Mitochondrial Metabolic Pathways Research Articles

Reactive oxygen species and related haem pathway components as possible epigenetic modifiers in neurobehavioural pathology

The neuroendocrine response to stress utilizes several bio-communicative pathways which also play a role in neurodevelopmental plasticity. The mechanism of action of steroidal compounds includes DNA alteration by reactive oxygen species (ROS) arising through redox cycling of reactive hormone derivatives. ROS and reactive nitrogen species play a significant role in signaling networks affecting gene transcriptional regulation during normal as well as stress-induced responses. ROS-associated synaptic and regulatory region plasticity may have been important for normal brain evolution, but probably simultaneously lowered the threshold for inducing neuropathology. A shift from 'plasticity' to 'instability' is likely to be associated with the emergence of complex effects depending on the timing, duration and intensity of the ROS insult, and is suggested to include heritable epigenetic chromatin/regulatory region remodeling differentially influencing expression levels of significant neuropsychiatric genes and their variant alleles. Neurobehavioural disorder clinical manifestations have been linked with ROS effects. The concepts discussed here relate to ROS-associated instability of DNA regulatory region sequences and a proposal that it may play an important modifying role in brain and neuro-behaviourally related gene expression. Genes encoding key steps in mitochondrial, haem, iron and bilirubin ROS metabolic pathways have been used as examples to illustrate how ROS-modified regulatory networks could possibly alter the context within which (even ostensibly unrelated) neuropsychiatric gene candidates may sometimes be recruited. Furthermore, reactions of certain radicals release sufficient energy to generate UV-photons. DNA conformational changes accompanied by changes in photon emission suggest that functional neuroimaging findings probably reflect interaction on the level of ROS/biophoton/genome regulatory region domains rather than the signatures of individual neurobehavioural disorder candidate genes.

Acute myocardial ischaemia induces specific alterations of ventricular mitochondrial function in experimental pigs

As cardiac metabolic flexibility is crucial, this study examined whether acute ischaemia can induce specific qualitative alterations of the mitochondrial metabolic pathways as well as energy transfer systems. Left descending coronary artery ligation was performed after sternotomy in eight pigs and the heart was excised after 45 min of ischaemia. Maximal O2 uptake (V(max), micromol O2 min(-1) g(-1) dry weight) of saponin-skinned myofibres were measured from ischaemic and non-ischaemic area of ventricular myocardium. V(max) decreased by approximately 20% in ischaemic myocardium with both glutamate-malate (18.1 +/- 1.3 vs. 22.1 +/- 1.7 in control, P < 0.05) and pyruvate substrates (19.3 +/- 1.0 vs. 23.3 +/- 2.0 in control, P < 0.05) whereas no difference was observed with palmitoyl carnitine (15.6 +/- 1.8 vs. 16.6 +/- 0.9 in control). The K(m) of mitochondrial respiration for ADP decreased in ischaemic heart by 24% (679 +/- 79 vs. 899 +/- 84 microm of ADP in control, P < 0.05). Moreover, the mitochondrial creatine kinase efficacy (K(m) without creatine/K(m) with creatine), representative of the coupling of oxidative phosphorylation process with the mitochondrial creatine kinase, was reduced in ischaemic heart (11.6 +/- 2.5 in ischaemic vs. 18.0 +/- 2.2 in control, P < 0.05). These findings argue for specific mitochondrial impairments at the level of pyruvate oxidation and creatine kinase channelling system after an acute period of in vivo ischaemia, whereas the lipid mitochondrial oxidation pathway seems to be preserved. Such a loss of metabolic flexibility following acute ischaemia could become an early feature of metabolic dysregulation of the heart.

Reduction of All-trans Retinal to All-trans Retinol in the Outer Segments of Frog and Mouse Rod Photoreceptors

The first step in the Visual Cycle, the series of reactions that regenerate the vertebrate visual pigment rhodopsin, is the reduction of all-trans retinal to all-trans retinol, a reaction that requires NADPH. We have used the fluorescence of all-trans retinol to study this reduction in living rod photoreceptors. After the bleaching of rhodopsin, fluorescence (excitation, 360nm; emission, 457 or 540nm) appears in frog and wild-type mouse rod outer segments reaching a maximum in 30–60min at room temperature. With this excitation and emission, the mitochondrial-rich ellipsoid region of the cells shows strong fluorescence as well. Fluorescence measurements at different emission wavelengths establish that the outer segment and ellipsoid signals originate from all-trans retinol and reduced pyridine nucleotides, respectively. Using outer segment fluorescence as a measure of all-trans retinol formation, we find that in frog rod photoreceptors the NADPH necessary for the reduction of all-trans retinal can be supplied by both cytoplasmic and mitochondrial metabolic pathways. Inhibition of the reduction reaction, either by retinoic acid or through suppression of metabolic activity, reduced the formation of retinol. Finally, there are no significant fluorescence changes after bleaching in the rod outer segments of Rpe65−/− mice, which lack 11-cis retinal.

The hydrogenosomes of the chytrid fungus Neocallimastix frontalis—Characterizing the functions of an anaerobic cousin of mitochondria through proteome analysis

Hydrogenosomes are anaerobic forms of mitochondria in at least four independent groups of anaerobic eukaryotes, the parabasalids, the heterolobosea, the ciliates and the anaerobic chytridiomycetes. Their hydrogenosomes generate ATP from the fermentative decomposition of pyruvate but lack the hallmark pathways of mitochondrial metabolism, the TCA cycle and membrane‐associated electron transport. They also, with one exception, do not contain their own genome. We are analysing the proteome of the hydrogenosomes from the chytridiomycete Neocallimastix frontalis through 2D electrophoresis and tandem mass spectroscopy to assess the functional capacities of these organelles and to further characterize their relationship with mitochondria.

The genome of the protist parasite Entamoeba histolytica

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Gender-specific proteomic alterations in glycolytic and mitochondrial pathways in aging monkey hearts

We utilized proteomic techniques in a primate model ( Macaca fascicularis) of aging to determine potential mechanisms to explain gender differences in protection of the aging heart. The majority of prior work in this field utilized rodent models, and importantly no prior study utilized a proteomic approach in the aging heart. We studied changes in proteins in seven monkeys in each group (young and old males and females (YMs, OMs, YFs, and OFs, respectively)), and used two-dimensional gel electrophoresis in combination with mass spectrometry in five monkeys in each group. We found decreases ( P < 0.05) in the expression of key enzymes in glycolysis (e.g. pyruvate kinase, α-enolase, triosephosphate isomerase), glucose oxidation (e.g. pyruvate dehydrogenase E1 β-subunit), and the tricarboxylic acid (TCA) cycle (2-oxoglutarate dehydrogenase) in left ventricular (LV) samples from OM monkeys; these changes in glycolytic, glucose oxidation, and TCA enzymes were not observed either in YMs, YFs or OFs. We found additional gender differences in the reduced expression and function of proteins that are responsible for electron transport and oxidative phosphorylation in mitochondria only in hearts from OM monkeys, with corresponding decreased oxidation rates with NADH and ascorbate- N,N,N′,N′ ́ ́-tetramethyl- p-phenylenediamine substrates. The changes in glycolytic and mitochondrial metabolic pathways in OM monkey hearts are similar to changes observed in hearts affected by diabetes or LV dysfunction, and could be involved in the mechanism for the cardiomyopathy of aging. The sparing of these changes in OF hearts could be involved in the mechanism mediating delayed cardiovascular risk in OFs.

Mitochondrial disorders.

In the medical literature the term 'mitochondrial disorders' is to a large extent applied to the clinical syndromes associated with abnormalities of the common final pathway of mitochondrial energy metabolism, i.e. oxidative phosphorylation (OXPHOS). Faulty oxidative phosphorylation may be due to overall dysfunction of the respiratory chain, a heteromultimeric structure embedded in the inner mitochondrial membrane, or can be associated with single or multiple defects of the five complexes forming the respiratory chain itself. From the genetic standpoint, the respiratory chain is a unique structure of the inner mitochondrial membrane formed by means of the complementation of two separate genetic systems: the nuclear genome and the mitochondrial genome. The nuclear genome encodes the large majority of the protein subunits of the respiratory complexes and most of the mitochondrial DNA (mtDNA) replication and expression systems, whereas the mitochondrial genome encodes only 13 respiratory complex subunits, and some RNA components of the mitochondrial translational apparatus. Accordingly, mitochondrial disorders due to defects in OXPHOS include both mendelian-inherited and cytoplasmic-inherited diseases. This review describes human genetic diseases associated with mtDNA and nuclear DNA mutations leading to impaired OXPHOS.

A cDNA microarray analysis of gene expression profiles in rat hippocampus following a ketogenic diet

The ketogenic diet (KD) is an effective therapy for medically intractable epilepsy, but its anticonvulsant mechanisms are unknown. Few studies to date have addressed the molecular changes following treatment with a KD. In the present study, we fed juvenile rats either a standard diet or a KD for 1 month, and then determined changes in hippocampal gene expression using cDNA microarray analysis (Clontech). To validate the microarray expression results, we also performed Northern blot and RT-PCR analysis on a small subset of affected genes. Among a total of 1176 cDNAs, 42 genes were strongly up- or down-regulated (>2-fold change over controls) by a KD. We found that the expression of mitochondrial ATP synthase β subunit, mitochondrial ATP synthase D subunit (ATP5H) and mitochondrial ATP synthase β subunit precursor (ATP5F) were especially increased in KD-treated group, whereas the KD down-regulated protein kinase C (PKC) β and ɛ isoforms. Thus, the most prominent changes were seen in genes encoding proteins involved in mitochondrial metabolic and intracellular signal transduction pathways. Our data provide some insights into the complex cascade of cellular changes in the hippocampus induced by a KD, some of which may contribute to its anticonvulsant effects.

A unifying concept of seizure onset and termination

Recent discoveries in molecular biology and human genetics have contributed greatly to an understanding of the nature of seizure (ictal) activity. However, two questions of fundamental clinical importance continue to resist scientific inquiry: when and why does a seizure begin; and when and why does a seizure end? This paper cites evidence from the medical literature in support of two counterintuitive concepts that address this issue. First, that despite the diversity of conditions that are associated with seizures, the ictal response results from disturbances of a mitochondrial metabolic pathway that is common to them all. Second, that the seizure is not inherently harmful but is, instead, associated with massive intracerebral circulatory changes that are intended to restore impaired mitochondrial function. We hypothesize that the protogenic pathophysiological condition leading to neuronal hyperexitability and seizures results from inadequate mitochondrial energy production due to hypoxia or a hypoxia-equivalent state. Failure to generate sufficient adenosine triphosphate compromises ionic pump function and the ability to maintain neuronal homeostasis and stability. The seizure cascade is a heroic effort to perfuse the brain when local mechanisms fail to restore energy production and ionic equilibrium. In summary, a seizure starts when the neuron's aerobic machinery fails to maintain effective ionic pump function and terminates when increased cerebral perfusion, associated with the seizure response, restores adequate supplies of metabolic nutrients required for mitochondrial respiration. This unorthodox unifying concept that views ictogenesis as part of a restorative process rather than as a life threatening event may provide the basis for a much needed paradigm shift in the management of seizures. Current antiepileptic drugs are associated with many serious side effects, including death, and fail to control seizures in 20% of patients with primary generalized epilepsy and 35% of patients with partial epilepsy. We propose that efforts to prevent and control seizures should be directed away from pharma-chemical suppression towards removing the causes of disturbed neuronal energy production and developing methods and bioactive agents that promote an optimized physiological milieu within the brain.

A Metabolic Model for Life Span Determination in Caenorhabditis elegans

Several studies with the nematode Caenorhabditis elegans have made the unexpected discovery that certain hypomorphic mutations in genes encoding mitochondrial proteins result in life span extension. These mutations appear to act independently of the other known pathway that regulates life span extension, the dauer-specifying insulin/IGF-1-like pathway. Here we present a hypothesis that unifies the effects of these two classes of genes on longevity. The central concept is that energy generation in C. elegans occurs by differential flux through two coexisting mitochondrial metabolic pathways—aerobic respiration and fermentative malate dismutation. In the latter process, fumarate is terminally reduced at complex II to succinate. We suggest that most, if not all, long-lived mutants in C. elegans utilize malate dismutation, a byproduct of which is the generation of fewer radical species.

Neuroprotective effects of L-carnitine in induced mitochondrial dysfunction.

The neuroprotective action of l-carnitine (LC) in the rat model of 3-nitropropionic acid (3-NPA)-induced mitochondrial dysfunction was examined. 3-NPA is known to produce decreases in neuronal ATP levels via inhibition of the succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain. SDH is involved in reactions of the Krebs cycle and oxidative phosphorylation, and its inhibition leads to both necrosis and apoptosis. LC enhances mitochondrial metabolism and, together with its acetylated form, acetyl-l-carnitine (ALC), via the LC-ALC-mediated transfer of acetyl groups, plays an important modulatory role in neurotransmitter signal transduction pathways and gene expression in neuronal cells. In the study described here, adult male Sprague-Dawley rats were injected with 3-NPA alone or treated with LC prior to 3-NPA administration. Pretreatment with LC totally prevented the 3-NPA-induced decrease in brain temperature measured using temperature probes implanted intracranially. It appears that the protective effects of LC against 3-NPA-induced neurotoxicity are achieved via compensatory enhancement of several pathways of mitochondrial energy metabolism. The results of this and previous studies conducted by our division in the 3-NPA model of mitochondrial dysfunction demonstrate that 3-NPA may be employed in vivo to evaluate enhancers of mitochondrial function that might exert neuroprotective effects.

Minimizing oxidative injury to the developing brain: the therapeutic quest continues

EDITOR-IN-CHIEF Oxidative injury to the developing central nervous system continues to be an area of concern and controversy in perinatal medicine. The clinical examples of hypoxia‐ ischemia-induced oxidative stress in the perinatal period include antepartum and intrapartum asphyxial events and neonatal respiratory distress. In the last scenario, oxidative stress may also be produced during resuscitation secondary to hyperoxygenation. The spectrum of oxidative stress-induced neurological syndromes extends from behavioral and learning disabilities to cerebral palsy. However, the causal relationship between clinically recognized episodes of hypoxia and perinatal brain lesions is often uncertain. Most cases of perinatal neurological compromise do not demonstrate any defined antecedents and most cases of perinatal asphyxia do not result in any demonstrable neurodevelopmental deficit. These controversies reflect deficiencies in our knowledge in this field. Recent investigations, however, have made significant contributions to the elucidation of this complex problem, especially in the underlying molecular mechanisms 1 . Hypoxia‐ ischemia-induced neuronal injury essentially starts with neuronal energy failure, resulting in the excessive production of both oxygen-free and other secondary radicals, and non-radical reactive species which can injure cell constituents. There is direct evidence of oxygen-free radical generation in the developing brain in response to hypoxia 2,3 . Cells generate oxygen-free radicals during normal respiratory and metabolic activities. Oxygen-free radicals are redox intermedates produced during the sequential reduction of oxygen to water, as an integral part of the energy-regenerating process of oxidative phosphorylation. Biologically significant examples of oxygen-free radicals include superoxide, hydroxyl, lipid hydroperoxide, lipid peroxyl and lipid alkoxyl radicals, and also nitric oxide. The non-radical intermediates include hydrogen peroxide and singlet oxygen. These radical and non-radical products are often collectively called reactive oxygen species. The mechanisms for the formation of these agents are complex and multiple, and include mitochondrial respiration and various metabolic pathways 4‐7 . Additional mechanisms of free radical generation in the brain include inflammatory and immunological reactions, expression of adhesion molecules, and stimulation of polymorphonuclear

Structural determinants of fluorochemical-induced mitochondrial dysfunction.

Perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) are thought to induce peroxisome proliferation and interfere with mitochondrial metabolic pathways. Direct measurements revealed that PFOA and the unsubstituted sulfonamide of perfluorooctane (FOSA) uncouple mitochondrial respiration by increasing proton conductance. The purpose of this investigation was to characterize structural determinants responsible for the mitochondrial uncoupling effect of several structurally related fluorochemicals. Included in the study were PFOA, PFOS, FOSA, the N-acetate of FOSA (perfluorooctanesulfonamidoacetate, FOSAA), N-ethylperfluorooctanesulfonamide (N-EtFOSA), and the N-ethyl alcohol [2-(N-ethylperfluorooctanesulfonamido)ethyl alcohol, N-EtFOSE] and N-acetic acid (N-ethylperfluorooctanesulfonamidoacetate, N-EtFOSAA) of N-EtFOSA. Each test compound was dissolved in ethanol and added directly to an incubation medium containing substrate-energized rat liver mitochondria. Mitochondrial respiration and membrane potential were measured concurrently using an oxygen electrode and a TPP+ -selective electrode, respectively. All of the compounds tested, at sufficiently high concentrations, had the capacity to interfere with mitochondrial respiration, albeit via different mechanisms and with varying potencies. At sufficiently high concentrations, the free acids PFOA and PFOS caused a slight increase in the intrinsic proton leak of the mitochondrial inner membrane, which resembled a surfactant-like change in membrane fluidity. Similar effects were observed with the sulfonamide N-EtFOSE. Another fully substituted sulfonamide, N-EtFOSAA, at high concentrations caused inhibition of respiration, the release of cytochrome c, and high-amplitude swelling of mitochondria. The swelling was prevented by cyclosporin A or by EGTA, indicating that this compound induced the mitochondrial permeability transition. The unsubstituted and mono-substituted amides FOSA, N-EtFOSA, and FOSAA all exerted a strong uncoupling effect on mitochondria resembling that of protonophoric uncouplers. Among these compounds, FOSA was a very potent uncoupler of oxidative phosphorylation, with an IC50 of approximately 1 microM. These data suggest that the protonated nitrogen atom with a favorable pKa is essential for the uncoupling action of perfluorooctane sulfonamides in mitochondria, which may be critical to the mechanism by which these compounds interfere with mitochondrial metabolism to induce peroxisome proliferation in vivo.

COMBINED DEFICIENCY OF PYRUVATE DEHYDROGENASE AND MITOCHONDRIAL RESPIRATOR CHAIN COMPLEX I: CASE REPORT

We described a male child of two years and three months affected by assial hypotonia, psicomotor retardation, muscular distal hypotrophy with hyporeflexia, dystonic movements and deafness. He was born from non‐consanguineous parents by spontaneous caesarian twin delivery at 38 weeks. His sister is healthy. He came to our observation at 16 months of age. Screenings for metabolic diseases and CSF neurotransmitters and lactate were normal, whereas the serum lactate was mildly increased. Brain MRI showed delayed myelination, with the normal lactate at the spectroscopic study. EEG showed interemispheric asymmetry with epileptic discharges. EMG showed a neurogenic pattern. Molecular DNA analysis for Spinal Muscular Atrophy was negative. Muscle biopsy showed neurogenic injury and an absence of ragged red fibers; enzymatic assay showed a severe deficiency of Complex I (2.8 nmol/min/mg protein, n.v. 17–33) and a mild deficiency of Pyruvate Dehydrogenase (PDHC=0.47 nmol/min/mg protein, n.v.0.8‐2).The clinical features of patients with a dysfunction in mitochondrial metabolic pathways are variable. A PDHC deficiency leads to a graduated spectrum of neurological involvement starting from severe forms with death in the neonatal period, Leigh disease and carbohydrate‐induced episodic ataxia. The most common features associated with a PDHC defect are delayed development and hypotonia. Patients with Complex I deficiency show a variable phenotype from fatal infantile encephalomyopathy to adult‐onset myopathy; neurodegenerative disorders are also described: Parkinson's disease, dystonia and Leber's optic neuropathy. A combined PHDC and Complex I deficiency is rarely reported. We can make the hypothesis that a defect of a single enzyme (Complex I) can play a role on the other enzyme deficiency. The presence of a primitive PHDC and Complex I deficiency is unlikely be hypothesized since the parents are not consanguineous and a double genetic defect is improbable.

Toxin-induced mitochondrial dysfunction

Publisher Summary This chapter reviews the mode of action of several well-characterized mitochondrial toxins, and discusses their effects in contrast with mechanisms of cell death in various degenerative disorders. The majority of known toxins specifically target components of mitochondrial metabolic pathways. The chapter describes the grouping of toxic agents in terms of their principal target site within the mitochondria, often reflecting effects on components of the mitochondrial respiratory system. In several cases, however, these agents have multiple sites of action because of their affinity for certain chemical moieties present in multiple mitochondrial proteins, demonstrated by cyanide's propensity for Fe 3+ containing enzymes which include both complexes II and IV of the electron-transport chain (ETC). The mitochondrial toxin 3-nitropropionic acid (3-NP) irreversibly inhibits the activity of succinate dehydrogenase, a metabolic enzyme that participates in both the tricarboxylic-acid (TCA) cycle and in complexes II–III of the ETC. Systemic administration of this agent to humans, nonhuman primates, and rodents results in central nervous system (CNS) lesions that selectively target subpopulations of striatal neurons, closely replicating the nature and regional specificity of pathological events seen in Huntington's disease (HD).

Consequence of restricted mitochondrial oxidative metabolism on photosynthetic carbon assimilation in mesophyll protoplasts: Decrease in light activation of four chloroplastic enzymes

The patterns of light activation of 4 chloroplastic enzymes were examined in mesophyll protoplasts of pea (Pisum sativum) in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM, inhibitor of alternative pathway). The results were compared with those of DCMU (inhibitor of photosynthetic electron transport). The light activation of NADP glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), fructose-1,6-bisphosphatase (FBPase), phosphoribulokinase (PRK) (enzymes of the Calvin cycle) and NADP malate dehydrogenase (NADP-MDH) (reflects chloroplast redox state) was more pronounced at limiting CO2 (0.1 mM NaHCO3) than that at optimal CO2 (1.0 mM NaHCO3). SHAM decreased markedly (up to 33%) the light activation of all 4 enzymes, while antimycin A or oligomycin exerted only a limited effect (<10% decrease). Antimycin A or oligomycin or SHAM had no significant effect on light activation of these 4 enzymes in isolated chloroplasts. However, DCMU caused a remarkable decrease in light activation of enzymes in both protoplasts (up to 78%) and chloroplasts (up to 69%). These results suggest that the restriction of alternative pathway of mitochondrial metabolism results in a marked decrease in the light activation of key chloroplastic enzymes in mesophyll protoplasts but not in isolated chloroplasts. Such a decrease in the light activation of enzymes could be also a secondary feedback effect because of the restriction on carbon assimilation.

An evaluation of thiram toxicity on cultured human skin fibroblasts

Thiram is widely used in agriculture as a fungicide and, to a lesser extent, as a vulcanizing agent in the rubber industry. In spite of the extensive use of thiram, knowledge on its toxicity and health risk remains limited, and few investigations have been performed to assess specific damage at the cellular and subcellular level. We report here the cytotoxic effects of thiram on cultured human skin fibroblasts. Our results demonstrated that thiram exposure induced a dose- and time-dependent decrease in the viable cell recovery with 100% cell death observed with a concentration of 5.0 mg/l. As judged by morphological changes and biochemical criteria, thiram-mediated cell death was not of the apoptotic but seemed to be of the necrotic type. This cell death was not associated with a modification of gene expression of different constituents of the extracellular matrix. A late increase of lactate production was evident after thiram treatment, suggesting a mitochondrial metabolic pathway dysfunction as reported by other authors using similar compounds. However, this phenomenon appeared as a secondary response to the toxic action of thiram. The cytotoxic effect of thiram is possibly due to an oxidant effect inherent to the structure of thiram and the interaction between thiram and vital cellular molecules.

Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.

Kidney proximal tubule cells developed severe energy deficits during hypoxia/reoxygenation not attributable to cellular disruption, lack of purine precursors, the mitochondrial permeability transition, or loss of cytochrome c. Reoxygenated cells showed decreased respiration with complex I substrates, but minimal or no impairment with electron donors at complexes II and IV. This was accompanied by diminished mitochondrial membrane potential (DeltaPsi(m)). The energy deficit, respiratory inhibition, and loss of DeltaPsi(m) were strongly ameliorated by provision of alpha-ketoglutarate plus aspartate (alphaKG/ASP) supplements during either hypoxia or only during reoxygenation. Measurements of (13)C-labeled metabolites in [3-(13)C]aspartate-treated cells indicated the operation of anaerobic pathways of alphaKG/ASP metabolism to generate ATP, yielding succinate as end product. Anaerobic metabolism of alphaKG/ASP also mitigated the loss of DeltaPsi(m) that occurred during hypoxia before reoxygenation. Rotenone, but not antimycin or oligomycin, prevented this effect, indicating that electron transport in complex I, rather than F(1)F(0)-ATPase activity, had been responsible for maintenance of DeltaPsi(m) by the substrates. Thus, tubule cells subjected to hypoxia/reoxygenation can have persistent energy deficits associated with complex I dysfunction for substantial periods of time before onset of the mitochondrial permeability transition and/or loss of cytochrome c. The lesion can be prevented or reversed by citric acid cycle metabolites that anaerobically generate ATP by intramitochondrial substrate-level phosphorylation and maintain DeltaPsi(m) via electron transport in complex I. Utilization of these anaerobic pathways of mitochondrial energy metabolism known to be present in other mammalian tissues may provide strategies to limit mitochondrial dysfunction and allow cellular repair before the onset of irreversible injury by ischemia or hypoxia.

Genética de las epilepsias mitocondriales

Recently the molecular basis of a series of clinical disorders associated with defects in the oxidative phosphorylation system (OXPHOS system) leading to ATP synthesis, the final pathway of mitochondrial energy metabolism, has been established. The polypeptide components of the OXPHOS system are codified in both nuclear and mitochondrial DNA. Therefore these mitochondrial diseases may be originated by mutations of genes found in both genetic systems. In recent years, several such neuromuscular diseases have been defined and associated with mitochondrial DNA mutations. One of the most striking of these is the syndrome of myoclonic epilepsy with ragged red fibres (MERRF), characterized by myoclonic epilepsy of maternal inheritance. This disorder is caused by a specific mutation on the mitochondrial tRNA(Lys) (position 8344), which gives rise to a reduction in the level of lysil-tRNA(Lys) and thus to premature termination of the translation of proteins codified in the mitochondrial DNA.

Structure and energy pathways of spermatozoa of the rove beetle Aleochara bilineata (Coleoptera, Staphylinidae)

The morphology of mature spermatozoa of the rove beetle Aleochara bilineata was examined by using scanning and transmission electron microscopy. They are about 1000 μm long and filiform. The acrosome and the nucleus are elongate and each about 20 μm long. A well-developed centriole adjunct region connects the nucleus with the sperm tail. The axoneme reveals the 9 + 9 + 2 pattern of the pterygote sperm flagellum. Two accessory bodies and two mitochondrial derivatives with paracrystalline inclusions are present. Cristae are reduced to the cortical zone of the derivatives. Cytochrome-c oxidase activity was detected within the cristae by DAB-reaction. The energy metabolism of the spermatozoa was investigated by using different inhibitors affecting the mitochondrial and cytoplasmic metabolic pathways. Sperm movement was used as an indicator for the utilization of ATP by the axoneme. In control experiments, the duration of motility was longer than 45 min. In the presence of atractyloside or potassium cyanide the motility duration was not affected. On the other hand, iodoacetic acid in the medium stopped sperm motility within 15 min. This indicates that sperm energy metabolism mainly depends on the glycolytic pathway.