A decrease in oxidative phosphorylation (OXPHOS) is characteristic of many cancer types and, in particular, of clear cell renal carcinoma (CCRC) deficient in von Hippel-Lindau (vhl) gene. In the absence of functional pVHL, hypoxia-inducible factor (HIF) 1-alpha and HIF2-alpha subunits are stabilized, which induces the transcription of many genes including those involved in glycolysis and reactive oxygen species (ROS) metabolism. Transfection of these cells with vhl is known to restore HIF-alpha subunit degradation and to reduce glycolytic genes transcription. We show that such transfection with vhl of 786-0 CCRC (which are devoid of HIF1-alpha) also increased the content of respiratory chain subunits. However, the levels of most transcripts encoding OXPHOS subunits were not modified. Inhibition of HIF2-alpha synthesis by RNA interference in pVHL-deficient 786-0 CCRC also restored respiratory chain subunit content and clearly demonstrated a key role of HIF in OXPHOS regulation. In agreement with these observations, stabilization of HIF-alpha subunit by CoCl(2) decreased respiratory chain subunit levels in CCRC cells expressing pVHL. In addition, HIF stimulated ROS production and mitochondrial manganese superoxide dismutase content. OXPHOS subunit content was also decreased by added H(2)O(2.) Interestingly, desferrioxamine (DFO) that also stabilized HIF did not decrease respiratory chain subunit level. While CoCl(2) significantly stimulates ROS production, DFO is known to prevent hydroxyl radical production by inhibiting Fenton reactions. This indicates that the HIF-induced decrease in OXPHOS is at least in part mediated by hydroxyl radical production.

• MeSH
• deferoxamin farmakologie   MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa * genetika   metabolismus   MeSH
• glykolýza genetika   MeSH
• homeostáza MeSH
• kobalt farmakologie   MeSH
• lidé MeSH
• nádory genetika   MeSH
• oxidativní fosforylace * MeSH
• peroxid vodíku farmakologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• reaktivní formy kyslíku * metabolismus   MeSH
• receptory aromatických uhlovodíků - jaderný translokátor * genetika   metabolismus   MeSH
• respirační vzplanutí fyziologie   účinky léků   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• transportní proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

The major finding of the present study concerns the marked decrease of respiratory chain complex I activity in the cerebral cortex of immature rats following seizures induced by bilateral intracerebroventricular infusion of dl-homocysteic acid (600 nmol/side). This decrease was already evident during the acute phase of seizures (60-90 min after infusion) and persisted for at least 20 h after the seizures. It was selective for complex I since activities of complex II and IV and citrate synthase remained unaffected. Inhibition of complex I activity was not associated with changes in complex I content. Based on enhanced lipoperoxidation and decreased aconitase activity, it can be postulated that oxidative modification is most likely responsible for the observed inhibition. Mitochondrial respiration, as well as cortical ATP levels remained in the control range, apparently due to excess capacity of the complex I documented by energy thresholds. On the other hand, the enhanced production of reactive oxygen species by inhibited complex I was observed in mitochondria from HCA-treated animals. The decrease of complex I activity was substantially attenuated when animals were treated with substances providing an anticonvulsant effect and also with selected free radical scavengers. We can assume that inhibition of complex I may elicit enhanced formation of reactive oxygen species and contribute thus to neuronal injury demonstrated in this model.

• MeSH
• akonitáthydratasa metabolismus   MeSH
• analýza rozptylu MeSH
• antioxidancia farmakologie   MeSH
• citrátsynthasa metabolismus   MeSH
• cyklické N-oxidy farmakologie   MeSH
• energetický metabolismus účinky léků   MeSH
• financování organizované MeSH
• homocystein analogy a deriváty   MeSH
• krysa rodu rattus MeSH
• lékové interakce MeSH
• metaloporfyriny farmakologie   MeSH
• mitochondrie účinky léků   MeSH
• mozková kůra enzymologie   růst a vývoj   ultrastruktura   MeSH
• novorozená zvířata MeSH
• peroxidace lipidů účinky záření   MeSH
• potkani Wistar MeSH
• respirační komplex I antagonisté a inhibitory   metabolismus   MeSH
• spinové značení MeSH
• spotřeba kyslíku účinky léků   MeSH
• záchvaty chemicky indukované   patologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH

Recently, the relationship of mitochondrial DNA (mtDNA) variants to metabolic risk factors for diabetes and other common diseases has begun to attract increasing attention. However, progress in this area has been limited because (1) the phenotypic effects of variation in the mitochondrial genome are difficult to isolate owing to confounding variation in the nuclear genome, imprinting phenomena, and environmental factors; and (2) few animal models have been available for directly investigating the effects of mtDNA variants on complex metabolic phenotypes in vivo. Substitution of different mitochondrial genomes on the same nuclear genetic background in conplastic strains provides a way to unambiguously isolate effects of the mitochondrial genome on complex traits. Here we show that conplastic strains of rats with identical nuclear genomes but divergent mitochondrial genomes that encode amino acid differences in proteins of oxidative phosphorylation exhibit differences in major metabolic risk factors for type 2 diabetes. These results (1) provide the first direct evidence linking naturally occurring variation in the mitochondrial genome, independent of variation in the nuclear genome and other confounding factors, to inherited variation in known risk factors for type 2 diabetes; and (2) establish that spontaneous variation in the mitochondrial genome per se can promote systemic metabolic disturbances relevant to the pathogenesis of common diseases.

• MeSH
• diabetes mellitus 2. typu genetika   MeSH
• financování organizované MeSH
• genetická variace MeSH
• genom MeSH
• genová dávka MeSH
• haplotypy MeSH
• krysa rodu rattus MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie enzymologie   genetika   MeSH
• polymorfismus genetický MeSH
• potkani inbrední BN MeSH
• potkani inbrední SHR MeSH
• respirační komplex IV genetika   MeSH
• rizikové faktory MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• substituce aminokyselin MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.

• MeSH
• antimycin A analogy a deriváty   farmakologie   MeSH
• buněčné dýchání MeSH
• elektronová paramagnetická rezonance MeSH
• ethidium analogy a deriváty   chemie   MeSH
• ferrikyanidy farmakologie   MeSH
• financování organizované MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• glycerolfosfáty metabolismus   MeSH
• hnědá tuková tkáň enzymologie   účinky léků   ultrastruktura   MeSH
• křečci praví MeSH
• mitochondrie enzymologie   metabolismus   účinky léků   MeSH
• reaktivní formy kyslíku analýza   metabolismus   MeSH
• respirační komplex III metabolismus   MeSH
• spotřeba kyslíku MeSH
• transport elektronů MeSH
• ubichinon metabolismus   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• mužské pohlaví MeSH
• zvířata MeSH

Isolated defects of mitochondrial ATPase due to diminished biosynthesis of the enzyme represent new class of severe mitochondrial diseases of nuclear origin. The primary cause of decreased cellular content of ATPase appears to be a problem in assembly of the F1 catalytic part of the enzyme. With the aim to elucidate how the low ATPase content affects mitochondrial energy provision and ROS production, we have investigated fibroblasts from patients with ATPase decrease to 10-30%. Measurements of cellular respiration showed pronounced decrease in ATPase capacity for basal respiration, mitochondrial ATP synthesis was decreased to 26-33%. Cytofluorometric analysis using TMRM revealed altered discharge of mitochondrial membrane potential (DeltaPsim) in patient cells, which was 20 mV increased at state 3-ADP. Analysis of ROS production by CM-H2DCFDA demonstrated 2-fold increase in ROS production in patient cells compared to controls. ROS production rate was sensitive to uncoupler (FCCP) and thus apparently related to increased DeltaPsim. Our studies clearly demonstrate that low ATPase content and decreased mitochondrial ATP production lead to high values of DeltaPsim and are associated with activation of ROS generation by the mitochondrial respiratory chain. In conclusion, both the energetic deprivation and increased oxidative stress are important components of the pathogenic mechanism of ATPase disorders.

• MeSH
• adenosintrifosfát biosyntéza   MeSH
• adenosintrifosfatasy * nedostatek   MeSH
• energetický metabolismus * MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové potenciály MeSH
• mitochondrie * metabolismus   MeSH
• reaktivní formy kyslíku * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

ATP synthase is a key enzyme of mitochondrial energy conversion. In mammals, it produces most of cellular ATP. Alteration of ATP synthase biogenesis may cause two types of isolated defects: qualitative when the enzyme is structurally modified and does not function properly, and quantitative when it is present in insufficient amounts. In both cases the cellular energy provision is impaired, and diminished use of mitochondrial DeltamuH+ promotes ROS production by the mitochondrial respiratory chain. The primary genetic defects have so far been localized in mtDNA ATP6 gene and nuclear ATP12 gene, however, involvement of other nuclear genes is highly probable.

• MeSH
• financování organizované MeSH
• lidé MeSH
• mitochondriální nemoci enzymologie   genetika   patologie   MeSH
• mitochondriální protonové ATPasy biosyntéza   genetika   metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• přehledy MeSH

Mitochondrial membrane potential (Deltapsi(m)) plays important roles in the normal function of cells and in pathobiochemical situations. The application of ion-selective electrodes for the measurement of Deltapsi(m) is important for studying normal biological reactions and pathways and mitochondrial diseases. We constructed and optimized a computerized device for real-time monitoring of the Deltapsi(m), which included modification of tetraphenylphosphonium (TPP(+))-selective membrane that improved reproducibility of the TPP(+)-selective electrode. Application of MATLAB software increased the sensitivity of the system. We tested our improved device for membrane potential measurements of isolated mitochondria (in absolute scale of millivolts). In addition, we assessed relative changes of Deltapsi(m) (as changes in TPP(+) concentration) of digitonin-permeabilized cells (hepatocytes, control transmitochondrial cybrids, HeLa G and BSC-40) after addition of substrates, inhibitors, and uncoupler of respiratory chain. Our system can be successfully used for studies of many aspects of the regulation of mitochondrial bioenergetics and as a diagnostic tool for mitochondrial oxidative phosphorylation disorders.

• MeSH
• elektrofyziologie metody   přístrojové vybavení   MeSH
• financování organizované MeSH
• indikátory a reagencie chemie   MeSH
• iontově selektivní elektrody MeSH
• jaterní mitochondrie metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• membránové potenciály fyziologie   MeSH
• mitochondriální membrány metabolismus   MeSH
• nádorové buňky kultivované MeSH
• oniové sloučeniny chemie   MeSH
• organofosforové sloučeniny chemie   MeSH
• periferní zařízení počítače MeSH
• počítačové systémy MeSH
• potkani Wistar MeSH
• reprodukovatelnost výsledků MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• hodnotící studie MeSH

Although mitochondrial deficiency in cancer has been described by Warburg, many years ago, the mechanisms underlying this impairment remain essentially unknown. Many types of cancer cells are concerned and, in particular, clear cell renal carcinoma (CCRC). In this cancer, the tumor suppressor gene, VHL (von Hippel-Lindau factor) is invalidated. Previous studies have shown that the transfection of the VHL gene in VHL-deficient cells originating from CCRCs could suppress their ability to form tumors when they were injected into nude mice. However, various additional genetic alterations are observed in such cancer cells. In order to investigate whether VHL invalidation was related to the mitochondrial impairment, we have studied the effects of wild-type VHL transfection into VHL-deficient 786-0 or RCC10 cells on their oxidative phosphorylation (OXPHOS) subunit contents and functions. We show that the presence of wild-type VHL protein (pVHL) increased mitochondrial DNA and respiratory chain protein contents and permitted the cells to rely on their mitochondrial ATP production to grow in the absence of glucose. In parallel to mtDNA increase, the presence of pVHL up regulated the mitochondrial transcription factor A, as shown by western blot analysis. In conclusion, in CCRCs, pVHL deficiency is one of the factors responsible for down-regulation of the biogenesis of OXPHOS complexes.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• DNA primery MeSH
• kultivované buňky MeSH
• lidé MeSH
• mitochondriální DNA metabolismus   MeSH
• mitochondrie * metabolismus   MeSH
• myši nahé MeSH
• myši MeSH
• nádorové supresorové proteiny fyziologie   genetika   MeSH
• nádorový supresorový protein VHL MeSH
• oxidativní fosforylace MeSH
• sekvence nukleotidů MeSH
• ubikvitinligasy fyziologie   genetika   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Dysfunctions of the F(1)F(o)-ATPase complex cause severe mitochondrial diseases affecting primarily the paediatric population. While in the maternally inherited ATPase defects due to mtDNA mutations in the ATP6 gene the enzyme is structurally and functionally modified, in ATPase defects of nuclear origin mitochondria contain a decreased amount of otherwise normal enzyme. In this case biosynthesis of ATPase is down-regulated due to a block at the early stage of enzyme assembly-formation of the F(1) catalytic part. The pathogenetic mechanism implicates dysfunction of Atp12 or other F(1)-specific assembly factors. For cellular energetics, however, the negative consequences may be quite similar irrespective of whether the ATPase dysfunction is of mitochondrial or nuclear origin.

• MeSH
• adenosintrifosfatasy genetika   MeSH
• buněčné jádro enzymologie   metabolismus   MeSH
• fibroblasty metabolismus   MeSH
• lidé MeSH
• mitochondriální DNA genetika   metabolismus   MeSH
• mitochondriální nemoci enzymologie   genetika   MeSH
• mitochondriální protonové ATPasy biosyntéza   genetika   nedostatek   MeSH
• mitochondrie enzymologie   MeSH
• mutace MeSH
• reaktivní formy kyslíku analýza   metabolismus   MeSH
• vakuolární protonové ATPasy * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH
• srovnávací studie MeSH

• MeSH
• acidóza laktátová MeSH
• biochemie MeSH
• chemické techniky analytické MeSH
• elektroforéza MeSH
• kojenec MeSH
• lidé MeSH
• mitochondriální DNA MeSH
• mitochondriální nemoci MeSH
• mitochondriální protonové ATPasy MeSH
• mutace genetika   MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• statistika jako téma MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• Publikační typ
• kazuistiky MeSH