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Adequate experimental animal models play an important role in an objective assessment of the effectiveness of medicines and functional foods enriched with biologically active substances. The aim of our study was a comparative assessment of the effect of consumption of 1 or 2% cholesterol with and without regular (two times a week), moderate running exercise on the main biomarkers of lipid and cholesterol metabolism, as well as the intestinal microbiota of male Wistar rats. In experimental rats, a response of 39 indicators (body weight, food consumption, serum biomarkers, liver composition, and changes in intestinal microbiota) was revealed. Total serum cholesterol level increased 1.8 times in animals consuming cholesterol with a simultaneous increase in low-density lipoprotein cholesterol (2 times) and decrease in high-density lipoprotein cholesterol (1.3 times) levels compared to the control animals. These animals had 1.3 times increased liver weight, almost 5 times increased triglycerides level, and more than 6 times increased cholesterol content. There was a tendency towards a decrease in triglycerides levels against the background of running exercise. The consumption of cholesterol led to a predominance of the Bacteroides family, due to a decrease in F. prausnitzii (1.2 times) and bifidobacteria (1.3 times), as well as an increase in Escherichia family (1.2 times). The running exercise did not lead to the complete normalization of microbiota.

We studied the effect of separate and combined influence of chronic forced physical activity reduction and high-fat and high-carbohydrate diet containing cholesterol on some indicators of carbohydrate, lipid, and cholesterol metabolism in growing male Wistar rats. Used combination of factors simulating a sedentary lifestyle and unhealthy diet did not have a synergistic effect on the selected biomarkers. On the contrary, the effect was antagonistic: body weight and appetite decreased and insulin resistance increased. The obtained results indicate certain prospects of hypercholesterolemia model using in preclinical studies of specialized food products to optimize the diet of individuals with disorders of carbohydrate and lipid metabolism., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Increasing the ability of the human body to adapt to physical stress is relevant from the standpoint of using foods for special uses containing functional food ingredients (FFI) with effectiveness proven in vivo. The purpose of this study was to evaluate the effect of FFI from Chenopodium quinoa grains with a high content of polyphenols and phytoecdysteroids on the physical endurance of male Wistar rats. Material and methods . The experiment was carried out during 36 days using 50 weaned male Wistar rats. The animals were randomly divided into 3 groups (n=12): Control, Run and Run-FFI. Rats of the Control and Run groups received a standard semisynthetic diet during the experiment. Rats of the Run-FFI group received a semi-synthetic diet with the addition of FFI in an amount of 0.055±0.003%, containing phytoecdysteroids (50.4±0.6 mg/g) and polyphenols (212.0±2.0 mg/g). During the experiment, the rats were assessed for their neuromotor function (grip strength of front paws), memory, and behavioral reactions in the "Elevated Plus Maze" (EPM), "Conditioned Passive Avoidance Reflex" (CPAR) and "Open Field" (OF) tests. Once a week, animals from the Run and Run-FFI groups were subjected to moderate physical load on a "Treadmill". On the 36th day of the experiment, the animals of these groups were subjected to exhausting physical load. Immediately after running, the animals were placed in metabolic cages to collect daily urine. At the end of the experiment, the content of corticosterone, the activity of catalase, indicators of protein, lipid and mineral metabolism, indexes of the liver functional state and antioxidant defense system parameters were analyzed in the blood serum; the level of prostaglandin E2 and dopamine were determined in daily urine. Results . Physiological tests (CRAR, OF) showed that weekly exercise increased anxiety in laboratory animals. The FFI introduction into the diet led to normalization of the assessed parameters (EPM). As a result of 36-day consumption of FFI against the background of physical loads, a significant decrease by 22% in the main stress marker, corticosterone, was revealed in the blood of rats, as well as significant increase by 23% in the stress inhibitor - prostaglandin E2 urinary excretion, compared with animals of the Run group to the level not differed from the indicators of the control animals. There were no differences in endurance performance between the Run and Run-FFI groups on the results of the exhaustive exercise. Consumption of FFI prevented the formation of excess ammonia, significantly reducing the level of urea in the blood and normalizing its excretion to control levels in the urine, which was increased in the Run group by 19%. Conclusion . The results obtained demonstrated the adaptogenic properties of the developed FFI in response to stress caused by weekly moderate and acute exhaustive physical activity. The obtained data on the biological effect of the developed FPI on the adaptive potential of laboratory animals will serve as an experimental basis for its inclusion in the composition of specialized foods., Competing Interests: The authors declare no conflicts of interest., (Copyright© GEOTAR-Media Publishing Group.)

Nowadays all over the world there is an active search for new promising bioactive compounds and evaluation of their efficiency and safety for the usage in food for special dietary uses in order to form balanced healthy diets for different groups of consumers (children, pregnant and lactating women, etc.) or inclusion in diet therapy in order to increase the effectiveness of prevention and treatment of non-communicable diseases and reduce the need for medical care. Such substances can be cyclic peptides - compounds with a unique structure, thanks to which the stability of their configuration and high bioavailability are achieved, which, in turn, allows them to exhibit a wide range of biological effects. Basically, these bioactive compounds are extracted from the leaves and stems of medicinal plants or plants that have no tradition of food use in our country. Common flax (Linum usitatissimum), a crop widely used in nutrition, is practically their only food source. The purpose of the research was to generalize information on biological activity of flax cyclic peptides in order to assess the prospects of their food application. Material and methods . Systematic analysis of scientific literature was carried out using keywords (Linseed, or Flaxseed, or Flax, or Linum usitatissimum) and (Orbitide, or Cyclolinopeptide, or Linusorb, or LOMIX) in Google Scholar, PubMed systems. A total of 500 publications were identified and analyzed. Publish or Perish software was used for analysis. The topics of the publications were analyzed using KH Coder software. Results . Cyclic peptides are among the least studied components of flax, and the profile of cyclic peptides of domestic flax seed varieties has not been described so far. At the same time, the results of preclinical studies of flax cyclic peptides represent promising data that open prospects for their use in the therapy of various diseases. Immunosuppressive and anti-inflammatory activity, as well as hypocholesterolemic and antioxidant effects of flax cyclic peptides have been demonstrated. Conclusion . The current information on the biological activity of cyclic peptides is incomplete and should be expanded by new data obtained, in particular, in in vivo models and clinical trials. The study of the composition of flax cyclic peptides from domestic raw materials, the development and implementation of the methods of their extraction will contribute to the sustainable development of the industry and will allow reorienting the production of oilseed raw materials from its export to the introduction of knowledgeintensive technologies., Competing Interests: The authors have no conflict of interest to declare., (Copyright© GEOTAR-Media Publishing Group.)

The choice of an optimal biological model of exogenous hypercholesterolemia is necessary for correct assessment of the cholesterol-lowering properties of bioactive substances and specialized food products and for validity of the experimental results. We studied the effects of cholesterol consumption in the composition of the standard semi-synthetic diet and high-fat high-carbohydrate diet for more than 3 months on lipid and cholesterol metabolism in male Wistar rats. Our findings suggest that cholesterol consumption in both diets led to a significant impairment of lipid and cholesterol metabolism, as well as an increase in insulin resistance in rats., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Special attention is being paid to the study of amaranth proteins. They are characterized by a high biological value that significantly exceeds those of grain crops. The production of protein concentrate from amaranth flour includes preliminary enzymatic hydrolysis, extraction of the resulting mixture, protein precipitation, microfiltration, and freeze-drying. In our study, the obtained amaranth protein concentrate was limited by valine, with an amino acid score of 74%. The true digestibility of the amaranth protein concentrate determined in vivo was 97.6 ± 0.3%, which was significantly lower than that of casein (99.3 ± 0.2%). The protein digestibility-corrected amino acid score value of the concentrate was 72.2%. The obtained concentrate was a rich source of selenium, copper, magnesium, manganese, and iron. Ferulic acid was the only polyphenolic compound found in the amaranth protein concentrate, but its content was significantly greater compared to the original flour. Saponins were not removed completely during the process of obtaining the amaranth protein concentrate. We identified 15 saponins in the concentrate, mainly of the bidesmoside type, the sapogenins of which are related derivatives of oleanolic acid. Thus, the developed amaranth protein concentrate can be used as an ingredient in functional food products, with a high biological value.

The aim of this study was to investigate the effects of soybean lecithin and plasmalogens concentrating on a variety of physiological tests and biochemical analyses in healthy Wistar rats. For six weeks, male Wistar rats were given a standard diet that included plasmalogens or soybean lecithin. We measured anxiety levels, overall exploratory activity, short- and long-term memory, cognitive abilities, and grip strength. Lecithin increased significantly anxiety and enhanced memory and cognitive functions. Plasmalogens significantly improved appetite and increased grip strength. When compared to plasmalogens, lecithin significantly raised HDL levels while lowering LDL levels. The plasmalogens group showed a significant increase in the C16:0DMA/C16:0 ratio, which led us to assume that plasmalogen consumption could increase their synthesis in neural tissue. The study's findings imply that, despite their various modes of action, soy lecithin and plasmalogens may both be significant nutritional components for enhancing cognitive functions.

Modern methods for extracting bioactive сompounds (BAC) from various raw materials are focused on efficacy and environmental awareness, involve the use of mathematical and statistical optimization methods, the choice of green solvents, and the use of additive extraction technology. The aim of this review was to present and briefly discuss up-to-date information on modern technological approaches to the production of plant BAС extracts for applying in food ingredients and foods for special dietary uses. Material and methods . For the main search of sources, the PubMed bibliographic database, Scopus and Web of Science databases, and the Google Scholar search engine were used. The search depth was 15 years. Results . The article presents a brief review of modern approaches to the extraction, concentration and purification of polyphenolic compounds from various plant materials. As an additive extraction technology aimed at destroying/increasing the permeability of the plant cell wall, a wide range of physical methods has been successfully used: ultrasound, microwave radiation, homogenization, application of a pulsed electric field, high hydrostatic pressure, cryo-crushing. A brief description of each method, its advantages and disadvantages are presented. Improving food safety and compliance with environmental regulations requires the choice of a safe, environmentally friendly and yet efficient extraction process. For these purposes enzymatic extraction, environmentally friendly solvents, such as deep eutectic solvents, supercritical fluid extraction and membrane technology are successfully used. The use of mathematical and statistical methods can reduce the total number of experimental trials and reduce the cost and time of experiments. The use of these methods together makes it possible to vary deliberately the process parameters in relation to a specific plant material and the final product, which provides the opportunity to isolate BAS concentrates from plant raw materials with a high yield. Conclusion . The introduction of innovative technological approaches for obtaining extracts of BAC of plant origin determines the prospects for the production of a wide range of foods for special dietary uses that meet high safety and efficiency requirements., Competing Interests: Authors declare no conflict of interests., (Copyright© GEOTAR-Media Publishing Group.)

Amaranth (Amaranthus L.), like other pseudocereals as quinoa (Chenopodium quinoa Willd.), chia (Salvia hispanica L.) and buckwheat (Fagopyrum sp.), is a promising source of dietary protein. Depending on the subspecies and breeds of amaranth, the protein content in its grain is estimated from 13.1 to 21.5%, and its amino acid score varies over a significant range and can be limited. The aim of this study was to obtain a protein concentrate from amaranth (Amaranthus L.) grain of the Voronezh breed, enrich it with chicken egg protein, determine the amino acid score of the obtained protein module, and experimentally evaluate in vivo its true digestibility and biological value. Material and methods . The amaranth protein concentrate was obtained from grain according to the technological scheme, including its enzymatic treatment, alkaline extraction, acid precipitation of proteins, microfiltration and lyophilization. The amino acid composition and amino acid score of the concentrate were determined. The protein module was obtained by mixing amaranth protein concentrate and chicken egg protein in a weight ratio of 58:42. The true digestibility and biological value of the protein module has been determined in vivo. The experiment was carried out on 32 Wistar male rats divided into 2 groups (n=16 rats): control group 1 with a body weight of 118.7±3.1 g and experimental group 2 with a body weight of 119.5±3.0 g. Animals of groups 1 and 2 received diets in which egg protein and a protein module were used as a protein source, respectively. Within 15 days of the experiment, individual indicators of food intake and body weight gain of each animal were determined. From the 14th to the 15th day food intake was determined and feces were collected. The amount of nitrogen in the food and feces was determined for each rat using the Kjeldahl method. The true digestibility of the protein was determined according to obtained data. Results . The resulting amaranth protein concentrate contained 70.4±0.6% of protein, 17.0±1.0% fat, 9.8±0.8% carbohydrates, 1.8±0.2% ash, its moisture content was 1.4±0.1%. There were no significant differences in food intake and body weight gain between animals of both groups. The calculated value of the true digestibility of chicken egg protein was 98.8±0.1% for the control group 1, of the protein module was 99.0±0.1% for the experimental group 2, the differences between the groups were not significant. Conclusion . The results of amino acid analysis and the in vivo study of the true digestibility of the protein module (composition amaranth protein/chicken egg protein) indicate the absence of limitation relative to the amino acid scale of the "ideal" protein (FAO/WHO, 2007) and high true digestibility. The biological value of the protein module, calculated according to PDCAAS, is 99.0±0.1%, which confirms the prospects for its inclusion in specialized foods., Competing Interests: Authors declare no conflict of interest., (Copyright© GEOTAR-Media Publishing Group.)

The variety of stressful conditions in daily human activity requires nutritional support with safe, specialized food products containing functional food ingredients (FFIs) enriched with biologically active plant substances with proven adaptogenic properties. In this in vivo study, by evaluating a set of physiological parameters and biochemical markers, we investigated the effectiveness of the developed FFIs from Chenopodium quinoa grains in stress conditions induced by daily episodes of immobilization for 36 days. The results of the evaluation of the anxiety-like functions, locomotor, and search activity of rats in the "open field" and "elevated plus maze" tests demonstrated the ability of FFIs to reduce stressful behavior induced by immobilization. The improvement in the long-term memory of animals treated with FFIs was noted in the passive avoidance test. Together with the hypolipidemic effect and compensation of transaminase levels, FFIs normalized the excretion of catecholamines in the urine and reduced the levels of malondialdehyde to values of the control group. According to the results of the assessment of FFI acute oral toxicity, the LD 50 value exceeded 5000 mg/kg of body weight, which categorizes the FFIs under hazard class 5-substances with low hazard. The conducted experiment demonstrated the effectiveness of nutritional support with FFIs on the selected stress model. The positive safety profile of FFIs makes them reasonable to study on other stress models and to conduct clinical testing as part of specialized food products in various categories of people exposed to chronic stress.

Extracts from bilberry leaves and blueberries containing a wide range of biologically active compounds, including polyphenols, are of particular interest due to their antioxidant, hypoglycemic and hypolipidemic properties. In this regard, The aim of this research was to investigate in vivo the effect of a concentrate of blueberrie polyphenolic compounds with buckwheat flour on some physiological and biochemical parameters in C57Bl/6 mice with impaired carbohydrate and lipid metabolism induced by the consumption of a high fat high carbohydrate (HFHC) diet. Material and methods . The polyphenol concentrate was obtained by sorption of blueberry extract on grinded buckwheat flour. Total polyphenol content was determined by Folin-Ciocalteu method, profiles of anthocyanins, flavonoids and easily digested carbohydrates were determined by HPLC. An in vivo experiment was carried out using 84 male mice C57Bl/6 for 109 days. Animals were divided into 3 groups: control fed standard semisynthetic diet, control treated with HFHC diet and experimental group treated with HFHC diet with addition of blueberry polyphenol concentrate (60 mg-eq. of gallic acid/kg body weight). Food intake, body weight gain and fasting blood glucose levels were measured during the experiment. Grip strength of the front paws of the animals was measured weekly. Oral glucose tolerance and insulin resistance tests were carried out twice. Common physiological tests (Elevated Plus Maze and Passive Avoidance Test) were used to assess the anxiety and memory of animals. Glycated hemoglobin level was determined in blood, plasma was collected for leptin and insulin level determination. The hepatic levels of triglycerides and cholesterol were assessed. Results . The concentrate of polyphenols extracted from blueberries and sorbed on grinded buckwheat flour was obtained under conditions that made it possible to exclude the sorption of easily digested carbohydrates - glucose, fructose and sucrose on the flour. The total concentrate content of polyphenols was 65.5±0.7 mg-eq. gallic acid/g, anthocyanins - 27.3±2.7 mg/g, flavonoids - 1.2±0.1 mg/g. The consumption of the concentrate by C57Bl/6 male mice with carbohydrate and lipid metabolism disorders induced by a HFHC diet had a significant (p<0.05) hypoglycemic and hypolipidemic effect, reducing the area under the curve in the insulin resistance test by 5.7% and decreasing the insulin and leptin levels by 31.3 and 15.9%, respectively (relative to the animals of comparison group fed HFHC diet). The consumption of the concentrate had a significant (p<0.05) anxiolytic effect, reducing the anxiety of animals by 2.2 times, as determined in the Elevated Plus Maze test. Conclusion . The results of the study indicate the prospects for using the developed blueberry polyphenol concentrate adsorbed on buckwheat flour as part of specialized foods for the prevention of such alimentary dependent diseases as metabolic syndrome, obesity, and diabetes mellitus., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

Increasing the ability of the human body to adapt in conditions of physical or emotional stress is promising from the standpoint of the use of preventive nutrition containing functional food ingredients (FFI) with proven effectiveness in complex physiological in vivo studies. In this work, we developed FFI from spinach leaves ( Spinacia oleracea L.) with a high content of polyphenols and adaptogens-phytoecdysteroids. Using in vivo models of increased physical activity and immobilization-induced emotional stress, we evaluated the nonspecific resistance of rats in response to the addition of the developed FFI to the diet. In the acute toxicity experiment, we found no signs of FFI toxicity up to 5000 mg/kg body weight. As a result of the daily 26-day consumption of FFI, we observed an anxiolytic effect in physiological studies. FFI prevented an increase in the content of biogenic amines in the blood, the main markers of the stress system, and had a positive effect on the lipid metabolism of the rats. The obtained results demonstrate a "smoothing" effect on the body's reaction in response to induced stress conditions.

For correct and reliable experimental in vivo assessment of antistress effect of various bioactive substances, appropriate biomodels reproducing stress and organism response to stress in laboratory animals should be chosen. We chose treadmill test for simulating exhaustive physical load and forced immobilization accompanied by disorders of physiological and psychological condition. Verification of the models used indicates their wide applicability for testing certain biological manifestations under reproduced stress exposure., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Plasmalogens and astaxanthin have a wide range of biological effects, including pronounced antioxidant properties. One of the main disadvantages of using these biologically active lipids is their low stability, which leads to a decrease in biological activity in vivo. The aim of the work was the study of the physiological and biochemical effectiveness of plasmalogens and astaxanthin in microencapsulated form. Methods. The experiment was conducted using 70 male Wistar rats during 60 days. The first 28 days of the experiment animals received modified diet with lowered content of fat-soluble vitamins A, D, and E, via excluding fat-soluble vitamin mixture and sunflower oil from the diet. On the 29th day of the experiment, the animals were divided into groups. Standard fat-soluble vitamin mixture and sunflower oil were added into the diet of one group (K2 group), two other groups received emulsions, containing plasmalogens (0.80%), astaxanthin (0.04%) and fat-soluble vitamins in native (G3 group) or microcapsulated (G4 group) forms instead of sunflower oil (5.0% of the diet). During the next 32 days of the experiment, the animals' grip strength was measured; anxiety and motor activity were assessed in the elevated plus maze and open field tests; cognitive functions were assessed in the passive avoidance and Morris water maze tests. In the blood serum, the level of corticosterone, triglycerides, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), malone dialdehyde, hydroperoxides, and total antioxidant activity were determined. Results . A significant increase in the grip strength in animals treated with an emulsion with encapsulated plasmalogens and astaxanthin indicates animal endurance growth. In the Morris water maze test, animals of the same group showed the best learning ability, which indicates an improvement in cognitive functions. A significant more than 3-fold decrease in blood corticosterone level in the animals treated with plasmalogens and astaxanthin, regardless of the form of administration, in comparison with the indicator of animals in the control groups, indicates an adaptogenic effect and requires further study. The consumption of the emulsions led to a significant improvement in lipid metabolism: a significant decrease in serum cholesterol by 20% was shown, against the background of a significant reduction in LDL cholesterol by 25%. Conclusion . The beneficial effect of including the experimental emulsion in the diet is expressed in improving memory and cognitive functions, increasing muscle tone and the static component of endurance in male Wistar rats., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

The effects of vanadium complex with enzymatic hydrolysate of soy protein (V-EHSPI) were studied in male Wistar rats with induced disorders of carbohydrate and lipid metabolism. The content of vanadium (IV) in the studied complex was 15.8 mg/g dry product. High-lipid high-carbohydrate diet was used to induce disorders of lipid and carbohydrate metabolism. Addition of vanadium in three different doses to the diet over 100-day experiment reduced body weight gain and the levels of glucose, insulin, leptin, and triglycerides. V-EHSPI produced beneficial effects on carbohydrate and lipid metabolism even in a dose 5 μg/kg body weight/day (calculated from the mean food consumption over the entire treatment period). Significant inhibition of growth and changes in the weight of organs in animals treated with V-EHSPI attested to toxicity of vanadium in the studied dose range.

Formally, phytoadaptogens extracted mainly from medicinal plants are not food substances, and their use in preventive nutrition is significantly limited. However, screening studies can detect phytoadaptogens in food plants, however in much smaller quantities. For example, phytoecdysteroids are biologically active substances (BAS), used to relieve chronic fatigue syndrome, reduce nervous and muscle fatigue, improve memory and attention processes. Phytoecdysteroids can be isolated not only from various medicinal plants, but also from food plants like spinach (Spinacia oleracea) and quinoa (Chenopōdium quīnoa). Researchers show interest in possible synergies in the manifestation of a wide range of pharmacological effects, determined by a combination of phytoecdysteroids and other biologically active substances of these food plants. The aim of this research is a brief analytical review of publications characterizing the spectrum of minor biologically active substances of spinach and quinoa, as well as those devoted to the experimental assessment of their pharmacological activity in experiments on laboratory animals. Results . An analysis of the publications presented in the review indicates that spinach and its extracts are promising food sources of phytoadaptogens, carotenoids, phenolic compounds, in particular quercetin, kempferol, phenolic acids (p-coumaric, ferulic), as well as lignans metabolized into biologically active phytoestrogens (enterolactone and enterodiol). The possible prophylactic effect of spinach is being actively studied in vivo in laboratory animals when modeling diseases such as diabetes mellitus, cardiovascular and cancer diseases. Numerous adaptogenic effects and the neuroprotective effect of spinach and its extracts are shown, expressed in improving the memory and learning of animals. Publications of the last decade indicate an increased interest in such a promising food source of high-grade protein and a wide spectrum of biologically active substances, including phytoecdysteroids, as quinoa grains. More than 20 phenolic compounds in free or conjugated forms were found in quinoa grains; tannins, saponins, sterols, phytic acid, and ecdysteroids are present in small quantities. The consumptionof quinoa can have a beneficial effect on the organism, normalizing carbohydrate and lipid metabolism, reducing body weight and preventing excessive lipid peroxidation. The anti-inflammatory effects and antioxidant effects of quinoa are widely discussed. Conclusion . Spinach, quinoa and their extracts are promising food sources of biologically active substances, their pharmacological action is confirmed from the standpoint of evidence-based medicine by in vivo experimental studies. Accordingly, it is advisable to expand the use of these food plants for inclusion in biologically active dietary supplements, functional food ingredients and specialized food products for various purposes., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

Preclinical physiological and biochemical studies of the effectiveness of the tested biologically active substances in the conditions of simulated pathology in laboratory animals are an important stage preceding the assessment of the clinical effectiveness of the created specialized foods for therapeutic or preventive nutrition. The aim of this work is a brief review of the developed algorithm for preclinical assessment of the effectiveness of specialized foods, which includes a set of sequential stages of testing the safety, stability, bioavailability and effectiveness of biologically active substances in experiments on laboratory animals. Results . The paper presents a brief review of methodological approaches for in vivo preclinical assessment of the effectiveness of minor plant biologically active substances - polyphenols from bilberry fruits and leaves and phytoecdysteroids extracted from spinach. A beneficial effect of bilberry leaves' polyphenols on carbohydrate and lipid metabolism of male Zucker and Wistar rats was shown, which determined the necessity to increase the effectiveness of the developed polyphenolic ingredient by sorption on a food carrier. When evaluating the in vivo effects of polyphenols from blueberries and leaves sorbed on buckwheat flour, a pronounced hypoglycemic effect was found. The experiments aimed to the assessment of the adaptogenic effects of phytoecdysteroids from spinach leaves showed a significant decrease in the content of the main biomarkers of the stress system, which indicates a «smoothing» of the response of the animal organism to a strong stress impact. Conclusion . The inclusion of minor biologically active substances into the composition of foods for preventive and therapeutic nutrition is the necessary condition to improve their effectiveness. The scientific and practical significance of the preclinical evaluation to a decisive extent depends on the presence of a correctly selected biomodel. Thus, the adequacy of the assessment of the effects of minor biologically active substances as functional food ingredients in the composition of the specialized food products depends on the reproducibility of pathological processes (clinical, biochemical and morphological disorders) characteristic for the studied disease on the selected in vivo model., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

The hypoglycemic properties of polyphenolic compounds of plant origin are confirmed by the results of numerous preclinical and clinical studies. However, the biological effects of these compounds are limited by their low bioavailability. This makes it urgent to develop methods for its increasing due to new methods of entering polyphenols into the organism, for example, by extracting them from natural sources in the form of extracts and concentrating extracts on food polymer matrices for subsequent use as a functional food ingredient (FFI). The aim of this study was to evaluate in vivo the possible effect of consumption of the obtained FFI in the form of a food matrix - buckwheat flour enriched with bilberry polyphenols - on carbohydrate metabolism disorders induced by a high-fat diet with a high content of easily digestible carbohydrates (sucrose) and anxiety level of male C57Bl/6c mice. Material and methods . The food matrix was obtained by sorption of the bilberry fruits polyphenol extract on buckwheat flour. The total polyphenol content in the composition of food matrix was 8.9±0.7 mg-eq gallic acid/g flour. Total anthocyanin content in the composition of food matrix was 4.6±0.1 mg/g flour. The experiment was conducted for 150 days using 48 male C57Bl/6c mice (weaners). The animals were divided into 3 groups: the control group K1 (n=16, the mice received a standard semi-synthetic diet (22.5% protein, 10% fat, 58% carbohydrates as starch, 362 kcal/100 g), the control group K2 (n=14) and the experimental group G3 (n=18). Disorders of carbohydrate and lipid metabolism in animals of groups K2 and G3 were modeled by feeding an iso-nitrogenous high-fat diet with a high content of easily digestible carbohydrates (HFHC-diet: 22.5% protein, 30% fat, 18% carbohydrates in the form of starch, 20% sucrose, 493 kcal/100 g). FFI, a food matrix in the amount of 6.6 g/100 g of feed, was introduced into the diet of animals of the experimental group G3, which corresponded to the amount of polyphenols equal to 58.7 mg-eq gallic acid/100 g of feed and the content of anthocyanins 30.4 mg/100 g of feed. Once every three weeks, the level of glucose in the blood of animals was monitored. On days 60 and 114 of the experiment, animals were tested on an elevated plus maze. Animals were decapitated under light ether anesthesia at the end of experiment. The content of glycated hemoglobin was determined in the blood. Results and discussion . Animals of both groups treated with HFHC-diet consumed significantly less feed compared with animals of the control group K1 (2.91±0.05 g/day per mouse). Moreover, animals of the experimental group G3 consumed significantly more food (2.51±0.04 g/day per mouse) compared with animals of the control group K2 (2.36±0.04 g/day per mouse). In contrast, the energy consumption of animals of both groups fed HFHC-diet was significantly higher compared to the K1 group (10.5±0.2 kcal/ day per mouse). Energy consumption by animals of group G3 (12.3±0.2 kcal/day per mouse) was significantly higher compared to animals of the control group K2 (11.5±0.2 kcal/day per mouse). The data obtained indicate that the consumption of FFI in the form of polyphenols adsorbed on the food matrix can contribute to increased appetite in animals treated with the high-fat diet. The results of the Elevated Plus Maze test indicated the absence of the effect of polyphenols in the composition of the food matrix on the anxiety level of animals. Starting from day 42 until the end of the experiment, the glucose level in animals of group G3 was significantly lower than the corresponding indicator for animals of the control group K2. Conclusion . In accordance with the results obtained, further studies of the safety and clinical efficacy of including the developed FFI in the form of a food matrix with polyphenols into the composition of specialized foods for the prevention of carbohydrate metabolism disorders are advisable., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

We investigate the long-term effect of very-low dose exposure to a mixture of six pesticides associated with hydrophilic vitamin deficiency on the neurobehavioral outcomes of rats. Two hundred Wistar rats were divided into four groups, two control groups, a vitamin sufficient control group and a vitamin deficiency control group and 2 test groups, a vitamin sufficient test group, and a vitamin deficiency group. The test groups were exposed for 9 months to a mixture of diquat, imazamox, imazethapyr, tepraloxydin, bentazone and acifluorfen in doses of 0.01xNOAEL (mg/kg bw/day). After 9 months of exposure, the behavior changes were evaluated by open field test and elevated plus maze test and the memory was assessed by passive avoidance test. Chronic vitamin deficiency decreased locomotor and special orientation activity and increased anxiety-like behavior in rats. Exposure to very low doses of a mixture of 6 pesticides caused central nervous effects, manifested as decreased locomotor activity, and increased anxiety levels. Vitamin deficiency and low dose chronic pesticides mixture exposure thus affected the central nervous system, especially long-term memory., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Among various food proteins, soybean proteins have the greatest traditions of application for the dietary correction and prevention of lipid metabolism disorders and related complications. Aim. In an in vivo experiment using male Wistar rats, the lipid-lowering properties of soy protein and its enzymatic hydrolysate were tested to evaluate their possible use as ingredients of specialized foods. Material and methods. Animals were randomly divided into 3 groups: control group G1 and 2 experimental groups G2 and G3. The total duration of the experiment was 70 days. The animals of the control group G1 were fed with high-lipid semi-synthetic diet. Animals of the experimental groups G2 and G3 received the same high-fat semi-synthetic diet, but with a 50% replacement of casein with soy protein isolate (SPI) and enzymatic hydrolyzate of SPI (EHSPI), respectively. The blood glucose was measured once per 2 weeks. At the end of the experiment on the 71st day the level of glycated hemoglobin was determined in the blood; the levels of triglycerides, cholesterol, high density lipoproteins (HDL), low density lipoproteins (LDL) and the concentration of malon dialdehyde were determined in the serum. Results and discussion. Starting from the 6th week of the experiment and prior to its completion, the average food intake of animals from the G3 group was significantly (р<0.05) lower compared to animals of the G1 control group. The food intake of animals of group G2 was significantly (р<0.05) reduced compared with this indicator for animals of group G1, starting from the week 8 of the experiment and prior to its completion. The monitoring of the body weight gain did not reveal significant differences between all groups of animals, despite differences in the food intake. Replacing casein in the diet by 50% with SPI had a pronounced antioxidant and cholesterol-lowering effect. The total cholesterol content (1.65±0.05 mmol/l) decreased significantly (р<0.05) due to a decrease in LDL (0.90±0.03 mmol/l), and malon dialdehyde level lowered (3.7±0.5 μmol/l, р<0.05) in the serum of group G2 rats compared with animals of the control group G1 (2.01±0.13 and 1.12±0.09 mmol/l; 5.1±0.4 μmol/l, respectively). Replacing casein by 50% with EHSPI in the diet of G3 rats was unfavorable, significantly (р<0.05) increasing the level of total cholesterol (2.76±0.16 mmol/l) and cholesterol in LDL (1.66±0.12 mmol/l) in blood of these animals compared with animals of both comparison groups G1 and G2. Conclusion. A preclinical comparative study of the cholesterol-lowering and antioxidant properties of the SPI substantiates the prospect of its following clinical trials with the aim of including into the composition of specialized foods for prevention and diet therapy of the disorders of endogenous cholesterol homeostasis., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

Blueberries, which contain a wide range of polyphenolic compounds, are used in traditional medicine for prevention and treatment of carbohydrate metabolism disorders. However, the high content of mono- and disaccharides in the juice significantly reduces the possibility of using in preventive nutrition by persons with impaired carbohydrate metabolism. In this regard, it is necessary to search for technological approaches aimed at obtaining functional food ingredients with a higher content of polyphenols and a low content of mono- and disaccharides, for subsequent inclusion in corresponding therapeutic foods. The aim of this study was to develop a technological approach to obtain a food matrix by enriching buckwheat flour with polyphenolic compounds extracted from blueberries. Material and methods . The work presents a method for producing a food matrix by sorption of polyphenolic compounds from an aqueous-alcoholic extract of blueberries on buckwheat flour. The concentration of total polyphenols and anthocyanins in the extract and their content in the food matrix was determined by spectrophotometric methods. The content of mono- and disaccharides and profile of individual anthocyanins was determined by HPLC. Results and discussion . The sorption on buckwheat flour was 45% of the total polyphenol content and 48% of anthocyanin content in the initial blueberry fruit extract. The profile of anthocyanins sorbed on a food matrix did not differ significantly from the profile of blueberry extract. The results of determining the content of mono- and disaccharides indicate the absence of their sorption from the extract of blueberries on buckwheat flour. Conclusion . The results obtained are of significant interest, indicating that the sorption approach allows targeted concentration of polyphenols in the food matrix with a minimum amount of easily digestible carbohydrates, which significantly increases the efficiency of its use as a functional food ingredient in dietetic products for people with carbohydrate metabolism disorders., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

The aim of this work was to study the impact of modification of fatty acids composition of laboratory animals' diet in the presence of plasmalogenes (PG), astaxanthin (ASTA) and their combination on animals' adaptation potential in stress conditions., Material and Methods: The fatty acids composition of diet was altered with the use of lipid module, containing 88.7% of high oleic sunflower oil, 6.3% of coconut oil and 5% of micralagae Schizochytrium sp. oil. The experiment was conducted with the use of 80 male Wistar rats with initial body weight 125±5 g. Selected animals (n=50) were divided into 5 groups according to their activity in tests «Open field» (OF) and «Elevated plus-maze» (EPM). The animals of control group 1st were not exposed to physiological tests - this was an intact control group. The animals of control groups 1st and 2nd were treated with standard half-synthetic diets for 30 days [381 kcal/100 g of dry food, 20.1% of casein on calories, 10% of fat (the mixture of lard and sunflower oil in 1:1 ratio)]. In the diet of 3d group animals the sunflower oil (50% of fat in the diet) was substituted with lipid module, enriched with ASTA (4.0±0.3 mg/day/kg b.w.); in the diet of group 4 animals - with lipid module, enriched with PG (79.0±2.0 mg/day/kg b.w.); in the diet of group 5 animals - with lipid module, enriched with ASTA and PG (the same doses). The evaluation of anxiety level and total exploration activity was conducted in «elevated plus maze» test. On the 31st day of experiment, animals were exposed to forced swim test for evaluation of their physical endurance and capacity in the conditions of high stress., Results and Discussion: The introduction into the animal diet of lipid module, enriched with PG and/or ASTA had pronounced hypolipidemic effect, lowered total serum cholesterol against a background of decrease in low-density lypoproteins (LDL) level. The concentration of ω-3 polyunsaturated fatty acid (PUFA) - docosahexaenoic acid in liver cells of animals treated with lipid module enriched with PG and/or ASTA increased more the 3 times, while ω-6 linoleic acid decreased twice. The consumption of enriched with ASTA lipid module inhibited the increase in blood corticosterone level after stress (exhausting physical exercise) and lowered it to the control animals values, thus showing adaptogenic effect. The animals treated with lipid module enriched with ASTA (group 3) spent significantly less time in open arms of the maze in comparison with the first test, what may show the increase in anxiety level. The introduction of PG into lipid module neutralized this effect. The forced swim test with load showed no increase in working ability and endurance of animals of all tested groups., Conclusion: The further study of adaptogenic action of PG in combination with ASTA in the composition of lipid module compared to the similar effect of traditional phospholipids is of special interest., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

Numerous experimental and clinical studies have shown high efficiency of plant polyphenolic compounds in restoring age-related memory and learning disorders. In the present study a functional food ingredient (FFI) was obtained by sorption of an aqueous solution of bilberry leaves extract on buckwheat flour, which allowed to concentrate polyphenols and increase their storage stability. The aim of this study was to evaluate the impact of a developed FFI, enriched with bilberry leaves` polyphenols, on the anxiety level, locomotor activity, memory and spatial learning of db/db mice with genetical type 2 diabetes. Material and methods . The experiment was conducted using 10 heterozygote male db/db mice and 10 homozygote male db/+ mice as the comparison control group (7 weeks of age). According to body weight, blood glucose level, the results of insulin resistance test and elevated plus-maze (EPM) test, animals were randomized into three groups: control group C1 - db/+ animals, control group C2 and experimental group G3 - obese db/db mice. Buckwheat flour was included into the diet of C2 group in a dose 22.5 g/100 g; FFI was included into the diet of G3 group in a dose 2.5 g/100 g (that was equal to 59.2± 1.4 mg-eq gallic acid per 100 g of the diet). The anxiety level and general locomotor activity were evaluated in the EPM test. The evaluation of behavior, memory and spatial learning was performed using passive avoidance test (PAT). Glycated hemoglobin level was determined in blood, insulin and leptin levels were determined in blood plasma, general antioxidant activity was determined in liver cytosolic fraction. Results and discussion . The obtained data on biochemical parameters and insulin resistance tests showed the absence of normalizing effects of developed FFI. However, the inclusion of polyphenol-containing FFI into the diet led to beneficial changes in physiological parameters. Animals of G3 group, provided with FFI, were significantly less anxious compared to both control groups. During PAT testing of short-term memory, no animals in G3 group entered to the dark compartment (0%), what demonstrated increased learning ability and well-established memory of these animals in comparison with C1 (50%) and C2 groups (80%). Conclusion . The results prove the effectiveness of bilberry leaves` polyphenols, sorbed on the brown buckwheat flour, in the correction of central nervous system disorders in db/ db mice with genetically altered type 2 diabetes, what points at possible prospect of FFI inclusion in therapeutic products for patients with type 2 diabetes mellitus., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

We assessed the effect of intake of easily digested carbohydrates for 133 days on quantitative parameters of neuromotorics and cognitive function in Wistar rats and C57Bl/6J mice. Neuromotorics (muscle tone) was assessed in rats and mice by the forelimb muscle force (grip strength) over 4 months. Anxiety was assessed in the elevated plus-maze test and cognitive function (short-term and long-term memory) was evaluated by conditioned passive avoidance response (CPAR) test over 3 months. The mice, in contrast to rats, receiving the diet with easily digested sugars demonstrated suppression of neuromotorics. Anxiety increased with age in female mice, but not in rats, irrespective of the diet. Cognitive function in rats receiving experimental rations did not change significantly in comparison with the control. In mice, consumption of equimolar mixture of fructose and glucose impared short-term, but not long-term memory, in comparison with the group receiving glucose alone. We revealed a small (by 14-17%), but statistically significant increase in the brain weight in mice receiving fructose and sucrose. The study demonstrates sufficient interspecies differences in the influence of carbohydrate rations on neuromotorics and behavioral responses in the in vivo metabolic syndrome model.

The aim of this study was the evaluation of hypoglycemic action of functional food ingredient (FFI) - a polyphenol food matrix in 3-month in vivo experiment with the use of fat male mice C57BL/6. The food matrix was produced by means of incubation of bilberry leaves extract (2% solution, pH 3.6) with buckwheat flour in ratio 1:50 during 45 min at 25 °C. The polyphenol content in experimental batch of matrix was 26.6±0.5 mg-equivalent of gallic acid per 1 g of flour. The experiment was conducted with the use of 46 male mice C57Bl/6c. Blood glucose level was estimated before starting the feeding experiment. An oral fasting glucose tolerance test (GTT) was also conducted, the area under the curve (AUC) was calculated. The animals were randomly divided into 4 groups (according to the body weight, glucose level and AUC): control group K1, fed with standard semisynthetic diet, control group K2 ( n =10) and experimental groups G3 and G4 ( n =13). The symptoms of type 2 diabetes in groups K2, G3 and G4 were modeled with the use of high-fat high-carbohydrate diet (HFHC diet, 30% fat, 20% sucrose). FFI was included into the diet of animals of experimental groups G3 and G4 in doses 2.5 and 5 g per 100 g of ration, respectively. The GTT test was repeated on the 40th and 82nd days of experiment. On the 15th, 54th and 91st days the insulin resistance test (IRT) was conducted. The consumption of HFHC diet by K2 group animals resulted in development of obesity in these animals to the week 11, which was characterized by increase in the body weight gain (37.1±4.9%) in comparison with control group K1 animals (23.4±2.2%). The introduction of FFI into animal's diet decreased significantly body weight gain of these animals in comparison with K2 group. On the week 8, the significant increase in blood glucose level of animals fed with HFHC diet was found (K2 - 8.2±0.3, G3 - 8.1±0.3, G4 - 8.5±0.3 mmol/L), in comparison with control group K1 (7.1±0.3 mmol/L). On the 91st day of experiment this difference remained significant only for group K2 (9.3±0.6 against K1 group - 7.7±0.3 mmol/L), what shows the beneficial hypoglycemic action of the FFI in both doses (for G3 group - 8.5±0.2, for G4 - 8.4±0.3 mmol/L). On the week 8 the disorder in glucose tolerance was found in animals of group K2, what is one of type 2 diabetes symptoms. The introduction of FFI in a dose 2.5 g/100 g into group G3 diet inhibited the development of these disease symptoms. The introduction of FFI in a higher dose (5 g/100 g of a diet) to a certain extent inhibited the development of insulin resistance. There was no effect of FFI, introduced into animal's diet, on cognitive functions and short-term and long-term memory., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

Rats of the experimental group for 30 days received a ration in which the fat component (sunflower oil) was completely substituted with a lipid module enriched with docosahexaenoic acid and astaxanthin. Anxiety of animals in elevated plus-maze test and muscle tone in the grip strength test were evaluated before and after the experiment. Exhausting treadmill exercise was simulated after 30 days of feeding the lipid module. Modification of the lipid component of the diet (enrichment with docosahexaenoic acid and astaxanthin) reduced fatigue during exhausting physical exercise, increased muscle tone, and produced an anxiolytic effect. The results indicate the prospects of using the lipid module as a functional food ingredient in the composition of specialized food products.

To investigate the effect of enrichment of the rats diet with polyunsaturated fatty acids (PUFA) ω-3 (220 mg docosahexaenoic acid per 1 kg of animal body weight per day) and astaxanthin (5 mg/kg body weight) on serum corticosterone concentration, physical fatigue, anxiety of rats after exhausting the load. During 30 days the rats of the test group received the diet in which the usual fat component comprising sunflower oil and lard (1:1) was completely replaced by the mixture of oils (high oleic sunflower (89%), coconut (6%), and marine oil from microalgae Schizochytrium sp. (5%) with a high content of docosahexaenoic acid with the addition of astaxanthin). Ratio of ω-6 and ω-3 PUFA in the lipid component of the experimental diet was 5.2:1 (n=12) and 135:1 in the diet of rats in the control group (n=12). DHA enrichment of the diet resulted in a significant 10-fold increase of the DHA liver content and ω-6 PUFA reducing (in particular of linoleic acid in 2.7-fold). No significant differences have been identified between the groups in terms of anxiety, estimated on the elevated plus maze at the beginning and on 24th day of the experiment. Results of the exhausting load on a treadmill (25th day) showed a significant reduction in physical fatigue in rats of the experimental group compared with the control group of rats: the number of contacts with the electrical grid was 4.2±0.9 versus 19.7±4.4, fulltime shock was 0.9±0.2 versus 3.3±0.8 sec. Significantly lower serum corticosterone concentration took place in the subjected to exhausting exertion animals receiving lipid module (15.0±3.9 ng/ml) compared to control animals (31.0±5.4 ng/ml). Thus, modification of the lipid component of the diet by its enrichment with DHA and astaxanthin led to decrease of the rat fatigue during exercise training (test treadmill) and prevent from the serum corticosterone raise, that indicates animal stress adaptation ability.

The aim of the study was to evaluate type 2 diabetes medicamental biomodel in 70-days experiment. Control group animals were provided with water ad libitum throughout the experiment, experimental group animals for the first two weeks were provided with 20% solution of fructose ad libitum instead of water. On the 15th day, experimental group animals (average body weight 257±8 g) were injected abdominally with streptozotocin (STZ) in dosage 40 mg/kg of body weight. For the next three weeks on the 22nd, 28th and 36th days, glucose level in blood taken from the tail vein was measured using portable electrochemical glucometer. On the 37th day animals with blood glucose level 11.0 mmol/L or higher were included in experimental group for further research. On the 44th and 60th day control measurements of glucose level were conducted. On the 70th day animals were taken out of experiment by decapitation under ether anesthesia. The concentration of glucose, glycosylated hemoglobin, triglycerides, cholesterine, HLD and LDL were measured in blood serum. Additionally anxiety level of animals was evaluated before and after STZ injection using Elevated plusmaze. The comparison of physical fatigue of control and experimental groups was performed using treadmill. On the 37th day blood glucose concentration of control group animals was 6.6±0.4 mmol/L. 33% of animals (13 of 40) with glucose level 11.0 mmol/L or higher formed the experimental group (average glucose level 16.2±1.3 mmol/L), other 27 rats had normal glucose level. The anxiety level of diabetic rats was higher than in control group. Diabetic rats showed significantly lower physical fatigue than control rats. On the 44th and 60th day of experiment glucose level in experimental rats from group 2 (15.5±1.4 и 14.8±1.2 mmol/L) was significantly higher than of control animals (7.0±0.5 и 6.8±0.3 mmol/L). Glycated hemoglobin level in blood serum of diabetic group (7.2±0.7%) was significantly higher than of control group (3.3±0.2%). This proves the progression of stable long-term hyperglycemia. According to results represented model can be used for initial experimental evaluation of tested antidiabetic biologically active substances.

Monkeypox virus (MPV) belongs to the orthopoxvirus genus of the family Poxviridae, is endemic in parts of Africa, and causes a human disease that resembles smallpox. The 196,858-bp MPV genome was analyzed with regard to structural features and open reading frames. Each end of the genome contains an identical but oppositely oriented 6379-bp terminal inverted repetition, which similar to that of other orthopoxviruses, includes a putative telomere resolution sequence and short tandem repeats. Computer-assisted analysis was used to identify 190 open reading frames containing >/=60 amino acid residues. Of these, four were present within the inverted terminal repetition. MPV contained the known essential orthopoxvirus genes but only a subset of the putative immunomodulatory and host range genes. Sequence comparisons confirmed the assignment of MPV as a distinct species of orthopoxvirus that is not a direct ancestor or a direct descendent of variola virus, the causative agent of smallpox.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.

Atypical puffing of polytene chromosomes of Chironomus plumosus (1 larvae, IV stage) from the Ivan lake in Chita region, southern part of Siberia, has been described. At the sites of ordinary localization of interdisks and puff-patterns typical of Ch. plumosus the puffs in our material displayed different levels of activity: from a light vacuolous spot to puffs of class 5. Most of these puffs were revealed in Ch. plumosus for the first time, namely, puffs IA10a-r, IB12v-y + 13a-d, IIC14p-z + 15a-z + 16a-e, IIC14p-z + 15a-h, IID14a-m + 13s-w, IID11-2a-d, IID1p-x + 2a-d, IIIE3g-a and IIIF13h-p + 14a-e. Some other puffs, such as IB16a-k, IB15m-r + 16a-m, IB21a-o, IIC20, IVG6 and IVG7, were described earlier (Maksimova, 1979, 1983). The majority of observed puffs turned out to be heterozygous. Only one puff-knob, IIIE3g-a of class 5 activity, was found in all cells of the studied salivary glands. Its origin may be due to the appearance of heterozygoous inversion pluE1.2. All other puffs were observed in some part of cells. It is supposed that the appearance of larvae with unusually high functional activity of chromosomes may be presumably induced by stress influence of certain environmental factors.

The interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with 24mer synthetic oligonucleotide duplexes having different purine base substitutions in the palindromic recognition sequence, GATC, was investigated by means of gel shift and methyl transfer assays. The substitutions were introduced in either the upper or lower strand: guanine by 7-deazaguanine (G-->D) or 2-aminopurine (G-->N) and target adenine by purine (A-->P) or 2-aminopurine (A-->N). The effects of each base modification on binding/methylation were approximately equivalent for both strands. G-->D and G-->N substitutions resulted in a sharp decrease in binary complex formation. This suggests that T4 Dam makes hydrogen bonds with either the N7- or O6-keto groups (or both) in forming the complex. In contrast, A-->P and A-->N substitutions were much more tolerant for complex formation. This confirms our earlier observations that the presence of intact 5'-G:C base pairs at both ends of the methylation site is critical, but that base substitutions within the central A:T base pairs show less inhibition of complex formation. Addition of T4 Dam to a complete substrate mixture resulted in a burst of [3H]methylated product. In all cases the substrate dependencies of bursts and methylation rates were proportional to each other. For the perfect 24mer k cat = 0.014/s and K m = 7.7 nM was obtained. In contrast to binary complex formation the two guanine substitutions exerted relatively minor effects on catalytic turnover (the k cat was reduced at most 2. 5-fold), while the two adenine substitutions showed stronger effects (5- to 15-fold reduction in k cat). The effects of base analog substitutions on K m(DNA) were more variable: A-->P (decreased); A-->N and G-->D (unchanged); G-->N (increased).

The DNA-[N 6-adenine]-methyltransferase (Dam MTase) of phage T4 catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have used a gel shift assay to monitor complex formation between T4 Dam and various synthetic duplex oligonucleotides, either native or modified/defective. The results are summarized as follows. (i) T4 Dam bound with approximately 100-fold higher affinity to a 20mer specific (GATC-containing) duplex containing the canonical palindromic methylation sequence, GATC, than to a non-specific duplex containing another palindrome, GTAC. (ii) Compared with the unmethylated duplex, the hemimethylated 20mer specific duplex had a slightly increased ( approximately 2-fold) ability to form complexes with T4 Dam. (iii) No stable complex was formed with a synthetic 12mer specific (GATC-containing) duplex, although T4 Dam can methylate it. This indicates that there is no relation between formation of a catalytically competent 12mer-Dam complex and one stable to gel electrophoresis. (iv) Formation of a stable complex did not require that both strands be contiguous or completely complementary. Absence of a single internucleotide phosphate strongly reduced complex formation only when missing between the T and C residues. This suggests that if T4 Dam makes critical contact(s) with a backbone phosphate(s), then the one between T and C is the only likely candidate. Having only one half of the recognition site intact on one strand was sufficient for stable complex formation provided that the 5'G.C base-pairs be present at both ends of the palindromic, GATC. Since absence of either a G or C abolished T4 Dam binding, we conclude that both strands are recognized by T4 Dam.

Primary structure of two parts of Karelian fever virus (KFV) genome (29-57 nt and 10507-11591 nt) cloned in recombinant plasmids has been studied and compared with that of Sindbis (HRSP strain) and Ockelbo viruses. Fifty-four nucleotide substitutes were revealed in the sequenced parts of KFV genome including partially 5' and 3'-nontranslated sites ( a total of 1613 nucleotides, or approximately 13.8% of the genome length), in comparison with the Sindbis virus prototype HRSP strain, this being in good correlation with strain variability. Eighteen nucleotide substitutes (96.4% homology) were detected in the NSP1 gene site (60-557 nt) of KFV in comparison with Sindbis virus and only 5 substitutes (98.8% homology) vs. Ockelbo virus. These data on primary structure of KFV genome reliably and unambiguously indicate the appurtenance of this virus to Sindbis-like viruses.

As a result of two successive recombinations of influenza A viruses: A/Leningrad/0139/76 (H3N2) with A/PR/8/34 (H1N1) strain and the resulting reassortant A/Leningrad/0139/76, R (H3N2) with a field isolate A/Kiev/59/79 (H1N1), a virus. A/Kiev/59/79, R (H1N1) was obtained. It was established by electrophoresis in polyacrylamide gel and sequencing of kDNA fragments of its genome that the genes PB2, PB1 and NP of this virus belonged to serosubtype H3N2, while genes PA, M, and NS to serosubtype H1N1 (like A/PR/8/34). The surface glycoproteins HA and NA belonged to serosubtype H1N1/like A/USSR/90/77.

Simultaneous circulation of different subtypes of influenza A viruses provides conditions for reassortant strains formation. A comparative investigation of genome of 47 influenza A virus strains (H1N1, H2N2, and H3N2) was carried out by competitive dot hybridization technique and sequence analysis of some of cDNA-copies of the virus genes. All the genes of 43 strains encoding nonglycolysed proteins corresponded to the serum subtype of surface glycoproteins. The reassortant pattern of genome for some genes of core proteins was revealed in 4 viruses. All the dot hybridization data were completely confirmed by sequence analysis of the genes.

Investigations of the antigenic structure and genome of influenza A (H1N1) viruses isolated in the Mongolian People's Republic in 1982, 1983, 1986 and 1987 from children with acute respiratory diseases using monoclonal antibodies and nucleotide sequencing revealed 4 strains identical to the prototype strain A/PR/8/34 (H1N1), variant M. Sinai, and 8 strains closely similar to the epidemic strain A/USSR/90/77 (H1N1) in the antigenic structure and A/Leningrad/54 (H1N1) in the primary structure of HA.

The authors own results on the variety of the genomic primary structures in human influenza A viruses participating in the epidemic process, including the atypical viruses. The comparative studies revealed new trends in the HA gene antigenic drift on the late stages and the PB1 gene shift. Modifications occurring in the primary structure of the influenza A viruses native genomes during laboratory treatment (adaptation to new hosts, vaccine preparation, egg passaging) have been analyzed. Sequencing of several types of "antigenic anachronisms" revealed the direct links between some of such viruses and the anthropogenic pollution of the biosphere by vaccine strains. Modifications in the HA genes of influenza A viruses during the persistent infection have also been studied.