culture model (CATH.a neurons, we demonstrate that both endogenous and adenovirus-mediated over-expressed CuZnSOD (AdCuZnSOD are present in mitochondria. Furthermore, we show that over-expression of CuZnSOD attenuates the AngII-mediated increase in mitochondrial O2·− levels and the AngII-induced inhibition of neuronal potassium current. Taken together, these data clearly show that over-expressed CuZnSOD in neurons localizes in mitochondria, scavenges AngII-induced mitochondrial O2·−, and inhibits AngII intra-neuronal signaling.

  1. Over-expressed copper/zinc superoxide dismutase localizes to mitochondria in neurons inhibiting the angiotensin II-mediated increase in mitochondrial superoxide.

    Science.gov (United States)

    Li, Shumin; Case, Adam J; Yang, Rui-Fang; Schultz, Harold D; Zimmerman, Matthew C

    2013-01-01

    Angiotensin II (AngII) is the main effector peptide of the renin-angiotensin system (RAS), and contributes to the pathogenesis of cardiovascular disease by exerting its effects on an array of different cell types, including central neurons. AngII intra-neuronal signaling is mediated, at least in part, by reactive oxygen species, particularly superoxide (O2 (•-)). Recently, it has been discovered that mitochondria are a major subcellular source of AngII-induced O2 (•-). We have previously reported that over-expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix-localized O2 (•-) scavenging enzyme, inhibits AngII intra-neuronal signaling. Interestingly, over-expression of copper/zinc superoxide dismutase (CuZnSOD), which is believed to be primarily localized to the cytoplasm, similarly inhibits AngII intra-neuronal signaling and provides protection against AngII-mediated neurogenic hypertension. Herein, we tested the hypothesis that CuZnSOD over-expression in central neurons localizes to mitochondria and inhibits AngII intra-neuronal signaling by scavenging mitochondrial O2 (•-). Using a neuronal cell culture model (CATH.a neurons), we demonstrate that both endogenous and adenovirus-mediated over-expressed CuZnSOD (AdCuZnSOD) are present in mitochondria. Furthermore, we show that over-expression of CuZnSOD attenuates the AngII-mediated increase in mitochondrial O2 (•-) levels and the AngII-induced inhibition of neuronal potassium current. Taken together, these data clearly show that over-expressed CuZnSOD in neurons localizes in mitochondria, scavenges AngII-induced mitochondrial O2 (•-), and inhibits AngII intra-neuronal signaling.

  2. Cloning, characterization, and expression of Cytochrome b ( Cytb)—a key mitochondrial gene from Prorocentrum donghaiense

    Science.gov (United States)

    Zhao, Liyuan; Mi, Tiezhu; Zhen, Yu; Yu, Zhigang

    2012-05-01

    Mitochondrial cytochrome b (Cytb), one of the few proteins encoded by the mitochondrial DNA, plays an important role in transferring electrons. As a mitochondrial gene, it has been widely used for phylogenetic analysis. Previously, a 949-bp fragment of the coding gene and mRNA editing were characterized from Prorocentrum donghaiense, which might prove useful for resolving P. donghaiense from closely related species. However, the full-length coding region has not been characterized. In this study, we used rapid amplification of cDNA ends (RACE) to obtain full-length, 1 124 bp cDNA. Cytb transcript contained a standard initiation codon ATG, but did not have a recognizable stop codon. Homology comparison showed that the P. donghaiense Cytb had a high sequence identity to Cytb sequences from other dinoflagellate species. Phylogenetic analysis placed Cytb from P. donghaiense in the clade of dinoflagellates and it clustered together strongly with that from P. minimum. Based on the full-length sequence, we inferred 32 editing events at different positions, accounting for 2.93% of the Cytb gene. 34.4% (11) of the changes were A to G, 25% (8) were T to C, and 25% (8) were C to U, with smaller proportions of G to C and G to A edits (9.4% (3) and 6.2% (2), respectively). The expression level of the Cytb transcript was quantified by real-time PCR with a TaqMan probe at different times during the whole growth phase. The average Cytb transcript was present at 39.27±7.46 copies of cDNA per cell during the whole growth cycle, and the expression of Cytb was relatively stable over the different phases. These results deepen our understanding of the structure and characteristics of Cytb in P. donghaiense, and confirmed that Cytb in P. donghaiense is a candidate reference gene for studying the expression of other genes.

  3. Characterization of a Dairy Gyr herd with respect to its mitochondrial DNA (mt DNA origin

    Directory of Open Access Journals (Sweden)

    Anibal Eugênio Vercesi Filho

    2010-01-01

    Full Text Available The Zebu breeds were introduced in Brazil mainly in the last century by imports from the Indian subcontinent. When the Zebu cattle arrived, the national herd suffered a significative change by backcrossing the national cows of taurine origin with Zebu sires. These processes created a polymorphism in the mitochondrial DNA (mtDNA in the Zebu animals with are in a major part derived from backcrossing and sharing mtDNA of taurine origin. To verify the maternal origin of cows belonging to the Dairy Gyr herd of APTA, Mococa 60 females were analyzed and 33 presented mtDNA from Bos taurus origin and 27 presented mtDNA from Bos indicus origin. None of these animals presented patterns of both mtDNA origins, indicating absence of heteroplasmy for these mitochondrial genotypes.

  4. Genetic characterization of Meigu goat (Capra hircus) based on the mitochondrial DNA.

    Science.gov (United States)

    Duan, Xiaoyue; Zhang, Hao; Li, Haijun; Niu, Lili; Wang, Linjie; Li, Li; Zhang, Hongping; Zhong, Tao

    2016-01-01

    Meigu goat (Capra hircus) is one of the indigenous goat breeds in China. Our research findings revealed that the entire mitochondrial genome of Meigu goat was 16,643 bp in length. The contents of A, C, T and G in the mitochondrial genome were 33.59%, 26.05%, 27.31% and 13.05%, respectively. The mitogenome of meigu goat contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 1 control region. Components of the Meigu goat's mitogenome were similar to those of other Capra hircus in gene arrangement and composition. These results could provide essential information for molecular phylogenetic and evolutionary analyses of domestic goats.

  5. Characterization of the complete mitochondrial genomes from Polycladida (Platyhelminthes) using next-generation sequencing.

    Science.gov (United States)

    Aguado, M Teresa; Grande, Cristina; Gerth, Michael; Bleidorn, Christoph; Noreña, Carolina

    2016-01-10

    The complete mitochondrial genomes of three polycladids, the acotylean Hoploplana elisabelloi and the cotyleans Enchiridium sp. and Prosthiostomum siphunculus have been assembled with high coverage from Illumina sequencing data. The mt genomes contain 36 genes including 12 of the 13 protein-coding genes characteristic for metazoan mitochondrial genomes, two ribosomal RNA genes, and 22 transfer RNA genes. Gene annotation, gene order, genetic code, start and stop codons and codon bias have been identified. In comparison with the well investigated parasitic Neodermata, our analysis reveals a great diversity of gene orders within Polycladida and Platyhelminthes in general. By analyzing representative genomes of the main groups of Platyhelminthes we explored the phylogenetic relationships of this group. The phylogenetic analyses strongly supported the monophyly of Polycladida, and based on a small taxon sampling suggest the monophyly of Acotylea and Cotylea.

  6. Complete sequence of the mitochondrial genome of Odontamblyopus rubicundus (Perciformes: Gobiidae): genome characterization and phylogenetic analysis.

    Science.gov (United States)

    Liu, Tianxing; Jin, Xiaoxiao; Wang, Rixin; Xu, Tianjun

    2013-12-01

    Odontamblyopus rubicundus is a species of gobiid fishes, inhabits muddy-bottomed coastal waters. In this paper, the first complete mitochondrial genome sequence of O. rubicundus is reported. The complete mitochondrial genome sequence is 17119 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, a control region and an L-strand origin as in other teleosts. Most mitochondrial genes are encoded on H-strand except for ND6 and seven tRNA genes. Some overlaps occur in protein-coding genes and tRNAs ranging from 1 to 7 bp. The possibly nonfunctional L-strand origin folded into a typical stem-loop secondary structure and a conserved motif (5'-GCCGG-3') was found at the base of the stem within the tRNACys gene. The TAS, CSB-2 and CSB-3 could be detected in the control region. However, in contrast to most of other fishes, the central conserved sequence block domain and the CSB-1 could not be recognized in O. rubicundus, which is consistent with Acanthogobius hasta (Gobiidae). In addition, phylogenetic analyses based on different sequences of species of Gobiidae and different methods showed that the classification of O. rubicundus into Odontamblyopus due to morphology is debatable.

  7. Characterization of the complete mitochondrial genome of the black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Wu, Qiu-Ling; Cui, Wen-Xia; Wei, Shu-Jun

    2015-02-01

    The complete mitochondrial genome of the black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae) was determined (GenBank accession No. KF163965). The length of this mitochondrial genome is 15,377 bp with an A + T content of 82.5%. There are 37 typical animal mitochondrial genes, that is, 13 protein-coding, 2 rRNA and 22 tRNA gene and an A + T-rich region. The tRNA gene trnM was rearranged to the upstream of the trnI-trnQ-trnM cluster compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN start codon except for the gene cox1, which uses CGA as in other lepidopteran species. Ten protein-coding genes stop with termination codon TAA, whereas three protein-coding gene use incomplete stop codon T. The A + T-region is located between rrnS and trnM with a length of 332 bp and A + T content of 94.88%.

  8. Complete sequence of the mitochondrial genome of Odontamblyopus rubicundus (Perciformes: Gobiidae): genome characterization and phylogenetic analysis

    Indian Academy of Sciences (India)

    Tianxing Liu; Xiaoxiao Jin; Rixin Wang; Tianjun Xu

    2013-12-01

    Odontamblyopus rubicundus is a species of gobiid fishes, inhabits muddy-bottomed coastal waters. In this paper, the first complete mitochondrial genome sequence of O. rubicundus is reported. The complete mitochondrial genome sequence is 17119 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, a control region and an L-strand origin as in other teleosts. Most mitochondrial genes are encoded on H-strand except for ND6 and seven tRNA genes. Some overlaps occur in protein-coding genes and tRNAs ranging from 1 to 7 bp. The possibly nonfunctional L-strand origin folded into a typical stem-loop secondary structure and a conserved motif (5′-GCCGG-3′) was found at the base of the stem within the $tRNA^{Cys}$ gene. The TAS, CSB-2 and CSB-3 could be detected in the control region. However, in contrast to most of other fishes, the central conserved sequence block domain and the CSB-1 could not be recognized in O. rubicundus, which is consistent with Acanthogobius hasta (Gobiidae). In addition, phylogenetic analyses based on different sequences of species of Gobiidae and different methods showed that the classification of O. rubicundus into Odontamblyopus due to morphology is debatable.

  9. Alterations of the mitochondrial proteome caused by the absence of mitochondrial DNA: A proteomic view

    Science.gov (United States)

    Chevallet, Mireille; Lescuyer, Pierre; Diemer, Hélène; van Dorsselaer, Alain; Leize-Wagner, Emmanuelle; Rabilloud, Thierry

    2006-01-01

    The proper functioning of mitochondria requires that both the mitochondrial and the nuclear genome are functional. To investigate the importance of the mitochondrial genome, which encodes only 13 subunits of the respiratory complexes, the mitochondrial rRNAs and a few tRNAs, we performed a comparative study on the 143B cell line and on its Rho-0 counterpart, i.e. devoid of mitochondrial DNA. Quantitative differences were found, of course in the respiratory complexes subunits, but also in the mitochondrial translation apparatus, mainly mitochondrial ribosomal proteins, and in the ion and protein import system, i.e. including membrane proteins. Various mitochondrial metabolic processes were also altered, especially electron transfer proteins and some dehydrogenases, but quite often on a few proteins for each pathway. This study also showed variations in some hypothetical or poorly characterized proteins, suggesting a mitochondrial localization for these proteins. Examples include a stomatin-like protein and a protein sharing homologies with bacterial proteins implicated in tyrosine catabolism. Proteins involved in apoptosis control are also found modulated in Rho-0 mitochondria. PMID:16548050

  10. Characterization of Black and Green Tea from Local Market

    Directory of Open Access Journals (Sweden)

    Sonia Ancuta Socaci

    2013-11-01

    Full Text Available The leaves from Camellia sinensis are used from ancient times for preparation of tea but also as raw material for different extracts which are used in food industry as well as in pharmaceutical or cosmetic products. Due to the increasing interest in tea health benefits, the aim of the present study was to characterize several brands of green and black tea found on local market, regarding their content in total phenolic compounds, flavonoids and antioxidant capacity. Total phenolics and flavonoids were determined spectrophotometrically using a modified Folin-Ciocalteu method, respectively a chromogenic system of NaNO2–Al(NO33–NaOH based method. The antioxidant capacity of each tea sample was assessed through the evaluation of free radical scavenging effect on 2,2-diphenyl-1-picrylhydrazyl. The results obtained for the green and black tea samples varied widely, depending on the tea variety. The antioxidant capacity of the analyzed teas ranged between 12.10 and 40.03%RSA, while the total phenolic content was within 2090 and 6080 mg GA/ 100g. The concentrantion in flavonoids was between 9.04 and 15.34 g/100g of tea.

  11. Characterization of local heat fluxes around ICRF antennas on JET

    Energy Technology Data Exchange (ETDEWEB)

    Campergue, A.-L. [Ecole Nationale des Ponts et Chaussées, F77455 Marne-la-Vallée (France); Jacquet, P.; Monakhov, I.; Arnoux, G.; Brix, M.; Sirinelli, A. [Euratom/CCFE Association, Culham Science Centre, Abingdon, OX14 3DB (United Kingdom); Bobkov, V. [Max-Planck-Institut für Plasmaphysik, EURATOM-Assoziation, Garching (Germany); Milanesio, D. [Politecnico di Torino, Department of Electronics, Torino (Italy); Colas, L. [CEA, IRFM, F-13108 Saint-Paul-Lez-Durance (France); Collaboration: JET-EFDA Contributors

    2014-02-12

    When using Ion Cyclotron Range of Frequency (ICRF) heating, enhanced power deposition on Plasma-Facing Components (PFCs) close to the antennas can occur. Experiments have recently been carried out on JET with the new ITER-Like-Wall (ILW) to characterize the heat fluxes on the protection of the JET ICRF antennas, using Infra-Red (IR) thermography measurement. The measured heat flux patterns along the poloidal limiters surrounding powered antennas were compared to predictions from a simple RF sheath rectification model. The RF electric field, parallel to the static magnetic field in front of the antenna, was evaluated using the TOPICA code, integrating a 3D flattened model of the JET A2 antennas. The poloidal density variation in front of the limiters was obtained from the mapping of the Li-beam or edge reflectometry measurements using the flux surface geometry provided by EFIT equilibrium reconstruction. In many cases, this simple model can well explain the position of the maximum heat flux on the different protection limiters and the heat-flux magnitude, confirming that the parallel RF electric field and the electron plasma density in front of the antenna are the main driving parameters for ICRF-induced local heat fluxes.

  12. Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei.

    Science.gov (United States)

    Kafková, Lucie; Ammerman, Michelle L; Faktorová, Drahomíra; Fisk, John C; Zimmer, Sara L; Sobotka, Roman; Read, Laurie K; Lukes, Julius; Hashimi, Hassan

    2012-10-01

    A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.

  13. Accuracy characterization and measurement point planning for workpiece localization

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    [1]Ding, H., Zhu, L. M., Xiong, Z. H., A survey on coordinate measurement, geometric modeling and PR or NC code generation from measured data points, Chinese Journal of Mechanical Engineering, 2003, 39(11): 28-37.[2]Zhu, L. M., Xiong, Z. H., Ding, H. et al., A distance function based approach for localization and profile error evaluation of complex surface, Transactions of ASME, Journal of Manufacturing Science & Engineering, 2004, 126(3): 542-554.[3]Zhu, L. M., Ding, H., Application of kinematic geometry to computational metrology: distance function based heirarchical algorithms for cylindricity evaluation, International Journal of Machine Tools & Manufacture, 2003, 43(2): 203-215.[4]Li, Z., Gou, J., Chu, Y., Geometric algorithms for workpiece localization, IEEE Transactions on Robotics and Automation, 1998, 14: 864-878.[5]Sourlier, D., Bucher, A., Surface-independent, theoretically exact bestfit for arbitrary sculptured, complex, or standard geometries, Precision Engineering, 1995, 17: 101-113.[6]Forbes, A. B., Least-squares best-fit geometric elements, in Algorithms for Approximation II (ed. Mason, J. C., Cox, M. G.), London: Chapman and Hall, 1990, 311-319.[7]Hong, J. W., Tan, X. L., Method and apparatus for determining position and orientation of mechanical objects, U.S. Patent 5208763, 1990.[8]Yan, Z. C., Meng, C. H., Uncertainty analysis and variation reduction of three-dimensional coordinate metrology, International Journal of Machine Tools & Manufacture, 1999, 39: 1199-1261.[9]Yau, H. T., Uncertainty analysis in geometric best fit, International Journal of Machine Tools and Manufacture, 1998, 38: 1323-1342.[10]Murray, R. M., Li, Z., Sastry, S. S., A Mathematical Introduction to Robotic Manipulation, Boca Raton: CRC Press, 1994.[11]Wang, M. Y., Characterizations of localization accuracy of fixtures, IEEE Transactions on Robotics and Automation, 2002, 18(6): 976-981.[12]Chu, Y. X., Gou, J. B., Li, Z. X

  14. Mitochondrial helicases and mitochondrial genome maintenance

    DEFF Research Database (Denmark)

    Aamann, Maria Diget; de Souza-Pinto, Nadja C; Kulikowicz, Tomasz;

    2010-01-01

    Helicases are essential enzymes that utilize the energy of nucleotide hydrolysis to drive unwinding of nucleic acid duplexes. Helicases play roles in all aspects of DNA metabolism including DNA repair, DNA replication and transcription. The subcellular locations and functions of several helicases...... have been studied in detail; however, the roles of specific helicases in mitochondrial biology remain poorly characterized. This review presents important recent advances in identifying and characterizing mitochondrial helicases, some of which also operate in the nucleus....

  15. Genetic characterization of clinical acanthamoeba isolates from Japan using nuclear and mitochondrial small subunit ribosomal RNA.

    Science.gov (United States)

    Rahman, Md Moshiur; Yagita, Kenji; Kobayashi, Akira; Oikawa, Yosaburo; Hussein, Amjad I A; Matsumura, Takahiro; Tokoro, Masaharu

    2013-08-01

    Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.

  16. Characterization of complete mitochondrial genome of Dezhou donkey (Equus asinus) and evolutionary analysis.

    Science.gov (United States)

    Sun, Yan; Jiang, Qiang; Yang, Chunhong; Wang, Xiuge; Tian, Fang; Wang, Yinchao; Ma, Yong; Ju, Zhihua; Huang, Jinming; Zhou, Xiangshan; Zhong, Jifeng; Wang, Changfa

    2016-05-01

    Mitochondrial DNA (mtDNA) has been widely used in species identification and genetic diversification. Comparisons among mtDNAs of closely related species are valuable for phylogenetic studies. However, only the partial mtDNA Cytb gene and the D-loop sequences were used to analysis the phylogenetic relationship between donkey breeds due to lack of complete mitochondrial genome. Dezhou donkey, as a bigger somatotype ass, is one of Chinese domestic donkey breeds, and used by many places as breeding stock. To further investigate the phylogenetic relationship of Dezhou donkey with other breeds, the complete mtDNA was firstly sequenced and de novo assembled using next generation sequence data from total genomic DNA. The genome was 16,813 bp in length (NCBI submission number: KT182635) and contained 13 protein coding genes, 2 ribosomal RNA genes, 25 transfer RNA genes, and 1 control region. Based on the novel complete mtDNA sequence, the sequences of 13 protein coding genes and 2 ribosomal RNA genes were amplifying in other 2 Dezhou donkeys and 3 Yunnan donkeys, respectively. The pattern of genetic variation in horse, wild ass and domestic donkeys among these 15 genes indicated the sequence polymorphisms. The more accurate phylogenetic relationships of donkey species (Dezhou donkey, Yunnan donkey and previously published donkeys) were first obtained using the combined sequences of 12S rRNA+16S rRNA+13 protein-coding genes. Molecular-based phylogeny supported the hypothesis that Chinese domestic donkey breeds may have originated from Somali wild ass, not from Asian wild ass by analyzing mitochondrial genomes.

  17. Sequence and characterization of six mitochondrial subgenomes from Globodera rostochiensis: multipartite structure is conserved among close nematode relatives.

    Science.gov (United States)

    Gibson, Tracey; Blok, Vivian C; Dowton, Mark

    2007-09-01

    Recently, a multipartite mitochondrial genome was characterized in the potato cyst nematode, Globodera pallida. Six subgenomic circles were detectable by PCR, while full-length genomes were not. We investigate here whether this subgenomic organization occurs in a close relative of G. pallida. We amplified and sequenced one entire mitochondrial subgenome from the cyst-forming nematode, Globodera rostochiensis. Comparison of the noncoding region of this subgenome with those reported previously for G. pallida facilitated the design of amplification primers for a range of subgenomes from G. rostochiensis. We then randomly sequenced five subgenomic fragments, each representative of a unique subgenome. This study indicates that the multipartite structure reported for G. pallida is conserved in G. rostochiensis. A comparison of subgenomic organization between these two Globodera species indicates a considerable degree of overlap between them. Indeed, we identify two subgenomes with an organization identical with that reported for G. pallida. However, other subgenomes are unique to G. rostochiensis, although some of these have blocks of genes comparable to those in G. pallida. Dot-plot comparisons of pairs of subgenomes from G. rostochiensis indicate that the different subgenomes share fragments with high sequence identity. We interpret this as evidence that recombination is operating in the mitochondria of G. rostochiensis.

  18. Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex

    Directory of Open Access Journals (Sweden)

    Gidda Satinder K

    2008-09-01

    Full Text Available Abstract Background Carnation Italian ringspot virus (CIRV is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36, are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined. Results Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM, but not with the sorting and assembly machinery (SAM. Conclusion Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer

  19. Cloning, Expression and Characterization of Mitochondrial Manganese Superoxide Dismutase from the Whitefly, Bemisia tabaci

    Directory of Open Access Journals (Sweden)

    Xian-Long Gao

    2013-01-01

    Full Text Available A mitochondrial manganese superoxide dismutase from an invasive species of the whitefly Bemisia tabaci complex (Bt-mMnSOD was cloned and analyzed. The full length cDNA of Bt-mMnSOD is 1210 bp with a 675 bp open reading frame, corresponding to 224 amino acids, which include 25 residues of the mitochondrial targeting sequence. Compared with various vertebrate and invertebrate animals, the MnSOD signature (DVWEHAYY and four conserved amino acids for manganese binding (H54, H102, D186 and H190 were observed in Bt-mMnSOD. Recombinant Bt-mMnSOD was overexpressed in Escherichia coli, and the enzymatic activity of purified mMnSOD was assayed under various temperatures. Quantitative real-time PCR analysis with whiteflies of different development stages showed that the mRNA levels of Bt-mMnSOD were significantly higher in the 4th instar than in other stages. In addition, the in vivo activities of MnSOD in the whitefly were measured under various conditions, including exposure to low (4 °C and high (40 °C temperatures, transfer from a favorable to an unfavorable host plant (from cotton to tobacco and treatment with pesticides. Our results indicate that the whitefly MnSOD plays an important role in cellular stress responses and anti-oxidative processes and that it might contribute to the successful worldwide distribution of the invasive whitefly.

  20. Cloning, expression and characterization of mitochondrial manganese superoxide dismutase from the Whitefly, Bemisia tabaci.

    Science.gov (United States)

    Gao, Xian-Long; Li, Jun-Min; Wang, Yong-Liang; Jiu, Min; Yan, Gen-Hong; Liu, Shu-Sheng; Wang, Xiao-Wei

    2013-01-07

    A mitochondrial manganese superoxide dismutase from an invasive species of the whitefly Bemisia tabaci complex (Bt-mMnSOD) was cloned and analyzed. The full length cDNA of Bt-mMnSOD is 1210 bp with a 675 bp open reading frame, corresponding to 224 amino acids, which include 25 residues of the mitochondrial targeting sequence. Compared with various vertebrate and invertebrate animals, the MnSOD signature (DVWEHAYY) and four conserved amino acids for manganese binding (H54, H102, D186 and H190) were observed in Bt-mMnSOD. Recombinant Bt-mMnSOD was overexpressed in Escherichia coli, and the enzymatic activity of purified mMnSOD was assayed under various temperatures. Quantitative real-time PCR analysis with whiteflies of different development stages showed that the mRNA levels of Bt-mMnSOD were significantly higher in the 4th instar than in other stages. In addition, the in vivo activities of MnSOD in the whitefly were measured under various conditions, including exposure to low (4 °C) and high (40 °C) temperatures, transfer from a favorable to an unfavorable host plant (from cotton to tobacco) and treatment with pesticides. Our results indicate that the whitefly MnSOD plays an important role in cellular stress responses and anti-oxidative processes and that it might contribute to the successful worldwide distribution of the invasive whitefly.

  1. Molecular characterization of Taenia multiceps isolates from Gansu Province, China by sequencing of mitochondrial cytochrome C oxidase subunit 1.

    Science.gov (United States)

    Li, Wen Hui; Jia, Wan Zhong; Qu, Zi Gang; Xie, Zhi Zhou; Luo, Jian Xun; Yin, Hong; Sun, Xiao Lin; Blaga, Radu; Fu, Bao Quan

    2013-04-01

    A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species.

  2. 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)

  3. The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae

    DEFF Research Database (Denmark)

    Hansson Petersen, Camilla A; Alikhani, Nyosha; Behbahani, Homira

    2008-01-01

    by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for Abeta in mitochondria and demonstrate both in vitro and in vivo that Abeta is located to the mitochondrial cristae. Importantly, we also show...

  4. Characterization of the mitochondrial genome of the threatened alpine butterfly, Parnassius nomion (Lepidoptera: Papilionidae).

    Science.gov (United States)

    Dong, Wan-Wei; Jiang, Guo-Fang

    2016-01-01

    The sequenced mitochondrial genome of the threatened alpine butterfly Parnassius nomion includes 13 protein-coding genes (ND1-6, COI-III, ATP6, ATP8, ND4, ND4L, CTYB), 2 ribosomal RNAs (12 S and 16 S), 22 transfer RNAs, which is 14,547 bp in length. Its gene order and orientation are identical to the common type found in most of other completely sequenced lepidopteran mitogenomes. All protein-coding genes are initiated by ATN codons, except for COI, which uses CGA as its start codon. Eleven PCGs use the standard TAA as their termination codon, and COI, COII use the incomplete termination codon T.

  5. Characterization of the mitochondrial genome of the cabbage webworm, Hellula undalis (Lepidoptera: Pyralidae).

    Science.gov (United States)

    Dong, Wan-Wei; Feng, Xiao-Jing; Huang, Guo-Hua; Jiang, Guo-Fang

    2016-01-01

    The sequenced mitochondrial genome of the cabbage webworm Hellula undalis includes 13 protein-coding genes (PCGs) (nad1-6, cox1-3, atp6, atp8, nad4L and cob), two ribosomal RNAs (12S and 16S) and 19 transfer RNAs, which is 14,678 bp in length. Its gene order and orientation are identical to the common types found in most of the other completely sequenced lepidopteran mitogenomes. Thirteen PCGs start with a typical ATN codon, while cox1 uses CGA as its start codon. Some PCGs use the standard TAA as their termination codon, while others use the incomplete termination codon T (cox1, cox2 and nad4).

  6. [Purification of the mitochondrial calcium uniporter from the beef heart and characterization of its properties].

    Science.gov (United States)

    Gritsenko, E N; Kutyshenko, V P; Saris, N E-L; Wahlsten, M; Jokela, J; Mironova, G D

    2010-01-01

    A low-molecular-weight component (LMC) inducing selective transport of calcium across the bilayer lipid membrane has been isolated from mitochondria of the bovine heart by the method developed in our laboratory, which excludes the use of detergents and proteolytic enzymes. It was shown that, in the presence of 10 mM CaCl2, LMC forms conduction channels in the membrane multiples of 5 pS. The specific inhibitor of mitochondrial calcium uniporter, ruthenium red, closes Ca2(+)-induced channels formed in the membrane by LMC. In the absence of calcium or in the presence of potassium ions only, the component is incapable of forming channels of conduction. It was shown using nuclear magnetic resonance that LMC is a complex consisting of lipids, amino acids, and sugars with a molecular weight of 1-2 kDa.

  7. Characterization of mitochondrial genome of Chinese wild mulberry silkworm, Bomyx mandarina(Lepidoptera:Bombycidae)

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The complete mitochondrial genome of Chinese Bombyx mandarina(ChBm) was determined.The cir-cular genome is 15682 bp long, and contains a typical gene complement, order, and arrangement iden-tical to that of Bombyx mori(B.mori) and Japanese Bombyx mandarina(JaBm) except for two addi-tional tRNA-like structures:tRNASer(TGA)-like and tRNAIle(TAT)-like.All protein-coding sequences are initi-ated with a typical ATN codon except for the COI gene, which has a 4-bp TTAG putative initiator codon.Eleven of 13 protein-coding genes(PCGs) have a complete termination codon(all TAA), but the re-maining two genes terminate with incomplete codons.All tRNAs have the typical clover-leaf structures of mitochondrial tRNAs, with the exception of tRNASer(TGA)-like, with a four stem-and-loop structure.The length of the A+T-rich region of ChBm is 484 bp, shorter than those of JaBm(747 bp) and B.mori(494―499 bp).Phylogenetic analysis among B.mori, ChBm, JaBm, and Antheraea pernyi(Anpe) showed that B.mori is more closely related to ChBm than JaBm.The earliest divergence time estimate for B.mori-ChBm and B.mori-JaBm is about 1.08±0.18―1.41±0.24 and 1.53±0.20―2.01±0.26 Mya, respec-tively.ChBm and JaBm diverged around 1.11±0.16―1.45±0.21 Mya.

  8. Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics.

    Science.gov (United States)

    Xiao, Lin; Xian, Hongxu; Lee, Kit Yee; Xiao, Bin; Wang, Hongyan; Yu, Fengwei; Shen, Han-Ming; Liou, Yih-Cherng

    2015-10-09

    Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death.

  9. Characterization of the complete mitochondrial genomes of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae).

    Science.gov (United States)

    Chai, Huan-Na; Du, Yu-Zhou; Zhai, Bao-Ping

    2012-01-01

    The complete mitochondrial genomes (mitogenomes) of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae) were determined and analyzed. The circular genomes were 15,388 bp long for C. medinalis and 15,395 bp long for C. suppressalis. Both mitogenomes contained 37 genes, with gene order similar to that of other lepidopterans. Notably, 12 protein-coding genes (PCGs) utilized the standard ATN, but the cox1 gene used CGA as the initiation codon; the cox1, cox2, and nad4 genes in the two mitogenomes had the truncated termination codons T, T, and TA, respectively, but the nad5 gene was found to use T as the termination codon only in the C. medinalis mitogenome. Additionally, the codon distribution and Relative Synonymous Codon Usage of the 13 PCGs in the C. medinalis mitogenome were very different from those in other pyralid moth mitogenomes. Most of the tRNA genes had typical cloverleaf secondary structures. However, the dihydrouridine (DHU) arm of the trnS1(AGN) gene did not form a stable stem-loop structure. Forty-nine helices in six domains, and 33 helices in three domains were present in the secondary structures of the rrnL and rrnS genes of the two mitogenomes, respectively. There were four major intergenic spacers, except for the A+T-rich region, spanning at least 12 bp in the two mitogenomes. The A+T-rich region contained an 'ATAGT(A)'-like motif followed by a poly-T stretch in the two mitogenomes. In addition, there were a potential stem-loop structure, a duplicated 25-bp repeat element, and a microsatellite '(TA)(13)' observed in the A+T-rich region of the C. medinalis mitogenome. A poly-T motif, a duplicated 31-bp repeat element, and a 19-bp triplication were found in the C. suppressalis mitogenome. However, there are many differences in the A+T-rich regions between the C. suppressalis mitogenome sequence in the present study and previous reports. Finally, the phylogenetic relationships of these insects were reconstructed based on amino acid

  10. Characterization of the Complete Mitochondrial Genomes of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae

    Directory of Open Access Journals (Sweden)

    Huan-Na Chai, Yu-Zhou Du, Bao-Ping Zhai

    2012-01-01

    Full Text Available The complete mitochondrial genomes (mitogenomes of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae were determined and analyzed. The circular genomes were 15,388 bp long for C. medinalis and 15,395 bp long for C. suppressalis. Both mitogenomes contained 37 genes, with gene order similar to that of other lepidopterans. Notably, 12 protein-coding genes (PCGs utilized the standard ATN, but the cox1 gene used CGA as the initiation codon; the cox1, cox2, and nad4 genes in the two mitogenomes had the truncated termination codons T, T, and TA, respectively, but the nad5 gene was found to use T as the termination codon only in the C. medinalis mitogenome. Additionally, the codon distribution and Relative Synonymous Codon Usage of the 13 PCGs in the C. medinalis mitogenome were very different from those in other pyralid moth mitogenomes. Most of the tRNA genes had typical cloverleaf secondary structures. However, the dihydrouridine (DHU arm of the trnS1(AGN gene did not form a stable stem-loop structure. Forty-nine helices in six domains, and 33 helices in three domains were present in the secondary structures of the rrnL and rrnS genes of the two mitogenomes, respectively. There were four major intergenic spacers, except for the A+T-rich region, spanning at least 12 bp in the two mitogenomes. The A+T-rich region contained an 'ATAGT(A'-like motif followed by a poly-T stretch in the two mitogenomes. In addition, there were a potential stem-loop structure, a duplicated 25-bp repeat element, and a microsatellite '(TA13' observed in the A+T-rich region of the C. medinalis mitogenome. A poly-T motif, a duplicated 31-bp repeat element, and a 19-bp triplication were found in the C. suppressalis mitogenome. However, there are many differences in the A+T-rich regions between the C. suppressalis mitogenome sequence in the present study and previous reports. Finally, the phylogenetic relationships of these insects were reconstructed based on

  11. Sequence Characterization of Mitochondrial 12S rRNA Gene in Mouse Deer (Moschiola indica for PCR-RFLP Based Species Identification

    Directory of Open Access Journals (Sweden)

    Chandra Mohan Siddappa

    2013-01-01

    Full Text Available Mitochondrial 12S rRNA has proven to be a useful molecular marker for better conservation and management of the endangered species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP of the mitochondrial 12S rRNA gene has proven to be a reliable and efficient tool for the identification of different Indian deer species of family cervidae. In the present study, mitochondrial 12S rRNA gene sequence of mouse deer (Moschiola indica belonging to the family Tragulidae was characterized and analysed in silico for its use in species identification. Genomic DNA was isolated from the hair follicles and mitochondrial 12S rRNA gene was amplified using universal primers. PCR product was cloned and sequenced for the first time. The sequence of mouse deer showed 90.04, 90.08, 90.04, 91.2, 90.04, and 90.08% identities with sika deer, sambar, hog deer, musk deer, chital, and barking deer, respectively. Restriction mapping in Lasergene (DNAstar Inc., Madison, WI, USA revealed that mouse deer mitochondrial 12S rRNA gene sequence can be differentiated from the other deer species in PCR-RFLP using RsaI, DdeI, BsrI, and BstSFI. With the help of predicted pattern, mouse deer can be identified using genomic DNA from a variety of biomaterials, thereby providing molecular aid in wildlife forensics and conservation of the species.

  12. Mutations of C19orf12, coding for a transmembrane glycine zipper containing mitochondrial protein, cause mis-localization of the protein, inability to respond to oxidative stress and increased mitochondrial Ca2+

    DEFF Research Database (Denmark)

    Venco, Paola; Bonora, Massimo; Giorgi, Carlotta;

    2015-01-01

    19orf12 protein was not exclusively present in mitochondria, but also in the Endoplasmic Reticulum (ER) and MAM (Mitochondria Associated Membrane), while mutant C19orf12 variants presented a different localization. Moreover, after induction of oxidative stress, a GFP-tagged C19orf12 wild-type protein...... was able to relocate to the cytosol. On the contrary, mutant isoforms were not able to respond to oxidative stress. High mitochondrial calcium concentration and increased H2O2 induced apoptosis were found in fibroblasts derived from one patient as compared to controls. C19orf12 protein is a 17 k...... to rearrange in a structural domain, which is homologs to the N-terminal regulatory domain of the magnesium transporter MgtE, suggesting that C19orf12 may act as a regulatory protein for human MgtE transporters. The mutations here described affect respectively one glycine residue of the glycine zipper motifs...

  13. Characterization and comparison of mitochondrial DNAs and rRNAs from Penicillium urticae and P. chrysogenum.

    Science.gov (United States)

    Sekiguchi, J; Ohsaki, T; Yamamoto, H; Koichi, K; Shida, T

    1990-03-01

    Mitochondrial DNA (mt DNA) from a patulin producer, Penicillium urticae (synonym P. griseofulvum), was 27.8 kb +/- 0.6 kb in size by electron microscopy and 27.2 kb by agarose gel electrophoresis. Restriction endonuclease maps for nine restriction enzymes were constructed, and eleven fragments which covered the total range of the mt DNA were cloned into the Escherichia coli plasmid vector pUC19. Southern analysis of the native genomes of P. urticae and P. chrysogenum with six of the cloned fragments as probes indicated similar genome arrangements as well as similar restriction maps. Both the large and small rRNA genes of P. urticae and P. chrysogenum were located on these restriction maps using Southern hybridization, and the result also supported the similar arrangement. Agarose/formaldehyde gel electrophoresis indicated that the small rRNA was 1.5 kb in size in both species; but, surprisingly, the large rRNA was 4.2 kb in size for P. urticae and 3.5 kb for P. chrysogenum. These sizes were, respectively, 1.1 kb and 0.4 kb larger than those from the very closely related Aspergillus nidulans.

  14. Characterization of the complete mitochondrial genomes of two whipworms Trichuris ovis and Trichuris discolor (Nematoda: Trichuridae).

    Science.gov (United States)

    Liu, Guo-Hua; Wang, Yan; Xu, Min-Jun; Zhou, Dong-Hui; Ye, Yong-Gang; Li, Jia-Yuan; Song, Hui-Qun; Lin, Rui-Qing; Zhu, Xing-Quan

    2012-12-01

    For many years, whipworms (Trichuris spp.) have been described with a relatively narrow range of both morphological and biometrical features. Moreover, there has been insufficient discrimination between congeners (or closely related species). In the present study, we determined the complete mitochondrial (mt) genomes of two whipworms Trichuris ovis and Trichuris discolor, compared them and then tested the hypothesis that T. ovis and T. discolor are distinct species by phylogenetic analyses using Bayesian inference, maximum likelihood and maximum parsimony) based on the deduced amino acid sequences of the mt protein-coding genes. The complete mt genomes of T. ovis and T. discolor were 13,946 bp and 13,904 bp in size, respectively. Both mt genomes are circular, and consist of 37 genes, including 13 genes coding for proteins, 2 genes for rRNA, and 22 genes for tRNA. The gene content and arrangement are identical to that of human and pig whipworms Trichuris trichiura and Trichuris suis. Taken together, these analyses showed genetic distinctiveness and strongly supported the recent proposal that T. ovis and T. discolor are distinct species using nuclear ribosomal DNA and a portion of the mtDNA sequence dataset. The availability of the complete mtDNA sequences of T. ovis and T. discolor provides novel genetic markers for studying the population genetics, diagnostics and molecular epidemiology of T. ovis and T. discolor.

  15. Genetic characterization of Golden mahseer (Tor putitora) populations using mitochondrial DNA markers.

    Science.gov (United States)

    Sati, Jyoti; Kumar, Rohit; Sahoo, Prabhati Kumari; Patiyal, Rabindar S; Ali, Shahnawaz; Barat, Ashoktaru

    2015-02-01

    Golden Mahseer (Tor putitora) is an economically important fish of India and Southeast Asia. The present study examined the genetic variations between seven geographically isolated populations of T. putitora using Cyt b (Cytochrome b) and ATPase6/8 gene sequences of mitochondrial DNA. Analysis of 133 sequences of Cyt b (1141 bp) and 130 sequences of ATPase6/8 gene (842 bp) revealed 47 and 44 haplotypes, respectively. The estimated haplotype and nucleotide diversity was high in River Jia Bhoreli (Bhalukpong) population (h = 1.00000, π = 0.007121 for Cyt b and h = 0.90441 π = 0.004867 for ATPase6/8). Results of AMOVA indicated that majority of the genetic variations in both genes were due to variation among populations (60.79% for Cyt b and 51.41% for ATPase6/8 gene). The pairwise F(ST) comparison and neighbor-joining tree revealed high genetic divergence of River Jia Bhoreli population from other populations. The understanding of genetic variations of T. putitora populations will play a key role in conservation and management of this endangered fish species.

  16. Genetic characterization of ticks from southwestern Romania by sequences of mitochondrial cox1 and nad5 genes.

    Science.gov (United States)

    Chitimia, Lidia; Lin, Rui-Qing; Cosoroaba, Iustin; Wu, Xiang-Yun; Song, Hui-Qun; Yuan, Zi-Guo; Zhu, Xing-Quan

    2010-11-01

    In the present study, samples representing three hard tick species and one soft tick species, namely Dermacentor marginatus, Haemaphysalis punctata, Ixodes ricinus and Argas persicus from southwestern Romania, and one hard tick, Haemaphysalis longicornis, from China were characterized genetically by a portion of mitochondrial cytochrome c oxidase subunit 1 gene (pcox1) and a portion of nicotinamide adenine dinucleotide dehydrogenase subunit 5 gene (pnad5). The pcox1 and pnad5 were amplified separately from individual ticks by PCR, sequenced and analyzed. The length of pcox1 and pnad5 sequences of all samples was 732 and 519 bp, respectively. The intra-specific sequence variation in De. marginatus was 0.1-1.0% for pcox1 and 0.2-1.2% for pnad5, whereas in Ha. punctata it was 0.4-1.9% for pcox1 and 0.4-1.0% for pnad5. For the tick species examined in the present study, sequence comparison revealed that the inter-specific sequence differences were higher: 15.9-27.6% for pcox1 and 20.3-42.4% for pnad5. This suggests that the cox1 and nad5 sequences could provide useful genetic markers for the specific identification and genetic characterization of ticks in Romania and elsewhere.

  17. Mitochondrial Dynamics in Mitochondrial Diseases

    Directory of Open Access Journals (Sweden)

    Juan M. Suárez-Rivero

    2016-12-01

    Full Text Available Mitochondria are very versatile organelles in continuous fusion and fission processes in response to various cellular signals. Mitochondrial dynamics, including mitochondrial fission/fusion, movements and turnover, are essential for the mitochondrial network quality control. Alterations in mitochondrial dynamics can cause neuropathies such as Charcot-Marie-Tooth disease in which mitochondrial fusion and transport are impaired, or dominant optic atrophy which is caused by a reduced mitochondrial fusion. On the other hand, mitochondrial dysfunction in primary mitochondrial diseases promotes reactive oxygen species production that impairs its own function and dynamics, causing a continuous vicious cycle that aggravates the pathological phenotype. Mitochondrial dynamics provides a new way to understand the pathophysiology of mitochondrial disorders and other diseases related to mitochondria dysfunction such as diabetes, heart failure, or Hungtinton’s disease. The knowledge about mitochondrial dynamics also offers new therapeutics targets in mitochondrial diseases.

  18. Molecular characterization of Fasciola hepatica from Sardinia based on sequence analysis of genomic and mitochondrial gene markers.

    Science.gov (United States)

    Farjallah, Sarra; Ben Slimane, Badreddine; Piras, Cristina Maria; Amor, Nabil; Garippa, Giovanni; Merella, Paolo

    2013-11-01

    The aim of the present study is to investigate for the first time the genetic diversity of samples identified morphologically as Fasciola hepatica (Platyhelminthes: Trematoda: Digenea) (n=66) from sheep and cattle from two localities of Sardinia and to compare them with available data from other localities by partial sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes, the mitochondrial cytochrome c oxidase subunit I (COI), and nicotinamide adenine dinucleotide dehydrogenase subunit I (ND1) genes. Comparison of the sequences from Sardinia with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. hepatica. The nucleotide sequencing of ITS rDNA showed no nucleotide variation in the ITS-1, 5.8S and ITS-2 rDNA sequences among all Sardinian samples, comparing with two ITS-2 haplotypes in standard F. hepatica, showing a substitution C/T in 20 position 859, reported previously from Tunisia, Algeria, Australia, Uruguay and Spain. The present study shows that in Sardinian sheep and cattle there is the most frequent haplotype (FhITS-H1) of F. hepatica species from South Europe. Considering NDI sequences, the phylogenetic trees showed reliable grouping among the haplotypes of F. hepatica from Sardinia and the mitochondrial lineage I, including the main N1 haplotype, observed previously from Europe (Russia, Belarus, Ukraine and Bulgaria), Armenia, West Africa (Nigeria), America (Uruguay and USA), Asia (Turkey, Japan, and China), Georgia, Turkmenistan, Azerbaijan and Australia. Furthermore, common haplotypes FhCOI-H1 and FhCOI-H2 of F. hepatica from Sardinia also corresponded mostly to the first lineage including the main C1 haplotype reported previously from Eastern European and Western Asian populations, they belonged just to a phylogenically distinguishable clade, as F. hepatica from Australia, France, Turkey, Uruguay, Russia, Armenia, Ukraine, Belarus

  19. Sirt3, Mitochondrial ROS, Ageing, and Carcinogenesis

    Directory of Open Access Journals (Sweden)

    David Gius

    2011-09-01

    Full Text Available One fundamental observation in cancer etiology is that the rate of malignancies in any mammalian population increases exponentially as a function of age, suggesting a mechanistic link between the cellular processes governing longevity and carcinogenesis. In addition, it is well established that aberrations in mitochondrial metabolism, as measured by increased reactive oxygen species (ROS, are observed in both aging and cancer. In this regard, genes that impact upon longevity have recently been characterized in S. cerevisiae and C. elegans, and the human homologs include the Sirtuin family of protein deacetylases. Interestingly, three of the seven sirtuin proteins are localized into the mitochondria suggesting a connection between the mitochondrial sirtuins, the free radical theory of aging, and carcinogenesis. Based on these results it has been hypothesized that Sirt3 functions as a mitochondrial fidelity protein whose function governs both aging and carcinogenesis by modulating ROS metabolism. Sirt3 has also now been identified as a genomically expressed, mitochondrial localized tumor suppressor and this review will outline potential relationships between mitochondrial ROS/superoxide levels, aging, and cell phenotypes permissive for estrogen and progesterone receptor positive breast carcinogenesis.

  20. Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm.

    Science.gov (United States)

    Cataldo, L; Baig, K; Oko, R; Mastrangelo, M A; Kleene, K C

    1996-11-01

    The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.

  1. Molecular characterization and mutational analysis of the human B17 subunit of the mitochondrial respiratory chain complex I.

    Science.gov (United States)

    Smeitink, J; Loeffen, J; Smeets, R; Triepels, R; Ruitenbeek, W; Trijbels, F; van den Heuvel, L

    1998-08-01

    Bovine NADH:ubiquinone oxidoreductase (complex 1) of the mitochondrial respiratory chain consists of about 36 nuclear-encoded subunits. We review the current knowledge of the 15 human complex I subunits cloned so far, and report the 598-bp cDNA sequence, the chromosomal localization and the tissue expression of an additional subunit, the B17 subunit. The cDNA open reading frame of B17 comprises 387 bp and encodes a protein of 128 amino acids (calculated Mr 15.5 kDa). There is 82.7% and 78.1% homology, respectively, at the cDNA and amino acid level with the bovine counterpart. The gene of the B17 subunit has been mapped to chromosome 2. Multiple-tissue dot-blots showed ubiquitous expression of the mRNA with relatively higher expression in tissues known for their high energy demand. Of these, kidney showed the highest expression. Mutational analysis of the subunit revealed no mutations or polymorphisms in 20 patients with isolated enzymatic complex I deficiency in cultured skin fibroblasts.

  2. Characterization of Fasciola spp. in Myanmar on the basis of spermatogenesis status and nuclear and mitochondrial DNA markers.

    Science.gov (United States)

    Ichikawa, Madoka; Bawn, Saw; Maw, Ni Ni; Htun, Lat Lat; Thein, Myint; Gyi, Aung; Sunn, Kyaw; Katakura, Ken; Itagaki, Tadashi

    2011-12-01

    Fasciola spp. in Myanmar were characterized on the basis of spermatogenesis status and DNA markers of nuclear internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1). We collected 88 adult flukes from Yangon, Lashio, and Myitkyina. Spermatogenesis status was analyzed by the presence of sperm in the seminal vesicles, and 8 aspermic and 80 spermic flukes were detected. The flukes were identified on the basis of spermatogenesis status and ITS1 types which were analyzed by a PCR-RFLP method, and 80 spermic flukes were identified as F. gigantica. A very low detection rate of aspermic Fasciola sp. indicated that they are not established in Myanmar. In phylogenetic analyses, the 7 aspermic Fasciola sp. from Myitkyina displayed a haplotype in nad1 sequence, which was identical to that of aspermic Fasciola sp. from other Asian countries including China. Therefore, they were probably introduced from China through an infected domestic ruminant. On the other hand, 17 nad1 haplotypes detected in F. gigantica belonged to 2 clades unique to Myanmar, each with a distinct founder haplotype in a network analysis. This indicated a unique history of F. gigantica introduction into Myanmar involving ancient artificial movements of domestic ruminants.

  3. Ampelographic and DNA characterization of local grapevine accessions of the Tuscia area (Latium,Italy)

    Science.gov (United States)

    The presence of local vines in the Tuscia area (Latium region, Italy) was documented by historical sources. Ampelographic and molecular characterization (35 morphological descriptors and 16 microsatellite loci) was carried out on accessions belonging to the most frequently mentioned local vines. S...

  4. Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7.

    Science.gov (United States)

    Lantsman, Yelena; Tan, Kevin S W; Morada, Mary; Yarlett, Nigel

    2008-09-01

    A mitochondrion-like organelle (MLO) was isolated from isotonic homogenates of Blastocystis. The organelle sedimented at 5000 g for 10 min, and had an isopycnic density in sucrose of 1.2 g ml(-1). Biochemical characterization enabled the demonstration of several key enzymes that allowed the construction of a metabolic pathway consisting of an incomplete Krebs cycle linked to the oxygen-sensitive enzymes pyruvate : NADP(+) oxidoreductase (PNO), acetate : succinate CoA transferase (ASCT) and succinate thiokinase (STK), which cumulatively are responsible for recycling CoA and generating ATP. The organelle differs from typical aerobic mitochondria in possessing an oxygen-sensitive PNO that can use FAD(+) or FMN(+) as electron acceptor but is inactive with NAD(+), Spinacia oleracea ferredoxin or Clostridium pasteurianum ferredoxin. A gene with 77 % sequence similarity to the PNO mitochondrion precursor cluster from Euglena gracilis sp[Q941N5] was identified in the Blastocystis genome database. A second cluster with 56 % sequence similarity to the pyruvate : ferredoxin oxidoreductase (PFOR) from Trichomonas vaginalis was also identified, which is in agreement with the concept that the PNO gene arose through the fusion of a eubacterial gene for PFOR with the gene for NADPH : cytochrome p450 reductase. Hydrogenase activity was not detected under the conditions used in this study. The Blastocystis oranelle therefore demonstrates significant biochemical differences from traditional mitochondria and hydrogenosomes, but possesses features of both. Based upon the results of this study, the Blastocystis organelle falls into the category of a MLO.

  5. A variant of Leber hereditary optic neuropathy characterized by recovery of vision and by an unusual mitochondrial genetic etiology

    Energy Technology Data Exchange (ETDEWEB)

    Mackey, D. (Royal Children' s Hospital, Melbourne (Australia)); Howell, N. (Univ. of Texas, Galveston (United States))

    1992-12-01

    The Tas2 and Vic2 Australian families are affected with a variant of Leber hereditary optic neuropathy (LHON). The risk of developing the optic neuropathy shows strict maternal inheritance, and the opthalmological changes in affected family members are characteristic of LHON. However, in contrast to the common form of the disease, members of these two families show a high frequency of vision recovery. To ascertain the mitochondrial genetic etiology of the LHON in these families, both (a) the nucleotide sequences of the seven mitochondrial genes encoding subunits of respiratory-chain complex I and (b) the mitochondrial cytochrome b gene were determined for representatives of both families. Neither family carries any of the previously identified primary mitochondrial LHON mutations: ND4/11778, ND1/3460, or ND1/4160. Instead, both LHON families carry multiple nucleotide changes in the mitochondrial complex I genes, which produce conservative amino acid changes. From the available sequence data, it is inferred that the Vic2 and Tas2 LHON families are phylogenetically related to each other and to a cluster of LHON families in which mutations in the mitochondrial cytochrome b gene have been hypothesized to play a primary etiological role. However, sequencing analysis establishes that the Vic2 and Tas2 LHON families do not carry these cytochrome b mutations. There are two hypotheses to account for the unusual mitochondrial genetic etiology of the LHON in the Tas2 and Vic2 LHON families. One possibility is that there is a primary LHON mutation within the mitochondrial genome but that it is at a site that was not included in the sequencing analyses. Alternatively, the disease in these families may result from the cumulative effects of multiple secondary LHON mutations that have less severe phenotypic consequences. 29 refs., 3 figs., 3 tabs.

  6. 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 ...

  7. Mitochondrial Myopathies

    Science.gov (United States)

    ... which stimulates normal beating of the heart. Cardiac muscle damage also may occur. People with mitochondrial disorders may need to have regular examina- tions by a cardiologist. Other potential health issues Some people with mitochondrial disease experience ...

  8. 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...

  9. Mitochondrial genetics

    OpenAIRE

    Chinnery, Patrick Francis; Hudson, Gavin

    2013-01-01

    Introduction In the last 10 years the field of mitochondrial genetics has widened, shifting the focus from rare sporadic, metabolic disease to the effects of mitochondrial DNA (mtDNA) variation in a growing spectrum of human disease. The aim of this review is to guide the reader through some key concepts regarding mitochondria before introducing both classic and emerging mitochondrial disorders. Sources of data In this article, a review of the current mitochondrial genetics literature was con...

  10. MitoMiner: a data warehouse for mitochondrial proteomics data.

    Science.gov (United States)

    Smith, Anthony C; Blackshaw, James A; Robinson, Alan J

    2012-01-01

    MitoMiner (http://mitominer.mrc-mbu.cam.ac.uk/) is a data warehouse for the storage and analysis of mitochondrial proteomics data gathered from publications of mass spectrometry and green fluorescent protein tagging studies. In MitoMiner, these data are integrated with data from UniProt, Gene Ontology, Online Mendelian Inheritance in Man, HomoloGene, Kyoto Encyclopaedia of Genes and Genomes and PubMed. The latest release of MitoMiner stores proteomics data sets from 46 studies covering 11 different species from eumetazoa, viridiplantae, fungi and protista. MitoMiner is implemented by using the open source InterMine data warehouse system, which provides a user interface allowing users to upload data for analysis, personal accounts to store queries and results and enables queries of any data in the data model. MitoMiner also provides lists of proteins for use in analyses, including the new MitoMiner mitochondrial proteome reference sets that specify proteins with substantial experimental evidence for mitochondrial localization. As further mitochondrial proteomics data sets from normal and diseased tissue are published, MitoMiner can be used to characterize the variability of the mitochondrial proteome between tissues and investigate how changes in the proteome may contribute to mitochondrial dysfunction and mitochondrial-associated diseases such as cancer, neurodegenerative diseases, obesity, diabetes, heart failure and the ageing process.

  11. cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

    Institute of Scientific and Technical Information of China (English)

    HUANG; Shengbing; SONG; Wei; LIN; Qishui

    2005-01-01

    A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5'-terminal and 3'-terminal were obtained by RACE technique. The full-length cDNA that encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-Qop. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.

  12. FASTKD2 Nonsense Mutation in an Infantile Mitochondrial Encephalomyopathy Associated with Cytochrome C Oxidase Deficiency

    OpenAIRE

    2008-01-01

    In two siblings we found a mitochondrial encephalomyopathy, characterized by developmental delay, hemiplegia, convulsions, asymmetrical brain atrophy, and low cytochrome c oxidase (COX) activity in skeletal muscle. The disease locus was identified on chromosome 2 by homozygosity mapping; candidate genes were prioritized for their known or predicted mitochondrial localization and then sequenced in probands and controls. A homozygous nonsense mutation in the KIAA0971 gene segregated with the di...

  13. Genetically Determined Insulin Resistance is Characterized by Down-Regulation of Mitochondrial Oxidative Metabolism in Human Skeletal Muscle

    DEFF Research Database (Denmark)

    Kristensen, Jonas M; Skov, Vibe; Wojtaszewski, Jørgen

    2010-01-01

    Transcriptional profiling of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated a co-ordinated down-regulation of oxidative phosphorylation (OxPhos) genes, suggesting a link between insulin resistance and mitochondrial dysfunction. However, whether...... mitochondrial dysfunction is a cause or consequence of insulin resistance remains to be clarified. In the present study, we tested the hypothesis that mitochondrial oxidative metabolism was down-regulated in skeletal muscle of patients with genetically determined insulin resistance. Skeletal muscle biopsies.......02), and complex V (ATP5B; p=0.005). Our data demonstrate that genetically determined insulin resistance is associated with a co-ordinated down-regulation of OxPhos components both at the transcriptional and translational level. These findings suggest that an impaired biological response to insulin in skeletal...

  14. Morphological characterization of local landraces of rapeseed (Brassica campestris L. var toria of Nepal

    Directory of Open Access Journals (Sweden)

    Salik Ram Gupta

    2015-12-01

    Full Text Available Rapeseed (Brassica campestris L. var toria is the main source of edible oil for Nepalese people. 54 rapeseed lines were collected from different hilly district of Nepal ranging from 987 m to 2550 m altitude. These lines were planted in augmented design for its traits characterization in Khumaltar 2013. Different traits of local rapeseed were characterized, and evaluated. NGRC 02778 performed better followed by SR-02 than local checks Morang-2, Chitwan Local and Unnati in terms of yield, days to maturity and pest infestation. Similarly, genotype SR-18 was late and SR-16 was earlier in terms of days to maturity. In conclusion, SR-02 was found better genotype based on different characteristics measured among all local rapeseeds planted in Khumaltar 2013. Thus SR-2 can be used as parents in crossing material for further breeding purposes and it can also be tested in further trial.

  15. Characterizing Curvilinear Features Using the Localized Normal-Score Ensemble Kalman Filter

    OpenAIRE

    Haiyan Zhou; Liangping Li; J. Jaime Gómez-Hernández

    2012-01-01

    The localized normal-score ensemble Kalman filter is shown to work for the characterization of non-multi-Gaussian distributed hydraulic conductivities by assimilating state observation data. The influence of type of flow regime, number of observation piezometers, and the prior model structure are evaluated in a synthetic aquifer. Steady-state observation data are not sufficient to identify the conductivity channels. Transient-state data are necessary for a good characterization of the hydraul...

  16. Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase: SUBSTRATE SPECIFICITY, DNA BRANCH MIGRATION, AND ABILITY TO OVERCOME BLOCKADES TO DNA UNWINDING.

    Science.gov (United States)

    Khan, Irfan; Crouch, Jack D; Bharti, Sanjay Kumar; Sommers, Joshua A; Carney, Sean M; Yakubovskaya, Elena; Garcia-Diaz, Miguel; Trakselis, Michael A; Brosh, Robert M

    2016-07-01

    Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5' or 3' single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5' to 3' directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.

  17. Local dependence in random graph models: characterization, properties and statistical inference.

    Science.gov (United States)

    Schweinberger, Michael; Handcock, Mark S

    2015-06-01

    Dependent phenomena, such as relational, spatial and temporal phenomena, tend to be characterized by local dependence in the sense that units which are close in a well-defined sense are dependent. In contrast with spatial and temporal phenomena, though, relational phenomena tend to lack a natural neighbourhood structure in the sense that it is unknown which units are close and thus dependent. Owing to the challenge of characterizing local dependence and constructing random graph models with local dependence, many conventional exponential family random graph models induce strong dependence and are not amenable to statistical inference. We take first steps to characterize local dependence in random graph models, inspired by the notion of finite neighbourhoods in spatial statistics and M-dependence in time series, and we show that local dependence endows random graph models with desirable properties which make them amenable to statistical inference. We show that random graph models with local dependence satisfy a natural domain consistency condition which every model should satisfy, but conventional exponential family random graph models do not satisfy. In addition, we establish a central limit theorem for random graph models with local dependence, which suggests that random graph models with local dependence are amenable to statistical inference. We discuss how random graph models with local dependence can be constructed by exploiting either observed or unobserved neighbourhood structure. In the absence of observed neighbourhood structure, we take a Bayesian view and express the uncertainty about the neighbourhood structure by specifying a prior on a set of suitable neighbourhood structures. We present simulation results and applications to two real world networks with 'ground truth'.

  18. Mitochondrial vasculopathy

    Institute of Scientific and Technical Information of China (English)

    Josef Finsterer; Sinda Zarrouk-Mahjoub

    2016-01-01

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

  19. Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation

    DEFF Research Database (Denmark)

    Lombard, David B; Alt, Frederick W; Cheng, Hwei-Ling;

    2007-01-01

    Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble...... mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial...... deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis...

  20. Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis.

    Directory of Open Access Journals (Sweden)

    David C Hess

    2009-03-01

    Full Text Available Mitochondria are central to many cellular processes including respiration, ion homeostasis, and apoptosis. Using computational predictions combined with traditional quantitative experiments, we have identified 100 proteins whose deficiency alters mitochondrial biogenesis and inheritance in Saccharomyces cerevisiae. In addition, we used computational predictions to perform targeted double-mutant analysis detecting another nine genes with synthetic defects in mitochondrial biogenesis. This represents an increase of about 25% over previously known participants. Nearly half of these newly characterized proteins are conserved in mammals, including several orthologs known to be involved in human disease. Mutations in many of these genes demonstrate statistically significant mitochondrial transmission phenotypes more subtle than could be detected by traditional genetic screens or high-throughput techniques, and 47 have not been previously localized to mitochondria. We further characterized a subset of these genes using growth profiling and dual immunofluorescence, which identified genes specifically required for aerobic respiration and an uncharacterized cytoplasmic protein required for normal mitochondrial motility. Our results demonstrate that by leveraging computational analysis to direct quantitative experimental assays, we have characterized mutants with subtle mitochondrial defects whose phenotypes were undetected by high-throughput methods.

  1. Recombinant Mitochondrial Transcription Factor A with N-terminal Mitochondrial Transduction Domain Increases Respiration and Mitochondrial Gene Expression

    OpenAIRE

    Iyer, Shilpa; Thomas, Ravindar R.; Portell, Francisco R.; Dunham, Lisa D.; Quigley, Caitlin K.; Bennett, James P

    2009-01-01

    We developed a scalable procedure to produce human mitochondrial transcription factor A (TFAM) modified with an N-terminal protein transduction domain (PTD) and mitochondrial localization signal (MLS) that allow it to cross membranes and enter mitochondria through its “mitochondrial transduction domain” (MTD=PTD+MLS). Alexa488-labeled MTD-TFAM rapidly entered the mitochondrial compartment of cybrid cells carrying the G11778A LHON mutation. MTD-TFAM reversibly increased respiration and levels ...

  2. AFM characterization of the shape of surface structures with localization factor.

    Science.gov (United States)

    Bonyár, Attila

    2016-08-01

    Although with the use of scanning probe microscopy (SPM) methods the topographical imaging of surfaces is now widely available, the characterization of surface structures, especially their shape, and the processes which change these features is not trivial with the existing surface describing parameters. In this work the application of a parameter called localization factor is demonstrated for the quantitative characterization of surface structures and for processes which alter the shape of these structures. The theory and optimal operation range of this parameter are discussed with three application examples: microstructure characterization of gold thin films, characterization of the changes in the grain structure of these films during thermal annealing, and finally, characterization of the oxidation processes on a polished tin surface.

  3. Comparing the effects of mitochondrial targeted and localized antioxidants with cellular antioxidants in human skin cells exposed to UVA and hydrogen peroxide.

    Science.gov (United States)

    Oyewole, Anne O; Wilmot, Marie-Claire; Fowler, Mark; Birch-Machin, Mark A

    2014-01-01

    Skin cancer and aging are linked to increased cellular reactive oxygen species (ROS), particularly following exposure to ultraviolet A (UVA) in sunlight. As mitochondria are the main source of cellular ROS, this study compared the protective effects of mitochondria-targeted and -localized antioxidants (MitoQ and tiron, respectively) with cellular antioxidants against oxidative stress-induced [UVA and hydrogen peroxide (H2O2)] mitochondrial DNA (mtDNA) damage in human dermal fibroblasts. With the use of a long quantitative PCR assay, tiron (EC50 10 mM) was found to confer complete (100%) protection (Pantioxidants investigated. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway provides cellular protection against oxidative stress. An ELISA assay for the Nrf2 target gene heme oxygenase-1 (HO-1) and studies using Nrf2 small interfering RNA both indicated that tiron's mode of action was Nrf2 independent. The comet assay showed that tiron's protective effect against H2O2-induced nuclear DNA damage was greater than the cellular antioxidants and MitoQ (Pantioxidants.

  4. Characterizing Curvilinear Features Using the Localized Normal-Score Ensemble Kalman Filter

    Directory of Open Access Journals (Sweden)

    Haiyan Zhou

    2012-01-01

    Full Text Available The localized normal-score ensemble Kalman filter is shown to work for the characterization of non-multi-Gaussian distributed hydraulic conductivities by assimilating state observation data. The influence of type of flow regime, number of observation piezometers, and the prior model structure are evaluated in a synthetic aquifer. Steady-state observation data are not sufficient to identify the conductivity channels. Transient-state data are necessary for a good characterization of the hydraulic conductivity curvilinear patterns. Such characterization is very good with a dense network of observation data, and it deteriorates as the number of observation piezometers decreases. It is also remarkable that, even when the prior model structure is wrong, the localized normal-score ensemble Kalman filter can produce acceptable results for a sufficiently dense observation network.

  5. Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization.

    Science.gov (United States)

    Lam, M; Oleinick, N L; Nieminen, A L

    2001-12-14

    Photodynamic therapy (PDT), a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in human epidermoid carcinoma A431 cells. However, the precise mechanism of PDT-induced apoptosis is not well characterized. To dissect the pathways of PDT-induced apoptosis, we investigated the involvement of mitochondrial damage by examining a second generation photosensitizer, the silicon phthalocyanine 4 (Pc 4). By using laser-scanning confocal microscopy, we found that Pc 4 localized to cytosolic membranes primarily, but not exclusively, in mitochondria. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes when cells were exposed to Pc 4 and 670-675 nm light. This was followed by mitochondrial inner membrane permeabilization, depolarization and swelling, cytochrome c release, and apoptotic death. Desferrioxamine prevented mitochondrial ROS production and the events thereafter. Cyclosporin A plus trifluoperazine, blockers of the mitochondrial permeability transition, inhibited mitochondrial inner membrane permeabilization and depolarization without affecting mitochondrial ROS generation. These data indicate that the mitochondrial ROS are critical in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death during PDT with Pc 4.

  6. Characterization of the Cardiac Overexpression of HSPB2 Reveals Mitochondrial and Myogenic Roles Supported by a Cardiac HspB2 Interactome.

    Directory of Open Access Journals (Sweden)

    Julianne H Grose

    Full Text Available Small Heat Shock Proteins (sHSPs are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2, which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait and a human cardiac library (prey coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 "cardiac interactome" to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID. A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is

  7. Hepatitis C Virus-Induced Mitochondrial Dysfunctions

    Directory of Open Access Journals (Sweden)

    Birke Bartosch

    2013-03-01

    Full Text Available Chronic hepatitis C is characterized by metabolic disorders and a microenvironment in the liver dominated by oxidative stress, inflammation and regeneration processes that lead in the long term to hepatocellular carcinoma. Many lines of evidence suggest that mitochondrial dysfunctions, including modification of metabolic fluxes, generation and elimination of oxidative stress, Ca2+ signaling and apoptosis, play a central role in these processes. However, how these dysfunctions are induced by the virus and whether they play a role in disease progression and neoplastic transformation remains to be determined. Most in vitro studies performed so far have shown that several of the hepatitis C virus (HCV proteins localize to mitochondria, but the consequences of these interactions on mitochondrial functions remain contradictory, probably due to the use of artificial expression and replication systems. In vivo studies are hampered by the fact that innate and adaptive immune responses will overlay mitochondrial dysfunctions induced directly in the hepatocyte by HCV. Thus, the molecular aspects underlying HCV-induced mitochondrial dysfunctions and their roles in viral replication and the associated pathology need yet to be confirmed in the context of productively replicating virus and physiologically relevant in vitro and in vivo model systems.

  8. Gestational diabetes is characterized by reduced mitochondrial protein expression and altered calcium signaling proteins in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Kristen E Boyle

    Full Text Available The rising prevalence of gestational diabetes mellitus (GDM affects up to 18% of pregnant women with immediate and long-term metabolic consequences for both mother and infant. Abnormal glucose uptake and lipid oxidation are hallmark features of GDM prompting us to use an exploratory proteomics approach to investigate the cellular mechanisms underlying differences in skeletal muscle metabolism between obese pregnant women with GDM (OGDM and obese pregnant women with normal glucose tolerance (ONGT. Functional validation was performed in a second cohort of obese OGDM and ONGT pregnant women. Quantitative proteomic analysis in rectus abdominus skeletal muscle tissue collected at delivery revealed reduced protein content of mitochondrial complex I (C-I subunits (NDUFS3, NDUFV2 and altered content of proteins involved in calcium homeostasis/signaling (calcineurin A, α1-syntrophin, annexin A4 in OGDM (n = 6 vs. ONGT (n = 6. Follow-up analyses showed reduced enzymatic activity of mitochondrial complexes C-I, C-III, and C-IV (-60-75% in the OGDM (n = 8 compared with ONGT (n = 10 subjects, though no differences were observed for mitochondrial complex protein content. Upstream regulators of mitochondrial biogenesis and oxidative phosphorylation were not different between groups. However, AMPK phosphorylation was dramatically reduced by 75% in the OGDM women. These data suggest that GDM is associated with reduced skeletal muscle oxidative phosphorylation and disordered calcium homeostasis. These relationships deserve further attention as they may represent novel risk factors for development of GDM and may have implications on the effectiveness of physical activity interventions on both treatment strategies for GDM and for prevention of type 2 diabetes postpartum.

  9. Synthetic Bichalcone TSWU-BR23 Induces Apoptosis of Human Colon Cancer HT-29 Cells by p53-Mediated Mitochondrial Oligomerization of BAX/BAK and Lipid Raft Localization of CD95/FADD.

    Science.gov (United States)

    Lin, Meng-Liang; Chen, Shih-Shun; Wu, Tian-Shung

    2015-10-01

    A synthetic bichalcone analog, (E)-1-(3-((4-(4-acetylphenyl)piperazin-1-yl)methyl)-4-hydroxy-5-methoxyphenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR23), has been shown to induce apoptosis in human colon cancer HT-29 cells involving the induction of CD95 and FAS-associated protein death domain (FADD), but its precise mechanism of action has not been fully elucidated. Using cell-surface biotinylation and sucrose density-gradient-based membrane flotation techniques, we showed that the disruption of TSWU-BR23-induced lipid raft localization of CD95/FADD by cholesterol-depleting agent (methyl-β-cyclodextrin) was reversed by cholesterol replenishment. Blockade of p53 expression by short-hairpin RNA (shRNA) suppressed oligomeric Bcl-2-associated x protein (BAX)/Bcl-2 antagonist killer 1 (BAK)-mediated mitochondrial apoptosis but did not inhibit lipid raft localization of CD95/FADD and pro-caspase-8 cleavage induced by TSWU-BR23. Co-expression of p53 shRNA and dominant-negative mutant of FADD completely inhibited TSWU-BR32-induced mitochondrial apoptotic cell death. Collectively, these data demonstrate that TSWU-BR23 leads to HT-29 cell apoptosis by inducing p53-mediated mitochondrial oligomerization of BAX/BAK and the localization of CD95/FADD with lipid rafts at the cell surface.

  10. In Situ Characterization of the Local Work Function along Individual Free Standing Nanowire by Electrostatic Deflection.

    Science.gov (United States)

    Chen, Yicong; Zhao, Chengchun; Huang, Feng; Zhan, Runze; Deng, Shaozhi; Xu, Ningsheng; Chen, Jun

    2016-02-17

    In situ characterization of the work function of quasi one dimensional nanomaterials is essential for exploring their applications. Here we proposed to use the electrostatic deflection induced by work function difference between nanoprobe and nanowire for in situ measuring the local work function along a free standing nanowire. The physical mechanism for the measurement was discussed in details and a parabolic relationship between the deflection and the potential difference was derived. As a demonstration, measurement of the local work functions on the tip and the sidewall of a ZnO nanowire with Au catalyst at its end and a LaB6 nanowire have been achieved with good accuracy.

  11. Statistical Characterization and Mitigation of NLOS Errors in UWB Localization Systems

    CERN Document Server

    Montorsi, Francesco; Vitetta, Giorgio M; 10.1109/ICUWB.2011.6058928

    2012-01-01

    In this paper some new experimental results about the statistical characterization of the non-line-of-sight (NLOS) bias affecting time-of-arrival (TOA) estimation in ultrawideband (UWB) wireless localization systems are illustrated. Then, these results are exploited to assess the performance of various maximum-likelihood (ML) based algorithms for joint TOA localization and NLOS bias mitigation. Our numerical results evidence that the accuracy of all the considered algorithms is appreciably influenced by the LOS/NLOS conditions of the propagation environment.

  12. Generation and characterization of transgenic mice expressing mitochondrial targeted red fluorescent protein selectively in neurons: modeling mitochondriopathy in excitotoxicity and amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Wang Yi

    2011-11-01

    Full Text Available Abstract Background Mitochondria have roles or appear to have roles in the pathogenesis of several chronic age-related and acute neurological disorders, including Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, Parkinson's disease, and cerebral ischemia, and could be critical targets for development of rational mechanism-based, disease-modifying therapeutics for treating these disorders effectively. A deeper understanding of neural tissue mitochondria pathobiologies as definitive mediators of neural injury, disease, and cell death merits further study, and the development of additional tools to study neural mitochondria will help achieve this unmet need. Results We created transgenic mice that express the coral (Discosoma sp. red fluorescent protein DsRed2 specifically in mitochondria of neurons using a construct engineered with a Thy1 promoter, specific for neuron expression, to drive expression of a fusion protein of DsRed2 with a mitochondrial targeting sequence. The biochemical and histological characterization of these mice shows the expression of mitochondrial-targeted DsRed2 to be specific for mitochondria and concentrated in distinct CNS regions, including cerebral cortex, hippocampus, thalamus, brainstem, and spinal cord. Red fluorescent mitochondria were visualized in cerebral cortical and hippocampal pyramidal neurons, ventrobasal thalamic neurons, subthalamic neurons, and spinal motor neurons. For the purpose of proof of principle application, these mice were used in excitotoxicity paradigms and double transgenic mice were generated by crossing Thy1-mitoDsRed2 mice with transgenic mice expressing enhanced-GFP (eGFP under the control of the Hlxb9 promoter that drives eGFP expression specifically in motor neurons and by crossing Thy1-mitoDsRed2 mice to amyotrophic lateral sclerosis (ALS mice expressing human mutant superoxide dismutase-1. Conclusions These novel transgenic mice will be a useful tool for better understanding

  13. A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

    OpenAIRE

    Wei, Lei; Yin, Li; Hu,Xiaole; Xu, Ying; Chen,Fang

    2014-01-01

    Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene ...

  14. Mitochondrial biogenesis: pharmacological approaches.

    Science.gov (United States)

    Valero, Teresa

    2014-01-01

    diseases do not have exclusively a mitochondrial origin but they might have an important mitochondrial component both on their onset and on their development. This is the case of type 2 diabetes or neurodegenerative diseases. Type 2 diabetes is characterized by a peripheral insulin resistance accompanied by an increased secretion of insulin as a compensatory system. Among the explanations about the origin of insulin resistance Mónica Zamora and Josep A. Villena (Department of Experimental and Health Sciences, Universitat Pompeu Fabra / Laboratory of Metabolism and Obesity, Universitat Autònoma de Barcelona, Spain) [5] consider the hypothesis that mitochondrial dysfunction, e.g. impaired (mitochondrial) oxidative capacity of the cell or tissue, is one of the main underlying causes of insulin resistance and type 2 diabetes. Although this hypothesis is not free of controversy due to the uncertainty on the sequence of events during type 2 diabetes onset, e.g. whether mitochondrial dysfunction is the cause or the consequence of insulin resistance, it has been widely observed that improving mitochondrial function also improves insulin sensitivity and prevents type 2 diabetes. Thus restoring oxidative capacity by increasing mitochondrial mass appears as a suitable strategy to treat insulin resistance. The effort made by researchers trying to understand the signaling pathways mediating mitochondrial biogenesis has uncovered new potential pharmacological targets and opens the perspectives for the design of suitable treatments for insulin resistance. In addition some of the current used strategies could be used to treat insulin resistance such as lifestyle interventions (caloric restriction and endurance exercise) and pharmacological interventions (thiazolidinediones and other PPAR agonists, resveratrol and other calorie restriction mimetics, AMPK activators, ERR activators). Mitochondrial biogenesis is of special importance in modern neurochemistry because of the broad spectrum

  15. Molecular characterization of Aedes aegypti (L. (Diptera: Culicidae of Easter Island based on analysis of the mitochondrial ND4 gene

    Directory of Open Access Journals (Sweden)

    Claudia Andrea Núñez

    2016-06-01

    Full Text Available ABSTRACT Aedes aegypti mosquitoes are the main vector of viruses Dengue, Zika and Chikungunya. Shortly after the first report of the dengue vector Ae. aegypti in Easter Island (Rapa Nui in late 2000, the first disease outbreak dengue occurred. Viral serotyping during the 2002 outbreak revealed a close relationship with Pacific DENV-1 genotype IV viruses, supporting the idea that the virus most likely originated in Tahiti. Mitochondrial NADH dehydrogenase subunit 4 (ND4 DNA sequences generated from 68 specimens of Ae. aegypti from Easter Island reporting a unique finding of a single maternal lineage of Ae. aegypti on Easter Island.

  16. Mitochondrial disorders.

    Science.gov (United States)

    Zeviani, M; Tiranti, V; Piantadosi, C

    1998-01-01

    Mitochondrial respiration, the most efficient metabolic pathway devoted to energy production, is at the crosspoint of 2 quite different genetic systems, the nuclear genome and the mitochondrial genome (mitochondrial DNA, mtDNA). The latter encodes a few essential components of the mitochondrial respiratory chain and has unique molecular and genetic properties that account for some of the peculiar features of mitochondrial disorders. However, the perpetuation, propagation, and expression of mtDNA, the majority of the subunits of the respiratory complexes, as well as a number of genes involved in their assembly and turnover, are contained in the nuclear genome. Although mitochondrial disorders have been known for more than 30 years, a major breakthrough in their understanding has come much later, with the discovery of an impressive, ever-increasing number of mutations of mitochondrial DNA. Partial deletions or duplications of mtDNA, or maternally inherited point mutations, have been associated with well-defined clinical syndromes. However, phenotypes transmitted as mendelian traits have also been identified. These include clinical entities defined on the basis of specific biochemical defects, and also a few autosomal dominant or recessive syndromes associated with multiple deletions or tissue-specific depletion of mtDNA. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogenous. They range from lesions of single tissues or structures, such as the optic nerve in Leber hereditary optic neuropathy or the cochlea in maternally inherited nonsyndromic deafness, to more widespread lesions including myopathies, encephalomyopathies, cardiopathies, or complex multisystem syndromes. The recent advances in genetic studies provide both diagnostic tools and new pathogenetic insights in this rapidly expanding area of human pathology.

  17. Oestrogens ameliorate mitochondrial dysfunction in Leber's hereditary optic neuropathy.

    Science.gov (United States)

    Giordano, Carla; Montopoli, Monica; Perli, Elena; Orlandi, Maurizia; Fantin, Marianna; Ross-Cisneros, Fred N; Caparrotta, Laura; Martinuzzi, Andrea; Ragazzi, Eugenio; Ghelli, Anna; Sadun, Alfredo A; d'Amati, Giulia; Carelli, Valerio

    2011-01-01

    Leber's hereditary optic neuropathy, the most frequent mitochondrial disease due to mitochondrial DNA point mutations in complex I, is characterized by the selective degeneration of retinal ganglion cells, leading to optic atrophy and loss of central vision prevalently in young males. The current study investigated the reasons for the higher prevalence of Leber's hereditary optic neuropathy in males, exploring the potential compensatory effects of oestrogens on mutant cell metabolism. Control and Leber's hereditary optic neuropathy osteosarcoma-derived cybrids (11778/ND4, 3460/ND1 and 14484/ND6) were grown in glucose or glucose-free, galactose-supplemented medium. After having shown the nuclear and mitochondrial localization of oestrogen receptors in cybrids, experiments were carried out by adding 100 nM of 17β-oestradiol. In a set of experiments, cells were pre-incubated with the oestrogen receptor antagonist ICI 182780. Leber's hereditary optic neuropathy cybrids in galactose medium presented overproduction of reactive oxygen species, which led to decrease in mitochondrial membrane potential, increased apoptotic rate, loss of cell viability and hyper-fragmented mitochondrial morphology compared with control cybrids. Treatment with 17β-oestradiol significantly rescued these pathological features and led to the activation of the antioxidant enzyme superoxide dismutase 2. In addition, 17β-oestradiol induced a general activation of mitochondrial biogenesis and a small although significant improvement in energetic competence. All these effects were oestrogen receptor mediated. Finally, we showed that the oestrogen receptor β localizes to the mitochondrial network of human retinal ganglion cells. Our results strongly support a metabolic basis for the unexplained male prevalence in Leber's hereditary optic neuropathy and hold promises for a therapeutic use for oestrogen-like molecules.

  18. FASTKD2 nonsense mutation in an infantile mitochondrial encephalomyopathy associated with cytochrome c oxidase deficiency.

    Science.gov (United States)

    Ghezzi, Daniele; Saada, Ann; D'Adamo, Pio; Fernandez-Vizarra, Erika; Gasparini, Paolo; Tiranti, Valeria; Elpeleg, Orly; Zeviani, Massimo

    2008-09-01

    In two siblings we found a mitochondrial encephalomyopathy, characterized by developmental delay, hemiplegia, convulsions, asymmetrical brain atrophy, and low cytochrome c oxidase (COX) activity in skeletal muscle. The disease locus was identified on chromosome 2 by homozygosity mapping; candidate genes were prioritized for their known or predicted mitochondrial localization and then sequenced in probands and controls. A homozygous nonsense mutation in the KIAA0971 gene segregated with the disease in the proband family. The corresponding protein is known as fas activated serine-threonine kinase domain 2, FASTKD2. Confocal immunofluorescence colocalized a tagged recombinant FASTKD2 protein with mitochondrial markers, and membrane-potential-dependent in vitro mitochondrial import was demonstrated in isolated mitochondria. In staurosporine-induced-apoptosis experiments, decreased nuclear fragmentation was detected in treated mutant versus control fibroblasts. In conclusion, we found a loss-of-function mutation in a gene segregating with a peculiar mitochondrial encephalomyopathy associated with COX deficiency in skeletal muscle. The corresponding protein is localized in the mitochondrial inner compartment. Preliminary data indicate that FASTKD2 plays a role in mitochondrial apoptosis.

  19. Statistical Characterization and Mitigation of NLOS Bias in UWB Localization Systems

    CERN Document Server

    Montorsi, Francesco; Vitetta, Giorgio M

    2012-01-01

    Propagation in non-line-of-sight (NLOS) conditions is one of the major impairments in ultrawideband (UWB) wireless localization systems based on time-of-arrival (TOA) measurements. In this paper the problem of the joint statistical characterization of the NLOS bias and of the most representative features of LOS/NLOS UWB waveforms is investigated. In addition, the performance of various maximum-likelihood (ML) estimators for joint localization and NLOS bias mitigation is assessed. Our numerical results evidence that the accuracy of all the considered estimators is appreciably influenced by the LOS/NLOS conditions of the propagation environment and that a statistical knowledge of multiple signal features can be exploited to mitigate the NLOS bias, reducing the overall localization error.

  20. The mitochondrial elongation factors MIEF1 and MIEF2 exert partially distinct functions in mitochondrial dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Tong; Yu, Rong [Department of Oncology–Pathology, Karolinska Institutet, CCK R8:05, Karolinska University Hospital Solna, SE-171 76 Stockholm (Sweden); Jin, Shao-Bo [Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Stockholm (Sweden); Han, Liwei [Department of Oncology–Pathology, Karolinska Institutet, CCK R8:05, Karolinska University Hospital Solna, SE-171 76 Stockholm (Sweden); Lendahl, Urban [Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Stockholm (Sweden); Zhao, Jian, E-mail: Jian.Zhao@ki.se [Department of Oncology–Pathology, Karolinska Institutet, CCK R8:05, Karolinska University Hospital Solna, SE-171 76 Stockholm (Sweden); Nistér, Monica [Department of Oncology–Pathology, Karolinska Institutet, CCK R8:05, Karolinska University Hospital Solna, SE-171 76 Stockholm (Sweden)

    2013-11-01

    Mitochondria are dynamic organelles whose morphology is regulated by a complex balance of fission and fusion processes, and we still know relatively little about how mitochondrial dynamics is regulated. MIEF1 (also called MiD51) has recently been characterized as a key regulator of mitochondrial dynamics and in this report we explore the functions of its paralog MIEF2 (also called MiD49), to learn to what extent MIEF2 is functionally distinct from MIEF1. We show that MIEF1 and MIEF2 have many functions in common. Both are anchored in the mitochondrial outer membrane, recruit Drp1 from the cytoplasm to the mitochondrial surface and cause mitochondrial fusion, and MIEF2, like MIEF1, can interact with Drp1 and hFis1. MIEF1 and MIEF2, however, also differ in certain aspects. MIEF1 and MIEF2 are differentially expressed in human tissues during development. When overexpressed, MIEF2 exerts a stronger fusion-promoting effect than MIEF1, and in line with this, hFis1 and Mff can only partially revert the MIEF2-induced fusion phenotype, whereas MIEF1-induced fusion is reverted to a larger extent by hFis1 and Mff. MIEF2 forms high molecular weight oligomers, while MIEF1 is largely present as a dimer. Furthermore, MIEF1 and MIEF2 use distinct domains for oligomerization: in MIEF1, the region from amino acid residues 109–154 is required, whereas oligomerization of MIEF2 depends on amino acid residues 1 to 49, i.e. the N-terminal end. We also show that oligomerization of MIEF1 is not required for its mitochondrial localization and interaction with Drp1. In conclusion, our data suggest that the mitochondrial regulators MIEF1 and MIEF2 exert partially distinct functions in mitochondrial dynamics. - Highlights: • MIEF1 and MIEF2 recruit Drp1 to mitochondria and cause mitochondrial fusion. • MIEF2, like MIEF1, can interact with Drp1 and hFis1. • MIEF1 and MIEF2 are differentially expressed in human tissues during development. • MIEF2 exerts a stronger fusion

  1. Characterizing wave propagation to improve indoor step-level person localization using floor vibration

    Science.gov (United States)

    Mirshekari, Mostafa; Pan, Shijia; Zhang, Pei; Noh, Hae Young

    2016-04-01

    The objective of this paper is to characterize frequency-dependent wave propagation of footstep induced floor vibration to improve robustness of vibration-based occupant localization. Occupant localization is an essential part of many smart structure applications (e.g., energy management, patient/customer tracking, etc.). Exist- ing techniques include visual (e.g. cameras and IR sensors), acoustic, RF, and load-based approaches. These approaches have many deployment and operational requirements that limits their adaptation. To overcome these limitations, prior work has utilized footstep-induced vibrations to allow sparse sensor configuration and non-intrusive detection. However, frequency dependent propagation characteristics and low signal-to-noise ratio (SNR) of footstep-induced vibrations change the shape of the signal. Furthermore, estimating the wave propagation velocity for forming the multilateration equations and localizing the footsteps is a challenging task. They, in turn, lead to large errors of localization. In this paper, we present a structural vibration based indoor occupant localization technique using improved time-difference-of-arrival between multiple vibration sensors. In particular we overcome signal distortion by decomposing the signal into frequency components and focusing on high energy components for accurate indoor localization. Such decomposition leverages the frequency-specific propagation characteristics and reduces the effect of low SNR (by choosing the components of highest energy). Furthermore, we develop a velocity calibration method that finds the optimal velocity which minimizes the localization error. We validate our approach through field experiments in a building with human participants. We are able to achieve an average localization error of less than 0.21 meters, which corresponds to a 13X reduction in error when compared to the baseline method using raw data.

  2. Mitochondrial Band-7 family proteins: scaffolds for respiratory chain assembly?

    OpenAIRE

    2014-01-01

    The band-7 protein family comprises a diverse set of membrane-bound proteins characterized by the presence of a conserved domain. The exact function of this band-7 domain remains elusive, but examples from animal and bacterial stomatin-type proteins demonstrate binding to lipids and the ability to assemble into membrane-bound oligomers that form putative scaffolds. Some members, such as prohibitins (PHB) and human stomatin-like protein 2 (HsSLP2), localize to the mitochondrial inner membrane ...

  3. The complete mitochondrial genome of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae): genome characterization and phylogenetic considerations.

    Science.gov (United States)

    Xu, Tian-Jun; Cheng, Yuan-Zhi; Liu, Xue-Zhu; Shi, Ge; Wang, Ri-Xin

    2011-01-01

    The complete mitochondrial genome sequence of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae) was determined and phylogenetic analysis was conducted to elucidate the evolutionary relationship of the marbled rockfish with other Sebastinae species. This mitochondrial genome, consisting of 17301 bp, is highly similar to that of most other vertebrates, containing the same gene order and an identical number of genes or regions, including 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and one putative control region. Most of the genes are encoded on the H-strand, while the ND6 and seven tRNA genes (for Gln, Ala, Asn, Tyr, Ser (UCA), Glu, and Pro) are encoded on the L-strand. The reading frame of two pairs of genes overlapped on the same strand (the ATPase 8 and 6 genes overlapped by ten nucleotides; ND4L and ND4 genes overlapped by seven nucleotides). The possibly nonfunctional light-strand replication origin folded into a typical stem-loop secondary structure and a conserved motif (5'-GCCGG-3') was found at the base of the stem within the tRNA(Cys) gene. An extent termination-associated sequence (ETAS) and conserved sequence blocks (CSB) were identified in the control region, except for CSB-1; unusual long tandem repeats were found at the 3' end of the control region. Phylogenetic analyses supported the view that Sebastinae comprises four genera (Sebates, Hozukius, Helicolenus, and Sebasticus).

  4. Molecular characterization of local maize varieties from the Biosphere Reserve La Sepultura, Mexico.

    Directory of Open Access Journals (Sweden)

    Manuel Antonio Hernández-Ramos

    2016-12-01

    Full Text Available The objective of this research was the molecular and phylogenetic characterization of local maize populations of the La Sepultura Biosphere Reserve (REBISE, Mexico. In nine communities from the REBISE, Mexico, nineteen populations of local maize were sampled during 2012-2013. Two improved commercial varieties (Vs-536 and V-424, a commercial hybrid (H-MX3, a local corn (Coastal recently introduced in the REBISE, two local materials from the state of Tlaxcala, Mexico; both from Chalqueño race and conical Elotes (young corns and a teosinte (Zea mays ssp. Mexicana were included. They totaled twenty six different samples. The characterization was done with the PCR technique and inter-type microsatellite genetic markers. In total 113, fragments were ampli ed and that ranged from 150-2200 bp, of which 85,8% were polymorphic. Sequences “AG”, “AC” and “GAA” showed a greater number of ampli ed bands and higher polymorphism. The primers comprised of UBC834, I9 and UBC868 ampli ed the best. The similarities found within the polymorphic bands may be due to their equality such as varieties, races, species or genetic combination effect, a result of their cross- pollination. The dendrogram obtained showed a relatively low cophenetic correlation (r= 0,721, with a low degree of reliability, however four major groups of varieties are clearly conformed. The rst was named Valles Altos Corns, integrated by the conical Chalqueño and the conical Elotes races; the second is identi ed as having a common ancestor with Teocintle; the third is an improved hybrid by the H-MX3; and the fourth includes the REBISE local maize varieties and other commercial varieties (Vs-536 y Vs-424.

  5. Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria.

    Science.gov (United States)

    Spelbrink, J N; Li, F Y; Tiranti, V; Nikali, K; Yuan, Q P; Tariq, M; Wanrooij, S; Garrido, N; Comi, G; Morandi, L; Santoro, L; Toscano, A; Fabrizi, G M; Somer, H; Croxen, R; Beeson, D; Poulton, J; Suomalainen, A; Jacobs, H T; Zeviani, M; Larsson, C

    2001-07-01

    The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

  6. Molecular Characterization of Pasteurella multocida: Its Implication with Epidemiology and The Development of Local Isolate Vaccines

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    Supar

    2007-12-01

    Full Text Available Pasteurella multocida strains are the causative agents of pasteurellosis attacking wide range domestic and wild animals. The important pasteurellosis in animals in Indonesia are Haemorrhagic septicaemic (HS or Septicaemia epizootica (SE in large and small ruminants, fowl cholera in poultry and water powls. HS associated with P. multocida in large ruminants was controlled by killed whole cell vaccines produced by the use of P. multocida Katha strain, whereas fowl cholera was controlled by antimicrobial drugs. At present, there are only a limited molecular biology techniques have been applied to investigate P. multocida isolates from different geographic locations in Indonesia. Genomic DNA of P. multocida from HS cases from various provinces which were treated with restriction endonuclease ApaI and analysed by means of pulsed-field gel electrophoreses (PFGE demonstrated the presence of high degree distinctive DNA pattern compared to that of the vaccine (Katha strain from Burma and other reference strains. Similar different patterns were found in genomic DNA of local P. multocida isolates from cholera disease of chicken and ducks. P. multocida isolates from some provinces showed different DNA patterns to each other. These DNA pattern differences were probably associated with the alteration of their pathogenicity, antigenicity and immunogenicity, but it has not been confirmed yet. Vaccines prepared from P. multocida isolate originated from local HS cases and local cholera demonstrated better protection in experimental animals against heterologous and homologous challenges, in terms of higher and consistency antibody responses compared to that of Katha strain or imported P. multocida poultry strains. This supports the potential aspects of molecular characterization of local P. multocida isolates kept at the BCC Unit. These isolates may play an important role in developing local master seeds to produce pasteurellosis local vaccines which would be more

  7. Frequent mitochondrial gene rearrangements at the hymenopteran nad3-nad5 junction.

    Science.gov (United States)

    Dowton, Mark; Castro, Lyda R; Campbell, Sarah L; Bargon, Sharmilla D; Austin, Andrew D

    2003-05-01

    We characterized the organization of mitochondrial genes from a diverse range of hymenopterans. Of the 21 taxa characterized, 12 had distinct, derived organizations. Some rearrangements were consistent with the duplication-random loss mechanism, while others were not. Local inversions were relatively common, i.e., rearrangements characterized by the movement of genes from one mitochondrial strand to the other, opposite or close to their ancestral position. This type of rearrangement is inconsistent with the duplication/random loss model of mitochondrial gene rearrangement. Instead, they are best explained by the operation of recombination. Taxa with derived organizations were restricted to a single, monophyletic group of wasps, the Apocrita, which comprise about 90% of all hymenopterans.

  8. Characterization of failure modes in deep UV and deep green LEDs utilizing advanced semiconductor localization techniques.

    Energy Technology Data Exchange (ETDEWEB)

    Tangyunyong, Paiboon; Miller, Mary A.; Cole, Edward Isaac, Jr.

    2012-03-01

    We present the results of a two-year early career LDRD that focused on defect localization in deep green and deep ultraviolet (UV) light-emitting diodes (LEDs). We describe the laser-based techniques (TIVA/LIVA) used to localize the defects and interpret data acquired. We also describe a defect screening method based on a quick electrical measurement to determine whether defects should be present in the LEDs. We then describe the stress conditions that caused the devices to fail and how the TIVA/LIVA techniques were used to monitor the defect signals as the devices degraded and failed. We also describe the correlation between the initial defects and final degraded or failed state of the devices. Finally we show characterization results of the devices in the failed conditions and present preliminary theories as to why the devices failed for both the InGaN (green) and AlGaN (UV) LEDs.

  9. Possible Implication of Local Immune Response in Darier's Disease: An Immunohistochemical Characterization of Lesional Inflammatory Infiltrate

    Directory of Open Access Journals (Sweden)

    Clelia Miracco

    2010-01-01

    Full Text Available Cell-mediated immunity is considered to be normal in Darier's Disease (DD, an inherited skin disorder complicated by skin infections. To date, there are no investigations on the local inflammatory infiltrate in DD skin lesions. In this immunohistochemical study we characterized and quantified it, making comparisons with two other inflammatory skin disorders, that is, pemphigus vulgaris (PV and lichen ruber planus (LRP, and with the normal skin (NSk. We found a significant (<.05 decrease of CD1a+ Langerhans cells (LCs in DD, compared to PV, LRP, and NSk, and of CD123+ plasmacytoid dendritic cells (pDCs, compared to PV and LRP. We hypothesize that the genetic damage of keratinocytes might result in a loss of some subsets of dendritic cells and, consequently, in an impaired local immune response, which might worsen the infections that inevitably occur in this disease.

  10. Identification, characterization and evolution of non-local quasi-Lagrangian structures in turbulence

    Institute of Scientific and Technical Information of China (English)

    Yue Yang

    2016-01-01

    The recent progress on non-local Lagrangian and quasi-Lagrangian structures in turbulence is reviewed. The quasi-Lagrangian structures, e.g., vortex surfaces in vis-cous flow, gas-liquid interfaces in multi-phase flow, and flame fronts in premixed combustion, can show essential Lagrangian following properties, but they are able to have topological changes in the temporal evolution. In addition, they can represent or influence the turbulent flow field. The challenges for the investigation of the non-local structures include their identification, characterization, and evolution. The improving understanding of the quasi-Lagrangian struc-tures is expected to be helpful to elucidate crucial dynamics and develop structure-based predictive models in turbulence.

  11. Aerosol retrieval algorithm for the characterization of local aerosol using MODIS L1B data

    Science.gov (United States)

    Wahab, A. M.; Sarker, M. L. R.

    2014-02-01

    Atmospheric aerosol plays an important role in radiation budget, climate change, hydrology and visibility. However, it has immense effect on the air quality, especially in densely populated areas where high concentration of aerosol is associated with premature death and the decrease of life expectancy. Therefore, an accurate estimation of aerosol with spatial distribution is essential, and satellite data has increasingly been used to estimate aerosol optical depth (AOD). Aerosol product (AOD) from Moderate Resolution Imaging Spectroradiometer (MODIS) data is available at global scale but problems arise due to low spatial resolution, time-lag availability of AOD product as well as the use of generalized aerosol models in retrieval algorithm instead of local aerosol models. This study focuses on the aerosol retrieval algorithm for the characterization of local aerosol in Hong Kong for a long period of time (2006-2011) using high spatial resolution MODIS level 1B data (500 m resolution) and taking into account the local aerosol models. Two methods (dark dense vegetation and MODIS land surface reflectance product) were used for the estimation of the surface reflectance over land and Santa Barbara DISORT Radiative Transfer (SBDART) code was used to construct LUTs for calculating the aerosol reflectance as a function of AOD. Results indicate that AOD can be estimated at the local scale from high resolution MODIS data, and the obtained accuracy (ca. 87%) is very much comparable with the accuracy obtained from other studies (80%-95%) for AOD estimation.

  12. Characterization of Miniature Millimeter-Wave Vivaldi Antenna for Local Multipoint Distribution Service

    Science.gov (United States)

    Simon, Rainee N.; Lee, Richard Q.

    1997-01-01

    The paper presents first, an efficient measurement technique to characterize the input impedance of a Vivaldi antenna, second, a simple technique to impedance match a Vivaldi antenna to a 50 Ohm feed line and lastly, a desktop arrangement to determine the directional gain of a Vivaldi antenna. The characterization is done using a microwave wafer probe station, a ground-signal microwave probe, impedance standard substrate and an automatic network analyzer. The Vivaldi antenna with a matching transformer has a VSVIR close to unity and a gain of about 10 dB over the frequency band of 27.5 to 28.35 GHz which is allocated for local multipoint distribution service (LMDS).

  13. Isolation and characterization of chitosan from different local insects in Egypt.

    Science.gov (United States)

    Marei, Narguess H; El-Samie, Emtithal Abd; Salah, Taher; Saad, Gamal R; Elwahy, Ahmed H M

    2016-01-01

    Chitin was extracted from four different local sources: the shrimp (Penaeus monodon), the desert locust (Schistocerca gregaria), the honey bee (Apis mellifera) and the beetles (Calosoma rugosa). Chitosan was then obtained by deacetylation of chitin and physicochemically characterized using the Fourier transform infrared (FTIR) and X-ray diffraction. The moisture content, water binding capacity, fats binding capacity, ash content were determined and chitosans morphology was visualized using the scanning electron microscope (SEM). The difference between the obtained chitosans from three insect sources and α-chitosan from shrimp in terms of crystallinity, fibrous structure was discussed.

  14. Mitochondrial myopathy induces a starvation-like response.

    Science.gov (United States)

    Tyynismaa, Henna; Carroll, Christopher J; Raimundo, Nuno; Ahola-Erkkilä, Sofia; Wenz, Tina; Ruhanen, Heini; Guse, Kilian; Hemminki, Akseli; Peltola-Mjøsund, Katja E; Tulkki, Valtteri; Oresic, Matej; Moraes, Carlos T; Pietiläinen, Kirsi; Hovatta, Iiris; Suomalainen, Anu

    2010-10-15

    Mitochondrial respiratory chain (RC) deficiency is among the most common causes of inherited metabolic disease, but its physiological consequences are poorly characterized. We studied the skeletal muscle gene expression profiles of mice with late-onset mitochondrial myopathy. These animals express a dominant patient mutation in the mitochondrial replicative helicase Twinkle, leading to accumulation of multiple mtDNA deletions and progressive subtle RC deficiency in the skeletal muscle. The global gene expression pattern of the mouse skeletal muscle showed induction of pathways involved in amino acid starvation response and activation of Akt signaling. Furthermore, the muscle showed induction of a fasting-related hormone, fibroblast growth factor 21 (Fgf21). This secreted regulator of lipid metabolism was also elevated in the mouse serum, and the animals showed widespread changes in their lipid metabolism: small adipocyte size, low fat content in the liver and resistance to high-fat diet. We propose that RC deficiency induces a mitochondrial stress response, with local and global changes mimicking starvation, in a normal nutritional state. These results may have important implications for understanding the metabolic consequences of mitochondrial myopathies.

  15. Mitochondrial respiration is sensitive to cytoarchitectural breakdown.

    Science.gov (United States)

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

    2016-11-07

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

  16. Molecular characterization of Echinococcus granulosus from Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene.

    Science.gov (United States)

    Sánchez, Elizabeth; Cáceres, Omar; Náquira, César; Garcia, David; Patiño, Gladys; Silvia, Herrera; Volotão, Aline C; Fernandes, Octavio

    2010-09-01

    Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosus worldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

  17. Characterization of population structure from the mitochondrial DNA vis-à-vis language and geography in Papua New Guinea.

    Science.gov (United States)

    Lee, Esther J; Koki, George; Merriwether, D Andrew

    2010-08-01

    Situated along a corridor linking the Asian continent with the outer islands of the Pacific, Papua New Guinea has long played a key role in understanding the initial peopling of Oceania. The vast diversity in languages and unique geographical environments in the region have been central to the debates on human migration and the degree of interaction between the Pleistocene settlers and newer migrants. To better understand the role of Papua New Guinea in shaping the region's prehistory, we sequenced the mitochondrial DNA (mtDNA) control region of three populations, a total of 94 individuals, located in the East Sepik Province of Papua New Guinea. We analyzed these samples with a large data set of Oceania populations to examine the role of geography and language in shaping population structure within New Guinea and between the region and Island Melanesia. Our results from median-joining networks, star-cluster age estimates, and population genetic analyses show that while highland New Guinea populations seem to be the oldest settlers, there has been significant gene flow within New Guinea with little influence from geography or language. The highest genetic division is between Papuan speakers of New Guinea versus East Papuan speakers located outside of mainland New Guinea. Our study supports the weak language barriers to genetic structuring among populations in close contact and highlights the complexity of understanding the genetic histories of Papua New Guinea in association with language and geography.

  18. Characterization of the Complete Mitochondrial Genome of Leucoma salicis (Lepidoptera: Lymantriidae) and Comparison with Other Lepidopteran Insects

    Science.gov (United States)

    Sun, Yu-Xuan; Wang, Lei; Wei, Guo-Qing; Qian, Cen; Dai, Li-Shang; Sun, Yu; Abbas, Muhammad Nadeem; Zhu, Bao-Jian; Liu, Chao-Liang

    2016-01-01

    The complete mitochondrial genome (mitogenome) of Leucoma salicis (Lepidoptera: Lymantriidae) was sequenced and annotated. It is a circular molecule of 15,334 bp, containing the 37 genes usually present in insect mitogenomes. All protein-coding genes (PCGs) are initiated by ATN codons, other than cox1, which is initiated by CGA. Three of the 13 PCGs had an incomplete termination codon, T or TA, while the others terminated with TAA. The relative synonymous codon usage of the 13 protein-coding genes (PCGs) was consistent with those of published lepidopteran sequences. All tRNA genes had typical clover-leaf secondary structures, except for the tRNASer (AGN), in which the dihydrouridine (DHU) arm could not form a stable stem-loop structure. The A + T-rich region of 325 bp had several distinctive features, including the motif ‘ATAGA’ followed by an 18 bp poly-T stretch, a microsatellite-like (AT)7 element, and an 11-bp poly-A present immediately upstream of tRNAMet. Relationships among 32 insect species were determined using Maximum Likelihood (ML), Neighbor Joining (NJ) and Bayesian Inference (BI) phylogenetic methods. These analyses confirm that L. salicis belongs to the Lymantriidae; and that Lymantriidae is a member of Noctuoidea, and is a sister taxon to Erebidae, Nolidae and Noctuidae, most closely related to Erebidae. PMID:27974854

  19. Genetic characterization of the Pacific sheath-tailed bat (Emballonura semicaudata rotensis) using mitochondrial DNA sequence data

    Science.gov (United States)

    Oyler-McCance, Sara J.; Valdez, Ernest W.; O'Shea, Thomas; Fike, Jennifer A.

    2013-01-01

    Emballonura semicaudata occurs in the southwestern Pacific and populations on many islands have declined or disappeared. One subspecies (E. semicaudata rotensis) occurs in the Northern Mariana Islands, where it has been extirpated from all but 1 island (Aguiguan). We assessed genetic similarity between the last population of E. s. rotensis and 2 other subspecies, and examined genetic diversity on Aguiguan. We sampled 12 E. s. rotensis, sequenced them at 3 mitochondrial loci, and compared them with published sequences from 2 other subspecies. All 12 E. s. rotensis had identical sequences in each of the 3 regions. Using cytochrome-b (Cytb) data E. s. rotensis was sister to E. s. palauensis in a clade separate from E. s. semicaudata. 12S ribosomal RNA (12S) sequences grouped all E. s. semicaudata in 1 clade with E. s. rotensis in a clade by itself. Genetic distances among the 3 subspecies at Cytb were smallest between E. s. palauensis and E. s. rotensis. Distance between E. s. semicaudata and the other 2 subspecies was not different from the distance between E. s. semicaudata and the full species E. raffrayana. A similar relationship was found using the 12S data. These distances are larger than those typically reported for mammalian subspecies using Cytb sequence and within the range of sister species.

  20. Molecular characterization of Echinococcus granulosusfrom Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

    Directory of Open Access Journals (Sweden)

    Elizabeth Sánchez

    2010-09-01

    Full Text Available Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10, have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44, sheep (41 and humans (14 from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4. The G1 genotype is considered the most widespread and infectious form of E. granulosusworldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

  1. Characterization of mitochondrial control region, two intergenic spacers and tRNAs of Zaprionus indianus (Diptera: Drosophilidae).

    Science.gov (United States)

    da Silva, Norma Machado; de Souza Dias, Aline; da Silva Valente, Vera Lúcia; Valiati, Victor Hugo

    2009-12-01

    The control region in insects is the major noncoding region in animal mitochondrial DNA (mtDNA), and is responsible for a large part of the variation in the DNA sequence and size of the genome of this organelle. In this study, the mtDNA control region, two intergenic spacers and tRNA genes of a Zaprionus indianus strain were cloned, sequenced and compared with other Drosophila species. The overall A+T content in the Z. indianus control region is 94.3%, and a comparison with other Drosophila species demonstrated that the most conserved region appears to be the 420 base pairs nearest to the tRNA(ile), similar to the findings of other authors. We also describe conserved sequence blocks, including a poly-T involved in the replication process of Drosophila mtDNA; a putative secondary structure also involved in the replication process and repeated sequences. tRNA(ile) sequence demonstrated the greatest variability when the tRNA sequences of species were compared.

  2. Molecular characterization of Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola sp. in China based on nuclear and mitochondrial DNA.

    Science.gov (United States)

    Peng, Mao; Ichinomiya, Mie; Ohtori, Maiko; Ichikawa, Madoka; Shibahara, Toshiyuki; Itagaki, Tadashi

    2009-09-01

    Parthenogenic Fasciola forms as well as bisexual Fasciola hepatica and Fasciola gigantica in mainland China have been identified on the basis of their spermatogenesis and genotypes in nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase I (NDI). The Chinese aspermic Fasciola would include forms originating in interspecific hybrids between F. hepatica and F. gigantica, since they showed the genotype of ITS1-Fh/Fg that had mixed sequences of the two Fasciola species or heterogeneous genotypes in ITS1 and NDI. Additionally, there were Chinese aspermic flukes in which the sequences of ITS1 and NDI genotypes completely coincided with those in aspermic forms from Japan, Korea, and Vietnam, suggesting that the aspermic forms from these four countries are offspring with a common provenance. The Fh-C4 haplotype in NDI was detected in both aspermic specimens and F. hepatica, indicating that aspermic forms showing the haplotype might come into existence in China. The ratio of body length and width in aspermic Fasciola specimens showed intermediate values between those of F. hepatica and F. gigantica.

  3. Adipose-Derived Mesenchymal Stromal/Stem Cells: Tissue Localization, Characterization, and Heterogeneity

    Directory of Open Access Journals (Sweden)

    Patrick C. Baer

    2012-01-01

    Full Text Available Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources. It is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose-derived mesenchymal stromal/stem cells (ASCs yields a high amount of stem cells, which is essential for stem-cell-based therapies and tissue engineering. Several studies have provided evidence that ASCs in situ reside in a perivascular niche, whereas the exact localization of ASCs in native adipose tissue is still under debate. ASCs are isolated by their capacity to adhere to plastic. Nevertheless, recent isolation and culture techniques lack standardization. Cultured cells are characterized by their expression of characteristic markers and their capacity to differentiate into cells from meso-, ecto-, and entodermal lineages. ASCs possess a high plasticity and differentiate into various cell types, including adipocytes, osteoblasts, chondrocytes, myocytes, hepatocytes, neural cells, and endothelial and epithelial cells. Nevertheless, recent studies suggest that ASCs are a heterogeneous mixture of cells containing subpopulations of stem and more committed progenitor cells. This paper summarizes and discusses the current knowledge of the tissue localization of ASCs in situ, their characterization and heterogeneity in vitro, and the lack of standardization in isolation and culture methods.

  4. The effect of neoadjuvant chemoradiotherapy on whole-body physical fitness and skeletal muscle mitochondrial oxidative phosphorylation in vivo in locally advanced rectal cancer patients--an observational pilot study.

    Directory of Open Access Journals (Sweden)

    Malcolm A West

    Full Text Available BACKGROUND: In the United Kingdom, patients with locally advanced rectal cancer routinely receive neoadjuvant chemoradiotherapy. However, the effects of this on physical fitness are unclear. This pilot study is aimed to investigate the effect of neoadjuvant chemoradiotherapy on objectively measured in vivo muscle mitochondrial function and whole-body physical fitness. METHODS: We prospectively studied 12 patients with rectal cancer who completed standardized neoadjuvant chemoradiotherapy, recruited from a large tertiary cancer centre, between October 2012 and July 2013. All patients underwent a cardiopulmonary exercise test and a phosphorus magnetic resonance spectroscopy quadriceps muscle exercise-recovery study before and after neoadjuvant chemoradiotherapy. Data were analysed and reported blind to patient identity and clinical course. Primary variables of interest were the two physical fitness measures; oxygen uptake at estimated anaerobic threshold and oxygen uptake at Peak exercise (ml.kg-1.min-1, and the post-exercise phosphocreatine recovery rate constant (min-1, a measure of muscle mitochondrial capacity in vivo. RESULTS: Median age was 67 years (IQR 64-75. Differences (95%CI in all three primary variables were significantly negative post-NACRT: Oxygen uptake at estimated anaerobic threshold -2.4 ml.kg-1.min-1 (-3.8, -0.9, p = 0.004; Oxygen uptake at Peak -4.0 ml.kg-1.min-1 (-6.8, -1.1, p = 0.011; and post-exercise phosphocreatine recovery rate constant -0.34 min-1 (-0.51, -0.17, p<0.001. CONCLUSION: The significant decrease in both whole-body physical fitness and in vivo muscle mitochondrial function raises the possibility that muscle mitochondrial mechanisms, no doubt multifactorial, may be important in deterioration of physical fitness following neoadjuvant chemoradiotherapy. This may have implications for targeted interventions to improve physical fitness pre-surgery. TRIAL REGISTRATION: Clinicaltrials.gov registration NCT01859442.

  5. Characterizing human skin blood flow regulation in response to different local skin temperature perturbations.

    Science.gov (United States)

    Wu, Y; Nieuwenhoff, M D; Huygen, F J P M; van der Helm, F C T; Niehof, S; Schouten, A C

    2017-05-01

    Small nerve fibers regulate local skin blood flow in response to local thermal perturbations. Small nerve fiber function is difficult to assess with classical neurophysiological tests. In this study, a vasomotor response model in combination with a heating protocol was developed to quantitatively characterize the control mechanism of small nerve fibers in regulating skin blood flow in response to local thermal perturbation. The skin of healthy subjects' hand dorsum (n=8) was heated to 42°C with an infrared lamp, and then naturally cooled down. The distance between the lamp and the hand was set to three different levels in order to change the irradiation intensity on the skin and implement three different skin temperature rise rates (0.03°C/s, 0.02°C/s and 0.01°C/s). A laser Doppler imager (LDI) and a thermographic video camera recorded the temporal profile of the skin blood flow and the skin temperature, respectively. The relationship between the skin blood flow and the skin temperature was characterized by a vasomotor response model. The model fitted the skin blood flow response well with a variance accounted for (VAF) between 78% and 99%. The model parameters suggested a similar mechanism for the skin blood flow regulation with the thermal perturbations at 0.03°C/s and 0.02°C/s. But there was an accelerated skin vasoconstriction after a slow heating (0.01°C/s) (p-value<0.05). An attenuation of the skin vasodilation was also observed in four out of the seven subjects during the slow heating (0.01°C/s). Our method provides a promising way to quantitatively assess the function of small nerve fibers non-invasively and non-contact.

  6. Localization and characterization of renal calcitonin receptors by in vitro autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Sexton, P.M.; Adam, W.R.; Moseley, J.M.; Martin, T.J.; Mendelsohn, F.A.

    1987-12-01

    Calcitonin receptors were identified in renal cortex and medulla using the radioligand /sup 125/I-salmon calcitonin. Microscopic localization of these receptors revealed binding over medullary and cortical thick ascending limb of the loop of Henle and in distal convoluted tubule. A number of receptor positive cells in the inner medulla were also identified. Characterization of the binding demonstrated a single class of high-affinity binding sites in both the medulla and the cortex with affinity constants of 0.74 +/- 0.09 x 10(9) M-1 and 0.32 +/- 0.05 x 10(9) M-1, respectively, and receptor concentrations of 205 +/- 45 fmol/mg protein and 453 +/- 54 fmol/mg protein, respectively. Competition for /sup 125/I-salmon calcitonin binding by a wide range of calcitonin analogs revealed a close correspondence between the reported biological potencies and activities in the current system. The localization of binding sites within the nephron corresponds to the reported localization of calcitonin-stimulated adenylate cyclase activity and suggests that the receptor mediated actions of calcitonin in the kidney utilize cyclic AMP as a second messenger. In addition, the microscopic identification of specific calcitonin receptors helps the delineation of direct actions of this hormone from those which are indirect.

  7. Subcellullar localization, developmental expression and characterization of a liver triacylglycerol hydrolase.

    Science.gov (United States)

    Lehner, R; Cui, Z; Vance, D E

    1999-03-15

    The mechanism and enzymic activities responsible for the lipolysis of stored cytosolic triacylglycerol in liver and its re-esterification remain obscure. A candidate enzyme for lipolysis, a microsomal triacylglycerol hydrolase (TGH), was recently purified to homogeneity from pig liver and its kinetic properties were determined [Lehner and Verger (1997) Biochemistry 36, 1861-1868]. We have characterized the enzyme with regard to its species distribution, subcellular localization, developmental expression and reaction with lipase inhibitors. The hydrolase co-sediments with endoplasmic reticulum elements and is associated with isolated liver fat droplets. Immunocytochemical studies localize TGH exclusively to liver cells surrounding capillaries. Both TGH mRNA and protein are expressed in rats during weaning. The enzyme covalently binds tetrahydrolipstatin, an inhibitor of lipases and of triacylglycerol hydrolysis. The enzyme is absent from liver-derived cell lines (HepG2 and McArdle RH7777) known to be impaired in very-low-density lipoprotein (VLDL) assembly and secretion. The localization and developmental expression of TGH are consistent with a proposed role in triacylglycerol hydrolysis and with the proposal that some of the resynthesized triacylglycerol is utilized for VLDL secretion.

  8. Lost region in amyloid precursor protein (APP) through TALEN-mediated genome editing alters mitochondrial morphology.

    Science.gov (United States)

    Wang, Yajie; Wu, Fengyi; Pan, Haining; Zheng, Wenzhong; Feng, Chi; Wang, Yunfu; Deng, Zixin; Wang, Lianrong; Luo, Jie; Chen, Shi

    2016-02-29

    Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) deposition in the brain. Aβ plaques are produced through sequential β/γ cleavage of amyloid precursor protein (APP), of which there are three main APP isoforms: APP695, APP751 and APP770. KPI-APPs (APP751 and APP770) are known to be elevated in AD, but the reason remains unclear. Transcription activator-like (TAL) effector nucleases (TALENs) induce mutations with high efficiency at specific genomic loci, and it is thus possible to knock out specific regions using TALENs. In this study, we designed and expressed TALENs specific for the C-terminus of APP in HeLa cells, in which KPI-APPs are predominantly expressed. The KPI-APP mutants lack a 12-aa region that encompasses a 5-aa trans-membrane (TM) region and 7-aa juxta-membrane (JM) region. The mutated KPI-APPs exhibited decreased mitochondrial localization. In addition, mitochondrial morphology was altered, resulting in an increase in spherical mitochondria in the mutant cells through the disruption of the balance between fission and fusion. Mitochondrial dysfunction, including decreased ATP levels, disrupted mitochondrial membrane potential, increased ROS generation and impaired mitochondrial dehydrogenase activity, was also found. These results suggest that specific regions of KPI-APPs are important for mitochondrial localization and function.

  9. Mitochondrial Myopathy

    Science.gov (United States)

    ... diseases are caused by CoQ10 deficiency, and CoQ10 supplementation is clearly beneficial in these cases. It might provide some relief from other mitochondrial diseases. Creatine, L-carnitine, and CoQ10 supplements often are combined into a “ ...

  10. Phenotypic and genotypic characterization of locally isolated Salmonella strains used in preparation of Salmonella antigens in Egypt

    OpenAIRE

    Hazem Mohammed Ibrahim; Dalia Ahmed Mohammed Abd El-moaty; Hanan Ali Ahmed; Mona Ibrahim El-Enbaawy

    2016-01-01

    Aim: This work was conducted to study the phenotypic and genotypic characterization of locally isolated Salmonella strains (Salmonella Pullorum, Salmonella Enteritidis, and Salmonella Typhimurium) from poultry used in the preparation of Salmonella antigens in Egypt. Materials and Methods: The phenotypic characterization of Salmonella strains was done using standard microbiological, biochemical, and serological techniques. Molecular identification was done using different sets of primers on...

  11. Fully automated software for quantitative measurements of mitochondrial morphology.

    Science.gov (United States)

    McClatchey, P Mason; Keller, Amy C; Bouchard, Ron; Knaub, Leslie A; Reusch, Jane E B

    2016-01-01

    Mitochondria undergo dynamic changes in morphology in order to adapt to changes in nutrient and oxygen availability, communicate with the nucleus, and modulate intracellular calcium dynamics. Many recent papers have been published assessing mitochondrial morphology endpoints. Although these studies have yielded valuable insights, contemporary assessment of mitochondrial morphology is typically subjective and qualitative, precluding direct comparison of outcomes between different studies and likely missing many subtle effects. In this paper, we describe a novel software technique for measuring the average length, average width, spatial density, and intracellular localization of mitochondria from a fluorescent microscope image. This method was applied to distinguish baseline characteristics of Human Umbilical Vein Endothelial Cells (HUVECs), primary Goto-Kakizaki rat aortic smooth muscle cells (GK SMCs), primary Wistar rat aortic smooth muscle cells (Wistar SMCs), and SH-SY5Ys (human neuroblastoma cell line). Consistent with direct observation, our algorithms found SH-SY5Ys to have the greatest mitochondrial density, while HUVECs were found to have the longest mitochondria. Mitochondrial morphology responses to temperature, nutrient, and oxidative stressors were characterized to test algorithm performance. Large morphology changes recorded by the software agreed with direct observation, and subtle but consistent morphology changes were found that would not otherwise have been detected. Endpoints were consistent between experimental repetitions (R=0.93 for length, R=0.93 for width, R=0.89 for spatial density, and R=0.74 for localization), and maintained reasonable agreement even when compared to images taken with compromised microscope resolution or in an alternate imaging plane. These results indicate that the automated software described herein allows quantitative and objective characterization of mitochondrial morphology from fluorescent microscope images.

  12. Complete kinetic and thermodynamic characterization of the unisite catalytic pathway of Escherichia coli F1-ATPase. Comparison with mitochondrial F1-ATPase and application to the study of mutant enzymes.

    Science.gov (United States)

    Al-Shawi, M K; Senior, A E

    1988-12-25

    the catalytic nucleotide-binding domain, reinforcing the view that impairment of catalysis was due to a localized effect. Such analyses confirmed that six other F1-beta-subunit mutants, previously generated and characterized in this laboratory and thought to have inhibitory side-chain substitutions in the catalytic nucleotide-binding domain, are also devoid of gross structural disruption.

  13. GnRH receptors in human granulosa cells: Anatomical localization and characterization by autoradiographic study

    Energy Technology Data Exchange (ETDEWEB)

    Latouche, J.; Crumeyrolle-Arias, M.; Jordan, D.; Kopp, N.; Augendre-Ferrante, B.; Cedard, L.; Haour, F. (Institut Pasteur, Paris (France))

    1989-09-01

    The presence of receptors for GnRH in human ovary has been investigated by quantitative autoradiography. Simultaneous visualization and characterization of specific receptors on frozen sections were obtained on six pairs of human ovaries. Among them only one exhibited a large preovulatory follicle. This dominant follicle exhibited a specific and high affinity binding capacity for {sup 125}I-GnRHa exclusively localized on the granulosa cell layer. Analysis of saturation curve indicates a Kd value of 0.22 nM and Bmax of 9.6 fmol/mg protein. In contrast LH-hCG binding sites were present in all antral follicles. These data demonstrate for the first time the presence of high affinity GnRH receptors in human granulosa cells at a late stage of follicular maturation.

  14. Full optical characterization of autostereoscopic 3D displays using local viewing angle and imaging measurements

    Science.gov (United States)

    Boher, Pierre; Leroux, Thierry; Bignon, Thibault; Collomb-Patton, Véronique

    2012-03-01

    Two commercial auto-stereoscopic 3D displays are characterized a using Fourier optics viewing angle system and an imaging video-luminance-meter. One display has a fixed emissive configuration and the other adapts its emission to the observer position using head tracking. For a fixed emissive condition, three viewing angle measurements are performed at three positions (center, right and left). Qualified monocular and binocular viewing spaces in front of the display are deduced as well as the best working distance. The imaging system is then positioned at this working distance and crosstalk homogeneity on the entire surface of the display is measured. We show that the crosstalk is generally not optimized on all the surface of the display. Display aspect simulation using viewing angle measurements allows understanding better the origin of those crosstalk variations. Local imperfections like scratches and marks generally increase drastically the crosstalk, demonstrating that cleanliness requirements for this type of display are quite critical.

  15. Genetic counseling in mitochondrial disease.

    Science.gov (United States)

    Vento, Jodie M; Pappa, Belen

    2013-04-01

    Mitochondrial diseases are a genetically and clinically diverse group of disorders that arise as a result of dysfunction of the mitochondria. Mitochondrial disorders can be caused by alterations in nuclear DNA and/or mitochondrial DNA. Although some mitochondrial syndromes have been described clearly in the literature many others present as challenging clinical cases with multisystemic involvement at variable ages of onset. Given the clinical variability and genetic heterogeneity of these conditions, patients and their families often experience a lengthy and complicated diagnostic process. The diagnostic journey may be characterized by heightened levels of uncertainty due to the delayed diagnosis and the absence of a clear prognosis, among other factors. Uncertainty surrounding issues of family planning and genetic testing may also affect the patient. The role of the genetic counselor is particularly important to help explain these complexities and support the patient and family's ability to achieve effective coping strategies in dealing with increased levels of uncertainty.

  16. Characterization of the mitochondrial genome of the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae) and phylogenetic analysis of advanced moths and butterflies.

    Science.gov (United States)

    Wei, Shu-Jun; Shi, Bao-Cai; Gong, Ya-Jun; Li, Qian; Chen, Xue-Xin

    2013-04-01

    Here we determined the mitochondrial genome sequence of a notorious pest, the diamondback moth Plutella xylostella (Lepidoptera: Yponomeutoidea: Plutellidae). The mitochondrial genome contains 37 typical animal mitochondrial genes and an A+T-rich region. The gene arrangement is identical to that of other ditrysian lepidopteran mitochondrial genomes, but different from the ancestral gene arrangement in the non-ditrysian Hepialidae of Lepidoptera. The start codon of the cox1 gene is CGA, which is dissimilar to its homologs in most other insects. In Lepidoptera, cox1 and cox2 have low nucleotide diversities, while the nad6, nad2, and nad3 genes are highly variable. Phylogenetic analyses uncovered the reciprocal monophyly of Ditrysia, Apoditrysia, Obtectomera, and Macrolepidoptera, and the placement of the Hesperiidae within Papilionoidea. Our analyses suggest that the complete mitochondrial genome sequences are a promising marker toward fully resolving the phylogenetic relationships within Lepidoptera.

  17. Characterizing local forces and rearrangements inside a gravity-driven granular flow

    Science.gov (United States)

    Thackray, Emma; Nordstrom, Kerstin

    While the gravity-driven flow of a granular material in a silo geometry can be modeled by the Beverloo equation, the mesoscale-level particle rearrangements and interactions that drive this flow are not well-understood. We have constructed a quasi-two-dimensional system of bidisperse, millimeter-scale disks with photoelastic properties that make force networks within the material visible. The system is contained in an acrylic box with an adjustable bottom opening. We can approach the clogging transition by adjusting this opening and by adding external forcing to the top of the flowing pile. By placing the system between cross-polarizers, we can obtain high-speed video of this system during flow, and extract intensity signals that can be used to identify and quantify localized, otherwise indeterminate forces. We can simultaneously track individual particle motions, which can be used to identify shear transformation zones in the system. We are therefore able to correlate local forces with rearrangements within the system, and characterize the evolution of this interplay on the approach to the clogging transition.

  18. Ketamine-Induced Apoptosis in Normal Human Urothelial Cells: A Direct, N-Methyl-d-Aspartate Receptor-Independent Pathway Characterized by Mitochondrial Stress.

    Science.gov (United States)

    Baker, Simon C; Shabir, Saqib; Georgopoulos, Nikolaos T; Southgate, Jennifer

    2016-05-01

    Recreational abuse of ketamine has been associated with the emergence of a new bladder pain syndrome, ketamine-induced cystitis, characterized by chronic inflammation and urothelial ulceration. We investigated the direct effects of ketamine on normal human urothelium maintained in organ culture or as finite cell lines in vitro. Exposure of urothelium to ketamine resulted in apoptosis, with cytochrome c release from mitochondria and significant subsequent caspase 9 and 3/7 activation. The anesthetic mode-of-action for ketamine is mediated primarily through N-methyl d-aspartate receptor (NMDAR) antagonism; however, normal (nonimmortalized) human urothelial cells were unresponsive to NMDAR agonists or antagonists, and no expression of NMDAR transcript was detected. Exposure to noncytotoxic concentrations of ketamine (≤1 mmol/L) induced rapid release of ATP, which activated purinergic P2Y receptors and stimulated the inositol trisphosphate receptor to provoke transient release of calcium from the endoplasmic reticulum into the cytosol. Ketamine concentrations >1 mmol/L were cytotoxic and provoked a larger-amplitude increase in cytosolic Ca(2+) concentration that was unresolved. The sustained elevation in cytosolic Ca(2+) concentration was associated with pathological mitochondrial oxygen consumption and ATP deficiency. Damage to the urinary barrier initiates bladder pain and, in ketamine-induced cystitis, loss of urothelium from large areas of the bladder wall is a reported feature. This study offers first evidence for a mechanism of direct toxicity of ketamine to urothelial cells by activating the intrinsic apoptotic pathway.

  19. Characterization of radish mitochondrial atpA: influence of nuclear background on transcription of atpA-associated sequences and relationship with male sterility.

    Science.gov (United States)

    Makaroff, C A; Apel, I J; Palmer, J D

    1990-11-01

    We have previously shown that the mitochondrial gene atpA, encoding the alpha subunit of F1 ATP synthase, is associated with DNA rearrangements and nuclear-specific transcript patterns in the male-sterile cytoplasm of Ogura radish. Here we present a detailed characterization of this gene from both the normal (fertile) and Ogura (male-sterile) cytoplasms of radish to determine if it is involved in Ogura cytoplasmic male sterility. The normal and Ogura radish atpA loci are virtually identical for 3.8 kb, including a 507 codon open reading frame whose product is approximately 92% identical to other plant ATPA polypeptides. Rearrangement breakpoints have been identified 613 bp 5' and 1663 bp 3' to the atpA coding region. The 5' rearrangement breakpoint is located within a repeated sequence that has been associated with other rearrangement events in radish mitochondria. The previously identified transcript difference results from transcription originating upstream of this rearrangement site. Although the presence of this transcript is affected by nuclear background, analyses in several different sterile and fertile nuclear backgrounds indicate that the presence of this transcript is not strictly correlated with male sterility. In addition, normal levels of ATPA polypeptide are present in sterile plants containing the Ogura cytoplasm.

  20. Characterization of an avian histidine decarboxylase and localization of histaminergic neurons in the chicken brain.

    Science.gov (United States)

    Bessho, Yuki; Iwakoshi-Ukena, Eiko; Tachibana, Tetsuya; Maejima, Sho; Taniuchi, Shusuke; Masuda, Keiko; Shikano, Kenshiro; Kondo, Kunihiro; Furumitsu, Megumi; Ukena, Kazuyoshi

    2014-08-22

    In mammals, it is established that histamine is a neurotransmitter and/or neuromodulator in the central nervous system. It is produced by the enzyme histidine decarboxylase (HDC) in the tuberomammillary nucleus of the posterior hypothalamus. However, HDC as well as histaminergic neurons have not yet been characterized in the avian brain. We have cloned the cDNA for HDC from the chicken hypothalamus and demonstrated that the chicken HDC sequence is highly homologous to the mammalian counterpart, and that the expressed protein shows high enzymatic activity. The expression of HDC mRNA at various sites in the brain was investigated using quantitative RT-PCR. The results showed that the HDC mRNA was highly expressed in the hypothalamic infundibulum. In situ hybridization analyses revealed that the cells containing HDC mRNA were localized in the medial mammillary nucleus of the hypothalamic infundibulum. Intracerebroventricular injection of histamine in chicks resulted in inhibition of feeding behavior. This is the first report of the characterization of histaminergic neurons in the avian brain, and our findings indicate that neuronal histamine exerts anorexigenic effects in chicks.

  1. Two Examples of Integrated Aquifer Characterization at Local and Regional Scales

    Science.gov (United States)

    Lefebvre, R.; Gloaguen, E.; Rivard, C.; Parent, M.; Morin, R. H.; Pugin, A.; Pullan, S.; Crow, H.; Paradis, D.; Tremblay, L.; Blouin, M.; Laurencelle, M.

    2012-12-01

    An integrated aquifer characterization approach was developed with the aim of efficiently providing detailed data that could be used to develop conceptual hydrogeological models and quantitatively describe the spatial continuity and heterogeneity of unconsolidated sediments. The approach involves the integration of geological, hydraulic, geophysical and geochemical data. The emphasis of the approach is placed on the acquisition of detailed and continuous indirect data and selective soil sampling and direct measurements of hydraulic properties covering the full range of materials present in the system. Direct data are used to establish relations between indirect hydrogeophysical measurements and hydrofacies (HF), which are material types with distinct hydraulic conductivity (K). Surface geophysical surveys are used to provide 1D or 2D definitions of sediment structures and material types. Hydraulic tests are used to define HF and estimate their ranges of K. Groundwater (GW) geochemistry (major, minor, isotopes, GW age) is used to support the definition of conceptual models and to provide constraints on numerical models of GW flow and transport (mass and GW age). The approach relies on the geostatistical integration of multi-source data to define aquifer boundaries, on the recognition of HF and estimation of K from CPT/SMR data using fuzzy clustering and relevant vector machines for HF classification and K regression, on the geostatistical simulation of HF and K to provide the spatial distribution of hydraulic parameters in GW flow and transport models, and on the validation of these models using geochemical data. The integrated characterization approach was first developed and tested at local scale for the study of a shallow granular aquifer within a 12 km2 sub-watershed where a former unlined landfill is located. Results are being applied to the assessment of the efficiency of natural attenuation as a site management approach. The integrated characterization

  2. 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.

  3. 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 ...

  4. Mitochondrial lipid transport and biosynthesis: A complex balance

    Science.gov (United States)

    2016-01-01

    Little is known about how mitochondrial lipids reach inner membrane–localized metabolic enzymes for phosphatidylethanolamine synthesis. Aaltonen et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201602007) and Miyata et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601082) now report roles for two mitochondrial complexes, Ups2–Mdm35 and mitochondrial contact site and cristae organizing system, in the biosynthesis and transport of mitochondrial lipids. PMID:27354376

  5. Granular vortices: Identification, characterization and conditions for the localization of deformation

    Science.gov (United States)

    Tordesillas, Antoinette; Pucilowski, Sebastian; Lin, Qun; Peters, John F.; Behringer, Robert P.

    2016-05-01

    We relate the micromechanics of vortex evolution to that of force chain buckling and, on this basis, formulate the conditions for strain localization in a continuum model of dense granular media. Using the traditional bifurcation analysis of shear bands, we show that kinematic vortex fields are in fact solutions to the boundary value problem satisfying null boundary conditions. To establish an empirical basis for our study, we first develop a method to identify the location of the core and boundary of each vortex from a given displacement field in two dimensions. We then employ this method to characterize the residual deformation field (i.e., the deviation of particle motions from the continuum deformation) in a physical experiment and a discrete element simulation of dense granular samples submitted to biaxial compression. Vortices in the failure regime are essentially confined to the shear band. Primary vortices, the clear majority, rotate in the same direction as the shear band; secondary vortices, the so-called wakes, rotate in the opposite direction. Primary vortices align in spatial succession along the central axis of the band; wakes form next to the band boundaries, in between and beside two adjacent primary vortices. Force chain buckling, the governing mechanism for shear bands, is responsible for vortex formation in the failure regime. Vortex dynamics are consistent with stick-slip dynamics. From quiescent conditions of jamming or stick, vortical motions arise from force chain buckling and associated relative particle rotations and sliding; these in turn precipitate intermittent periods of unjamming or slip, evident in the attendant drops in stress ratio and bursts in both kinetic energy and local nonaffine deformation. A kinematic vortex field inside shear bands is proposed that is consistent with the equations of continuum mechanics and the underlying instability of force chain buckling: such a field is periodic with a repeating unit cell comprising a

  6. Human phenol sulfotransferase STP2 gene: Molecular cloning, structural characterization, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, C.; Raftogianis, R.; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States)

    1996-05-01

    Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of {open_quotes}simple{close_quotes} planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs, STP2, as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans. STP2 spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of most STP2 exon-intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans, STM; a rat PST gene; a human estrogen ST (EST) gene, STE; and a guinea pig EST gene. The two initial STP2 exons, IA and IB, were identified by performing 5{prime}-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5{prime}untranslated region sequences. The two apparent 5{prime}-ons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements. STP2 was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization of STP2 will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissues. 63 refs., 7 figs., 1 tab.

  7. Mitochondrial ABC transporters.

    Science.gov (United States)

    Lill, R; Kispal, G

    2001-01-01

    In contrast to bacteria, mitochondria contain only a few ATP binding cassette (ABC) transporters in their inner membrane. The known mitochondrial ABC proteins fall into two major classes that, in the yeast Saccharomyces cerevisiae, are represented by the half-transporter Atm1p and the two closely homologous proteins Mdl1p and Mdl2p. In humans two Atm1p orthologues (ABC7 and MTABC3) and two proteins homologous to Mdll/2p have been localized to mitochondria. The Atm1p-like proteins perform an important function in mitochondrial iron homeostasis and in the maturation of Fe/S proteins in the cytosol. Mutations in ABC7 are causative of hereditary X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A). MTABC3 may be a candidate gene for the lethal neonatal syndrome. The function of the mitochondrial Mdl1/2p-like proteins is not clear at present with the notable exception of murine ABC-me that may transport intermediates of heme biosynthesis from the matrix to the cytosol in erythroid tissues.

  8. Mutations of C19orf12, coding for a transmembrane glycine zipper containing mitochondrial protein, cause mis-localization of the protein, inability to respond to oxidative stress and increased mitochondrial Ca2+

    DEFF Research Database (Denmark)

    Venco, Paola; Bonora, Massimo; Giorgi, Carlotta;

    2015-01-01

    Mutations in C19orf12 have been identified in patients affected by Neurodegeneration with Brain Iron Accumulation (NBIA), a clinical entity characterized by iron accumulation in the basal ganglia. By using western blot analysis with specific antibody and confocal studies, we showed that wild-type...

  9. Preparation and characterization of poly(ε-caprolactone) nanospheres containing the local anesthetic lidocaine.

    Science.gov (United States)

    Ramos Campos, Estefânia Vangelie; Silva de Melo, Nathalie Ferreira; Guilherme, Viviane Aparecida; de Paula, Eneida; Rosa, André Henrique; de Araújo, Daniele Ribeiro; Fraceto, Leonardo Fernandes

    2013-01-01

    The objective of this work was to develop a modified release system for the local anesthetic lidocaine (LDC), using poly(ε-caprolactone) (PCL) nanospheres (NSs), to improve the pharmacological properties of the drug when administered by the infiltration route. In vitro experiments were used to characterize the system and investigate the release mechanism. The NSs presented a polydispersion index of 0.072, an average diameter of 449.6 nm, a zeta potential of -20.1 mV, and an association efficiency of 93.3%. The release profiles showed that the release of associated LDC was slower than that of the free drug. Atomic force microscopy analyses showed that the spherical structure of the particles was preserved as a function of time, as well as after the release experiments. Cytotoxicity and pharmacological tests confirmed that association with the NSs reduced the toxicity of LDC, and prolonged its anesthetic action. This new formulation could potentially be used in applications requiring gradual anesthetic release, especially dental procedures.

  10. Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells.

    Science.gov (United States)

    Jaramillo, Blanca Estela; Ponce, Arturo; Moreno, Jacqueline; Betanzos, Abigail; Huerta, Miriam; Lopez-Bayghen, Esther; Gonzalez-Mariscal, Lorenza

    2004-07-01

    ZO-2 is a MAGUK protein that in confluent epithelial sheets localizes at tight junctions (TJ) whereas in sparse cultures accumulates in clusters at the nucleus. Here, we have characterized several nuclear properties of ZO-2. We observe that ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B(1) and actin from the nuclei of sparse cultures. We show that ZO-2 presents several NLS at its amino region, that when deleted, diminish the nuclear import of the ZO-2 amino segment and impair the ability of the region to regulate the transcriptional activity of promoters controlled by AP-1. Several RS repeats are detected in the ZO-2 amino segment, however, their deletion does not preclude the display of a speckled nuclear pattern. ZO-2 displays two putative NES. However, only the second one appears to be functional, as when conjugated to ovalbumin (OV), it is able to translocate this protein from the nucleus to the cytoplasm in a leptomycin B-sensitive way.

  11. Characterization and localization of c-kit and epidermal growth factor receptor in different patterns of adenoid cystic carcinoma

    Directory of Open Access Journals (Sweden)

    Anshi Jain

    2016-01-01

    Conclusions: C-kit and EGFR biomarkers can be used to enhance the characterization of ACC and to determine the localization of dual cell population which could suggest the dual origin of ACC and provides evidence for the new therapeutic strategy in ACC.

  12. Mitochondrial DNA variants of respiratory complex I that uniquely characterize haplogroup T2 are associated with increased risk of age-related macular degeneration.

    Directory of Open Access Journals (Sweden)

    John Paul SanGiovanni

    Full Text Available BACKGROUND: Age-related macular degeneration (AMD, a chronic neurodegenerative and neovascular retinal disease, is the leading cause of blindness in elderly people of western European origin. While structural and functional alterations in mitochondria (mt and their metabolites have been implicated in the pathogenesis of chronic neurodegenerative and vascular diseases, the relationship of inherited variants in the mitochondrial genome and mt haplogroup subtypes with advanced AMD has not been reported in large prospective cohorts. METHODOLOGY/PRINICIPAL FINDINGS: We examined the relationship of inherited mtDNA variants with advanced AMD in 1168 people using a three-stage design on samples from 12-year and 10-year prospective studies on the natural history of age-related eye disease. In Stage I we resequenced the entire genome in 99 elderly AMD-free controls and 215 people with advanced AMD from the 12-year study. A consistent association with AMD in 14 of 17 SNPs characterizing the mtDNA T haplogroup emerged. Further analysis revealed these associations were driven entirely by the T2 haplogroup, and characterized by two variants in Complex I genes (A11812G of MT-ND4 and A14233G of MT-ND6. We genotyped T haplogroups in an independent sample of 490 cases and 61 controls from the same study (Stage II and in 56 cases and 246 controls from the 10-year study (Stage III. People in the T2 haplogroup were approximately 2.5 times more likely to have advanced AMD than their peers (odds ratio [OR] = 2.54, 95%CI 1.36-4.80, P

  13. Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank, E-mail: fkempken@bot.uni-kiel.de

    2013-08-30

    Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

  14. Seismic local site effects characterization in the Andarax River Valley (SE Spain) from ambient seismic noise

    Science.gov (United States)

    Carmona, Enrique; García-Jerez, Antonio; Luzón, Francisco; Sánchez-Martos, Francisco; Sánchez-Sesma, Francisco J.; Piña, José

    2014-05-01

    This work is focused on the characterization of seismic local effects in the Low Andarax River Valley (SE Spain). The Low Andarax River valley is located in an active seismic region, with the higher seismic hazard values in Spain. The landform is composed mainly by sedimentary materials which increase its seismic hazard due to the amplification of the seismic inputs and spectral resonances. We study seismic local effects in the Low Andarax River by analyzing the Horizontal-to-Vertical Spectral Ratio (HVSR) of ambient noise records. The noise data were recorded during two field campaigns in 2012 and 2013. There have been a total of 374 noise measurements with 15 and 30 minutes duration. The acquisition was performed with a Digital Broadband Seismometer Guralp CMG-6TD. The distance between measurements was about 200 meters, covering an area around 40 km2. There have been 6 significant peak frequencies between 0.3 Hz and 5 Hz. It was possible to find interesting areas with similar spectral peaks that coincide with zones with similar microgravimetric anomalies at the alluvial valley. It is also observed a decrease in the frequency peaks from West to East suggesting increased sediment layer. We also compute the soil models at those sites where geotechnical information is available, assuming that the seismic noise is diffuse. We invert the HVSR for these places using horizontally layered models and in the imaginary part the Green functions at the source. It is observed that the S wave velocity inverted models are consistent with the known geotechnical information obtained from drilled boreholes. We identify the elastodynamic properties of the limestone-dolomite materials with a formation of phyllites and quartzite that form the basement of the depression, and those properties of the Miocene and Pliocene detrital deposits (marls, sandy silts, sands and conglomerates) that fill the valley. These results together with the observed resonant frequencies along the Andarax

  15. Mitochondrial DNA Alterations and Reduced Mitochondrial Function in Aging

    OpenAIRE

    Hebert, Sadie L.; Lanza, Ian R.; Nair, K. Sreekumaran

    2010-01-01

    Oxidative damage to mitochondrial DNA increases with aging. This damage has the potential to affect mitochondrial DNA replication and transcription which could alter the abundance or functionality of mitochondrial proteins. This review describes mitochondrial DNA alterations and changes in mitochondrial function that occur with aging. Age-related alterations in mitochondrial DNA as a possible contributor to the reduction in mitochondrial function are discussed.

  16. Remote Sensing-Based Characterization of Settlement Structures for Assessing Local Potential of District Heat

    Directory of Open Access Journals (Sweden)

    Michael Nast

    2011-07-01

    Full Text Available In Europe, heating of houses and commercial areas is one of the major contributors to greenhouse gas emissions. When considering the drastic impact of an increasing emission of greenhouse gases as well as the finiteness of fossil resources, the usage of efficient and renewable energy generation technologies has to be increased. In this context, small-scale heating networks are an important technical component, which enable the efficient and sustainable usage of various heat generation technologies. This paper investigates how the potential of district heating for different settlement structures can be assessed. In particular, we analyze in which way remote sensing and GIS data can assist the planning of optimized heat allocation systems. In order to identify the best suited locations, a spatial model is defined to assess the potential for small district heating networks. Within the spatial model, the local heat demand and the economic costs of the necessary heat allocation infrastructure are compared. Therefore, a first and major step is the detailed characterization of the settlement structure by means of remote sensing data. The method is developed on the basis of a test area in the town of Oberhaching in the South of Germany. The results are validated through detailed in situ data sets and demonstrate that the model facilitates both the calculation of the required input parameters and an accurate assessment of the district heating potential. The described method can be transferred to other investigation areas with a larger spatial extent. The study underlines the range of applications for remote sensing-based analyses with respect to energy-related planning issues.

  17. Characterization and localization of metal-responsive-element-binding transcription factors from tilapia

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, Andrew Pok-Lap; Au, Candy Yee-Man; Chan, William Wai-Lun [Department of Biochemistry, Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong (Hong Kong); Chan, King Ming, E-mail: kingchan@cuhk.edu.hk [Department of Biochemistry, Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong (Hong Kong)

    2010-08-01

    Two isoforms of MTF-1, MTF-1L (long form) and MTF-1S (short form), were cloned in tilapia (Ti) and characterized in a tilapia liver cell line, Hepa-T1. The cloned tiMTF-1L has the characteristics of all of the tiMTF-1S identified so far with the zinc finger domain having six fingers, the acidic-rich, proline-rich, and serine/threonine-rich domains; however, the short form encodes for the zinc finger domain with five zinc fingers only and no other domains. The transient transfection of tiMTF-1L into human HepG2 cells showed both constitutive and zinc-induced metal-responsive-element (MRE)-driven reporter gene expression. However, the transfection of tiMTF-1S (which lacks all three transactivation domains) into a human cell line showed reduced transcriptional activities compared with an endogenous control in both basal- and Zn{sup 2+}-induced conditions. The tiMTF-1 isoforms were tagged with GFP and transfected into Hepa-T1 cells (tilapia hepatocytes). The nuclear translocation of tiMTF-1L was observed when the cells were exposed to a sufficient concentration of metals for 6 h. However, tiMTF-1S, was localized in the nucleus with or without metal treatment. Electrophoretic mobility shift assay (EMSA) confirmed that both of the isoforms were able to bind to the MRE specifically in vitro. Tissue distribution studies showed that tiMTF-1L was more abundant than tiMTF-1S in all of the tissues tested.

  18. Localization and functional characterization of a novel adipokinetic hormone in the mollusk, Aplysia californica.

    Directory of Open Access Journals (Sweden)

    Joshua I Johnson

    Full Text Available Increasing evidence suggests that gonadotropin-releasing hormone (GnRH, corazonin, adipokinetic hormone (AKH, and red pigment-concentrating hormone all share common ancestry to form a GnRH superfamily. Despite the wide presence of these peptides in protostomes, their biological effects remain poorly characterized in many taxa. This study had three goals. First, we cloned the full-length sequence of a novel AKH, termed Aplysia-AKH, and examined its distribution in an opisthobranch mollusk, Aplysia californica. Second, we investigated in vivo biological effects of Aplysia-AKH. Lastly, we compared the effects of Aplysia-AKH to a related A. californica peptide, Aplysia-GnRH. Results suggest that Aplysia-AKH mRNA and peptide are localized exclusively in central tissues, with abdominal, cerebral, and pleural ganglia being the primary sites of Aplysia-AKH production. However, Aplysia-AKH-positive fibers were found in all central ganglia, suggesting diverse neuromodulatory roles. Injections of A. californica with Aplysia-AKH significantly inhibited feeding, reduced body mass, increased excretion of feces, and reduced gonadal mass and oocyte diameter. The in vivo effects of Aplysia-AKH differed substantially from Aplysia-GnRH. Overall, the distribution and biological effects of Aplysia-AKH suggest it has diverged functionally from Aplysia-GnRH over the course of evolution. Further, that both Aplysia-AKH and Aplysia-GnRH failed to activate reproduction suggest the critical role of GnRH as a reproductive activator may be a phenomenon unique to vertebrates.

  19. Localization and Functional Characterization of a Novel Adipokinetic Hormone in the Mollusk, Aplysia californica

    Science.gov (United States)

    Johnson, Joshua I.; Kavanaugh, Scott I.; Nguyen, Cindy; Tsai, Pei-San

    2014-01-01

    Increasing evidence suggests that gonadotropin-releasing hormone (GnRH), corazonin, adipokinetic hormone (AKH), and red pigment-concentrating hormone all share common ancestry to form a GnRH superfamily. Despite the wide presence of these peptides in protostomes, their biological effects remain poorly characterized in many taxa. This study had three goals. First, we cloned the full-length sequence of a novel AKH, termed Aplysia-AKH, and examined its distribution in an opisthobranch mollusk, Aplysia californica. Second, we investigated in vivo biological effects of Aplysia-AKH. Lastly, we compared the effects of Aplysia-AKH to a related A. californica peptide, Aplysia-GnRH. Results suggest that Aplysia-AKH mRNA and peptide are localized exclusively in central tissues, with abdominal, cerebral, and pleural ganglia being the primary sites of Aplysia-AKH production. However, Aplysia-AKH-positive fibers were found in all central ganglia, suggesting diverse neuromodulatory roles. Injections of A. californica with Aplysia-AKH significantly inhibited feeding, reduced body mass, increased excretion of feces, and reduced gonadal mass and oocyte diameter. The in vivo effects of Aplysia-AKH differed substantially from Aplysia-GnRH. Overall, the distribution and biological effects of Aplysia-AKH suggest it has diverged functionally from Aplysia-GnRH over the course of evolution. Further, that both Aplysia-AKH and Aplysia-GnRH failed to activate reproduction suggest the critical role of GnRH as a reproductive activator may be a phenomenon unique to vertebrates. PMID:25162698

  20. THE STRUCTURAL CHARACTERIZATION AND LOCALLY SUPPORTED BASES FOR BIVARIATE SUPER SPLINES

    Institute of Scientific and Technical Information of China (English)

    Zhi-qiang Xu; Ren-hong Wang

    2004-01-01

    Super splines are bivariate splines defined on triangulations, where the smoothness enforced at the vertices is larger than the smoothness enforced across the edges. In this paper, the smoothness conditions and conformality conditions for super splines are presented.Three locally supported super splines on type-1 triangulation are presented. Moreover, the criteria to select local bases is also given. By using local supported super spline function, avariation-diminishing operator is built. The approximation properties of the operator are also presented.

  1. On characterizing non-locality and anisotropy for the magnetorotational instability

    DEFF Research Database (Denmark)

    Nauman, Farrukh; Blackman, Eric G.

    2014-01-01

    The extent to which angular momentum transport in accretion discs is primarily local or non-local and what determines this is an important avenue of study for understanding accretion engines. Taking a step along this path, we analyse simulations of the magnetorotational instability (MRI...

  2. Data on the characterization of follicle-stimulating hormone monoclonal antibodies and localization in Japanese eel pituitary

    Directory of Open Access Journals (Sweden)

    Dae-Jung Kim

    2016-09-01

    In support of our recent publication, "Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica" [1], it was important to characterize the specificity of eel follicle-stimulating hormone antibodies. Here, the production and ELISA system of these monoclonal antibodies are presented. The affinity-purified monoclonal antibodies specifically detected eel rec-FSH in ELISA and on western blots of rec-FSH produced from CHO cells. Immunohistochemical analysis revealed that FSH staining was specifically localized in the eel pituitary.

  3. Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins.

    NARCIS (Netherlands)

    Schwab, M.A.; Sauer, S.W.; Okun, J.G.; Nijtmans, L.G.J.; Rodenburg, R.J.T.; Heuvel, L.P.W.J. van den; Drose, S.; Brandt, U.; Hoffmann, G.F.; Laak, H.J. ter; Kolker, S.; Smeitink, J.A.M.

    2006-01-01

    Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of propionyl-CoA and metabolites of alternat

  4. Characterizing localization properties of two spinless electrons in a one-dimensional Harper model with concurrence

    Institute of Scientific and Technical Information of China (English)

    Gong Long-Yan; Tong Pei-Qing

    2008-01-01

    By mapping the Fock space of many local fermionic modes isomorphically onto a many-qubit space and using the measure of concurrence,this paper studies numerically the mode entanglement of two spinless electrons with on-site interaction U moving in the one-dimensional Harper model.Generally speaking,for electrons in extended regimes (potential parameter λ< 2),the spectrum-averaged concurrence N first decreases slowly as λ increases until its local minimum,then increases with λ until its peak at λ = 2,while for electrons in localized regimes (λ> 2),Ndecreases drastically as λ increases.The functions of N versus λ are different for electrons in extended and localized regimes.The maximum of Noccurs at the point λ = 2,which is the critical value in the one-dimensional singleparticle Harper model.From these studies it can distinguish extended,localized and critical regimes for the two-particle system.It is also found for the same λ that the interaction U always induce the decreases of concurrence,i.e.,the concurrence can reflect the localization effect due to the interaction.All these provide us a new quantity to understand the localization properties of eigeustates of two interacting particles.

  5. Isoenzyme characterization of Leishmania isolated from human cases with localized cutaneous leishmaniasis from the State of Campeche, Yucatan Peninsula, Mexico.

    Science.gov (United States)

    Canto-Lara, S B; Cardenas-Maruffo, M F; Vargas-Gonzalez, A; Andrade-Narvaez, F

    1998-04-01

    Seventy-five isolates from the State of Campeche, Mexico, an area endemic for localized cutaneous leishmaniasis (LCL), were characterized by isoenzyme markers (glucose phosphate isomerase, mannose phospate isomerase, nucleoside hydrolase, phosphoglucomutase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase). Seventy (93.3%) were identified as Leishmania (Leishmania) mexicana and 5 (6.7%) as L. (Viannia) braziliensis. This is the first report of authochthonus human LCL caused by L. (V.) braziliensis in the State of Campeche, Yucatan Peninsula, Mexico.

  6. Isolation and Characterization of Pollulan Produced by a Local Isolate of Aureobasidium Pullulans

    OpenAIRE

    Kassim, Mohammad Bashir Ismail [محمد بشيراسماعيل قاسم

    2000-01-01

    A local isolate of Aureobasidium pullulans was obtained from fig leaves surface. The isolate was identified as A. pullulans on the basis of appearance of polymorphism in the colonies, melanin pigment and pullulan production in shake flask cultures. Pullulan production by the local isolate of A pullulans was two thirds of that produced by ATCC 42023. The studies of physico — chemical properties, chemical analysis, sugar composition periodate oxidation and smith degradation showed that the pull...

  7. Characterizing emerging European stock markets through complex networks: From local properties to self-similar characteristics

    Science.gov (United States)

    Caraiani, Petre

    2012-07-01

    We investigate the properties of the returns of the main emerging stock markets from Europe by means of complex networks. We transform the series of daily returns into complex networks, and analyze the local properties of these networks with respect to degree distributions, clustering, or average line length. We further use the clustering coefficients as quantities describing the local structure of the network, and approach them by using multifractal analysis. We find evidence of scale-free networks and multifractality of clustering coefficients.

  8. Characterization of a nuclear localization signal in the foot-and-mouth disease virus polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez-Aparicio, Maria Teresa; Rosas, Maria Flora [Centro de Biología Molecular, “Severo Ochoa” (CSIC-UAM), Cantoblanco 28049, Madrid (Spain); Sobrino, Francisco, E-mail: fsobrino@cbm.uam.es [Centro de Biología Molecular, “Severo Ochoa” (CSIC-UAM), Cantoblanco 28049, Madrid (Spain); Centro de Investigación en Sanidad Animal, INIA, Valdeolmos, 28130 Madrid (Spain)

    2013-09-15

    We have experimentally tested whether the MRKTKLAPT sequence in FMDV 3D protein (residues 16 to 24) can act as a nuclear localization signal (NLS). Mutants with substitutions in two basic residues within this sequence, K18E and K20E, were generated. A decreased nuclear localization was observed in transiently expressed 3D and its precursor 3CD, suggesting a role of K18 and K20 in nuclear targeting. Fusion of MRKTKLAPT to the green fluorescence protein (GFP) increased the nuclear localization of GFP, which was not observed when GFP was fused to the 3D mutated sequences. These results indicate that the sequence MRKTKLAPT can be functionally considered as a NLS. When introduced in a FMDV full length RNA replacements K18E and K20E led to production of revertant viruses that replaced the acidic residues introduced (E) by K, suggesting that the presence of lysins at positions 18 and 20 of 3D is essential for virus multiplication. - Highlights: • The FMDV 3D polymerase contains a nuclear localization signal. • Replacements K18E and K20E decrease nuclear localization of 3D and its precursor 3CD. • Fusion of the MRKTKLAPT 3D motif to GFP increases the nuclear localization of GFP. • Replacements K18E and K20E abolish the ability of MRKTKLAPT to relocate GFP. • RNAs harboring replacements K18E and K20E lead to recovery of revertant FMDVs.

  9. ER-associated mitochondrial division links the distribution of mitochondria and mitochondrial DNA in yeast.

    Science.gov (United States)

    Murley, Andrew; Lackner, Laura L; Osman, Christof; West, Matthew; Voeltz, Gia K; Walter, Peter; Nunnari, Jodi

    2013-05-14

    Mitochondrial division is important for mitochondrial distribution and function. Recent data have demonstrated that ER-mitochondria contacts mark mitochondrial division sites, but the molecular basis and functions of these contacts are not understood. Here we show that in yeast, the ER-mitochondria tethering complex, ERMES, and the highly conserved Miro GTPase, Gem1, are spatially and functionally linked to ER-associated mitochondrial division. Gem1 acts as a negative regulator of ER-mitochondria contacts, an activity required for the spatial resolution and distribution of newly generated mitochondrial tips following division. Previous data have demonstrated that ERMES localizes with a subset of actively replicating mitochondrial nucleoids. We show that mitochondrial division is spatially linked to nucleoids and that a majority of these nucleoids segregate prior to division, resulting in their distribution into newly generated tips in the mitochondrial network. Thus, we postulate that ER-associated division serves to link the distribution of mitochondria and mitochondrial nucleoids in cells. DOI:http://dx.doi.org/10.7554/eLife.00422.001.

  10. Mitochondrial targeted β-lapachone induces mitochondrial dysfunction and catastrophic vacuolization in cancer cells.

    Science.gov (United States)

    Ma, Jing; Lim, Chaemin; Sacher, Joshua R; Van Houten, Bennett; Qian, Wei; Wipf, Peter

    2015-11-01

    Mitochondria play important roles in tumor cell physiology and survival by providing energy and metabolites for proliferation and metastasis. As part of their oncogenic status, cancer cells frequently produce increased levels of mitochondrial-generated reactive oxygen species (ROS). However, extensive stimulation of ROS generation in mitochondria has been shown to be able to induce cancer cell death, and is one of the major mechanisms of action of many anticancer agents. We hypothesized that enhancing mitochondrial ROS generation through direct targeting of a ROS generator into mitochondria will exhibit tumor cell selectivity, as well as high efficacy in inducing cancer cell death. We thus synthesized a mitochondrial targeted version of β-lapachone (XJB-Lapachone) based on our XJB mitochondrial targeting platform. We found that the mitochondrial targeted β-lapachone is more efficient in inducing apoptosis compared to unconjugated β-lapachone, and the tumor cell selectivity is maintained. XJB-Lapachone also induced extensive cellular vacuolization and autophagy at a concentration not observed with unconjugated β-lapachone. Through characterization of mitochondrial function we revealed that XJB-Lapachone is indeed more capable of stimulating ROS generation in mitochondria, which led to a dramatic mitochondrial uncoupling and autophagic degradation of mitochondria. Taken together, we have demonstrated that targeting β-lapachone accomplishes higher efficacy through inducing ROS generation directly in mitochondria, resulting in extensive mitochondrial and cellular damage. XJB-Lapachone will thus help to establish a novel platform for the design of next generation mitochondrial targeted ROS generators for cancer therapy.

  11. Mitochondrial biogenesis and turnover.

    Science.gov (United States)

    Diaz, Francisca; Moraes, Carlos T

    2008-07-01

    Mitochondrial biogenesis is a complex process involving the coordinated expression of mitochondrial and nuclear genes, the import of the products of the latter into the organelle and turnover. The mechanisms associated with these events have been intensively studied in the last 20 years and our understanding of their details is much improved. Mitochondrial biogenesis requires the participation of calcium signaling that activates a series of calcium-dependent protein kinases that in turn activate transcription factors and coactivators such as PGC-1alpha that regulates the expression of genes coding for mitochondrial components. In addition, mitochondrial biogenesis involves the balance of mitochondrial fission-fusion. Mitochondrial malfunction or defects in any of the many pathways involved in mitochondrial biogenesis can lead to degenerative diseases and possibly play an important part in aging.

  12. Local characterization of hindered Brownian motion by using digital video microscopy and 3D particle tracking

    CERN Document Server

    Dettmer, Simon L; Pagliara, Stefano

    2014-01-01

    In this article we present methods for measuring hindered Brownian motion in the confinement of complex 3D geometries using digital video microscopy. Here we discuss essential features of automated 3D particle tracking as well as diffusion data analysis. By introducing local mean squared displacement-vs-time curves, we are able to simultaneously measure the spatial dependence of diffusion coefficients, tracking accuracies and drift velocities. Such local measurements allow a more detailed and appropriate description of strongly heterogeneous systems as opposed to global measurements. Finite size effects of the tracking region on measuring mean squared displacements are also discussed. The use of these methods was crucial for the measurement of the diffusive behavior of spherical polystyrene particles (505 nm diameter) in a microfluidic chip. The particles explored an array of parallel channels with different cross sections as well as the bulk reservoirs. For this experiment we present the measurement of local...

  13. Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artefacts and reveals no contribution to basal proton leak.

    Science.gov (United States)

    Jastroch, Martin; Hirschberg, Verena; Klingenspor, Martin

    2012-09-01

    Mechanistic studies on uncoupling proteins (UCPs) not only are important to identify their cellular function but also are pivotal to identify potential drug targets to manipulate mitochondrial energy transduction. So far, functional and comparative studies of uncoupling proteins in their native environment are hampered by different mitochondrial, cellular and genetic backgrounds. Artificial systems such as yeast ectopically expressing UCPs or liposomes with reconstituted UCPs were employed to address crucial mechanistic questions but these systems also produced inconsistencies with results from native mitochondria. We here introduce a novel mammalian cell culture system (Human Embryonic Kidney 293 - HEK293) to study UCP1 function. Stably transfected HEK293 cell lines were derived that contain mouse UCP1 at concentrations comparable to tissue mitochondria. In this cell-based test system UCP1 displays native functional behaviour as it can be activated with fatty acids (palmitate) and inhibited with purine nucleotides guanosine-diphosphate (GDP). The catalytic centre activity of the UCP1 homodimer in HEK293 is comparable to activities in brown adipose tissue supporting functionality of UCP1. Importantly, at higher protein levels than in yeast mitochondria, UCP1 in HEK293 cell mitochondria is fully inhibitable and does not contribute to basal proton conductance, thereby emphasizing the requirement of UCP1 activation for therapeutic purposes. These findings and resulting analysis on UCP1 characteristics demonstrate that the mammalian HEK293 cell system is suitable for mechanistic and comparative functional studies on UCPs and provides a non-confounding mitochondrial, cellular and genetic background.

  14. Mitochondrial aging and age-related dysfunction of mitochondria.

    Science.gov (United States)

    Chistiakov, Dimitry A; Sobenin, Igor A; Revin, Victor V; Orekhov, Alexander N; Bobryshev, Yuri V

    2014-01-01

    Age-related changes in mitochondria are associated with decline in mitochondrial function. With advanced age, mitochondrial DNA volume, integrity and functionality decrease due to accumulation of mutations and oxidative damage induced by reactive oxygen species (ROS). In aged subjects, mitochondria are characterized by impaired function such as lowered oxidative capacity, reduced oxidative phosphorylation, decreased ATP production, significant increase in ROS generation, and diminished antioxidant defense. Mitochondrial biogenesis declines with age due to alterations in mitochondrial dynamics and inhibition of mitophagy, an autophagy process that removes dysfunctional mitochondria. Age-dependent abnormalities in mitochondrial quality control further weaken and impair mitochondrial function. In aged tissues, enhanced mitochondria-mediated apoptosis contributes to an increase in the percentage of apoptotic cells. However, implementation of strategies such as caloric restriction and regular physical training may delay mitochondrial aging and attenuate the age-related phenotype in humans.

  15. Evidence of a bigenomic regulation of mitochondrial gene expression by thyroid hormone during rat brain development

    Energy Technology Data Exchange (ETDEWEB)

    Sinha, Rohit Anthony; Pathak, Amrita; Mohan, Vishwa; Babu, Satish; Pal, Amit; Khare, Drirh [Department of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014 (India); Godbole, Madan M., E-mail: madangodbole@yahoo.co.in [Department of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014 (India)

    2010-07-02

    Hypothyroidism during early mammalian brain development is associated with decreased expression of various mitochondrial encoded genes along with evidence for mitochondrial dysfunction. However, in-spite of the similarities between neurological disorders caused by perinatal hypothyroidism and those caused by various genetic mitochondrial defects we still do not know as to how thyroid hormone (TH) regulates mitochondrial transcription during development and whether this regulation by TH is nuclear mediated or through mitochondrial TH receptors? We here in rat cerebellum show that hypothyroidism causes reduction in expression of nuclear encoded genes controlling mitochondrial biogenesis like PGC-1{alpha}, NRF-1{alpha} and Tfam. Also, we for the first time demonstrate a mitochondrial localization of thyroid hormone receptor (mTR) isoform in developing brain capable of binding a TH response element (DR2) present in D-loop region of mitochondrial DNA. These results thus indicate an integrated nuclear-mitochondrial cross talk in regulation of mitochondrial transcription by TH during brain development.

  16. Structural and functional characterization of human telomerase RNA processing and cajal body localization signals.

    Science.gov (United States)

    Theimer, Carla A; Jády, Beáta E; Chim, Nicholas; Richard, Patricia; Breece, Katherine E; Kiss, Tamás; Feigon, Juli

    2007-09-21

    The RNA component of human telomerase (hTR) includes H/ACA and CR7 domains required for 3' end processing, localization, and accumulation. The terminal loop of the CR7 domain contains the CAB box (ugAG) required for targeting of scaRNAs to Cajal bodies (CB) and an uncharacterized sequence required for accumulation and processing. To dissect out the contributions of the CR7 stem loop to hTR processing and localization, we solved the solution structures of the 3' terminal stem loops of hTR CR7 and U64 H/ACA snoRNA, and the 5' terminal stem loop of U85 C/D-H/ACA scaRNA. These structures, together with analysis of localization, processing, and accumulation of hTRs containing nucleotide substitutions in the CR7 domain, identified the sequence and structural requirements of the hTR processing and CB localization signals and showed that these signals are functionally independent. Further, 3' end processing was found to be a prerequisite for translocation of hTR to CBs.

  17. Characterization, subcellular localization and nuclear targeting of casein kinase 2 from Zea mays

    DEFF Research Database (Denmark)

    Peracchia, G; Jensen, A B; Culiáñez-Macià, F A

    1999-01-01

    by using in-frame fusions of the maize CK2alpha subunit to the reporter gene encoding beta-glucuronidase (GUS) which were assayed in transiently transformed onion epidermal cells. Analysis of chimeric constructs identified one region containing a nuclear localization signal (NLS) that is highly conserved...

  18. Women with inoperable or locally advanced breast cancer -- what characterizes them?

    DEFF Research Database (Denmark)

    El-Charnoubi, Waseem Asim Ghulam; Svendsen, Jesper Brink; Tange, Ulla Brix;

    2012-01-01

    Breast cancer is the most common cancer among Danish women. Locally advanced breast cancer occurs in a relatively large proportion of all new primary breast cancer diagnoses and for unexplained reasons 20-30% of women with breast cancer wait more than eight weeks from the initial breast cancer...

  19. Isolation, characterization and localization of extracellular polymeric substances from the cyanobacterium Arthrospira platensis strain MMG-9

    NARCIS (Netherlands)

    Ahmed, M.; Moerdijk-Poortvliet, T.C.W.; Wijnholds, A.; Stal, L.J.; Hasnain, S.

    2014-01-01

    Arthrospira platensis is a cyanobacterium known for its nutritional value and secondary metabolites. Extracellular polymeric substances (EPS) are an important trait of most cyanobacteria, including A. platensis. Here, we extracted and analysed different fractions of EPS from a locally isolated strai

  20. Isolation, characterization and localization of extracellular polymeric substances from the cyanobacterium

    NARCIS (Netherlands)

    Ahmed, M.; Wijnholds, A.; Stal, L.J.; Hasnain, S.

    2014-01-01

    Arthrospira platensis is a cyanobacterium known for its nutritional value and secondary metabolites. Extracellular polymeric substances (EPS) are an important trait of most cyanobacteria, including A. platensis. Here, we extracted and analysed different fractions of EPS from a locally isolated strai

  1. Miro, MCU, and calcium: bridging our understanding of mitochondrial movement in axons

    Directory of Open Access Journals (Sweden)

    Robert eNiescier

    2013-09-01

    Full Text Available Neurons are extremely polarized structures with long axons and dendrites, which require proper distribution of mitochondria and maintenance of mitochondrial dynamics for neuronal functions and survival. Indeed, recent studies show that various neurological disorders are linked to mitochondrial transport in neurons. Mitochondrial anterograde transport is believed to deliver metabolic energy to synaptic terminals where energy demands are high, while mitochondrial retrograde transport is required to repair or remove damaged mitochondria in axons. It has been suggested that Ca2+ plays a key role in regulating mitochondrial transport by altering the configuration of mitochondrial protein, miro. However, molecular mechanisms that regulate mitochondrial transport in neurons still are not well characterized. In this review, we will discuss the roles of miro in mitochondrial transport and how the recently identified components of the mitochondrial calcium uniporter add to our current model of mitochondrial mobility regulation.

  2. Miro, MCU, and calcium: bridging our understanding of mitochondrial movement in axons.

    Science.gov (United States)

    Niescier, Robert F; Chang, Karen T; Min, Kyung-Tai

    2013-09-10

    Neurons are extremely polarized structures with long axons and dendrites, which require proper distribution of mitochondria and maintenance of mitochondrial dynamics for neuronal functions and survival. Indeed, recent studies show that various neurological disorders are linked to mitochondrial transport in neurons. Mitochondrial anterograde transport is believed to deliver metabolic energy to synaptic terminals where energy demands are high, while mitochondrial retrograde transport is required to repair or remove damaged mitochondria in axons. It has been suggested that Ca(2) (+) plays a key role in regulating mitochondrial transport by altering the configuration of mitochondrial protein, miro. However, molecular mechanisms that regulate mitochondrial transport in neurons still are not well characterized. In this review, we will discuss the roles of miro in mitochondrial transport and how the recently identified components of the mitochondrial calcium uniporter add to our current model of mitochondrial mobility regulation.

  3. Mitochondrial sirtuins as therapeutic targets for age-related disorders.

    Science.gov (United States)

    Shih, Jennifer; Donmez, Gizem

    2013-03-01

    Sirtuins are a class of histone deacetylases that have a wide range of regulatory roles in the cell. Three sirtuins, SIRT3 to SIRT5, localize to and function within the mitochondria. Mitochondrial dysfunction is thought to be the underlying mechanism of several age-related diseases, such as metabolic syndrome, cancer, and neurodegeneration. This review examines current evidence that mitochondrial sirtuins are involved in regulating mitochondrial function and pathogenesis.

  4. 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.

  5. Characterization of immune cells and cytokine localization in the rat utero-placental unit mid- to late gestation.

    Science.gov (United States)

    Tessier, Daniel R; Raha, Sandeep; Holloway, Alison C; Yockell-Lelièvre, Julien; Tayade, Chandrakant; Gruslin, Andrée

    2015-08-01

    The success of pregnancy is dependent on the precise regulation of the immune response within the utero-placental environment. Rats are beginning to be widely used as a model for human immune-related pregnancy complications. However, our knowledge of immune cells and cytokine localization in the rat utero-placental tissue is limited. The current study aimed to localize the immune cell populations, including uterine natural killer (uNK) cells, neutrophils, and macrophages within the rat utero-placental unit at two crucial gestational ages, gestational days 15.5 and 18.5. In addition, we characterized the distribution of the cytokines TNFα, IFNγ, and IL-10 in the utero-placental regions at both the above-mentioned gestational ages. Our study has demonstrated co-localization TNFα and IFNγ with uNK cells in perivascular regions of the rat mesometrial triangle at both gestational ages. Neutrophils and IL-10-positive cells were localized at the maternal-fetal interface and in the spiral artery lumen of the rat mesometrial triangle at both gestational ages. TNFα and IL-10 demonstrated a temporal change in the localization from GD15.5 to GD18.5, which coincides with the leading edge of trophoblast invasion into the mesometrial triangle. The current study furthers our knowledge of the localization of uterine immune cells and relevant cytokines, and provides a base from which to research the function of these immune cells and cytokines during rat pregnancy as a model to study human immune-related pregnancy complications.

  6. Optical extinction spectrum of a single metal nanoparticle: Quantitative characterization of a particle and of its local environment

    Science.gov (United States)

    Muskens, O. L.; Billaud, P.; Broyer, M.; Del Fatti, N.; Vallée, F.

    2008-11-01

    Optical absorption spectroscopy of a single metal nanoparticle is used to characterize its properties and to obtain quantitative information on its local environment. Experiments were performed using the spatial modulation spectroscopy (SMS) technique on 16 nm mean diameter gold nanoparticles embedded in different medium (i.e., deposited on glass or embedded in a polymer layer). Extraction of the nanoparticle characteristics and determination of the dielectric constant of its environment are discussed, focusing on the impact of the particle shape assumption. The refractive index of the local environment deduced from these measurements shows large particle-to-particle variation, yielding information about fluctuations of the dielectric properties of the surrounding medium on a nanometric scale, inaccessible in ensemble measurements. The influence of the environment of a nanoparticle on its optically extracted geometry and its surface plasmon resonance broadening by surface effect are also studied at a single-particle level.

  7. Selective ion changes during spontaneous mitochondrial transients in intact astrocytes.

    Directory of Open Access Journals (Sweden)

    Guillaume Azarias

    Full Text Available The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na(+ concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na(+ concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca(2+ concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg(2+ concentration accompanying mitochondrial Na(+ spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na(+-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.

  8. Selective ion changes during spontaneous mitochondrial transients in intact astrocytes.

    Science.gov (United States)

    Azarias, Guillaume; Chatton, Jean-Yves

    2011-01-01

    The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na(+) concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na(+) concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca(2+) concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg(2+) concentration accompanying mitochondrial Na(+) spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na(+)-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.

  9. Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Koh, Ai Leen [Department of Materials Science and Engineering, Stanford University, Durand Building Room 139, 496 Lomita Mall, Stanford, CA 94305 (United States); Stanford Nanocharacterization Laboratory, Stanford University, Stanford, CA 94305 (United States); Department of Mechanical Engineering, Stanford University, Stanford, CA 94305 (United States)], E-mail: alkoh@stanford.edu; Shachaf, Catherine M.; Elchuri, Sailaja; Nolan, Garry P. [Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305 (United States); Sinclair, Robert [Department of Materials Science and Engineering, Stanford University, Durand Building Room 139, 496 Lomita Mall, Stanford, CA 94305 (United States); Stanford Nanocharacterization Laboratory, Stanford University, Stanford, CA 94305 (United States)

    2008-12-15

    We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

  10. The pseudo-mitochondrial genome influences mistakes in heteroplasmy interpretation

    Directory of Open Access Journals (Sweden)

    Wittock Roy

    2006-07-01

    Full Text Available Abstract Background Nuclear mitochondrial pseudogenes (numts are a potential source of contamination during mitochondrial DNA PCR amplification. This possibility warrants careful experimental design and cautious interpretation of heteroplasmic results. Results Here we report the cloning and sequencing of numts loci, amplified from human tissue and rho-zero (ρ0 cells (control with primers known to amplify the mitochondrial genome. This paper is the first to fully sequence 46 paralogous nuclear DNA fragments that represent the entire mitochondrial genome. This is a surprisingly small number due primarily to the primer sets used in this study, because prior to this, BLAST searches have suggested that nuclear DNA harbors between 400 to 1,500 paralogous mitochondrial DNA fragments. Our results indicate that multiple numts were amplified simultaneously with the mitochondrial genome and increased the load of pseudogene signal in PCR reactions. Further, the entire mitochondrial genome was represented by multiple copies of paralogous nuclear sequences. Conclusion These findings suggest that mitochondrial genome disease-associated biomarkers must be rigorously authenticated to preclude any affiliation with paralogous nuclear pseudogenes. Importantly, the common perception that mitochondrial template "swamps" numts loci precluding detectable amplification, depends on the region of the mitochondrial genome targeted by the PCR reaction and the number of pseudogene loci that may co-amplify. Cloning and relevant sequencing data will facilitate the correct interpretation. This is the first complete, wet-lab characterization of numts that represent the entire mitochondrial genome.

  11. Identification and characterization of a highly variable region in mitochondrial genomes of fusarium species and analysis of power generation from microbial fuel cells

    Science.gov (United States)

    Hamzah, Haider Mousa

    In the microbial fuel cell (MFC) project, power generation from Shewanella oneidensis MR-1 was analyzed looking for a novel system for both energy generation and sustainability. The results suggest the possibility of generating electricity from different organic substances, which include agricultural and industrial by-products. Shewanella oneidensis MR-1 generates usable electrons at 30°C using both submerged and solid state cultures. In the MFC biocathode experiment, most of the CO2 generated at the anodic chamber was converted into bicarbonate due the activity of carbonic anhydrase (CA) of the Gluconobacter sp.33 strain. These findings demonstrate the possibility of generation of electricity while at the same time allowing the biomimetic sequestration of CO2 using bacterial CA. In the mitochondrial genomes project, the filamentous fungal species Fusarium oxysporum was used as a model. This species causes wilt of several important agricultural crops. A previous study revealed that a highly variable region (HVR) in the mitochondrial DNA (mtDNA) of three species of Fusarium contained a large, variable unidentified open reading frame (LV-uORF). Using specific primers for two regions of the LV-uORF, six strains were found to contain the ORF by PCR and database searches identified 18 other strains outside of the Fusarium oxysporum species complex. The LV-uORF was also identified in three isolates of the F. oxysporum species complex. Interestingly, several F. oxysporum isolates lack the LV-uORF and instead contain 13 ORFs in the HVR, nine of which are unidentified. The high GC content and codon usage of the LV-uORF indicate that it did not co-evolve with other mt genes and was horizontally acquired and was introduced to the Fusarium lineage prior to speciation. The nonsynonymous/synonymous (dN/dS) ratio of the LV-uORFs (0.43) suggests it is under purifying selection and the putative polypeptide is predicted to be located in the mitochondrial membrane. Growth assays

  12. Cellular localization and biochemical characterization of a novel calcium-dependent protein kinase from tobacco

    Institute of Scientific and Technical Information of China (English)

    Yun WANG; Mei ZHANG; Ke KE; Ying Tang LU

    2005-01-01

    By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 (accession number AY971376), which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526 nmol/min/mg and 210 μg/ml respectively with calf thymus histone (fraction Ⅲ, abbreviated to histone Ⅲs) as substrate. For substrate syntide-2, NtCPK5 showed a higher. Vmax of 2008 nmol/min/mg and a lower Km of 30 μM. The K0.5 of calcium activation was 0.04 μM or 0.06 μM for histone Ⅲs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein.

  13. Characterization of aquaporin 4 protein expression and localization in tissues of the dogfish (Squalus acanthias.

    Directory of Open Access Journals (Sweden)

    Christopher P Cutler

    2012-02-01

    Full Text Available The role of aquaporin water channels in Elasmobanchs such as the dogfish Squalus acanthias is completely unknown. This investigation determines the expression and cellular and sub-cellular localization of AQP4 protein in dogfish tissues. Two polyclonal antibodies were generated (AQP4/1 and AQP4/2. Western blots using the AQP4/1 antibody showed two bands (35.5kDa and 49.5kDa in most tissues similar to mammals. Liver and rectal gland showed further bands. However, unlike in mammals, AQP4 protein was expressed in all tissues including respiratory tract and liver. The AQP4/2 antibody appeared much less specific in blots. Both antibodies were used in immunohistochemistry and showed similar cellular localizations, although the AQP4/2 antibody had a more restricted sub-cellular distribution compared to AQP4/1 and therefore appeared to be more specific. In kidney a sub-set of tubules were stained which may represent intermediate tubule segments. AQP4/1 and AQP4/2 antibodies localized to the same tubules segments in serial sections although the intensity and sub-cellular distribution were different. AQP4/2 showed a basal or basolateral membrane distribution whereas AQP4/1 was often distributed throughout the cell including the nucleus. In rectal gland and cardiac stomach AQP4 was localized to secretary tubules but again AQP/1 and AQP/2 showed different sub-cellular distributions. In gill, both antibodies stained large cells in the primary filament and secondary lamellae. Again AQP4/1 antibody stained most or all the cell including the nucleus, whereas AQP4/2 had a plasma membrane and sometimes cytoplasmic distribution. Two types of large mitochondria-rich cells are known to exist in elasmobranches, that express either Na,K ATPase or V-type ATPase. Using Na,K-ATPase and V-type ATPase antibodies, AQP4 was colocalized with these proteins using the AQP4/1 antibody. Results show AQP4 is expressed in both (and all branchial Na,K ATPase and V-type ATPase

  14. Differential localization and characterization of functional calcitonin gene-related peptide receptors in human subcutaneous arteries

    DEFF Research Database (Denmark)

    Edvinsson, L; Ahnstedt, H; Larsen, R;

    2014-01-01

    Calcitonin gene-related peptide (CGRP) and its receptor are widely distributed within the circulation and the mechanism behind its vasodilation not only differs from one animal species to another but is also dependent on the type and size of vessel. The present study examines the nature of CGRP......-induced vasodilation, characteristics of the CGRP receptor antagonist telcagepant and localization of the key components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) of the CGRP receptor in human subcutaneous arteries....

  15. Functional characterization and localization of a gill-specific claudin isoform in Atlantic salmon

    DEFF Research Database (Denmark)

    Engelund, Morten Buch; Yu, Alan S L; Li, Jiahua;

    2012-01-01

    , with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na(+)-K(+)-ATPase was negligible, suggesting limited association with mitochondria...... is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon...

  16. Dynamics of mitochondrial transport in axons

    Directory of Open Access Journals (Sweden)

    Robert Francis Niescier

    2016-05-01

    Full Text Available The polarized structure and long neurites of neurons pose a unique challenge for proper mitochondrial distribution. It is widely accepted that mitochondria move from the cell body to axon ends and vice versa; however, we have found that mitochondria originating from the axon ends moving in the retrograde direction never reach to the cell body, and only a limited number of mitochondria moving in the anterograde direction from the cell body arrive at the axon ends of mouse hippocampal neurons. Furthermore, we have derived a mathematical formula using the Fokker-Planck equation to characterize features of mitochondrial transport, and the equation could determine altered mitochondrial transport in axons overexpressing parkin. Our analysis will provide new insights into the dynamics of mitochondrial transport in axons of normal and unhealthy neurons.

  17. Neonatal muscular manifestations in mitochondrial disorders.

    Science.gov (United States)

    Tulinius, Már; Oldfors, Anders

    2011-08-01

    During the last decade rapid development has occurred in defining nuclear gene mutations causing mitochondrial disease. Some of these newly defined gene mutations cause neonatal or early infantile onset of disease, often associated with severe progressive encephalomyopathy combined with other multi-organ involvement such as cardiomyopathy or hepatopathy and with early death. Findings suggesting myopathy in neonates are hypotonia, muscle weakness and wasting, and arthrogryposis. We aim to describe the clinical findings of patients with mitochondrial disease presenting with muscular manifestations in the neonatal period or in early infancy and in whom the genetic defect has been characterized. The majority of patients with neonatal onset of mitochondrial disease have mutations in nuclear genes causing dysfunction of the mitochondrial respiratory chain, leading to defective oxidative phosphorylation.

  18. Determination of relationship between some Turkish local tomato genotypes by using phenotypic characterization

    Directory of Open Access Journals (Sweden)

    Asu OĞUZ

    2014-06-01

    Full Text Available Turkey is the fourth producer country among the other countries in the world and tomato are produced around 11 million tons per year. Over many years, tomato adapted to the geography of Turkey has shown a high biodiversity. In this research, with 76 local tomato genotypes collected from 52 different provience, 4 foreign and 8 wild species, total 88 tomato genotypes were used. Morphological variations among these materials were investigated. Some of the local genotypes were determined to be accessions increasing variations. A cluster diagram obtained from the morphological descriptors produced ten main sub-cluster groups of tomato accessions at a coefficient of 0.15. Accessions were put into cluster groups based on certain qualities unique. It was observed that 86 out of 88 tomato accessions under study were distinct accessions. G80 and G83 were recorded similar (94% accessions in all accessions. Similarity coefficient values among the 88 accessions ranged from -0.11 to 0.94. Accessions with similar quantitative and qualitative morphological characters appeared well grouped in the same cluster. These accessions are considered as important genetic resources in tomato breeding studies.

  19. Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species.

    Science.gov (United States)

    Kuipers, A G J; Kamstra, S A; de Jeu, M J; Visser, R G F

    2002-01-01

    Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68-127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed.

  20. Dynamic morphometric characterization of local connective tissue network structure in humans using ultrasound

    Directory of Open Access Journals (Sweden)

    Konofagou Elisa E

    2007-06-01

    Full Text Available Abstract Background In humans, connective tissue forms a complex, interconnected network throughout the body that may have mechanosensory, regulatory and signaling functions. Understanding these potentially important phenomena requires non-invasive measurements of collagen network structure that can be performed in live animals or humans. The goal of this study was to show that ultrasound can be used to quantify dynamic changes in local connective tissue structure in vivo. We first performed combined ultrasound and histology examinations of the same tissue in two subjects undergoing surgery: in one subject, we examined the relationship of ultrasound to histological images in three dimensions; in the other, we examined the effect of a localized tissue perturbation using a previously developed robotic acupuncture needling technique. In ten additional non-surgical subjects, we quantified changes in tissue spatial organization over time during needle rotation vs. no rotation using ultrasound and semi-variogram analyses. Results 3-D renditions of ultrasound images showed longitudinal echogenic sheets that matched with collagenous sheets seen in histological preparations. Rank correlations between serial 2-D ultrasound and corresponding histology images resulted in high positive correlations for semi-variogram ranges computed parallel (r = 0.79, p Conclusion The combination of ultrasound and semi-variogram analyses allows quantitative assessment of dynamic changes in the structure of human connective tissue in vivo.

  1. Characterization of local complex structures in a recurrence plot to improve nonlinear dynamic discriminant analysis.

    Science.gov (United States)

    Ding, Hang

    2014-01-01

    Structures in recurrence plots (RPs), preserving the rich information of nonlinear invariants and trajectory characteristics, have been increasingly analyzed in dynamic discrimination studies. The conventional analysis of RPs is mainly focused on quantifying the overall diagonal and vertical line structures through a method, called recurrence quantification analysis (RQA). This study extensively explores the information in RPs by quantifying local complex RP structures. To do this, an approach was developed to analyze the combination of three major RQA variables: determinism, laminarity, and recurrence rate (DLR) in a metawindow moving over a RP. It was then evaluated in two experiments discriminating (1) ideal nonlinear dynamic series emulated from the Lorenz system with different control parameters and (2) data sets of human heart rate regulations with normal sinus rhythms (n = 18) and congestive heart failure (n = 29). Finally, the DLR was compared with seven major RQA variables in terms of discriminatory power, measured by standardized mean difference (DSMD). In the two experiments, DLR resulted in the highest discriminatory power with DSMD = 2.53 and 0.98, respectively, which were 7.41 and 2.09 times the best performance from RQA. The study also revealed that the optimal RP structures for the discriminations were neither typical diagonal structures nor vertical structures. These findings indicate that local complex RP structures contain some rich information unexploited by RQA. Therefore, future research to extensively analyze complex RP structures would potentially improve the effectiveness of the RP analysis in dynamic discrimination studies.

  2. Characterization of a nuclear localization signal of canine parvovirus capsid proteins.

    Science.gov (United States)

    Vihinen-Ranta, M; Kakkola, L; Kalela, A; Vilja, P; Vuento, M

    1997-12-01

    We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N-terminal residues 4-13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of Lys6, Arg7, or Arg9 with glycine abolished it. The targeting activity was found to residue in a cluster of basic residues, Lys5, Arg7, and Arg9. Nuclear import was saturated by excess of unlabelled peptide conjugates (showing that it was a receptor-mediated process). Transport into the nucleus was an energy-dependent and temperature-dependent process actively mediated by the nuclear pores and inhibited by wheat germ agglutinin.

  3. Improved characterization of local seismicity using the Dubai Seismic Network, United Arab Emirates

    Science.gov (United States)

    Al Khatibi, Eman; Abou Elenean, K. M.; Megahed, A. S.; El-Hussain, I.

    2014-08-01

    In April 2006, Dubai Municipality established a broadband seismological network in Dubai Emirate, United Arab Emirates (UAE). This network was the first seismic network in UAE and consists of four remote seismic stations to observe local and regional seismic activity that may have an effect on Dubai Emirate and the surrounding areas. The network exchanges real-time data with the National Center of Meteorology and Seismology in Abu Dhabi, the Earthquake Monitoring Center in Oman and imports in real-time data from few Global Seismic Network stations, which increases the aperture of the network. In April 2012, Dubai Municipality installed an additional five free-field strong motion stations inside the urban area to estimate and publish real-time ShakeMaps for public and decision makers. Although the local seismic activity from April 2006 to June 2013 reflects low seismic activity with the Emirate, it indicates active tectonics in the relatively aseismic northern Oman Mountains region. A few inland clusters of micro-to-small earthquakes have been identified with the new network. A clear cluster of small-to-moderate earthquakes took place in the eastern part of UAE to the east of Masafi, while two clusters of micro-to-small earthquakes took place at Wadi Nazwa and northern Huwaylat. Focal mechanisms of few well recorded earthquakes in this region indicate normal faulting, generally trending NE in parallel to the transition shear zone between the collision at Zagros and the subduction at the Makran zone.

  4. Characterizing the Locality of Diabatic States for Electronic Excitation Transfer by Decomposing the Diabatic Coupling

    Energy Technology Data Exchange (ETDEWEB)

    Vura-Weis, Josh; Newton, M. D.; Wasielewski, Michael R; Subotnik, J.E.

    2010-12-09

    A common strategy to calculate electronic coupling matrix elements for charge or energy transfer is to take the adiabatic states generated by electronic structure computations and rotate them to form localized diabatic states. In this paper, we show that, for intermolecular transfer of singlet electronic excitation, usually we cannot fully localize the electronic excitations in this way. Instead, we calculate putative initial and final states with small excitation tails caused by weak interactions with high energy excited states in the electronic manifold. These tails do not lead to substantial changes in the total diabatic coupling between states, but they do lead to a different partitioning of the total coupling between Coulomb (Förster), exchange (Dexter), and one-electron components. The tails may be reduced by using a multistate diabatic model or eliminated entirely by truncation (denoted as “chopping”). Without more information, we are unable to conclude with certainty whether the observed diabatic tails are a physical reality or a computational artifact. This research suggests that decomposition of the diabatic coupling between chromophores into Coulomb, exchange, and one-electron components may depend strongly on the number of states considered, and such results should be treated with caution.

  5. Localization and characterization of fatigue cracks around fastener holes using spherically focused ultrasonic probes

    Science.gov (United States)

    Hopkins, Deborah; Datuin, Marvin; Aldrin, John; Warchol, Mark; Warchol, Lyudmila; Forsyth, David

    2017-02-01

    Results are presented from laboratory experiments and simulations that demonstrate the ability to localize fatigue cracks around fastener holes using spherically focused ultrasonic probes for shear-wave inspections. For the experiments, fatigue cracks were created in aluminum plates in a testing frame under cyclic loading. With the exceptions of one specimen with a mid-bore crack and another with a "through" crack, the remaining specimens contain surface-breaking cracks. All of the specimens were inspected for the cracks intersecting the back wall, and some were flipped over and re-inspected with the crack intersecting the front surface. Parameter and variable sensitivity studies were performed using CIVA Simulation Software. In contrast to C-scans where detection and localization of small cracks can be very difficult, modeling and initial experimental results demonstrate that cracks can be accurately located in "True" B-scans (B-scans projected in the part along the beam path). Initial results show that small-amplitude diffracted/scattered signals from the crack tips and edges are essential in obtaining clear crack traces in the True B-scans. It is important therefore that experimental data be acquired with sufficient gain to capture the diffracted/scattered signals. In all of the cases studied here, saturating the high-amplitude specular reflections from the fastener hole and crack enhanced the crack trace in the True B-scans.

  6. Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from Arabidopsis.

    Science.gov (United States)

    Ebert, Berit; Rautengarten, Carsten; Guo, Xiaoyuan; Xiong, Guangyan; Stonebloom, Solomon; Smith-Moritz, Andreia M; Herter, Thomas; Chan, Leanne Jade G; Adams, Paul D; Petzold, Christopher J; Pauly, Markus; Willats, William G T; Heazlewood, Joshua L; Scheller, Henrik Vibe

    2015-04-01

    Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis.

  7. Identification and characterization of multiple conserved nuclear localization signals within adenovirus E1A

    Energy Technology Data Exchange (ETDEWEB)

    Marshall, Kris S.; Cohen, Michael J.; Fonseca, Greg J.; Todorovic, Biljana; King, Cason R. [Department of Microbiology and Immunology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada); Yousef, Ahmed F. [Department of Chemical and Environmental Engineering, Masdar Institute, Abu Dhabi (United Arab Emirates); Zhang, Zhiying [College of Animal Science and Technologies, Northwest A and F University, Yangling, Shaanxi 712100 (China); Mymryk, Joe S., E-mail: jmymryk@uwo.ca [Department of Microbiology and Immunology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada); Department of Oncology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada)

    2014-04-15

    The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell. - Highlights: • HAdV E1A uses multiple mechanisms for nuclear import. • We identified an additional non-canonical NLS in the N-terminal/CR1 portion of E1A. • The new NLS does not contact importin-alpha directly. • All NLSs are functionally conserved in the E1A proteins of all 6 HAdV species.

  8. Characterization of local complex structures in a recurrence plot to improve nonlinear dynamic discriminant analysis

    Science.gov (United States)

    Ding, Hang

    2014-01-01

    Structures in recurrence plots (RPs), preserving the rich information of nonlinear invariants and trajectory characteristics, have been increasingly analyzed in dynamic discrimination studies. The conventional analysis of RPs is mainly focused on quantifying the overall diagonal and vertical line structures through a method, called recurrence quantification analysis (RQA). This study extensively explores the information in RPs by quantifying local complex RP structures. To do this, an approach was developed to analyze the combination of three major RQA variables: determinism, laminarity, and recurrence rate (DLR) in a metawindow moving over a RP. It was then evaluated in two experiments discriminating (1) ideal nonlinear dynamic series emulated from the Lorenz system with different control parameters and (2) data sets of human heart rate regulations with normal sinus rhythms (n = 18) and congestive heart failure (n = 29). Finally, the DLR was compared with seven major RQA variables in terms of discriminatory power, measured by standardized mean difference (DSMD). In the two experiments, DLR resulted in the highest discriminatory power with DSMD = 2.53 and 0.98, respectively, which were 7.41 and 2.09 times the best performance from RQA. The study also revealed that the optimal RP structures for the discriminations were neither typical diagonal structures nor vertical structures. These findings indicate that local complex RP structures contain some rich information unexploited by RQA. Therefore, future research to extensively analyze complex RP structures would potentially improve the effectiveness of the RP analysis in dynamic discrimination studies.

  9. Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

    DEFF Research Database (Denmark)

    Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-bjergaard, Ulrik

    2016-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...

  10. Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

    DEFF Research Database (Denmark)

    Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik

    2016-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...

  11. Prevalence and clinical characterization of Japanese diabetes mellitus with an A-to-G mutation at nucleotide 3243 of the mitochondrial tRNA{sup Leu (UUR)} gene

    Energy Technology Data Exchange (ETDEWEB)

    Odawara, Masato; Sasaki, Kayoko; Yamashita, Kamejiro [Univ. of Tsukuba (Japan)

    1995-04-01

    An A-to-G mutation at nucleotide position 3243 of the mitochondrial genome has been associated with insulin-dependent diabetes mellitus (IDDM) and with noninsulin-dependent diabetes mellitus (NIDDM) with deafness. We investigated the prevalence of this mutation in Japanese patients with IDDM, NIDDM, and impaired glucose tolerance (IGT) and in nondiabetic control individuals, and we identified it in 3 of 300 patients with NIDDM or IGT (1.0%). None of these individuals had significant sensorineural hearing loss. None of the 94 IDDM or the 115 nondiabetic control subjects was positive for this mutation. Oral glucose tolerance test revealed that a 57-yr-old male with this mutation was rather hyperinsulinemic in the fasting state. The insulin secretion in this patient decreased with age; he did not complain of any hearing disorder, although audiometry revealed a slight elevation of hearing threshold at high frequencies. In conclusion, we found that a mitochondrial gene mutation at nucleotide position 3243 was present in about 1% of NIDDM patients including those patients with IGT. The subtype of diabetes mellitus with this mutation may have a clinical profile similar to that found in patients with NIDDM commonly seen in outpatient clinics. 25 refs., 2 figs., 1 tab.

  12. Characterization and expression of a cDNA, AmphiSDHD,encoding the amphioxus cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase

    Institute of Scientific and Technical Information of China (English)

    MA Lifang; ZHANG Shicui; ZHUANG Zhimeng; LIU Zhenhui; LI Hongyan; XIA Jianjun

    2005-01-01

    In this study, an amphioxus cDNA, AmphiSDHD, encoding the cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 1429 bp in length, with an open reading frame of 465 bp coding for a protein of 154 amino acids. The deduced protein contains a mitochondrial targeting presequence of 65 amino acids rich in basic residues like arginine and hydroxy residues such as serine and threonine. Alignment of the amino acid sequences of AmphiSDHD and other eukaryotic SDHD proteins showed that AmphiSDHD has three transmembrane segments, and includes two histidine residues in the second transmembrane segment that are the putative binding sites for the heme b molecule. The phylogenetic tree constructed suggests that AmphiSDHD appears more closely related to vertebrate SDHD proteins than invertebrate ones. Northern blotting demonstrated that AmphiSDHD is ubiquitously expressed in amphioxus, being in line with the fact that SDHD is a house-keeping protein.

  13. Mitochondrial phospholipids: role in mitochondrial function.

    Science.gov (United States)

    Mejia, Edgard M; Hatch, Grant M

    2016-04-01

    Mitochondria are essential components of eukaryotic cells and are involved in a diverse set of cellular processes that include ATP production, cellular signalling, apoptosis and cell growth. These organelles are thought to have originated from a symbiotic relationship between prokaryotic cells in an effort to provide a bioenergetic jump and thus, the greater complexity observed in eukaryotes (Lane and Martin 2010). Mitochondrial processes are required not only for the maintenance of cellular homeostasis, but also allow cell to cell and tissue to tissue communication (Nunnari and Suomalainen 2012). Mitochondrial phospholipids are important components of this system. Phospholipids make up the characteristic outer and inner membranes that give mitochondria their shape. In addition, these membranes house sterols, sphingolipids and a wide variety of proteins. It is the phospholipids that also give rise to other characteristic mitochondrial structures such as cristae (formed from the invaginations of the inner mitochondrial membrane), the matrix (area within cristae) and the intermembrane space (IMS) which separates the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). Phospholipids are the building blocks that make up these structures. However, the phospholipid composition of the OMM and IMM is unique in each membrane. Mitochondria are able to synthesize some of the phospholipids it requires, but the majority of cellular lipid biosynthesis takes place in the endoplasmic reticulum (ER) in conjunction with the Golgi apparatus (Fagone and Jackowski 2009). In this review, we will focus on the role that mitochondrial phospholipids play in specific cellular functions and discuss their biosynthesis, metabolism and transport as well as the differences between the OMM and IMM phospholipid composition. Finally, we will focus on the human diseases that result from disturbances to mitochondrial phospholipids and the current research being performed to help

  14. Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings

    Science.gov (United States)

    Kim, S. H.; Terry, M. E.; Hoops, P.; Dauwalder, M.; Roux, S. J.

    1988-01-01

    A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

  15. Characterization of nucleocytoplasmic shuttling and intracellular localization signals in Duck Enteritis Virus UL54.

    Science.gov (United States)

    Liu, Chaoyue; Cheng, Anchun; Wang, Mingshu; Chen, Shun; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Chen, Xiaoyue

    2016-08-01

    Duck Enteritis virus (DEV) UL54 is a homolog of herpes simplex virus-1 (HSV-1) trafficking protein ICP27, which plays an essential role in infection. In this study, DEV UL54 shuttling between the nucleus and cytoplasm was verified with a heterokaryon assay. One predicted nuclear export sequence (NES) (339-348 aa) was shown to be functional and chromosomal region maintenance 1 (CRM1)-dependent; however, the insensitivity of UL54 to Leptomycin B (LMB) and NES mutation suggests that other mechanisms are responsible for the observed nuclear export. Next, three non-classical nuclear localization sequences (NLSs), referred to as NLS1 (105-122 aa), NLS2 (169-192 aa) and NLS3 (257-274 aa), were identified. Furthermore, a recombinant DEV with the UL54 NLSs deleted (DEV- UL54 mNLSs) was constructed and showed that UL54 NLSs moderately affected DEV growth.

  16. Localized Quantitative Characterization of Chemical Functionalization Effects on Adhesion Properties of SWNT

    Directory of Open Access Journals (Sweden)

    Hao Lu

    2011-01-01

    Full Text Available Chemical modification of single-walled carbon nanotubes (SWNT has been found to be an excellent method to promote SWNT dispersion, and possibly to improve interaction with matrices via covalent bonding. It is thus a quite promising technique to enhance the mechanical properties of SWNT-reinforced nanocomposites. However, the underlying mechanism of SWNT chemical functionalization effects on interfacial strength is not quantitatively understood, limiting their usefulness in the design of nanocomposites. In this work, an atomic force microscopy (AFM- based adhesive force mapping technique combined with a statistical analysis method were developed and implemented to study adhesive interactions of small SWNT bundles functionalized by amino, epoxide, and hydroperoxide groups as compared to SDS-treated SWNT in controlled environment. Finally, the importance of such localized quantitative measurements in SWNT-reinforced nanocomposites design and fabrication was also discussed.

  17. Characterizing the local population of star-forming and passive galaxies with analytical models of chemical evolution

    CERN Document Server

    Spitoni, E; Matteucci, F

    2016-01-01

    Analytical models of chemical evolution, including inflow and outflow of gas, are important tools to study how the metal content in galaxies evolves as a function of time. In this work, we present new analytical solutions for the evolution of the gas mass, total mass and metallicity of a galactic system, when a decaying exponential infall rate of gas and galactic winds are assumed. We apply our model to characterize a sample of local star-forming and passive galaxies from the Sloan Digital Sky Survey data, with the aim of reproducing their observed mass-metallicity relation; in this way, we can derive how the two populations of star-forming and passive galaxies differ in their particular distribution of ages, formation time scales, infall masses and mass loading factors. We find that the local passive galaxies are on average older and assembled on shorter typical time-scales than the local star-forming ones; on the other hand, the larger mass star-forming galaxies show generally older ages and longer typical ...

  18. Local configuration pattern features for age-related macular degeneration characterization and classification.

    Science.gov (United States)

    Mookiah, Muthu Rama Krishnan; Acharya, U Rajendra; Fujita, Hamido; Koh, Joel E W; Tan, Jen Hong; Noronha, Kevin; Bhandary, Sulatha V; Chua, Chua Kuang; Lim, Choo Min; Laude, Augustinus; Tong, Louis

    2015-08-01

    Age-related Macular Degeneration (AMD) is an irreversible and chronic medical condition characterized by drusen, Choroidal Neovascularization (CNV) and Geographic Atrophy (GA). AMD is one of the major causes of visual loss among elderly people. It is caused by the degeneration of cells in the macula which is responsible for central vision. AMD can be dry or wet type, however dry AMD is most common. It is classified into early, intermediate and late AMD. The early detection and treatment may help one to stop the progression of the disease. Automated AMD diagnosis may reduce the screening time of the clinicians. In this work, we have introduced LCP to characterize normal and AMD classes using fundus images. Linear Configuration Coefficients (CC) and Pattern Occurrence (PO) features are extracted from fundus images. These extracted features are ranked using p-value of the t-test and fed to various supervised classifiers viz. Decision Tree (DT), Nearest Neighbour (k-NN), Naive Bayes (NB), Probabilistic Neural Network (PNN) and Support Vector Machine (SVM) to classify normal and AMD classes. The performance of the system is evaluated using both private (Kasturba Medical Hospital, Manipal, India) and public domain datasets viz. Automated Retinal Image Analysis (ARIA) and STructured Analysis of the Retina (STARE) using ten-fold cross validation. The proposed approach yielded best performance with a highest average accuracy of 97.78%, sensitivity of 98.00% and specificity of 97.50% for STARE dataset using 22 significant features. Hence, this system can be used as an aiding tool to the clinicians during mass eye screening programs to diagnose AMD.

  19. Mitochondrial carbonic anhydrase CA VB: Differences in tissue distribution and pattern of evolution from those of CA VA suggest distinct physiological roles

    OpenAIRE

    Shah, Gul N.; Hewett-Emmett, David; Grubb, Jeffrey H.; Migas, Mary C.; Fleming, Robert E.; Waheed, Abdul; Sly, William S.

    2000-01-01

    A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitoc...

  20. Mitochondrial Biogenesis and Turnover

    OpenAIRE

    Diaz, Francisca; Moraes, Carlos T.

    2008-01-01

    Mitochondrial biogenesis is a complex process involving the coordinated expression of mitochondrial and nuclear genes, the import of the products of the latter into the organelle and turnover. The mechanisms associated with these events have been intensively studied in the last twenty years and our understanding of their details is much improved. Mitochondrial biogenesis requires the participation of calcium signaling that activates a series of calcium dependent protein kinases that in turn a...

  1. Single spore isolation and morphological characterization of local Malaysian isolates of rice blast fungus Magnoporthe grisea

    Science.gov (United States)

    Mishra, Ankitta; Ratnam, Wickneswari; Bhuiyan, Md Atiqur Rahman; Ponaya, Ariane; Jena, Khisord K.

    2015-09-01

    Rice blast is a destructive disease, caused by the fungal pathogen Magnaporthe grisea. It causes considerable damage to rice and leads to crop loss in rice growing regions worldwide. Although fungicides can be used to control rice blast, they generate additional cost in rice production and contamination of environment and food. Therefore, the use of resistant varieties is thought to be one of the most economically and environmentally efficient ways of crop protection from the disease. Six new local Malaysian isolates of M. grisea were isolated using single spore isolation method. Five isolates were from infected leaf samples collected from Kompleks Latihan MADA, Kedah and one was from Kelantan. These isolates were identified using morphological characteristics and microscopic studies and later confirmed by ITSequences. These isolates were induced to sporulate and used for greenhouse screening on two differential rice varieties: Mahsuri (susceptible) and Pongsu Seribu 2 (resistant). Among the 6 isolates, isolate number 3 was found to be the most virulent showing high sporulation while isolate number 4 was very slow growing, and the least virulent.

  2. Molecular characterization of Wolbachia infection in bed bugs (Cimex lectularius collected from several localities in France

    Directory of Open Access Journals (Sweden)

    Akhoundi Mohammad

    2016-01-01

    Full Text Available Wolbachia symbionts are maternally inherited intracellular bacteria that have been detected in numerous insects including bed bugs. The objective of this study, the first epidemiological study in Europe, was to screen Wolbachia infection among Cimex lectularius collected in the field, using PCR targeting the surface protein gene (wsp, and to compare obtained Wolbachia strains with those reported from laboratory colonies of C. lectularius as well as other Wolbachia groups. For this purpose, 284 bed bug specimens were caught and studied from eight different regions of France including the suburbs of Paris, Bouches-du-Rhône, Lot-et-Garonne, and five localities in Alpes-Maritimes. Among the samples, 166 were adults and the remaining 118 were considered nymphs. In all, 47 out of 118 nymphs (40% and 61 out of 166 adults (37% were found positive on wsp screening. Among the positive cases, 10 samples were selected randomly for sequencing. The sequences had 100% homology with wsp sequences belonging to the F-supergroup strains of Wolbachia. Therefore, we confirm the similarity of Wolbachia strains detected in this epidemiological study to Wolbachia spp. reported from laboratory colonies of C. lectularius.

  3. Molecular characterization of Wolbachia infection in bed bugs (Cimex lectularius) collected from several localities in France

    Science.gov (United States)

    Akhoundi, Mohammad; Cannet, Arnaud; Loubatier, Céline; Berenger, Jean-Michel; Izri, Arezki; Marty, Pierre; Delaunay, Pascal

    2016-01-01

    Wolbachia symbionts are maternally inherited intracellular bacteria that have been detected in numerous insects including bed bugs. The objective of this study, the first epidemiological study in Europe, was to screen Wolbachia infection among Cimex lectularius collected in the field, using PCR targeting the surface protein gene (wsp), and to compare obtained Wolbachia strains with those reported from laboratory colonies of C. lectularius as well as other Wolbachia groups. For this purpose, 284 bed bug specimens were caught and studied from eight different regions of France including the suburbs of Paris, Bouches-du-Rhône, Lot-et-Garonne, and five localities in Alpes-Maritimes. Among the samples, 166 were adults and the remaining 118 were considered nymphs. In all, 47 out of 118 nymphs (40%) and 61 out of 166 adults (37%) were found positive on wsp screening. Among the positive cases, 10 samples were selected randomly for sequencing. The sequences had 100% homology with wsp sequences belonging to the F-supergroup strains of Wolbachia. Therefore, we confirm the similarity of Wolbachia strains detected in this epidemiological study to Wolbachia spp. reported from laboratory colonies of C. lectularius. PMID:27492563

  4. Production and characterization of violacein by locally isolated Chromobacterium violaceum grown in agricultural wastes.

    Science.gov (United States)

    Ahmad, Wan Azlina; Yusof, Nur Zulaikha; Nordin, Nordiana; Zakaria, Zainul Akmar; Rezali, Mohd Fazlin

    2012-07-01

    The present work highlighted the production of violacein by the locally isolated Chromobacterium violaceum (GenBank accession no. HM132057) in various agricultural waste materials (sugarcane bagasse, solid pineapple waste, molasses, brown sugar), as an alternative to the conventional rich medium. The highest yield for pigment production (0.82 g L⁻¹) was obtained using free cells when grown in 3 g of sugarcane bagasse supplemented with 10% (v/v) of L-tryptophan. A much lower yield (0.15 g L⁻¹) was obtained when the cells were grown either in rich medium (nutrient broth) or immobilized onto sugarcane bagasse. Violacein showed similar chemical properties as other natural pigments based on the UV-Vis, Fourier transform infrared spectroscopy, thin-layer chromatography, nuclear magnetic resonance, and mass spectrometry analysis. The pigment is highly soluble in acetone and methanol, insoluble in water or non-polar organic solvents, and showed good stability between pH 5-9, 25-100 °C, in the presence of light metal ions and oxidant such as H₂O₂. However, violacein would be slowly degraded upon exposure to light. This is the first report on the use of cheap and easily available agricultural wastes as growth medium for violacein-producing C. violaceum.

  5. Characterization of local thermodynamic equilibrium in a laser-induced aluminum alloy plasma.

    Science.gov (United States)

    Zhang, Yong; Zhao, Zhenyang; Xu, Tao; Niu, GuangHui; Liu, Ying; Duan, Yixiang

    2016-04-01

    The electron temperature was evaluated using the line-to-continuum ratio method, and whether the plasma was close to the local thermodynamic equilibrium (LTE) state was investigated in detail. The results showed that approximately 5 μs after the plasma formed, the changes in the electron and excitation temperatures, which were determined using a Boltzmann plot, overlapped in the 15% error range, which indicated that the LTE state was reached. The recombination of electrons and ions and the free electron expansion process led to the deviation from the LTE state. The plasma's expansion rate slowed over time, and when the expansion time was close to the ionization equilibrium time, the LTE state was almost reached. The McWhirter criterion was adopted to calculate the threshold electron density for different species, and the results showed that experimental electron density was greater than the threshold electron density, which meant that the LTE state may have existed. However, for the nonmetal element N, the threshold electron density was greater than the value experimental value approximately 0.8 μs after the plasma formed, which meant that LTE state did not exist for N.

  6. Characterization of Bacillus cereus isolates from local dairy farms in China.

    Science.gov (United States)

    Cui, Yifang; Liu, Xiaoye; Dietrich, Richard; Märtlbauer, Erwin; Cao, Jie; Ding, Shuangyang; Zhu, Kui

    2016-06-01

    Bacillus cereus is an important opportunistic foodborne pathogen. In the present work, a total of 306 milk and environmental samples were collected from 10 local dairy farms in Beijing, China. Of the 92 B. cereus-like isolates, 88 and 4 belonged to B. cereus and B. thuringiensis, respectively. The prevalence of B. cereus isolates in bedding, feces, feed, liquid manure and raw milk was 93.3%, 78.9%, 41.2%, 100.0% and 9.8%, respectively. Three main toxin genes nhe, hbl and ces were detected with rates of 100.0%, 78.3% and 1.1%, but no strain harbored cytK1 The production of Nhe, Hbl and cereulide could be confirmed by specific monoclonal antibodies-based enzyme immunoassays in 94.6%, 70.7% and 1.1% of all isolates, respectively. Cytotoxicity tests were used to further corroborate the results of genetic and protein-based assays; 91.3% of the isolates showed cytotoxicity to Vero cells. All isolates were tested for antimicrobial resistance against 17 antibiotics. All isolates were resistant to lincomycin, retapamulin, tiamulin and valnemulin, while two strains were susceptible to ampicillin and ceftiofur. A total of 16 isolated strains were resistant to tetracycline. Since spores of B. cereus are not inactivated during manufacturing of most milk products, contamination of milk with B. cereus on the farm level may represent a potential hazard, particularly with respect to emetic toxin-producing strains.

  7. The mouse Fau gene: genomic structure, chromosomal localization, and characterization of two retropseudogenes.

    Science.gov (United States)

    Casteels, D; Poirier, C; Guénet, J L; Merregaert, J

    1995-01-01

    The Fau gene is the cellular homolog of the fox sequence of the Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV). FBR-MuSV acquired the Fau gene by transduction in a transcriptional orientation opposite to that of the genomic Fau gene. The genomic structure of the mouse Fau gene (MMFAU) and its upstream elements have been determined and are similar to those of the human FAU gene. The gene consists of five exons and is located on chromosome 19. The first exon is not translated. The promoter region has no well-defined TATA box but contains the polypyrimidine initiator flanked by regions of high GC content (65%) and shows all of the characteristics of a housekeeping gene. The 5' end of the mRNA transcript was determined by 5' RACE analysis and is located, as expected, in the polypyrimidine initiator site. Furthermore, the sequences of two retropseudogenes (Fau-ps1 and Fau-ps2) are reported. Both pseudogenes are approximately 75% identical to the Fau cDNA, but both are shorter due to a deletion at the 5' end and do not encode a functional protein. Fau-prs is interrupted by an AG-rich region of about 350 bp within the S30 region of the Fau cDNA. Fau-ps1 was localized on chromosome 1 and Fau-ps2 on chromosome 7.

  8. Genetic characterization of Kenai brown bears (Ursus arctos): Microsatellite and mitochondrial DNA control region variation in brown bears of the Kenai Peninsula, south central Alaska

    Science.gov (United States)

    Jackson, J.V.; Talbot, S.L.; Farley, S.

    2008-01-01

    We collected data from 20 biparentally inherited microsatellite loci, and nucleotide sequence from the maternally inherited mitochondrial DNA (mtDNA) control region, to determine levels of genetic variation of the brown bears (Ursus arctos L., 1758) of the Kenai Peninsula, south central Alaska. Nuclear genetic variation was similar to that observed in other Alaskan peninsular populations. We detected no significant inbreeding and found no evidence of population substructuring on the Kenai Peninsula. We observed a genetic signature of a bottleneck under the infinite alleles model (IAM), but not under the stepwise mutation model (SMM) or the two-phase model (TPM) of microsatellite mutation. Kenai brown bears have lower levels of mtDNA haplotypic diversity relative to most other brown bear populations in Alaska. ?? 2008 NRC.

  9. Characterization of splice variants of the genes encoding human mitochondrial HMG-CoA lyase and HMG-CoA synthase, the main enzymes of the ketogenesis pathway.

    Science.gov (United States)

    Puisac, Beatriz; Ramos, Mónica; Arnedo, María; Menao, Sebastián; Gil-Rodríguez, María Concepción; Teresa-Rodrigo, María Esperanza; Pié, Angeles; de Karam, Juan Carlos; Wesselink, Jan-Jaap; Giménez, Ignacio; Ramos, Feliciano J; Casals, Nuria; Gómez-Puertas, Paulino; Hegardt, Fausto G; Pié, Juan

    2012-04-01

    The genes HMGCS2 and HMGCL encode the two main enzymes for ketone-body synthesis, mitochondrial HMG-CoA synthase and HMG-CoA lyase. Here, we identify and describe possible splice variants of these genes in human tissues. We detected an alternative transcript of HMGCS2 carrying a deletion of exon 4, and two alternative transcripts of HMGCL with deletions of exons 5 and 6, and exons 5, 6 and 7, respectively. All splice variants maintained the reading frame. However, Western blot studies and overexpression measurements in eukaryotic or prokaryotic cell models did not reveal HL or mHS protein variants. Both genes showed a similar distribution of the inactive variants in different tissues. Surprisingly, the highest percentages were found in tissues where almost no ketone bodies are synthesized: heart, skeletal muscle and brain. Our results suggest that alternative splicing might coordinately block the two main enzymes of ketogenesis in specific human tissues.

  10. Characterizing the relationship between hyperstoichiometry, defect structure and local corrosion kinetics of uranium dioxide

    Energy Technology Data Exchange (ETDEWEB)

    He Heming; Qin, Z. [Department of Chemistry, University of Western Ontario, 1151 Richmond Street, London, Ontario, N6A 5B7 (Canada); Shoesmith, D.W., E-mail: dwshoesm@uwo.c [Department of Chemistry, University of Western Ontario, 1151 Richmond Street, London, Ontario, N6A 5B7 (Canada)

    2010-12-15

    The ability of the UO{sub 2} fluorite structure to accommodate large amounts of interstitial oxygen in various lattice sites leads to the formation of hyper-stoichiometric phases. The defect structures occurring in hyper-stoichiometric UO{sub 2+x} over the range 0.02 {<=} x {<=} 0.1 have been characterized by SEM/EDX and Raman analyses. The results demonstrate that as the nominal stoichiometry increases from 2.002 to 2.1, the diversity of defective structures existing on the UO{sub 2+} surface also increases. Scanning electrochemical microscopy (SECM) measurements combined with a theoretical model were used to determine the rate constant for the reduction of the redox mediator ferrocene methanol, acting as a cathodic oxidant to corrode the four UO{sub 2+x} specimens. The rate constant was found to vary with location on the surface. Stoichiometric locations, with a well defined fluorite structure, exhibited very low corrosion rates. Higher rates were observed at more non-stoichiometric locations with the highest rates being obtained on locations exhibiting tetragonal distortions as their composition approached UO{sub 2.33}. The distribution of rates increases with the degree of nominal non-stoichiometry as the diversity of microstructures existing on the UO{sub 2+x} surface increases.

  11. Local versus field scale soil heterogeneity characterization - a challenge for representative sampling in pollution studies

    Science.gov (United States)

    Kardanpour, Z.; Jacobsen, O. S.; Esbensen, K. H.

    2015-06-01

    This study is a contribution to development of a heterogeneity characterisation facility for "next generation" sampling aimed at more realistic and controllable pesticide variability in laboratory pots in experimental environmental contaminant assessment. The role of soil heterogeneity on quantification of a set of exemplar parameters, organic matter, loss on ignition (LOI), biomass, soil microbiology, MCPA sorption and mineralization is described, including a brief background on how heterogeneity affects sampling/monitoring procedures in environmental pollutant studies. The Theory of Sampling (TOS) and variographic analysis has been applied to develop a fit-for-purpose heterogeneity characterization approach. All parameters were assessed in large-scale profile (1-100 m) vs. small-scale (0.1-1 m) replication sampling pattern. Variographic profiles of experimental analytical results concludes that it is essential to sample at locations with less than a 2.5 m distance interval to benefit from spatial auto-correlation and thereby avoid unnecessary, inflated compositional variation in experimental pots; this range is an inherent characteristic of the soil heterogeneity and will differ among soils types. This study has a significant carrying-over potential for related research areas e.g. soil science, contamination studies, and environmental monitoring and environmental chemistry.

  12. [Mitochondrial and oocyte development].

    Science.gov (United States)

    Deng, Wei-Ping; Ren, Zhao-Rui

    2007-12-01

    Oocyte development and maturation is a complicated process. The nuclear maturation and cytoplasmic maturation must synchronize which can ensure normal oocyte fertilization and following development. Mitochondrial is the most important cellular organell in cytoplasm, and the variation of its distribution during oocyte maturation, the capacity of OXPHOS generating ATP as well as the content or copy number or transcription level of mitochondrial DNA play an important role in oocyte development and maturation. Therefore, the studies on the variation of mitochondrial distribution, function and mitochondrial DNA could enhance our understanding of the physiology of reproduction and provide new insight to solve the difficulties of assisted reproduction as well as cloning embryo technology.

  13. Progress in mitochondrial epigenetics.

    Science.gov (United States)

    Manev, Hari; Dzitoyeva, Svetlana

    2013-08-01

    Mitochondria, intracellular organelles with their own genome, have been shown capable of interacting with epigenetic mechanisms in at least four different ways. First, epigenetic mechanisms that regulate the expression of nuclear genome influence mitochondria by modulating the expression of nuclear-encoded mitochondrial genes. Second, a cell-specific mitochondrial DNA content (copy number) and mitochondrial activity determine the methylation pattern of nuclear genes. Third, mitochondrial DNA variants influence the nuclear gene expression patterns and the nuclear DNA (ncDNA) methylation levels. Fourth and most recent line of evidence indicates that mitochondrial DNA similar to ncDNA also is subject to epigenetic modifications, particularly by the 5-methylcytosine and 5-hydroxymethylcytosine marks. The latter interaction of mitochondria with epigenetics has been termed 'mitochondrial epigenetics'. Here we summarize recent developments in this particular area of epigenetic research. Furthermore, we propose the term 'mitoepigenetics' to include all four above-noted types of interactions between mitochondria and epigenetics, and we suggest a more restricted usage of the term 'mitochondrial epigenetics' for molecular events dealing solely with the intra-mitochondrial epigenetics and the modifications of mitochondrial genome.

  14. Genetic localization of diuron- and mucidin-resistant mutants relative to a group of loci of the mitochondrial DNA controlling coenzyme QH2-cytochrome c reductase in Saccharomyces cerevisiae.

    Science.gov (United States)

    Colson, A M; Slonimski, P P

    1979-01-02

    Diuron-resistance, DIU (Colson et al., 1977), antimycin-resistance, ANA (Michaelis, 1976; Burger et al., 1976), funiculosin-resistance, FUN (Pratje and Michaelis, 1977; Burger et al., 1977) and mucidin-resistance, MUC (Subik et al., 1977) are each coded by a pair of genetic loci on the mit DNA of S. cerevisiae. In the present paper, these respiratiory-competent, drug-resistant loci are localized relative to respiratory-deficient BOX mutants deficient in coenzyme QH2-cytochrome c reductase (Kotylak and Slonimski, 1976, 1977) using deletion and recombination mapping. Three drug-resistant loci possessing distinct mutated allelic forms are distinguished. DIU1 is allelic or closely linked to ANA2, FUN1 and BOX1; DIU2 is allelic or closely linked to ANA1, MUC1 and BOX4/5; MUC2 is allelic to BOX6. The high recombinant frequencies observed between the three loci (13% on the average for 33 various combinations analyzed) suggest the existence of either three genes coding for three distinct polypeptides or of a single gene coding for a single polypeptide but subdivided into three easily separable segments. The resistance of the respiratory-chain observed in vitro in the drug-resistant mutants and the allelism relationships between respiratory-competent, drug-resistant loci and coQH2-cyt c reductase deficient, BOX, loci strongly suggest that each of the three drug-resistant loci codes for a structural gene-product which is essential for the normal coQH2-cyt c reductase activity and is obviously a good candidate for a gene product of the drug-resistant loci mapped in this paper. Polypeptide length modifications of cytochrome b were observed in mutants deficient in the coQH2-cyt c red and localized at the BOX1, BOX4 and BOX6 genetic loci (Claisse et al., 1977, 1978) which are precisely the loci allelic to drug resistant mutants as shown in the present work. Taken together these two sets of data provide a strong evidence in favor of the idea that there exist three non contiguous

  15. Proteomic Dissection of the Mitochondrial DNA Metabolism Apparatus in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    SAlly A. Mackenzie

    2004-01-06

    This study involves the investigation of nuclear genetic components that regulate mitochondrial genome behavior in higher plants. The approach utilizes the advanced plant model system of Arabidopsis thaliana to identify and functionally characterize multiple components of the mitochondrial DNA replication, recombination and mismatch repair system and their interaction partners. The rationale for the research stems from the central importance of mitochondria to overall cellular metabolism and the essential nature of the mitochondrial genome to mitochondrial function. Relatively little is understood about mitochondrial DNA maintenance and transmission in higher eukaryotes, and the higher plant mitochondrial genome displays unique properties and behavior. This investigation has revealed at least three important properties of plant mitochondrial DNA metabolism components. (1) Many are dual targeted to mitochondrial and chloroplasts by novel mechanisms, suggesting that the mitochondria a nd chloroplast share their genome maintenance apparatus. (2)The MSH1 gene, originating as a component of mismatch repair, has evolved uniquely in plants to participate in differential replication of the mitochondrial genome. (3) This mitochondrial differential replication process, termed substoichiometric shifting and also involving a RecA-related gene, appears to represent an adaptive mechanism to expand plant reproductive capacity and is likely present throughout the plant kingdom.

  16. A Biophysical Model of the Mitochondrial ATP-Mg/Pi Carrier

    OpenAIRE

    2012-01-01

    Mitochondrial adenine nucleotide (AdN) content is regulated through the Ca2+-activated, electroneutral ATP-Mg/Pi carrier (APC). The APC is a protein in the mitochondrial carrier super family that localizes to the inner mitochondrial membrane (IMM). It is known to modulate a number of processes that depend on mitochondrial AdN content, such as gluconeogenesis, protein synthesis, and citrulline synthesis. Despite this critical role, a kinetic model of the underlying mechanism has not been devel...

  17. Mitochondrial oxidative stress causes hyperphosphorylation of tau.

    Directory of Open Access Journals (Sweden)

    Simon Melov

    Full Text Available Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD: tau phosphorylation, and beta-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2 die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a well-characterized mouse model of AD (Tg2576 with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Ass load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD.

  18. Mitochondrial oxidative stress causes hyperphosphorylation of tau.

    Science.gov (United States)

    Melov, Simon; Adlard, Paul A; Morten, Karl; Johnson, Felicity; Golden, Tamara R; Hinerfeld, Doug; Schilling, Birgit; Mavros, Christine; Masters, Colin L; Volitakis, Irene; Li, Qiao-Xin; Laughton, Katrina; Hubbard, Alan; Cherny, Robert A; Gibson, Brad; Bush, Ashley I

    2007-06-20

    Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD): tau phosphorylation, and beta-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2) die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau) in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a well-characterized mouse model of AD (Tg2576) with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Ass load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD.

  19. Use of stationary focused ultrasound fields for characterization of tissue and localized tissue ablation

    Science.gov (United States)

    Winey, Brian Andrew

    Ultrasound-induced blood stasis has been observed for more than 30 years. The physical understanding of the phenomenon has not been fully explored. Analytical descriptions of the acoustic interaction with spheres in suspension have been derived but the physical implications and limitations have not been demonstrated. The analytical expressions will be tested against physical observations using numerical simulations. The simulations will begin with stationary spheres and continue with the inclusion of moving spheres and a moving suspending fluid. To date, experimental observations of acoustically induced blood stasis have been either in vitro or invasive. We demonstrate ultrasound-induced blood stasis in murine normal leg muscle versus tumor-bearing legs, observed through noninvasive measurements of optical spectroscopy, and discuss possible diagnostic uses for this effect of ultrasound. We derive the optimal optical wavelengths for measuring the effects of the ultrasound at small source detector separations. Using optical oximetry performed at the optimal wavelengths, we demonstrate that effects of ultrasound can be used to differentiate tumor from normal leg muscle tissue in mice. To provide a statistical analysis of the experiments, we propose a novel diagnostic algorithm that quantitatively differentiates tumor from nontumor with maximum specificity 0.83, maximum sensitivity 0.79, and area under receiver-operating-characteristics curve 0.90. Ultrasound has long been known to cause tissue heating when applied in high intensities. More recently, interest has arisen in the area of High Intensity Focused Ultrasound (HIFU) for localized tissue heating effects, specifically thermal ablation. All present techniques employ focused traveling high intensity acoustic waves to create a region of elevated temperature. Such high intensity traveling waves can be damaging to normal tissue in the vicinity of the focal region, and have demonstrated surface burns and caused

  20. Petrological and Geochemical characterization of central Chihuahua basalts: a possible local sign of rifting activity

    Science.gov (United States)

    Espejel-Garcia, V. V.; Garcia-Rascon, M.; Villalobos-Aragon, A.; Morton-Bermea, O.

    2012-12-01

    Gusano and Rancho El Milagro, report an age of 28.7 ± 0.6 and 28.4 Ma respectively, but it is possible that the basalts of La Guajolota are younger, for being considered as small local eruptions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kobashigawa, Shinko, E-mail: kobashin@nagasaki-u.ac.jp [Atomic Bomb Disease Institute, Course of Life Sciences and Radiation Research, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Suzuki, Keiji; Yamashita, Shunichi [Atomic Bomb Disease Institute, Course of Life Sciences and Radiation Research, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer We report first time that ionizing radiation induces mitochondrial dynamic changes. Black-Right-Pointing-Pointer Radiation-induced mitochondrial fission was caused by Drp1 localization. Black-Right-Pointing-Pointer We found that radiation causes delayed ROS from mitochondria. Black-Right-Pointing-Pointer 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 O{sub 2}{sup {center_dot}-} 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 O{sub 2}{sup {center_dot}-}. 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.

  2. Comparing Perception-Based and Geographic Information System (GIS)-based characterizations of the local food environment.

    Science.gov (United States)

    Moore, Latetia V; Diez Roux, Ana V; Brines, Shannon

    2008-03-01

    Measuring features of the local food environment has been a major challenge in studying the effect of the environment on diet. This study examined associations between alternate ways of characterizing the local food environment by comparing Geographic Information System (GIS)-derived densities of various types of stores to perception-based measures of the availability of healthy foods. Survey questions rating the availability of produce and low-fat products in neighborhoods were aggregated into a healthy food availability score for 5,774 residents of North Carolina, Maryland, and New York. Densities of supermarkets and smaller stores per square mile were computed for 1 mile around each respondent's residence using kernel estimation. The number of different store types in the area was used to measure variety in the food environment. Linear regression was used to examine associations of store densities and variety with reported availability. Respondents living in areas with lower densities of supermarkets rated the selection and availability of produce and low-fat foods 17% lower than those in areas with the highest densities of supermarkets (95% CL, -18.8, -15.1). In areas without supermarkets, low densities of smaller stores and less store variety were associated with worse perceived availability of healthy foods only in North Carolina (8.8% lower availability, 95% CL, -13.8, -3.4 for lowest vs. highest small-store density; 10.5% lower 95% CL, -16.0, -4.7 for least vs. most store variety). In contrast, higher smaller store densities and more variety were associated with worse perceived healthy food availability in Maryland. Perception- and GIS-based characterizations of the environment are associated but are not identical. Combinations of different types of measures may yield more valid measures of the environment.

  3. Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite.

    Directory of Open Access Journals (Sweden)

    Rajesh Prasad

    Full Text Available Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4. Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5, suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3 to moderate (KP4 preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease

  4. Characterization of plasma jet ejected from a parallel-plate rail gun for simulating edge localized mode

    Energy Technology Data Exchange (ETDEWEB)

    Chung, K.S., E-mail: alsk3@snu.ac.kr; Chung, Kyoung-Jae, E-mail: jkjlsh1@snu.ac.kr; Jung, B.K.; Hwang, Y.S., E-mail: yhwang@snu.ac.kr

    2013-10-15

    Highlights: • A small plasma gun is constructed to study edge localized mode. • A plasma jet ejected from the gun is characterized with a quadruple Langmuir probe. • The device and diagnostics are suitable for research about the control of plasma jet. -- Abstract: A small plasma gun with parallel-plate configuration is fabricated to generate a bunch of plasma which is similar to ELM (edge localized mode) plasma, by taking advantages of its simplicity and cost-effectiveness. Prior to explore how to control the ELM-like plasma so as to relieve heat load on the divertor target, characteristics of a plasma jet ejected from the plasma gun are investigated using a quadruple Langmuir probe which is appropriate for measuring rapidly varying plasma parameters such as electron density, temperature, and ion velocity at the same time. The plasma density and ion velocity measured at 112 mm away from the exit are 3 × 10{sup 19} m{sup −3} and 11 km/s, respectively, which seem to be suitable for investigating next step research on the control of ELM-like plasma using various methods such as electromagnetic waves and high-voltage pulses. Also, the quadruple Langmuir probe is proven to be adequate for use in such experiments.

  5. Characterization of local hydrophobicity on sapphire (0001) surfaces in aqueous environment by colloidal probe atomic force microscopy

    Science.gov (United States)

    Wada, Tomoya; Yamazaki, Kenji; Isono, Toshinari; Ogino, Toshio

    2017-02-01

    Sapphire (0001) surfaces exhibit a phase-separation into hydrophobic and hydrophilic domains upon high-temperature annealing, which were previously distinguished by the thickness of adsorbed water layers in air using atomic force microscopy (AFM). To characterize their local surface hydrophobicity in aqueous environment, we used AFM equipped with a colloidal probe and measured the local adhesive force between each sapphire domain and a hydrophilic SiO2 probe surface, or a hydrophobic polystyrene one. Two data acquisition modes for statistical analyses were used: one is force measurements at different positions of the surface and the other repeated measurement at a fixed position. We found that adhesive force measurements using the polystyrene probe allow us to distinctly separate the hydrophilic and hydrophobic domains. The dispersion in the force measurement data at different positions of the surface is larger than that in the repeated measurements at a fixed position. It indicates that the adhesive force measurement is repeatable although their data dispersion for the measurement positions is relatively large. From these results, we can conclude that the hydrophilic and hydrophobic domains on the sapphire (0001) surfaces are distinguished by a difference in their hydration degrees.

  6. Functional characterization, localization, and inhibitor sensitivity of the TPR-FGFR1 fusion in 8p11 myeloproliferative syndrome.

    Science.gov (United States)

    Malli, Theodora; Buxhofer-Ausch, Veronika; Rammer, Melanie; Erdel, Martin; Kranewitter, Wolfgang; Rumpold, Holger; Marschon, Renate; Deutschbauer, Sabine; Simonitsch-Klupp, Ingrid; Valent, Peter; Muellner-Ammer, Kirsten; Sebesta, Christian; Birkner, Thomas; Webersinke, Gerald

    2016-01-01

    Myeloid and lymphoid neoplasms with fibroblast growth factor receptor 1 (FGFR1) abnormalities, also known as 8p11 myeloproliferative syndrome (EMS), represent rare and aggressive disorders, associated with chromosomal aberrations that lead to the fusion of FGFR1 to different partner genes. We report on a third patient with a fusion of the translocated promoter region (TPR) gene, a component of the nuclear pore complex, to FGFR1 due to a novel ins(1;8)(q25;p11p23). The fact that this fusion is a rare but recurrent event in EMS prompted us to examine the localization and transforming potential of the chimeric protein. TPR-FGFR1 localizes in the cytoplasm, although the nuclear pore localization signal of TPR is retained in the fusion protein. Furthermore, TPR-FGFR1 enables cytokine-independent survival, proliferation, and granulocytic differentiation of the interleukin-3 dependent myeloid progenitor cell line 32Dcl3, reflecting the chronic phase of EMS characterized by myeloid hyperplasia. 32Dcl3 cells transformed with the TPR-FGFR1 fusion and treated with increasing concentrations of the tyrosine kinase inhibitors ponatinib (AP24534) and infigratinib (NVP-BGJ398) displayed reduced survival and proliferation with IC50 values of 49.8 and 7.7 nM, respectively. Ponatinib, a multitargeted tyrosine kinase inhibitor, is already shown to be effective against several FGFR1-fusion kinases. Infigratinib, tested only against FGFR1OP2-FGFR1 to date, is also efficient against TPR-FGFR1. Taking its high specificity for FGFRs into account, infigratinib could be beneficial for EMS patients and should be further investigated for the treatment of myeloproliferative neoplasms with FGFR1 abnormalities.

  7. Characterization of Extracellular Penicilin G Acylase Produced by A New Local Strain of Bacillus subtilis BAC4

    Directory of Open Access Journals (Sweden)

    SUPARTONO

    2008-06-01

    Full Text Available Penicillin G acylase (PGA which catalyses penicillin G hydrolysis reaction is a key enzyme for the industrial production of penicilin G derivatives used in therapeutics. A new local strain of Bacillus subtilis BAC4 was found capable of producing extracellular PGA. However, characteristics of this extracellular PGA are not known. The goal of this research was to characterize the extracellular PGA produced by B. subtilis BAC4. Enzyme production was carried out by batch fermentation, followed by enzyme purification and characterization of the PGA. The PGA activity was determined by the Kornfeld method, with optimal activity for hydrolysing penicillin G observed at 43 oC and pH 8.5. The activation energy of penicillin G hydrolysis by the PGA of B. subtilis BAC4 was determined as 4.9 kcal.mol-1 and Vmax and Km values were found to be 0.7 µmole.min-1.mg-1 and 3.5 mM respectively. PGA catalytic activity was competitively inhibited by phenylacetic acid with an inhibition constant, Ki(PAA, of 347.2 mM. It was concluded that the extracellular PGA of B. subtilis BAC4 can hydrolyse penicillin G efficiently.

  8. Nanoscale characterization of local structures and defects in photonic crystals using synchrotron-based transmission soft X-ray microscopy

    Science.gov (United States)

    Nho, Hyun Woo; Kalegowda, Yogesh; Shin, Hyun-Joon; Yoon, Tae Hyun

    2016-04-01

    For the structural characterization of the polystyrene (PS)-based photonic crystals (PCs), fast and direct imaging capabilities of full field transmission X-ray microscopy (TXM) were demonstrated at soft X-ray energy. PS-based PCs were prepared on an O2-plasma treated Si3N4 window and their local structures and defects were investigated using this label-free TXM technique with an image acquisition speed of ~10 sec/frame and marginal radiation damage. Micro-domains of face-centered cubic (FCC (111)) and hexagonal close-packed (HCP (0001)) structures were dominantly found in PS-based PCs, while point and line defects, FCC (100), and 12-fold symmetry structures were also identified as minor components. Additionally, in situ observation capability for hydrated samples and 3D tomographic reconstruction of TXM images were also demonstrated. This soft X-ray full field TXM technique with faster image acquisition speed, in situ observation, and 3D tomography capability can be complementally used with the other X-ray microscopic techniques (i.e., scanning transmission X-ray microscopy, STXM) as well as conventional characterization methods (e.g., electron microscopic and optical/fluorescence microscopic techniques) for clearer structure identification of self-assembled PCs and better understanding of the relationship between their structures and resultant optical properties.

  9. Molecular characterization and genetic diversity in an avocado collection of cultivars and local Spanish genotypes using SSRs.

    Science.gov (United States)

    Alcaraz, M L; Hormaza, J I

    2007-12-01

    In this work, 75 avocado accessions maintained in an ex situ germplasm collection at the E.E. la Mayora in Málaga (Spain) were characterized with 16 microsatellites previously developed in this species. This avocado collection includes both local Spanish genotypes obtained through prospection and genotypes obtained by exchange with different countries. A total of 156 different amplification fragments were detected ranging from 4 to 16 per locus with an average of 9.75 alleles per locus. All the microsatellites were highly informative with an expected heterozygosity higher than 0.5 and a probability of identity below 0.36. The total probability of identity was 2.85x10(-14). Fifteen of the 16 loci studied showed a positive Wright's fixation index (F) indicating a deficit of heterozygotes with an average over all the SSRs of 0.18. A dendrogram was generated using UPGMA (unweighted pair group method with arithmetic averages) based on the Nei and Li similarity index. This dendrogram classified most of the genotypes analyzed into three major groups which mainly differed in racial origin although with low bootstrap support probably due to the presence of many interracial hybrids in the collection. All the genotypes studied could be unequivocally distinguished with the combination of SSRs used except some putative mutations of 'Hass' and an additional group of two cultivars. The results obtained indicate that the set of SSRs used is highly informative and are discussed in terms of their implications for avocado germplasm characterization and management.

  10. Vaginal inserts based on chitosan and carboxymethylcellulose complexes for local delivery of chlorhexidine: preparation, characterization and antimicrobial activity.

    Science.gov (United States)

    Bigucci, Federica; Abruzzo, Angela; Vitali, Beatrice; Saladini, Bruno; Cerchiara, Teresa; Gallucci, Maria Caterina; Luppi, Barbara

    2015-01-30

    The aim of this work was to prepare vaginal inserts based on chitosan/carboxymethylcellulose polyelectrolyte complexes for local delivery of chlorhexidine digluconate. Complexes were prepared with different chitosan/carboxymethylcellulose molar ratios at a pH value close to pKa interval of the polymers and were characterized in terms of physico-chemical properties, complexation yield and drug loading. Then complexes were used to prepare inserts as vaginal dosage forms and their physical handling, morphology, water-uptake ability and drug release properties as well as antimicrobial activity toward Candida albicans and Escherichia coli were evaluated. Results confirmed the ionic interaction between chitosan and carboxymethylcellulose and the influence of the charge amount on the complexation yield. Complexes were characterized by high values of drug loading and showed increasing water-uptake ability with the increase of carboxymethylcellulose amount. The selection of appropriate chitosan/carboxymethylcellulose molar ratios allowed to obtain cone-like shaped solid inserts, easy to handle and able to hydrate releasing the drug over time. Finally, the formulated inserts showed antimicrobial activity against common pathogens responsible for vaginal infections.

  11. Nanoscale characterization of local structures and defects in photonic crystals using synchrotron-based transmission soft X-ray microscopy

    Science.gov (United States)

    Nho, Hyun Woo; Kalegowda, Yogesh; Shin, Hyun-Joon; Yoon, Tae Hyun

    2016-01-01

    For the structural characterization of the polystyrene (PS)-based photonic crystals (PCs), fast and direct imaging capabilities of full field transmission X-ray microscopy (TXM) were demonstrated at soft X-ray energy. PS-based PCs were prepared on an O2-plasma treated Si3N4 window and their local structures and defects were investigated using this label-free TXM technique with an image acquisition speed of ~10 sec/frame and marginal radiation damage. Micro-domains of face-centered cubic (FCC (111)) and hexagonal close-packed (HCP (0001)) structures were dominantly found in PS-based PCs, while point and line defects, FCC (100), and 12-fold symmetry structures were also identified as minor components. Additionally, in situ observation capability for hydrated samples and 3D tomographic reconstruction of TXM images were also demonstrated. This soft X-ray full field TXM technique with faster image acquisition speed, in situ observation, and 3D tomography capability can be complementally used with the other X-ray microscopic techniques (i.e., scanning transmission X-ray microscopy, STXM) as well as conventional characterization methods (e.g., electron microscopic and optical/fluorescence microscopic techniques) for clearer structure identification of self-assembled PCs and better understanding of the relationship between their structures and resultant optical properties. PMID:27087141

  12. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    Energy Technology Data Exchange (ETDEWEB)

    Bhaskar,; Kumari, Neeti [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India); Goyal, Neena, E-mail: neenacdri@yahoo.com [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  13. Characterization of a Leber's hereditary optic neuropathy (LHON) family harboring two primary LHON mutations m.11778G>A and m.14484T>C of the mitochondrial DNA.

    Science.gov (United States)

    Catarino, Claudia B; Ahting, Uwe; Gusic, Mirjana; Iuso, Arcangela; Repp, Birgit; Peters, Katrin; Biskup, Saskia; von Livonius, Bettina; Prokisch, Holger; Klopstock, Thomas

    2016-10-06

    Leber's hereditary optic neuropathy (LHON) is an inherited mitochondrial disease that usually leads to acute or subacute bilateral central vision loss. In 95% of cases, LHON is caused by one of three primary mutations of the mitochondrial DNA (mtDNA), m.11778G>A in the MT-ND4 gene, m.14484T>C in the MT-ND6 gene, or m.3460G>A in the MT-ND1 gene. Here we characterize clinically, genetically, and biochemically a LHON family with multiple patients harboring two of these primary LHON mutations, m.11778G>A homoplasmic and m.14484T>C heteroplasmic. The unusually low male-to-female ratio of affected family members is also seen among the other patients previously reported with two primary LHON mutations m.11778G>A and m.14484T>C. While the index patient had very late onset of symptoms at 75years and severe visual loss, her two daughters had both onset in childhood (6 and 9years), with moderate to mild visual loss. A higher degree of heteroplasmy of the m.14484T>C mutation was found to correlate with an earlier age at onset in this family. Ours is the first LHON family harboring two primary LHON mutations where functional studies were performed in several affected family members. A more pronounced bioenergetic defect was found to correlate with an earlier age at onset. The patient with the earliest age at onset had a more significant complex I dysfunction than all controls, including the LHON patient with only the m.11778G>A mutation, suggesting a synergistic effect of the two primary LHON mutations in this patient.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Ya-Wen eLu

    2015-02-01

    Full Text Available The human nuclear and mitochondrial genomes co-exist within each cell. While the mitochondrial genome encodes for a limited number of proteins, transfer RNAs, and ribosomal RNAs, the vast majority of mitochondrial proteins are encoded in the nuclear genome. Of the multitude of mitochondrial disorders known to date, only a fifth are maternally inherited. The recent characterization of the mitochondrial proteome therefore serves as an important step towards delineating the nosology of a large spectrum of phenotypically heterogeneous diseases. Following the identification of the first nuclear gene defect to underlie a mitochondrial disorder, a plenitude of genetic variants that provoke mitochondrial pathophysiology have been molecularly elucidated and classified into six categories that impact: 1 oxidative phosphorylation (subunits and assembly factors; 2 mitochondrial DNA maintenance and expression; 3 mitochondrial protein import and assembly; 4 mitochondrial quality control (chaperones and proteases; 5 iron-sulfur cluster homeostasis; and 6 mitochondrial dynamics (fission and fusion. Here, we propose that an additional class of genetic variant be included in the classification schema to acknowledge the role of genetic defects in phospholipid biosynthesis, remodeling, and metabolism in mitochondrial pathophysiology. This seventh class includes a small but notable group of nuclear-encoded proteins whose dysfunction impacts normal mitochondrial phospholipid metabolism. The resulting human disorders present with a diverse array of pathologic consequences that reflect the variety of functions that phospholipids have in mitochondria and highlight the important role of proper membrane homeostasis in mitochondrial biology.

  16. 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.

  17. 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.

  18. Localized environment characterization device

    KAUST Repository

    Alzain, Hashim

    2016-07-21

    Various apparatuses and methods are provided for measuring the likely environmental impact of a particular geographic location on power generation properties of potential solar installations at the particular location. In an example embodiment of one such apparatus, a measurement device is provided. The measurement device includes a base portion comprising a base frame element disposed on a plurality of supporting legs, and a top panel comprising a series of connected members and one or more measurement modules whose planar dimensions are defined by the series of connected members. The top panel is connected to the base portion by a joint such that the top panel can rotate about the joint, and a panel support element is configured to fasten the top panel immovably at a desired degree of rotation in relation to the base portion.

  19. Mitochondrially targeted fluorescent redox sensors.

    Science.gov (United States)

    Yang, Kylie; Kolanowski, Jacek L; New, Elizabeth J

    2017-04-06

    The balance of oxidants and antioxidants within the cell is crucial for maintaining health, and regulating physiological processes such as signalling. Consequently, imbalances between oxidants and antioxidants are now understood to lead to oxidative stress, a physiological feature that underlies many diseases. These processes have spurred the field of chemical biology to develop a plethora of sensors, both small-molecule and fluorescent protein-based, for the detection of specific oxidizing species and general redox balances within cells. The mitochondrion, in particular, is the site of many vital redox reactions. There is therefore a need to target redox sensors to this particular organelle. It has been well established that targeting mitochondria can be achieved by the use of a lipophilic cation-targeting group, or by utilizing natural peptidic mitochondrial localization sequences. Here, we review how these two approaches have been used by a number of researchers to develop mitochondrially localized fluorescent redox sensors that are already proving useful in providing insights into the roles of reactive oxygen species in the mitochondria.

  20. Sheet resistance characterization of locally anisotropic transparent conductive films made of aligned metal-enriched single-walled carbon nanotubes.

    Science.gov (United States)

    Kang, Hosung; Kim, Duckjong; Baik, Seunghyun

    2014-09-21

    One-dimensional conductive fillers such as single-walled carbon nanotubes (SWNTs) can be aggregated and aligned during transparent conductive film (TCF) formation by the vacuum filtration method. The potential error of analysing the average sheet resistance of these anisotropic films, using the four-point probe in-line method and the conversion formula developed assuming uniform isotropic material properties, was systematically investigated by finite element analysis and experiments. The finite element analysis of anisotropic stripe-patterned TCFs with alternating low (ρ1) and high (ρ2) resistivities revealed that the estimated average sheet resistance approached ρ1/t when the probes were parallel to the aligned nanotubes. The thickness of the film is t. It was more close to ρ2/t when the probes were perpendicular to the aligned tubes. Indeed, TCFs fabricated by the vacuum filtration method using metal-enriched SWNTs exhibited highly anisotropic local regions where tubes were aggregated and aligned. The local sheet resistances of randomly oriented, aligned, and perpendicular tube regions of the TCF at a transmittance of 89.9% were 5000, 2.4, and 12 300 Ω □(-1), respectively. Resistivities of the aggregated and aligned tube region (ρ1 = 1.2 × 10(-5) Ω cm) and the region between tubes (ρ2 = 6.2 × 10(-2) Ω cm) could be approximated with the aid of finite element analysis. This work demonstrates the potential error of characterizing the average sheet resistance of anisotropic TCFs using the four-point probe in-line method since surprisingly high or low values could be obtained depending on the measurement angle. On the other hand, a better control of aggregation and alignment of nanotubes would realize TCFs with a very small anisotropic resistivity and a high transparency.

  1. Olefin epoxidation by molybdenum peroxo compound: molecular mechanism characterized by the electron localization function and catastrophe theory.

    Science.gov (United States)

    Berski, Slawomir; Sensato, Fabrício R; Polo, Victor; Andrés, Juan; Safont, V S

    2011-02-03

    The oxygen atom transfer reaction from the Mimoun-type complex MoO(η(2)-O(2))(2)OPH(3) to ethylene C(2)H(4) affording oxirane C(2)H(4)O has been investigated within the framework of the Bonding Evolution Theory in which the corresponding molecular mechanism is characterized by the topological analysis of the electron localization function (ELF) and Thom's catastrophe theory (CT). Topological analysis of ELF and electron density analysis reveals that all Mo-O bonds in MoO(η(2)-O(2))(2)OPH(3) and MoO(2)(η(2)-O(2))OPH(3) belong to closed-shell type interactions though negative values of total energy densities E(e)(r(BCP)) imply some covalent contribution. The peroxo O(i)-O(j) bonds are characterized as charge-shift or protocovalent species in which pairs of monosynaptic basins V(3)(O(i)), V(3)(O(j)) with a small electron population of ~0.25e each, are localized between core basins C(O(i)), C(O(j)). The oxygen transfer reaction from molybdenum diperoxo complex MoO(η(2)-O(2))(2)OPH(3) to C(2)H(4) system can be described by the following consecutive chemical events: (a) protocovalent peroxo O(2)-O(1) bond breaking, (b) reduction of the double C(1)=C(2) bond to single C(1)-C(2) bond in ethylene, (c) displacement of oxygen O(1) with two nonbonding basins, V(i=1,2)(O(1)), (d) increase of a number of the nonbonding basins to three (V(i=1,2,4)(O(1))); (e) reorganization and reduction in the number of nonbonding basis to two basins (V(i=1,4)(O(1))) resembling the ELF-topology of the nonbonding electron density in oxirane, (e) formation of the first O(1)-C(2) bond in oxirane, (f) C(2)-O(1)-C(2) ring closure, (g) formation of singular nonbonding basin V(O(2)) in new Mo=O(2) bond. The oxygen atom is transferred as an anionic moiety carrying a rather small electronic charge ranging from 0.5 to 0.7e.

  2. Infantile encephalopathy and defective mitochondrial DNA translation in patients with mutations of mitochondrial elongation factors EFG1 and EFTu.

    Science.gov (United States)

    Valente, Lucia; Tiranti, Valeria; Marsano, Rene Massimiliano; Malfatti, Edoardo; Fernandez-Vizarra, Erika; Donnini, Claudia; Mereghetti, Paolo; De Gioia, Luca; Burlina, Alberto; Castellan, Claudio; Comi, Giacomo P; Savasta, Salvatore; Ferrero, Iliana; Zeviani, Massimo

    2007-01-01

    Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)-encoded RNAs and nuclear DNA-encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis.

  3. Infantile Encephalopathy and Defective Mitochondrial DNA Translation in Patients with Mutations of Mitochondrial Elongation Factors EFG1 and EFTu

    Science.gov (United States)

    Valente, Lucia; Tiranti, Valeria; Marsano, René Massimiliano; Malfatti, Edoardo; Fernandez-Vizarra, Erika; Donnini, Claudia; Mereghetti, Paolo; De Gioia, Luca; Burlina, Alberto; Castellan, Claudio; Comi, Giacomo P.; Savasta, Salvatore; Ferrero, Iliana; Zeviani, Massimo

    2007-01-01

    Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)–encoded RNAs and nuclear DNA–encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis. PMID:17160893

  4. Highly selective luminescent nanostructures for mitochondrial imaging and targeting

    Science.gov (United States)

    Fanizza, E.; Iacobazzi, R. M.; Laquintana, V.; Valente, G.; Caliandro, G.; Striccoli, M.; Agostiano, A.; Cutrignelli, A.; Lopedota, A.; Curri, M. L.; Franco, M.; Depalo, N.; Denora, N.

    2016-02-01

    Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino functionalized luminescent silica coated QD nanoparticles (QD@SiO2 NPs) provides a versatile nanoplatform to anchor a potent and selective TSPO ligand, characterized by a 2-phenyl-imidazo[1,2-a]pyridine acetamide structure along with a derivatizable carboxylic end group, useful to conjugate the TSPO ligand and achieve TSPO-QD@SiO2 NPs by means of a covalent amide bond. The colloidal stability and optical properties of the proposed nanomaterials are comprehensively investigated and their potential as mitochondrial imaging agents is fully assessed. Sub-cellular fractionation, together with confocal laser scanning fluorescence microscopy and co-localization analysis of targeted TSPO-QD@SiO2 NPs in C6 glioma cells overexpressing the TSPO, proves the great potential of these multifunctional nanosystems as in vitro selective mitochondrial imaging agents.Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino

  5. Cathodoluminescence characterization of quartz grains from the Upper Cretaceous of dinosaur fossil localities in the Gobi desert, Mongolia

    Science.gov (United States)

    Saneyoshi, M.; Nishido, H.; Masuda, R.; Tsogtbaatar, K.; Chinzorig, T.

    2013-12-01

    The Upper Cretaceous eolian sediments in Mongolia's Gobi desert are one of the most important occurrences of the dinosaurs in the world. Large numbers of confiscated dinosaur fossils illegally worked out by poachers has been stored in the Mongolian Paleontological Center at Ulaanbaatar. In most cases, their localities are unknown. The purpose of this study is to identify their localities by cathodoluminescence (CL) features of quartz grains attached to the dinosaur specimens by comparing to the quartz samples collected from the sediments of circumjacent resources in this area. This study focuses on the confiscated specimen which makes up the nest with the babies' Protoceratops. Most of all Protoceratops in every growth process, have been discovered from the Djadokhta Formation in the Gobi desert. This formation crops out at Tugrikin Shireh and Bayn Dzak in the central part of the Gobi desert, and is derived from medium- to fine-grained sand mainly composed of quartz grains, of which sedimentary environments should be obvious to be eolian. The formation age of the sand beds at Tugrikin Shireh and Bayn Dzak has been estimated to be Middle Campanian. CL spectra of quartz have been demonstrated to show different features between the quartz from hydrothermal, plutonic, volcanic and metamorphic origins, suggesting the spectra reflect the condition of the quartz formation and the local environment. Therefore, we have applied the CL characterization of quartz grains to the evaluation of the provenance of the desert sediments. The quartz grains after sieving (#60-80 mesh size) were embedded in the brass holders with non-luminescent epoxy resin, and their surfaces were polished with 1 μm diamond abrasive. Color CL images obtained by the Luminoscope exhibit blue, violet and red emissions in the grains, suggesting various types of emission centers in the quartz. SEM-CL analysis was conducted using an SEM (JSM-5410) combined with a grating monochromator (Mono CL2) to measure

  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. 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.

  8. Ubiquitination of specific mitochondrial matrix proteins.

    Science.gov (United States)

    Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G; Ciechanover, Aaron

    2016-06-17

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems - at least partially - in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins.

  9. Mitochondrial genome instability in colorectal adenoma and adenocarcinoma.

    Science.gov (United States)

    de Araujo, Luiza F; Fonseca, Aline S; Muys, Bruna R; Plaça, Jessica R; Bueno, Rafaela B L; Lorenzi, Julio C C; Santos, Anemari R D; Molfetta, Greice A; Zanette, Dalila L; Souza, Jorge E S; Valente, Valeria; Silva, Wilson A

    2015-11-01

    Mitochondrial dysfunction is regarded as a hallmark of cancer progression. In the current study, we evaluated mitochondrial genome instability and copy number in colorectal cancer using Next Generation Sequencing approach and qPCR, respectively. The results revealed higher levels of heteroplasmy and depletion of the relative mtDNA copy number in colorectal adenocarcinoma. Adenocarcinoma samples also presented an increased number of mutations in nuclear genes encoding proteins which functions are related with mitochondria fusion, fission and localization. Moreover, we found a set of mitochondrial and nuclear genes, which cooperate in the same mitochondrial function simultaneously mutated in adenocarcinoma. In summary, these results support an important role for mitochondrial function and genomic instability in colorectal tumorigenesis.

  10. Molecular cloning, subcellular localization and characterization of two adenylate kinases from cassava, Manihot esculenta Crantz cv. KU50.

    Science.gov (United States)

    Boonrueng, Channarong; Tangpranomkorn, Surachat; Yazhisai, Uthaman; Sirikantaramas, Supaart

    2016-10-01

    Adenylate kinase (ADK) is a phosphotransferase that plays an important role in cellular energy homeostasis. Many isozymes located in different subcellular compartments have been reported. In this study, we focus on the characterization of cassava (Manihot esculenta) ADKs. We found 15 ADKs that are publicly available in the African cassava genome database. We cloned two ADKs, namely MeADK1 and MeADK2, which are phylogenetically grouped together with the plastidial ADK in potato. Both MeADK1 and MeADK2 showed 66% identity in the amino acid sequences with plastidial ADK in potato. However, we demonstrated that they are localized to mitochondria using GFP fusions of MeADK1 and MeADK2. The Escherichia coli-produced recombinant MeADK1 and MeADK2 preferred forward reactions that produce ATP. They exhibited similar specific activities. The semi-quantitative RT-PCR analysis showed that MeADK1 and MeADK2 in 2-month-old leaves have similar expression patterns under a diurnal light-dark cycle. However, MeADK2 transcripts were expressed at much higher levels than MeADK1 in 5-month-old leaves and roots. Thus, we conclude that MeADK2 might play a vital role in energy homeostasis in cassava mitochondria.

  11. Acoustic radiation force impulse (ARFI) imaging: Characterizing the mechanical properties of tissues using their transient response to localized force

    Science.gov (United States)

    Nightingale, Kathryn R.; Palmeri, Mark L.; Congdon, Amy N.; Frinkely, Kristin D.; Trahey, Gregg E.

    2004-05-01

    Acoustic radiation force impulse (ARFI) imaging utilizes brief, high energy, focused acoustic pulses to generate radiation force in tissue, and conventional diagnostic ultrasound methods to detect the resulting tissue displacements in order to image the relative mechanical properties of tissue. The magnitude and spatial extent of the applied force is dependent upon the transmit beam parameters and the tissue attenuation. Forcing volumes are on the order of 5 mm3, pulse durations are less than 1 ms, and tissue displacements are typically several microns. Images of tissue displacement reflect local tissue stiffness, with softer tissues (e.g., fat) displacing farther than stiffer tissues (e.g., muscle). Parametric images of maximum displacement, time to peak displacement, and recovery time provide information about tissue material properties and structure. In both in vivo and ex vivo data, structures shown in matched B-mode images are in good agreement with those shown in ARFI images, with comparable resolution. Potential clinical applications under investigation include soft tissue lesion characterization, assessment of focal atherosclerosis, and imaging of thermal lesion formation during tissue ablation procedures. Results from ongoing studies will be presented. [Work supported by NIH Grant R01 EB002132-03, and the Whitaker Foundation. System support from Siemens Medical Solutions USA, Inc.

  12. Purification and characterization of native and recombinant SaPIN2a, a plant sieve element-localized proteinase inhibitor.

    Science.gov (United States)

    Wang, Zhen-Yu; Ding, Ling-Wen; Ge, Zhi-Juan; Wang, Zhaoyu; Wang, Fanghai; Li, Ning; Xu, Zeng-Fu

    2007-01-01

    SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.

  13. Cloning, structural characterization, and chromosomal localization of the gene encoding the human prostaglandin E(2) receptor EP2 subtype.

    Science.gov (United States)

    Smock, S L; Pan, L C; Castleberry, T A; Lu, B; Mather, R J; Owen, T A

    1999-09-17

    Northern blot analysis of human placental RNA using a probe to the 5' end of the human prostaglandin E(2) (PGE(2)) EP2 receptor subtype coding region revealed the existence of a high abundance, low molecular weight transcript. To investigate the origin of this transcript, and its possible relationship to the human EP2 mRNA, we have cloned and characterized the gene encoding the human PGE(2) EP2 receptor subtype, identified transcriptional initiation and termination sites in two tissues (spleen and thymus), and determined its chromosomal localization. The human EP2 gene consists of two exons separated by a large intron, utilizes a common initiation site in both spleen and thymus at 1113 bp upstream of the translation initiation site, and has 3' transcript termini at 1140 bp and 1149 bp downstream of the translation stop site in spleen and thymus respectively. Southern and fluorescence in situ hybridization analysis demonstrated the human EP2 gene to be a single copy gene located in band 22 of the long arm of chromosome 14 (14q22). Though our initial interest in this gene was to investigate potential differential splicing of the human EP2 gene in placenta, this work demonstrates that the atypical transcript observed in placenta probably arises from a distinct, yet related, gene. Knowledge of the sequence, structure, and transcription events associated with the human EP2 gene will enable a broader understanding of its regulation and potential role in normal physiology and disease.

  14. Quasi-local energy-momentum and two-surface characterization of the pp-wave spacetimes

    CERN Document Server

    Szabados, L B

    1995-01-01

    In the present paper the determination of the {\\it pp}-wave metric form the geometry of certain spacelike two-surfaces is considered. It has been shown that the vanishing of the Dougan--Mason quasi-local mass m_{\\}, associated with the smooth boundary \\:=\\partial\\Sigma\\approx S^2 of a spacelike hypersurface \\Sigma, is equivalent to the statement that the Cauchy development D(\\Sigma) is of a {\\it pp}-wave type geometry with pure radiation, provided the ingoing null normals are not diverging on \\ and the dominant energy condition holds on D(\\Sigma). The metric on D(\\Sigma) itself, however, has not been determined. Here, assuming that the matter is a zero-rest-mass-field, it is shown that both the matter field and the {\\it pp}-wave metric of D(\\Sigma) are completely determined by the value of the zero-rest-mass-field on \\ and the two dimensional Sen--geometry of \\ provided a convexity condition, slightly stronger than above, holds. Thus the {\\it pp}-waves can be characterized not only by the usual Cauchy data on...

  15. Characterization of the complete mitochondrial genome of the storage mite pest Tyrophagus longior (Gervais) (Acari: Acaridae) and comparative mitogenomic analysis of four acarid mites.

    Science.gov (United States)

    Yang, Banghe; Li, Chaopin

    2016-02-01

    Mites of the genus Tyrophagus are economically important polyphagous pest commonly living on stored products and also responsible for allergic reactions to humans. Complete mitochondrial genomes (mitogenomes) and the gene features therein are widely used as molecular markers in the study of population genetics, phylogenetics as well as molecular evolution. However, scarcity on the sequence data has greatly impeded the studies in these areas pertaining to the Acari (mites and ticks). Information on the Tyrophagus mitogenomes is quite critical for phylogenetic evaluation and molecular evolution of the mitogenomes within Acariformes. Herein, we reported the complete mitogenome of the allergenic acarid storage mite Tyrophagus longior (Astigmata: Acaridae), an important member of stored food pests, and compared with those of other three acarid mites. The complete mitogenome of T. longior was a circular molecule of 13,271 bp. Unexpectedly, only 19 transfer RNA genes (tRNAs) were present, lacking trnF, trnS1 and trnQ. Furthermore, it also contained 13 protein-coding genes (PCGs) and 2 genes for rRNA (rrnS and rrnL) commonly detected in metazoans. The four mitogenomes displayed similar characteristics with respect to the gene content, nucleotide comparison, and codon usages. Yet, the gene order of T. longior was different from that in other Acari. The J-strands of the four mitogenomes possessed high A+T content (67.4-70.0%), and exhibited positive GC-skews and negative AT-skews. Most inferred tRNAs of T. longior were extremely truncated, lacking either a D- or T-arm, as found in other acarid mites. In T. longior mitogenome the A+T-rich region was just 50 bp in length and can be folded as a stable stem-loop structure, whereas in the region some structures of microsatellite-like (AT)n and palindromic sequences was not present. Besides, reconstructing of the phylogenetic relationship based on concatenated amino acid sequences of 13 PCGs supported that monophyly of the family

  16. Molecular cloning and characterization of a cytoplasmic manganese superoxide dismutase and a mitochondrial manganese superoxide dismutase from Chinese mitten crab Eriocheir sinensis.

    Science.gov (United States)

    Wang, Mengqiang; Wang, Lingling; Yi, Qilin; Gai, Yunchao; Song, Linsheng

    2015-11-01

    Superoxide dismutase (SOD) functions as the first and essential enzyme in the antioxidant system and is ubiquitously existed in both prokaryotes and eukaryotes. In the present study, both cytoplasmic and mitochondrial manganese SOD were identified from Chinese mitten crab Eriocheir sinensis (designed as EscytMnSOD and EsmtMnSOD). The complete nucleotide sequence of EscytMnSOD comprised 1349 bp and consisted of a 5' untranslated regions (UTR) of 43 bp, a 3' UTR of 445 bp and an open reading frame (ORF) of 861 bp encoding a polypeptide of 286 amino acid residues. The full-length cDNA sequence of EsmtMnSOD comprised 990 bp, containing a 5' UTR of 55 bp, a 3' UTR of 278 bp and an ORF of 657 bp encoding a polypeptide of 218 amino acid residues. The deduced amino acid sequences of EscytMnSOD and EsmtMnSOD contained highly conserved MnSOD signature and typical functional domain, and exhibited high similarity with their reported homologues. In the phylogenetic tree, EscytMnSOD and EsmtMnSOD were clustered with their homologues from the land crab Cardisoma armatum. The EscytMnSOD and EsmtMnSOD transcripts were constitutively expressed in haemocytes, muscle, heart, gill, haepatopancreas and gonad, with the highest expression level in gills and haepatopancreas, respectively. The mRNA expression levels of them were all up-regulated in haemocytes with similar profiles after the stimulation of Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. The EsmtMnSOD with low basal expression level responded to invading microbes intensely, while the EscytMnSOD with high basal expression level exhibited mild responses against stimulating microbes. The purified rEscytMnSOD and rEsmtMnSOD proteins exhibited specific Mn(2+)-dependent enzymatic activities, while rEscytMnSOD with lower basic activity displayed higher stability than rEsmtMnSOD. All these results indicated that EscytMnSOD and EsmtMnSOD were efficiently antioxidant enzymes and potentially involved in the innate immune

  17. Cytonuclear Interactions in the Evolution of Animal Mitochondrial tRNA Metabolism.

    Science.gov (United States)

    Pett, Walker; Lavrov, Dennis V

    2015-06-27

    The evolution of mitochondrial information processing pathways, including replication, transcription and translation, is characterized by the gradual replacement of mitochondrial-encoded proteins with nuclear-encoded counterparts of diverse evolutionary origins. Although the ancestral enzymes involved in mitochondrial transcription and replication have been replaced early in eukaryotic evolution, mitochondrial translation is still carried out by an apparatus largely inherited from the α-proteobacterial ancestor. However, variation in the complement of mitochondrial-encoded molecules involved in translation, including transfer RNAs (tRNAs), provides evidence for the ongoing evolution of mitochondrial protein synthesis. Here, we investigate the evolution of the mitochondrial translational machinery using recent genomic and transcriptomic data from animals that have experienced the loss of mt-tRNAs, including phyla Cnidaria and Ctenophora, as well as some representatives of all four classes of Porifera. We focus on four sets of mitochondrial enzymes that directly interact with tRNAs: Aminoacyl-tRNA synthetases, glutamyl-tRNA amidotransferase, tRNA(Ile) lysidine synthetase, and RNase P. Our results support the observation that the fate of nuclear-encoded mitochondrial proteins is influenced by the evolution of molecules encoded in mitochondrial DNA, but in a more complex manner than appreciated previously. The data also suggest that relaxed selection on mitochondrial translation rather than coevolution between mitochondrial and nuclear subunits is responsible for elevated rates of evolution in mitochondrial translational proteins.

  18. 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 ...

  19. 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....

  20. [Mitochondrial diseases and stroke].

    Science.gov (United States)

    Irimia, P; Oliveros-Cid, A; Martínez-Vila, E

    1998-04-01

    We review the mitochondrial diseases in which cerebrovascular changes are seen, such as the MERRF syndrome (myoclonic epilepsy and ragged red fibers) or the Kearns-Sayre syndrome (progressive external ophthalmoplegia, retinitis pigmentaria, cerebellar disorders and disorders of cardiac conduction), focusing on the syndrome involving mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). We consider the different clinical aspects, diagnostic methods, pathophysiological mechanisms of the cerebrovascular involvement as well as therapeutic approaches.

  1. Mitochondrial protection by resveratrol.

    Science.gov (United States)

    Ungvari, Zoltan; Sonntag, William E; de Cabo, Rafael; Baur, Joseph A; Csiszar, Anna

    2011-07-01

    Mitochondrial dysfunction and oxidative stress are thought to play important roles in mammalian aging. Resveratrol is a plant-derived polyphenol that exerts diverse antiaging activities, mimicking some of the molecular and functional effects of dietary restriction. This review focuses on the molecular mechanisms underlying the mitochondrial protective effects of resveratrol, which could be exploited for the prevention or amelioration of age-related diseases in the elderly.

  2. Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein.

    Science.gov (United States)

    Woellhaf, Michael W; Sommer, Frederik; Schroda, Michael; Herrmann, Johannes M

    2016-10-15

    Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker's yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries.

  3. Mitochondrial transit peptide exhibits cell penetration ability and efficiently delivers macromolecules to mitochondria.

    Science.gov (United States)

    Jain, Aastha; Chugh, Archana

    2016-09-01

    Mitochondrial malfunction under various circumstances can lead to a variety of disorders. Effective targeting of macromolecules (drugs) is important for restoration of mitochondrial function and treatment of related disorders. We have designed a novel cell-penetrating mitochondrial transit peptide (CpMTP) for delivery of macromolecules to mitochondria. Comparison between properties of cell-penetrating peptides (CPPs) and mitochondrial signal sequences enabled prediction of peptides with dual ability for cellular translocation and mitochondrial localization. Among the predicted peptides, CpMTP translocates across HeLa cells and shows successful delivery of noncovalently conjugated cargo molecules to mitochondria. CpMTP may have applications in transduction and transfection of mitochondria for therapeutics.

  4. Mitochondrial gene therapy augments mitochondrial physiology in a Parkinson's disease cell model.

    Science.gov (United States)

    Keeney, Paula M; Quigley, Caitlin K; Dunham, Lisa D; Papageorge, Christina M; Iyer, Shilpa; Thomas, Ravindar R; Schwarz, Kathleen M; Trimmer, Patricia A; Khan, Shaharyar M; Portell, Francisco R; Bergquist, Kristen E; Bennett, James P

    2009-08-01

    Neurodegeneration in Parkinson's disease (PD) affects mainly dopaminergic neurons in the substantia nigra, where age-related, increasing percentages of cells lose detectable respiratory activity associated with depletion of intact mitochondrial DNA (mtDNA). Replenishment of mtDNA might improve neuronal bioenergetic function and prevent further cell death. We developed a technology ("ProtoFection") that uses recombinant human mitochondrial transcription factor A (TFAM) engineered with an N-terminal protein transduction domain (PTD) followed by the SOD2 mitochondrial localization signal (MLS) to deliver mtDNA cargo to the mitochondria of living cells. MTD-TFAM (MTD = PTD + MLS = "mitochondrial transduction domain") binds mtDNA and rapidly transports it across plasma membranes to mitochondria. For therapeutic proof-of-principle we tested ProtoFection technology in Parkinson's disease cybrid cells, using mtDNA generated from commercially available human genomic DNA (gDNA; Roche). Nine to 11 weeks after single exposures to MTD-TFAM + mtDNA complex, PD cybrid cells with impaired respiration and reduced mtDNA genes increased their mtDNA gene copy numbers up to 24-fold, mtDNA-derived RNAs up to 35-fold, TFAM and ETC proteins, cell respiration, and mitochondrial movement velocities. Cybrid cells with no or minimal basal mitochondrial impairments showed reduced or no responses to treatment, suggesting the possibility of therapeutic selectivity. Exposure of PD but not control cybrid cells to MTD-TFAM protein alone or MTD-TFAM + mtDNA complex increased expression of PGC-1alpha, suggesting activation of mitochondrial biogenesis. ProtoFection technology for mitochondrial gene therapy holds promise for improving bioenergetic function in impaired PD neurons and needs additional development to define its pharmacodynamics and delineate its molecular mechanisms. It also is unclear whether single-donor gDNA for generating mtDNA would be a preferred therapeutic compared with the pooled

  5. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion.

    Science.gov (United States)

    Spinazzola, Antonella; Viscomi, Carlo; Fernandez-Vizarra, Erika; Carrara, Franco; D'Adamo, Pio; Calvo, Sarah; Marsano, René Massimiliano; Donnini, Claudia; Weiher, Hans; Strisciuglio, Pietro; Parini, Rossella; Sarzi, Emmanuelle; Chan, Alicia; DiMauro, Salvatore; Rötig, Agnes; Gasparini, Paolo; Ferrero, Iliana; Mootha, Vamsi K; Tiranti, Valeria; Zeviani, Massimo

    2006-05-01

    The mitochondrial (mt) DNA depletion syndromes (MDDS) are genetic disorders characterized by a severe, tissue-specific decrease of mtDNA copy number, leading to organ failure. There are two main clinical presentations: myopathic (OMIM 609560) and hepatocerebral (OMIM 251880). Known mutant genes, including TK2, SUCLA2, DGUOK and POLG, account for only a fraction of MDDS cases. We found a new locus for hepatocerebral MDDS on chromosome 2p21-23 and prioritized the genes on this locus using a new integrative genomics strategy. One of the top-scoring candidates was the human ortholog of the mouse kidney disease gene Mpv17. We found disease-segregating mutations in three families with hepatocerebral MDDS and demonstrated that, contrary to the alleged peroxisomal localization of the MPV17 gene product, MPV17 is a mitochondrial inner membrane protein, and its absence or malfunction causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion, not only in affected individuals but also in Mpv17-/- mice.

  6. 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.

  7. Peripheral neuropathy in mitochondrial disorders.

    Science.gov (United States)

    Pareyson, Davide; Piscosquito, Giuseppe; Moroni, Isabella; Salsano, Ettore; Zeviani, Massimo

    2013-10-01

    Why is peripheral neuropathy common but mild in many mitochondrial disorders, and why is it, in some cases, the predominant or only manifestation? Although this question remains largely unanswered, recent advances in cellular and molecular biology have begun to clarify the importance of mitochondrial functioning and distribution in the peripheral nerve. Mutations in proteins involved in mitochondrial dynamics (ie, fusion and fission) frequently result in a Charcot-Marie-Tooth phenotype. Peripheral neuropathies with different phenotypic presentations occur in mitochondrial diseases associated with abnormalities in mitochondrial DNA replication and maintenance, or associated with defects in mitochondrial respiratory chain complex V. Our knowledge of mitochondrial disorders is rapidly growing as new nuclear genes are identified and new phenotypes described. Early diagnosis of mitochondrial disorders, essential to provide appropriate genetic counselling, has become crucial in a few treatable conditions. Recognising and diagnosing an underlying mitochondrial defect in patients presenting with peripheral neuropathy is therefore of paramount importance.

  8. Ocular manifestations of mitochondrial disease

    Directory of Open Access Journals (Sweden)

    S. D. Mathebula

    2012-12-01

    Full Text Available Mitochondrial disease caused by mutations in mitochondrial DNA is recognized as one of the most common causes of inherited neurological disease. Neuro-ophthalmic manifestations are a common feature of mitochondrial disease.  Optic atrophy causing central visual loss is the dominant feature of mitochondrial DNA diseases. Nystagmus is also encountered in mitochondrial disease.Although optometrists are not involved with the management of mitochondrial disease, they are likely to see more patients with this disease. Oph-thalmic examination forms part of the clinical assessment of mitochondrial disease. Mitochondrial disease should be suspected in any patient with unexplained optic neuropathy, ophthalmoplegia, pigmentary retinopathy or retrochiasmal visual loss. Despite considerable advances in the under-standing of mitochondrial genetics and the patho-genesis of mtDNA diseases, no effective treatment options are currently available for patients withmitochondrial dysfunction. (S Afr Optom 201271(1 46-50

  9. 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.

  10. Characterization of L1 ORF1p self-interaction and cellular localization using a mammalian two-hybrid system.

    Directory of Open Access Journals (Sweden)

    Mark Sokolowski

    Full Text Available Long INterspersed Element-1 (LINE-1, L1 is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers in vitro. While valuable for understanding ORF1p function, these in vitro approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction in trans leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p cis-preference in L1 mobilization is established, the impact of ability of ORF1pto interact in trans on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu RNP nuclear access in the host cell.

  11. Comparison and characterization of α-amylase inducers in Aspergillus nidulans based on nuclear localization of AmyR

    OpenAIRE

    Murakoshi, Yuriko; Makita, Tomohiro; Kato, Masashi; Kobayashi, Tetsuo

    2012-01-01

    AmyR, a fungal transcriptional activator responsible for induction of amylolytic genes in Aspergillus nidulans, localizes to the nucleus in response to the physiological inducer isomaltose. Maltose, kojibiose, and d-glucose were also found to trigger the nuclear localization of GFP-AmyR. Isomaltose- and kojibiose-triggered nuclear localization was not inhibited by the glucosidase inhibitor, castanospermine, while maltose-triggered localization was inhibited. Thus, maltose itself does not appe...

  12. Mitochondrial dynamics and inherited peripheral nerve diseases.

    Science.gov (United States)

    Pareyson, Davide; Saveri, Paola; Sagnelli, Anna; Piscosquito, Giuseppe

    2015-06-02

    Peripheral nerves have peculiar energetic requirements because of considerable length of axons and therefore correct mitochondria functioning and distribution along nerves is fundamental. Mitochondrial dynamics refers to the continuous change in size, shape, and position of mitochondria within cells. Abnormalities of mitochondrial dynamics produced by mutations in proteins involved in mitochondrial fusion (mitofusin-2, MFN2), fission (ganglioside-induced differentiation-associated protein-1, GDAP1), and mitochondrial axonal transport usually present with a Charcot-Marie-Tooth disease (CMT) phenotype. MFN2 mutations cause CMT type 2A by altering mitochondrial fusion and trafficking along the axonal microtubule system. CMT2A is an axonal autosomal dominant CMT type which in most cases is characterized by early onset and rather severe course. GDAP1 mutations also alter fission, fusion and transport of mitochondria and are associated either with recessive demyelinating (CMT4A) and axonal CMT (AR-CMT2K) and, less commonly, with dominant, milder, axonal CMT (CMT2K). OPA1 (Optic Atrophy-1) is involved in fusion of mitochondrial inner membrane, and its heterozygous mutations lead to early-onset and progressive dominant optic atrophy which may be complicated by other neurological symptoms including peripheral neuropathy. Mutations in several proteins fundamental for the axonal transport or forming the axonal cytoskeleton result in peripheral neuropathy, i.e., CMT, distal hereditary motor neuropathy (dHMN) or hereditary sensory and autonomic neuropathy (HSAN), as well as in hereditary spastic paraplegia. Indeed, mitochondrial transport involves directly or indirectly components of the kinesin superfamily (KIF5A, KIF1A, KIF1B), responsible of anterograde transport, and of the dynein complex and related proteins (DYNC1H1, dynactin, dynamin-2), implicated in retrograde flow. Microtubules, neurofilaments, and chaperones such as heat shock proteins (HSPs) also have a fundamental

  13. Mitochondrial diseases: therapeutic approaches.

    Science.gov (United States)

    DiMauro, Salvatore; Mancuso, Michelangelo

    2007-06-01

    Therapy of mitochondrial encephalomyopathies (defined restrictively as defects of the mitochondrial respiratory chain) is woefully inadequate, despite great progress in our understanding of the molecular bases of these disorders. In this review, we consider sequentially several different therapeutic approaches. Palliative therapy is dictated by good medical practice and includes anticonvulsant medication, control of endocrine dysfunction, and surgical procedures. Removal of noxious metabolites is centered on combating lactic acidosis, but extends to other metabolites. Attempts to bypass blocks in the respiratory chain by administration of electron acceptors have not been successful, but this may be amenable to genetic engineering. Administration of metabolites and cofactors is the mainstay of real-life therapy and is especially important in disorders due to primary deficiencies of specific compounds, such as carnitine or coenzyme Q10. There is increasing interest in the administration of reactive oxygen species scavengers both in primary mitochondrial diseases and in neurodegenerative diseases directly or indirectly related to mitochondrial dysfunction. Aerobic exercise and physical therapy prevent or correct deconditioning and improve exercise tolerance in patients with mitochondrial myopathies due to mitochondrial DNA (mtDNA) mutations. Gene therapy is a challenge because of polyplasmy and heteroplasmy, but interesting experimental approaches are being pursued and include, for example, decreasing the ratio of mutant to wild-type mitochondrial genomes (gene shifting), converting mutated mtDNA genes into normal nuclear DNA genes (allotopic expression), importing cognate genes from other species, or correcting mtDNA mutations with specific restriction endonucleases. Germline therapy raises ethical problems but is being considered for prevention of maternal transmission of mtDNA mutations. Preventive therapy through genetic counseling and prenatal diagnosis is

  14. Molecular characterization, functional expression, tissue localization and protective potential of a Taenia solium fatty acid-binding protein.

    Science.gov (United States)

    Illescas, Oscar; Carrero, Julio C; Bobes, Raúl J; Flisser, Ana; Rosas, Gabriela; Laclette, Juan P

    2012-12-01

    The fatty acid-binding proteins (FABPs) comprise a family of proteins that are widely expressed in animal cells and perform a variety of vital functions. Here, we report the identification, characterization, recombinant expression, tissue localization and protective potential of a Taenia solium FABP (TsFABP1). The TsFABP1 primary structure showed all the conserved residues characteristic of the subfamily iv of the intracellular Lipid-Binding Proteins (iLBPs), including those involved in the binding stabilization of the fatty acid molecule. Through a competitive binding assay we found that TsFABP1 is able to bind at least six different fatty acids with preference toward palmitic and stearic acid, suggesting that TsFABP1 is a member of the iLBP subfamily iv. Immunolocalization assays carried out on larval and adult tissues of four species of taeniids using anti-TsFABP1 hyperimmune sera produced in mice and rabbit, showed intense labeling in the tegument of the spiral canal and in subtegumental cytons of the larvae. These findings suggest that the spiral canal might be a major place for FA uptake in the developing scolex. In contrast, only subtegumental cytons in the adult worms stained positive. We propose that TsFABP1 is involved in the mechanism to mobilize fatty acids between compartments in the extensive syncytial tissue of taeniids. Protection assays carried out in a murine model of cysticercosis showed that subcutaneous immunization with TsFABP1 resulted in about 45% reduction of parasite load against an intraperitoneal challenge with Taenia crassiceps cysts. This reduction in parasite load correlated with the level of cellular and humoral immune responses against TsFABP1, as determined in spleen lymphocyte proliferation and ELISA testing.

  15. Characterization, localization, and seasonal changes of the sucrose transporter FeSUT1 in the phloem of Fraxinus excelsior.

    Science.gov (United States)

    Öner-Sieben, Soner; Rappl, Christine; Sauer, Norbert; Stadler, Ruth; Lohaus, Gertrud

    2015-08-01

    Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker's yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior.

  16. Hyperglycemic switch from mitochondrial nitric oxide to superoxide production in endothelial cells.

    Science.gov (United States)

    Brodsky, Sergey V; Gao, Shujuan; Li, Hong; Goligorsky, Michael S

    2002-11-01

    The accumulated ultrastructural and biochemical evidence is highly suggestive of the existence of mitochondrial nitric oxide (NO) synthase (mtNOS), where local production of NO regulates the electron transport along the respiratory chain. Here, the functional competence of mtNOS in situ in a living cell was examined using an intravital fluorescent NO indicator, 4,5-diaminofluorescein, employing a new procedure for loading it into the mitochondria to demonstrate local NO generation in undisrupted endothelial cells and in isolated mitochondria as well as in human embryonic kidney cells stably expressing endothelial NOS. With the use of this approach, we showed that endothelial cells incubated in the presence of high concentration of D-glucose (but not L-glucose) are characterized by the reduced NO synthetic function of mitochondria despite the unaltered abundance of the enzyme. In parallel, mitochondrial generation of superoxide was augmented in endothelial cells incubated in the presence of a high concentration of D-glucose. Both the NO generation and superoxide production in hyperglycemic environment could be restored to control levels by treating cells with a cell-permeable superoxide dismutase mimetic. In addition, enhanced mitochondrial superoxide production could be suppressed with an inhibitor of NOS in stimulated endothelial cells. In conclusion, the data 1) provide direct evidence of mitochondrial NO production in endothelial cells, 2) demonstrate its suppression and enhanced superoxide generation in hyperglycemic environment, and 3) provide evidence that "uncoupled" mtNOS represents an important source of superoxide anions in endothelial cells incubated in high glucose-containing medium.

  17. Large-scale mitochondrial DNA analysis of the domestic goat reveals six haplogroups with high diversity.

    Directory of Open Access Journals (Sweden)

    Saeid Naderi

    Full Text Available BACKGROUND: From the beginning of domestication, the transportation of domestic animals resulted in genetic and demographic processes that explain their present distribution and genetic structure. Thus studying the present genetic diversity helps to better understand the history of domestic species. METHODOLOGY/PRINCIPAL FINDINGS: The genetic diversity of domestic goats has been characterized with 2430 individuals from all over the old world, including 946 new individuals from regions poorly studied until now (mainly the Fertile Crescent. These individuals represented 1540 haplotypes for the HVI segment of the mitochondrial DNA (mtDNA control region. This large-scale study allowed the establishment of a clear nomenclature of the goat maternal haplogroups. Only five of the six previously defined groups of haplotypes were divergent enough to be considered as different haplogroups. Moreover a new mitochondrial group has been localized around the Fertile Crescent. All groups showed very high haplotype diversity. Most of this diversity was distributed among groups and within geographic regions. The weak geographic structure may result from the worldwide distribution of the dominant A haplogroup (more than 90% of the individuals. The large-scale distribution of other haplogroups (except one, may be related to human migration. The recent fragmentation of local goat populations into discrete breeds is not detectable with mitochondrial markers. The estimation of demographic parameters from mismatch analyses showed that all groups had a recent demographic expansion corresponding roughly to the period when domestication took place. But even with a large data set it remains difficult to give relative dates of expansion for different haplogroups because of large confidence intervals. CONCLUSIONS/SIGNIFICANCE: We propose standard criteria for the definition of the different haplogroups based on the result of mismatch analysis and on the use of sequences of

  18. 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.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    It has been suggested that human mitochondrial variants influence maximal oxygen uptake (VO2max). Whether mitochondrial respiratory capacity per mitochondrion (intrinsic activity) in human skeletal muscle is affected by differences in mitochondrial variants is not known. We recruited 54 males and...

  20. 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.

  1. Complete mitochondrial genome of Esox reichertii (Amur pike).

    Science.gov (United States)

    Liu, Yu; Yang, Jun

    2015-01-01

    The whole mitochondrial genome of Esox reichertii (fish) was first sequenced and characterized. It was determined to be 16,909 bp long, which contains the control region (CR), the origin of light-strand replication (OL), 22 transfer RNA genes, 2 ribosomal genes and 13 protein-coding genes. Overall base composition of the complete mitochondrial DNA was 28.65% A, 28.67% T, 27.28% C and 15.41% G, with 57.32%AT.

  2. Mitochondrial Myopathy with DNA Deletions

    OpenAIRE

    J Gordon Millichap

    1992-01-01

    Deletions of mitochondrial DNA (mtDNA) are reported in 19 of 56 patients with mitochondrial myopathy examined in the Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, MN.

  3. 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

  4. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation.

    Science.gov (United States)

    Münch, Christian; Harper, J Wade

    2016-06-30

    The mitochondrial matrix is unique in that it must integrate the folding and assembly of proteins derived from the nuclear and mitochondrial genomes. In Caenorhabditis elegans, the mitochondrial unfolded protein response (UPRmt) senses matrix protein misfolding and induces a program of nuclear gene expression, including mitochondrial chaperonins, to promote mitochondrial proteostasis. While misfolded mitochondrial-matrix-localized ornithine transcarbamylase induces chaperonin expression, our understanding of mammalian UPRmt is rudimentary, reflecting a lack of acute triggers for UPRmt activation. This limitation has prevented analysis of the cellular responses to matrix protein misfolding and the effects of UPRmt on mitochondrial translation to control protein folding loads. Here we combine pharmacological inhibitors of matrix-localized HSP90/TRAP1 (ref. 8) or LON protease, which promote chaperonin expression, with global transcriptional and proteomic analysis to reveal an extensive and acute response of human cells to UPRmt. This response encompasses widespread induction of nuclear genes, including matrix-localized proteins involved in folding, pre-RNA processing and translation. Functional studies revealed rapid but reversible translation inhibition in mitochondria occurring concurrently with defects in pre-RNA processing caused by transcriptional repression and LON-dependent turnover of the mitochondrial pre-RNA processing nuclease MRPP3 (ref. 10). This study reveals that acute mitochondrial protein folding stress activates both increased chaperone availability within the matrix and reduced matrix-localized protein synthesis through translational inhibition, and provides a framework for further dissection of mammalian UPRmt.

  5. 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.

  6. Characterization of the Localized Immune Response in the Respiratory Tract of Ferrets following Infection with Influenza A and B Viruses

    Science.gov (United States)

    Carolan, Louise A.; Rockman, Steve; Borg, Kathryn; Guarnaccia, Teagan; Reading, Patrick; Mosse, Jennifer; Kelso, Anne; Barr, Ian

    2015-01-01

    ABSTRACT The burden of infection with seasonal influenza viruses is significant. Each year is typically characterized by the dominance of one (sub)type or lineage of influenza A or B virus, respectively. The incidence of disease varies annually, and while this may be attributed to a particular virus strain or subtype, the impacts of prior immunity, population differences, and variations in clinical assessment are also important. To improve our understanding of the impacts of seasonal influenza viruses, we directly compared clinical symptoms, virus shedding, and expression of cytokines, chemokines, and immune mediators in the upper respiratory tract (URT) of ferrets infected with contemporary A(H1N1)pdm09, A(H3N2), or influenza B virus. Gene expression in the lower respiratory tract (LRT) was also assessed. Clinical symptoms were minimal. Overall cytokine/chemokine profiles in the URT were consistent in pattern and magnitude between animals infected with influenza A and B viruses, and peak expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-12p40, alpha interferon (IFN-α), IFN-β, and tumor necrosis factor alpha (TNF-α) mRNAs correlated with peak levels of viral shedding. MCP1 and IFN-γ were expressed after the virus peak. Granzymes A and B and IL-10 reached peak expression as the virus was cleared and seroconversion was detected. Cytokine/chemokine gene expression in the LRT following A(H1N1)pdm09 virus infection reflected the observations seen for the URT but was delayed 2 or 3 days, as was virus replication. These data indicate that disease severities and localized immune responses following infection with seasonal influenza A and B viruses are similar, suggesting that other factors are likely to modulate the incidence and impact of seasonal influenza. IMPORTANCE Both influenza A and B viruses cocirculate in the human population, and annual influenza seasons are typically dominated by an influenza A virus subtype or an influenza B virus lineage

  7. Design and characterization of HER-2-targeted gold nanoparticles for enhanced X-radiation treatment of locally advanced breast cancer.

    Science.gov (United States)

    Chattopadhyay, Niladri; Cai, Zhongli; Pignol, Jean-Philippe; Keller, Brian; Lechtman, Eli; Bendayan, Reina; Reilly, Raymond M

    2010-12-06

    Our purpose was to develop a human epidermal growth factor receptor-2 (HER-2) targeted nanotechnology-based radiosensitizer. HER-2 is overexpressed in 20-30% of all breast cancers and up to 2-fold higher in locally advanced disease (LABC). Trastuzumab was derivatized with a polyethylene glycol (OPSS-PEG-SVA) cross-linker to produce trastuzumab-PEG-OPSS. These immunoconjugates were analyzed by SDS-PAGE, and their immunoreactivity was assessed by flow cytometry using HER-2 overexpressing SK-BR-3 breast cancer cells. Reacting trastuzumab with increasing ratios of PEG resulted in an increase in molecular weight from approximately 148 kDa to 243 kDa, associated with increasing PEG substitution (0.6 to 18.9 PEG chains per trastuzumab). Attachment of approximately 7 PEG chains per trastuzumab resulted in 56% retention in immunoreactivity assessed by flow cytometry. The conjugates were then linked to 30 nm AuNPs. Using a novel (123)iodine-radiotracer based assay that overcomes the current limitations of spectrophotometric quantification of biological molecules on AuNPs we estimate 14.3 ± 2.7 antibodies were attached to each AuNP when 2 × 10(11) AuNPs were reacted with 20 μg of trastuzumab-PEG-OPSS. Specificity of trastuzumab-PEG-AuNPs for HER-2 and internalization in SK-BR-3 cells was demonstrated by comparing the uptake of trastuzumab-PEG-AuNPs or PEG-AuNPs by darkfield microscopy. The ability of trastuzumab-PEG-AuNPs in combination with 300 kVp X-rays to enhance DNA double strand breaks (DSBs) in SK-BR-3 cells was assessed by immunofluorescence using the γ-H2AX assay. γ-H2AX assay results revealed 5.1-fold higher DNA-DSBs with trastuzumab-PEG-AuNPs and X-radiation as compared to treatment with X-radiation alone. The trastuzumab-PEG-AuNPs are a promising targeted nanotechnology-based radiosensitizer for improving LABC therapy. The design and systematic approaches taken to surface modify and characterize trastuzumab-PEG-AuNPs described in this study would have

  8. Composition and Dynamics of Human Mitochondrial Nucleoids

    OpenAIRE

    Garrido, Nuria; Griparic, Lorena; Jokitalo, Eija; Wartiovaara, Jorma; van der Bliek, Alexander M.; Spelbrink, Johannes N.

    2003-01-01

    The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied in Saccharomyces cerevisiae. Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA. However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm. In this article we have used immunocytochemistry and bioche...

  9. Pharmacologic Effects on Mitochondrial Function

    Science.gov (United States)

    Cohen, Bruce H.

    2010-01-01

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

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

    Science.gov (United States)

    Ortiz-Avila, Omar; Esquivel-Martínez, Mauricio; Olmos-Orizaba, Berenice Eridani; Saavedra-Molina, Alfredo; Rodriguez-Orozco, Alain R; Cortés-Rojo, Christian

    2015-01-01

    Diabetic encephalopathy is a diabetic complication related to the metabolic alterations featuring diabetes. Diabetes is characterized by increased lipid peroxidation, altered glutathione redox status, exacerbated levels of ROS, and mitochondrial dysfunction. Although the pathophysiology of diabetic encephalopathy remains to be clarified, oxidative stress and mitochondrial dysfunction play a crucial role in the pathogenesis of chronic diabetic complications. Taking this into consideration, the aim of this work was to evaluate the effects of 90-day avocado oil intake in brain mitochondrial function and oxidative status in streptozotocin-induced diabetic rats (STZ rats). Avocado oil improves brain mitochondrial function in diabetic rats preventing impairment of mitochondrial respiration and mitochondrial membrane potential (ΔΨ m ), besides increasing complex III activity. Avocado oil also decreased ROS levels and lipid peroxidation and improved the GSH/GSSG ratio as well. These results demonstrate that avocado oil supplementation prevents brain mitochondrial dysfunction induced by diabetes in association with decreased oxidative stress.

  11. 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 (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. PMID:26180820

  12. 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.

  13. Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology.

    Science.gov (United States)

    Niemann, Moritz; Wiese, Sebastian; Mani, Jan; Chanfon, Astrid; Jackson, Christopher; Meisinger, Chris; Warscheid, Bettina; Schneider, André

    2013-02-01

    Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.

  14. Mitochondrial morphology, topology, and membrane interactions in skeletal muscle: a quantitative three-dimensional electron microscopy study.

    Science.gov (United States)

    Picard, Martin; White, Kathryn; Turnbull, Douglass M

    2013-01-15

    Dynamic remodeling of mitochondrial morphology through membrane dynamics are linked to changes in mitochondrial and cellular function. Although mitochondrial membrane fusion/fission events are frequent in cell culture models, whether mitochondrial membranes dynamically interact in postmitotic muscle fibers in vivo remains unclear. Furthermore, a quantitative assessment of mitochondrial morphology in intact muscle is lacking. Here, using electron microscopy (EM), we provide evidence of interacting membranes from adjacent mitochondria in intact mouse skeletal muscle. Electron-dense mitochondrial contact sites consistent with events of outer mitochondrial membrane tethering are also described. These data suggest that mitochondrial membranes interact in vivo among mitochondria, possibly to induce morphology transitions, for kiss-and-run behavior, or other processes involving contact between mitochondrial membranes. Furthermore, a combination of freeze-fracture scanning EM and transmission EM in orthogonal planes was used to characterize and quantify mitochondrial morphology. Two subpopulations of mitochondria were studied: subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibited significant differences in morphological descriptors, including form factor (means ± SD for SS: 1.41 ± 0.45 vs. IMF: 2.89 ± 1.76, P mitochondrial size and morphological parameters were highly skewed, suggesting the presence of mechanisms to influence mitochondrial size and shape. In addition, physical continuities between SS and IMF mitochondria indicated mixing of both subpopulations. These data provide evidence that mitochondrial membranes interact in vivo in mouse skeletal muscle and that factors may be involved in regulating skeletal muscle mitochondrial morphology.

  15. 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.

  16. The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter.

    Science.gov (United States)

    Pan, Xin; Liu, Jie; Nguyen, Tiffany; Liu, Chengyu; Sun, Junhui; Teng, Yanjie; Fergusson, Maria M; Rovira, Ilsa I; Allen, Michele; Springer, Danielle A; Aponte, Angel M; Gucek, Marjan; Balaban, Robert S; Murphy, Elizabeth; Finkel, Toren

    2013-12-01

    Mitochondrial calcium has been postulated to regulate a wide range of processes from bioenergetics to cell death. Here, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU(-/-) mice have no apparent capacity to rapidly uptake calcium. Whereas basal metabolism seems unaffected, the skeletal muscle of MCU(-/-) mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU(-/-) mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU(-/-) mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. The lack of PTP opening does not seem to protect MCU(-/-) cells and tissues from cell death, although MCU(-/-) hearts fail to respond to the PTP inhibitor cyclosporin A. Taken together, these results clarify how acute alterations in mitochondrial matrix calcium can regulate mammalian physiology.

  17. Mutation in the novel nuclear-encoded mitochondrial protein CHCHD10 in a family with autosomal dominant mitochondrial myopathy.

    Science.gov (United States)

    Ajroud-Driss, Senda; Fecto, Faisal; Ajroud, Kaouther; Lalani, Irfan; Calvo, Sarah E; Mootha, Vamsi K; Deng, Han-Xiang; Siddique, Nailah; Tahmoush, Albert J; Heiman-Patterson, Terry D; Siddique, Teepu

    2015-01-01

    Mitochondrial myopathies belong to a larger group of systemic diseases caused by morphological or biochemical abnormalities of mitochondria. Mitochondrial disorders can be caused by mutations in either the mitochondrial or nuclear genome. Only 5% of all mitochondrial disorders are autosomal dominant. We analyzed DNA from members of the previously reported Puerto Rican kindred with an autosomal dominant mitochondrial myopathy (Heimann-Patterson et al. 1997). Linkage analysis suggested a putative locus on the pericentric region of the long arm of chromosome 22 (22q11). Using the tools of integrative genomics, we established chromosome 22 open reading frame 16 (C22orf16) (later designated as CHCHD10) as the only high-scoring mitochondrial candidate gene in our minimal candidate region. Sequence analysis revealed a double-missense mutation (R15S and G58R) in cis in CHCHD10 which encodes a coiled coil-helix-coiled coil-helix protein of unknown function. These two mutations completely co-segregated with the disease phenotype and were absent in 1,481 Caucasian and 80 Hispanic (including 32 Puerto Rican) controls. Expression profiling showed that CHCHD10 is enriched in skeletal muscle. Mitochondrial localization of the CHCHD10 protein was confirmed using immunofluorescence in cells expressing either wild-type or mutant CHCHD10. We found that the expression of the G58R, but not the R15S, mutation induced mitochondrial fragmentation. Our findings identify a novel gene causing mitochondrial myopathy, thereby expanding the spectrum of mitochondrial myopathies caused by nuclear genes. Our findings also suggest a role for CHCHD10 in the morphologic remodeling of the mitochondria.

  18. Preventing Mitochondrial Fission Impairs Mitochondrial Function and Leads to Loss of Mitochondrial DNA

    OpenAIRE

    Parone, Philippe A.; Sandrine Da Cruz; Daniel Tondera; Yves Mattenberger; James, Dominic I.; Pierre Maechler; François Barja; Jean-Claude Martinou

    2008-01-01

    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 t...

  19. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    Energy Technology Data Exchange (ETDEWEB)

    Simarro, Maria [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Gimenez-Cassina, Alfredo [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Kedersha, Nancy [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A. [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Rhee, Kirsten [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Tisdale, Sarah; Danial, Nika [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Benarafa, Charaf [Theodor Kocher Institute, University of Bern, 3012 Bern (Switzerland); Orduna, Anonio [Unidad de Investigacion, Hospital Clinico Universitario de Valladolid, 47005 Valladolid (Spain); Anderson, Paul, E-mail: panderson@rics.bwh.harvard.edu [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States)

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  20. Genetic dissection of methylcrotonyl CoA carboxylase indicates a complex role for mitochondrial leucine catabolism during seed development and germination.

    Science.gov (United States)

    Ding, Geng; Che, Ping; Ilarslan, Hilal; Wurtele, Eve S; Nikolau, Basil J

    2012-05-01

    3-methylcrotonyl CoA carboxylase (MCCase) is a nuclear-encoded, mitochondrial-localized biotin-containing enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism, and it may also play a role in the catabolism of isoprenoids and the mevalonate shunt. In Arabidopsis, two MCCase subunits (the biotinylated MCCA subunit and the non-biotinylated MCCB subunit) are each encoded by single genes (At1g03090 and At4g34030, respectively). A reverse genetic approach was used to assess the physiological role of MCCase in plants. We recovered and characterized T-DNA and transposon-tagged knockout alleles of the MCCA and MCCB genes. Metabolite profiling studies indicate that mutations in either MCCA or MCCB block mitochondrial Leu catabolism, as inferred from the increased accumulation of Leu. Under light deprivation conditions, the hyper-accumulation of Leu, 3-methylcrotonyl CoA and isovaleryl CoA indicates that mitochondrial and peroxisomal Leu catabolism pathways are independently regulated. This biochemical block in mitochondrial Leu catabolism is associated with an impaired reproductive growth phenotype, which includes aberrant flower and silique development and decreased seed germination. The decreased seed germination phenotype is only observed for homozygous mutant seeds collected from a parent plant that is itself homozygous, but not from a parent plant that is heterozygous. These characterizations may shed light on the role of catabolic processes in growth and development, an area of plant biology that is poorly understood.

  1. Melatonin mitigates mitochondrial malfunction.

    Science.gov (United States)

    León, Josefa; Acuña-Castroviejo, Darío; Escames, Germane; Tan, Dun-Xian; Reiter, Russel J

    2005-01-01

    Melatonin, or N-acetyl-5-methoxytryptamine, is a compound derived from tryptophan that is found in all organisms from unicells to vertebrates. This indoleamine may act as a protective agent in disease conditions such as Parkinson's, Alzheimer's, aging, sepsis and other disorders including ischemia/reperfusion. In addition, melatonin has been proposed as a drug for the treatment of cancer. These disorders have in common a dysfunction of the apoptotic program. Thus, while defects which reduce apoptotic processes can exaggerate cancer, neurodegenerative disorders and ischemic conditions are made worse by enhanced apoptosis. The mechanism by which melatonin controls cell death is not entirely known. Recently, mitochondria, which are implicated in the intrinsic pathway of apoptosis, have been identified as a target for melatonin actions. It is known that melatonin scavenges oxygen and nitrogen-based reactants generated in mitochondria. This limits the loss of the intramitochondrial glutathione and lowers mitochondrial protein damage, improving electron transport chain (ETC) activity and reducing mtDNA damage. Melatonin also increases the activity of the complex I and complex IV of the ETC, thereby improving mitochondrial respiration and increasing ATP synthesis under normal and stressful conditions. These effects reflect the ability of melatonin to reduce the harmful reduction in the mitochondrial membrane potential that may trigger mitochondrial transition pore (MTP) opening and the apoptotic cascade. In addition, a reported direct action of melatonin in the control of currents through the MTP opens a new perspective in the understanding of the regulation of apoptotic cell death by the indoleamine.

  2. 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.

  3. Mitochondrial and Cell Death Mechanisms in Neurodegenerative Diseases

    Directory of Open Access Journals (Sweden)

    Lee J. Martin

    2010-03-01

    Full Text Available Alzheimer’s disease (AD, Parkinson’s disease (PD and amyotrophic lateral sclerosis (ALS are the most common human adult-onset neurodegenerative diseases. They are characterized by prominent age-related neurodegeneration in selectively vulnerable neural systems. Some forms of AD, PD, and ALS are inherited, and genes causing these diseases have been identified. Nevertheless, the mechanisms of the neuronal cell death are unresolved. Morphological, biochemical, genetic, as well as cell and animal model studies reveal that mitochondria could have roles in this neurodegeneration. The functions and properties of mitochondria might render subsets of selectively vulnerable neurons intrinsically susceptible to cellular aging and stress and overlying genetic variations, triggering neurodegeneration according to a cell death matrix theory. In AD, alterations in enzymes involved in oxidative phosphorylation, oxidative damage, and mitochondrial binding of Aβ and amyloid precursor protein have been reported. In PD, mutations in putative mitochondrial proteins have been identified and mitochondrial DNA mutations have been found in neurons in the substantia nigra. In ALS, changes occur in mitochondrial respiratory chain enzymes and mitochondrial cell death proteins. Transgenic mouse models of human neurodegenerative disease are beginning to reveal possible principles governing the biology of selective neuronal vulnerability that implicate mitochondria and the mitochondrial permeability transition pore. This review summarizes how mitochondrial pathobiology might contribute to neuronal death in AD, PD, and ALS and could serve as a target for drug therapy.

  4. Audiological evaluation in Chinese patients with mitochondrial encephalomyopathies

    Institute of Scientific and Technical Information of China (English)

    Liu Yuhe; Xue Junfang; Zhao Danhua; Chen Li; Yuan Yun; Wang Zhaoxia

    2014-01-01

    Background Hearing impairment has been reported to be common in patients with mitochondrial disorders,a group of diseases characterized by pleiomorphic clinical manifestations due to defects in oxidative phosphorylation of mitochondria.This study aimed to investigate the audiological characteristics in a large cohort of patients with mitochondrial disease.Methods Comprehensive audiological evaluations,including pure tone audiometry,tympanometry,speech audiometry,otoacoustic emissions,electrocochleography and auditory brainstem evoked potentials,were performed in 73 Chinese patients with mitochondrial encephalomyopathy and with confirmed mitochondrial DNA (mtDNA) defects.Results Among the patients,71% had hearing impairment.However,the incidence rate and severity of hearing impairment were much less in the chronic progressive external ophthalmoplegia (CPEO) subtype than in the mitochondrial encephalomyopathy,lactic acidosis,and stroke-like episodes (MELAS),myoclonic epilepsy with ragged red fibers (MERRF) and Kearns-Sayre syndrome (KSS) subtypes.While most of our patients had a predominantly cochlea origin for the hearing deficit,five patients had an auditory neuropathy spectrum disorder and three patients had impairment of both cochlea and auditory codex.Conclusions Various portions of the auditory system could be involved in patients with mitochondrial diseases,including cochlea,auditory nerve,auditory pathway and cortex.Hearing loss was more associated with multisystem involvement.Genotype,mutant load of mtDNA and other unknown factors could contribute to heterogeneity of hearing impairment in mitochondrial disease.

  5. Mitochondrial diseases caused by toxic compound accumulation: from etiopathology to therapeutic approaches

    OpenAIRE

    2015-01-01

    Mitochondrial disorders are a group of highly invalidating human conditions for which effective treatment is currently unavailable and characterized by faulty energy supply due to defective oxidative phosphorylation (OXPHOS). Given the complexity of mitochondrial genetics and biochemistry, mitochondrial inherited diseases may present with a vast range of symptoms, organ involvement, severity, age of onset, and outcome. Despite the wide spectrum of clinical signs and biochemical underpinnings ...

  6. Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease

    OpenAIRE

    2011-01-01

    Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle-specific expression of a novel mitochondrial-targeted superoxide biosensor (mt-cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and aden...

  7. Mitochondrial morphology is altered in atrophied skeletal muscle of aged mice.

    Science.gov (United States)

    Leduc-Gaudet, Jean-Philippe; Picard, Martin; St-Jean Pelletier, Félix; Sgarioto, Nicolas; Auger, Marie-Joëlle; Vallée, Joanne; Robitaille, Richard; St-Pierre, David H; Gouspillou, Gilles

    2015-07-20

    Skeletal muscle aging is associated with a progressive decline in muscle mass and strength, a process termed sarcopenia. Evidence suggests that accumulation of mitochondrial dysfunction plays a causal role in sarcopenia, which could be triggered by impaired mitophagy. Mitochondrial function, mitophagy and mitochondrial morphology are interconnected aspects of mitochondrial biology, and may coordinately be altered with aging. However, mitochondrial morphology has remained challenging to characterize in muscle, and whether sarcopenia is associated with abnormal mitochondrial morphology remains unknown. Therefore, we assessed the morphology of SubSarcolemmal (SS) and InterMyoFibrillar (IMF) mitochondria in skeletal muscle of young (8-12wk-old) and old (88-96wk-old) mice using a quantitative 2-dimensional transmission electron microscopy approach. We show that sarcopenia is associated with larger and less circular SS mitochondria. Likewise, aged IMF mitochondria were longer and more branched, suggesting increased fusion and/or decreased fission. Accordingly, although no difference in the content of proteins regulating mitochondrial dynamics (Mfn1, Mfn2, Opa1 and Drp1) was observed, a mitochondrial fusion index (Mfn2-to-Drp1 ratio) was significantly increased in aged muscles. Our results reveal that sarcopenia is associated with complex changes in mitochondrial morphology that could interfere with mitochondrial function and mitophagy, and thus contribute to aging-related accumulation of mitochondrial dysfunction and sarcopenia.

  8. Fabrication and characterization of nuclear localization signal-conjugated glycol chitosan micelles for improving the nuclear delivery of doxorubicin

    Directory of Open Access Journals (Sweden)

    Zhao J

    2012-09-01

    Full Text Available Jingmou Yu,1 Xin Xie,1 Meirong Zheng,1 Ling Yu,2 Lei Zhang,1 Jianguo Zhao,1 Dengzhao Jiang,1 Xiangxin Che11Key Laboratory of Systems Biology Medicine of Jiangxi Province, College of Basic Medical Science, Jiujiang University, Jiujiang, 2Division of Nursing, 2nd Affiliated Hospital, Yichun University, Yichun, People's Republic of ChinaBackground: Supramolecular micelles as drug-delivery vehicles are generally unable to enter the nucleus of nondividing cells. In the work reported here, nuclear localization signal (NLS-modified polymeric micelles were studied with the aim of improving nuclear drug delivery.Methods: In this research, cholesterol-modified glycol chitosan (CHGC was synthesized. NLS-conjugated CHGC (NCHGC was synthesized and characterized using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX, an anticancer drug with an intracellular site of action in the nucleus, was chosen as a model drug. DOX-loaded micelles were prepared by an emulsion/solvent evaporation method. The cellular uptake of different DOX formulations was analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity of blank micelles, free DOX, and DOX-loaded micelles in vitro was investigated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay in HeLa and HepG2 cells.Results: The degree of substitution was 5.9 cholesterol and 3.8 NLS groups per 100 sugar residues of the NCHGC conjugate. The critical aggregation concentration of the NCHGC micelles in aqueous solution was 0.0209 mg/mL. The DOX-loaded NCHGC (DNCHGC micelles were observed as being almost spherical in shape under transmission electron microscopy, and the size was determined as 248 nm by dynamic light scattering. The DOX-loading content of the DNCHGC micelles was 10.1%. The DOX-loaded micelles showed slow drug-release behavior within 72 hours in vitro. The DNCHGC micelles exhibited greater

  9. Loss of Dendritic Complexity Precedes Neurodegeneration in a Mouse Model with Disrupted Mitochondrial Distribution in Mature Dendrites

    Directory of Open Access Journals (Sweden)

    Guillermo López-Doménech

    2016-10-01

    Full Text Available Correct mitochondrial distribution is critical for satisfying local energy demands and calcium buffering requirements and supporting key cellular processes. The mitochondrially targeted proteins Miro1 and Miro2 are important components of the mitochondrial transport machinery, but their specific roles in neuronal development, maintenance, and survival remain poorly understood. Using mouse knockout strategies, we demonstrate that Miro1, as opposed to Miro2, is the primary regulator of mitochondrial transport in both axons and dendrites. Miro1 deletion leads to depletion of mitochondria from distal dendrites but not axons, accompanied by a marked reduction in dendritic complexity. Disrupting postnatal mitochondrial distribution in vivo by deleting Miro1 in mature neurons causes a progressive loss of distal dendrites and compromises neuronal survival. Thus, the local availability of mitochondrial mass is critical for generating and sustaining dendritic arbors, and disruption of mitochondrial distribution in mature neurons is associated with neurodegeneration.

  10. 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.

  11. Yeast mitochondrial RNAP conformational changes are regulated by interactions with the mitochondrial transcription factor.

    Science.gov (United States)

    Drakulic, Srdja; Wang, Liping; Cuéllar, Jorge; Guo, Qing; Velázquez, Gilberto; Martín-Benito, Jaime; Sousa, Rui; Valpuesta, José M

    2014-01-01

    Mitochondrial RNA polymerases (MtRNAPs) are members of the single-subunit RNAP family, the most well-characterized member being the RNAP from T7 bacteriophage. MtRNAPs are, however, functionally distinct in that they depend on one or more transcription factors to recognize and open the promoter and initiate transcription, while the phage RNAPs are capable of performing these tasks alone. Since the transcriptional mechanisms that are conserved in phage and mitochondrial RNAPs have been so effectively characterized in the phage enzymes, outstanding structure-mechanism questions concern those aspects that are distinct in the MtRNAPs, particularly the role of the mitochondrial transcription factor(s). To address these questions we have used both negative staining and cryo-EM to generate three-dimensional reconstructions of yeast MtRNAP initiation complexes with and without the mitochondrial transcription factor (MTF1), and of the elongation complex. Together with biochemical experiments, these data indicate that MTF1 uses multiple mechanisms to drive promoter opening, and that its interactions with the MtRNAP regulate the conformational changes undergone by the latter enzyme as it traverses the template strand.

  12. Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA.

    Directory of Open Access Journals (Sweden)

    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.

  13. Switch from ER-mitochondrial to SR-mitochondrial calcium coupling during muscle differentiation.

    Science.gov (United States)

    Yi, Muqing; Weaver, David; Eisner, Verónica; Várnai, Péter; Hunyady, László; Ma, Jianjie; Csordás, György; Hajnóczky, György

    2012-11-01

    Emerging evidence indicates that mitochondria are locally coupled to endoplasmic reticulum (ER) Ca2+ release in myoblasts and to sarcoplasmic reticulum (SR) Ca2+ release in differentiated muscle fibers in order to regulate cytoplasmic calcium dynamics and match metabolism with cell activity. However, the mechanism of the developmental transition from ER to SR coupling remains unclear. We have studied mitochondrial sensing of IP3 receptor (IP3R)- and ryanodine receptor (RyR)-mediated Ca2+ signals in H9c2 myoblasts and differentiating myotubes, as well as the attendant changes in mitochondrial morphology. Mitochondria in myoblasts were largely elongated, luminally connected and relatively few in number, whereas the myotubes were densely packed with globular mitochondria that displayed limited luminal continuity. Vasopressin, an IP3-linked agonist, evoked a large cytoplasmic Ca2+ ([Ca2+]c) increase in myoblasts, whereas it elicited a smaller response in myotubes. Conversely, RyR-mediated Ca2+ release induced by caffeine, was not observed in myoblasts, but triggered a large [Ca2+]c signal in myotubes. Both the IP3R and the RyR-mediated [Ca2+]c rise was closely associated with a mitochondrial matrix Ca2+ ([Ca2+]m) signal. Every myotube that showed a [Ca2+]c spike also displayed a [Ca2+]m response. Addition of IP3 to permeabilized myoblasts and caffeine to permeabilized myotubes also resulted in a rapid [Ca2+]m rise, indicating that Ca2+ was delivered via local coupling of the ER/SR and mitochondria. Thus, as RyRs are expressed during muscle differentiation, the local connection between RyR and mitochondrial Ca2+ uptake sites also appears. When RyR1 was exogenously introduced to myoblasts by overexpression, the [Ca2+]m signal appeared together with the [Ca2+]c signal, however the mitochondrial morphology remained unchanged. Thus, RyR expression alone is sufficient to induce the steps essential for their alignment with mitochondrial Ca2+ uptake sites, whereas the

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  15. Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence

    Science.gov (United States)

    The human mitochondrial glutamate dehydrogenase isozymes (hGDH1 and 2) are abundant matrix-localized proteins encoded by nuclear genes. The proteins are synthesized in the cytoplasm, with an atypically long N-terminal mitochondrial targeting sequence (MTS). The results of secondary structure predi...

  16. NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease.

    Science.gov (United States)

    Cerutti, Raffaele; Pirinen, Eija; Lamperti, Costanza; Marchet, Silvia; Sauve, Anthony A; Li, Wei; Leoni, Valerio; Schon, Eric A; Dantzer, Françoise; Auwerx, Johan; Viscomi, Carlo; Zeviani, Massimo

    2014-06-03

    Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD(+)-dependent protein deacetylase. As NAD(+) boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD(+) play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD(+) precursor, or reduction of NAD(+) consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans.

  17. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    Science.gov (United States)

    He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

    2012-07-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

  18. Local structures in ionic liquids probed and characterized by microscopic thermal diffusion monitored with picosecond time-resolved Raman spectroscopy.

    Science.gov (United States)

    Yoshida, Kyousuke; Iwata, Koichi; Nishiyama, Yoshio; Kimura, Yoshifumi; Hamaguchi, Hiro-o

    2012-03-14

    Vibrational cooling rate of the first excited singlet (S(1)) state of trans-stilbene and bulk thermal diffusivity are measured for seven room temperature ionic liquids, C(2)mimTf(2)N, C(4)mimTf(2)N, C(4)mimPF(6), C(5)mimTf(2)N, C(6)mimTf(2)N, C(8)mimTf(2)N, and bmpyTf(2)N. Vibrational cooling rate measured with picosecond time-resolved Raman spectroscopy reflects solute-solvent and solvent-solvent energy transfer in a microscopic solvent environment. Thermal diffusivity measured with the transient grating method indicates macroscopic heat conduction capability. Vibrational cooling rate of S(1) trans-stilbene is known to have a good correlation with bulk thermal diffusivity in ordinary molecular liquids. In the seven ionic liquids studied, however, vibrational cooling rate shows no correlation with thermal diffusivity; the observed rates are similar (0.082 to 0.12 ps(-1) in the seven ionic liquids and 0.08 to 0.14 ps(-1) in molecular liquids) despite large differences in thermal diffusivity (5.4-7.5 × 10(-8) m(2) s(-1) in ionic liquids and 8.0-10 × 10(-8) m(2) s(-1) in molecular liquids). This finding is consistent with our working hypothesis that there are local structures characteristically formed in ionic liquids. Vibrational cooling rate is determined by energy transfer among solvent ions in a local structure, while macroscopic thermal diffusion is controlled by heat transfer over boundaries of local structures. By using "local" thermal diffusivity, we are able to simulate the vibrational cooling kinetics observed in ionic liquids with a model assuming thermal diffusion in continuous media. The lower limit of the size of local structure is estimated with vibrational cooling process observed with and without the excess energy. A quantitative discussion with a numerical simulation shows that the diameter of local structure is larger than 10 nm. If we combine this lower limit, 10 nm, with the upper limit, 100 nm, which is estimated from the transparency (no light

  19. Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (I. A Model of Adhesives Based on Chemical Characterization and Localization of Polysaccharides from the Marine Diatom Achnanthes longipes and Other Diatoms).

    Science.gov (United States)

    Wustman, B. A.; Gretz, M. R.; Hoagland, K. D.

    1997-04-01

    Extracellular adhesives from the diatoms Achnanthes longipes, Amphora coffeaeformis, Cymbella cistula, and Cymbella mexicana were characterized by monosaccharide and methylation analysis, lectin-fluorescein isothiocyanate localization, and cytochemical staining. Polysaccharide was the major component of adhesives formed during cell motility, synthesis of a basal pad, and/or production of a highly organized shaft. Hot water-insoluble/hot 0.5 M NaHCO3-soluble anionic polysaccharides from A. longipes and A. coffeaeformis adhesives were primarily composed of galactosyl (64-70%) and fucosyl (32-42%) residues. In A. longipes polymers, 2,3-, t-, 3-, and 4-linked/substituted galactosyl, t-, 3-, 4-, and 2-linked fucosyl, and t- and 2-linked glucuronic acid residues predominated. Adhesive polysaccharides from C. cistula were EDTA-soluble, sulfated, consisted of 83% galactosyl (4-, 4,6-, and 3,4-linked/substituted) and 13% xylosyl (t-, 4f/5p-, and 3p-linked/substituted) residues, and contained no uronosyl residues. Ulex europaeus agglutinin uniformly localized [alpha](1,2)-L-fucose units in C. cistula and Achnanthes adhesives formed during motility and in the pads of A. longipes. D-Galactose residues were localized throughout the shafts of C. cistula and capsules of A. coffeaeformis. D-Mannose and/or D-glucose, D-galactose, and [alpha](t)-L-fucose residues were uniformly localized in the outer layers of A. longipes shafts by Cancavalia ensiformis, Abrus precatorius, and Lotus tetragonolobus agglutinin, respectively. A model for diatom cell adhesive structure was developed from chemical characterization, localization, and microscopic observation of extracellular adhesive components formed during the diatom cell-attachment process.

  20. MITOCHONDRIAL NEUROGASTROINTESTINAL ENCEPHALOMYOPATHY (MNGIE

    Directory of Open Access Journals (Sweden)

    P. Ayatollahi

    2006-06-01

    Full Text Available Mitochondrial neurogastrointestinal encephalo-myopathy (MNGIE is a rare autosomal recessive disease caused by thymidine phosphorylase (TP gene mutation. Here we report a patient with MNGIE in whom sensorimotor polyneuropathy was the first presenting symptom and had a fluctuating course. This 26-year-old female patient developed acute-onset demyelinating polyneuropathy from the age of 6 with two relapses later on. In addition, she had gastrointestinal symptoms (diarrhea, recurrent abdominal pain, progressive weight loss and ophthalmoparesis. Brain magnetic resonance imaging showed white matter abnormalities, and muscle biopsy showed ragged red fibers. This constellation of clinical and laboratory findings raised the diagnosis of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE. This report highlights the uncommon clinical characteristics of this rare disease.

  1. Mitochondrial cholesterol: mechanisms of import and effects on mitochondrial function.

    Science.gov (United States)

    Martin, Laura A; Kennedy, Barry E; Karten, Barbara

    2016-04-01

    Mitochondria require cholesterol for biogenesis and membrane maintenance, and for the synthesis of steroids, oxysterols and hepatic bile acids. Multiple pathways mediate the transport of cholesterol from different subcellular pools to mitochondria. In steroidogenic cells, the steroidogenic acute regulatory protein (StAR) interacts with a mitochondrial protein complex to mediate cholesterol delivery to the inner mitochondrial membrane for conversion to pregnenolone. In non-steroidogenic cells, several members of a protein family defined by the presence of a StAR-related lipid transfer (START) domain play key roles in the delivery of cholesterol to mitochondrial membranes. Subdomains of the endoplasmic reticulum (ER), termed mitochondria-associated ER membranes (MAM), form membrane contact sites with mitochondria and may contribute to the transport of ER cholesterol to mitochondria, either independently or in conjunction with lipid-transfer proteins. Model systems of mitochondria enriched with cholesterol in vitro and mitochondria isolated from cells with (patho)physiological mitochondrial cholesterol accumulation clearly demonstrate that mitochondrial cholesterol levels affect mitochondrial function. Increased mitochondrial cholesterol levels have been observed in several diseases, including cancer, ischemia, steatohepatitis and neurodegenerative diseases, and influence disease pathology. Hence, a deeper understanding of the mechanisms maintaining mitochondrial cholesterol homeostasis may reveal additional targets for therapeutic intervention. Here we give a brief overview of mitochondrial cholesterol import in steroidogenic cells, and then focus on cholesterol trafficking pathways that deliver cholesterol to mitochondrial membranes in non-steroidogenic cells. We also briefly discuss the consequences of increased mitochondrial cholesterol levels on mitochondrial function and their potential role in disease pathology.

  2. Mitochondrial Energetics and Therapeutics

    OpenAIRE

    2010-01-01

    Mitochondrial dysfunction has been linked to a wide range of degenerative and metabolic diseases, cancer, and aging. All these clinical manifestations arise from the central role of bioenergetics in cell biology. Although genetic therapies are maturing as the rules of bioenergetic genetics are clarified, metabolic therapies have been ineffectual. This failure results from our limited appreciation of the role of bioenergetics as the interface between the environment and the cell. A systems app...

  3. Sealing the Mitochondrial Respirasome

    OpenAIRE

    Winge, Dennis R.

    2012-01-01

    The mitochondrial respiratory chain is organized within an array of supercomplexes that function to minimize the generation of reactive oxygen species (ROS) during electron transfer reactions. Structural models of supercomplexes are now known. Another recent advance is the discovery of non-OXPHOS complex proteins that appear to adhere to and seal the individual respiratory complexes to form stable assemblages that prevent electron leakage. This review highlights recent advances in our underst...

  4. Localization and Characterization of 170 BAC-derived clones and mapping of Ninety-Four Microsatellites in the Hessian Fly

    Science.gov (United States)

    Ninety-four microsatellites from enriched genomic libraries of Hessian fly (Mayetiola destructor (Say)) were localized to 170 cognate clones in a Hessian fly bacterial artificial chromosome (BAC) library. These microsatellite-positive BAC clones were physically mapped to polytene chromosomes by fl...

  5. Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Li-juan LI; Hua-jun ZHANG; Cong ZHANG; Zheng-li SHI

    2009-01-01

    The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS 1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLSI were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.

  6. Sphingolipids and mitochondrial apoptosis.

    Science.gov (United States)

    Patwardhan, Gauri A; Beverly, Levi J; Siskind, Leah J

    2016-04-01

    The sphingolipid family of lipids modulate several cellular processes, including proliferation, cell cycle regulation, inflammatory signaling pathways, and cell death. Several members of the sphingolipid pathway have opposing functions and thus imbalances in sphingolipid metabolism result in deregulated cellular processes, which cause or contribute to diseases and disorders in humans. A key cellular process regulated by sphingolipids is apoptosis, or programmed cell death. Sphingolipids play an important role in both extrinsic and intrinsic apoptotic pathways depending on the stimuli, cell type and cellular response to the stress. During mitochondrial-mediated apoptosis, multiple pathways converge on mitochondria and induce mitochondrial outer membrane permeabilization (MOMP). MOMP results in the release of intermembrane space proteins such as cytochrome c and Apaf1 into the cytosol where they activate the caspases and DNases that execute cell death. The precise molecular components of the pore(s) responsible for MOMP are unknown, but sphingolipids are thought to play a role. Here, we review evidence for a role of sphingolipids in the induction of mitochondrial-mediated apoptosis with a focus on potential underlying molecular mechanisms by which altered sphingolipid metabolism indirectly or directly induce MOMP. Data available on these mechanisms is reviewed, and the focus and limitations of previous and current studies are discussed to present important unanswered questions and potential future directions.

  7. MITOCHONDRIAL BKCa CHANNEL

    Directory of Open Access Journals (Sweden)

    Enrique eBalderas

    2015-03-01

    Full Text Available Since its discovery in a glioma cell line 15 years ago, mitochondrial BKCa channel (mitoBKCa has been studied in brain cells and cardiomyocytes sharing general biophysical properties such as high K+ conductance (~300 pS, voltage-dependency and Ca2+-sensitivity. Main advances in deciphering the molecular composition of mitoBKCa have included establishing that it is encoded by the Kcnma1 gene, that a C-terminal splice insert confers mitoBKCa ability to be targeted to cardiac mitochondria, and evidence for its potential coassembly with β subunits. Notoriously, β1 subunit directly interacts with cytochrome c oxidase and mitoBKCa can be modulated by substrates of the respiratory chain. mitoBKCa channel has a central role in protecting the heart from ischemia, where pharmacological activation of the channel impacts the generation of reactive oxygen species and mitochondrial Ca2+ preventing cell death likely by impeding uncontrolled opening of the mitochondrial transition pore. Supporting this view, inhibition of mitoBKCa with Iberiotoxin, enhances cytochrome c release from glioma mitochondria. Many tantalizing questions remain. Some of them are: how is mitoBKCa coupled to the respiratory chain? Does mitoBKCa play non-conduction roles in mitochondria physiology? Which are the functional partners of mitoBKCa? What are the roles of mitoBKCa in other cell types? Answers to these questions are essential to define the impact of mitoBKCa channel in mitochondria biology and disease.

  8. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  9. Reductive stress impairs myoblasts mitochondrial function and triggers mitochondrial hormesis.

    Science.gov (United States)

    Singh, François; Charles, Anne-Laure; Schlagowski, Anna-Isabel; Bouitbir, Jamal; Bonifacio, Annalisa; Piquard, François; Krähenbühl, Stephan; Geny, Bernard; Zoll, Joffrey

    2015-07-01

    Even though oxidative stress damage from excessive production of ROS is a well known phenomenon, the impact of reductive stress remains poorly understood. This study tested the hypothesis that cellular reductive stress could lead to mitochondrial malfunction, triggering a mitochondrial hormesis (mitohormesis) phenomenon able to protect mitochondria from the deleterious effects of statins. We performed several in vitro experiments on L6 myoblasts and studied the effects of N-acetylcysteine (NAC) at different exposure times. Direct NAC exposure (1mM) led to reductive stress, impairing mitochondrial function by decreasing maximal mitochondrial respiration and increasing H₂O₂production. After 24h of incubation, the reactive oxygen species (ROS) production was increased. The resulting mitochondrial oxidation activated mitochondrial biogenesis pathways at the mRNA level. After one week of exposure, mitochondria were well-adapted as shown by the decrease of cellular ROS, the increase of mitochondrial content, as well as of the antioxidant capacities. Atorvastatin (ATO) exposure (100μM) for 24h increased ROS levels, reduced the percentage of live cells, and increased the total percentage of apoptotic cells. NAC exposure during 3days failed to protect cells from the deleterious effects of statins. On the other hand, NAC pretreatment during one week triggered mitochondrial hormesis and reduced the deleterious effect of statins. These results contribute to a better understanding of the redox-dependant pathways linked to mitochondria, showing that reductive stress could trigger mitochondrial hormesis phenomenon.

  10. Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9

    DEFF Research Database (Denmark)

    Schmitz-Abe, Klaus; Ciesielski, Szymon J; Schmidt, Paul J

    2015-01-01

    The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases, characterized by defects in mitochondrial heme synthesis, iron-sulfur cluster (Fe-S) biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homologue located in the 5q...

  11. Prevalence and Severity of Voice and Swallowing Difficulties in Mitochondrial Disease

    Science.gov (United States)

    Read, Jennifer L.; Whittaker, Roger G.; Miller, Nick; Clark, Sue; Taylor, Robert; McFarland, Robert; Turnbull, Douglass

    2012-01-01

    Background: Mutations of mitochondrial DNA (mtDNA) cause a broad spectrum of clinical phenotypes. Anecdotal evidence suggests that voice and swallow problems are a common feature of these diseases. Aims: To characterize accurately the prevalence and severity of voice and swallow problems in a large cohort of patients with mitochondrial disease.…

  12. Characterization of optical turbulence at the GREGOR solar telescope: temporal and local behavior and its influence on the solar observations

    Science.gov (United States)

    Sprung, D.; Sucher, E.; Stein, K.; von der Lühe, O.; Berkefeld, Th.

    2016-10-01

    Local atmospheric turbulence at the telescope level is regarded as a major reason for affecting the performance of the adaptive optics systems using wavelengths in the visible and infrared for solar observations. During the day the air masses around the telescope dome are influenced by flow distortions. Additionally heating of the infrastructure close to telescope causes thermal turbulence. Thereby optical turbulence is produced and leads to quality changes in the local seeing throughout the day. Image degradation will be yielded affecting the performance of adaptive optical systems. The spatial resolution of the solar observations will be reduced. For this study measurements of the optical turbulence, represented by the structure function parameter of the refractive index Cn2 were performed on several locations at the GREGOR telescope at the Teide observatory at Tenerife at the Canary Islands / Spain. Since September 2012 measurements of Cn2 were carried out between the towers of the Vacuum Tower Telescope (VTT) and of GREGOR with a laser-scintillometer. The horizontal distance of the measurement path was about 75 m. Additional from May 2015 up to March 2016 the optical turbulence was determined at three additional locations close to the solar telescope GREGOR. The optical turbulence is derived from sonic anemometer measurements. Time series of the sonic temperature are analyzed and compared to the direct measurements of the laser scintillometer. Meteorological conditions are investigated, especially the influence of the wind direction. Turbulence of upper atmospheric layers is not regarded. The measured local turbulence is compared to the system performance of the GREGOR telescopes. It appears that the mountain ridge effects on turbulence are more relevant than any local causes of seeing close to the telescope. Results of these analyses and comparison of nearly one year of measurements are presented and discussed.

  13. Development, characterization, and in vivo assessment of mucoadhesive nanoparticles containing fluconazole for the local treatment of oral candidiasis.

    Science.gov (United States)

    Rençber, Seda; Karavana, Sinem Yaprak; Yılmaz, Fethiye Ferda; Eraç, Bayri; Nenni, Merve; Özbal, Seda; Pekçetin, Çetin; Gurer-Orhan, Hande; Hoşgör-Limoncu, Mine; Güneri, Pelin; Ertan, Gökhan

    2016-01-01

    This study aimed to develop a suitable buccal mucoadhesive nanoparticle (NP) formulation containing fluconazole for the local treatment of oral candidiasis. The suitability of the prepared formulations was assessed by means of particle size (PS), polydispersity index, and zeta potential measurements, morphology analysis, mucoadhesion studies, drug entrapment efficiency (EE), in vitro drug release, and stability studies. Based on the optimum NP formulation, ex vivo drug diffusion and in vitro cytotoxicity studies were performed. Besides, evaluation of the antifungal effect of the optimum formulation was evaluated using agar diffusion method, fungicidal activity-related in vitro release study, and time-dependent fungicidal activity. The effect of the optimum NP formulation on the healing of oral candidiasis was investigated in an animal model, which was employed for the first time in this study. The zeta potential, mucoadhesion, and in vitro drug release studies of various NP formulations revealed that chitosan-coated NP formulation containing EUDRAGIT(®) RS 2.5% had superior properties than other formulations. Concerning the stability study of the selected formulation, the formulation was found to be stable for 6 months. During the ex vivo drug diffusion study, no drug was found in receptor phase, and this is an indication of local effect. The in vitro antifungal activity studies showed the in vitro efficacy of the NP against Candida albicans for an extended period. Also, the formulation had no cytotoxic effect at the tested concentration. For the in vivo experiments, infected rabbits were successfully treated with local administration of the optimum NP formulation once a day. This study has shown that the mucoadhesive NP formulation containing fluconazole is a promising candidate with once-a-day application for the local treatment of oral candidiasis.

  14. Development, characterization, and in vivo assessment of mucoadhesive nanoparticles containing fluconazole for the local treatment of oral candidiasis

    Science.gov (United States)

    Rençber, Seda; Karavana, Sinem Yaprak; Yılmaz, Fethiye Ferda; Eraç, Bayri; Nenni, Merve; Özbal, Seda; Pekçetin, Çetin; Gurer-Orhan, Hande; Hoşgör-Limoncu, Mine; Güneri, Pelin; Ertan, Gökhan

    2016-01-01

    This study aimed to develop a suitable buccal mucoadhesive nanoparticle (NP) formulation containing fluconazole for the local treatment of oral candidiasis. The suitability of the prepared formulations was assessed by means of particle size (PS), polydispersity index, and zeta potential measurements, morphology analysis, mucoadhesion studies, drug entrapment efficiency (EE), in vitro drug release, and stability studies. Based on the optimum NP formulation, ex vivo drug diffusion and in vitro cytotoxicity studies were performed. Besides, evaluation of the antifungal effect of the optimum formulation was evaluated using agar diffusion method, fungicidal activity-related in vitro release study, and time-dependent fungicidal activity. The effect of the optimum NP formulation on the healing of oral candidiasis was investigated in an animal model, which was employed for the first time in this study. The zeta potential, mucoadhesion, and in vitro drug release studies of various NP formulations revealed that chitosan-coated NP formulation containing EUDRAGIT® RS 2.5% had superior properties than other formulations. Concerning the stability study of the selected formulation, the formulation was found to be stable for 6 months. During the ex vivo drug diffusion study, no drug was found in receptor phase, and this is an indication of local effect. The in vitro antifungal activity studies showed the in vitro efficacy of the NP against Candida albicans for an extended period. Also, the formulation had no cytotoxic effect at the tested concentration. For the in vivo experiments, infected rabbits were successfully treated with local administration of the optimum NP formulation once a day. This study has shown that the mucoadhesive NP formulation containing fluconazole is a promising candidate with once-a-day application for the local treatment of oral candidiasis. PMID:27358561

  15. The Potato Tuber Mitochondrial Proteome

    DEFF Research Database (Denmark)

    Salvato, Fernanda; Havelund, Jesper F; Chen, Mingjie;

    2014-01-01

    manner using normalized spectral counts including as many as 5-fold more “extreme” proteins (low mass, high isoelectric point, hydrophobic) than previous mitochondrial proteome studies. We estimate that this compendium of proteins represents a high coverage of the potato tuber mitochondrial proteome...... that more than 50% of the identified proteins harbor at least one modification. The most prominently observed class of posttranslational modifications was oxidative modifications. This study reveals approximately 500 new or previously unconfirmed plant mitochondrial proteins and outlines a facile strategy...... for unbiased, near-comprehensive identification of mitochondrial proteins and their modified forms....

  16. Characterizing the correlations between local phase fractions of gas-liquid two-phase flow with wire-mesh sensor.

    Science.gov (United States)

    Tan, C; Liu, W L; Dong, F

    2016-06-28

    Understanding of flow patterns and their transitions is significant to uncover the flow mechanics of two-phase flow. The local phase distribution and its fluctuations contain rich information regarding the flow structures. A wire-mesh sensor (WMS) was used to study the local phase fluctuations of horizontal gas-liquid two-phase flow, which was verified through comparing the reconstructed three-dimensional flow structure with photographs taken during the experiments. Each crossing point of the WMS is treated as a node, so the measurement on each node is the phase fraction in this local area. An undirected and unweighted flow pattern network was established based on connections that are formed by cross-correlating the time series of each node under different flow patterns. The structure of the flow pattern network reveals the relationship of the phase fluctuations at each node during flow pattern transition, which is then quantified by introducing the topological index of the complex network. The proposed analysis method using the WMS not only provides three-dimensional visualizations of the gas-liquid two-phase flow, but is also a thorough analysis for the structure of flow patterns and the characteristics of flow pattern transition. This article is part of the themed issue 'Supersensing through industrial process tomography'.

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

    OpenAIRE

    2006-01-01

    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 c...

  18. Characterization by a time-frequency method of classical waves propagation in one-dimensional lattice : effects of the dispersion and localized nonlinearities

    CERN Document Server

    Richoux, Olivier; Hardy, Jean

    2009-01-01

    This paper presents an application of time-frequency methods to characterize the dispersion of acoustic waves travelling in a one-dimensional periodic or disordered lattice made up of Helmholtz resonators connected to a cylindrical tube. These methods allow (1) to evaluate the velocity of the wave energy when the input signal is an acoustic pulse ; (2) to display the evolution of the spectral content of the transient signal ; (3) to show the role of the localized nonlinearities on the propagation .i.e the emergence of higher harmonics. The main result of this paper is that the time-frequency methods point out how the nonlinearities break the localization of the waves and/or the filter effects of the lattice.

  19. Synthesis and Characterization of Poly(lactic-co-glycolic Acid Nanoparticles-Loaded Chitosan/Bioactive Glass Scaffolds as a Localized Delivery System in the Bone Defects

    Directory of Open Access Journals (Sweden)

    K. Nazemi

    2014-01-01

    Full Text Available The functionality of tissue engineering scaffolds can be enhanced by localized delivery of appropriate biological macromolecules incorporated within biodegradable nanoparticles. In this research, chitosan/58S-bioactive glass (58S-BG containing poly(lactic-co-glycolic acid (PLGA nanoparticles has been prepared and then characterized. The effects of further addition of 58S-BG on the structure of scaffolds have been investigated to optimize the characteristics of the scaffolds for bone tissue engineering applications. The results showed that the scaffolds had high porosity with open pores. It was also shown that the porosity decreased with increasing 58S-BG content. Furthermore, the PLGA nanoparticles were homogenously distributed within the scaffolds. According to the obtained results, the nanocomposites could be considered as highly bioactive bone tissue engineering scaffolds with the potential of localized delivery of biological macromolecules.

  20. Microtubules are Essential for Mitochondrial Dynamics-Fission, Fusion, and Motility- in Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Laken C. Woods

    2016-03-01

    Full Text Available Mitochondrial function is dependent upon mitochondrial structure which is in turn dependent upon mitochondrial dynamics, including fission, fusion, and motility. Here we examined the relationship between mitochondrial dynamics and the cytoskeleton in Dictyostelium discoideum. Using time-lapse analysis, we quantified mitochondrial fission, fusion, and motility in the presence of cytoskeleton disrupting pharmaceuticals and the absence of the potential mitochondria-cytoskeleton linker protein, CluA. Our results indicate that microtubules are essential for mitochondrial movement, as well as fission and fusion; actin plays a less significant role, perhaps selecting the mitochondria for transport. We also suggest that CluA is not a linker protein but plays an unidentified role in mitochondrial fission and fusion. The significance of our work is to gain further insight into the role the cytoskeleton plays in mitochondrial dynamics and function. By better understanding these processes we can better appreciate the underlying mitochondrial contributions to many neurological disorders characterized by altered mitochondrial dynamics, structure, and/or function.

  1. Mitochondrial haplogroup H1 in north Africa: an early holocene arrival from Iberia.

    Directory of Open Access Journals (Sweden)

    Claudio Ottoni

    Full Text Available The Tuareg of the Fezzan region (Libya are characterized by an extremely high frequency (61% of haplogroup H1, a mitochondrial DNA (mtDNA haplogroup that is common in all Western European populations. To define how and when H1 spread from Europe to North Africa up to the Central Sahara, in Fezzan, we investigated the complete mitochondrial genomes of eleven Libyan Tuareg belonging to H1. Coalescence time estimates suggest an arrival of the European H1 mtDNAs at about 8,000-9,000 years ago, while phylogenetic analyses reveal three novel H1 branches, termed H1v, H1w and H1x, which appear to be specific for North African populations, but whose frequencies can be extremely different even in relatively close Tuareg villages. Overall, these findings support the scenario of an arrival of haplogroup H1 in North Africa from Iberia at the beginning of the Holocene, as a consequence of the improvement in climate conditions after the Younger Dryas cold snap, followed by in situ formation of local H1 sub-haplogroups. This process of autochthonous differentiation continues in the Libyan Tuareg who, probably due to isolation and recent founder events, are characterized by village-specific maternal mtDNA lineages.

  2. Faithful transcription initiation from a mitochondrial promoter in transgenic plastids.

    Science.gov (United States)

    Bohne, Alexandra-Viola; Ruf, Stephanie; Börner, Thomas; Bock, Ralph

    2007-01-01

    The transcriptional machineries of plastids and mitochondria in higher plants exhibit striking similarities. All mitochondrial genes and part of the plastid genes are transcribed by related phage-type RNA polymerases. Furthermore, the majority of mitochondrial promoters and a subset of plastid promoters show a similar structural organization. We show here that the plant mitochondrial atpA promoter is recognized by plastid RNA polymerases in vitro and in vivo. The Arabidopsis phage-type RNA polymerase RpoTp, an enzyme localized exclusively to plastids, was found to recognize the mitochondrial atpA promoter in in vitro assays suggesting the possibility that mitochondrial promoters might function as well in plastids. We have, therefore, generated transplastomic tobacco plants harboring in their chloroplast genome the atpA promoter fused to the coding region of the bacterial nptII gene. The chimeric nptII gene was found to be efficiently transcribed in chloroplasts. Mapping of the 5' ends of the nptII transcripts revealed accurate recognition of the atpA promoter by the chloroplast transcription machinery. We show further that the 5' untranslated region (UTR) of the mitochondrial atpA transcript is capable of mediating translation in chloroplasts. The functional and evolutionary implications of these findings as well as possible applications in chloroplast genome engineering are discussed.

  3. Isolating the segment of the mitochondrial electron transport chain responsible for mitochondrial damage during cardiac ischemia

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Qun; Yin, Guotian; Stewart, Sarah; Hu, Ying [Department of Medicine, Division of Cardiology, Case Western Reserve University, Cleveland, OH 44106 (United States); Lesnefsky, Edward J., E-mail: edward.lesnefsky@va.gov [Department of Medicine, Division of Cardiology, Case Western Reserve University, Cleveland, OH 44106 (United States); Medical Service, Louis Stokes Veterans Affairs Medical Center, Cleveland, OH 44106 (United States)

    2010-07-09

    Ischemia damages the mitochondrial electron transport chain (ETC), mediated in part by damage generated by the mitochondria themselves. Mitochondrial damage resulting from ischemia, in turn, leads to cardiac injury during reperfusion. The goal of the present study was to localize the segment of the ETC that produces the ischemic mitochondrial damage. We tested if blockade of the proximal ETC at complex I differed from blockade distal in the chain at cytochrome oxidase. Isolated rabbit hearts were perfused for 15 min followed by 30 min stop-flow ischemia at 37 {sup o}C. Amobarbital (2.5 mM) or azide (5 mM) was used to block proximal (complex I) or distal (cytochrome oxidase) sites in the ETC. Time control hearts were buffer-perfused for 45 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated. Ischemia decreased cytochrome c content in SSM but not in IFM compared to time control. Blockade of electron transport at complex I preserved the cytochrome c content in SSM. In contrast, blockade of electron transport at cytochrome oxidase with azide did not retain cytochrome c in SSM during ischemia. Since blockade of electron transport at complex III also prevented cytochrome c loss during ischemia, the specific site that elicits mitochondrial damage during ischemia is likely located in the segment between complex III and cytochrome oxidase.

  4. LRPPRC is a mitochondrial matrix protein that is conserved in metazoans

    Energy Technology Data Exchange (ETDEWEB)

    Sterky, Fredrik H. [Department of Laboratory Medicine, Karolinska Institutet, Retzius vaeg 8, SE-171 77 Stockholm (Sweden); Ruzzenente, Benedetta [Max-Planck-Institut fuer Biologie des Alterns, Gleueler Str. 50a, D-50931 Cologne (Germany); Department of Biology, University of Padova, Via Ugo Bassi 58B, I-35127 Padova (Italy); Gustafsson, Claes M. [Max-Planck-Institut fuer Biologie des Alterns, Gleueler Str. 50a, D-50931 Cologne (Germany); Department of Biochemistry and Cell Biology, University of Gothenburg, P.O. Box 440, SE-405 30 Gothenburg (Sweden); Samuelsson, Tore [Department of Biochemistry and Cell Biology, University of Gothenburg, P.O. Box 440, SE-405 30 Gothenburg (Sweden); Larsson, Nils-Goeran, E-mail: larsson@age.mpg.de [Max-Planck-Institut fuer Biologie des Alterns, Gleueler Str. 50a, D-50931 Cologne (Germany)

    2010-08-06

    Research highlights: {yields} LRPPRC orthologs are restricted to metazoans. {yields} LRPPRC is imported to the mitochondrial matrix. {yields} No evidence of nuclear isoform. -- Abstract: LRPPRC (also called LRP130) is an RNA-binding pentatricopeptide repeat protein. LRPPRC has been recognized as a mitochondrial protein, but has also been shown to regulate nuclear gene transcription and to bind specific RNA molecules in both the nucleus and the cytoplasm. We here present a bioinformatic analysis of the LRPPRC primary sequence, which reveals that orthologs to the LRPPRC gene are restricted to metazoan cells and that all of the corresponding proteins contain mitochondrial targeting signals. To address the subcellular localization further, we have carefully analyzed LRPPRC in mammalian cells and identified a single isoform that is exclusively localized to mitochondria. The LRPPRC protein is imported to the mitochondrial matrix and its mitochondrial targeting sequence is cleaved upon entry.

  5. "Stiff neonate" with mitochondrial DNA depletion and secondary neurotransmitter defects.

    LENUS (Irish Health Repository)

    Moran, Margaret M

    2011-12-01

    Mitochondrial disorders comprise a heterogenous group. A neonate who presented with episodes of severe truncal hypertonia and apnea progressed to a hypokinetic rigid syndrome characterized by hypokinesia, tremulousness, profound head lag, absent suck and gag reflexes, brisk deep tendon reflexes, ankle and jaw clonus, and evidence of autonomic dysfunction. Analysis of cerebrospinal fluid neurotransmitters from age 7 weeks demonstrated low levels of amine metabolites (homovanillic acid and 5-hydroxyindoleacetic acid), tetrahydrobiopterin, and pyridoxal phosphate. Mitochondrial DNA quantitative studies on muscle homogenate demonstrated a mitochondrial DNA depletion disorder. Respiratory chain enzymology demonstrated decreased complex IV activity. Screening for mitochondrial DNA rearrangement disorders and sequencing relevant mitochondrial genes produced negative results. No clinical or biochemical response to treatment with pyridoxal phosphate, tetrahydrobiopterin, or l-dopa occurred. The clinical course was progressive, and the patient died at age 19 months. Mitochondrial disorders causing secondary neurotransmitter diseases are usually severe, but are rarely reported. This diagnosis should be considered in neonates or infants who present with hypertonia, hypokinesia rigidity, and progressive neurodegeneration.

  6. Excitotoxins, Mitochondrial and Redox Disturbances in Multiple Sclerosis

    Science.gov (United States)

    Rajda, Cecilia; Pukoli, Dániel; Bende, Zsuzsanna; Majláth, Zsófia; Vécsei, László

    2017-01-01

    Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). There is increasing evidence that MS is not only characterized by immune mediated inflammatory reactions, but also by neurodegenerative processes. There is cumulating evidence that neurodegenerative processes, for example mitochondrial dysfunction, oxidative stress, and glutamate (Glu) excitotoxicity, seem to play an important role in the pathogenesis of MS. The alteration of mitochondrial homeostasis leads to the formation of excitotoxins and redox disturbances. Mitochondrial dysfunction (energy disposal failure, apoptosis, etc.), redox disturbances (oxidative stress and enhanced reactive oxygen and nitrogen species production), and excitotoxicity (Glu mediated toxicity) may play an important role in the progression of the disease, causing axonal and neuronal damage. This review focuses on the mechanisms of mitochondrial dysfunction (including mitochondrial DNA (mtDNA) defects and mitochondrial structural/functional changes), oxidative stress (including reactive oxygen and nitric species), and excitotoxicity that are involved in MS and also discusses the potential targets and tools for therapeutic approaches in the future. PMID:28208701

  7. 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.

  8. Giardia mitosomal protein import machinery differentially recognizes mitochondrial targeting signals.

    Science.gov (United States)

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Estraño, Carlos E

    2014-01-01

    Giardia lamblia mitosomes are believed to be vestigial mitochondria which lack a genome. Similar to higher eukaryotes, mitosomal proteins possess either N-terminal or internal mitosomal targeting sequences. To date, some components of the higher eukaryote archetypal mitochondrial protein import apparatus have been identified and characterized in Giardia mitosomes; therefore, it is expected that mitochondrial signals will be recognized by the mitosomal protein import system. To further determine the level of conservation of the Giardia mitosome protein import apparatus, we expressed mitochondrial proteins from higher eukaryotes in Giardia. These recombinant proteins include Tom20 and Tom22; two components of the mitochondrial protein import machinery. Our results indicate that N-terminal mitochondrial targeting sequence is recognized by the mitosomal protein import machinery; however, interestingly the internal mitochondrial targeting sequences of higher eukaryotes are not recognized by the mitosome. Our results indicate that Giardia mitosome protein transport machinery shows differential recognition of higher eukaryotic mitochondria transfer signals, suggesting a divergence of the transport system in G. lamblia. Therefore, our data support the hypothesis that the protein import machinery in Giardia lamblia mitosome is an incomplete vestigial derivative of mitochondria components.

  9. Mitochondrial calcium uptake capacity modulates neocortical excitability.

    Science.gov (United States)

    Sanganahalli, Basavaraju G; Herman, Peter; Hyder, Fahmeed; Kannurpatti, Sridhar S

    2013-07-01

    Local calcium (Ca(2+)) changes regulate central nervous system metabolism and communication integrated by subcellular processes including mitochondrial Ca(2+) uptake. Mitochondria take up Ca(2+) through the calcium uniporter (mCU) aided by cytoplasmic microdomains of high Ca(2+). Known only in vitro, the in vivo impact of mCU activity may reveal Ca(2+)-mediated roles of mitochondria in brain signaling and metabolism. From in vitro studies of mitochondrial Ca(2+) sequestration and cycling in various cell types of the central nervous system, we evaluated ranges of spontaneous and activity-induced Ca(2+) distributions in multiple subcellular compartments in vivo. We hypothesized that inhibiting (or enhancing) mCU activity would attenuate (or augment) cortical neuronal activity as well as activity-induced hemodynamic responses in an overall cytoplasmic and mitochondrial Ca(2+)-dependent manner. Spontaneous and sensory-evoked cortical activities were measured by extracellular electrophysiology complemented with dynamic mapping of blood oxygen level dependence and cerebral blood flow. Calcium uniporter activity was inhibited and enhanced pharmacologically, and its impact on the multimodal measures were analyzed in an integrated manner. Ru360, an mCU inhibitor, reduced all stimulus-evoked responses, whereas Kaempferol, an mCU enhancer, augmented all evoked responses. Collectively, the results confirm aforementioned hypotheses and support the Ca(2+) uptake-mediated integrative role of in vivo mitochondria on neocortical activity.

  10. Sugarcane genes related to mitochondrial function

    Directory of Open Access Journals (Sweden)

    Fonseca Ghislaine V.

    2001-01-01

    Full Text Available Mitochondria function as metabolic powerhouses by generating energy through oxidative phosphorylation and have become the focus of renewed interest due to progress in understanding the subtleties of their biogenesis and the discovery of the important roles which these organelles play in senescence, cell death and the assembly of iron-sulfur (Fe/S centers. Using proteins from the yeast Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana we searched the sugarcane expressed sequence tag (SUCEST database for the presence of expressed sequence tags (ESTs with similarity to nuclear genes related to mitochondrial functions. Starting with 869 protein sequences, we searched for sugarcane EST counterparts to these proteins using the basic local alignment search tool TBLASTN similarity searching program run against 260,781 sugarcane ESTs contained in 81,223 clusters. We were able to recover 367 clusters likely to represent sugarcane orthologues of the corresponding genes from S. cerevisiae, H. sapiens and A. thaliana with E-value <= 10-10. Gene products belonging to all functional categories related to mitochondrial functions were found and this allowed us to produce an overview of the nuclear genes required for sugarcane mitochondrial biogenesis and function as well as providing a starting point for detailed analysis of sugarcane gene structure and physiology.

  11. Characterization of long-range functional connectivity in epileptic networks by neuronal spike-triggered local field potentials

    Science.gov (United States)

    Lopour, Beth A.; Staba, Richard J.; Stern, John M.; Fried, Itzhak; Ringach, Dario L.

    2016-04-01

    Objective. Quantifying the relationship between microelectrode-recorded multi-unit activity (MUA) and local field potentials (LFPs) in distinct brain regions can provide detailed information on the extent of functional connectivity in spatially widespread networks. These methods are common in studies of cognition using non-human animal models, but are rare in humans. Here we applied a neuronal spike-triggered impulse response to electrophysiological recordings from the human epileptic brain for the first time, and we evaluate functional connectivity in relation to brain areas supporting the generation of seizures. Approach. Broadband interictal electrophysiological data were recorded from microwires adapted to clinical depth electrodes that were implanted bilaterally using stereotactic techniques in six presurgical patients with medically refractory epilepsy. MUA and LFPs were isolated in each microwire, and we calculated the impulse response between the MUA on one microwire and the LFPs on a second microwire for all possible MUA/LFP pairs. Results were compared to clinical seizure localization, including sites of seizure onset and interictal epileptiform discharges. Main results. We detected significant interictal long-range functional connections in each subject, in some cases across hemispheres. Results were consistent between two independent datasets, and the timing and location of significant impulse responses reflected anatomical connectivity. However, within individual subjects, the spatial distribution of impulse responses was unique. In two subjects with clear seizure localization and successful surgery, the epileptogenic zone was associated with significant impulse responses. Significance. The results suggest that the spike-triggered impulse response can provide valuable information about the neuronal networks that contribute to seizures using only interictal data. This technique will enable testing of specific hypotheses regarding functional connectivity

  12. Calcium-induced alteration of mitochondrial morphology and mitochondrial-endoplasmic reticulum contacts in rat brown adipocytes.

    Science.gov (United States)

    Golic, I; Velickovic, K; Markelic, M; Stancic, A; Jankovic, A; Vucetic, M; Otasevic, V; Buzadzic, B; Korac, B; Korac, A

    2014-09-09

    Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca2+]i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ® drinking or tap water (control) drinking for three days. Alizarin Red S staining showed increased Ca2+ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca2+ diet affected mitochondrial fusion as mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were increased, and mitochondrial fission as dynamin related protein 1 (DRP1) was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER). The level of uncoupling protein-1 (UCP1) was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes.

  13. Calcium-induced alteration of mitochondrial morphology and mitochondrial-endoplasmic reticulum contacts in rat brown adipocytes

    Directory of Open Access Journals (Sweden)

    I. Golic

    2014-09-01

    Full Text Available Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca2+]i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ® drinking or tap water (control drinking for three days. Alizarin Red S staining showed increased Ca2+ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca2+ diet affected mitochondrial fusion as mitofusin 1 (MFN1 and mitofusin 2 (MFN2 were increased, and mitochondrial fission as dynamin related protein 1 (DRP1 was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER. The level of uncoupling protein-1 (UCP1 was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes

  14. Characterization, sub-cellular localization and expression profiling of the isoprenylcysteine methylesterase gene family in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Ma Wujun

    2010-09-01

    Full Text Available Abstract Background Isoprenylcysteine methylesterases (ICME demethylate prenylated protein in eukaryotic cell. Until now, knowledge about their molecular information, localization and expression pattern is largely unavailable in plant species. One ICME in Arabidopsis, encoded by At5g15860, has been identified recently. Over-expression of At5g15860 caused an ABA hypersensitive phenotype in transgenic Arabidopsis plants, indicating that it functions as a positive regulator of ABA signaling. Moreover, ABA induced the expression of this gene in Arabidopsis seedlings. The current study extends these findings by examining the sub-cellular localization, expression profiling, and physiological functions of ICME and two other ICME-like proteins, ICME-LIKE1 and ICME-LIKE2, which were encoded by two related genes At1g26120 and At3g02410, respectively. Results Bioinformatics investigations showed that the ICME and other two ICME-like homologs comprise a small subfamily of carboxylesterase (EC 3.1.1.1 in Arabidopsis. Sub-cellular localization of GFP tagged ICME and its homologs showed that the ICME and ICME-like proteins are intramembrane proteins predominantly localizing in the endoplasmic reticulum (ER and Golgi apparatus. Semi-quantitative and real-time quantitative PCR revealed that the ICME and ICME-like genes are expressed in all examined tissues, including roots, rosette leaves, cauline leaves, stems, flowers, and siliques, with differential expression levels. Within the gene family, the base transcript abundance of ICME-LIKE2 gene is very low with higher expression in reproductive organs (flowers and siliques. Time-course analysis uncovered that both ICME and ICME-like genes are up-regulated by mannitol, NaCl and ABA treatment, with ICME showing the highest level of up-regulation by these treatments. Heat stress resulted in up-regulation of the ICME gene significantly but down-regulation of the ICME-LIKE1 and ICME-LIKE2 genes. Cold and dehydration

  15. Molecular characterization of five new S alleles associated with self-incompatibility in local Spanish almond cultivars

    OpenAIRE

    2011-01-01

    Almond is a highly heterozygous species with a high number of S-alleles controlling its gametophytic self-incompatibility system (GSI). In this work we have analysed Spanish local almond cultivars for S-RNase allele diversity. By cloning and sequencing five new S-RNase alleles were identified: S31 (804 bp) in 'Pou de Felanitx' and 'Totsol', S32 (855 bp) in 'Taiatona', S33 (1165 bp) in 'Pou d'Establiments' and 'Muel', S34 (1663 bp) in 'Pané-Barquets', and S35 (1658 bp) in 'Planeta de les Garri...

  16. Development, characterization, and in vivo assessment of mucoadhesive nanoparticles containing fluconazole for the local treatment of oral candidiasis

    Directory of Open Access Journals (Sweden)

    Rençber S

    2016-06-01

    Full Text Available Seda Rençber,1 Sinem Yaprak Karavana,1 Fethiye Ferda Yilmaz,2 Bayri Eraç,2 Merve Nenni,3 Seda Özbal,4 Çetin Pekçetin,4 Hande Gurer-Orhan,3 Mine Hoşgör Limoncu,2 Pelin Güneri,5 Gökhan Ertan11Faculty of Pharmacy, Department of Pharmaceutical Technology, 2Department of Pharmaceutical Microbiology, 3Department of Pharmaceutical Toxicology, Ege University, Bornova, Turkey; 4Department of Histology and Embryology, School of Medicine, Dokuz Eylul University, Inciralti, Turkey; 5Faculty of Dentistry, Department of Oral Diagnosis and Radiology, Ege University, Bornova, TurkeyAbstract: This study aimed to develop a suitable buccal mucoadhesive nanoparticle (NP formulation containing fluconazole for the local treatment of oral candidiasis. The suitability of the prepared formulations was assessed by means of particle size (PS, polydispersity index, and zeta potential measurements, morphology analysis, mucoadhesion studies, drug entrapment efficiency (EE, in vitro drug release, and stability studies. Based on the optimum NP formulation, ex vivo drug diffusion and in vitro cytotoxicity studies were performed. Besides, evaluation of the antifungal effect of the optimum formulation was evaluated using agar diffusion method, fungicidal activity-related in vitro release study, and time-dependent fungicidal activity. The effect of the optimum NP formulation on the healing of oral candidiasis was investigated in an animal model, which was employed for the first time in this study. The zeta potential, mucoadhesion, and in vitro drug release studies of various NP formulations revealed that chitosan-coated NP formulation containing EUDRAGIT® RS 2.5% had superior properties than other formulations. Concerning the stability study of the selected formulation, the formulation was found to be stable for 6 months. During the ex vivo drug diffusion study, no drug was found in receptor phase, and this is an indication of local effect. The in vitro antifungal

  17. Inheritance of the yeast mitochondrial genome

    DEFF Research Database (Denmark)

    Piskur, Jure

    1994-01-01

    Mitochondrion, extrachromosomal genetics, intergenic sequences, genome size, mitochondrial DNA, petite mutation, yeast......Mitochondrion, extrachromosomal genetics, intergenic sequences, genome size, mitochondrial DNA, petite mutation, yeast...

  18. Systematic Site Characterization at Seismic Stations combined with Empirical Spectral Modeling: critical data for local hazard analysis

    Science.gov (United States)

    Michel, Clotaire; Hobiger, Manuel; Edwards, Benjamin; Poggi, Valerio; Burjanek, Jan; Cauzzi, Carlo; Kästli, Philipp; Fäh, Donat

    2016-04-01

    The Swiss Seismological Service operates one of the densest national seismic networks in the world, still rapidly expanding (see http://www.seismo.ethz.ch/monitor/index_EN). Since 2009, every newly instrumented site is characterized following an established procedure to derive realistic 1D VS velocity profiles. In addition, empirical Fourier spectral modeling is performed on the whole network for each recorded event with sufficient signal-to-noise ratio. Besides the source characteristics of the earthquakes, statistical real time analyses of the residuals of the spectral modeling provide a seamlessly updated amplification function w.r. to Swiss rock conditions at every station. Our site characterization procedure is mainly based on the analysis of surface waves from passive experiments and includes cross-checks of the derived amplification functions with those obtained through spectral modeling. The systematic use of three component surface-wave analysis, allowing the derivation of both Rayleigh and Love waves dispersion curves, also contributes to the improved quality of the retrieved profiles. The results of site characterisation activities at recently installed strong-motion stations depict the large variety of possible effects of surface geology on ground motion in the Alpine context. Such effects range from de-amplification at hard-rock sites to amplification up to a factor of 15 in lacustrine sediments with respect to the Swiss reference rock velocity model. The derived velocity profiles are shown to reproduce observed amplification functions from empirical spectral modeling. Although many sites are found to exhibit 1D behavior, our procedure allows the detection and qualification of 2D and 3D effects. All data collected during the site characterization procedures in the last 20 years are gathered in a database, implementing a data model proposed for community use at the European scale through NERA and EPOS (www.epos-eu.org). A web stationbook derived from it

  19. Characterizing the Locality of Diabatic States forElectronic Excitation Transfer By Decomposing theDiabatic Coupling

    Energy Technology Data Exchange (ETDEWEB)

    Newton, M.D.; Vura-Weis, J.; Wasielewski, M.R.; Subotnik, J.E.

    2010-10-19

    A common strategy to calculate electronic coupling matrix elements for charge or energy transfer is to take the adiabatic states generated by electronic structure computations and rotate them to form localized diabatic states. In this paper, we show that, for intermolecular transfer of singlet electronic excitation, usually we cannot fully localize the electronic excitations in this way. Instead, we calculate putative initial and final states with small excitation tails caused by weak interactions with high energy excited states in the electronic manifold. These tails do not lead to substantial changes in the total diabatic coupling between states, but they do lead to a different partitioning of the total coupling between Coulomb (Forster), exchange (Dexter), and one-electron components. The tails may be reduced by using a multistate diabatic model or eliminated entirely by truncation (denoted as 'chopping'). Without more information, we are unable to conclude with certainty whether the observed diabatic tails are a physical reality or a computational artifact. This research suggests that decomposition of the diabatic coupling between chromophores into Coulomb, exchange, and one-electron components may depend strongly on the number of states considered, and such results should be treated with caution.

  20. Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from Arabidopsis[OPEN

    Science.gov (United States)

    Ebert, Berit; Rautengarten, Carsten; Guo, Xiaoyuan; Xiong, Guangyan; Stonebloom, Solomon; Smith-Moritz, Andreia M.; Herter, Thomas; Chan, Leanne Jade G.; Adams, Paul D.; Petzold, Christopher J.; Pauly, Markus; Willats, William G.T.; Heazlewood, Joshua L.; Scheller, Henrik Vibe

    2015-01-01

    Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis. PMID:25804536

  1. Identification and characterization of a nuclear localization signal of TRIM28 that overlaps with the HP1 box

    Energy Technology Data Exchange (ETDEWEB)

    Moriyama, Tetsuji; Sangel, Percival [Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Yamaguchi, Hiroki [School of Medicine, Osaka University, Osaka 565-0871 (Japan); Obuse, Chikashi [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan); Miyamoto, Yoichi [Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Oka, Masahiro, E-mail: moka@nibiohn.go.jp [Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Yoneda, Yoshihiro, E-mail: y-yoneda@nibiohn.go.jp [National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan)

    2015-07-03

    Tripartite motif-containing 28 (TRIM28) is a transcription regulator, which forms a repressor complex containing heterochromatin protein 1 (HP1). Here, we report identification of a nuclear localization signal (NLS) within the 462-494 amino acid region of TRIM28 that overlaps with its HP1 binding site, HP1 box. GST-pulldown experiments revealed the interaction of the arginine-rich TRIM28 NLS with various importin α subtypes (α1, α2 and α4). In vitro transport assay demonstrated that nuclear localization of GFP-TRIM28 NLS is mediated by importin αs, in conjunction with importin β1 and Ran. Further, we demonstrated that HP1 and importin αs compete for binding to TRIM28. Together, our findings suggest that importin α has an essential role in the nuclear delivery and preferential HP1 interaction of TRIM28. - Highlights: • TRIM28 contains an NLS within the 462-494 amino acid region. • The nuclear import of TRIM28 is mediated by importin α/importin β1. • TRIM28 NLS overlaps with HP1 Box. • HP1 and importin α compete for binding to TRIM28.

  2. Sealing the mitochondrial respirasome.

    Science.gov (United States)

    Winge, Dennis R

    2012-07-01

    The mitochondrial respiratory chain is organized within an array of supercomplexes that function to minimize the generation of reactive oxygen species (ROS) during electron transfer reactions. Structural models of supercomplexes are now known. Another recent advance is the discovery of non-OXPHOS complex proteins that appear to adhere to and seal the individual respiratory complexes to form stable assemblages that prevent electron leakage. This review highlights recent advances in our understanding of the structures of supercomplexes and the factors that mediate their stability.

  3. Scanning capacitance microscopy and spectroscopy applied to local charge modifications and characterization of nitride-oxide-silicon heterostructures

    Science.gov (United States)

    Dreyer, M.; Wiesendanger, R.

    1995-10-01

    We have combined a home-built capacitance sensor with a commercial scanning force microscope to obtain a Scanning Capacitance Microscope (SCM). The SCM has been used to study Nitride-Oxide-Silicon (NOS) heterostructures which offer potential applications in charge storage technology. Charge writing and reading on a submicrometer scale is demonstrated with our SCM setup. In addition, SCM appears to be very useful for the characterization of subsurface defects in semiconductor devices which are inaccessible by most of the other scanning probe microscopies. Finally, we introduce a novel spectroscopic mode of SCM operation which offers combined voltage-dependent and spatially resolved information about inhomogeneous charge distributions in semiconductor devices.

  4. Mitochondrial iron metabolism and sideroblastic anemia.

    Science.gov (United States)

    Sheftel, Alex D; Richardson, Des R; Prchal, Josef; Ponka, Prem

    2009-01-01

    Sideroblastic anemias are a heterogeneous group of disorders, characterized by mitochondrial iron overload in developing red blood cells. The unifying characteristic of all sideroblastic anemias is the ring sideroblast, which is a pathological erythroid precursor containing excessive deposits of non-heme iron in mitochondria with perinuclear distribution creating a ring appearance. Sideroblastic anemias may be hereditary or acquired. Hereditary sideroblastic anemias are caused by defects in genes present on the X chromosome (mutations in the ALAS2, ABCB7, or GRLX5 gene), genes on autosomal chromosomes, or mitochondrial genes. Acquired sideroblastic anemias are either primary (refractory anemia with ring sideroblasts, RARS, representing one subtype of the myelodysplastic syndrome) or secondary due to some drugs, toxins, copper deficiency, or chronic neoplastic disease. The pathogenesis of mitochondrial iron loading in developing erythroblasts is diverse. Ring sideroblasts can develop as a result of a heme synthesis defect in erythroblasts (ALAS2 mutations), a defect in iron-sulfur cluster assembly, iron-sulfur protein precursor release from mitochondria (ABCB7 mutations), or by a defect in intracellular iron metabolism in erythroid cells (e.g. RARS).

  5. Mitochondrial transcription: is a pattern emerging?

    Science.gov (United States)

    Jaehning, J A

    1993-04-01

    Despite the striking similarities of RNA polymerases and transcription signals shared by eubacteria, archaebacteria and eukaryotes, there has been little indication that transcription in mitochondria is related to any previously characterized model. Only in yeast has the subunit structure of the mitochondrial RNA polymerase been determined. The yeast enzyme is composed of a core related to polymerases from bacteriophage T7 and T3, and a promoter recognition factor similar to bacterial sigma factors. Soluble systems for studying mitochondrial transcript initiation in vitro have been described from several organisms, and used to determine consensus sequences at or near transcription start sites. Comparison of these sequences from fungi, plants, and amphibians with the T7/T3 promoter suggests some intriguing similarities. Mammalian mitochondrial promoters do not fit this pattern but instead appear to utilize upstream sites, the target of a transcriptional stimulatory factor, to position the RNA polymerase. The recent identification of a possible homologue of the mammalian upstream factor in yeast mitochondria may indicate that a pattern will eventually be revealed relating the transcriptional machineries of all eukaryotic mitochondria.

  6. 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.

  7. A whole mitochondrial genome screening in a MELAS patient: A novel mitochondrial tRNA{sup Val} mutation

    Energy Technology Data Exchange (ETDEWEB)

    Mezghani, Najla [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Mnif, Mouna [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Kacem, Maha [Service de Medecine interne, C.H.U. Fattouma Bourguiba de Monastir (Tunisia); Mkaouar-Rebai, Emna, E-mail: emna_mkaouar@mail2world.com [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Hadj Salem, Ikhlass [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Kallel, Nozha; Charfi, Nadia; Abid, Mohamed [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Fakhfakh, Faiza [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia)

    2011-04-22

    Highlights: {yields} We report a young Tunisian patient with clinical features of MELAS syndrome. {yields} Reported mitochondrial mutations were absent after a mutational screening of the whole mtDNA. {yields} We described a novel m.1640A>G mutation in the tRNA{sup Val} gene which was absent in 150 controls. {yields} Mitochondrial deletions and POLG1 gene mutations were absent. {yields} The m.1640A>G mutation could be associated to MELAS syndrome. -- Abstract: Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA{sup Val}. This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.

  8. Using Locally Generated Magnetic Indices to Characterize the Ionosphere From Magnetic Data Acquisition System (Magdas Ground Based Observatories in Nigeria.

    Directory of Open Access Journals (Sweden)

    U.C. Rabiu

    2013-06-01

    Full Text Available This work presents an attempt to establish a baseline for geomagnetic indices inNigeria. This is particularly very crucial since these indices give indications of theseverity of magnetic fluctuations, and hence the level of disturbances in theionosphere. K (an index which measures the magnetic perturbations of theplanetary field and A (a linear measure of the Earth's field that provides a dailyaverage level for geomagnetic activity geomagnetic indices were generated locallyfrom geomagnetic data obtained using ground based MAGDAS magnetometerslocated at Abuja (9 ̊ 40’N, 7 ̊ 29’E, Ilorin (8 ̊30’N, 4 ̊33’E and Lagos (6 ̊27’N,3 ̊23’E in Nigeria using Computer-based derivation. The indices generated wereused to characterize the ionosphere over the Magdas magnetometer Nigerianetwork stations. Results obtained showed average K values of 3.5 (ABU, 4.60(LAG and 4.13 (ILR, the ionosphere over the three stations was found to berelatively active (4.08 thus setting the baseline for characterizing the ionosphereover Nigeria from ground based magnetometers.

  9. Molecular Genetics of Mitochondrial Disorders

    Science.gov (United States)

    Wong, Lee-Jun C.

    2010-01-01

    Mitochondrial respiratory chain (RC) disorders (RCDs) are a group of genetically and clinically heterogeneous diseases because of the fact that protein components of the RC are encoded by both mitochondrial and nuclear genomes and are essential in all cells. In addition, the biogenesis, structure, and function of mitochondria, including DNA…

  10. Redox Homeostasis and Mitochondrial Dynamics

    NARCIS (Netherlands)

    Willems, P.H.G.M.; Rossignol, R.; Dieteren, C.E.J.; Murphy, M.P.; Koopman, W.J.H.

    2015-01-01

    Within living cells, mitochondria are considered relevant sources of reactive oxygen species (ROS) and are exposed to reactive nitrogen species (RNS). During the last decade, accumulating evidence suggests that mitochondrial (dys)function, ROS/RNS levels, and aberrations in mitochondrial morphology

  11. Mitochondrial disorders and the eye

    Directory of Open Access Journals (Sweden)

    O’Neill EC

    2011-09-01

    Full Text Available Nicole J Van Bergen, Rahul Chakrabarti, Evelyn C O'Neill, Jonathan G Crowston, Ian A TrounceCentre for Eye Research Australia, Department of Ophthalmology, University of Melbourne, Victoria, AustraliaAbstract: The clinical significance of disturbed mitochondrial function in the eye has emerged since mitochondrial DNA (mtDNA mutation was described in Leber's hereditary optic neuropathy. The spectrum of mitochondrial dysfunction has become apparent through increased understanding of the contribution of nuclear and somatic mtDNA mutations to mitochondrial dynamics and function. Common ophthalmic manifestations of mitochondrial dysfunction include optic atrophy, pigmentary retinopathy, and ophthalmoplegia. The majority of patients with ocular manifestations of mitochondrial disease also have variable central and peripheral nervous system involvement. Mitochondrial dysfunction has recently been associated with age-related retinal disease including macular degeneration and glaucoma. Therefore, therapeutic targets directed at promoting mitochondrial biogenesis and function offer a potential to both preserve retinal function and attenuate neurodegenerative processes.Keywords: mitochondria, disease, retina, eye, aging, neuroprotection

  12. Defects in mitochondrial fission protein dynamin-related protein 1 are linked to apoptotic resistance and autophagy in a lung cancer model.

    Directory of Open Access Journals (Sweden)

    Kelly Jean Thomas

    Full Text Available Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549 cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype-mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1. A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy. A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.

  13. Characterization of atmospheric aerosols in Ile-de-France: Local contribution and Long range transport; Caracteisation des aeosols atmospheiques en Ile-de-France: contribution locale et transport a longues distances

    Energy Technology Data Exchange (ETDEWEB)

    Cuesta, J.E

    2006-06-15

    Atmospheric aerosols interact directly in a great number of processes related to climate change and public health, modifying the energy budget and partly determining the quality of the air we breathe. In my PhD, I chose to study the perturbation, if not the aggravation, of the living conditions in Ile-de-France associated to aerosol transport episodes in the free troposphere. This situation is rather frequent and still badly known. To achieve my study, I developed the observation platform 'TReSS' Transportable Remote Sensing Station, whose instruments were developed at the Laboratoire de Meteorology Dynamique by the LiMAG team. 'TReSS' consists of a new high-performance 'Mini-Lidar' and of two standard radiometers: a sun photometer and a thermal infrared radiometer. The principle of my experimental approach is the synergy of the vertical Lidar profiles and the particle size distributions over the column, obtained by the 'Almucantar' inversion of sun photometer data. The new 'Lidar and Almucantar' method characterizes the vertical distribution by layer and the optical micro-physical properties of the local and transported aerosols. Firstly, I undertook the characterization of the Paris aerosol, mainly of anthropogenic origin. Their radiative properties were analyzed in the daily and yearly scales. Then, I conducted a statistical multi-year study of transport episodes and a two-week study case, representative of a succession of desert dust intrusion in Ile-de-France. My PhD work concludes by a study on the impact of biomass burning aerosols during the heat wave on August 2003. I study the impact of the transported aerosols into the local radiative budget and the possible consequences on the diurnal cycle of the atmospheric boundary layer. (author)

  14. Characterization and interpretation of the Edge Snake in between type-I edge localized modes at ASDEX Upgrade

    Energy Technology Data Exchange (ETDEWEB)

    Sommer, F; Guenter, S; Kallenbach, A; Maraschek, M; Boom, J; Fischer, R; Hicks, N; Reiter, B; Wolfrum, E [Max-Planck-Institut fuer Plasmaphysik, Boltzmannstrasse 2, D-85748 Garching, EURATOM Association (Germany); Luhmann, N C Jr [University of California at Davis, Davis, CA 95616 (United States); Park, H K [POSTECH, Pahang, Gyeongbuk 790-784 (Korea, Republic of); Wenninger, R, E-mail: fabian.sommer@ipp.mpg.de [Universitaetssternwarte der Ludwig-Maximilians-Universitaet, D-81679 Muenchen (Germany)

    2011-08-15

    A new magnetohydrodynamic instability called the 'Edge Snake', which was found in 2006 at the tokamak ASDEX Upgrade during type-I ELMy H-modes, is investigated. It is located within the separatrix in the region of high temperature and density gradients and has a toroidal mode number of n = 1. The Edge Snake consists of a radially and poloidally strongly localized current wire, in which the temperature and density profiles flatten. This significant reduction in pressure gradient leads to a reduction in the neoclassical Bootstrap current and can plausibly explain the drive of the instability. The experimental observations point towards a magnetic island with a defect current inside the O-point of the island. The Edge Snake is compared with similar instabilities at JET, DIII-D and ASDEX Upgrade.

  15. Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

    Science.gov (United States)

    De Wolf, M; Lagrou, A; Hilderson, H J; Dierick, W

    1978-12-01

    In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.

  16. Characterization and sub-cellular localization of GalNAc-binding proteins isolated from human hepatic stellate cells.

    Science.gov (United States)

    Zhong, Yaogang; Zhang, Jing; Yu, Hanjie; Zhang, Jiaxu; Sun, Xiu-Xuan; Chen, Wentian; Bian, Huijie; Li, Zheng

    2015-12-25

    Although the expression levels of total GalNAc-binding proteins (GNBPs) were up-regulated significantly in human hepatic stellate cells (HSCs) activated with transforming growth factor-β1(TGF-β1), yet little is known about the precise types, distribution and sub-cellular localization of the GNBPs in HSCs. Here, 264 GNBPs from the activated HSCs and 257 GNBPs from the quiescent HSCs were identified and annotated. A total of 46 GNBPs were estimated to be significantly up-regulated and 40 GNBPs were estimated to be significantly down-regulated in the activated HSCs. For example, the GNBPs (i.e. BTF3, COX17, and ATP5A1) responsible for the regulation of protein binding were up-regulated, and those (i.e. FAM114A1, ENO3, and TKT) responsible for the regulation of protein binding were down-regulated in the activated HSCs. The motifs of the isolated GNBPs showed that Proline residue had the maximum preference in consensus sequences. The western blotting showed the expression levels of COX17, and PRMT1 were significantly up-regulated, while, the expression level of CLIC1(B5) was down-regulated in the activated HSCs and liver cirrhosis tissues. Moreover, the GNBPs were sub-localized in the Golgi apparatus of HSCs. In conclusion, the precision alteration of the GNBPs referred to pathological changes in liver fibrosis/cirrhosis may provide useful information to find new molecular mechanism of HSC activation and discover the biomarkers for diagnosis of liver fibrosis/cirrhosis as well as development of new anti-fibrotic strategies.

  17. Mitochondrial Dysfunction in Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    P. C. Keane

    2011-01-01

    Full Text Available Parkinson's disease (PD is a progressive, neurodegenerative condition that has increasingly been linked with mitochondrial dysfunction and inhibition of the electron transport chain. This inhibition leads to the generation of reactive oxygen species and depletion of cellular energy levels, which can consequently cause cellular damage and death mediated by oxidative stress and excitotoxicity. A number of genes that have been shown to have links with inherited forms of PD encode mitochondrial proteins or proteins implicated in mitochondrial dysfunction, supporting the central involvement of mitochondria in PD. This involvement is corroborated by reports that environmental toxins that inhibit the mitochondrial respiratory chain have been shown to be associated with PD. This paper aims to illustrate the considerable body of evidence linking mitochondrial dysfunction with neuronal cell death in the substantia nigra pars compacta (SNpc of PD patients and to highlight the important need for further research in this area.

  18. Muscle regeneration in mitochondrial myopathies

    DEFF Research Database (Denmark)

    Krag, T O; Hauerslev, S; Jeppesen, T D;

    2013-01-01

    Mitochondrial myopathies cover a diverse group of disorders in which ragged red and COX-negative fibers are common findings on muscle morphology. In contrast, muscle degeneration and regeneration, typically found in muscular dystrophies, are not considered characteristic features of mitochondrial...... myopathies. We investigated regeneration in muscle biopsies from 61 genetically well-defined patients affected by mitochondrial myopathy. Our results show that the perturbed energy metabolism in mitochondrial myopathies causes ongoing muscle regeneration in a majority of patients, and some were even affected...... by a dystrophic morphology. The results add to the complexity of the pathogenesis underlying mitochondrial myopathies, and expand the knowledge about the impact of energy deficiency on another aspect of muscle structure and function....

  19. Mitochondrial dynamics and peripheral neuropathy.

    Science.gov (United States)

    Baloh, Robert H

    2008-02-01

    Peripheral neuropathy is perhaps the archetypal disease of axonal degeneration, characteristically involving degeneration of the longest axons in the body. Evidence from both inherited and acquired forms of peripheral neuropathy strongly supports that the primary pathology is in the axons themselves and points to disruption of axonal transport as an important disease mechanism. Recent studies in human genetics have further identified abnormalities in mitochondrial dynamics--the fusion, fission, and movement of mitochondria--as a player in the pathogenesis of inherited peripheral neuropathy. This review provides an update on the mechanisms of mitochondrial trafficking in axons and the emerging relationship between the disruption of mitochondrial dynamics and axonal degeneration. Evidence suggests mitochondria are a "critical cargo" whose transport is necessary for proper axonal and synaptic function. Importantly, understanding the regulation of mitochondrial movement and the consequences of decreased axonal mitochondrial function may define new paths for therapeutic agents in peripheral neuropathy and other neurodegenerative diseases.

  20. Endocrine disorders in mitochondrial disease.

    Science.gov (United States)

    Schaefer, Andrew M; Walker, Mark; Turnbull, Douglass M; Taylor, Robert W

    2013-10-15

    Endocrine dysfunction in mitochondrial disease is commonplace, but predominantly restricted to disease of the endocrine pancreas resulting in diabetes mellitus. Other endocrine manifestations occur, but are relatively rare by comparison. In mitochondrial disease, neuromuscular symptoms often dominate the clinical phenotype, but it is of paramount importance to appreciate the multi-system nature of the disease, of which endocrine dysfunction may be a part. The numerous phenotypes attributable to pathogenic mutations in both the mitochondrial (mtDNA) and nuclear DNA creates a complex and heterogeneous catalogue of disease which can be difficult to navigate for novices and experts alike. In this article we provide an overview of the endocrine disorders associated with mitochondrial disease, the way in which the underlying mitochondrial disorder influences the clinical presentation, and how these factors influence subsequent management.

  1. Fatal hypertensive crisis as presentation of mitochondrial complex I deficiency.

    NARCIS (Netherlands)

    Lohmeier, K.; Distelmaier, F.; Heuvel, L.P.W.J. van den; Rodenburg, R.J.T.; Smeitink, J.A.M.; Mayatepek, E.; Hoehn, T.

    2007-01-01

    Complex I deficiency is a frequent defect of the mitochondrial electron transport chain. We report on a 3-year-old boy, who rapidly deteriorated after sudden flushing and collapse. This fatal and unusual case was characterized by widely uncontrollable arterial hypertension. It might indicate that hy

  2. Characterization of the local density-of-states fluctuations near the integer quantum Hall transition in a quantum-dot array

    Science.gov (United States)

    Jug, Giancarlo; Ziegler, Klaus

    1997-10-01

    We present a calculation for the second moment of the local density of states in a model of a two-dimensional quantum dot array near the quantum Hall transition. The quantum dot array model is a realistic adaptation of the lattice model for the quantum Hall transition in the two-dimensional electron gas in an external magnetic field proposed by Ludwig, Fisher, Shankar, and Grinstein. We make use of a Dirac fermion representation for the Green's functions in the presence of fluctuations for the quantum dot energy levels. A saddle-point approximation yields nonperturbative results for the first and second moments of the local density of states, showing interesting fluctuation behavior near the quantum Hall transition. To our knowledge we discuss here one of the first analytic characterizations of chaotic behavior for a two-dimensional mesoscopic structure. The connection with possible experimental investigations of the local density of states in the quantum dot array structures (by means of NMR Knight-shift or single-electron-tunneling techniques) and our work is also established.

  3. Paclitaxel loaded biodegradable poly (sebacic acid-co-ricinoleic acid cylindrical implants for local delivery-in vitro characterization

    Directory of Open Access Journals (Sweden)

    Jagadeesh G Hiremath

    2013-01-01

    Full Text Available The aim of the present research work was to develop the biodegradable polymeric implant for the delivery of antineoplastic drug, paclitaxel (PTX using poly (sebacic-co-recinoleic acid 70:30 w/w. PTX loaded implants were prepared by indigenously developed melt molding technique. Implants were characterized in terms of physico-chemical evaluations, drug content, drug stability and intactness, thermal analysis, drug physical state and crystallinity, surface morphology, hydrolytic degradation, drug release and its kinetics. Prepared implants were yellow and cylindrical in shape with smooth surfaces. Drug in the implants was found to be stable, intact and uniformly dispersed as amorphous state within the polymer matrix. In vitro release, kinetic studies showed zero order and Korsmeyer-Peppas model release being exhibited. Drug release from the polymeric implants was occurred could be as results of diffusion.

  4. Topical Tacrolimus and Periodontal Therapy in the Management of a Case of Oral Chronic GVHD Characterized by Specific Gingival Localization

    Directory of Open Access Journals (Sweden)

    Davide Conrotto

    2014-01-01

    Full Text Available Background. Chronic graft versus host disease (cGVHD is a complication following bone marrow transplantation. The oral lesions are difficult to control with a systemic pharmacological therapy. Case Description. A 63-year-old female patient, who underwent an allogeniec transplantation for acute myeloid leukemia, developed a chronic oral and cutaneous GVHD. The patient was treated with topical tacrolimus 0.1%, twice daily for two months, and underwent a protocol of oral hygiene characterized by 3 appointments of scaling, root planning, and daily oral hygiene instructions. The patient showed marked resolution of gingival lesions and a significant improvement of related pain and gingival inflammatory indexes. Clinical Implications. This case report suggests that treatment with topical tacrolimus and professional oral hygiene may be helpful in the management of chronic oral GVHD with severe gingival involvement.

  5. Topical tacrolimus and periodontal therapy in the management of a case of oral chronic GVHD characterized by specific gingival localization.

    Science.gov (United States)

    Conrotto, Davide; Broccoletti, Roberto; Carcieri, Paola; Giaccone, Luisa; Arduino, Paolo G

    2014-01-01

    Background. Chronic graft versus host disease (cGVHD) is a complication following bone marrow transplantation. The oral lesions are difficult to control with a systemic pharmacological therapy. Case Description. A 63-year-old female patient, who underwent an allogeniec transplantation for acute myeloid leukemia, developed a chronic oral and cutaneous GVHD. The patient was treated with topical tacrolimus 0.1%, twice daily for two months, and underwent a protocol of oral hygiene characterized by 3 appointments of scaling, root planning, and daily oral hygiene instructions. The patient showed marked resolution of gingival lesions and a significant improvement of related pain and gingival inflammatory indexes. Clinical Implications. This case report suggests that treatment with topical tacrolimus and professional oral hygiene may be helpful in the management of chronic oral GVHD with severe gingival involvement.

  6. Mitochondrial dysfunction in neuromuscular disorders.

    Science.gov (United States)

    Katsetos, Christos D; Koutzaki, Sirma; Melvin, Joseph J

    2013-09-01

    This review deciphers aspects of mitochondrial (mt) dysfunction among nosologically, pathologically, and genetically diverse diseases of the skeletal muscle, lower motor neuron, and peripheral nerve, which fall outside the traditional realm of mt cytopathies. Special emphasis is given to well-characterized mt abnormalities in collagen VI myopathies (Ullrich congenital muscular dystrophy and Bethlem myopathy), megaconial congenital muscular dystrophy, limb-girdle muscular dystrophy type 2 (calpainopathy), centronuclear myopathies, core myopathies, inflammatory myopathies, spinal muscular atrophy, Charcot-Marie-Tooth neuropathy type 2, and drug-induced peripheral neuropathies. Among inflammatory myopathies, mt abnormalities are more prominent in inclusion body myositis and a subset of polymyositis with mt pathology, both of which are refractory to corticosteroid treatment. Awareness is raised about instances of phenotypic mimicry between cases harboring primary mtDNA depletion, in the context of mtDNA depletion syndrome, and established neuromuscular disorders such as spinal muscular atrophy. A substantial body of experimental work, derived from animal models, attests to a major role of mitochondria (mt) in the early process of muscle degeneration. Common mechanisms of mt-related cell injury include dysregulation of the mt permeability transition pore opening and defective autophagy. The therapeutic use of mt permeability transition pore modifiers holds promise in various neuromuscular disorders, including muscular dystrophies.

  7. Identification and characterization of human cDNAs specific to BCS1, PET112, SCO1, COX15, and COX11, five genes involved in the formation and function of the mitochondrial respiratory chain.

    Science.gov (United States)

    Petruzzella, V; Tiranti, V; Fernandez, P; Ianna, P; Carrozzo, R; Zeviani, M

    1998-12-15

    We have successfully applied a strategy based on the "cyberscreening" of the expressed sequence tags database using yeast protein sequences as "probes" to identify the human gene orthologs to BCS1, COX15, PET112, COX11, and SCO1, five yeast genes involved in the biogenesis of the mitochondrial respiratory chain complexes. In yeast, BCS1 is involved mainly in the assembly of complex III, while the other genes appear to control the structure/function of cytochrome-c oxidase. Significant amino acid identity and similarity were demonstrated by comparison of the human with the corresponding yeast polypeptides. Sequence alignment revealed numerous colinear identical regions and the conservation of functional domains. Mitochondrial targeting of the human gene products, suggested by computer analysis of the protein sequences, was confirmed by an in vitro import and protease-protection assay. These data strongly suggest that the human gene products share similar or identical functions with their yeast homologues. Genes controlling the structure/function of the respiratory chain complexes are attractive candidates for human mitochondrial disorders such as Leigh disease. However, both sequence analysis and functional complementation assays on an index patient do not support an etiological role for any of these genes.

  8. PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.

    Science.gov (United States)

    Hafez, Mohamed; Guha, Tuhin Kumar; Shen, Chen; Sethuraman, Jyothi; Hausner, Georg

    2014-01-01

    Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.

  9. ES1 is a mitochondrial enlarging factor contributing to form mega-mitochondria in zebrafish cones.

    Science.gov (United States)

    Masuda, Takamasa; Wada, Yasutaka; Kawamura, Satoru

    2016-03-01

    Total mass of mitochondria increases during cell proliferation and differentiation through mitochondrial biogenesis, which includes mitochondrial proliferation and growth. During the mitochondrial growth, individual mitochondria have been considered to be enlarged independently of mitochondrial fusion. However, molecular basis for this enlarging process has been poorly understood. Cone photoreceptor cells in the retina possess large mitochondria, so-called mega-mitochondria that have been considered to arise via the enlarging process. Here we show that ES1 is a novel mitochondria-enlarging factor contributing to form mega-mitochondria in cones. ES1 is specifically expressed in cones and localized to mitochondria including mega-mitochondria. Knockdown of ES1 markedly reduced the mitochondrial size in cones. In contrast, ectopic expression of ES1 in rods significantly increased both the size of individual mitochondria and the total mass of the mitochondrial cluster without changing the number of them. RNA-seq analysis showed that ERRα and its downstream mitochondrial genes were significantly up-regulated in the ES1-expressing rods, suggesting facilitation of mitochondrial enlargement via ERRα-dependent processes. Furthermore, higher energy state was detected in the ES1-expressing rods, indicating that the enlarged mitochondria by ES1 are capable of producing high energy. ES1 is the mitochondrial protein that is first found to promote enlargement of individual mitochondria.

  10. G alpha12 is targeted to the mitochondria and affects mitochondrial morphology and motility.

    Science.gov (United States)

    Andreeva, Alexandra V; Kutuzov, Mikhail A; Voyno-Yasenetskaya, Tatyana A

    2008-08-01

    G alpha12 constitutes, along with G alpha13, one of the four families of alpha subunits of heterotrimeric G proteins. We found that the N terminus of G alpha12, but not those of other G alpha subunits, contains a predicted mitochondrial targeting sequence. Using confocal microscopy and cell fractionation, we demonstrated that up to 40% of endogenous G alpha12 in human umbilical vein endothelial cells colocalize with mitochondrial markers. N-terminal sequence of G alpha12 fused to GFP efficiently targeted the fusion protein to mitochondria. G alpha12 with mutated mitochondrial targeting sequence was still located in mitochondria, suggesting the existence of additional mechanisms for mitochondrial localization. Lysophosphatidic acid, one of the known stimuli transduced by G alpha12/13, inhibited mitochondrial motility, while depletion of endogenous G alpha12 increased mitochondrial motility. G alpha12Q229L variants uncoupled from RhoGEFs (but not fully functional activated G alpha12Q229L) induced transformation of the mitochondrial network into punctate mitochondria and resulted in a loss of mitochondrial membrane potential. All examined G alpha12Q229L variants reduced phosphorylation of Bcl-2 at Ser-70, while only mutants unable to bind RhoGEFs also decreased cellular levels of Bcl-2. These G alpha12 mutants were also more efficient Hsp90 interactors. These findings are the first demonstration of a heterotrimeric G protein alpha subunit specifically targeted to mitochondria and involved in the control of mitochondrial morphology and dynamics.

  11. The mitochondrial Ca2+ uniporter: regulation by auxiliary subunits and signal transduction pathways.

    Science.gov (United States)

    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.

  12. Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals.

    Science.gov (United States)

    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.

  13. Pim-1 preserves mitochondrial morphology by inhibiting dynamin-related protein 1 translocation.

    Science.gov (United States)

    Din, Shabana; Mason, Matthew; Völkers, Mirko; Johnson, Bevan; Cottage, Christopher T; Wang, Zeping; Joyo, Anya Y; Quijada, Pearl; Erhardt, Peter; Magnuson, Nancy S; Konstandin, Mathias H; Sussman, Mark A

    2013-04-09

    Mitochondrial morphological dynamics affect the outcome of ischemic heart damage and pathogenesis. Recently, mitochondrial fission protein dynamin-related protein 1 (Drp1) has been identified as a mediator of mitochondrial morphological changes and cell death during cardiac ischemic injury. In this study, we report a unique relationship between Pim-1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemic stress. Transgenic hearts overexpressing cardiac Pim-1 display reduction of total Drp1 protein levels, increased phosphorylation of Drp1-(S637), and inhibition of Drp1 localization to the mitochondria. Consistent with these findings, adenoviral-induced Pim-1 neonatal rat cardiomyocytes (NRCMs) retain a reticular mitochondrial phenotype after simulated ischemia (sI) and decreased Drp1 mitochondrial sequestration. Interestingly, adenovirus Pim-dominant negative NRCMs show increased expression of Bcl-2 homology 3 (BH3)-only protein p53 up-regulated modulator of apoptosis (PUMA), which has been previously shown to induce Drp1 accumulation at mitochondria and increase sensitivity to apoptotic stimuli. Overexpression of the p53 up-regulated modulator of apoptosis-dominant negative adenovirus attenuates localization of Drp1 to mitochondria in adenovirus Pim-dominant negative NRCMs promotes reticular mitochondrial morphology and inhibits cell death during sI. Therefore, Pim-1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to sI.

  14. A specific isoform of poly(ADP-ribose) glycohydrolase is targeted to the mitochondrial matrix by a N-terminal mitochondrial targeting sequence

    Energy Technology Data Exchange (ETDEWEB)

    Whatcott, Clifford J. [Department of Pharmacology and Toxicology, College of Pharmacy, Arizona Cancer Center, University of Arizona, Tucson, AZ 85728 (United States); Meyer-Ficca, Mirella L.; Meyer, Ralph G. [Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, NBC Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, Kennett Square, PA 19348 (United States); Jacobson, Myron K., E-mail: mjacobson@pharmacy.arizona.edu [Department of Pharmacology and Toxicology, College of Pharmacy, Arizona Cancer Center, University of Arizona, Tucson, AZ 85728 (United States)

    2009-12-10

    Poly(ADP-ribose) polymerases (PARPs) convert NAD to polymers of ADP-ribose that are converted to free ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG). The activation of the nuclear enzyme PARP-1 following genotoxic stress has been linked to release of apoptosis inducing factor from the mitochondria, but the mechanisms by which signals are transmitted between nuclear and mitochondrial compartments are not well understood. The study reported here has examined the relationship between PARG and mitochondria in HeLa cells. Endogenous PARG associated with the mitochondrial fraction migrated in the range of 60 kDa. Transient transfection of cells with PARG expression constructs with amino acids encoded by exon 4 at the N-terminus was targeted to the mitochondria as demonstrated by subcellular fractionation and immunofluorescence microscopy of whole cells. Deletion and missense mutants allowed identification of a canonical N-terminal mitochondrial targeting sequence consisting of the first 16 amino acids encoded by PARG exon 4. Sub-mitochondrial localization experiments indicate that this mitochondrial PARG isoform is targeted to the mitochondrial matrix. The identification of a PARG isoform as a component of the mitochondrial matrix raises several