Synaptopathy under conditions of altered gravity: changes in synaptic vesicle fusion and glutamate release.

Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO ( approximately 10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[(14)C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca(2+) or Mg(2+)/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.

[Release of glutamate from cytosol of synaptosomes under conditions of experimental hypergravity].

The release of L-[14C]glutamate via Na+-dependent glutamate transporters functioned in the reverse mode was investigated in cortical synaptosomes under centrifuge-induced hypergravity. The protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) induced increase in [Na+]i, depolarized the plasma membrane, dissipated the proton gradient across synaptic vesicles and mitochondrial membrane, caused a fall in both the ATP level and the ATP/ADP ratio. 35 mM KCl-stimulated L-[14C]glutamate release from synaptosomes preliminary treated with 1 microM FCCP considerably increased from 27.0+/-2.2 % to 35.0+/-2.3 % of total accumulated synaptosomal label after centrifuge-induced hypergravity as compared to control animals (P< or =0.05). We found the competitive nontransportable glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate to inhibit FCCP and high KCL-stimulated release of L-[14C]glutamate. The release would be expected to occur via plasma membrane glutamate transporters. Transportable inhibitor of glutamate transporters-DL-threo-beta-hydroxyaspartate (DL-THA) induced heteroexchange of L-[14C]glutamate from enlarged by FCCP cytosolic pool of the neurotransmitter. DL-THA-evoked release of L-[14C]glutamate was also increased significantly after hypergravity. Combined application of KCl, DL-THA and FCCP unmasked dramatic changes in the activity of the glutamate transporters functioning in the reverse mode after centrifuge-induced G-loading.

[Methyl-beta-cyclodextrin influences glutamate transport in the rat brain nerve terminals by depletion of membrane cholesterol].

Role of membrane cholesterol in direct and reversed function of Na+ -dependent glutamate transporters and exocytosis was investigated. The depletion of membrane cholesterol by methyl-beta-cyclodextrin (MebetaCD) resulted in a dose-dependent significant reduction of the L-[14C]glutamate uptake by synaptosomes. Treatment of synaptosomes with 15 mM MebetaCD caused a decrease in the velocity of L-[14C]glutamate uptake by 49 +/- 4% (P < or = 0.05). The depolarization stimulated Ca2+ -dependent glutamate release that occurred via reverse functioning of glutamate transporters decreased insignificantly for 1 min from 8.0 +/- 0.4% to 6.7 +/- 0.4% of total accumulated synaptosomal label after MebetaCD treatment. The depletion of membrane cholesterol resulted in a reduction of the depolarization evoked exocytotic release from 8.0 +/- 1.0% to 4.2 +/- 1.0% of total synaptosomal label. Thus, cholesterol depletion was found to decrease significantly the Na+ -dependent uptake and exocytotic release of glutamate.

[Study on efficacy and toxicity of new combined regimen "taxoter + cisplatin + 5-fluorouracil" in disseminated and locally-spread stomach cancer. Comparative analysis of tolerance and efficacy in patients younger and older than 65 years].

The aim of our study was to evaluate the efficacy and safety of 3-drugs regimen: T 75 mg/m2 d2 + P 75 mg/m2 d2 + F 500 mg/m2 x 3h d 1-3 every 28 days. 31 patients (pts) with morphologically proven advanced gastric cancer of the age 29-77 years (median 61.0) have been treated with this regimen. They received 138 cycles (1-10, median 4.0 cycles per pt). The response rate was evaluated in pts received > or =2 cycles: CR 1/27 (3.7%), PR 12/27 (44.4%), SD 7/27 (25.9%), PD 7/27 (25.9%). The median duration of CR+PR--4.5 mon (1.1-9.9), of SD--6.8 mon (3.0-10.7). Median TTP--5.5 mon. Overall survival: median--11.5 mon, 1-year--46.6%. PS improvement was observed in 54.8%pts, symptomatic improvement--in 71% pts. Toxicity per pt (per cycle) was moderate. There were 11 elderly among these pts. We didn't receive any significant differences in efficacy and severe toxicity in this group compared to non-elderly pts. We observed 55.6% PR, 33.3% SD, 11.1% PD, TTP--4.6 mon, median OS-7.5 mon. in elderly and 5.6% CR, 38.9% PR, 22.2% SD, 33.3 % PD, TTP--6.1 mon, median OS-12.3 mon for non-elderly pts. But dose reduction was performed more frequently in the elderly then non-elderly: 63.6% vs 30.0% pts (p = 0.07) in 64.8% vs 19.1% cycles (p < 0.0001). We consider this regimen to be effective and well tolerated both for elderly and for non-elderly patients.

Phenylarsine oxide is able to dissipate synaptic vesicle acidic pool.

Phenylarsine oxide (PAO) has a number of targets in the neurons, one of them is exocytotic process. In this study, we have focused on the mechanisms of phenylarsine oxide action on Ca(2+)-dependent and Ca(2+)-independent neurotransmitter release from rat brain synaptosomes. We investigated the influence of phenylarsine oxide on: (i) l-[(14)C]glutamate and [(3)H]GABA release and uptake; (ii) plasma membrane potential using a potential-sensitive fluorescent probe rhodamine 6G; (iii) exo/endocytotic process using a pH-sensitive fluorescent probe acridine orange (AO). It has been found that phenylarsine oxide induced deacidification of synaptic vesicles. This effect was completely abolished by preliminary treatment of synaptosomes with a protonophore FCCP indicating that both reagents injured a proton electrochemical gradient. Dissipation of the proton gradient by low concentrations of phenylarsine oxide (not exceed 1 microM) did not prevent KCl-triggered exocytotic response, but essentially modified endocytotic one. At higher concentrations of phenylarsine oxide (up to 10 microM), the proton gradient dissipation was intensified and the exocytotic response was fully abolished. The reagent did not change plasma membrane potential, but depolarized mitochondria. It also caused potent inhibition of the Ca(2+)-stimulated l-[(14)C]glutamate and [(3)H]GABA release and increase the Ca(2+)-independent release of l-[(14)C]glutamate, but not of [(3)H]GABA. Disulfide-reducing reagents (dithiothreitol and beta-mercaptoethanol) completely prevented phenylarsine oxide-evoked injuries. They could also restore the initial levels of the mitochondrial potential, the exocytotic response to KCl and the release and uptake of neurotransmitters. Our data provide the evidence that phenylarsine oxide causes dissipation of synaptic vesicle acidic pool resulting in the reduction of vesicle filling and as consequence in attenuation of Ca(2+)-stimulated neurotransmitter release.

[Modulating effect of glutamate transporter inhibitors on accumulation and release of the neuromediator by the brain nerve terminals in rats].

L-[14C]glutamate uptake and release processes in nerve terminals has been investigated using the nontransportable and transportable competitive inhibitors of glutamate transport as tools. The effects of DL-threo-beta-benzyloxyaspartate (DL-TBOA) and DL-threo-beta-hydroxyaspartate (DL-THA) on the accumulation of L-[14C]glutamate have been evaluated after the exposure of rats to centrifuge-induced hypergravity. Both analogs potently inhibited the L-[14C]glutamate uptake in a dose-dependent manner (100 microM glutamate, 30 s incubation period). The IC50 values for DL-TBOA calculated on the basis of curves of non-linear regression kinetic analysis was 18 +/- 2 microM and 11 +/- 2 microM (p < or = 0.05) before and after the exposure to artificial gravity, respectively. L-THA, transportable analog, exhibited similar inhibitory characteristics (18 +/- 2 and 12 +/- 2 microM, respectively). We have also demonstrated that DL-TBOA exerted slighter effect on depolarization-evoked carrier-mediated L-[14C]glutamate release in control rats in comparison with gravity-loaded ones. Thus, DL-TBOA had complex effect on glutamatergic transmission, inhibited uptake and release of L-glutamate, and perhaps, became more potent under centrifuge-induced hypergravity.

Glutamatergic transmission in the rat brain and gravitational stress.

The effects of hypergravity stress on L-[14C]-glutamate release from synaptosomes obtained from the rat brain and on the kinetic parameters of high-affinity glutamate transport activity were investigated. We found that hypergravity stress affected only the Ca(2+)-dependent component of L-[14C]-glutamate release. It did not modify the transporter affinity, but the maximum rate of uptake dropped from 12.5 +/- 3.2 to 5.6 +/- 0.9 nmol/min/mg of protein (in control rats and in animals subjected to hypergravity, respectively).

[Study on the effect of hypergravity stress on L-glutamate uptake by rat brain nerve endings]. / Issledovanie vliianiia gipergravitatsionnogo stressa na aktivnoe pogloshchenie L-glutamata nervnymi okonchaniiami golovnogo mozga krys.

Using rat brain synaptosomes, we have investigated the effect of hypergravity on the kinetic parameters of Na(+)-dependent, high-affinity L-glutamate transport activity. The time-course of L-[14C]-glutamate uptake and dependence of L-[14C]-glutamate uptake velocity on glutamate concentrations were analyzed. K(m) and Vmax of this process have been determined. The hypergravity stress was created by centrifugation of rats for 1 hour at 10 g. We observed no differences in K(m) values between the control rats (10.7 +/- 2.5 microM) and animals exposed to hypergravity (6.7 +/- 1.5 microM). The similarity of this parameter for the two studied groups of animals showed that affinity of glutamate transporter to substrate was not sensitive to hypergravity stress. In contrast, the maximal velocity of glutamate uptake changed in hypergravity conditions. Vmax reduced from 12.5 +/- +/- 3.2 nmol/min per 1 mg of protein (control group) to 5.6 +/- 0.9 nmol/min per 1 mg of protein (animals, exposed to hypergravity stress). The possible mechanisms of attenuation of the glutamate transporter activity without modifying K(m) of glutamate uptake were discussed.

[Interaction of ribosomes with membranes in vitro]. / Vzaimodeistvie ribosom s membranami in vitro.

Fluorescent "fusion-reporting" probe (R18) was used to study the interaction of ribosomes with membranes in vitro. The latter was incorporated both in the membranes, and ribosomes. The interaction of R18-labeled ribosomes with non-labeled liposomes (of different size and composition) or microsomes increased the fluorescence observed, i.e., the dilution of fluorescent probe took place. The dependence of interaction process on the change of liposomes indicates that the interaction between ribosomes and negatively charged liposomes was more efficient, than that with neutral ones. It is shown that ribosomes can interact with phospholipid membranes even after degradation of protein components accessible for proteins. The interaction of R18-labeled membranes with non-labeled ribosomes results in the increase of fluorescence too. Results obtained indicate to the possibility of direct interaction between ribosomes and membranes.

[Interaction of precursors of secretory proteins and components of a cell-free protein-synthesizing system with phospholipid membranes]. / Vzaimodeistvie predshestvennikov sekretiruemykh belkov i komponentov beskletochnoi beloksinteziruiushchei sistemy s fosfolipidnymi membranami.

Translocation of eucaryotic secretory proteins across the phospholipid membrane containing, no protein components has been studied on the model system. The level of translocation was found to depend critically on physical and chemical properties of membranes. It was found that the cell-free system components can exhibit membrane activity.

[Binding of secreted protein mRNA translation products with artificial phospholipid vesicles]. / Sviazyvanie produktov transliatsii mRNK sekretiruemykh belkov s iskusstvennymi fosfolipidnymi vezikulami.

Translocation of eucaryotic secretory proteins across the phospholipid membrane containing no protein components has been studied. The level of translocation was found to depend critically on the physico-chemical properties of the membranes. It was found also that eucaryotic secretory proteins can pass through the phospholipid bilayer by both co- and post-translational interactions.

[Determination of isoelectric points of oligomeric forms of mitochondrial creatine kinase]. / Opredelenie izoélektricheskikh tochek oligomernykh form mitokhondrial'noi kreatinkinazy.

Using isoelectrofocusing in three pH gradients differing in the initial pH value of the ampholyte gel mixture and in gradient pH range, the isoelectric points for the dimeric and octameric forms of mitochondrial creatine kinase from bovine heart and pigeon breast muscle were determined. The isoelectric points for the dimer and octamer are equal to 9.67 +/- 0.01 and 8.93 +/- 0.05 for the heart enzyme and to 9.56 +/- 0.08 and 8.91 +/- 0.23 for the skeletal muscle enzyme. The correctness of identification of the oligomeric forms of mitochondrial creatine kinase was confirmed by ultracentrifugation in a sucrose density linear gradient. Since creatine kinase is known to bind to mitochondrial membrane cardiolipin by electrostatic forces, it can be assumed that both oligomeric forms of the enzymes can bind to the membranes. However, the properties of the creatine kinase dimer suggest its greater ability to bind to mitochondrial membranes.

[Features of the associative function of the brains of children with high and low lability of nervous system processes]. / Osobennosti assotsiativnoi funktsii mozga detei s vysokoi i nizkoi podvizhnost'iu nervnykh protsessov.

Characteristics of brain associative functions in children of 6 years old with different lability of nervous processes, were compared by methods of conditioning and informational analysis. In children with a low lability, these functions were 1.6 time slower than in other children. Enhanced demands to lability (limitation of time, acceleration of signals presentation) lead to their further slowing and successive inhibition. In children with a high lability, in such conditions an activation of brain associative functions was recorded followed by expressed successive excitation. These differences may be considered as typological. In cases of learned inertness of motor reactions, the characteristics of these functions corresponded to those in children with a high lability of nervous processes.

[Age and biosynthesis and breakdown of thrombocyte proteins]. / Vozrastnye osobennosti biosinteza i raspada belkov trombotsitov

Platelet protein biosynthesis and catabolism in adult and one-month-old rabbits was studied with lysine-14C in vivo. Free radiolysine-14C failed to pass through the membrane of the circulating blood platelets, and its appearance within the platelets was possible only at the stage of platelet production. Platelet protein synthesis in one-month-old rabbits was shown to be slower than that of adults. The half-life period of platelet protein in adults averaged 2 days and in one-month-old rabbits-3 days. Slowing down the platelet protein circulation was noted at the early postnatal ontogenesis.

[Comparative characteristics of ribonuclease isolated from the lliver and pancreas of burned and intact rats]. / Sravnitel'naia kharakteristika ribonukleazy, vydelennoi iz pecheni i podzheludochnoi zhelezy obozhzhennykh i intaktnykh krys

Alkaline RNA-ase was isolated from the pancreas and liver of intact and burnt rats, and purified 1400 and 2000 times accordingly. The optimum of the enzyme activity was achieved at pH 7.6-7.8. Normal and post-burn RNA-ase biosynthesis was studied using radioamino acids (C14-lysine and C14-glycine). Burnt animals displayed a slowed down alkaline RNA-ase biosynthesis, both in the liver and in the pancreas. Ribonuclease "half-life periods" of the liver of intact rats was 65 min, whereas in that of the burnt animals it was prolonged to 100.7 min. The end-preparation activity of the liver and pancreas alkaline RNA-ase of the intact and burnt animals showed no significant difference.