Glutamate uptake in blood platelets from epileptic patients

Glutamate, the major excitatory amino acid neurotransmitter, is involved in epileptogenesis and initiation and spread of seizures. We studied glutamate uptake into blood platelets from patients with distinct epileptic syndromes: included were 20 patients with temporal lobe epilepsy and hippocampal sclerosis (TLE+HS), 20 with juvenile myoclonus epilepsy (JME) and 20 healthy volunteers matched for age and sex. The affinity of glutamate for the transporters was highest in patients with TLE+HS, but the maximal velocity of transport was highest in controls. There were no differences in the plasma levels of glutamate. Carbamazepine (CBZ), valproate (VPA) and lamotrigine (LTG) did not affect the uptake in vitro. The alterations observed in the uptake of glutamate in TLE+HS patients may reflect an up-regulated uptake of glutamate in the brain.     

Mitochondrial respiration of the photosynthesizing cell

Current notions on respiration of photosynthesizing cells are reviewed. Over the past three decades, the modern methods based on isotope techniques and reverse and molecular genetics provided convincing evidence that mitochondrial respiration is functional in the light and contributes to the creation of optimal conditions for photosynthesis and for protection of cells from photodegradation. Novel data are presented on the substrates that are used for respiration in the light. Individual respiration steps are considered in the context of their possible role in photosynthesizing cells. The mechanisms and carriers mediating the export of reducing equivalents from chloroplasts for their subsequent oxidation in the mitochondrial electron-transport chain are discussed. The regulation of nonphosphorylating (unrelated to energy generation) electron transport pathways mediated by alternative oxidase and alternative type II NADPH-dehydrogenases, as well as the role of uncoupling proteins in plant mitochondria, are analyzed. These components were shown to play a significant role in NAD(P)H oxidation for maintaining the redox balance in mitochondria and whole green cells. A generalized scheme of biochemical interactions between organelles—chloroplasts, mitochondria, and peroxisomes—is presented. The directions for future research are outlined.     

Mitochondrial fatty acid synthesis and respiration

Recent studies have revealed that mitochondria are able to synthesize fatty acids in a malonyl-CoA/acyl carrier protein (ACP)-dependent manner. This pathway resembles bacterial fatty acid synthesis (FAS) type II, which uses discrete, nuclearly encoded proteins. Experimental evidence, obtained mainly through using yeast as a model system, indicates that this pathway is essential for mitochondrial respiratory function. Curiously, the deficiency in mitochondrial FAS cannot be complemented by inclusion of fatty acids in the culture medium or by products of the cytosolic FAS complex. Defects in mitochondrial FAS in yeast result in the inability to grow on nonfermentable carbon sources, the loss of mitochondrial cytochromes a/a3 and b, mitochondrial RNA processing defects, and loss of cellular lipoic acid. Eukaryotic FAS II generates octanoyl-ACP, a substrate for mitochondrial lipoic acid synthase. Endogenous lipoic acid synthesis challenges the hypothesis that lipoic acid can be provided as an exogenously supplied vitamin. Purified eukaryotic FAS II enzymes are catalytically active in vitro using substrates with an acyl chain length of up to 16 carbon atoms. However, with the exception of 3-hydroxymyristoyl-ACP, a component of respiratory complex I in higher eukaryotes, the fate of long-chain fatty acids synthesized by the mitochondrial FAS pathway remains an enigma. The linkage of FAS II genes to published animal models for human disease supports the hypothesis that mitochondrial FAS dysfunction leads to the development of disorders in mammals.     

Mitochondrial dysfunction in platelets and hippocampi of senescence-accelerated mice

Senescence-accelerated mice (SAM) strains are useful models to understand the mechanisms of age-dependent degeneration. In this study, measurements of the mitochondrial membrane potential (Δψ_m) of platelets and the Adenosine 5^′-triphosphate (ATP) content of hippocampi and platelets were made, and platelet mitochondria were observed in SAMP8 (faster aging mice) and SAMR1 (aging resistant control mice) at 2, 6 and 9 months of age. In addition, an Aβ-induced (Amyloid beta-protein) damage model of platelets was established. After the addition of Aβ, the Δψ_m of platelets of SAMP8 at 1and 6 months of age were measured. We found that platelet Δψ_m, and hippocampal and platelet ATP content of SAMP8 mice decreased at a relatively early age compared with SAMR1. The platelets of 6 month-old SAMP8 showed a tolerance to Aβ-induced damages. These results suggest that mitochondrial dysfunction might be one of the mechanisms leading to age-associated degeneration in SAMP mice at an early age and the platelets could serve as a biomarker for detection of mitochondrial function and age related disease.     

Mitochondrial respiration - an important therapeutic target in melanoma

The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research. However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma. In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS. Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated. We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol. We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis. Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma.     

Mitochondrial DNA topoisomerase I from human platelets.

An anucleated cell system has been used for the first time to study mitochondrial topoisomerase activity. Mitochondrial extracts from human blood platelets contained type I topoisomerase. The type I classification was based on ATP-independent activity, inhibition by ATP or camptothecin, and the lack of inhibition by novobiocin. Platelet mitochondrial topoisomerase I relaxation activity was inhibited linearly by increasing concentrations of EGTA. Topoisomerase activity greater than 90% inhibited by 175 microM EGTA was partially restored to 16 and 50% of the initial level of activity by the subsequent addition of 50 and 100 microM Ca2+, respectively. Additionally, results from studies of partially purified platelet mitochondrial topoisomerase I were consistent with the crude extract data. This work supports the hypothesis that platelet mitochondria contain a type I topoisomerase that is biochemically distinct from that previously isolated and characterized from cell nuclei.     

Adenosine and blood platelets

Adenosine is an important regulatory metabolite and an inhibitor of platelet activation. Adenosine released from different cells or generated through the activity of cell-surface ectoenzymes exerts its effects through the binding of four different G-protein-coupled adenosine receptors. In platelets, binding of A₂ subtypes (A_2A or A_2B) leads to consequent elevation of intracellular cyclic adenosine monophosphate, an inhibitor of platelet activation. The significance of this ligand and its receptors for platelet activation is addressed in this review, including how adenosine metabolism and its A₂ subtype receptors impact the expression and activity of adenosine diphosphate receptors. The expression of A₂ adenosine receptors is induced by conditions such as oxidative stress, a hallmark of aging. The effect of adenosine receptors on platelet activation during aging is also discussed, as well as potential therapeutic applications.