Biomedical subjects

J Houstek

Publications and source records attributed to J Houstek.

At least 19 recordsLinked to original sources

PubMed2006

Deficiency of mitochondrial ATP synthase of nuclear genetic origin.

We present clinical and laboratory data from 14 cases with an isolated deficiency of the mitochondrial ATP synthase (7-30% of control) caused by nuclear genetic defects. A quantitative decrease of the ATP synthase complex was documented by Blue-Native electrophoresis and Western blotting and was supported by the diminished activity of oligomycin/aurovertin-sensitive ATP hydrolysis in fibroblasts (10 cases), muscle (6 of 7 cases), and liver (one case). All patients had neonatal onset and elevated plasma lactate levels. In 12 patients investigated 3-methyl-glutaconic aciduria was detected. Seven patients died, mostly within the first weeks of life and surviving patients showed psychomotor and various degrees of mental retardation. Eleven patients had hypertrophic cardiomyopathy; other clinical signs included hypotonia, hepatomegaly, facial dysmorphism and microcephaly. This phenotype markedly differs from the severe central nervous system changes of ATP synthase disorders caused by mitochondrial DNA mutations of the ATP6 gene presenting mostly as NARP and MILS.

Adenosine Triphosphate↗

PubMed2005

Polyunsaturated fatty acids of marine origin upregulate mitochondrial biogenesis and induce beta-oxidation in white fat.

AIMS/HYPOTHESIS: Intake of n-3 polyunsaturated fatty acids reduces adipose tissue mass, preferentially in the abdomen. The more pronounced effect of marine-derived eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on adiposity, compared with their precursor alpha-linolenic acid, may be mediated by changes in gene expression and metabolism in white fat. METHODS: The effects of EPA/DHA concentrate (6% EPA, 51% DHA) admixed to form two types of high-fat diet were studied in C57BL/6J mice. Oligonucleotide microarrays, cDNA PCR subtraction and quantitative real-time RT-PCR were used to characterise gene expression. Mitochondrial proteins were quantified using immunoblots. Fatty acid oxidation and synthesis were measured in adipose tissue fragments. RESULTS: Expression screens revealed upregulation of genes for mitochondrial proteins, predominantly in epididymal fat when EPA/DHA concentrate was admixed to a semisynthetic high-fat diet rich in alpha-linolenic acid. This was associated with a three-fold stimulation of the expression of genes encoding regulatory factors for mitochondrial biogenesis and oxidative metabolism (peroxisome proliferator-activated receptor gamma coactivator 1 alpha [Ppargc1a, also known as Pgc1alpha] and nuclear respiratory factor-1 [Nrf1] respectively). Expression of genes for carnitine palmitoyltransferase 1A and fatty acid oxidation was increased in epididymal but not subcutaneous fat. In the former depot, lipogenesis was depressed. Similar changes in adipose gene expression were detected after replacement of as little as 15% of lipids in the composite high-fat diet with EPA/DHA concentrate, while the development of obesity was reduced. The expression of Ppargc1a and Nrf1 was also stimulated by n-3 polyunsaturated fatty acids in 3T3-L1 cells. CONCLUSIONS/INTERPRETATION: The anti-adipogenic effect of EPA/DHA may involve a metabolic switch in adipocytes that includes enhancement of beta-oxidation and upregulation of mitochondrial biogenesis.

Adipocytes↗

PubMed2005

Time-course of hormonal induction of mitochondrial glycerophosphate dehydrogenase biogenesis in rat liver.

Thyroid hormones are important regulators of mitochondrial metabolism. Due to their complex mechanism of action, the timescale of different responses varies from minutes to days. In this work, we studied selective T3 induction of the inner mitochondrial membrane enzyme-glycerophosphate dehydrogenase (mGPDH) in liver of euthyroid rats. We correlated the kinetics of the T3 level in blood, the mRNA level in liver, the activity and amount of mGPDH in liver mitochondria after a single dose of T3. The T3 level reached maximum after 1 h (80 nmol/l) and subsequently rapidly decreased. mGPDH mRNA increased also relatively fast, reaching a maximum after 12 h and fell to the control level after 72 h. An increase of mGPDH activity could be already found after 6 h and reached a maximum after 24 h in accordance with the increase in mGPDH content (2.4-fold vs. 2.7-fold induction). After 72 h, the mGPDH activity showed a significant 30% decrease. When the rats received three subsequent doses of T3, the increase of mGPDH activity was 2-fold higher than after a single T3 dose. The results demonstrate that mGPDH displays rapid induction as well as decay upon disappearance of a hormonal stimulus, indicating a rather short half-life of this inner mitochondrial membrane enzyme.

Animals↗

PubMed2005

Specific properties of heavy fraction of mitochondria from human-term placenta - glycerophosphate-dependent hydrogen peroxide production.

Mitochondrial respiratory chain enzyme Complexes are present in placenta at proportion similar to other tissues with exception of glycerophosphate dehydrogenase (mGPDH) which is expressed at a very high rate. As shown by Western blot quantification and respiratory chain enzyme activity measurements, the specific content of mGPDH is similar to that of succinate dehydrogenase or NADH dehydrogenase. Using fluorometric probe dichlorodihydrofluorescein diacetate we found that placental mitochondria display high rate of glycerophosphate-dependent hydrogen peroxide production. This was confirmed by oxygraphic detection of glycerophosphate-induced, KCN- or antimycin A-insensitive oxygen uptake. Hydrogen peroxide production by mGPDH was highly activated by one-electron acceptor, potassium ferricyanide and it was depressed by inhibitors of mGPDH and by cytochrome c. Our results indicate that mGPDH should be considered as an additional source of reactive oxygen species participating in induction of oxidative stress in placenta.

Animals↗

PubMed2005

Flow-cytometric monitoring of mitochondrial depolarisation: from fluorescence intensities to millivolts.

Redistribution potentiometric dyes represent a powerful tool for monitoring membrane potential of mitochondria, especially when these dyes are used with flow cytometry. In particular, tetramethylrhodamine methyl ester proved to be suitable for the screening of mitochondrial membrane potential in cultured human skin fibroblasts from patients suffering from different defects of oxidative phosphorylation. We have developed a method that makes it possible to measure the changes in mitochondrial membrane potential, or to assess the differences between respective mitochondrial membrane potentials in investigated cells and controls in the absolute scale of millivolts. Our approach employs the fact that a logarithmic transformation of Nernst equation-controlled intensity of fluorescence from potentiometric dyes accumulated in mitochondria leads to a linear scale for mitochondrial membrane potentials.

Adenosine Diphosphate↗

PubMed2005

Development of cytochrome-c oxidase activity in rat heart: downregulation in newborn rats.

Cytochrome-c oxidase (COX) activity of the rat heart was two- to sevenfold activated when the membrane integrity was disrupted by hypotonic swelling, freezing-thawing, or a detergent, indicating that a large portion of COX capacity in intact mitochondria is not active. The effect of detergent was tested in heart mitochondria isolated from 1-, 5-, 15-, 29-, and 60-d-old rats; activation by detergent was up to 20-fold in 1-d-old animals, whereas in mitochondria from 60-d-old rats it was only 7-fold. Our data indicate that the rat heart exhibits significant developmental changes dependent on downregulation of COX activity in the neonatal period.

Aging↗

PubMed2005

Tert-butyl hydroperoxide selectively inhibits mitochondrial respiratory-chain enzymes in isolated rat hepatocytes.

Sensitivity of various mitochondrial enzymes to oxidative damage was tested on isolated rat liver hepatocytes permeabilized by digitonin. In permeabilized hepatocytes normal respiratory control values were obtained and mitochondrial membranes remained intact. Respiratory rates of NADH-dependent (glutamate + malate, palmitylcarnitine + malate) and flavoprotein-dependent (succinate) substrates were determined in hepatocytes exposed for 5 min to 0.5-3 mM tert-butyl hydroperoxide before addition of digitonin. Our data showed that oxidation of NADH-dependent substrates is much more sensitive to oxidative stress than oxidation of flavoprotein-dependent ones, evidently due to the modification of iron-sulfur clusters or SH groups in the NADH dehydrogenase enzyme complex (Complex I).

Animals↗

PubMed2004

GUG is an efficient initiation codon to translate the human mitochondrial ATP6 gene.

A maternally inherited and practically homoplasmic mitochondrial (mtDNA) mutation, 8527A>G, changing the initiation codon AUG into GUG, normally coding for a valine, was observed in the ATP6 gene encoding the ATPase subunit a. No alternate Met codon could replace the normal translational initiator. The patient harboring this mutation exhibited clinical symptoms suggesting a mitochondrial disease but his mother who carried the same mtDNA mutation was healthy. The mutation was absent from 100 controls and occurred once amongst 44 patients suspected of Leber Hereditary Optic Neuropathy (LHON) but devoid of typical LHON mutations. In patient fibroblasts, no effect of 8527A>G mutation could be demonstrated on the biosynthesis of mtDNA-encoded proteins, on size and the content of ATPase subunit a, on ATP hydrolysis and on mitochondrial membrane potential. In addition, ATP synthesis was barely decreased. Therefore, GUG is a functional initiation codon for the human ATP6 gene.

Adenosine Triphosphatases↗

PubMed2004

Clinical, biochemical and molecular analyses of six patients with isolated cytochrome c oxidase deficiency due to mutations in the SCO2 gene.

BACKGROUND AND AIM: Cytochrome c oxidase (COX) deficiency represents a heterogeneous group of disorders. Numerous proteins are required for efficient COX assembly and maintenance. In 26 children with isolated COX deficiency, we studied mutations in the SCO2 gene, which is involved in the copper transport into the inner mitochondrial membrane, and we analysed the clinical and biochemical consequences of SCO2 mutations. METHODS: The activities of respiratory chain complexes were measured spectrophotometrically in isolated mitochondria and/or crude cell extracts in all available tissues. Two-dimensional polyacrylamide electrophoresis (2D-PAGE) was used to separate the complexes and their subunits. The mutations were detected by sequencing and RFLP analysis. RESULTS: Mutations in the SCO2 gene were found in six children. Early neonatal onset of hypertrophic cardiomyopathy and encephalopathy were observed in one boy with compound heterozygous mutations C1280T and G1541A. In all five children with homozygous mutation G1541A, progressive encephalopathy developed between 2 and 6 mo of age. Isolated COX deficiency was found in the skeletal muscle, heart, liver and brain but not in fibroblasts. 2D-PAGE in the skeletal muscle showed markedly decreased amounts of all COX subunits. CONCLUSION: Our results suggest that mutations in the SCO2 gene are not rare, at least in our population. Although clinical symptoms may rely on the type of SCO2 mutation, the prognosis is unfavourable in all patients.

Brain Diseases↗

PubMed2004

Developmental changes of cytochrome c oxidase and citrate synthase in rat heart homogenate.

Activity of cytochrome c oxidase and citrate synthase in rat heart homogenates was determined in 5-, 15-, 28- and 60-day-old rats. The activity of both enzymes increased during postnatal development but their changes followed different kinetics. The membrane-bound cytochrome c oxidase reached its adult values during the early postnatal period, i.e. between days 5 and 15, whereas soluble matrix-localized citrate synthase also continued to increase between days 15 and 60. Our data indicate a relative excess of cytochrome c oxidase in neonatal cardiocytes.

Age Factors↗

PubMed2004

Genetic defects of cytochrome c oxidase assembly.

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, is one of the key functional and regulatory sites of the mammalian energy metabolism. Owing to the importance of the enzyme, pathogenetic mutations affecting COX frequently result in severe, often fatal metabolic disorders. No satisfactory therapy is currently available so that the treatment remains largely symptomatic and does not improve the course of the disease. While only few genetic defects of COX are caused by mutations in mitochondrial genome, during the last five years a large number of pathogenetic mutations in nuclear genes have been discovered. All these mutations are located in genes encoding COX-specific assembly proteins including SURF1, SCO1, SCO2, COX10, and COX15. Despite the identification of increasing number of mutations, their precise etiopathogenetic mechanisms, which are necessary for the development of future therapeutic protocols, still remain to be elucidated. This review summarizes recent developments, including our efforts in elucidation of the molecular basis of human mitochondrial diseases due to specific defects of COX with special focus on SURF1 assembly protein.

Alkyl and Aryl Transferases↗

PubMed2004

Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria.

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.

Animals↗

PubMed2003

Glycerophosphate-dependent peroxide production by brown fat mitochondria from newborn rats.

Glycerophosphate (GP)-dependent, ferricyanide-induced hydrogen peroxide production was studied in brown adipose tissue mitochondria from newborn rats. Relations between the rate of hydrogen peroxide production and total amount of hydrogen peroxide produced at different GP and ferricyanide concentrations were determined. It was found that the rate of hydrogen peroxide production increases with increasing GP concentration and decreases with increasing ferricyanide concentration. Total amount of hydrogen peroxide produced increases with increasing ferricyanide concentration, however, not proportionally, and the efficiency of this process (oxygen/ferricyanide ratio) strongly declines. Data presented provide further information on the character and kinetics of hydrogen peroxide production by mammalian mitochondrial glycerophosphate dehydrogenase.

Adipose Tissue, Brown↗

PubMed2003

Polarographic evaluation of mitochondrial enzymes activity in isolated mitochondria and in permeabilized human muscle cells with inherited mitochondrial defects.

Inherited disturbances of the mitochondrial energy generating system represent a heterogeneous group of disorders associated with a broad spectrum of metabolic abnormalities and clinical symptoms. We used the polarographic and spectrophotometric method for detection of mitochondrial disorders, because these two techniques provide a different insight into mitochondrial function. In six patients suspected of mitochondrial disease we found defects of complex I (two patients), complex III (one patient), complex IV (two patients) and a combination of defect of complex III and IV (one patient). Citrate synthase activity, used as the reference enzyme, was not changed. A comparison of the two methods showed several differences in evaluation of mitochondrial enzymes activity due to the fact that both methods used different conditions for enzyme activity measurements. In contrast to oxygen consumption measurements, where the function of the whole-integrated respiratory chain is characterized, spectrophotometric measurements characterize activities of isolated complexes in disintegrated membranes. However, it may be concluded from our experiments that both methods provide useful and complementary data about mitochondrial energetic functions. Whereas spectrophotometric data are suitable for evaluation of maximal enzyme activities of mitochondrial enzyme complexes, polarographic data provide better information about enzyme activities in cells with mitochondrial defects under in situ conditions.

Adolescent↗

PubMed2002

Activities of cytochrome c oxidase and citrate synthase in lymphocytes of obese and normal-weight subjects.

BACKGROUND: Obesity represents a heterogeneous group of disorders associated with broad spectrum of metabolic and endocrine abnormalities. The metabolic changes in obesity may also concern the efficacy of mitochondrial system of energy provision. The aim of our study was to analyse activities of mitochondrial enzymes cytochrome c oxidase (COX) and citrate synthase (CS) in isolated lymphocytes of obese and normal-weight subjects. RESULTS: In the group of 304 non-obese controls, differences between men and women were found neither in the COX and CS activities nor in the COX/CS ratio in isolated lymphocytes. The activity of COX did not change even with age, whereas the activity of CS decreased significantly resulting in age-dependent increase of the COX/CS ratio (P<0.01). In the group of 60 obese patients aged 17-75 y, the COX activity was 1.2-fold higher (P<0.01) and the CS activity was 1.3-fold lower (P<0.01) compared to 151 non-obese healthy age-matched controls. Consequently, the COX/CS ratio became 1.7-fold higher (P<0.01) in the obese patients compared to the non-obese population, which indicates that both the absolute and relative oxidative capacity are increased. CONCLUSION: Isolated lymphocytes from peripheral blood contribute very little to the overall metabolic turnover, but they may serve as easily available marker cells for studying the changes of mitochondrial energy converting systems in obesity.

Adolescent↗

PubMed2001

A novel mutation in SURF1 causes skipping of exon 8 in a patient with cytochrome c oxidase-deficient leigh syndrome and hypertrichosis.

Leigh syndrome is a rare pediatric neurodegenerative disorder attributed to impaired mitochondrial energy metabolism. Mutations in SURF1 have been described in several patients with Leigh syndrome associated with cytochrome c oxidase deficiency. We report a new 18-bp deletion (821del18), spanning the splice donor junction of exon 8 of SURF1, in an infant presenting with cytochrome c oxidase-deficient Leigh syndrome and hypertrichosis. cDNA sequencing demonstrated that this deletion results in a messenger lacking exon 8. RT-PCR experiments suggested a rapid degradation of the aberrant mRNA species from the 5'-end.

Alternative Splicing↗

PubMed2000

A novel principle for conferring selectivity to poly(A)-binding proteins: interdependence of two ATP synthase beta-subunit mRNA-binding proteins.

Based on electrophoretic mobility-shift assays and UV cross-linking experiments, we present evidence in the present work for the existence of two mammalian cytosolic proteins that selectively interact with the 3'-untranslated region of the mRNA coding for the catalytic beta-subunit of mitochondrial ATP synthase (beta-mtATPase). One of the proteins, beta-mtATPase mRNA-binding protein (BARB)1, is a novel poly(A)-binding protein that specifically binds the poly(A) tail of the beta-mtATPase transcript. BARB1 achieves this mRNA selectivity through its interaction with a second protein, BARB2, that binds the beta-mtATPase mRNA through a 22-bp element with a uridylate core, located 75 bp upstream of the poly(A) tail. Conversely, in the absence of BARB1, BARB2 is still able to bind the beta-mtATPase mRNA, but does so with lower affinity. Thus the interaction between BARB1 and BARB2 and beta-mtATPase mRNA involves the formation of a complex between the two BARB proteins. We conclude that BARB1 and BARB2 selectively bind the 3'-untranslated region of beta-mtATPase mRNA in a novel and interdependent manner. The complex between these two proteins may be involved in post-transcriptional regulation of gene expression.

3' Untranslated Regions↗

PubMed2000

Activities of mitochondrial oxidative phosphorylation enzymes in cultured amniocytes.

Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9-22 independent cultures of amniocytes.

Adenosine Diphosphate↗