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Hrynevich S.V.,Institute of Biophysics and Cell Engineering | Pekun T.G.,Institute of Biophysics and Cell Engineering | Waseem T.V.,Institute of Biophysics and Cell Engineering | Waseem T.V.,Aix - Marseille University | Fedorovich S.V.,Institute of Biophysics and Cell Engineering
Neurochemical Research | Year: 2015

Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K+-induced Ca2+-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound’s fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms. © 2015, Springer Science+Business Media New York. Source

Pekun T.G.,Institute of Biophysics and Cell Engineering | Hrynevich S.V.,Institute of Biophysics and Cell Engineering | Waseem T.V.,Aix - Marseille University | Fedorovich S.V.,Institute of Biophysics and Cell Engineering
Journal of the Korean Physical Society | Year: 2014

Brain ischemia leads to a decrease in pHo. We have shown previously in synaptosomes that the extracellular acidification induces depolarization of mitochondria followed by synthesis of superoxide anions and oxidative stress. Here, we investigated the effects of lowered pHo on oxidative stress and membrane potentials in synaptosomes treated by the iron chelator deferoxamine and zinc chelator TPEN. We demonstrated that chelating of metals has no impact on superoxide anion synthesis and intrasynaptosomal mitochondria depolarization. Meanwhile, deferoxamine was able to inhibit oxidative stress induced by low pHo and hydrogen peroxide application. Compared to deferoxamine, TPEN was less effective but it decreased the DCF fluorescence induced by pHo 6.0 which had no effects in other oxidative stress models. We found that the chelators were able to inhibit slightly plasma membrane depolarization. Synaptosomes preincubation at low pHo caused no effects on the reduced glutathione level. Depletion of glutathione by CDNB produced no additional increase in the DCF fluorescence induced by pHo 7.0. Our results suggest that free iron is crucial for the development of oxidative stress elicited by acidification in synaptosomes. Chelating of this metal seems to be a promising strategy for protecting the neuronal presynaptic terminals against oxidative stress developed at stroke. © 2014, Pekun et al.; licensee Springer. Source

Malkov A.,French Institute of Health and Medical Research | Malkov A.,Russian Academy of Sciences | Ivanov A.I.,French Institute of Health and Medical Research | Popova I.,French Institute of Health and Medical Research | And 8 more authors.
Journal of Cerebral Blood Flow and Metabolism | Year: 2014

Excessive accumulation of reactive oxygen species (ROS) underlies oxidative damage. We find that in hippocampal slices, decreased activity of glucose-based antioxidant system induces a massive, abrupt, and detrimental change in cellular functions. We call this phenomenon metabolic collapse (MC). This collapse manifested in long-lasting silencing of synaptic transmission, abnormal oxidation of NAD(P)H and FADH2 associated with immense oxygen consumption, and massive neuronal depolarization. MC occurred without any preceding deficiency in neuronal energy supply or disturbances of ionic homeostasis and spread throughout the hippocampus. It was associated with a preceding accumulation of ROS and was largely prevented by application of an efficient antioxidant Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). The consequences of MC resemble cortical spreading depression (CSD), a wave of neuronal depolarization that occurs in migraine, brain trauma, and stroke, the cellular initiation mechanisms of which are poorly understood. We suggest that ROS accumulation might also be the primary trigger of CSD. Indeed, we found that Tempol strongly reduced occurrence of CSD in vivo, suggesting that ROS accumulation may be a key mechanism of CSD initiation. © 2014 ISCBFM All rights reserved. Source

Mukhtarov M.,Aix - Marseille University | Mukhtarov M.,Kazan Federal University | Liguori L.,National Research Council Italy | Waseem T.,Aix - Marseille University | And 6 more authors.
Frontiers in Molecular Neuroscience | Year: 2013

Monitoring of the intracellular concentrations of Cl- and H+ requires sensitive probes that allow reliable quantitative measurements without perturbation of cell functioning. For these purposes the most promising are genetically encoded fluorescent biosensors, which have become powerful tools for non-invasive intracellular monitoring of ions, molecules and enzymatic activity. A ratiometric CFP/YFP-based construct with a relatively good sensitivity to Cl- has been developed (Markova et al., 2008; Waseem et al., 2010). Recently, a combined Cl-/pH sensor (ClopHensor) opened the way for simultaneous ratiometric measurement of these two ions (Arosio et al., 2010). ClopHensor was obtained by fusion of a red-fluorescent protein (DsRed-monomer) to the E2GFP variant that contains a specific Cl--binding site. This construct possesses pKa = 6.8 for H+ and Kd in the 40-50 mM range for Cl- at physiological pH (~7.3) As in the majority of cell types the intracellular Cl- concentration ([Cl-]i) is about 10 mM, the development of sensors with higher sensitivity is highly desirable. Here we report the intracellular calibration and functional characterization of ClopHensor and its two derivatives: the membrane targeting PalmPalm-ClopHensor and the H148G/V224L mutant with improved Cl- affinity, reduced pH dependence and pKa shifted to more alkaline values. For functional analysis, constructs were expressed in CHO cells and [Cl-]i was changed by using pipettes with different Cl- concentrations during whole-cell recordings. Kd values for Cl- measured at 33°C and pH ~ 7.3 were, respectively, 39 mM, 47 mM and 21 mM for ClopHensor, PalmPalm-ClopHensor and the H148G/V224L mutant. PalmPalm-ClopHensor resolved responses to activation of Cl--selective glycine receptor channels better than did ClopHensor. Our observations indicate that these different ClopHensor constructs are promising tools for non- invasive measurement of [Cl-]i in various living cells. © 2013 Mukhtarov, Liguori, Waseem, Rocca, Buldakova, Arosio and Bregestovski. Source

Lysenko E.A.,RAS Timiryazev Institute of Plant Physiology | Klaus A.A.,RAS Timiryazev Institute of Plant Physiology | Pshybytko N.L.,Institute of Biophysics and Cell Engineering | Kusnetsov V.V.,RAS Timiryazev Institute of Plant Physiology
Photosynthesis Research | Year: 2015

Data on cadmium accumulation in chloroplasts of terrestrial plants are scarce and contradictory. We introduced CdSO4 in hydroponic media to the final concentrations 80 and 250 μM and studied the accumulation of Cd in chloroplasts of Hordeum vulgare and Zea mays. Barley accumulated more Cd in the chloroplasts as compared to maize, whereas in the leaves cadmium accumulation was higher in maize. The cadmium content in the chloroplasts of two species varied from 49 to 171 ng Cd/mg chlorophyll, which corresponds to one Cd atom per 728-2,540 chlorophyll molecules. Therefore, Mg2+ can be substituted by Cd2+ in a negligible amount of antenna chlorophylls only. The percentage of chloroplastic cadmium can be estimated as 0.21-1.32 % of all the Cd in a leaf. Photochemistry (Fv/Fm, ΦPSII, qP) was not influenced by Cd. Non-photochemical quenching of chlorophyll-excited state (NPQ) was greatly reduced in barley but not in maize. The decrease in NPQ was due to its fast relaxing component; the slow relaxing component rose slightly. In chloroplasts, Cd did not affect mRNA levels, but content of some photosynthetic proteins was reduced: slightly in the leaves of barley and heavily in the leaves of maize. In all analyzed C3-species, the effect of Cd on the content of photosynthetic proteins was mild or absent. This is most likely the first evidence of severe reduction of photosynthetic proteins in leaves of a Cd-treated C4-plant. © 2014 Springer Science+Business Media. Source

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