Mechanical and signaling responses of unloaded rat soleus muscle to chronically elevated ß-myosin activity.

It has been reported that muscle functional unloading is accompanied by an increase in motoneuronal excitability despite the elimination of afferent input. Thus, we hypothesized that pharmacological potentiation of spontaneous contractile soleus muscle activity during hindlimb unloading could activate anabolic signaling pathways and prevent the loss of muscle mass and strength. To investigate these aspects and underlying molecular mechanisms, we used ß-myosin allosteric effector Omecamtiv Mekarbil (OM). We found that OM partially prevented the loss of isometric strength and intrinsic stiffness of the soleus muscle after two weeks of disuse. Notably, OM was able to attenuate the unloading-induced decrease in the rate of muscle protein synthesis (MPS). At the same time, the use of drug neither prevented the reduction in the markers of translational capacity (18S and 28S rRNA) nor activation of the ubiquitin-proteosomal system, which is evidenced by a decrease in the cross-sectional area of fast and slow muscle fibers. These results suggest that chemically-induced increase in low-intensity spontaneous contractions of the soleus muscle during functional unloading creates prerequisites for protein synthesis. At the same time, it should be assumed that the use of OM is advisable with pharmacological drugs that inhibit the expression of ubiquitin ligases.

N-Terminal Fragment of Cardiac Myosin Binding Protein-C Increases the Duration of Actin-Myosin Interaction.

Cardiac myosin binding protein-C (cMyBP-C) located in the C-zone of myocyte sarcomere is involved in the regulation of myocardial contraction. Its N-terminal domains C0, C1, C2, and the m-motif between C1 and C2 can bind to the myosin head and actin of the thin filament and affect the characteristics of their interaction. Measurements using an optical trap showed that the C0-C2 fragment of cMyBP-C increases the interaction time of cardiac myosin with the actin filament, while in an in vitro motility assay, it dose-dependently reduces the sliding velocity of actin filaments. Thus, it was found that the N-terminal part of cMyBP-C affects the kinetics of the myosin cross-bridge.

Catalyst-free 1 : 2 annulation of quinolines with trifluoroacetylacetylenes: an access to functionalized oxazinoquinolines.

Metal- and solvent-free reaction of quinolines with two molecules of aryltrifluoroacetylacetylenes afforded 3-arylethynyl-3-trifluoromethyl-1,3-oxazinoquinolines in up to 92% yields. The formation of a zwitterionic intermediate in the first step triggered a multistep domino reaction. This one-pot synthesis opens an easy access to novel quinoline derivatives bearing trifluoromethyl, acetylene and ketone functions, thus providing a powerful tool for drug design.

Transition metal-free one-pot double C-H functionalization of quinolines with disubstituted electron-deficient acetylenes.

Transition metal-free one-pot reaction of quinolines with acylarylacetylenes and water proceeds in the presence of KOH (55-60 °C, MeCN, 48 h) to afford 2-aryl-3-acylquinolines in up to 66% yield. Here, a formal replacement of the acetylene moiety by the aryl and acyl substituents in the quinoline scaffold takes place. In fact, it has been proved experimentally that the reaction involves the ring cleavage, accompanied by the rearrangement and insertion of the electron-deficient acetylene moiety to form a dihydroquinoline intermediate with an aldehyde functional group in position 4. This intermediate gives the corresponding doubly functionalized quinolines.

Metal-free stereoselective annulation of quinolines with trifluoroacetylacetylenes and water: an access to fluorinated oxazinoquinolines.

Metal-free reaction between quinolines, aryltrifluoroacetylacetylenes and water at -18 °C-rt in MeCN resulted in stereoselective assembly of trifluoromethylated oxazinoquinolines with up to 99% yield that was essentially in contrast to a similar reaction with pyridines. The annulation proceeded via the 1,3-dipolar adducts of quinolines with trifluoroacetylacetylenes followed by intramolecular cyclization involving the trifluoroacetyl group and a molecule of water.

Analysis of ERK1/2 kinases in the inferior colliculus of rats genetically prone to audiogenic seizures during postnatal development.

The activity of ERK1/2 kinases in the quadrigemina inferior colliculus of Krushinsky-Molodkina rats of different age, which are characterized by an increased seizure readiness compared to Wistar rats, was analyzed. An increased (probably genetically determined) activity of these enzymes during the development of epileptiform activity in ontogeny was found, which may be the cause of abnormalities in the neurotransmitter system functioning.

Regulation of the peritoneal macrophage functional activity by the MP-5 and MP-6 myelopeptides under stress.

In mice, two-hour immobilization stress inhibited zymosan-induced production by macrophages of the oxygen radicals and cytokine IL-1ß. After myelopeptides MP-5 and MP-6 were administered into mice, the stress-induced inhibition of the reactive oxygen species (ROS) and IL-1ß was abrogated. MP-5 peptide stimulated spontaneous ROS production by macrophages and reduced IL-10 production under stress. Thus, under in vivo conditions and under stress, the effect of MP-5 and MP-6 myelopeptides modulates the peritoneal macrophage activity.

Effect of Cardiac Myosin-Binding Protein C on Tropomyosin Regulation of Actin-Myosin Interaction Using In Vitro Motility Assay.

We studied the modulating role of cardiac myosin-binding protein C (cMyBP-C) in tropomyosin regulation of the actin-myosin interaction. The effect of cMyBP-C on the velocity of actin-tropomyosin filament sliding over cardiac and slow skeletal myosins was evaluated using in vitro motility assay. The effect of cMyBP-C on the actin-tropomyosin filaments sliding depended on the type of myosin. The regulatory effect of cMyBP-C differs for cardiac and slow skeletal myosin because of the presence of specific essential light chain (LC1sa) in slow skeletal myosin isoform.

The properties of the actin-myosin interaction in the heart muscle depend on the isoforms of myosin but not of α-actin.

In myocardium of mammals there are two isoforms of myosin heavy chains, α and ß. In ventricle, together with ventricular isoforms of light chains they form two isomyosins: V1 and V3, homodimers of α- and ß-heavy chains, respectively. In atria, α- and ß-heavy chains together with atrial light chains form A1 (αα) and A2 (ßß) isomyosins. Besides in myocardium two isoforms of α-actin, skeletal and cardiac, are expressed. We assume that the differences in the amino acid sequence of cardiac and skeletal actin may affect its interaction with myosin. To test this hypothesis, we investigated characteristics of actin-myosin interactions of cardiac and skeletal isoforms of α-actin with the isoforms of cardiac myosin using an optical trap technique and an in vitro motility assay. It was found that the mechanical and kinetic characteristics of the interactions of the isoforms of cardiac myosin with actin depend on the isoforms of myosin not α-actin.

[The etiologic structure and biologic characteristics of agents of infections of bloodstream.]

As before, the infections of blood flow remain actual because of absence of universal approaches to their diagnostic and high lethality. Taking into account the fact that most frequently the sepsisogenic strains are selected in blood, studying of biological characteristics of hemocultures in comparison with strains isolated from other biotopes is very important for understanding the mechanisms of survival of pathogens in blood and prognosis development of septic complications. The article presents the results of microbiological analysis of blood carried out in multi-field clinic of Khanti-Mansiisk during 2007-2015. The following biological characteristics of S.aureus, E.coli, P.aeruginosa, C.albicans: hemolytic, catalase, anti-lysozyme, anti-complementary, biofilm forming, activity of hemocultures and strains isolated from other biotopes. The detection of hemolytic, catalase, anti-lysozyme, anti-complementary activities was implemented using photo-metric technique by capacity to lyse/inactivate corresponding substrate. The biofilm forming activity was evaluated according alteration of contact angle of moistening of surface of biofilm. The priority pathogens of infections of blood stream are coagulase-positive staphylococci - 38.3% (S.aureus), coagulase-negative staphylococci (S.epidermidis, S.hominis etc.), enterobacteria - 12.3% (E.coli, K.pneumonia), Pseudomonas aeruginosa - 4.1%, among fungi - C.albicans (2.5%). It is demonstrated that, in comparison with strains isolated from other biotops, hemocultures have statistically reliable higher values of catalase, anti-lysozyme, anti-complementary, biofilm forming activities. The conclusion is made concerning significance of these biological characteristics of pathogens for their survival in blood. It is proposed to use these indices as markers of sepsisogenic strains in prognosis of generalization of inflectional process.

[COMPARATIVE STUDY OF NIGROSTRIATAL SYSTEMS IN WISTAR RATS AND RATS PRONE TO SEIZURES].

In this work we analyzed the levels of functional activity of dopaminergic, GABA-ergic and glutamatergic neurons in the nigrostriatal system of control Wistar rats and Krushinsky-Molodkina (KM) rats prone to audiogenic seizures. In KM rats we have revealed disturbed activity of GABA- and dopaminergic neurons in substania nigra whereas the level of glutamatergic neurotransmission remained unchanged. We have also observed no significant differences in GAD65/67 and phospho-tyrosine hydroxylase contents in the striatum of KM and control Wistar rats. However, a high level of D1 dopamine receptor and a decreased level of D2 receptor found can mediate the upregulation of glutamatergic neurotransmission. Indeed, the expression of vesicular glutamate transporter type 2 (VGlut2) and NR2B subunit of NMDA receptor was increased in the striatum of KM rats. In striatal glutamatergic fibers phosphorylated ERK1/2 kinases have been revealed; at the same time, in KM rats an increased ERK1/2 activity has been detected both in striatum and substantia nigra. This finding correlated with activation of exocytosis rate as evidenced by downregulation of SNAP25 level. Apart from other reasons, the activation of glutamatergic system may be a result of disruption of the inhibitory effect of the dopamine- and GABAergic systems of substantia nigra that innervate striatum. We suppose that the increased activity of striatal glutamatergic neurons of KM rats without an adequate inhibition by GABA- and dopaminergic systems may be one of the reasons of high convulsive susceptibility in KM rats.

[THE INVESTIGATION OF VASOPRESSIN SECRETION IN NORMAL CONDITION AND DURING SEIZURE ONSET IN KRUSHINSKY-MOLODKINA RATS].

The aim of our study was to analyze vasopressin secretion rate from hypothalamic neurons in Krushinsky-Molodkina (KM) rats prone to audiogenic epilepsy in control and during audiogenic seizures. We evaluated vasopressin content in blood serum and neurophysin II amount in the neurohypophysis in KM rats as well as in Wistar rats. Obtained data demonstrated decreasing of vasopressin in the blood and at the same time increasing of neurophysin II content in the neurohy- pophysis of KM rats that revealed an inhibition of vasopressin release into blood circuit. The analysis of vasopressin content in blood on the different stages of audiogenic seizure showed significant increasing of vasopressin at clonustonus. Thus, we obtained first demonstration that in KM rats prone to audiogenic seizures vasopressin secretion rate is decreased in comparison to Wistar rats. Significantly upregulated vasopressin in blood at cloniconic stages reveals a participation of vasopressinergic neurosecretory system in the expression of audiogenic seizures.

[MOLECULAR MECHANISMS OF ERK1/2 KINASES REGULATION IN THE GLUTAMATE- AND GABA-ERGIC NEURONS DURING SEIZURE EXPRESSION IN KRUSHINSKY-MOLODKINA RATS].

The aim of the present study was to analyze a role of the ERK1/2 signaling pathway in the regulation of excitation and inhibitory neurons in the hippocampus and the temporal cortex of Krushinsky-Molodkina rats during seizure development finalizing with ataxia. Analysis was done by Western bloting as well as by immunohistochemistry. The results demonstrated significant up-regulation of ERK1/2 activity in the hippocampus in several seconds after sound stimulation. At the same time increased ERK1/2 activity was correlated with enhanced level of SNARE protein SNAP-25 and activation of synapsin I, the proteins which regulate exocytosis machinery. Decreased level of VGLUT2 associated with activation of ERK1/2 and exocytosis proteins supposed activation of glutamate release in the hippocampus, while in the temporal cortex diminished activity of ERK1/2 and synapsin I associated with VGLUT2 up-regulation assumed inhibition of glutamatergic transmission. Our data let us supposed that decreasing of glutamate release in th& temporal cortex could be a trigger for the inhibition of hippocampal glutamatergic system and the beginning of further ataxia stage. Our data demonstrated correlation between expression and activity of exocytosis proteins and ERK1/2 mainly in the glutamategic neurons of the hippocampus and the temporal cortex that let us proposed significant role of ERK1/2 kinases as a positive regulator of glutamate release and as a result initiation of seizure expression.

Investigations of Molecular Mechanisms of Actin-Myosin Interactions in Cardiac Muscle.

The functional characteristics of cardiac muscle depend on the composition of protein isoforms in the cardiomyocyte contractile machinery. In the ventricular myocardium of mammals, several isoforms of contractile and regulatory proteins are expressed - two isoforms of myosin (V1 and V3) and three isoforms of tropomyosin chains (α, ß, and κ). Expression of protein isoforms depends on the animal species, its age and hormonal status, and this can change with pathologies of the myocardium. Mutations in these proteins can lead to cardiomyopathies. The functional significance of the protein isoform composition has been studied mainly on intact hearts or on isolated preparations of myocardium, which could not provide a clear comprehension of the role of each particular isoform. Present-day experimental techniques such as an optical trap and in vitro motility assay make it possible to investigate the phenomena of interactions of contractile and regulatory proteins on the molecular level, thus avoiding effects associated with properties of a whole muscle or muscle tissue. These methods enable free combining of the isoforms to test the molecular mechanisms of their participation in the actin-myosin interaction. Using the optical trap and the in vitro motility assay, we have studied functional characteristics of the cardiac myosin isoforms, molecular mechanisms of the calcium-dependent regulation of actin-myosin interaction, and the role of myosin and tropomyosin isoforms in the cooperativity mechanisms in myocardium. The knowledge of molecular mechanisms underlying myocardial contractility and its regulation is necessary for comprehension of cardiac muscle functioning, its disorders in pathologies, and for development of approaches for their correction.

Role of the ERK signaling pathway in regulating vasopressin secretion in dehydrated rats.

Dehydration activates the vasopressinergic system of the hypothalamus. We analyzed the effects of dehydration induced by water deprivation for 3 days on the activities of ERK1/2 and transcription factors, Elk1 and cAMP response element-binding protein (CREB) in vasopressinergic neurons, as well as the distribution and level of the motor protein, kinesin, in the hypothalamo-neurohypophyseal system. We showed that dehydration resulted in enhanced vasopressin (VP) expression and activation of CREB, and increased the activity of the MEK/ERK/Elk1 pathway in magnocellular neurons of the supraoptic nucleus. The activation of VP secretion was associated also with accumulation of phospho-ERK1/2 in the VP-ergic terminals of the posterior lobe of the pituitary. Analysis of the amount and distribution of kinesin and SNAP25, the proteins associated with transport and secretion, demonstrated that dehydration enhanced kinesin content in the perikarya of magnocellular neurons in the supraoptic nucleus and decreased kinesin and SNAP25 levels in the posterior pituitary. ERK1/2 and ERK1/2-dependent transcription factors, Elk1 and CREB, participate in the regulation of dehydration-evoked VP expression. We propose that ERK1/2 and kinesin participate in regulation of anterograde transport of VP dense core vesicles.

[Inactivation of p53 leads to enhancement of tyrosine hydroxylase biosynthesis in the dopaminergic brain neurons].

In the present work we analyzed a possibility of interaction of protein p53, family members of the ERK1/2 signaling cascade, and the transcription factor CREB in regulation of functional activity of dopaminergic neurons. There were considered neurons of Substantia nigra and Zona incerta of control rats and of rats injected intraperitoneally with chemical inhibitor of p53 Pifithrin-alpha blocking transcription activity ofproapoptotic protein p53. We have shown the p53 inactivation to lead to an increase in the content of tyrosine'hydroxylase both in cell bodies and in terminal parts of axons. At the same time, activity of the transcription factor CREB is enhanced in the brain dopaminergic neurons. No significant differences in the content of phospho-ERK1/2 kinases were revealed in the cell bodies at use of Pifithrin-alpha as compared with control group. Thus, we have shown that action of p53 on biosynthesis of tyrosine hydroxylase is of inhibitory character and seems to be mediated by the transcription factor CREB.

Study of regulatory effect of tropomyosin on actin-myosin interaction in skeletal muscle by in vitro motility assay.

The interaction between myosin and actin in striated muscle tissue is regulated by Ca2+ via thin filament regulatory proteins. Skeletal muscle possesses a whole pattern of myosin and tropomyosin isoforms. The regulatory effect of tropomyosin on actin-myosin interaction was investigated by measuring the sliding velocity of both actin and actin-tropomyosin filaments over fast and slow skeletal myosins using the in vitro motility assay. The actin-tropomyosin filaments were reconstructed with tropomyosin isoforms from striated muscle tissue. It was found that tropomyosins with different content of α-, ß-, and γ-chains added to actin filaments affect the sliding velocity of filaments in different ways. On the other hand, the sliding velocity of filaments with the same content of α-, ß-, and γ-chains depends on myosin isoforms of striated muscle. The reciprocal effects of myosin and tropomyosin on actin-myosin interaction in striated muscle may play a significant role in maintenance of effective work of striated muscle both during ontogenesis and under pathological conditions.

RNA degradation differentially affects quantitative mRNA measurements of endogenous reference genes in human placenta.

It has been highlighted that RNA quality and appropriate reference gene selection is crucial for the interpretation of RT-qPCR results in human placental samples. In this context we investigated the effect of RNA degradation on the mRNA abundance of seven frequently used reference genes in 119 human placental samples. Combining RNA integrity measurements, RT-qPCR analysis and mathematical modeling we found major differences regarding the effect of RNA degradation on the measured expression levels between the different reference genes. Furthermore, we demonstrated that a modified RNA extraction method significantly improved RNA quality and consequently increased transcript levels of all reference genes.

Effect of p53 inhibition by pifithrin-alpha on functional activity of vasopressin neurones in rat hypothalamus.

In the present work we investigated the effects of p53 inhibition by pifithrin-alpha (PFT) in vitro and in vivo on functioning vasopressinergic magnocellular neurones of rat hypothalamus. In vivo treatments with PFT were done by intra-hypothalamic microinjections or by intra peritoneal injections. In in vitro experiments hypothalamic slices containing supraoptic nuclei and intact pituitary were incubated with or without PFT. In all experiments we observed accumulation of vasopressin (VP) in the cell perikarya after PFT injections, however expression of VP mRNA was not changed. Analysis of VP content in the posterior pituitary demonstrated that amount of VP was significantly decreased after PFT treatments. Additionally, long-term inhibition of p53 in experiments with intra-hypothalamic injections of PFT resulted in an increased diuresis rate. The obtained results demonstrated that in all experiments PFT treatments inhibited VP anterograde transport from the cells of supraoptic nuclei. Moreover, analysis of MEK/ERK activities revealed that phosphorylation levels of MEK1/2 and ERK1/2 were decreased after PFT treatments. Our findings provide new evidences that p53 could be involved in the control of VP secretion from hypothalamo-hypophyseal system and that this action probably can be mediated by ERK signalling pathway.

[Inhibition of Bcl-2 stimulates neuronal stem proliferation in organotypic cultures of mice hippocampus].

In the current study, we investigated the participation of Bcl-2 in both processes of hippocampus neuronal stem cells (NSC) proliferation and differentiation. Present experiments are performed on organotypic cultures of mice hippocampus. A selective inhibitor Bcl-2 HA14-1 (10 µM) is supplied in incubating medium and the concentration is maintained at a constant level. Our data demonstrate that per cent of surviving cells is significantly higher in the group with the supplement HA14-1 then in the control group. In additional, expressions both phospho-histone H3 and Oct3/4 significantly increase in the group with supplement HA14-1. The facts suggest about activation of NSCs proliferation. After 6 weeks incubation, formation of embryoid bodies is observed in the group with HA14-1, that also suggest about NSCs proliferation, but not their differentiation. Also we estimate the level of NSCs differentiation. Our data have shown that the level of CRMP-2 (a protein which participates in axon growth during NSCs differentiation) decreases in the group with HA14-1. We also estimate level of ERK1/2 kinase activity of the MAPK signaling pathway, which immediately regulates neuronal differentiation. Decreasing of both activities ERK1/2 and CRMP-2 indicates diminution of neuronal differentiation in the experimental group. Thus, we demonstrate that inhibition of Bcl-2 increasingly stimulates NSCs proliferation, so that, it suggests that Bcl-2 controls NSCs differentiation to neurons.