[Sweet protein brazzein as a promising sweetener].

The excessive consumption of sugar-containing foods contributes to the development of a number of diseases, including obesity, diabetes mellitus, etc. As a substitute for sugar, people with diabetes mellitus and obesity most often use sweeteners. Sweet proteins, in particular brazzein, are an alternative to synthetic sweeteners that have natural origin, are broken down in the intestines along with food proteins, and do not affect blood sugar and insulin levels. The purpose of the review was to analyze the available data on the sweet protein brazzein, its physical and chemical properties, existing biotechnological methods of production, and prospects for application in the food industry in order to further develop an optimized heterologous expression system. Material and methods. Google Scholar, Scopus, Web of Science, PubMed, RSCI and eLibrary.ru databases were used for collecting and analyzing literature. Search depth - 30 years. Results. Numerous studies of the physical and chemical properties of brazzein have demonstrated its high potential for use in the food industry. In particular, a short amino acid sequence, thermal stability, the ability to maintain its structure and sweet properties in a wide pH range, hypoallergenicity, lack of genotoxicity, and an extremely high level of sweetness compared to sucrose allow us to conclude that its use is promising. Mutant variants of brazzein have been generated, the sweetest of which (with three amino acid substitutions H31R/E36D/E41A) exceeds sucrose sweetness by 22 500 times. To date, various systems for the expression of recombinant brazzein have already been developed, in which bacteria (Escherichia coli, Lactococcus lactis, Bacillus licheniformis), yeast (Komagataella phaffii, Kluyveromyces lactis, Saccharomyces cerevisiae), plants (Zea mays, Oryza sativa, Lactuca sativa, Nicotiana tabacum, Daucus carota) and animals (Mus musculus) have been used. Conclusion. Due to its high sweetness, organoleptic properties and long history of human consumption, brazzein can be considered as a promising natural sweetener. Despite the short peptide sequence, the production of the recombinant protein faced a number of problems, including low protein yield (for example, it could only be detected in mouse milk by Western blot hybridization) and loss of sweetness. Thus, further optimization of the process is necessary for widespread brazzein use in the food industry, which includes the selection of an adequate producer and the use of extracellular expression systems to reduce the final cost of the product.

Effect of M2 Macrophage-Derived Soluble Factors on Behavioral Patterns and Cytokine Production in Various Brain Structures in Depression-Like Mice.

We studied the effect of soluble factors derived from human macrophages polarized to M2 phenotype under conditions of serum deprivation (M2-SF) on behavioral pattern and cytokine production in various brain structures in mice with modeled stress-induced depression. Intranasal administration of M2-SF for 7 days led to stimulation of locomotor and exploratory activities and a decrease in emotional reactivity in the open-field test as well as reduction in depression-like behavior in Porsolt forced swimming test and a decrease in anxiety and anhedonia. Correction of depression-like behavior was accompanied by down-regulation of proinflammatory cytokines (IL-1ß, IL-6, TNFα, and IFNγ) in pathogenetically important brain structures (striatum, hippocampus, and frontal cortex). These data indicate that the antidepressant potential of M2 type macrophages can be mediated by the anti-inflammatory effects of M2-SF.

Cytokine Production by Splenic Cells in C57BL/6J Mice with Depression-Like Behavior Depends on the Duration of Social Stress.

We studied the influence of depression-like behavior developed in C57BL/6J mice under conditions of social stress of different duration on cytokine production by splenic cells. Imbalance of the pro- and anti-inflammatory cytokines was detected at the early stage of depression-like behavior (10-day experience of defeats): increased production of proinflammatory IL-2 and IL-6 cytokines along with a decrease in anti-inflammatory IL-10 level; the levels of IL-1ß, TNFα, IFNγ, and IL-4 remained unaffected. At later terms (20 days of confrontations), we revealed more pronounced changes in spontaneous production of proinflammatory cytokines that were not detected after shorter social stress. These findings suggest that cytokine profile depends on duration of social stress. Possible mechanisms of cytokine production during formation of depression-like state are discussed.

Changes in Production of Cytokines by C57Bl/6J Mouse Spleen during Aggression Provoked by Social Stress.

The effect of aggressive behavior shaped under social stress of various durations on the production of proinflammatory cytokines by splenic cells was examined on C57BL/6J mice. Aggressive mice were characterized by enhanced production of IL-2 and IFN-γ (released by T helper type 1 cells) and reduced secretion of TNF-α, whose major producers are monocytes and macrophages. Elevation of IL-2 and IFN-γ in aggressive mice resulted from enhancement of spontaneous and Con A-stimulated production, the most pronounced effect was demonstrated by the with a longer period (20 days) of victories. In contrast, spontaneous production of TNF-α was similar in control and aggressive mice, although LPS-stimulated production of this cytokine decreased after 10- and 20-day stress. The possible mechanisms of the changes in cytokine production are discussed.

[Sperm DNA fragmentation: association with semen parameters in young men].

AIM: Abnormal sperm DNA integrity is an important risk factor for male infertility. The aim of this work was to examine sperm DNA fragmentation in a cohort of young male volunteers (n=111; age 21.0+/-0.2 years) from the general population and establish the association between the level of sperm DNA fragmentation and sperm functional parameters. MATERIALS AND METHODS: Sperm DNA fragmentation index (DFI) was determined by SCSA (sperm chromatin structure assay) using flow cytometry. Standard semen parameters (concentration, motility, and morphology) were evaluated according to the WHO guidelines (2010). RESULTS: and conclusions. In the study cohort, 79.0%, 12.4% and 8.6% of men had normal (DFI<15%), borderline (15 less or equal DFI<27%) and high (DFI more or equal 27%) levels of fragmentation, respectively. Men with impaired spermatogenesis had greater IDF values (14.53+/-1.43%) than men with normal semen parameters (8.88+/-0.77%, p<0.05). There was a statistically significant negative correlation between IFD and ejaculate concentration (r=-0.21, p<0.05), fractions of mobile (r=-0.41, p<0.05) and morphologically normal sperm (r=- 0.34, p<0.05). Testing sperm DNA fragmentation using SCSA technique can be employed in epidemiological studies of male fertility.

[THE OPTIMAL CONDITIONS OF STORAGE OF SPERMATOZOA FOR ANALYSIS OF DNA FRAGMENTATION].

The analysis of fragmentation of DNA of spermatozoons using technique of flow cytometry to evaluate male fertility more and more often begins to be applied in clinical diagnostic. However, development of optimal protocol of storage and preparation of spermatozoons for analysis still is at the stage of experimental elaboration. The studv was carried out to analyse effect of different conditions of preparation of ejaculate for adequate evaluation of index of fragmentation of DNA of spermatozoons using sperm chromatin structure assay technique. The sampling consisted of 20 patients of the Krasnoyarsk center of reproductive medicine. The sperm chromatin structure assay technique was applied to evaluate index of fragmentation of DNA of spermatozoons in fresh native ejaculate and after storage of spermatozoons under different temperature (37, 25 and 4 degrees C) and duration (1-2 and 1-3 days) and conditions of storage (-20 or -70 degrees C) of frozen spermatozoons (as native ejaculate or in TNE-buffer). It is demonstrated that index of fragmentation of DNA of spermatozoons has no significant alterations in ejaculate stored under 4 degrees C during 48 hours. In case of storage of ejaculate under 25 or 37 degrees C index of fragmentation of DNA of spermatozoons significantly increases already after first day of storage. The incubation of ejaculate under 37 degrees C results in increasing of index of fragmentation of DNA of spermatozoons already after first hour. The individual differences are established related to degree of increasing of index of fragmentation of DNA of spermatozoons because of impact of studied temperatures of ejaculate incubation. The storage of spermatozoons under temperature of - 20 and -70 degrees C in native ejaculate or in TNE-buffer has no effect of index of fragmentation of DNA of spermatozoons with measurement during 1-2 hours. Therefore, storage and transportation of native ejaculate under 4 degrees C during 1-2 days or in frozen condition under temperature of -20 degrees C or -70 degrees C can be recommended for adequate evaluation of level of fragmentation of DNA of spermatozoons.

[Level of DNA fragmentation in human sperm cells in varicocele and prostatitis].

Varicocele and prostatitis are the most common andrological diseases, which may be accompanied by a decrease in the production of sperm cells, the deterioration of their quality and increased risk of infertility. This work was aimed to the evaluation of sperm DNA fragmentation index (DFI) and main indices of sperm fertility (concentration, motility and morphology), and the relationship between these parameters in the men of active reproductive age suffering from prostatitis or varicocele. Assessment of sperm DNA fragmentation was performed by SCSA (sperm chromatin structure assay) using flow cytometry; sperm parameters were evaluated according to WHO recommendations. It was shown that men with prostatitis (n = 9) and varicocele (n = 22) had significantly higher DFI compared with men in the control group (n = 22). Negative influence of these diseases on the concentration and the percentage of motile sperm cells in the ejaculate was revealed. These data suggest that the deterioration in the quality of semen in varicocele and prostatitis may be caused not only by pathospermia, but also, at least partially, by violation of the integrity of the sperm DNA. Evaluation of sperm DNA fragmentation can be recommended for use in laboratory diagnostics for prediction of fertility in infertile men.

[Combined low-dose antihypertensive therapy in pregnant women with hypertension and preeclampsia].

The effect of low-dose combination antihypertensive therapy in fixed form, containing 2.5 mg of bisoprolol and 6.25 mg of hydrochlorothiazide, the clinical condition, the daily profile of blood pressure, body adrenoreactivity structural and functional parameters of the left ventricle in 28 pregnant women with preeclampsia in the background chronic hypertension (hypertension stage II, 2 nd degree) and 28 pregnant women with preeclampsia and mild to moderate severity in terms of 22-28 weeks of pregnancy. After 16 weeks of receiving a fixed form, containing 2.5 mg of bisoprolol and 6.25 mg hydrochlorothiazide, the target BP level reached 76% of pregnant women with preeclampsia with chronic hypertension and 81% - with preeclampsia. In both groups, the parameters are optimized daily profile of blood pressure, the value of the total peripheral vascular resistance.

[Hypertensive disease and metabolic syndrome: application of metoprolol].

The aim of this work was to study effects of metoprolol on structural and functional characteristics of left ventricle (LV), cerebral circulation (CC), microcirculation (MC), lipid spectrum, rheologic and viscous properties of blood in patients with grade II-III hypertensive disease (HD) and metabolic syndrome. 65 patients in this condition (ESH/ESC, 2007) were given metoprolol (100-150 mg/d) and indapamide (1.5 mg/d) for 6 months. Daily AP profile, structural and functional characteristics of left ventricle were evaluated before and after therapy by echocardiography, cerebral dopplerography, conjunctival biomicroscopy, measurement of MC, hemorheologic parameters, blood lipid spectrum and glucose level. The target AP level and normalization of AP daily profile were achieved in 65% of the patients. The treatment improved myocardial contractility and diastolic function, decreased LV mass, corrected autoregulation of cerebral blood flow and MC. Metoprolol was shown to be metabolically inert. The study confirmed benefits of its application to the treatment of HD with metabolic syndrome.

[Spontaneous aneuploidy level in blood cells of fertile females].

In this study we present the results of molecular-cytogenetic investigation of blood cells of fertile women of reproductive age. We used cultivated and uncultivated cells and two sets of FISH probes with direct and indirect labeling. The mean aneuploidy levels of 4 chromosomes were determined and statistical limits were scored. Aneuploidy levels determined in cultivated lymphocytes by multicolor FISH varied from 0.1 to 1.3%. The mean aneuploidy level for all chromosomes (13, 18, 21 and X) was 1.39%. The limit mutation detection was 3.4%. We found differences in results obtained with the use of direct and indirect labeling. It was shown that cultivating process influenced the aneuploidy level. Absolute error of FISH technique was 0.13%, relative error - 4.08%.

Effect of transplantation of splenic lymphoid cells on functional activity of the immune and nervous system in experimental animals.

We studied the effect of transplantation of splenic lymphoid cells on functional activity of the nervous (orientation and exploratory behavior and expression of genes for interleukin-1beta, type 1 interleukin-1 receptor, and erythropoietin in the brain) and immune system (cellular and humoral immune response, proliferative activity of immunocompetent cells, and expression of cytokine genes in splenocytes) in syngeneic animals with different behavioral characteristics. Intravenous injection of the lymphoid fraction of splenocytes from donor mice with a certain behavioral pattern in the open-field test had a modulatory effect on vertical locomotor activity of syngeneic recipient mice. The increase or decrease in vertical locomotor activity due to transplantation of immunocompetent cells was accompanied by specific changes in mRNA level for erythropoietin receptor and type 1 interleukin-1 receptor in brain cells of recipient mice. The regulation of orientation and exploratory behavior was also accompanied by changes in functional activity of the immune system in recipient animals. It was manifested in modulation of proliferative activity of thymic and splenic cells and cytokine gene expression in splenocytes.

Intersphincteric resection with partial removal of external anal sphincter for low rectal cancer.

Abdominoperineal resection (APR) remains the standard procedure for rectal cancer located within 0.5 cm from dentate line (DL). In this study, we present a new type of restorative surgery: intersphincteric resection with partial removal of external anal sphincter (EAS) and anorectal reconstruction for-ultra low rectal cancer. Between March 2003 and May 2008 fifty patients (28 males, aged between 39 and 71) were operated on for ultra low rectal cancer uT2-3N0M0 with partial preservation of EAS and total anorectal reconstruction (smooth-muscle neosphincter and colonic pouch). A protective stoma was performed in all cases. Functional outcome and quality of life were recorded at 3, 6, 12, 18, 24 months after stoma closure using Wexner score and FIQL respectively. Anal manometry, vectrum volumetry and myography data were taken as well. Results. Postoperative complications developed in 2 patients, but no secondary surgery was required. Carcinomas were staged as pT2 (n = 14) and pT3 (n = 36). The distal clearance was 2.00.4 (range 1.5-2.8) cm, lateral clearance was 0.80.3 (range 0.2-1.4) cm. After a median follow-up of 24 (range 2-61) months, 2 local recurrences were occurred and salvaged by APR. Contractive activity of saved elements of EAS improved with a course of time and squeezing anal pressure increased as well. Perfect functional outcome was achieved in 25 of 34 patients at 12 months after stoma closure, and all the patients were satisfied with procedure. Good functional results of suggested surgery seems to be an acceptable alternative to APR with permanent stoma in selected patients.

[Proctectomy with external sphincter elements' preservation in surgery of low-ampullary rectal cancer].

Operational technique for the rectum tumors, localized at the level of dentate line, developed in the State center of coloproctology, allows preservation of external anal sphincter elements and thus, partial preservation of defecation control. Technique is described and proved, preliminary treatment results of 42 patients are analysed. The operation is oncologically effective (local recurrence rate 4,8%). Preservation of external sphincter elements has a high functional value in complex with "neorectum" and "neosphincter". A majority of operated patients can keep the stool, 80% of able-bodied patients continued their professional activities.

[Comparative analysis of molecular genetic studies using DNA samples isolated from blood treated with EDTA or heparin].

A molecular genetic investigation of five genes: CFTR, GSTM1, GCTT1, MTHFR, and TP53 using DNA samples isolated from blood treated with either EDTA or stabilized heparin was conducted. Twenty DNA samples obtained from 10 persons. The results prove that heparin used as an anticoagulant instead of EDTA had no inhibitory effect on PCR and PCR/RFLP results, at least when DNA was isolated with a DNA-sorb-B kit. The results suggest that heparin-treated blood samples can be also used to study other genes.

[Lymphocyte metabolism in patients with acute pancreatitis with different genotypes of GSTM1 and GSTT1 genes].

In this study, we have investigated correlation between enzymatic activity of NAD(P)-dependent dehydrogenases of lymphocytes and polymorphic variants of glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genes in the group of unrelated patients with acute pancreatitis in comparison with healthy Russians from Krasnoyarsk. Thus, genotype GSTM1 0/0 is the marker of predisposition to the acute pancreatitis, wheras polymorphism of the GSTT1 gene is not involved in the development of the pancreatitis, at least in our group. The bioluminescence analysis showed statistically significant decrease of the levels of G3PD, NAD(+)MDH and the increase of NADH(+)LDH, NAD(+)GDH, NADH(+)GDH in lymphocytes of pancreatic group. Development of pancreatitis in patients with different genotypes GSTM1 and GSTT1 genes showed the rearrangement of the basic intracellular processes: dominance of a plastic metabolism in the patients with GSTM1--deletions and predominance of energetic processes at GSTT1 0 - pancreatitis.

Effect of transplantation of immunocompetent cell on orientation and exploratory behavior and cytokine gene expression in the brain of experimental animals.

Intravenous injection of adherent splenocyte fraction from donor (CBAxC57Bl/6)F(1) mice characterized by specific open-field behavior modified this behavior in syngeneic recipient mice. This was paralleled by appropriate changes in the levels of IL-1b and type 1 IL-1 receptor mRNA in the brain cells of recipient mice. Hence, we demonstrated the possibility of directed regulation of orientation and exploratory behavior in mice by transplantation of immunocompetent cells. Mononuclear phagocytes play an important role in this phenomenon.

Stimulation of cell component of the immune response activates exploratory behavior in senescence accelerated OXYS rats.

Suppression of the cell component of the immune system and open-field behavior developing in OXYS rats by the age of 3 months are regarded as manifestations of accelerated aging. Stimulation of cell-mediated immune response with BCG vaccine caused a dose-dependent increase of orientation and exploratory activity of OXYS rats in the open field test to virtually the same level as in Wistar rats.

Immunomorphological characteristics of animals with different levels of orientation and exploratory behavior.

We revealed some relationships between the level of orientation and exploratory behavior, functional activity of the immune system, and structural and functional organization of the CNS in animals. Significant differences in brain morphology and expression of cytokine IL-1 beta, type I IL-1 receptor, and erythropoietin receptor genes in brain cells were detected in (CBA x C57Bl/6)F1 mice with different initial levels of orientation and exploratory behavior. Immunocompetent cells of mice with high and low levels of exploratory behavior differ by spontaneous and mitogen-induced proliferative activity. The formation of humoral and cellular immune response in these animals causes opposite changes in exploratory behavior and the type of these changes depends on the initial level of this behavioral reaction.

Activity of cell immune response and open field behavior in Wistar and OXYS rats.

The state of the thymus, activity of cell-mediated immunity, and open field behavior were compared in Wistar and OXYS rats (premature aging). Early involution of the thymus was revealed in OXYS rats, which by the type of morphological changes corresponded to accidental involution and was associated with low level of delayed hypersensitivity reaction and decreased motor and exploratory behavior.