Search

The introduction of methods for food production using microbial synthesis, including those obtained with the help of genetically modified (GM) microorganisms, at the present stage, allows to increase production volumes and reduce the cost of food. At the same time, such products in accordance with TR CU 021/2011 "On food safety" are classified as a "novel food"» and can be placed on the market only after its risk estimation for health. The emergence of new data and research methods in the last years has made it necessary to improve the risk assessment system for this category of food. The aim of the research is to develope risk assessment approaches of food obtained by microbial synthesis on the example of the GM strain Aspergillus awamori Xyl T-15 and the enzyme preparation (EP) (a complex of glucoamylase and xylanase) produced by it. Material and methods . Outbred ICR mice (CD-1) and Wistar rats (males and females) were used in the experimental studies. Investigations of GM strain A. awamori Xyl T-15 virulence and its ability to disseminate internal organs have been carried out. Acute and subacute (during 80 days) toxicity of EP (a complex of glucoamylase and xylanase) have been studied. Results . The presented experimental data allow us to make a conclusion about the avirulence of the A. awamori Xyl T-15 strain, the lack of ability to disseminate internal organs (invasiveness). At the same time, the strain is characterized by the ability to produce mycotoxins (ochratoxin, fumonisin B2, T-2 and HT-2 toxins). The EP, a complex of glucoamylase and xylanase from A. awamori Xyl T-15, has a low oral acute toxicity for rats (LD 50 >5000 mg/kg). I ntragastric EP administration at doses of 10, 100 and 1000 mg/kg of body weight during 80 days had not revealed adversely affect on the rate of weight gain in animals, indicators of anxiety and cognitive function, and some studied biochemical indicators. At a dose of 100 mg/kg b.w. or more, there were changes in the relative mass of organs (lungs, kidneys, adrenal glands), small shifts in the parameters of erythropoiesis and leukocyte formula, at a dose of 1000 mg/kg b.w. - an increase in oxidative DNA destruction. T he most pronounced and dose-dependent was the effect of the EP on hepatocyte apoptosis. According to this indicator, the not observed adverse effect level (NOAEL) for EP is not more than 100 mg/kg b.w. in terms of protein. The main target organ for the toxic effect of EP is the liver. Conclusion . The data obtained demonstrate the necessity to conduct an additional analysis of the risks of possible negative effects of EP, namely, to study its impact on the gut microbiocenosis and the immune status of experimental animals, to analyze the presence of determinants of pathogenicity and antibiotic resistance, DNA of selective marker genes of A. awamori Xyl T-15 strain by PCR analysis and DNA sequencing methods., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

Due to wide spreading of inflammatory disease and imperfection of available anti-inflammatory drugs, mainly associated with their serious side effects, searching for new anti-inflammatory agents is a pressing problem. Natural triterpenoids and their synthetic analogs are a promising source of new drugs. In this study, we have investigated the anti-inflammatory and antitumor effects in vivo of the glycyrrhetinic acid derivative soloxolone methyl (SM), or methyl 2-cyano-3,12-dioxo-18βH-olean-9(ll),l(2)-dien-30-oate. SM was shown to efficiently suppress the development of edema in a mouse model of carrageenan- or histamine-induced acute inflammation. SM also inhibited the tumor growth and reduced the tumor cell count in the ascitic fluid in mice bearing Krebs-2 carcinoma, the development of which is accompanied by an inflammatory process in the surrounding tissues.

A time dynamic of LTP development in the first minutes after high frequency stimulation has been investigated by using a cell model of synaptic potentiation. An experimental assessment of dependence of potentiation and maintenance of a stable level of synaptic transmission in the CA1 area of mice hippocampus on the intracellular transport has been made with brefeldin A (the blocker of transport vesicle forming). The integrative activity of the systems, ensuring the delivery of glutamate receptors to the dendrite spines in amounts and in time intervals necessary for fast changing and long maintenance of transmission efficiency is discussing.

Mice of the ICR outbred population were infected intranasally (i/n) with the variola virus (VARV, strain Ind-3a). Clinical signs of the disease did not appear even at the maximum possible dose of the virus 5.2 lg PFU/head (plaque-forming units per head). In this case, 50% infective dose (ID50) of VARV estimated by the presence or absence of the virus in the lungs three days after infection (p.i.) was equal to 2.7 ± 0.4 lg PFU/head. Taking into account the 10% application of the virus in the lungs during the intranasal infection of the mice, it was adequate to 1.7 lg PFU/lungs. This indicates a high infectivity of the VARV for mice comparable to its infectivity for humans. After the i/n infection of mice with the VARV at a dose 30 ID50/ head the highest concentration of the virus detected in the lungs (4.9 ± 0.0 lg PFU/ml of homogenate) and in nasal cavity tissues (4.8 ± 0.0 lg PFU/ml) were observed. The pathomorphological changes in the respiratory organs of the mice infected with the VARV appeared at 3-5 days p.i., and the VARV reproduction noted in the epithelial cells and macrophages were noticed. When the preparations ST-246 and NIOCH-14 were administered orally at a dose of 60 μg/g of mouse weight up to one day before infection, after 2 hours, 1 and 2 days p.i., the VARV reproduction in the lungs after 3 days p.i. decreased by an order of magnitude. Thus, outbred ICR mice infected with the VARV can be used as a laboratory model of the smallpox when evaluating the therapeutic and prophylactic efficacy of the antismallpox drugs.

The experiment was performed with outbred ICR (CD-1). female mice (SPF). The animals were irradiated by 171 MeV protons at a dose of 20 cGy. The spacecraft radiation protection elements used in the experiment were a construction of wet hygiene wipes called a "protective blind", and a glass plate imitating an ISS window. Physical obstacles on the path of 171 MeV protons increase their linear energy transfer leading to the absorbed dose elevation and strengthening of the radiobiological effect. In the experiment, two types of obstacles together raised the absorbed dose from 20 to 23.2 cGy. Chemically different materials (glass and water in the wipes) were found to exert unequal modifying effects on physical and biological parameters of the proton-irradiated mice. There was a distinct dose-dependent reduction of bone marrow cellularity within the dose range from 20 cGy to 23.2 cGy in 24 hours after exposure. No modifying effect of the radiation protection elements on spontaneous motor activity was discovered when compared with entrance protons. The group of animals protected by the glass plate exhibited normal orientative-trying reactions and weakened grip with the forelimbs. Rationalization of physical methods of spacecrew protection should be based as on knowledge in physical dosimetry (ionizing chambers, thermoluminescent, track detectors etc.), so the radiobiological criteria established in experiments with animals.

Outbred CD-1 mice females aged 4 to 4.5-months were investigated in 21-22 hours following total γ-irradiation at 10, 25, 50, 75, 100 and 200 mGy. Loss in bone marrow karyocytes, as well as spleen and thymus mass reductions were significant in the group of animals irradiated at 50 and 200 mGy and less dramatic in mice irradiated at 75 mGy. The orientative-trying behavior reaction (OTBR) in the open field tested in 19-20 hours after exposure to 10 and 25 mGy was reliably stronger than in the group of biological control; however, emotional status (ES) in the animals that received 10 mGy dropped significantly. Mice irradiated at 50 mGy were found to weaken the grip of their front limbs. Dose levels differing in opposite radiobiological effects on the parameters under study were established. Doses in the range from 10 to 25 mGy maximized OTBR and ES, while doses of 50, 100 and 200 mGy produced high reactions of the immune and hemopoietic organs.

The modifying effect of the one compound on carcinogenicity of the other in the combined application is attributed usually to some changes in the carcinogen metabolism, i.e. its activation or inactivation. In this paper, when used separately, diethylnitrosamine (DENA) induced 4-6 times more neoplastic lesions in the liver of suckling mice than ortho-aminoazotoluene (OAT) did. However, after combined treatment with both carcinogens the total number of hepatic lesions was significantly lower than that in mice treated with DENA only. Similar effect was observed when OAT was administered 3 days before or 3 days after DENA injection. The observed protective effect is not mediated at metabolic level, because OAT has no effect on metabolism of DENA in mouse liver. Our findings can be unequivocally explained by the competition of the carcinogens for target protein molecules, presumably transcription factors, participating in hepatocyte differentiation, which differently interact with and are diversely impaired by different compounds.

It is found that after administration of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB,) which was hepatocarcinogenic to rats, in suckling mice, the number of neoplastic lesions in the liver of mice was 3 times higher than after analogous administration of equimolar dose of ortho-aminoazotoluene (OAT)). However, in the Ames test (TA-98 strain of Salmonella typhimurium) with activation by hepatic enzymes (S-9 fraction) of both intact and Aroclor-1254-induced mice and rats OAT contributed by an order of magnitude to revertant colonies compared to 3'-Me-DAB. In vivo inhibition of sulfotransferase activity, the enzyme which catalyzes the final stage of the mutagenic activation of aminoazo dyes, had no effect on carcinogenicity of 3'-Me-DAB but more than 4 times elevated that of OAT. It was concluded that the mechanism of carcinogenic action of aminoazo dyes studied is not genotoxic and that the carcinogenic potential of OAT is lost in the process of mutagenic activation.

It is considered that diethylnitrosamine (DENA) is metabolized mostly in the liver with producing highly reactive compounds alkylating cellular macromolecules and causing toxic and carcinogenic effects. However, competitive inhibition of cytochrome P450 2E1, which metabolizes DENA, by its other substrate, isopropanol, does not weaken, but enhances the toxicity of DENA effect on mice and reduces the number of preneoplastic nodules and liver tumors induced by it. In short-term tests for mutagenicity (in the Ames test and the SOS-hromotest) DENA causes moderate damage of DNA that reduces liver microsomal enzymes, that metabolize nitrosamines. The obtained data indicate the predominant role of the initial compound as compared to its metabolites in toxic, and probably hepatocarcinogenic, effects on DNA in mice.

In previous experimental studies on laboratory mice, it was shown that activation of specific immunity by injection with sheep red blood cells (SRBC) lessens males' sexual olfactory attractiveness for intact females. However, reduced attractiveness can decrease males' reproductive efficiency only under the conditions of free mating, which is not obligatory for natural populations. The goal of this work was to study the influence of immunoenhancement on sexual behavior and reproductive output of outbred ICR male mice. Males, either injected with saline (control group) or SRBC-treated, were kept with intact females during 5 days after injection. While the number of fertile copulations was practically equal in both groups, the potential (ovulated ova) and actual (number of embryos) fecundity was significantly higher in females having been paired off with SRBC-treated males. Main reproductive effects were registered at 3-5th day after injection, when specific antibody-forming process starts and males' scent becomes less attractive for females. On the base of previous and present data, the hypothesis is proposed that if the quality of a non-alternative mating partner is compromised by activation of specific immunity, a female tries to maximize its reproductive output (due to low chance of repeated copulation). This responsibility for the next generation is reminiscent of the Bible story about Lot and his daughters, and may help to sustain the species existence under conditions of parasitic press.

The investigators studied the ability of adult ICR mice (a laboratory model that was most approximated to the wildtype populations of mice) to maintain Ebola virus (EV) reproduction in the organism. The adult ICR mice inoculated with EV during 23 passages were shown to maintain viral reproduction in the liver. The elevated levels of platelets and the early generation of fibrin and fibrinogen degradation products suggested there were hemostatic changes that did not, however, progress to severe coagulopathy. The animals were in appearance apparently, other than adynamia observed on days 5-7. Thus, the susceptibility of the adult ICR mice to EV is characterized by their ability to maintain virus reproduction in the liver without evident signs of the infection. This pattern of susceptibility in the mice shows a possible role of this rodent species in the transmissive cycle of EV.

The carcinogenic effects of estragole in mice of the earlier unexplored strain ICR has been studied. It has been shown that there is a distinct correlation between the extent of inhibition of glucocorticoid-mediated induction of tyrosine aminotransferase and trypthophan oxygenase after acute administration of estragole and the frequency of liver tumors after estragole exposure. Estragole inhibits the induction of these enzymes only in female mice, but not in male mice and rats. DNA-binding activities of liver-enriched transcription factors were investigated on carcinogen-susceptible and -resistant animals. Estragole decreases the HNF4 (hepatic nuclear factor 4) and FOXA DNA-binding activities only in susceptible female mice, but not in nonsusceptible male mice and rats and does not influence the C/EBP and HNF1 activities. Pentachlorophenol, which prevents the hepatocarcinogenic effect of estragole, abolishes its inhibitory effect on tyrosine aminotransferase and trypthophan oxygenase glucocorticoid induction and restores the FOXA and HNF4 DNA-binding activities. The parallelism between the hepatocarcinogenic effects of estragole and the inhibition of FOXA and HNF4 DNA-binding activities serves as an additional argument for the involvement of these factors in the mechanisms of tumor suppression in the liver.

Circadian rhythms of horizontal and vertical motor activity and characteristic features of stress dysrhythmia caused by territorial conflict were assessed in ICR outbred mice with polar zoosocial status. The rhythms of diurnal motility of non-stressed dominant and submissive mice do not differ considerably. The territorial conflict causes desynchronosis in both high- and low-ranking animals, manifesting itself through the shift of the acrophase of motor activity and change in spectral parameters of a chronogram.

When used separately, diethylnitrosamine (DENA) initiates 4-6 times more neoplastic lesions in the liver of suckling mice than ortho-aminoazotoluene (OAT) lower. However, after combined treatment with either carcinogen the total number of hepatic lesions was significantly than that in mice injected with DENA only. Similar inhibition of DENA-induced hepatocarcinogenesis was observed when OAT was administered 8-12 hrs after DENA. Our findings cannot be adequately explained except by competition of the carcinogens for supposedly target molecules of protein origin, presumably, transcription factors, which contribute to generation of genetic information. They have different affinities for different compounds and, therefore, suffer different damage from the latter functioning.

The paper presents results of testing a modified algorithm for predicting virus ID50 values in a host of interest by extrapolation from a model host taking into account immune neutralizing factors and thermal inactivation of the virus. The method was tested for A/Aichi/2/68 influenza virus in SPF Wistar rats, SPF CD-1 mice and conventional ICR mice. Each species was used as a host of interest while the other two served as model hosts. Primary lung and trachea cells and secretory factors of the rats' airway epithelium were used to measure parameters needed for the purpose of prediction. Predicted ID50 values were not significantly different (p = 0.05) from those experimentally measured in vivo. The study was supported by ISTC/DARPA Agreement 450p.

The influence of subchronic administration of nootropic drugs (piracetam, phenotropil, meclophenoxate, pantocalcine, semax, nooglutil) on the brain-derived neurotrophic factor (BDNF) content in hippocampal and cortical tissues in mice with different exploratory behavior--high efficacy (HE) against low efficacy (LE)--in cross-maze test has been studied. The initial BDNF concentration in hippocamp (but not in cortex) of control HE mice was higher than that in LE mice (LE, 0.091 +/- 0.005 pg/microg; HE, 0.177 +/- 0.005 pg/microg; p < 0.0005). After drug administration, changes in the BDNF level were only observed in the hippocamp of LE mice, where it reached (pg/microg) 0.115 +/- 0.004 (for piracetam); 0.119 +/- 0.006 (for phenotropil); 0.123 +/- 0.007 (for semax); and 0.122 +/- 0.009 (for meclophenoxate). In the LE mice cortex, the BDNF content increased only after piracetam and semax injections (to 0.083 +/- 0.003 and 0.093 +/- 0.008, respectively, vs. 0.071 +/- 0.003 pg/microg in the control group; p < 0.0005). No changes were observed in the cortex of HE mice. Thus, the obtained results demonstrate that clinically used drugs piracetam, phenotropil, meclophenoxate, and semax realize their nootrope effects, at least partially, via increase in hippocampal BDNF level, which is achieved only under conditions of cognitive deficiency.

Hypothesis of reproductive compensation (Gowaty et al., 2007) suggests that constraining of free mating preference leads to reduction of the viability of progenies, which could be, partially, compensated by higher fecundity of the constrained parents. We consider infection as one of natural causes constraining female mating choice, because infection or immune response to infection can modulate male sexual demonstrations. Here we studied influence of LPS (bacterial endotoxin, activating non-specific immune response) on chemical attractiveness, sexual behavior and reproductive success in the outbreed male mice mated with the non-treated females. Single or repeated LPS administrations lead to increase of scent attractiveness of the male urine and soiled bedding for the non-estrus females. Injection of LPS (dose 50 mkg/kg) did not suppress the male sexual behavior. Time from pairing to successful mating correlates positively with the body mass of 16 day embryo. Embryos development, assessed by their body mass, was reduced in the females mated with the LPS-treated males. Higher level of plasma progesterone found in the females mated with the LPS-treated males, and shift of successful mating to the later time did not compensate reduction of embryo mass. At the same time the females mated with the LPS-treated males showed lower embryo lost in comparison with the females mated with the control males.

5 groups of 14-day ICR male mice received intraperitoneally diethylnitrosamine in the dose 50 mcg/g body weight. After weaning, they were kept in standard laboratory conditions. When aged 6 months, the mice of the 1st group were sacrificed and the number of iron-negative premalignant nodules in their livers as well as adenomas in the lungs were recordered. Since then, mice of the 3rd and 5th groups received 0.0002% L-thyroxine as drinking water throughout the experiment. Control mice of the 2nd and 4th groups were given tap water. At 9 months of age, total number of hepatic nodules in mice of group 2 was slightly (40%) higher whereas the number of gross nodules (2-5 mm in diameter) and extremely gross ones (> 5 mm) were 3- and 6-fold higher than in mice of group 1. After additional 2 months, the number of tumor nodules in the liver of control mice of group 5 could not be counted because of their mixed growth. By contrast, in thyroxine-treated mice, the number of gross nodules, as well as total number of tumor nodules in the livers were not higher than those in control mice of group 1. The data obtained indicate that exogeneous thyroxine inhibits growth of liver tumors even at the late stages of carcinogenic process. In addition, it increases the food intake and relative weight of kidneys and decreases relative weight of testes but does affect growth of pulmonary adenomas in male mice under study.

Activities of matrix metalloproteinases and chitotriosidase were measured in blood serum from male and female ICR mice with moderate hyperlipidemia receiving atorvastatin (75 mg/kg). Hyperlipidemia in male and female mice was characterized by increased serum concentration of cholesterol and, especially, triglycerides. The observed changes were more pronounced in female mice. Administration of atorvastatin decreased cholesterol (but not triglyceride) level in intact males, but had no effect on these parameters in females; chitotriosidase activity increased in male and female mice, while activity of matrix metalloproteinases increased only in males. Administration of atorvastatin produced similar effects in male and female mice with moderate hyperlipidemia: decrease in the concentration of cholesterol and, particularly, of triglycerides. Activities of matrix metalloproteinases and chitotriosidase increased in males and females, this increase being more pronounced in males. The existence of a negative correlation between cholesterol and triglyceride concentrations and activities of matrix metalloproteinases and chitotriosidase in males suggests that these enzymes can serve as a therapeutic target during hyperlipidemia.

Using originally designed low inertial open circuit respirometer, we studied influence of the bacterial endotoxin on the bioenergetic gain of male mice, which they showed after 15 min pair-wise test. Injection of lipopolysaccharide (LPS) provided increase of the post pair-wise test energy metabolism and increase of the relative energy cost of each agonistic act in dominant males. Significant correlation between energy response and number of agonistic acts in the pair-wise test was found only for LPS treated males. Spontaneous activity measured before social conflict and non-agonistic behavior during pair-wise test did not correlate with energy metabolism. Since all behavioral acts need energy and since the significant correlation between energy response and behavioral activity was found only for the agonistic behavior we assume these relationships being a result of synergetic influence on the energy metabolism of both the nonspecific immune response on LPS and neuroendocrine accompaniment of the intermale aggression.

The effect of atorvastatin on the activity of chitotriosidase (CTO) and total matrix metalloproteases (MMPs)-new markers of cardiovascular disorder-was studied on the model of murine lipemia induced by single administration of Triton WR 1339 in two doses, 500 mg/kg (mild lipemia) and 850 mg/kg (severe lipemia). A hypolipidemic effect of atorvastatin was observed in mice with mild lipemia, but not in those with severe lipemia. In both mild and severe lipemia cases, the serum CTO activity was increased upon the combined administration of atorvastatin and Triton WR 1339, correlating with cholesterol and triglyceride concentration. The total serum MMP activity decreased only in experiments with atorvastatin administration to intact mice. In mice with experimental lipemia induced by Triton WR 1339, the administration of atorvastatin also increased the ALT and AST activity in the blood serum.

The injection of outbred ICR strain male mice with SRBC affected their dominance ability in pair-wise test. In pair-wise tests done without previous presentation of the smell of a contestant, the SRBC-treated males submitted to control males. This effect occurred within 3 days after the injection. However, on the third day after treatment, under conditions of getting pre-test olfactory information about a contestant, the dominance of SRBC treated-males over the control animals was observed. Perception of the contestant smell is known to lead to emotional reaction and could be accompanied by an increase in energy metabolism. This increase was significant in both the control submissive and SRBC-treated dominate males. Thus, the modification of mice smell perception due to antigen injection can affect the pair-wise competition.

Since scent marks of mice are harbored by parasites, their sniffing during olfactory search of the mating partner leads to increase of the infection risk. A hypothesis that sexual signals can induce, along with the reproductive behavior, non-specific immune defense against respiratory infections is tested in the present paper. It was found in the experiments on outbred ICR mice that the scent of soiled bedding from cages with mature females stimulated leukocyte intervention to the upper air-ways. Migration of the white blood cells to lung tissue was accompanied with a more prominent immune and endocrine responeses to intranasal application of the bacterial lipopolysacharide (LPS). In particular, LPS administration to male mice treated by female scent was resulted in much greater amount of leukocyte aggregations in the peribronchial areas than that was found in the males kept isolated from the female signals. The female scent also enhanced adrenocortical response to LPS administration, which was coincided with statistically significant increase of IL-1beta concentration in hypothalamus. So, chemical signals of the mature female induce travel of white blood cells to the upper air-waya in the scent treated male mice. It can increase resistance to respiratory infections, on the one hand, and aggravates stress response to inhalation of the bacterial compounds, on the other hand.

The efficacy of the therapeutic, prophylactic and urgent prophylactic schemes for the use of Reaferon-ES lipint, a liposomal human recombinant alpha-interferon for oral use, was studied on mice infected with the avian influenza virus. Strain A/Chicken/Kurgan /05/2005 (subtype H5N1) of the avian influenza virus showed high virulence with respect to mice ICR. Theoretically-based calculations allowed to design an optimal therapeutic and prophylactic dose of the drug for the mice (1000 units/animal). It was observed that only after prophylactic use of Reaferon-ES lipint it was effective in protection of the mice infected with 10 LD50 of the avian influenza virus (A/Chicken/Kurgan/05/2005, H5N1). The protection coefficient was 0.35. Under such conditions the drug efficacy was comparable with that of Tamiflu. Therefore, Reaferon-ES lipint could be recommended for prophylaxis of the infection due not only to the season strains of the influenza virus, but also to the strains of the avian influenza virus.

A population of outbred mice of the ICR strain was divided into two subpopulations according to their high (EH mice) or low (EL mice) exploratory efficacy in the closed cross maze test. In addition, the EH and EL mice differed in the number of binding sites of (i) [G-3H]-MK-801 with NMDA receptors from hippocampus and (ii) [G-3H]-nicotine with nicotine cholinoreceptors (nACh) from neocortex. A subchronic administration of the cognition enhancer piracetam (200 mg/kg, once per day for 5 days) increased by 70% the number of binding sites of NMDA receptors in the EL mice. At the same time, this treatment decreased the density of neocortical nACh receptors in both EL and EH mice (by 55% and 40%, respectively). A subchronic administration of the cognition enhancer and anti-oxidant meclofenoxate (100 mg/kg, once per day for 5 days) also decreased the density of neocortical nACh receptors in both EL and EH mice (by 48% and 20%, respectively). However, meclofenoxate also increased by 41% the number of binding sites of NMDA receptors in the EH mice.

A novel approach to discovery of the peptide with an internal immunological image of carcinogen is suggested in this work. The hybridomas producing monoclonal antibodies (mAb) B2 against benzo[a]pyrene and benz[a]antracene were prepared. Polyclonal Ab against benzo[a]pyrene (Bp) and benz[a]antracene (Ba), antracene, chrysene, and pyrene were also prepared. We identified the related peptide-presenting phage specificity binding to mAT B2 and polyclonal Ab against Bp from 12 large random peptide libraries using phage display method. ICR mice were immunized with specific binding of positive phage clone for studying its immunogenicity. The Bp antibodies were found in the blood serum. All positive phage clones carried the sequence. Thus, the unique peptide with an internal immunological image of Bp may be the new candidate for anticarcinogenic vaccine.

Since non-territorial mice have as good chances for reproduction as territorial mice, we suppose that low territorial compatibility is compensated by higher breeding activity. Based on the scent war hypothesis, proteinuria was used as the criterion of territorial compatibility for male mice. Correlation between proteinuria, assessed as the protein/creatinine ratio, and reproductive output was studied in 22 male mice. Each male was caged with two females for five days. HPLC of urinary samples showed that more than 70 per cent of the proteins lay within the range 15-20 kDa. This result is typical for the major urinary proteins (MURs) which play the key role in chemical signalling im mice. Individual variation of the protein/creatinine ratio had good repeatability in the resampled urinary samples. Male proteinuria correlated negatively with early behavioural response to females, with mating success during the first two days, and with prenatal development of the progeny. Thus, the tradeoff between scent-marking efficiency determined by MUPs and breeding efficiency equalized the reproductive success of male mice with different ability of territorial competition.

Changes in the activities of NAD+- and NADP-dependent dehydrogenases of lymphocytes and tumor cells were studied in mice with Ehrlich ascites carcinoma, as well as changes in the concentrations of oxaloacetate, lactate, and NAD+ in the course of tumor growth. During the major period of tumor growth, conditions are produced in the lymphocytes for increased intensity of aerobic reactions directed at energy reproduction combined with a somewhat decreased intensity of synthetic processes. In the tumor cells, conditions predominantly arise for intensification of plastic metabolic reactions and reactions related to anaerobic energy reproduction.

We have developed a new bioluminescent method for determination of concentrations of pyruvate, lactate, malate, glutamate and NAD+ in the periferal blood lymphocytes from mice with Ehrlich ascites carcinoma. There were blood samples of intact mice for control. We found that variations of these substrates, depend on stage of malignant growth.

One of the problem in the selection of the most effective antiviral preparations with a broad spectrum of antiviral protective activity, is the "continuity" of assays of different level of complexity so, that the most effective antiviral therapeutic, selected by in vitro assays would be the most effective in vivo. Comparative study of the efficacy of the influenza virus inhibitor in the assays of inhibition of virus binding with fetuin, inhibition of infectious focus forming units in MDCK cells, inhibition of virus yield in infected MDCK cells, and inhibition of influenza virus infectivity in mice infected by viral aerosol are presented. The value of 50% inhibiting concentration IC50 for the pare "influenza virus strain A/NIB/23/89-MA-inhibitor tetra-Aca6-6'SLN" corresponded to 6-10 microM and was invariant for three different tests--in vitro assay of inhibition of virus binding with fetuin, inhibition of yield in infected MDCK cell culture, and inhibition of virus infectivity in mice, but not for the assay of inhibition of infectious focus forming units in cell culture.

The C to T substitution in position 311 n. p. of K-ras gene intron 2 in mice resistant to lung cancer (M. spretus) attenuates NF-Y transcription factor binding site in comparison with sensitive ICR mice (M. musculus). Appreciable differences between ICR and M. spretus in general pattern of binding of nuclear proteins to K-ras gene DNA within 248-332 n. p. fragment are demonstrated.

Using transplantable Ehrlich ascites tumor, hepatoma HA-1 and Lewis carcinoma it was shown, that preparations of fragmented genomic DNA can more or less effectively inhibit such tumors as well as the growth of their metastases. Such effects were produced by DNA preparations derived from tissues of mice, both syngeneic or allogeneic to tumor-bearer, as well as from human tissues.

13-14-day old mice of ICR and CBA strains were given a single intraperitoneal injection of nitrosoethylurea (80 mg/kg) or diethylnitrosamine (50 mg/kg). 2 weeks later, they were given drinking water containing phenobarbital (1 g/L) or thyroxine (2 mg/L). The control mice were given only tap water. 29.4% of male and 42.1% of female ICR mice who had received nitrosoethylurea died of leukemia within 3-6 months after the carcinogen treatment. There was no case of leukemia in mice treated with diethylnitrosamine. Nitrosoethylurea induced 3-more often lung adenomas than diethylnitrosamine. Phenobarbital and thyroxine did not affect development of either leukemias or lung adenomas. By contrast, phenobarbital significantly elevated the number and size of hepatic lesions, whereas thyroxine markedly decreased them in all the experiments. The total and free thyroxine levels were significantly decreased in the blood of mice given phenobarbital and increased in mice given thyroxine. The data obtained indicate that thyroid hormones suppress tumor development in the mouse liver and that the promotion of hepatic tumoro-genesis by phenobarbital is presumably caused by the elimination of this suppressing effect of the thyroid hormones.

A single intraperitoneal injection of Estragole (300 mg/kg) to female ICR mice 19 hours prior to Dexamethasone induction decreased induced activities of tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) nearly to 50% of the control values. In these mice, activities of the marker enzymes of liver damage: alanine aminotransferase (ALAT) and aspartate aminotransferase (AAT) increased in the blood 1.7-2.3-fold as compared with the untreated controls. By contrast, carbon tetrachloride (100 mg/kg) increased the blood AIAT and AsAT activities 135- and 30-fold as compared with the control, but inhibited the TAT and TO induction much less than Estragole did. Estragole seems to inhibit the glucocorticoid induction of these hepatic enzymes not via the unspecific toxic damage of the liver.

The finding of amitotic division of trophoblast cell nuclei in blastocysts of the American mink (Mustela vison), which has an obligatory period of delay in implantation (obligatory embryonic diapause) in its ontogenesis, led us to study the mechanisms and frequencies of division of trophoblast and decidua cell nuclei during the postimplantation embryogenesis of mouse (Mus musculus), which does not exhibit an obligatory diapause nor amitosis in blastocysts. It has been established that the main mechanism underlying the cell nuclei division in both tissues (trophoblast and decidua) forming the placenta is amitosis. These data suggest that the occurrence of an obligatory embryonic diapause in ontogenesis of certain animal species is related not only to the delay in implantation, but also to the alteration in the chronology of all processes of embryogenesis.

The susceptibility of the ICR, DD/He and CC57/BR mice to urethane-induced lung tumors was analyzed in comparison with the A/He (highly susceptible) and AKR (resistant) lines of mice. Allelic variants of the K-Ras gene intron 2 in these lines have been determined. Susceptibility of the ICR mice was similar to that of the A mice, and intron 2 of the K-Ras ICR gene carried the 37-bp deletion analogous to that described in the A/He line. The DD mice intron 2 also contained the deletion, but despite the presence of the "susceptible" K-Ras allele, the DD/He mice were resistant to urethane induction of lung tumors. The CC57BR line carried the deletion and demonstrated relatively high susceptibility. Our findings indicate that the K-Ras gene may be important in the chemical induction of lung tumors.

The drug targeting to the brain by polysorbate 80-coated nanoparticles was studied. The leu-enkephalin analog dalargin was used as a model drug for investigating the drug penetration through the blood-brain barrier. The nociceptive threshold was measured by the tail flick test. The intravenous injection of dalargin bound by sorption to poly(butylcyanoacrylate) nanoparticles subsequently coated with polysorbate 80 induced the analgesic effect in 5.0 and 7.5 mg/kg doses. The pretreatment with naloxone prevented this effect No other controls exhibited the analgesic activity, including the dalargin solution (10 mg/kg, i.v.), dalargin bound to nanoparticles not coated with polysorbate 80; and a simple mixture of dalargin, nanoparticles, and polysorbate 80 mixed directly before the intravenous injection. The luminescent and electron microscopy demonstrated the presence of separate nanoparticles in the capillary endothelium and cerebral neurons, as well as luminescent-labeled polymer in Purkinje's cells of the cerebellum.

Antiepileptic effects of a novel amino acid-containing 1,4-dihydropyridine glutapyrone and sodium valproate during combined therapy on generalized pentylenetetrazol- and focal 4-aminopyridine-induced epileptic activity in rat brain cortex were studied, as were combined effects of glutapyrone and phenobarbital on maximal electroshock in mice. The results of these investigations suggest that combined treatment by glutapyrone and sodium valproate or phenobarbital is reasonable and helps potentiate the effect of each drug, thus significantly reducing their doses, and minimize the risk of side effects of the drugs id used in higher doses in case of long treatment.

The experiments on focal penicillin-induced epileptic activity in the brain cortex (Wistar rats) and bicuculline- and thiosemicarbazide-induced seizures (Icr:Icl mice) showed that the glutapyrone possessed a significant antiepileptic activity. As previously shown, that glutapyrone has an influence on 45Ca2+ uptake by rat cortical synaptosomes (evoked by K+ depolarization) as compared with nifedipine and nimodipine, and it was effective in pentylenetetrazol-induced seizures in rats and mice. The mechanism of action of convulsants is associated with the disturbance of different links of GABAergic inhibition. It is suggested that the antiepileptic effects of glutapyrone are realized at least in part by the participation of GABAergic system.

Experiments on male Wistar rats and Icr:Icl mice studied the influence of the novel compound--amino acid-containing 1,4-dihydropyridine derivative glutapyrone (G) on acute generalized seizures, arecoline and nicotine tremor, and 45Ca2+ uptake in brain synaptosomes. It was shown that G produced significant antiepileptic effects on models of acute pentylenetetrazole seizures on rats and mice. Efficiency of antiepileptic effect depended on a dose and method of modeling seizures: it was more effective in case of intravenously pentylenetetrazole-induced seizure tested by clonic and tonic seizure components and death. The results suggest the participation of GABAergic system in realization of antiepileptic effect of G. Glutapyrone did not influence the 45Ca2+ uptake by rat cortical synaptosomes (evoked by a 1-min depdariration with 55 mM K+), this suggests that G lacked calcium antagonist properties characteristic of 1,4-dihydropyridine compounds such as nifedipine, nimodipine. In addition, G does not affect N- and M-cholinergic processes.

The effect of methylazoxymethanol (MAM), an antimitotic drug, on granule and Purkinje cells of the rat cerebellum was studied. MAM was injected subcutaneously into ICR mice on day 5 after birth. The number of nucleoli and DNA content in nuclei of cerebellar neurons were determined 2 and 12 days after the injection. The number of nucleoli served as a criterion of maturity of neurons. On day 2 after MAM administration the proportion of Purkinje cells containing a smaller number of nucleoli increased suggesting a direct effect of MAM on maturation of Purkinje neurons. On day 12 no difference in the number of nucleoli was found between experimental and control animals but the number of granule cells containing multiple nucleoli increased significantly. The latter suggests that the studied cell population was supplemented with less mature cells. As a result, development of Purkinje and granule cells is desynchronized and that appears to determine the secondary effect of MAM on Purkinje cell differentiation.

The effect of lipophilic cAMP analogs on the sensitivity of preimplantation mouse embryos of two strains to cytotoxic serotonin and adrenalin antagonists was studied. Dioctanoyl-cAMP significantly decreased the sensitivity of embryos to inmecarb and cyproheptadine: experimental embryos developed to the stage of morula or blastocyst, in contrast to control embryos incubated without this protector. A somewhat weaker effect was observed in experiments with propranolol: embryos incubated in the propranolol-containing medium after the addition of dioctanoyl-cAMP were capable of one to two cleavage divisions. 8-bromomonobutyryl-cAMP partially suppressed the inhibitory effect of cyproheptadine and did not affect the sensitivity of embryos to propranolol. These data suggest cAMP involvement in the regulatory activity of neurotransmitters in the early mouse embryos.

The pattern of purine derivatives was studied in the erythrocytes of C3HA and ICR mice during Hepatoma 22 and Ehrlich ascites tumor cells growth. Host erythrocytes purine nucleotides, nucleosides and bases were affected by the implanted tumors. The results indicated that the host erythrocytes markedly concentrated adenine and guanine nucleotides on the 5th and 11th-12th day of tumor growth. By contrast, content of nucleosides and bases were sharply decreased during the log growth phase (5th day) with the restoring of these precursors within the 11th-12th day (plateau phase). These observations indicate that aspects of the purine compounds metabolism in host erythrocytes are linked with tumor development.

Several variants of mouse embryos were received by polyethylene glycol-induced fusion of ovulated oocytes, blastomeres from various developmental stages, zygotes and their fragments. Cytological analysis and observation in culture have been carried out. The nuclear dynamics during the process of fusion, onset of mitosis in fusion products, their cleavage and initial morphogenetic processes in developing embryos have been followed. The possibility of directed constitution of embryos with varying quantitative and qualitative combination of nuclei and cytoplasm is, thus, shown. Some aspects of application of these models in studying the controlling mechanisms of early embryogenesis are considered.