A rise of mutation level in tissues of monkey goby and sturgeon fry under conditions of block by antibodies to the serotonin-modulated anticonsolidation protein

The work presents data on studies of effect of a decrease in the serotoninergic system activity on the level of mutagenic changes (the micronuclear test) in the goby Neogobius fluviatilis and the fry of sturgeon Acipenser güldenstädti persicus. It has been shown that the long exposure of the animals to conditions of industrial and oil pollution leads to a significant decrease in their liver of the level of serotonin-modulated anticonsolidation protein (SMAP) correlating directly with the serotonin level as well as to a sharp rise of the level of micronuclear in erythrocytes. The intramuscular administration of anti-SMAP polyclonal antibodies to the sturgeon fry produces a significant increase of the amount of micronuclear as compared with that in the animals injected with non-immune γ-globulin. The obtained results allow concluding that the decrease of activity of the serotoninergic system is the mechanism that is triggered with adverse environmental factors and realizes mutagenic damages in the modified genetic apparatus.     

The role of serotonin-modulated anticonsolidation protein in memory formation in rats in a shuttle box

Two series of experiments were carried out in Wistar male rats. In the first series, rats were trained to acquire conditioning in a shuttle box to 50% and 80% learning criteria. In the animals of the experimental group that achieved 50% learning criterion, a significant decrease in the levels of serotonin-modulated anticonsolidation protein (SMAP) (solid phase, indirect ELISA-test) was observed in the temporal cortex as compared to the animals of the active control group. In the animals of the experimental group that achieved 80% learning criterion, such a decrease was found in the occipital and temporal cortex. In the second series of the experiments, animals of the experimental group were injected with SMAP in saline at a concentration of 1.5 mg/ml in a volume of 10 microl through the cannula implanted into the left lateral ventricle of the brain. Control animals were administered with heating-inactivated SMAP in the same amount. The substances were injected to the animals under light ether anesthesia daily 40 min prior to learning sessions. Learning sessions were carried out in the shuttle box for several days to 50% learning criterion. The experimental rats achieved learning criterion within 7-8 days, whereas intact and control animals reached the same criterion within 4 days. Furthermore, the experimental group of animals differed in increased levels of fear, anxiety and aggression which did not decline throughout the whole learning period. The conclusion was made that SMAP participated in negative regulation of the memory trace formation.     

Adaptive increase of level of serotonin-modulated anticonsolidational protein in tissues of semimigratory and migratory fish at increase of water salinity

The article deals with studies of serotonergic system activity in different tissues of semimigratory fish—the Caspian roach (Rutilus rutilus caspicus) and the carpbream (Abramis brama orientalis)—and of migratory fish—shemaya (Chalcalburnus chalcoides) caught in fresh and brackish waters, as well as in the common carp (Cyprinus carpio L.) tissues under effect of brackish water in model experiments. Using the indirect solid-phase ELISA-test, the serotonergic system activity was evaluated by measuring in the tissues of the studied fish the serotonin-modulated anticonsolidation protein (SMAP) which is in linear relationship with the serotonin level. There was found a significant elevation of SMAP levels in brains of the Caspian roach, carpbream, shemaya, and the common carp under action of increased water salinity. A revealed increase of the SMAP content in brain of the Caspian roach, carpbream, shemaya and the common carp under action of increased water salinity reflects the corresponding elevated activity of the serotoninergic system and indicates involvement of adaptive readjustments in the animals’ body.     

Effect of serotonin-modulated anticonsolidation protein on formation of long-term memory in the learning model of active avoidance in the carp (Cyprinus carpio)

Effects of serotonin-modulated anticonsolidation protein (SMAP) that has property to impair formation of memory trace in mammals and of learning and memory in teleost fish was studied in the learning model of active avoidance. The experiment was performed in three stages: (1) fry of carp Cyprinus carpio L. was injected intracerebroventricularly with the SMAP at a dose of 0.3 μg/g; control individuals were administered with equal amount of the buffered saline for poikilothermic animals; (2) 24 h after injection, fish were learned the conditioned reflex of active avoidance; (3) 48 h after the learning the testing of the skill was perfomed. The administration of the protein was shown to lead to impairment of reproduction of the skill in the fish: the latent time of the skill reproduction in experimental individuals exceeded that in control fish more than two times, while the number of individuals succeeding the task in the expreimental group was insignificantly lower than in the control group. However, unlike mammals, injection of the SMAP in this model produced no effect on the process of learning in carps. Thus, there was first demonstrated the inhibiting effect of the SMAP whose concenration correlated positively with the content of the neurotransmitter serotonin in brain on consolidation of memory traces in teleost fish.     

Increase in expression level of alpha-tubulin gene in Arabidopsis seedlings under hypergravity conditions.

Under hypergravity conditions, elongation growth of plant shoots is suppressed. The analysis of the changes in gene expression by hypergravity treatment in Arabidopsis hypocotyls by the differential display method showed that a gene encoding alpha-tubulin, which is a component of microtubules, was up-regulated by hypergravity. In Arabidopsis six genes encoding alpha-tubulin (TUA1-TUA6) have been identified. In the present study, we examined the dose-response and the time course relations of the changes in the expression of all six alpha-tubulin genes in Arabidopsis hypocotyls grown under hypergravity conditions. The expression levels of all six alpha-tubulin genes, TUA1-TUA6, were increased by increasing gravity, although the extent was variable among genes. The increase in expression of all alpha-tubulin genes was detected within a few hours, when the seedlings grown at 1 g were transferred to 300 g condition. These results suggest that Arabidopsis hypocotyls regulate the expression level of six alpha-tubulin genes promptly in response to gravity stimuli. The increase in the amount of microtubules due to the activation of tubulin gene expression may be involved in the regulation by gravity signal of shoot growth.     

Increase in synthesis of human monoclonal antibodies by transfected Sp2/0 myeloma mouse cell line under conditions of microgravity

Microgravity can influence cell growth and function. A transfected Sp2/0 myeloma cell line P3A2 producing a human IgG1 anti-TNF monoclonal antibody was cultivated in static culture, spinner flasks and simulated microgravity using a rotating wall vessel bioreactor. Microgravity significantly decreased cell growth (from 1.7×10⁶ to 7.9×10⁵ cells/ml), but facilitated the synthesis of antibodies, (1.8, 1.3 and 0.5 g of anti-TNF hmAb per 10⁶ viable cells for cells cultivated under microgravity, in spinner flasks and static cultures, respectively). The results suggest that microgravity could be applied to improve the specific productivity of cell lines producing potentially important therapeutic proteins.     

Antimutagenic activity of serotoninergic system and underlying mechanisms in fry of sturgeon and goldfish

The paper deals with antimutagenic body activity and its underlying mechanisms. The experiments carried out on the one-year old sturgeons (Acipenser gueldenstaedti persicus) and goldfish (Carassius auratus) have shown that intramuscular administration of serotonin-modulated anticonsolidation protein (SMAP) leads to a twofold decrease of erythrocyte mutagenic alterations (the micronuclear test, p < 0.01) caused by action of benthic deposits (0.8 ml/l, 3 days) polluted with industrial wastes. Exposure of goldfish in water contaminated with crude oil (500 mg/l, 3 days) led to a sharp rise of the content of the 70 kDa brain protein fraction (p < 0.001); these water-soluble proteins are assumed to belong to heat shock proteins (HSP). At the same time, in the brain of the studied animals there was observed a simultaneous increase of the SMAP content (p < 0.001). After 3 h, intracerebral SMAP administration to goldfish increased significantly the 90 kDa protein fraction content (p < 0.01), probably HSP90, in the electrophoretic profiles of the brain water-soluble proteins. Thus, the obtained results indicate that the body serotoninergic system has the antimutagenic activity providing protection of cells from action of harmful environmental factors by an enhancement of synthesis of proteins suggested to belong to HSP.     

Feeding pattern and diet of first feeding brown trout fry under natural conditions

The feeding habits of brown trout Salmo trutta fry were studied during the critical first feeding period in a natural spawning and nursery stream. A low proportion of the fry initiated exogenous feeding before emergence from the gravel, and while having nearly 30% of the yolk sac remaining. This probably reflected low feeding motivation or limited feeding opportunities within the gravel environment. The majority of the fry started feeding after emergence, and after most or all yolk was absorbed. Some fry emerged with large amounts of yolk remaining, while others emerged after yolk exhaustion. The degree of stomach fullness revealed that feeding was more efficient after a territory had been acquired. The diets of the young fry were dominated by chironomid larvae, followed by zooplankton and Plecoptera larvae. Fry dispersing downstream and out of the nursery area were significantly smaller than resident fry, indicating displacement due to competition for territories. The majority of the downstream dispersing fry had initiated feeding, and there was at this point no evidence of starvation in any of the fry. It therefore appeared that the later emerging fry actively migrated out of the overpopulated nursery area to find available territories further downstream.     

Comparative analysis of uncoupling protein 4 distribution in various tissues under physiological conditions and during development

UCP4 is a member of the mitochondrial uncoupling protein subfamily and one of the three UCPs (UCP2, UCP4, UCP5), associated with the nervous system. Its putative functions include thermogenesis, attenuation of reactive oxidative species (ROS), regulation of mitochondrial calcium concentration and involvement in cell differentiation and apoptosis. Here we investigate UCP4's subcellular, cellular and tissue distribution, using an antibody designed specially for this study, and discuss the findings in terms of the protein's possible functions. Western blot and immunohistochemistry data confirmed that UCP4 is expressed predominantly in the central nervous system (CNS), as previously shown at mRNA level. No protein was found in heart, spleen, stomach, intestine, lung, thymus, muscles, adrenal gland, testis and liver. The reports revealing UCP4 mRNA in kidney and white adipose tissue were not confirmed at protein level. The amount of UCP4 varies in the mitochondria of different brain regions, with the highest protein content found in cortex. We show that UCP4 is present in fetal murine brain tissue as early as embryonic days 12-14 (E12-E14), which coincides with the beginning of neuronal differentiation. The UCP4 content in mitochondria decreases as the age of mice increases. UCP4 preferential expression in neurons and its developmental expression pattern under physiological conditions may indicate a specific protein function, e.g. in neuronal cell differentiation.     

Character of changes in serotonin-modulating anticonsolidation protein and cytochrome P-450 in tissues of Alburnoides bipunctatus eichwaldi caught in the rivers of Azerbaijan Republic

The work was designed to investigate the levels of a novel serotonin-modulating anticonsolidation protein (SMAP), which activity directly depends on serotonin level, and biomarker cytochrome P-450 in the liver, gills and brain of Alburnoides bipunctatus eichwaldi caught in Khudat, Agstafachay, Kura and Araz rivers running over the territory of Azerbaijan. Noticeable downregulation of cytochrome P-450 and SMAP was observed in the liver and gills of fish caught in Agstafachay river relatively to the values obtained for the fish from unpolluted Khudat river. The levels of cytochrome P-450 and SMAP in liver of fish from Kura and Araz rivers exhibited significant opposite changes: downregulation of cytochrome P-450 level vs. upregulation of SMAP level. In the brain of fish from Agstafachay river the cytochrome P-450 was found to be somewhat downregulated, whereas in the brain of fish from Kura and Araz rivers SMAP was significantly upregulated. The results obtained are analyzed from the standpoint of adaptation and disadaptation processes of water organisms to the effects of pollutants.     

Expression level of Ubc9 protein in rat tissues

Ubc9 is a homologue of the E2 ubiquitin conjugating enzyme and participates in the covalent linking of SUMO-1 molecule to the target protein. In this report we describe a simple and efficient method for obtaining pure human recombinant Ubc9 protein. The purified Ubc9 retained its native structure and was fully active in an in vitro sumoylation assay with the promyelocytic leukaemia (PML) peptide as a substrate. In order to better understand the physiology of Ubc9 protein we examined its levels in several rat tissues. Immunoblot analyses performed on tissue extracts revealed quantitative and qualitative differences in the expression pattern of Ubc9. The Ubc9 protein was present at a high level in spleen and lung. Moderate level of Ubc9 was detected in kidney and liver. Low amount of Ubc9 was observed in brain, whereas the 18 kDa band of Ubc9 was barely visible or absent in heart and skeletal muscle. In heart and muscle extracts the Ubc9 antibodies recognized a 38 kDa protein band. This band was not visible in extracts of other rat tissues. A comparison of the relative levels of Ubc9 mRNA and protein indicated that the overall expression level of Ubc9 was the highest in spleen and lung. In spleen, lung, kidney, brain, liver and heart there was a good correlation between the 18 kDa protein and Ubc9 mRNA levels. In skeletal muscle the Ubc9 mRNA level was unproportionally high comparing to the level of the 18 kDa protein. The presented data indicate that in the rat the expression of the Ubc9 protein appears to have some degree of tissue specificity.     

Increase in the level of arabinoxylan–hydroxycinnamate network in cell walls of wheat coleoptiles grown under continuous hypergravity conditions

Changes in the amount and composition of cell wall constituents in response to continuous hypergravity stimuli were studied in wheat ( Triticum aestivum L.) coleoptiles. The lengths of coleoptiles grown under hypergravity (300  g ) conditions for 2–4 days from germination stage were 60–70% of those of 1  g control. However, the net amounts of hemicellulosic polysaccharides and cellulose in hypergravity-treated coleoptiles increased progressively as much as those in the control coleoptiles. As a result, their contents per unit length of coleoptile largely increased under hypergravity conditions. In the hemicellulose fraction, the amounts of arabinose and xylose, the major components of the fraction, prominently increased in response to hypergravity. When hemicellulosic polysaccharides were separated into neutral and acidic polymers by an anion-exchange column, the amounts of the acidic fraction consisting of (glucurono)arabinoxylans were higher in hypergravity-treated coleoptiles than in control coleoptiles. The amounts of cell wall-bound ferulic acid and diferulic acid (DFA) increased dramatically in both 1  g control and hypergravity-treated coleoptiles. Particularly, the amounts of DFA in hypergravity-treated coleoptiles were significantly higher than those in control coleoptiles during the incubation period. These results suggest that continuous hypergravity increases the rigid network structures via arabinoxylan–hydroxycinnamate cross-links within cell wall architecture in wheat coleoptiles. These structures may have a load-bearing function and contribute to construct the stable cell wall against the gravitational force.     

Purification of monoclonal antibodies by hydrophobic interaction chromatography under no-salt conditions

Hydrophobic interaction chromatography (HIC) is commonly used as a polishing step in monoclonal antibody purification processes. HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. HIC, however, suffers from the limitation of use of high concentrations of kosmotropic salts to achieve the desired separation. These salts often pose a disposal concern in manufacturing facilities and at times can cause precipitation of the product. Here, we report an unconventional way of operating HIC in the flowthrough (FT) mode with no kosmotropic salt in the mobile phase. A very hydrophobic resin is selected as the stationary phase and the pH of the mobile phase is modulated to achieve the required selectivity. Under the pH conditions tested (pH 6.0 and below), antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. Optimum pH conditions were chosen under which the antibody product of interest flowed through while impurities such as aggregates and host cell proteins bound to the column. This strategy was tested with a panel of antibodies with varying pI and surface hydrophobicity. Performance was comparable to that observed using conventional HIC conditions with high salt.     

The effect of the delta-sleep peptide on the adrenaline level in rat tissues under normal conditions and during cold stress

Adrenalin content in the brain, liver and adrenal glands under the effect of cold stress grows by 314, 500 and 56% as compared to the control. A single administration of the delta-sleep inducing peptide (DSIP) in a dose of 12 microgram/100 g to intact animals makes the adrenalin content in the brain higher 1, 3, 6 and 24h after administration; two and three days later the adrenalin content in the brain does not change. The amount of adrenalin in the liver of the same animals increases 1, 3, 6 h and 1, 2, 3 days after DSIP administration. Intraperitoneal administration of DSIP induces an increase of the adrenalin level in the adrenal glands of rats an hour and a day after administration. Two days later the level of adrenaline decreases by 41%; 3, 6 h and 3 days after DSIP administration the content of adrenaline remains unchanged. As a result of the DSIP administration in a dose of 12 micrograms/100 g to the animals in the state of cold stress, the content of adrenalin increases in the rat brain by 129, in the liver--by 300, adrenal glands--by 44% as compared with the control.     

Degradation of Cry1Ac protein within transgenic Bacillus thuringiensis rice tissues under field and laboratory conditions

To clarify the environmental fate of the Cry1Ac protein from Bacillus thuringiensis subsp. kurstaki (Bt) contained in transgenic rice plant stubble after harvest, degradation was monitored under field conditions using an enzyme-linked immunosorbent assay. In stalks, Cry1Ac protein concentration decreased rapidly to 50% of the initial amount during the first month after harvest; subsequently, the degradation decreased gradually reaching 21.3% when the experiment was terminated after 7 mo. A similar degradation pattern of the Cry1Ac protein was observed in rice roots. However, when the temperature increased in April of the following spring, protein degradation resumed, and no protein could be detected by the end of the experiment. In addition, a laboratory experiment was conducted to study the persistence of Cry1Ac protein released from rice tissue in water and paddy soil. The protein released from leaves degraded rapidly in paddy soil under flooded conditions during the first 20 d and plateaued until the termination of this trial at 135 d, when 15.3% of the initial amount was still detectable. In water, the Cry1Ac protein degraded more slowly than in soil but never entered a relatively stable phase as in soil. The degradation rate of Cry1Ac protein was significantly faster in nonsterile water than in sterile water. These results indicate that the soil environment can increase the degradation of Bt protein contained in plant residues. Therefore, plowing a field immediately after harvest could be an effective method for decreasing the persistence of Bt protein in transgenic rice fields.     

Monoclonal antibodies against 27.8 kDa protein receptor efficiently block lymphocystis disease virus infection in flounder Paralichthys olivaceus gill cells

In previous research using co-immunoprecipitation, a 27.8 kDa protein in flounder Paralichthys olivaceus gill (FG) cells was found to bind lymphocystis disease virus (LCDV). In this paper, 13 hybridomas secreting monoclonal antibodies (MAbs) against the 27.8 kDa protein were obtained, and 2 MAbs designated as 2G11 and 3D9 were cloned by limiting dilution. Analyzed by indirect enzyme-linked immunosorbent assay (ELISA) and western blotting, the MAbs specifically reacted with the 27.8 kDa protein of FG cells. Confocal fluorescence microscopy and immunogold electron microscopy (IEM) provided evidence that the epitopes recognized by these MAbs were located primarily on the cell membrane and occasionally in the cytoplasm near the cell membrane of FG cells. The MAbs could block LCDV binding after MAbs were pre-incubated with isolated membrane proteins of FG cells in a blocking ELISA, and MAbs also could inhibit LCDV infection of FG cells in culture. Moreover, several target tissues of LCDV in flounder, including gill, stomach, intestine and liver, displayed the presence of the LCDV receptor-27.8 kDa. These results strongly supported the possibility that the 27.8 kDa protein is the putative receptor specific for LCDV infection of FG cells in flounder.     

Increase in efficacy of antibiotic therapy of anthrax under experimental conditions]

The results of experimental therapy of antraxis infected mice with cefazoline (kefzol) and ampicillin incapsulated into liposomes are presented. Protective activity of the same free antibiotics combinated with amixine and leukinferone was evaluated also. Treatment with liposomal cefazoline enhanced mice survival upto 60 per cent, and life period upto 1.3 +/- 0.3 days. After liposomal ampicillin administration for 3 times the same indices were 60 per cent and 6.5 +/- 0.9 days, after 2 times administration--80 per cent and 14 +/- 1.8 days when compared to the groups of the animals treated with free antibiotics. It was shown that administration of ampicillin with amixine or with leukinferone provided enhanced mice survival upto 20 per cent, administration of cefazoline with leukinferone--upto 30 per cent.