Autophagy lipidation machinery regulates axonal microtubule dynamics but is dispensable for survival of mammalian neurons.

Neurons maintain axonal homeostasis via employing a unique organization of the microtubule (MT) cytoskeleton, which supports axonal morphology and provides tracks for intracellular transport. Abnormal MT-based trafficking hallmarks the pathology of neurodegenerative diseases, but the exact mechanism regulating MT dynamics in axons remains enigmatic. Here we report on a regulation of MT dynamics by AuTophaGy(ATG)-related proteins, which previously have been linked to the autophagy pathway. We find that ATG proteins required for LC3 lipid conjugation are dispensable for survival of excitatory neurons and instead regulate MT stability via controlling the abundance of the MT-binding protein CLASP2. This function of ATGs is independent of their role in autophagy and requires the active zone protein ELKS1. Our results highlight a non-canonical role of ATG proteins in neurons and suggest that pharmacological activation of autophagy may not only promote the degradation of cytoplasmic material, but also impair axonal integrity via altering MT stability.

Perturbed Ca2+-dependent signaling of DYT2 hippocalcin mutant as mechanism of autosomal recessive dystonia.

A recent report of autosomal-recessive primary isolated dystonia (DYT2 dystonia) identified mutations in HPCA, a gene encoding a neuronal calcium sensor protein, hippocalcin (HPCA), as the cause of this disease. However, how mutant HPCA leads to neuronal dysfunction remains unknown. Using a multidisciplinary approach, we demonstrated the failure of dystonic N75K HPCA mutant to decode short bursts of action potentials and theta rhythms in hippocampal neurons by its Ca2+-dependent translocation to the plasma membrane. This translocation suppresses neuronal activity via slow afterhyperpolarization (sAHP) and we found that the N75K mutant could not control sAHP during physiologically relevant neuronal activation. Simulations based on the obtained experimental results directly demonstrated an increased excitability in neurons expressing N75K mutant instead of wild type (WT) HPCA. In conclusion, our study identifies sAHP as a downstream cellular target perturbed by N75K mutation in DYT2 dystonia, demonstrates its impact on neuronal excitability, and suggests a potential therapeutic strategy to efficiently treat DYT2.

Porous structure of ion exchange membranes investigated by various techniques.

A comparative review of various techniques is provided: mercury intrusion porosimetry, nitrogen sorption porosimetry, differential scanning calorimetry (DSC)-based thermoporosimetry, and standard contact porosimetry (SCP), which allows determining pore volume distribution versus pore radius/water binding energy in ion-exchange membranes (IEMs). IEMs in the swollen state have a labile structure involving micro-, meso- and macropores, whose size is a function of the external water vapor pressure. For such materials, the most appropriate methods for quantifying their porosity are DSC and SCP. Especially significant information is given by the SCP method allowing measuring porosimetric curves in a very large pore size range from 1 to 105nm. Experimental results of water distribution in homogeneous and heterogeneous commercial and modified IEMs are presented. The effect of various factors on water distribution is reviewed, i.e. nature of polymeric matrix and functional groups, method for membrane preparation, membrane ageing. A special attention is given to the effect of membrane modification by embedding nanoparticles in their structure. The porosimetric curves are considered along with the results of electrochemical characterization involving the measurements of membrane conductivity, as well as diffusion and electroosmotic permeability. It is shown that addition of nanoparticles may lead to either increase or decrease of water content in IEMs, different ranges of pore size being affected. Hybrid membranes modified with hydrated zirconium dioxide exhibit much higher permselectivity in comparison with the pristine membranes. The diversity of the responses of membrane properties to their modification allows for formation of membranes suitable for fuel cells, electrodialysis or other applications.

The study of the circannual relationship between the activity of the epiphysis and gonads in rats of different sex and age.

BACKGROUND: The relationship between the activity of the epiphysis and gonads in rats of different sex and age in different seasons of the year was determined by studying the levels of melatonin and testosterone in the blood plasma. Determination of the levels of melatonin and testosterone in the serum of rats was carried out by enzyme-linked immunosorbent assay. To assess the relationship between the levels of melatonin and testosterone the correlation coefficient was calculated. Based on the study of the levels of melatonin and testosterone in serum the circannual relationship between the activity of the pineal gland and gonads in males of reproductive age has been determined. In females, the relationship between the levels of melatonin and testosterone without the circannual dependence has been determined. The strongest correlation between melatonin and testosterone is present in males at the age of 9 months in autumn, and it corresponds to the human age of 29-30 years.

AGE AND SEX CHARACTERISTICS OF MELATONIN-POSITIVE-LABELED CELLS OF THE GASTRIC MUCOSA IN DESYNCHRONOSIS IN RATS.

The aim of the research was to study the state of melatonin-positive-labeled cells (MPLC) of GM in desynchronosis in rats of different age and gender. 780 sections of the pyloric part of the gastric mucosa were studied in rats of both genders at the age of 9, 15 and 20 months. Animals were divided into intact control groups and the groups of the animals kept under the conditions of continuous light for 14 days - desynchronosis. The study was performed by the method of immunohistochemical staining with the primary antibodies to melatonin (Biorbyt, UK) and the secondary Alexa Fluor 488-conjugated antibody (Abcam, UK). In the course of the research it was found that MPLC in all experimental groups were mainly located in the basal and middle segments of the tubular glands of gastric mucosa and were represented by three types of cells. In desynchronosis the number of melatonin-positive-labeled cells significantly reduced in almost every age group, with the exception of females at the age of 20 months. Thus in elderly males and females the number of melatonin-positive-labeled cells of type III increases, whereas in young and mature males it decreases, and cells of type I predominate.

THE STUDY OF THE RELATIONSHIP BETWEEN THE ACTIVITY OF EPIPHYSIS AND GONADS IN MALE RATS IN DIFFERENT SEASONS.

In male rats of different ages, we studied the functional activity of the pineal gland and the gonads during different seasons by determining the levels of melatonin and testosterone in the blood serum. It was found that the highest levels of melatonin observed in male rats in summer and winter, and the lowest - in autumn. The lowest level of melatonin has been established in the group of rats aged 9 months - 127.28+5.11 pmol/l that was 22% lower than the level of melatonin in 3 months old rats (P!0,05), and 20% below the level of melatonin in rats of 15 months old (Ps0,05). The highest level determined in rats aged 3 months (corresponds to the age of man -14 years old), the lowest - in rats 20 months-(corresponds to the age 55-56 years old). At the same time, the low level of melatonin was defined in rats aged 9 months in the autumn (corresponds to the age of man 29-30 years). In the study of testosterone levels in different seasons it was found that the highest level in all age groups presents in autumn, the lowest - in winter. When comparing the levels of testosterone in different age groups, it was determined that the fall of the highest level of testosterone present in the blood of males age 9 months and 15 months, which corresponds to the age of 29-30 and 43-44 of human years. Significant difference between the levels of testosterone in the groups that correspond to the reproductive age - rats of age 3, 9 and 15 months, are present only in the autumn; spring and summer - significant differences between the levels of testosterone absent. The degree of correlation differed in rats of different ages in different seasons: the highest correlation between the levels of melatonin and testosterone was determined in rats aged 9 months in autumn. The correlation coefficients in rats aged 20 months old have been weak - from 0.05 in the spring to 0.17 in autumn that shows the changes in neurohormonal regulation which occur with age.

Structure of perfluorinated membranes investigated by method of standard contact porosimetry.

The results of investigation of various factors influencing water distribution in perfluorinated membrane structure by method of standard contact porosimetry are summarized. The Nafion membranes (Dupon de Nemoure, USA) and MF-4SK membranes ("Plastpolymer", Russia) were the objects of the research. The influence of production process and conditioning method on porosimetric curves of perfluorinated membrane is discussed. New results related to the porosity of perfluorinated membranes after reinforcing fabric introduction and processing by organic solvents are reported. The role of the modifying components of various nature in the shaping of transport channels in perfluorinated membrane is studied. The influence of polyaniline and hydrogen zirconium phosphate on water distribution in membrane structure is revealed. The correlation between the maximum porosity value of the membrane and its diffusion and electroosmotic permeability, as well as between the fraction of the gel pore volume and membrane selectivity is established. It allows the prediction of possible changes in the structural characteristics and also in the transport properties of the membranes under the influence of the modifying components of different types and various operating conditions.

[Abnormal floral meristem development in transgenic tomato plants do not depend on the expression of genes encoding defense-related PR-proteins and antimicrobial peptides].

In this study, the morphological and cytoembryological analyses of the tomato plants transformed with the genes encoding chitin-binding proteins (ac and RS-intron-Shir) from Amaranthus caudatus L. andA. retroflexus L., respectively, as well as the gene amp2 encoding hevein-like antimicrobial peptides from Stellaria media L., have been performed. The transgenic lines were adapted to soil and grown the greenhouse. The analysis of putative transgenic tomato plants revealed several lines that did not differ phenotypically from the wild type plants and three lines with disruption in differentiation of the inflorescence shoot and the flower, as well as the fruit formation (modified plants of each line were transformed with a single gene as noted before). Abnormalities in the development of the generative organs were maintained for at least six vegetative generations. These transgenic plants were shown to be defective in the mail gametophyte formation, fertilization, and, consequently, led to parthenocarpic fruits. The detailed analysis of growing ovules in the abnormal transgenic plants showed that the replacement tissue was formed and proliferated instead of unfertilized embryo sac. The structure of the replacement tissue differed from both embryonic and endosperm tissue of the normal ovule. The formation of the replacement tissue occurred due to continuing proliferation of the endothelial cells that lost their ability for differentiation. The final step in the development of the replacement tissue was its death, which resulted in the cell lysis. The expression of the genes used was confirmed by RT-PCR in all three lines with abnormal phenotype, as well as in several lines that did not phenotypically differ from the untransformed control. This suggests that abnormalities in the organs of the generative sphere in the transgenic plants do not depend on the expression of the foreign genes that were introduced in the tomato genome. Here, we argue that agrobacterial transformation affects, directly or indirectly, expression of genes encoding for transcription factors that can activate a gene cascade responsible for the normal plant development.

[Optimization of accounting for radiation exposure of patients as a basis for assessing the risk of stochastic effects due to medical X-ray diagnostic irradiation].

The work is devoted to optimization of the control and accounting for radiation exposure of patients in the X-ray examinations by means of improving existing forms of state statistical survey (form #3-DOZ.). The analysis of the radiation exposure of patients in medical institutions of the Rostov region for the period of 2005-2011 was carried out. It is proposed to exclude the information about the individual patient doses received by calculation; differentiate a group of "adult patients' on age and gender, selecting the reproductive age, discriminate as a separate category of dose-generating, intervention methods of research that will allow to more accurately calculate the risk of the development of stochastic effects of the medical radiation exposure to population.

[Antipodal complex development in the embryo sac of wheat].

Dynamics of an antipodal complex formation in wheat (Triticum aestivum L.) has been observed in detail using a reconstruction of serial semifine sections. Three consecutive crucial stages have been identified in the development of the antipodal complex: (1) proliferation of initial cells, (2) growth and functional differentiation of antipodal cells, and (3) cell apoptosis. Specific features of the mitotic division of antipodal cells have been characterized. It has been shown that the structure of interphase nuclei and mitotic chromosomes of proliferating antipodal cells is similar to that of nucellar cells surrounding the embryo sac. According to the reconstruction of appropriately oriented serial sections, the division of antipodal cells is asynchronous. DNA content in differentiated antipodal cells has been determined by a cytophotometric analysis; in the case of a mature embryo sac, the ploidy of antipodal cells varied from 8 to 32C. Proliferation and DNA endoreduplication processes in the antipodal complex proceed at different time; the second process starts only after the termination of the first one. DNA endoreduplication is accompanied by total chromatin remodeling; as a result, giant chromosomes are formed in the nuclei of antipodal cells. The final stage of the antipodal complex development is programmed cell death or apoptosis. A model for the structural organization of an antipodal complex has been proposed based on the layer arrangement of cells. The secretory activity of antipodal cells directed towards the endosperm syncytium has been detected for the first time. The analysis of "truncated" ovules with an undeveloped endosperm has shown that developing endosperm can be a possible inductor, which stimulates the functional activity of antipodal cells and triggers their terminal differentiation. The obtained results evidence the functional role of antipodal cells in the development of the endosperm and embryo.

New isoform HvNHX3 of vacuolar Na+/H+-antiporter in barley: expression and immunolocalization.

The gene HvNHX3 encoding a new isoform of vacuolar Na+/H+-antiporter was identified in barley. This gene is expressed in roots and leaves of barley seedlings, and it encodes a protein consisting of 541 amino acid residues with predicted molecular weight 59.7 kDa. It was found that by its amino acid sequence HvNHX3 is closest to the Na+/H+-antiporter HbNHX1 of wild type from Hordeum brevisibulatum that grows on salt-marsh (solonchak) soils (95% homology). The expression of HvNHX3 during salt stress is increased several-fold in roots and leaves of barley seedlings. At the same time, the amount of HvNHX3 protein in roots does not change, but in leaves it increases significantly. It was shown using HvNHX3 immunolocalization in roots that this protein is present in all tissues, but in control plants it was clustered and in experimental plants after salt stress it was visualized as small granules. It has been proposed that HvNHX3 is converted into active form during declusterization. The conversion of HvNHX3 into its active form along with its quantitative increase in leaves during salt stress activates Na+/H+-exchange across the vacuolar membrane and Na+ release from cytoplasm, and, as a consequence, an increase of salt stress tolerance.

Characterization of ion-exchange membrane materials: properties vs structure.

This review focuses on the preparation, structure and applications of ion-exchange membranes formed from various materials and exhibiting various functions (electrodialytic, perfluorinated sulphocation-exchange and novel laboratory-tested membranes). A number of experimental techniques for measuring electrotransport properties as well as the general procedure for membrane testing are also described. The review emphasizes the relationships between membrane structures, physical and chemical properties and mechanisms of electrochemical processes that occur in charged membrane materials. The water content in membranes is considered to be a key factor in the ion and water transfer and in polarization processes in electromembrane systems. We suggest the theoretical approach, which makes it possible to model and characterize the electrochemical properties of heterogeneous membranes using several transport-structural parameters. These parameters are extracted from the experimental dependences of specific electroconductivity and diffusion permeability on concentration. The review covers the most significant experimental and theoretical research on ion-exchange membranes that have been carried out in the Membrane Materials Laboratory of the Kuban State University. These results have been discussed at the conferences "Membrane Electrochemistry", Krasnodar, Russia for many years and were published mainly in Russian scientific sources.

Persistent calcium current in rat suprachiasmatic nucleus neurons.

The suprachiasmatic nuclei contain the primary circadian clock, and suprachiasmatic nuclei neurons exhibit a diurnal modulation of spontaneous firing rate. The present study examined the voltage-gated persistent Ca(2+) current, in acutely isolated rat suprachiasmatic nuclei neurons using a ramp-type voltage-clamp protocol. Slow triangular voltage-clamp commands from a holding potential of -85 mV to +5 mV elicited inward current (100-400 pA) that was completely blocked by Cd(2+). This current showed little or no hysteresis, and was identified as persistent Ca(2+) current. The threshold for persistent Ca(2+) current ranged between -60 and -45 mV, and it was maximal at about -8 mV. Nifedipine at 10-20 microM blocked 80-100%. To assess the role of persistent Ca(2+) current in the generation of spontaneous action potentials in both acutely isolated and intact suprachiasmatic nuclei neurons, the effect of Cd(2+) and nifedipine on firing rate was studied using on-cell recording. Application of Cd(2+) exerted a weak excitatory effect and nifedipine had no significant effect on the spontaneous firing rate of isolated suprachiasmatic nuclei neurons. In all intact suprachiasmatic nuclei neurons in slice preparations (n=15), Cd(2+) slowly inhibited spontaneous firing; in high-frequency firing cells (four of 15), a transient increase of firing rate preceded inhibition. No significant effect of nifedipine on firing rate of intact suprachiasmatic nuclei neurons was found. Therefore, persistent Ca(2+) current itself (as carrier of charge) does not appear to contribute significantly to spontaneous firing of suprachiasmatic nuclei neurons. A slowly developing inhibitory effect of Cd(2+) on spontaneous firing of intact suprachiasmatic nuclei neurons in slice preparations may be due to penetration of Cd(2+) through Ca(2+) channels, and its subsequent effect on intracellular mechanisms, while the transient increase of firing rate in high-frequency firing neurons is probably due to inhibition of Ca(2+)-activated K(+) current.

[The GABA-ergic regulation of the Lymnaea stagnalis (L.) dorsal longitudinal muscle contractions].

Presence of GABA-immunoreactive fibres in the nerve cervicalis inferior was shown as well as their regulatory effects upon contractions of the dorsal longitudinal muscle in Lymnaea stagnalis. The exogenous GABA affected the amplitude of evoked muscle contractions diminishing them at concentrations from 10(-8) M and augmenting them from above 10(-5) M. Picrotoxin produced opposite effects on the muscle contractions in the same concentration range. Bicucullin did not affect the contractions. The findings are discussed from the standpoint of the supposed functional heterogeneity of the GABA receptors in the mollusc nerve-muscle junction.

Persistent subthreshold voltage-dependent cation single channels in suprachiasmatic nucleus neurons.

The hypothalamic suprachiasmatic nucleus (SCN) contains the primary circadian pacemaker in mammals, and transmits circadian signals by diurnal modulation of neuronal firing frequency. The ionic mechanisms underlying the circadian regulation of firing frequency are unknown, but may involve changes in membrane potential and voltage-gated ion channels. Here we describe novel tetrodotoxin- and nifedipine-resistant subthreshold, voltage-dependent cation (SVC) channels that are active at resting potential of SCN neurons and increase their open probability (P(o)) with membrane depolarization. The increased P(o) reflects changes in the kinetics of the slow component of the channel closed-time, but not the channel open-time or fast closed-time. This study provides a background for investigation of the possible role of SVC channels in regulation of circadian oscillations of membrane excitability in SCN neurons.

A new human gene KCNRG encoding potassium channel regulating protein is a cancer suppressor gene candidate located in 13q14.3.

We report the primary characterization of a new gene KCNRG mapped at chromosome band 13q14.3. This gene includes three exons and has two alternatively spliced isoforms that are expressed in normal tissues and in some tumor cell lines. Protein KCNRG has high homology to tetramerization domain of voltage-gated K+ channels. Using the patch-clamp technique we determined that KCNRG suppresses K+ channel activity in human prostate cell line LNCaP. It is known that selective blockers of K+ channels suppress lymphocyte and LNCaP cell line proliferation. We suggest that KCNRG is a candidate for a B-cell chronic lymphocytic leukemia and prostate cancer tumor suppressor gene.

Dynamics of structural and functional association of nucleolar chromosomes in cells of the hexaploid wheat Triticum aestivum L. at different stages of the cell cycle and during genome polyploidization.

Quantitative analysis of interphase association of the nucleolar chromosomes at different stages of the cell cycle and during genome polyploidization was carried out. Cells of various tissues of hexaploid wheat Triticum aestivum L. (Moskovskaya-35) were used, including diploid root meristematic cells, endopolyploid root cells, triploid endosperm cells and antipodal cells with polytene chromosomes. Interphase nucleoli impregnated with silver or stained with autoimmune antibodies to 53 kDa nucleolar protein served as markers of the nucleolar chromosome association. The following data were obtained: (1) silver-staining revealed two pairs of homologous chromosomes 1B and 6B with active nucleolus-organizing regions in the root meristematic cells; (2) maximal number of nucleoli in diploid meristematic cells reaches four, which corresponds to the number of chromosomes with active organizers; (3) analysis of cells at different stages of the cell cycle has shown that the tendency to the nucleoli association is observed as soon as cells pass individual stages of the cycle; (4) after DNA and chromosome reduplication, the nucleolus-organizing regions in sister chromatids function as a common structure-functional complex; (5) in endopolyploid root cells and antipodal cells with polytene chromosomes, the number of nucleoli does not correlate with ploidy level, and an additional nucleolus revealed in some cells is the result of activation of the latent organizer in one of the nucleolar chromosomes; (6) in the triploid endosperm nucleologenesis, the stage of prenucleolar bodies is missing. Our data suggest that "fusion" of nucleoli and reduction of their number due to the "satellite" association of the nucleolar chromosomes are two independent processes regulated by different mechanisms.

Role of the axodendritic tree in the functioning of helix bursting neurons: generation of pacemaker activity and propagation of action potentials along the axon.

The purpose of this study was to determine the role of the axodendritic tree in the generation of bursting pacemaker activity in the identified Helix RPa1 neuron, which is homologous to the Aplysia R15 cell, and propagation of action potentials along the axons. In doing so, I used recording of RPa1 neuron electrical activity after cutting off the right or left parietovisceral connections, the two-electrode voltage-clamp technique, registration of electrical activity of visceral nerve containing RPa1 axon branches, isolation of the RPa1 neuron and puff application of oxytocin on it. Cutting of the right (but not left) parietovisceral connection in all cases (more than 15 preparations) evoked complete disappearance of bursting pacemaker activity in the RPa1 neuron and hyperpolarization of its membrane potential up to -65 to -67 mV. Such silent state of the RPa1 neuron was maintained after its complete isolation from the ganglion. The described cutting did not result in a change of bursting activity of the pacemaker neuron V7 located in the visceral ganglion, although isolation of the V7 neuron also eliminated its own activity. Puff application of oxytocin (10 microM in a micropipette) on to the RPa1 neuron both after cutting the right parietovisceral connection or isolation of the neuron from the ganglion resulted in all cases (more than 10 cells) in transient depolarization with development of beating, oscillatory or bursting activity. Voltage clamping of RPa1 soma in the intact ganglion at a level close to zero membrane current sometimes, and, as a rule, at a more depolarized level, revealed bursting-like oscillations of membrane current, reflecting electrical bursting activity in the unclamped remote region of a neuron, most likely in the dendritic tree. Voltage clamping of RPa1 soma possessing bursting activity reveals bursting-like oscillation of membrane current and prevents propagation of corresponding axon action potentials in the visceral nerve. Controversially, clamping of RPa1 soma possessing beating activity exhibits a beating-like oscillation of membrane current and does not prevent propagation of corresponding action potentials in the nerve. Within the framework of the developed hypothesis that persistent bursting pacemaker activity of the RPa1 neuron is due to a constant activation of its peptidergic synaptic inputs [Kononenko N. I. (1993) Comp. Biochem. Physiol. 106A, 135-147], the experimental results were interpreted in the manner that these synapses and, correspondingly, the locus of electrical bursting activity generation, are localized on the dendritic tree of the RPa1 neuron mainly or possibly exclusively in the visceral ganglion. It is hypothesized that bursting and beating neuronal activities are due to functioning of different loci of the dendritic tree, regarding their electrical relations with axon branches.

[Relaparotomy]. / Relaparotomiia.

For a period of 1987-1995 yrs. the operations on a peritoneal cavity organs were performed in 14,718 patients. In 87 reoperated patients 93 relaparotomies were conducted. After relaparotomy 36 (41.3%) patients died.