scholarly journals Dihydrorhodamine 123 identifies impaired mitochondrial respiratory chain function in cultured cells harboring mitochondrial DNA mutations.

1996 ◽  
Vol 44 (6) ◽  
pp. 571-579 ◽  
Author(s):  
C Sobreira ◽  
M Davidson ◽  
M P King ◽  
A F Miranda
Keyword(s):  
Mitochondrial Dna ◽  
Respiratory Chain ◽  
Mitochondrial Function ◽  
Cultured Cells ◽  
Mitochondrial Respiratory Chain ◽  
Mtdna Mutations ◽  
Dihydrorhodamine 123 ◽  
Very High ◽  
Mitochondrial Respiratory ◽  
In Cells

Several human diseases have been found to be caused by mitochondrial DNA (mtDNA) mutations. Pathogenic mutated (mut) mtDNAs are usually "heteroplasmic," coexisting intracellularly with wild-type (wt) mtDNAs. For some mtDNA mutations, cells have normal levels of respiratory chain function unless the percentage of mut-mtDNA is very high. Although progress in understanding the molecular basis of mitochondrial diseases has been remarkable, the heterogeneity of mut-mtDNA distribution, even among cells of the same tissue, makes it difficult to clearly delineate the relationships between mtDNA mutations, gene dosage, and clinical phenotypes. In a search for screening methods for identifying cultured cells with deficient mitochondrial function, we incubated living cells harboring mut-mtDNAs with dihydrorhodamine 123 (DHR123), an uncharged, nonfluorescent agent that can be converted by oxidation to the fluorescent laser dye rhodamine 123 (R123). Bright mitochondrial staining was observed in cells that respired normally. Fluorescence was significantly reduced in cells with mitochondrial respiratory chain dysfunction resulting from very high levels of mut-mtDNAs. The data show that DHR123 is useful for assessing mitochondrial function in single cells, and can be used for isolating viable, respiratory chain-deficient cells from heterogeneous cultures.

Assessment of mitochondrial respiratory chain enzymes in cells and tissues

2020 ◽  
pp. 121-156
Author(s):  
Ann E. Frazier ◽  
Amy E. Vincent ◽  
Doug M. Turnbull ◽  
David R. Thorburn ◽  
Robert W. Taylor
Keyword(s):  
Respiratory Chain ◽  
Mitochondrial Respiratory Chain ◽  
Mitochondrial Respiratory ◽  
In Cells

scholarly journals Case Report: A Novel Mutation in the Mitochondrial MT-ND5 Gene Is Associated With Leber Hereditary Optic Neuropathy (LHON)

2021 ◽  
Vol 12 ◽  
Author(s):  
Martin Engvall ◽  
Aki Kawasaki ◽  
Valerio Carelli ◽  
Rolf Wibom ◽  
Helene Bruhn ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Respiratory Chain ◽  
Optic Neuropathy ◽  
Complex I ◽  
Novel Mutation ◽  
Mitochondrial Respiratory Chain ◽  
Leber Hereditary Optic Neuropathy ◽  
Mild Defect ◽  
Hereditary Optic Neuropathy ◽  
Mitochondrial Respiratory

Leber hereditary optic neuropathy (LHON) is a mitochondrial disease causing severe bilateral visual loss, typically in young adults. The disorder is commonly caused by one of three primary point mutations in mitochondrial DNA, but a number of other rare mutations causing or associated with the clinical syndrome of LHON have been reported. The mutations in LHON are almost exclusively located in genes encoding subunits of complex I in the mitochondrial respiratory chain. Here we report two patients, a mother and her son, with the typical LHON phenotype. Genetic investigations for the three common mutations were negative, instead we found a new and previously unreported mutation in mitochondrial DNA. This homoplasmic mutation, m.13345G>A, is located in the MT-ND5 gene, encoding a core subunit in complex I in the mitochondrial respiratory chain. Investigation of the patients mitochondrial respiratory chain in muscle found a mild defect in the combined activity of complex I+III. In the literature six other mutations in the MT-ND5 gene have been associated with LHON and by this report a new putative mutation in the MT-ND5 can be added.

scholarly journals Trib1 deficiency causes brown adipose respiratory chain depletion and mitochondrial disorder

2021 ◽  
Vol 12 (12) ◽  
Author(s):  
Xuelian Zhang ◽  
Bin Zhang ◽  
Chenyang Zhang ◽  
Guibo Sun ◽  
Xiaobo Sun
Keyword(s):  
Adipose Tissue ◽  
Respiratory Chain ◽  
Mitochondrial Function ◽  
Knockout Mice ◽  
Mitochondrial Respiratory Chain ◽  
Complex Iii ◽  
Adrenoceptor Agonist ◽  
Brown Adipose ◽  
Mitochondrial Respiratory

AbstractTribbles homolog 1 (TRIB1) belongs to the Tribbles family of pseudokinases, which plays a key role in tumorigenesis and inflammation. Although genome-wide analysis shows that TRIB1 expression is highly correlated with blood lipid levels, the relationship between TRIB1 and adipose tissue metabolism remains unclear. Accordingly, the aim of the present study was to explore the role of TRIB1 on mitochondrial function in the brown adipose tissue (BAT). Trib1-knockout mice were established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. The metabolic function of the BAT was induced by a β3-adrenoceptor agonist and the energy metabolism function of mitochondria in the BAT of mice was evaluated. Trib1-knockout mice exhibited obesity and impaired BAT thermogenesis. In particular, Trib1 knockout reduced the ability of the BAT to maintain body temperature, inhibited β3-adrenoceptor agonist-induced thermogenesis, and accelerated lipid accumulation in the liver and adipose tissues. In addition, Trib1 knockout reduced mitochondrial respiratory chain complex III activity, produced an imbalance between mitochondrial fusion and fission, caused mitochondrial structural damage and dysfunction, and affected heat production and lipid metabolism in the BAT. Conversely, overexpression of Trib1 in 3T3-L1 adipocytes increased the number of mitochondria and improved respiratory function. These findings support the role of Trib1 in regulating the mitochondrial respiratory chain and mitochondrial dynamics by affecting mitochondrial function and thermogenesis in the BAT.

scholarly journals Age Modulates Fe3O4Nanoparticles Liver Toxicity: Dose-Dependent Decrease in Mitochondrial Respiratory Chain Complexes Activities and Coupling in Middle-Aged as Compared to Young Rats

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Yosra Baratli ◽  
Anne-Laure Charles ◽  
Valérie Wolff ◽  
Lotfi Ben Tahar ◽  
Leila Smiri ◽  
...  
Keyword(s):  
Respiratory Chain ◽  
Rat Liver ◽  
Mitochondrial Function ◽  
Mitochondrial Respiratory Chain ◽  
Liver Mitochondria ◽  
Aged Rats ◽  
Middle Aged ◽  
Respiratory Chain Complexes ◽  
Chain Complexes ◽  
Mitochondrial Respiratory

We examined the effects of iron oxide nanoparticles (IONPs) on mitochondrial respiratory chain complexes activities and mitochondrial coupling in young (3 months) and middle-aged (18 months) rat liver, organ largely involved in body iron detoxification. Isolated liver mitochondria were extracted using differential centrifugations. Maximal oxidative capacities (Vmax, complexes I, III, and IV activities),Vsucc(complexes II, III, and IV activities), andVtmpd, (complex IV activity), together with mitochondrial coupling (Vmax/V0) were determined in controls conditions and after exposure to 250, 300, and 350 μg/ml Fe3O4in young and middle-aged rats. In young liver mitochondria, exposure to IONPs did not alter mitochondrial function. In contrast, IONPs dose-dependently impaired all complexes of the mitochondrial respiratory chain in middle-aged rat liver:Vmax(from 30 ± 1.6 to 17.9 ± 1.5;P<0.001),Vsucc(from 33.9 ± 1.7 to 24.3 ± 1.0;P<0.01),Vtmpd(from 43.0 ± 1.6 to 26.3 ± 2.2 µmol O2/min/g protein;P<0.001) using Fe3O4350µg/ml. Mitochondrial coupling also decreased. Interestingly, 350 μg/ml Fe3O4in the form of Fe3+solution did not impair liver mitochondrial function in middle-aged rats. Thus, IONPs showed a specific toxicity in middle-aged rats suggesting caution when using it in old age.

scholarly journals Respiratory-deficient human fibroblasts exhibiting defective mitochondrial DNA replication

1995 ◽  
Vol 305 (3) ◽  
pp. 817-822 ◽  
Author(s):  
A G Bodnar ◽  
J M Cooper ◽  
J V Leonard ◽  
A H V Schapira
Keyword(s):  
Mitochondrial Dna ◽  
Respiratory Chain ◽  
Cultured Cells ◽  
Human Fibroblasts ◽  
Genetic Defect ◽  
Mitochondrial Protein ◽  
Mitochondrial Respiratory Chain ◽  
Human Cell Line ◽  
Respiratory Chain Complexes ◽  
Respiratory Deficient

We have characterized cultured skin fibroblasts from two siblings affected with a fatal mitochondrial disease caused by a nuclear genetic defect. Mitochondrial respiratory-chain function was severely decreased in these cells. Southern-blot analysis showed that the fibroblasts had reduced levels of mitochondrial DNA (mtDNA). The mtDNA was unstable and was eliminated from the cultured cells over many generations, generating the rho0 genotype. As the mtDNA level decreased, the cells became more dependent upon pyruvate and uridine for growth. Nuclear-encoded subunits of respiratory-chain complexes were synthesized and imported into the mitochondria of the mtDNA-depleted cells, albeit at reduced levels compared with the controls. Mitochondrial protein synthesis directed by the residual mtDNA indicated that the mtDNA was expressed and that the defect specifically involves the replication or maintenance of mtDNA. This is a unique example of a respiratory-deficient human cell line exhibiting defective mtDNA replication.

scholarly journals Mitochondrial DNA alterations underlie an irreversible shift to aerobic glycolysis in fumarate hydratase–deficient renal cancer

2021 ◽  
Vol 14 (664) ◽  
pp. eabc4436
Author(s):  
Daniel R. Crooks ◽  
Nunziata Maio ◽  
Martin Lang ◽  
Christopher J. Ricketts ◽  
Cathy D. Vocke ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Respiratory Chain ◽  
Human Cancer ◽  
Aerobic Glycolysis ◽  
Krebs Cycle ◽  
Fumarate Hydratase ◽  
Mitochondrial Respiratory Chain ◽  
Respiratory Chain Complexes ◽  
Cycle Enzyme ◽  
Mtdna Mutations

Understanding the mechanisms of the Warburg shift to aerobic glycolysis is critical to defining the metabolic basis of cancer. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive cancer characterized by biallelic inactivation of the gene encoding the Krebs cycle enzyme fumarate hydratase, an early shift to aerobic glycolysis, and rapid metastasis. We observed impairment of the mitochondrial respiratory chain in tumors from patients with HLRCC. Biochemical and transcriptomic analyses revealed that respiratory chain dysfunction in the tumors was due to loss of expression of mitochondrial DNA (mtDNA)–encoded subunits of respiratory chain complexes, caused by a marked decrease in mtDNA content and increased mtDNA mutations. We demonstrated that accumulation of fumarate in HLRCC tumors inactivated the core factors responsible for replication and proofreading of mtDNA, leading to loss of respiratory chain components, thereby promoting the shift to aerobic glycolysis and disease progression in this prototypic model of glucose-dependent human cancer.

scholarly journals Mitochondrial respiratory chain inhibition and Na+K+ATPase dysfunction are determinant factors modulating the toxicity of nickel in the brain of indian catfish Clarias batrachus L.

2018 ◽  
Vol 11 (4) ◽  
pp. 306-315 ◽  
Author(s):  
Arpan Kumar Maiti ◽  
Nimai Chandra Saha ◽  
Goutam Paul ◽  
Kishore Dhara
Keyword(s):  
Respiratory Chain ◽  
Mitochondrial Function ◽  
Mitochondrial Respiratory Chain ◽  
Clarias Batrachus ◽  
Contributing Factors ◽  
Functional Changes ◽  
Partial Inhibition ◽  
Mitochondrial Functions ◽  
The Impact ◽  
Mitochondrial Respiratory

Abstract Nickel is a potential neurotoxic pollutant inflicting damage in living organisms, including fish, mainly through oxidative stress. Previous studies have demonstrated the impact of nickel toxicity on mitochondrial function, but there remain lacunae on the damage inflicted at mitochondrial respiratory level. Deficient mitochondrial function usually affects the activities of important adenosinetriphosphatases responsible for the maintenance of normal neuronal function, namely Na+K+ATPase, as explored in our study. Previous reports demonstrated the dysfunction of this enzyme upon nickel exposure but the contributing factors for the inhibition of this enzyme remained unexplored. The main purpose of this study was to elucidate the impact of nickel neurotoxicity on mitochondrial respiratory complexes and Na+K+ATPase in the piscine brain and to determine the contributing factors that had an impact on the same. Adult Clarias batrachus were exposed to nickel treated water at 10% and 20% of the 96 h LC50 value (41 mg.l−1) respectively and sampled on 20, 40 and 60 days. Exposure of fish brain to nickel led to partial inhibition of complex IV of mitochondrial respiratory chain, however, the activities of complex I, II and III remained unaltered. This partial inhibition of mitochondrial respiratory chain might have been sufficient to lower mitochondrial energy production in mitochondria that contributed to the partial dysfunction of Na+K+ATPase. Besides energy depletion other contributing factors were involved in the dysfunction of this enzyme, like loss of thiol groups for enzyme activity and lipid peroxidation-derived end products that might have induced conformational and functional changes. However, providing direct evidence for such conformational and functional changes of Na+K+ATPase was beyond the scope of the present study. In addition, immunoblotting results also showed a decrease in Na+K+ATPase protein expression highlighting the impact of nickel neurotoxicity on the expression of the enzyme itself. The implication of the inhibition of mitochondrial respiration and Na+K+ATPase dysfunction was the neuronal death as evidenced by enhanced caspase-3 and caspase-9 activities. Thus, this study established the deleterious impact of nickel neurotoxicity on mitochondrial functions in the piscine brain and identified probable contributing factors that can act concurrently in the inhibition of Na+K+ATPase. This study also provided a vital clue about the specific areas that the therapeutic agents should target to counter nickel neurotoxicity.

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