Functional reconstitution of sphingomyelin synthase in Chinese hamster ovary cell membranes.

Sphingomyelin synthase (phosphatidylcholine:ceramide phosphocholinetransferase) activity in the membranes of Chinese hamster ovary cells was found to be detectable with a fluorescent ceramide analog, containing a short acyl chain, as a substrate. We developed a method for the functional reconstitution of sphingomyelin synthase in detergent-treated membranes. Treatment of membranes with 1.5% octyl glucoside in the absence of exogenous phosphatidylcholine resulted in almost complete loss of sphingomyelin synthase activity, even after removal of the detergent by dialysis. In contrast, membranes treated with the detergent in the presence of exogenous phosphatidylcholine showed partial activity and, after dialysis of this mixture, enzyme activity was restored to almost the same level as the activity in dialyzed intact membranes. The effects of various lipids on enzyme activity in this reconstitution system suggested that L-alpha-phosphatidylcholine was the environmental lipid essential for the functional reconstitution of the enzyme. Furthermore, diacylglycerol was suggested to serve as an inhibitory regulator of sphingomyelin synthesis.     

The construction of a simultaneous functional order in nervous systems

The signal activity in a neural net will be constrained both by its physical structure and by environmental constraints. By monitoring its signal activity a neural system can build up a simultaneous functional order that encodes these constraints. We have previously (Part I) presented two models that construct a simultaneous functional order in a collection of neural elements using either signal-covariances or signal-coincides. In this paper we present the results of simulation experiments that were performed to study the influence of the physical constraints of a neural system on the simultaneous functional order produced by both models. In the simulation experiments we used a one-dimensional detector array. We delineate the physical constraints such an array has to satisfy in order to induce a functional order relation that allows an isomorphism with a geometrical order. We show that for an appropriate choice of the system parameters both models can produce a simultaneous functional order with sufficient internal coherence to allow isomorphisms with a triangulation. In this case the dimensionality and the coherence of the detector array are objectively available to the system itself.     

A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters

We describe here a novel proteoliposome reconstitution system for functional analysis of plant membrane transporters that is based on a modified wheat germ cell-free translation system. We established optimized conditions for the reconstitution system with Arabidopsis thaliana phosphoenolpyruvate/phosphate translocator 1 (AtPPT1) as a model transporter. A high activity of AtPPT1 was achieved by synthesis of the protein in the presence of both a detergent such as Brij35 and liposomes. We also determined the substrate specificities of three putative rice PPT homologs with this system. The cell-free proteoliposome reconstitution system provides a valuable tool for functional analysis of transporter proteins.     

Psychophysiological condition of first-year students with different levels of physical activity

The psychophysiological condition of first-year students (males) going or not going in for sport in the course of training was investigated. Baevskii’s method was used to determine the level of stress of regulatory mechanisms. The functional level of the nervous system, the stability of neural responses, and the level of functional abilities of the developed functional system were evaluated using the variation chronoreflexometric method. It was established that an improvement in the mental capacity parameters (increased mental capacity, reduction in the number of errors, an increase in the level of functional abilities, and shortening of the latent period of visuomotor response) was accompanied by an increase of stress of the body regulatory systems, which was more marked in individuals with a low level of physical activity. This fact indicates that physical activity reduces the cost of the adaptation of the body to permanently changing environmental conditions. The optimal human adaptive systemic reactions are ensured by the dynamic interaction of the functional systems forming complex correlations in the somatic, autonomic, motor, and psychoemotional spheres of the body’s activity.     

The psychophysiological condition of first year students with different levels of physical activity

The psychophysiological condition of (male) first year students who participate in sport or don't participate in sport during studies was studied. R. M. Bayevsky's method was used to establish the level of stress on regulatory mechanisms. The functional level of the nervous system, stability of neural responses, and the level of functional abilities of the developed functional system were evaluated using the variation chronoreflexometric method. It was established that an improvement of mental capacity indicators (increase of memory capacity, reduction of the amount of mistakes made, growth of the level of functional abilities and reduction of the latency period of the visual-motor response) was accompanied by a growth of stress on the body's regulatory systems which was more pronounced with regard to those test participants whose level of physical activity was low. This fact is proof that physical activity reduces the body's "adaptation price" to the changing conditions of the environment. The optimal realisation of a person's adaptive systemic reactions is provided for by the dynamic interaction of the functional systems, which form a complex correlation in the body's somatovegetative, motor and psychoemotional spheres of activity.     

Correction of the functional state of aircraft-carrier aviation pilots by a course of inhalation of therapeutic doses of xenon during a long sea campaign

The article considers the efficacy of the courses of xenon-oxygen inhalation for the correction of the functional state of aircraft-carrier aviation pilots in a distant sea campaign. The course of inhalation procedures was shown to stabilize the vagosympathetic balance of the body, to restore the level of the functional state of the cardiovascular system, and to retain its functional reserve at an adequate level, thus considerably improving the subjective psychoemotional state of the pilots, which manifests itself in the improvement of self-esteem, an increase in activity, well-being, and mood, as well as in an adequate decrease in the level of reactive anxiety. The results obtained give evidence of the benefits of the use of special xenon-based gas mixtures for correcting the pilots’ functional state in the most stressful periods of their professional activity. Recommendations on the application of this method by military medicine have been given.     

Changes in the activity of angiotensin converting enzyme in the structures of the rat brain after frontal lobectomy]

The functional state of the angiotensin system (the activity of the angiotensin converting enzyme--ACE) was studied in different rat brain regions during disturbance of the integrative activity of CNS after frontal lobectomy. The most prominent increase of the ACE activity (in four times) on the ninth day after lobectomy was detected in crown cortex and hippocamp. Considerable differences were discovered in striatum, thalamus and hypothalamus also. The variations of the ACE activity were not shown in the medulla oblongata and midbrain. It is possible to conclude, that ACE activity alternations have regional disposition and functional dependence.     

The NADH oxidase system (external) of muscle mitochondria and its role in the oxidation of cytoplasmic NADH.

An exo-NADH oxidase system [NADH oxidase system (external)], effecting intact-mitochondrial oxidation of added NADH, was studied in pigeon heart mitochondria. Breast muscle mitochondria showed an equal specific activity of the system. The exo-NADH oxidase activity (200 micron mol of NADH/min per g of protein) equalled two-thirds of the State-3 respiratory activity with malate + pyruvate or one-seventh of the total NADH oxidase activity of heart mitochondria. The activity was not caused by use of proteinase in the preparation procedure and all measured parameters were very reproducible from preparation to preparation. The activity is therefore most likely not due to preparation artefacts. The exo-NADH oxidase system is present in all mitochondria in the preparation and is not confined to a subpopulation. The system reduced all cytochrome anaerobically and direct interaction with all cytochrome oxidase was demonstrated by interdependent cyanide inhibition. The exo-NADH oxidase system seems to be located at the outer surface of the mitochondrial inner membrane because, for instance, only this system was rapidly inhibited by rotenone, and ferricyanide could act as acceptor in the rotenone-inhibited system (reductase activity = 20 times oxidase activity). In the presence of antimycin, added NADH reduced only a part of the b-cytochromes. Freezing and thawing the mitochondria, one of the methods used for making them permeable to NADH, destroyed this functional compartmentation. The characteristics of the exo-NADH oxidase system and the malate-aspartate shuttle are compared and the evidence for the shuttle's function in heart in vivo is re-evaluated. It is proposed that oxidation of cytoplasmic NADH in red muscles primarily is effected by the exo-NADH oxidase system.     

Hypophyseal-adrenocortical system reaction to the prolonged action of low doses of selenomethionine-75Se that form during internal contamination

A method of radiometry in vivo was applied to study the regularities attending distribution of the absorbed dose and its formation in the course of one year after the administration of 0.033 muCu/g of selenomethionine-75Se to rats, as well as the functional condition of the adaptive hypophysis-adrenal glands system by the determination of plasma corticosterone. A high tropicity of this preparation to the endocrine system organs was demonstrated. Intensification of the functional activity of the adaptive system was indicated by an increase of the blood plasma corticosterone concentration following the administration of a, radioactive preparation and its statistically significant increase (in comparison with control) 3, 6, and 10 months from the beginning of the experiment. The action of acute stress (histamine and formalin) 10 months after the administration of selenomethionine-75Se caused no sharp elevation of the plasma corticosterone concentration characteristic of control rats, this indicating an extreme strain of the hypophysis-adrenal gland system and its incapacity to respond to additional loads. It is supposed that analysis of functional condition of the adaptive system could serve as the key in the assessment of the biological effect of low radiation doses.     

Regulation of the synthesis of lactate dehydrogenase-X during spermatogenesis in the mouse

Total mouse testis RNA directs the synthesis of the sperm-specific C subunit of lactate dehydrogenase-X (LDH-X) when translated in a cell- free system derived from rabbit reticulocytes. The newly synthesized C subunits were isolated by immunoprecipitation with antibody specific for this isozyme, and quantitated by electrophoresis on SDS polyacrylamide gels. The amount of radioactivity incorporated into the enzyme subunit was directly proportional to the amount of testis RNA added to the translational system, thereby providing a sensitive and reliable method for assessing relative LDH-X mRNA activity. A combination of sucrose gradient centrifugation and oligo(dT)-cellulose chromatography resulted in a 23-fold purification of LDH-X mRNA over total cytoplasmic testis RNA. Analysis of LDH-X mRNA activity in the developing testis indicated that the appearance of functional LDH-X mRNA activity coincides with the appearance of LDH-X catalytic activity at 14 d postpartum. Measurement of LDH-X mRNA levels in separated testis cell populations prepared by centrifugal elutriation demonstrated that LDH-X mRNA represents 0.17-0.18% of the total functional mRNA activity in fractions enriched in pachytene spermatocytes and round spermatids, but only 0.09-0.10% of the translation products of elongated spermatids.     

Features of the morphofunctional state of the organs of the thymico-lymphatic system during various adaptive reactions induced by electric stimulation of emotiogenic structures of the brain

Weak electrical stimulation of emotiogenic brain structures was found to lead to the development of different general non-specific adaptation reactions. Stimulation of nucleus lateralis septi was found to evoke mainly the development of activation reaction, while stimulation of globus pallidum caused primarily the development of training reactions. Stress reactions were considerably less frequent in electrical stimulation of both kinds of structures than in the control. This can be explained by small values of the electrical current applied. Morphofunctional activity of thymus lymphatic system depended not only on the type of non-specific reaction but also on the character of the stimulated emotiogenic structure--the functional activity was higher with the stimulation of nucleus lateralis-septi. However, the functional activity of thymus lymphatic system in rats with globus pallidum stimulation was higher than in control rats that were not subject to any stimulation.     

Quantity of functionally changed cells as an identificator of the moment of the organizmus transfer to the next period of development

As "the threshold gears are peculiar to all processes which are flowing past in the world", the problem of looking up of quantitative criterion determining transition of a biological system from one condition to another, is one of the main problems of natural sciences. For analysis of the given problem we investigated some stages of forwardness of marine aster Asterias rubens (mean and late blastula, early and late gastrula, early larva), dis- tinguishing among themselves by morphological characters. Change of structure of cell populations at each stage of development A. rubens analyzed with the help of method of quantitative assessment of functional condition of cells genome. The method is based on cytophotometryc analysis of cell populations coloured by Feulgen (quantifying of DNA in cell) and Naftol yellow S (quantifying of histones in a cell). As the criterion of estimation of functional condition of the cell was used the parameter CFAGEN, coefficient of functional activity of cell genome deduced from histone/DNA ratio after estimation of the absorbency of the nucleus coloured by two chromophores. The data obtained showed that at the moment of organism transition from one stage of development (late blastula) to another (early gastrula) the difference in CFAGEN values between these stages was statistically authentic. Thus comparison of cell distribution histograms has show that cell population conforming to the early gastrula contains 33 % cells with the level of genome functional activity that differs from this one in the cell population conforming to the late blastula. Just in the same moment, the transition of an organism from the stage of late gastrula to the stage of larva is accompanied by statistically authentic distinction in CFAGEN between these stages and availability of more than 1/3 cells (34 %) of the population at the larva stage showing genome functional activity different from that at the late gastrula stage. Thus, it is established, that the biological system passes to qualitatively new condition, if quantity of members of this system changes (increaser or decreases comparatively to a standard point of reference: the norm, previous system status and etc.) not less than on 1/3.     

Existence of antiopiate systems as illustrated by MIF-1/Tyr-MIF-1

Evidence is presented that the small peptides MIF-1/Tyr-MIF-1 are part of an endogenous antiopiate system that may function to balance the opiate system. We review the biological activity, behavioral activity, and functional effects of this proposed opiate antagonist system. In addition, we suggest, based on antinociceptive mechanisms, that the individual components of the antiopiate system might function differently from naloxone.     

Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair

We developed a novel system to create DNA double-strand breaks (DSBs) at defined endogenous sites in the human genome, and used this system to detect protein recruitment and loss at and around these breaks by chromatin immunoprecipitation (ChIP). The detection of human ATM protein at site-specific DSBs required functional NBS1 protein, ATM kinase activity and ATM autophosphorylation on Ser 1981. DSB formation led to the localized disruption of nucleosomes, a process that depended on both functional NBS1 and ATM. These two proteins were also required for efficient recruitment of the repair cofactor XRCC4 to DSBs, and for efficient DSB repair. These results demonstrate the functional importance of ATM kinase activity and phosphorylation in the response to DSBs, and support a model in which ordered chromatin structure changes that occur after DNA breakage depend on functional NBS1 and ATM, and facilitate DNA DSB repair.     

Characteristics of visual perception in seven-year-old children differing in functional maturity of brain structures

Individual and age-related characteristics of visual perception as a whole and its individual components were studied in seven-year-old children as related to their brain functional development. A dependence was found between the success of visual perception and the characteristics determining the functional maturity of both the cerebral cortex and the brain regulatory structures. Difficulties in noise resistance, visuospatial perception, and visual analysis/synthesis were greater in first-year school students with signs of immaturity of the bioelectrical activity of the cerebral cortex. Poor development of the visual perception system in schoolchildren was also determined by an underdeveloped frontothalamic regulatory system and deviations in the functional state of the nonspecific activation system.     

Determination of beta-glucosidase enzymatic function of the histoplasma capsulatum H antigen using a native expression system

The Histoplasma capsulatum H antigen is a major secreted glycoprotein of this pathogenic fungus that is a target of humoral and cell-mediated host responses. Its predicted protein sequence displays homology to beta-glucosidases of other organisms, but a recombinant antigen expressed in a prokaryotic system showed no enzymatic activity. We expressed a recombinant form of the protein carrying a carboxyl-terminus oligohistidine tag in the native fungal background to facilitate proper glycosylation and folding of a product that could then be purified from culture supernatants using nickel affinity chromatography. The recombinant protein was expressed and secreted by a transformant carrying the modified gene under the control of its native promoter. The purified protein from the native expression system showed beta-glucosidase enzymatic activity in substrate gels and quantitative microplate assays. This activity was blocked by glucosidase-specific inhibitors. These results are the first direct demonstration of the function of this protein, and show the utility of expression in a native system to achieve post-translational modification necessary for structural and functional integrity.     

Effect of interlok, remantadine and biotop on normal monocyte functional activity and in acute viral hepatitis

The influence of interlok, biotop and remantadine on the functional activity of monocytes was determined with the use of the NBT test permitting the quantitative evaluation of this characteristic. In this study interlok was found to give promising results in the treatment of acute viral hepatitis, due both to its antiviral action and to the stimulation of the functional activity of monocytes. biotop proved to render a sharply pronounced stimulating effect on the functional activity of monocytes, thus enhancing antiviral resistance. Remantadine suppressed the functional activity of monocytes and increased their lesion by the virus, thus creating favorable conditions for virus persistence in monocytes.     

Validation of long-term primary neuronal cultures and network activity through the integration of reversibly bonded microbioreactors and MEA substrates

In vitro recording of neuronal electrical activity is a widely used technique to understand brain functions and to study the effect of drugs on the central nervous system. The integration of microfluidic devices with microelectrode arrays (MEAs) enables the recording of networks activity in a controlled microenvironment. In this work, an integrated microfluidic system for neuronal cultures was developed, reversibly coupling a PDMS microfluidic device with a commercial flat MEA through magnetic forces. Neurons from mouse embryos were cultured in a 100 μm channel and their activity was followed up to 18 days in vitro. The maturation of the networks and their morphological and functional characteristics were comparable with those of networks cultured in macro-environments and described in literature. In this work, we successfully demonstrated the ability of long-term culturing of primary neuronal cells in a reversible bonded microfluidic device (based on magnetism) that will be fundamental for neuropharmacological studies.