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Modes of Metabolic Compensation during Mitochondrial Disease Using the Drosophila Model of ATP6 Dysfunction  [PDF]
Alicia M. Celotto, Wai Kan Chiu, Wayne Van Voorhies, Michael J. Palladino
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0025823
Abstract: Numerous mitochondrial DNA mutations cause mitochondrial encephalomyopathy: a collection of related diseases for which there exists no effective treatment. Mitochondrial encephalomyopathies are complex multisystem diseases that exhibit a relentless progression of severity, making them both difficult to treat and study. The pathogenic and compensatory metabolic changes that are associated with chronic mitochondrial dysfunction are not well understood. The Drosophila ATP61 mutant models human mitochondrial encephalomyopathy and allows the study of metabolic changes and compensation that occur throughout the lifetime of an affected animal. ATP61animals have a nearly complete loss of ATP synthase activity and an acute bioenergetic deficit when they are asymptomatic, but surprisingly we discovered no chronic bioenergetic deficit in these animals during their symptomatic period. Our data demonstrate dynamic metabolic compensatory mechanisms that sustain normal energy availability and activity despite chronic mitochondrial complex V dysfunction resulting from an endogenous mutation in the mitochondrial DNA. ATP61animals compensate for their loss of oxidative phosphorylation through increases in glycolytic flux, ketogenesis and Kreb's cycle activity early during pathogenesis. However, succinate dehydrogenase activity is reduced and mitochondrial supercomplex formation is severely disrupted contributing to the pathogenesis seen in ATP61 animals. These studies demonstrate the dynamic nature of metabolic compensatory mechanisms and emphasize the need for time course studies in tractable animal systems to elucidate disease pathogenesis and novel therapeutic avenues.
Evaluatoin of Biochemical Changes Associated with Replacement of Maize with Whole Cassava Root Meal in the Diet of Hybrid Catfish
O.M.G. Abu,U.U. Gabriel,L.O. Sanni,O.A. Akinrotimi
Journal of Aquaculture Feed Science and Nutrition , 2012,
Abstract: This study was done to evaluate the biochemical changes associated with the replacement of maize with varying levels of whole cassava root meal in the diet of hybrid catfish. Four diets with varying replacement levels of maize with whole cassava root meal at 0% (Ao), 33% (B33), 66% (C66) and 100% (D100) were formulated and fed to hybrid catfish fingerlings for a period of 32 weeks. And the following biochemical properties were evaluated: serum metabolites namely creatinine, urea, total protein, bilurubin a. and b., while for serum enzymes aspartate aminotransferase (AST), alanine amino transferase (ALT), Alkaline phosphatase (ALP) and Lactate Dehydrogenase (LDH). The results indicated that creative and total protein were significant (p>0.05) in all the treated diets and had a concentration depended increase above the control values. Whereas the reverse was the case with bilirubin b., which was higher in the control than in the treated group. For the serum enzymes evaluated in this trial, replacement of maize with whole cassava root meal caused a significant declined (p<0.05) in serum aspartate aminotransferase (AST), alkaline phoslatase (ALP) and Lactate Dehydrogenase (LDH) at all levels. While the values of Alamine amino transferase (ALT) increased considerably (p<0.05). The mean values of these metabolites and enzymes were within the acceptable range for normal metabolism of hybrid catfish. It can therefore be concluded that whole cassava root meal can adequately replace maize without any adverse effects on the biochemical properties of the fish.
Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences
Liane Fendt, Bettina Zimmermann, Martin Daniaux, Walther Parson
BMC Genomics , 2009, DOI: 10.1186/1471-2164-10-139
Abstract: We amplified whole mitochondrial genomes as two overlapping PCR-fragments comprising each about 8500 bases in length. We developed a set of 96 primers that can be applied to a (manual) 96 well-based technology, which resulted in at least double strand sequence coverage of the entire coding region (codR).This elaborated sequencing strategy is straightforward and allows for an unambiguous sequence analysis and interpretation including sometimes challenging phenomena such as point and length heteroplasmy that are relevant for the investigation of forensic and clinical samples.Investigations of the human mt genome are in the focus of biological and medical scientific disciplines. Compared to nuclear DNA (nDNA), mitochondrial DNA (mtDNA) is more vulnerable to oxidative damage and undergoes a higher rate of mutation [1]. Because of these features the analysis of the mt genome has become a proven tool in population genetics. A multi-copy genome without recombination which accumulates mutations allows for the establishment of phylogenetic trees [2]. It was the information from the highly variable mitochondrial control region (CR) that lifted the secret of human evolution starting in Africa about 150000 years ago and gave an insight in human migration all over the world within the past 60000 years [3,4]. Sequences of full mt genomes are necessary to decipher yet not defined haplotypes and assign them to their phylogeographic environment.Mitochondrial DNA mutations in the coding region (codR) have been associated with several pathologies [5] including cancer [6-9]. During oxidative phosphorylation (OXPHOS) mitochondria produce reactive oxidative species (ROS) that potentially induce DNA mutations. Such an initial mutation is heteroplasmic with the mutated variant constituting a minority [10]. In the course of several replications the heteroplasmic mutation may become dominant leading to cancer [8]. This theory is based on the results of several investigations on cancer tissue
Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis  [PDF]
Thierry Rabilloud
Quantitative Biology , 2008, DOI: 10.1007/978-1-59745-028-7_6
Abstract: Mitochondria are complex organelles, and their proteomics analysis requires a combination of techniques. The emphasis in this chapter is made first on mitochondria preparation from cultured mammalian cells, then on the separation of the mitochondrial proteins with two-dimensional electrophoresis (2DE), showing some adjustment over the classical techniques to improve resolution of the mitochondrial proteins. This covers both the protein solubilization, the electrophoretic part per se, and the protein detection on the gels, which makes the interface with the protein identification part relying on mass spectrometry.
Nonlinear Impedance of Whole Cells Near an Electrode as a Probe of Mitochondrial Activity  [PDF]
Akilan Palanisami,George T. Mercier,Jie Fang,John H. Miller Jr.
Biosensors , 2011, DOI: 10.3390/bios1020046
Abstract: By simultaneously measuring the bulk media and electrode interface voltages of a yeast ( Saccharomyces cerevisiae) suspension subjected to an AC voltage, a yeast-dependent nonlinear response was found only near the current injection electrodes. Computer simulation of yeast near a current injection electrode found an enhanced voltage drop across the yeast near the electrode due to slowed charging of the electrode interfacial capacitance. This voltage drop is sufficient to induce conformation change in membrane proteins. Disruption of the mitochondrial electron transport chain is found to significantly change the measured nonlinear current response, suggesting nonlinear impedance can be used as a non-invasive probe of cellular metabolic activity.
A pilot study for channel catfish whole genome sequencing and de novo assembly
Yanliang Jiang, Jianguo Lu, Eric Peatman, Huseyin Kucuktas, Shikai Liu, Shaolin Wang, Fanyue Sun, Zhanjiang Liu
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-629
Abstract: A combination of low sequence coverage of 454 and Illumina sequencing appeared to provide effective assembly as reflected by a high N50 value. Using 454 sequencing alone, a sequencing depth of 18 X was sufficient to obtain the good quality assembly, whereas a 70 X Illumina appeared to be sufficient for a good quality assembly. Additional sequencing coverage after 18 X of 454 or after 70 X of Illumina sequencing does not provide significant improvement of the assembly. Considering the cost of sequencing, a 2 X 454 sequencing, when coupled to 70 X Illumina sequencing, provided an assembly of reasonably good quality. With several software tested, Newbler with a seed length of 16 and ABySS with a K-value of 60 appear to be appropriate for the assembly of 454 reads alone and Illumina paired-end reads alone, respectively. Using both 454 and Illumina paired-end reads, a hybrid assembly strategy using Newbler for initial 454 sequence assembly, Velvet for initial Illumina sequence assembly, followed by a second step assembly using MIRA provided the best assembly of the physical map contig, resulting in 193 contigs with a N50 value of 13,123 bp.A hybrid sequencing strategy using low sequencing depth of 454 and high sequencing depth of Illumina provided the good quality assembly with high N50 value and relatively low cost. A combination of Newbler, Velvet, and MIRA can be used to assemble the 454 sequence reads and the Illumina reads effectively. The assembled sequence can serve as a resource for comparative genome analysis. Additional long reads using the third generation sequencing platforms are needed to sequence through repetitive genome regions that should further enhance the sequence assembly.Channel catfish, Ictalurus punctatus, is the major aquaculture species in the United States, accounting for over 60% of all U.S. aquaculture production. Channel catfish is regarded as one of the best characterized species serving as a model for teleost immune studies [1], and an imp
Whole mitochondrial genome scan for population structure and selection in the Atlantic herring  [cached]
Teacher Amber GF,André Carl,Meril? Juha,Wheat Christopher W
BMC Evolutionary Biology , 2012, DOI: 10.1186/1471-2148-12-248
Abstract: Background Marine fish, such as the Atlantic herring (Clupea harengus), often show a low degree of differentiation over large geographical regions. Despite strong environmental gradients (salinity and temperature) in the Baltic Sea, population genetic studies have shown little genetic differentiation among herring in this area, but some evidence for environmentally-induced selection has been uncovered. The mitochondrial genome is a likely target for selection in this system due to its functional role in metabolism. Results We sequenced whole mitochondrial genomes for herring from throughout the Baltic region (n=98) in order to investigate evidence for geographical structuring, selection, and associations between genetic and environmental variation. Three well-supported clades that predate the formation of the Baltic Sea were identified, but geographic structuring of this variation was weak (ΦST = 0.036). There was evidence for significant positive selection, particularly in the ND2, ND4 and ND5 genes, and amino acids under significant selection in these genes explained some of the clade formation. Despite uncovering evidence for selection, correlations between genetic diversity or differentiation with environmental factors (temperature, salinity, latitude) were weak. Conclusions The results indicate that most of the current mtDNA diversity in herring predates the formation of the Baltic Sea, and that little structuring has evolved since. Thus, fisheries management units in this region cannot be determined on the basis of mtDNA variability. Preliminary evidence for selection underlying clade formation indicates that the NADH complex may be useful for examining adaptation and population structuring at a broader geographical scale.
Chemical Composition and Cyanide Levels of Hybrid Catfish Fed Whole Cassava Root Meal in Replacement of Maize
O.M.G. Abu,L.O. Sanni,E.S. Erondu,O.A. Akinrotimi
Journal of Food Technology , 2013, DOI: 10.3923/jftech.2010.52.57
Abstract: The chemical composition and cyanide levels of hybrid catfish fed with whole cassava root meal as a replacement for maize were analyzed and evaluated. Four different diets with varying levels of replacement of maize meal with whole cassava root meal were formulated and designated Ao, B33, C66 and D100 with 0, 33, 66 and 100% of whole cassava root meal inclusion, respectively. The fish were fed for a period of 32 weeks. The result from the study indicated there was no significant different in chemical composition of the experimental diets except in their cyanide levels, Ao (control) had 0.00 mg kgˉ1; diets B33 (0.02), C66 0.04 and D100 had 0.09 cyanide levels. The chemical composition of the experimental fish shown that the there were no significant different in the proximate level of the fish fed with different diets. The cyanide levels increased as the level of whole cassava root meal inclusion in the diets increased. Diet Ao (control), had 0.02 mg kgˉ1; diet B33 0.03 mg kgˉ1; while diet C66 0.05 mg kgˉ1 and diet D100 0.08. These values are within tolerable range for the normal metabolism of the fish. Therefore, it can be concluded from the results obtained in this trial that whole cassava root meal can replace maize successfully without any adverse effect.
Mitochondrial Oxidative Phosphorylation Compensation May Preserve Vision in Patients with OPA1-Linked Autosomal Dominant Optic Atrophy  [PDF]
Nicole J. Van Bergen, Jonathan G. Crowston, Lisa S. Kearns, Sandra E. Staffieri, Alex W. Hewitt, Amy C. Cohn, David A. Mackey, Ian A. Trounce
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0021347
Abstract: Autosomal Dominant Optic Atrophy (ADOA) is the most common inherited optic atrophy where vision impairment results from specific loss of retinal ganglion cells of the optic nerve. Around 60% of ADOA cases are linked to mutations in the OPA1 gene. OPA1 is a fission-fusion protein involved in mitochondrial inner membrane remodelling. ADOA presents with marked variation in clinical phenotype and varying degrees of vision loss, even among siblings carrying identical mutations in OPA1. To determine whether the degree of vision loss is associated with the level of mitochondrial impairment, we examined mitochondrial function in lymphoblast cell lines obtained from six large Australian OPA1-linked ADOA pedigrees. Comparing patients with severe vision loss (visual acuity [VA]<6/36) and patients with relatively preserved vision (VA>6/9) a clear defect in mitochondrial ATP synthesis and reduced respiration rates were observed in patients with poor vision. In addition, oxidative phosphorylation (OXPHOS) enzymology in ADOA patients with normal vision revealed increased complex II+III activity and levels of complex IV protein. These data suggest that OPA1 deficiency impairs OXPHOS efficiency, but compensation through increases in the distal complexes of the respiratory chain may preserve mitochondrial ATP production in patients who maintain normal vision. Identification of genetic variants that enable this response may provide novel therapeutic insights into OXPHOS compensation for preventing vision loss in optic neuropathies.
Selective Enrichment and Sequencing of Whole Mitochondrial Genomes in the Presence of Nuclear Encoded Mitochondrial Pseudogenes (Numts)  [PDF]
Jonci N. Wolff, Deborah C. A. Shearman, Rob C. Brooks, John W. O. Ballard
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0037142
Abstract: Numts are an integral component of many eukaryote genomes offering a snapshot of the evolutionary process that led from the incorporation of an α-proteobacterium into a larger eukaryotic cell some 1.8 billion years ago. Although numt sequence can be harnessed as molecular marker, these sequences often remain unidentified and are mistaken for genuine mtDNA leading to erroneous interpretation of mtDNA data sets. It is therefore indispensable that during the process of amplifying and sequencing mitochondrial genes, preventive measures are taken to ensure the exclusion of numts to guarantee the recovery of genuine mtDNA. This applies to mtDNA analyses in general but especially to studies where mtDNAs are sequenced de novo as the launch pad for subsequent mtDNA-based research. By using a combination of dilution series and nested rolling circle amplification (RCA), we present a novel strategy to selectively amplify mtDNA and exclude the amplification of numt sequence. We have successfully applied this strategy to de novo sequence the mtDNA of the Black Field Cricket Teleogryllus commodus, a species known to contain numts. Aligning our assembled sequence to the reference genome of Teleogryllus emma (GenBank EU557269.1) led to the identification of a numt sequence in the reference sequence. This unexpected result further highlights the need of a reliable and accessible strategy to eliminate this source of error.
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