A peptide containing residues 26-44 of tau protein impairs mitochondrial oxidative phosphorylation acting at the level of the adenine nucleotide translocator.

Having confirmed that adenovirus-mediated overexpression of NH(2)-tau fragment lacking the first 25 aminoacids evokes a potent neurotoxic effect, sustained by protracted stimulation of NMDA receptors, in primary neuronal cultures we investigated whether and how chemically synthesized NH(2)-derived tau peptides, i.e. NH(2)-26-44 and NH(2)-1-25 fragments, affect mitochondrial function. We tested both fragments on each step of the processes leading to ATP synthesis via oxidative phosphorylation: i) electron flow via the respiratory chain from physiological substrates to oxygen with the activity of each individual complex of the respiratory chain investigated in some detail, ii) membrane potential generation arising from externally added succinate and iii) the activity of both the adenine nucleotide translocator and iv) ATP synthase. Oxidative phosphorylation is not affected by NH(2)-1-25 tau fragment, but dramatically impaired by NH(2)-26-44 tau fragment. Both cytochrome c oxidase and the adenine nucleotide translocator are targets of NH(2)-26-44 tau fragment, but adenine nucleotide translocator is the unique mitochondrial target responsible for impairment of oxidative phosphorylation by the NH(2)-26-44 tau fragment, which then exerts deleterious effects on cellular availability of ATP synthesized into mitochondria.

Hydrogen peroxide and superoxide anion production during acetic acid-induced yeast programmed cell death.

Hydrogen peroxide production in yeast cells undergoing programmed cell death in response to acetic acid occurred in the majority of live cells 15 min after death induction and was no longer detectable after 60 min. Superoxide anion production was found later, 60 and 90 min after death induction when cells viability was 60 and 30%, respectively. In cells protected from death due to acid stress adaptation neither hydrogen peroxide nor superoxide anion could be observed after acetic acid treatment. The early production of hydrogen peroxide in cells in which survival was 100% could play a major role in acetic acid-induced programmed cell death signaling. Superoxide anion is assumed to be generated in cells already en route to acetic acid-induced programmed cell death.

Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality.

This study examines whether and how helium-neon laser irradiation (at fluences of 3.96-9 J/cm(2)) of cryopreserved ram sperm helps improve semen quality. Pools (n = 7) of cryopreserved ram sperm were divided into four aliquots and subjected to the treatments: no irradiation (control) or irradiation with three different energy doses. After treatment, the thawed sperm samples were compared in terms of viability, mass and progressive sperm motility, osmotic resistance, as well as DNA and acrosome integrity. In response to irradiation at 6.12 J/cm(2), mass sperm motility, progressive motility and viability increased (P < 0.05), with no significant changes observed in the other investigated properties. In parallel, an increase (P < 0.05) in ATP content was detected in the 6.12 J/cm(2)-irradiated semen samples. Because mitochondria are the main cell photoreceptors with a major role played by cytochrome c oxidase (COX), the COX reaction was monitored using cytochrome c as a substrate in both control and irradiated samples. Laser treatment resulted in a general increase in COX affinity for its substrate as well as an increase in COX activity (Vmax values), the highest activity obtained for sperm samples irradiated at 6.12 J/cm(2) (P < 0.05). Interestingly, in these irradiated sperm samples, COX activity and ATP contents were positively correlated, and, more importantly, they also showed positive correlation with motility, suggesting that the improved sperm quality observed was related to mitochondria-laser light interactions.

Mechanism of inhibition of the dicarboxylate carrier of mitochondria by thiol reagents.

The nature of the inhibition of the dicarboxylate carrier by compounds reacting with SH groups has been investigated. (1) Mersalyl and p-hydroxymercuribenzoate increase the Km without changing the V of malonate/Pi exchange, when they are added simultaneously with the dicarboxylate. If, on the other hand, the mitochondria are preincubated with SH reagents prior to the addition of malonate, the mersalyl inhibition of malonate/Pi exchange becomes predominantly non-competitive with respect to malonate. (2) In the case of Pi/Pi exchange, catalyzed by the dicarboxylate carrier, the mersalyl inhibition is competitive with respect to Pi (as indicated by Lineweaver-Burk plots), even when mersalyl is added before the substrate. Dixon plots of the rate of Pi uptake against mersalyl concentration are, however, non-linear, suggesting that the inhibition is partially competitive. (3) Dicarboxylates and dicarboxylate analogous protect against SH reagent inhibition of both dicarboxylate and Pi uptake via the dicarboxylate carrier. The protectors are effective when added before, or together with the SH reagents, but do not reverse the inhibition once it has been established. Protection by substrate analogues progressively decreases, as the time of incubation with the SH reagent increases. (4) The presence of Pi does not protect against the SH reagent inhibition of the Pi uptake. (5) The rate of SH reagent inhibition of the dicarboxylate carrier is competively inhibited by dicarboxylates. (6) It is concluded that SH reagents bind at or near the dicarboxylate specific binding site and distant from the Pi binding site. As a result of this reaction these inhibitors prevent dicarboxylate binding directly and decrease the affinity for Pi by an indirect conformational change.

Uptake of aspartate aminotransferase into mitochondria in vitro causes efflux of malate dehydrogenase and vice versa.

Incubation of intact mitochondria with aspartate aminotransferase results in efflux of malate dehydrogenase and vice versa. The export process is specific and rapid. It shows saturation kinetics with respect to the effector enzyme consistent with involvement of a receptor for the effector in the mitochondrial membrane system. Export is inhibited by both beta-mercaptoethanol and by the metal chelating agent bathophenanthroline; both substances inhibit release of malate dehydrogenase by aspartate aminotransferase competitively whereas for release of aspartate aminotransferase by malate dehydrogenase inhibition is non-competitive. The efflux process is dependent on a trans-membrane pH gradient. Exported enzymes differ from the native forms in their dependence of activity on pH. Export of both aspartate aminotransferase and malate dehydrogenase is effected by incubation of mitochondria with the newly-synthesised precursor of aspartate aminotransferase; this observation provides supporting evidence for the physiological significance of the other results reported here. It is speculated that exported enzymes are on a pathway to degradation, and that coupled uptake and export is involved in the co-ordination of synthesis and breakdown of mitochondrial proteins.

Transport of proteins into mitochondria.

There is still much that is obscure concerning the transport of proteins into or through the mitochondrial membrane systems. In addition, as pointed out previously, it is unlikely that the details of the process are the same for proteins destined for different compartments of the organelle. A brief summary of the process for matrix proteins might be as follows: The proteins are synthesized on free polysomes as precursors of higher molecular weight than the native forms. These precursors are liberated into the cell cytosol and subsequently translocated into the mitochondria. This timing might be different in yeast under some circumstances, synthesis being completed in association with the mitochondria. The precursors interact with a receptor in the outer mitochondrial membrane interaction being mediated by the presequences of the precursors. The presequences therefore act as addressing signals as well as possibly playing a role in one or all of (a) solubilization of precursors, (b) prevention of premature assembly into multimeric structures, or (c) maintenance of nonnative configurations required for transport. Interaction occurs with a second receptor, this time in the inner membrane of the mitochondria, interaction being with multiple sites in the polypeptide chain. Transport across the inner membrane then occurs, this transport depending on a transmembrane electrochemical gradient of which the proton component is the essential part. Transport is accompanied or followed by proteolysis of the prepiece, and formation of the native structure. While steps 1 and 2 of this sequence can be considered well established, the remaining steps are still poorly understood or purely hypothetical. Nevertheless, this sequence of events is consistent with known facts about the process and provides a framework for future investigations.

Improvement of stored turkey semen quality as a result of He-Ne laser irradiation.

Two experiments were carried out to evaluate the effects of He-Ne laser irradiation at various energy doses on the quality of stored turkey semen. Four semen pools were used in Experiment 1. Each pool was divided into 10 aliquots, nine of which were irradiated with energy doses ranging from 0.144 to 10.8 J/cm2 while the tenth one was not irradiated (control). Each sample was evaluated for motility immediately after irradiation, 24 and 48 h later. Energy doses ranging from 3.24 to 5.4 J/cm2 had higher (P <0.01) sperm motility index (SMI) value compared to the control and samples irradiated with lower and higher laser doses. The energy dose of 3.96 J/cm2 was selected for Experiment 2 to obtain further insight on its effects on turkey sperm preservation for up to 60 h. Each pool of four semen was divided into two aliquots: one represented the control and the other one was irradiated with He-Ne laser at an energy dose of 3.96 J/cm2. Each sample was evaluated for motility and viability immediately after irradiation and then at 12 h intervals up to 60 h. The cell energy charge was also measured by HPLC. Exposure to 3.96 J/cm2 increased the SMI and viability of turkey semen stored for 60 h compared to the control (P <0.05). The cell energy charge of irradiated samples was 200% higher than in the control. Laser irradiation increased the longevity of stored turkey spermatozoa, and might be a useful technique to enhance semen quality in long-term storage.

Metabolite transport in isolated yeast mitochondria: fumarate/malate and succinate/malate antiports.

In this study, we investigated the metabolite permeability of isolated coupled Saccharomyces cerevisiae mitochondria. The occurrence of a fumarate/malate antiporter activity was shown. The activity differs from that of the dicarboxylate carrier (which catalyses the succinate/malate antiport) in (a) kinetics (Km and Vmax values are about 27 microM and 22 nmol min(-1) mg protein(-1) and 70 microM and 4 nmol min(-1) mg protein(-1), respectively), (b) sensitivity to inhibitors, (c) Ki for the competitive inhibitor phenylsuccinate and (d) pH profiles.

Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3'-azido-3'-deoxythymidine.

The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase.

Effects of fatty acids, nucleotides and reactive oxygen species on durum wheat mitochondria.

Linoleic acid (LA) and other fatty acids added to respiring durum wheat mitochondria (DWM) were found to cause a remarkable membrane potential (deltaPsi) decrease, as monitored by measuring safranin fluorescence. The rate of deltaPsi decrease showed (i) saturation dependence on LA concentration; (ii) fatty acid specificity; (iii) inhibition by externally added ATP, GDP, GTP and Mg(2+) and (iv) sigmoid dependence upon initial DeltaPsi, thus suggesting the existence of an active plant mitochondrial uncoupling protein (PUMP) in mitochondria from monocotyledonous species (durum wheat, Triticum durum Desf.). Surprisingly, the rate of the linoleate dependent DeltaPsi decrease was found to be activated by reactive oxygen species (ROS) (hydrogen peroxide and superoxide anion) and, moreover, linoleate proved to lower the mitochondrial generation of superoxide anion. These results suggest that ROS can activate PUMP, thus protecting the cell against mitochondrial ROS production.

The role of metal ions in the uptake of aspartate aminotransferase and malate dehydrogenase into isolated rat liver mitochondria in vitro.

To gain further insight into the mitochondrial receptor area which allows selective uptake of both purified aspartate aminotransferase and malate dehydrogenase into mitochondria, the inhibition of metal complexing agents such as bathophenanthroline and tiron on the uptake of both enzymes has been investigated. In view of the nature of the inhibition found, we propose the existence of metal ion(s) at or near the aspartate aminotransferase, but far from the malate dehydrogenase binding site.

Rat liver mitochondria can hydrolyse thiamine pyrophosphate to thiamine monophosphate which can cross the mitochondrial membrane in a carrier-mediated process.

We show here that TPP --> TMP conversion can take place in rat liver mitochondria. This occurs via the novel, putative TPP pyrophosphatase localised in the mitochondrial matrix, as shown both by digitonin titration and by an HPLC enzyme assay carried out on the mitochondrial matrix fraction. Certain features of the reaction, including the substrate and pH dependence, are reported. Additional evidence is given that externally added TMP can cross the mitochondrial membrane in a manner consistent with the occurrence of a carrier-mediated process. This can occur both via the TPP translocator and via a novel translocator, inhibited by CAT but different from the ADP/ATP carrier.

Saccharomyces cerevisiae mitochondria can synthesise FMN and FAD from externally added riboflavin and export them to the extramitochondrial phase.

Evidence is given that mitochondria isolated from Saccharomyces cerevisiae can take up externally added riboflavin and synthesise from it both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) probably due to the existence of the mitochondrial riboflavin kinase already reported and the novel mitochondria FAD synthetase. Moreover Saccharomyces cerevisiae mitochondria can export the newly synthesised flavin derivatives to the extramitochondrial phase. This has been proven to take place with 1:1 stoichiometry with riboflavin decrease outside mitochondria, thus showing that flavin traffic occurs across the mitochondrial membranes.

Carrier-mediated transport controls hydroxyproline catabolism in heart mitochondria from spontaneously hypertensive rat.

In this study we have investigated hydroxyproline transport in rat heart mitochondria and, in particular, in heart left ventricle mitochondria isolated from both spontaneously hypertensive and Wistar-Kyoto rats. Hydroxyproline uptake by mitochondria, where its catabolism takes place, occurs via a carrier-mediated process as demonstrated by the occurrence of both saturation kinetics and the inhibition shown by phenylsuccinate and the thiol reagent mersalyl. In any case, hydroxyproline transport was found to limit the rate of mitochondrial hydroxyproline catabolism. A significant change in Vmax and Km values was found in mitochondria from hypertensive/hypertrophied rats in which the Km value decreases and the Vmax value increases with respect to normotensive rats, thus accounting for the increase of hydroxyproline metabolism due to its increased concentration in a hypertrophic/hypertensive state.

Glutamate neurotoxicity, oxidative stress and mitochondria.

The excitatory neurotransmitter glutamate plays a major role in determining certain neurological disorders. This situation, referred to as 'glutamate neurotoxicity' (GNT), is characterized by an increasing damage of cell components, including mitochondria, leading to cell death. In the death process, reactive oxygen species (ROS) are generated. The present study describes the state of art in the field of GNT with a special emphasis on the oxidative stress and mitochondria. In particular, we report how ROS are generated and how they affect mitochondrial function in GNT. The relationship between ROS generation and cytochrome c release is described in detail, with the released cytochrome c playing a role in the cell defense mechanism against neurotoxicity.

3'-Azido-3'-deoxythmidine uptake into isolated rat liver mitochondria and impairment of ADP/ATP translocator.

To gain some insight into the mechanism by which 3'-azido-3'-deoxythymidine (AZT) impairs mitochondrial metabolism, [14C]AZT uptake by rat liver mitochondria (RLM) in vitro was investigated. AZT accumulated in mitochondria in a time-dependent manner and entered the mitochondrial matrix. The rate of AZT uptake into mitochondria showed a hyperbolic dependence on the drug concentration and was inhibited by mersalyl, a thiol reagent that cannot enter mitochondria, thus showing that a membrane protein is involved in AZT transport. Investigation into the capability of AZT to affect certain mitochondrial carriers demonstrated that AZT was able to impair the ADP/ATP translocator, but had no effect on Pi, dicarboxylate, tricarboxylate, or oxodicarboxylate carriers. AZT inhibited ADP/ATP antiport in either mitochondria or mitoplasts in a competitive manner with different sensitivity (Ki values were 18.3 +/- 2.9 and 70.2 +/- 5.8 microM, respectively). Consistent with this were isotopic measurements showing that AZT accumulates in the intermembrane space. AZT does not use ADP/ATP carrier to enter mitochondria, as shown by the failure of both carboxyatractyloside (CAT) to inhibit AZT transport into mitochondria and AZT to induce ATP efflux from ATP-loaded mitochondria. ADP/ATP translocator impairment by AZT as one of the biochemical processes responsible for the ATP deficiency syndrome is discussed.

Mechanisms of toxicity of 3'-azido-3'-deoxythymidine. Its interaction with adenylate kinase.

Recent experiments from our laboratory have indicated that the inhibitory effect of 3'-azido-3'-deoxythymidine (AZT) on oxidative phosphorylation may occur directly, in addition to being brought about by its inhibition of mtDNA replication. We report here studies on the effect of AZT on adenylate kinase, an enzyme crucial to oxidative phosphorylation. AZT decreased the aromatic residues fluorescence of rabbit muscle adenylate kinase, indicating binding of AZT to the enzyme. Of three other enzymes studied as controls, AZT bound only to those that possessed ATP/ADP binding sites. Up to concentrations of 15 microM, AZT was a more potent effector of fluorescence quenching than were ATP, ADP, AMP, and the AZT control, deoxythymidine. AZT strongly inhibited adenylate kinase in the direction of ATP synthesis (Ki, 8 microM), the inhibition being of the partial competitive type, whereas deoxythymidine inhibition, also partially competitive, was much weaker (Ki, 90 microM). When measured in the direction of ADP synthesis, AZT failed to demonstrate any inhibition at concentrations up to 10 microM. Experiments on isolated intact rat liver mitochondria with the enzyme activity measured in both directions confirmed the isolated enzyme results. Respiratory control by these mitochondria was not affected by AZT. The finding of AZT affinity for ATP/ADP binding sites may open new avenues of approach to the study of AZT toxicity.

Rapid uncoupling of oxidative phosphorylation accompanies glutamate toxicity in rat cerebellar granule cells.

A 100 microM glutamate pulse administered to rat cerebellar granule cells causes a very rapid and progressive decrease in both cell and mitochondrial oxygen consumption caused by glucose and succinate addition, respectively. The respiratory control ratio, which reflects the ability of mitochondria to produce ATP, is reduced by 50% within the first 30 min after glutamate addition. Subsequent to glutamate exposure, a progressive decrease of respiratory control ratio to almost 1 was found within the following 3-5 h. The addition of extra calcium had no effect per se on oxygen consumption by cell homogenate.

Detection of reactive oxygen species in primary cultures of cerebellar granule cells.

The aim of this work was to develop a novel procedure useful to detect the formation of two reactive oxygen species, i.e. superoxide and singlet oxygen, in neuron monolayer primary cultures, thus, making possible the investigation of the effect of certain compounds on reactive oxygen species formation. Thus, use was made of two reactive oxygen species detecting systems consisting of ferricytochrome c (Fe-cyt c) and imidazole-RNO (N, N-dimethyl-4-nitrosoaniline) which allow for the photometric detection of superoxide anion and singlet oxygen, respectively. Both of them were used to assess the formation of reactive oxygen species in cerebellar granule cells exposed to glutamate: both superoxide anion and singlet oxygen proved to be generated in glutamate neurotoxicity in a way sensitive to glutamate NMDA-receptor inhibitor, MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a, d)cyclohepten-5,10-imine hydrogen maleate), to Ca(2+) complexing agent, EGTA, and to certain antioxidants. In principle, the reported protocol can be applied to any cell type in culture.

A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells.

Since xanthine oxidase (XO, Xanthine:oxidoreductase, E.C.1.2.3.22) is a key enzyme in reactive oxygen specie formation which plays a major role in cell oxidative stress, the availability of a sensitive and simple assay useful to detect its activity in monolayer cell cultures is worthwhile. In order to achieve this, we developed a method in which the conversion of pterine into isoxanthopterin is monitored fluorimetrically. Temperature assay was 50 degrees C. The activity of XO was detected in cerebellar granule cells exposed to glutamate. Since XO is formed from protease-dependent xanthine dehydrogenase processing, its activity appearance was found to be prevented by the protease inhibitor, leupeptin, as well as the glutamate NMDA-receptor inhibitor, MK-801, and the Ca(++) complexing agent, EGTA. The reported novel protocol, at variance with a conventional method, is shown to be a simple, fast, sensitive and relatively cheap method to assay XO activity. In addition, the reported assay can be applied to any cell type in culture.