m-Calpain is released from striatal synaptosomes.

Purpose of the study: We aimed to investigate whether m-calpain (a Ca2+-dependent neutral cysteine protease) is released from synaptosomes.Materials and methods: This research was carry on Wistar male rats and isolated nerve endings - synaptosomes. The synaptosomal integrity was checked by the method of measuring LDH activity. Activity of calpains was measured by the casein zymography in gel and in solution. Extracellular calpain was detected by immunoprecipitation and immunoblotting procedures Prediction of secreted proteins peptide on a protein sequence through a local version of the PrediSi tool (http://www.predisi.de). The probability of calpain isoform nonclassical secretion was analyzed by using SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP2.0) software.Results: It has been shown that calcium- and time-dependent m-calpain is released from synaptosomes in an activated form or in a form capable of activation, and this process is not a result of a violation of the integrity of synaptosomes. Analysis of the probability of secretion of the small catalytic subunit of rat m-calpain along a nonclassical pathway showed a high probability of its secretion. Additionally, the release of calpain from synaptosomes revealed by us is suppressed by the addition of glyburide, an ABC transporter inhibitor, to the incubation medium. Among extracellular proteins, potential substrates of calpains are of calpains are found, for example, matrix metalloprotease-2 and -9, alpha-synuclein, etc.Conclusions: Active m-calpain is present in the media generated from striatal synaptosomes. Glyburide prevents m-calpain release from striatal synaptosomes.

HighlightsActive m-calpain is present in the media generated from striatal synaptosomes.Glyburide prevents m-calpain release from striatal synaptosomes.

Glibenclamide alters serotonin and dopamine levels in the rat striatum and hippocampus, reducing cognitive impairment.

RATIONALE: Glibenclamide (GD) is a widely used medical drug; therefore, identifying the mechanisms underlying its pleiotropic effects in the central nervous system is urgent. OBJECTIVES: The aim of this work was to determine the ability of GD to modulate serotonin (5-hydroxytryptamine, 5-HT) and dopamine (DA) transmission and to assess the dose-dependent effect of GD on cognitive function in rats during natural ageing. METHODS: In Experiment 1, rats received 10, 25, or 50 µg/kg GD intraperitoneally for 10 days. In Experiment 2, rats received 50 µg/kg GD intraperitoneally for 30 days. Spatial and working memory was assessed in the MWM and Y-maze tests, respectively. In both experiments, the levels of DA and 5-HT, their metabolites, and turnover rate were analysed by HPLC-ED in the rat hippocampus and striatum. RESULTS: Changes in DA and 5-HT levels occurred only with a dose of 50 µg/kg GD. Therefore, in the second experiment, we administered a dose of 50 µg/kg GD. At this dose, GD prevented the development of impairments in spatial and working memory. The hippocampal concentrations of DA and DOPAC decreased, and the striatal concentrations of DA, DOPAC, 5-HT, and 5-HIAA increased. CONCLUSION: One of the possible mechanisms of the precognitive effect of GD is its ability to modulate monoamine transmission. Thus, in translating our results to humans, GD can be recommended as a prophylactic agent for natural ageing to reduce the risk of developing cognitive impairments.

Glibenclamide as a neuroprotective antidementia drug.

In the view of progressively aging human population and increased occurrence of dysmetabolic disorders, such as diabetes mellitus, cognitive impairment becomes a major threat to the national health. To date, the molecular mechanisms of cognitive dysfunction are partially described for diabetes and diseases of different nature, such as Alzheimer disease or Parkinson disease. The emergence of data pointing towards pleotropic effects of hypoglycaemic medicines indicates involvement of their targets in pathogenesis of cognitive impairment. We are aiming here to review available data on the most widely used hypoglycaemic drug, glibenclamide and find possible relationship of its targets to the pathogenesis of cognitive impairment.

The relationship between serum interleukin-1ß, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-α levels and clinical features in essential tremor.

BACKGROUND: In recent years, there has been discussion that essential tremor (ET) might be a neurodegenerative disease. Indicators of inflammation are considered as possible biomarkers of neurodegeneration. In this connection, the aim of our study was to identify the relationship between serum inflammation markers and clinical features in ET, including the severity of tremor, cognitive decline, depression. METHODS: The serum interleukin-1ß (IL-1ß), IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) levels were measured in 90 ET patients and 90 healthy control people of the corresponding age and gender. Fahn-Tolosa-Marin scale was used for the severity of the tremor. Cognitive function was assessed using the MoCA. Affective symptoms were measured by the Beck Depression Inventory. RESULTS: ET patients had significantly lower serum TNF-α (p < 0.01) but higher serum IL-8 (p < 0.02) and IL-10 (p < 0.01) levels compared to the control patients. The severity of tremor positively correlated with the serum IL-8 level, R = 0.3 (p < 0.01). The serum IL-6 level was higher in ET patients with cognitive impairment compared with normal cognitive ability (p < 0.01). ROC analysis showed that an IL-8 level of 4 pg/ml and higher related with a high risk of severe tremor in ET (AUC-ROC = 0.761). CONCLUSIONS: Our findings demonstrate that neuroinflammation makes a certain contribution to the development of ET.

The Dynamics of Mycoplasma gallisepticum Nucleoid Structure at the Exponential and Stationary Growth Phases.

The structure and dynamics of bacterial nucleoids play important roles in regulating gene expression. Bacteria of class Mollicutes and, in particular, mycoplasmas feature extremely reduced genomes. They lack multiple structural proteins of the nucleoid, as well as regulators of gene expression. We studied the organization of Mycoplasma gallisepticum nucleoids in the stationary and exponential growth phases at the structural and protein levels. The growth phase transition results in the structural reorganization of M. gallisepticum nucleoid. In particular, it undergoes condensation and changes in the protein content. The observed changes corroborate with the previously identified global rearrangement of the transcriptional landscape in this bacterium during the growth phase transition. In addition, we identified that the glycolytic enzyme enolase functions as a nucleoid structural protein in this bacterium. It is capable of non-specific DNA binding and can form fibril-like complexes with DNA.

Changes in activity of µ- and m-calpains and signs of neuroinflammation in the hippocampus and striatum of rats after single intraperitoneal injection of subseptic dose of endotoxin.

Some mechanisms of neuronal degeneration in endotoxinemia are already well described, but need to be detailed. In this study, we tested the effect of a single intraperitoneal injection of a LPS sub-septic dose (1 mg/kg of animal weight) on calpain activity in the striatum and hippocampus. We showed, that in the hippocampus the day after LPS administration an increase in production of IL-1ß and TNF-α mRNA, followed by elevated mRNA expression and activity of µ- and m-calpains without signs of microglia activation is observed. In striatal cells, the day after LPS injection an increase in expression of IL-1ß, TNF-α, IBA-1, m-calpain and calpastatin mRNA is revealed, which only intensifies over time. The elicited changes are accompanied by a decrease in motor behavior, which can be considered as a sign of sickness behavior. In the hippocampus, 180 days after LPS administration expression of TNF-α, content and activity of µ-calpain are increased. In the striatum, elevation in expression of TNF-α, IBA-1, µ- and m-calpain mRNA, with hyperactivation of only m-calpain, is observed. Significantly reduced motor activity can be a consequence of LPS-induced neuronal death. A long-lasting endotoxin activates microglia that damage neurons via proinflammation cytokines and calpain hyperactivation. The endotoxin hypothesis of neurodegeneration is unproven, but if correct, then neurodegeneration may be reduced by decreasing endotoxin-induced neuroinflammation and m-calpain hyperactivation. Therefore, the drugs, that decrease endotoxin-induced neuroinflammation and differently inhibit µ- or m-calpain, can be used to prevent or reduce the severity of neurodegeneration.

Calpain activity in plasma of patients with essential tremor and Parkinson's disease: a pilot study.

Background: Essential tremor (ET) and Parkinson's disease (PD) are the two most common movement disorders in adults with similar clinical symptoms, which is hinting towards existence of coincident pathogenesis steps.Objectives: The objective of this report is to characterize the relationship between ET and PD severity and the activity of calcium-dependent proteases calpain in plasma.Methods: The study enrolled 12 volunteers for each condition: ET, PD, healthy. We evaluated the stage of PD on the H&Y scale in patients with PD, and the severity of tremor in patients with ET on the FTMS scale. IL-1ß, TNFα, IL6, IL10 were determined in plasma using ELISA. Calpain activity was measured using fluorescent substrate and zymography methods.Results: We demonstrated that the activity of calpains in plasma of patients with PD and ET increased 5.1 and 4.3 times, respectively. The increase of calpain activity in plasma of PD patients correlated with the content of IL-1ß, for ET such a connection was not found. At the advanced stages of PD calpain activity in plasma was significantly higher than that of the PD group at the early stage, and this increase was mediated by the increase in m-calpain activity. The increase in the tremor severity in ET did not lead to an increase in the activity of calpains in plasma.Conclusions: We observed general increase in the activity of calpains in plasma of both PD and ET patients that hints towards presence of the common steps in the pathogenesis of these diseases.

Intranasal exposure of manganese induces neuroinflammation and disrupts dopamine metabolism in the striatum and hippocampus.

Prolonged exposure to manganese (Mn) may lead to toxic effects on the central nervous system (CNS). The mechanisms underlying neuronal death from exposure to Mn are not well understood but undoubtedly involve inflammatory processes. The aim of this study was to explore the effects of long-lasting intranasal Mn exposure in rats focusing on inflammatory processes and catecholamine (dopamine, norepinephrine) levels in the striatum and hippocampus. It was found that intranasal administration by instillation of MnCl2 solution once a day for 90 days leads to impaired movement and gait. We also observed that Mn concentration increased in the hippocampus (by 30 %) and in the striatum (by 220 %), dopamine (24 %) and DOPAC (35 %) were reduced in the striatum, and dopamine (190 %) and DOPAC (220 %) levels increased with simultaneously norepinephrine reduction (30 %) in the hippocampus. Observation of cytokine mRNA revealed increased expression of both assayed cytokines (IL-1ß and TNF-α) in the hippocampus. There was a 3-fold increase in the expression of IBA-1 mRNA, 2-fold increase in NFκB mRNA, and dramatic reduction in IkB mRNA in the striatum. Taken together, intranasal exposure to a high dose of MnCl2 induces neuroinflammation and neurotransmission disturbance, but the effects are specific for each studied brain region.

Anthropoaesthetics as an aspect of anthropoecology.

The source of activity of every individual or group is to a great extent determined by the necessity of interaction with other individuals or groups (Afanasiev, 1990), i.e. with the diversity of anthropological surroundings. This manifests itself in the participation of individuals in the organization of their ecosystems, including the ethnocultural and physical conditions of life (Sukharev, 1998). Therefore it is possible to single out certain ecological aspects of perception of anthropological specificity of the surrounding population by an individual (group), leaning upon the definition of anthropoecology as a discipline, studying the laws of interaction between human communities and the system of natural, social and other factors, (Anthropological dictionary, 2004) as well as the coevolution of humans with their environment, in the process of adaptation (Lisseyev, 2001). At the same time it is usually emphasized that the ecological approach is best of all realized on the base of the principle of reciprocity of development of an individual and the surrounding reality, on the perception of the objects of this reality (including other people) with due regard to the reasons for the preference and corresponding values orientation. This represents the subject of ecological psychology (Sergeyenko, 2002; Pavlenko, 2002). In this context the relations between the individual and the environment (autoecology) as well as between the individual and a group (sinecology) are considered (Lisseyev, 2001). The study of the aesthetic perception of anthropological types by individuals belonging to different ethnoterritorial and age groups is the subject matter of a special branch of physical anthropology-anthropoaesthetics, which studies the peculiarities of the aesthetic preference of human facial features in modern populations, analyzing the dependence of human perception on the anthropological environment (Haldeyeva, 2004).

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells.

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms.

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.