BEHAVIORAL AND NEUROCHEMICAL CHANGES DURING INTRANASAL ADMINISTRATION OF ALPHA-GLUTAMYL-TRYPTOPHAN AND CHELATE COMPLEX OF ZINC ARGINYL-GLYCINATE ON MONOAMINE SYSTEMS DYSFUNCTIONS KNOCK-OUT MODELS.

Monoamine neurotransmitter system dysfunctions lead to behavioral disorders, cognitive metabolic, and other pathological conditions. In this case, different amino acids are precursors of monoamines, while the parenteral path of monoamine administration has pharmacological restrictions. Therefore, intranasal administration one of the most promising methods of delivering an active substance is. The purpose of the work is to study the effect of intranasal administration of a chelate complex of zinc arginyl-glycinate and alpha-glutamyl-tryptophan dipeptide on behavioral and neurochemical changes in acute and chronic experiments. MATERIAL AND METHODS: The studies used outbred Wistar and DAT-KO rats, and inbred C57Bl6 and TAAR1-KO mice. Using intranasal administration of a chelate complex of zinc arginyl-glycinate and alpha-glutamyl-tryptophan dipeptide we tested methods for evaluating different behavioral indicators and the level of cerebral monoamines and their metabolites. RESULTS: An anxiolytic effect of zinc arginyl-glycinate and its combination with alpha-glutamyl-tryptophan was revealed. Both drugs have a physiological effect on the autonomic nervous system, but the determination of their operating mechanisms requires further research. CONCLUSION: Thus, these data indicate that intranasal delivery of the dipeptides is effective during acute and chronic intranasal administration in rodents, the latter showed a change in the anxiety indicator. Acute AG intranasal administration demonstrated signs of lower anxiety and depressive-like behavior in C57Bl6 mice. The acute intranasal administration of a chelate complex zinc arginyl-glycinate and combination with alpha-glutamyl-tryptophan in doses of 50-100 mg/kg of body weight may be used for pre-clinical studies as a new anxiolytic/antidepressant.

Minimal Age-Related Alterations in Behavioral and Hematological Parameters in Trace Amine-Associated Receptor 1 (TAAR1) Knockout Mice.

Since the discovery in 2001, the G protein-coupled trace amine-associated receptor 1 (TAAR1) has become an important focus of research targeted on evaluation of its role in the central nervous system (CNS). Meanwhile, impact of TAAR1 in the peripheral organs is less investigated. Expression of TAAR1 was demonstrated in different peripheral tissues: pancreatic ß-cells, stomach, intestines, white blood cells (WBC), and thyroid. However, the role of TAAR1 in regulation of hematological parameters has not been investigated yet. In this study, we performed analysis of anxiety-related behaviors, a complete blood count (CBC), erythrocyte fragility, as well as FT3/FT4 thyroid hormones levels in adult and middle-aged TAAR1 knockout mice. Complete blood count analysis was performed on a Siemens Advia 2120i hematology analyzer and included more than 35 measured and calculated parameters. Erythrocyte fragility test evaluated spherocytosis pathologies of red blood cells (RBC). No significant alterations in essentially all these parameters were found in mice without TAAR1. However, comparative aging analysis has revealed a decreased neutrophils level in the middle-aged TAAR1 knockout mouse group. Minimal alterations in these parameters observed in TAAR1 knockout mice suggest that future TAAR1-based therapies should exert little hematological effect and thus will likely have a good safety profile.

[Pancreatic duct stenting in the treatment of acute pancreatitis]. / Vliyanie stentirovaniya glavnogo pankreaticheskogo protoka na rezul'taty lecheniya bol'nykh s ostrym pankreatitom.

OBJECTIVE: To study the effect of pancreatic duct stenting on the results of treatment of acute pancreatitis. MATERIAL AND METHODS: Analysis included 99 patients with acute pancreatitis. Endoscopic stentingwas appliedin the study group, conventional approach - in the control group. RESULTS: Pancreatic duct stenting at theearly stages ofacute pancreatitis results decreased level of enzyme toxemia and manifestations of systemic inflammatory response. CONCLUSION: Stenting interrupts course of pathological process and minimizes the possibility of transition of aseptic to infected pancreatic necrosis. Therefore, reduced early mortalityis observed.

[Clinical evaluation of endoscopic hemostasis in patients with ulcerative gastroduodenal bleeding]. / Klinicheskaia otsenka éndoskopicheskii gemostaz v lechenii bol'nykh s gastroduodenal'nym krovotecheniem iazvennoi étiologii.

AIM: To compare the effectiveness of various methods of endoscopic hemostasis in patients with ulcerative gastroduodenal bleeding. MATERIAL AND METHODS: Several surgical techniques in treatment of 117 patients with ulcerative gastroduodenal bleeding were anayzed. Isolated and combined methods of endoscopic hemostasis were used in control and study groups. RESULTS: Combined endoscopic hemostasis is bettercompared with isolated techniques. It reduces the incidence of recurrence, surgical activity and mortality. CONCLUSION: The effectiveness of combined endoscopic hemostasis was 88.2% for primary hemostasis in acute ulcerative gastroduodenal bleeding. Herewith, surgical activity and mortality were decreased.

[ASF virus replication features in the presence of recombinant proteins CD2v, pX69R and pE248R.]

INTRODUCTION: African swine fever (ASF), sever hemorrhagic disease of swine caused by a large DNA virus of the Asfaviridae family. Since there are no effective and safe vaccines against ASF yet, it is urgent to study the functions of its proteins, which is applicable by analyzing the features of ASF virus replication in the presence of recombinant proteins in vitro. PURPOSE: To study the effect of ASFV recombinant proteins CD2v, pE248R and pX69R on the speed and level of reproduction of ASF virus in vitro. Thus, obtain the necessary knowledge to develop approaches for creating a vaccine against ASF. MATERIALS AND METHODS: ASFV isolate Krasnodar 07/17 and strain ASF/ARRIAH/CV-1 were used. Cloning of X69R, EP402R, and E248R genes was performed in the pJET1.2 / blunt vector and pCI-neo in E. coli JM-109 cells, according to the manufacturer's manual. Localization of recombinant proteins in CV-1 cell line carried out by direct immunofluorescence reaction (DIF) using polyclonal antibodies conjugated to FITC. The ASF virus reproduction level was assessed by hemadsorption reaction and qPCR kit (Central Research Institute of Epidemiology). RESULTS: Recombinant plasmids pCI-neo / E248R, pCI-neo / EP402R and pCI-neo / X69R were constructed. The localization and the specificity of the obtained recombinant proteins CD2v, pE248R and pX69R was confirmed. It was established that these recombinant proteins induce the level of ASF virus reproduction on days 3-5 of the experiment by ~ 1.2-1.5 lgHADU50/cm3 in comparison with the negative control. DISCUSSION: The data obtained demonstrate the important role of CD2v, pX69R and pE248R proteins in the reproduction of the virus, since they significantly affect its level. The exact function of pX69R protein was not determined, however, in the experiments its positive effect on ASF virus reproduction was established, manifested in an increase in its reproduction level. CONCLUSION: This methodology allows us to study the nature of the effect of proteins with unknown function on ASF virus replication.

Revised stability constant, spectroscopic properties and binding mode of Zn(II) to FluoZin-3, the most common zinc probe in life sciences.

2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-4-(2,7-difluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3) is used very broadly in life sciences as intra- and extracellular Zn(II) sensor selective for Zn(II) over Co(II), Ca(II) and Mg(II) ions at their physiological concentrations. It has been used for determination of relative and absolute levels of exchangeable Zn(II) in cells and extracellular fluids. Despite its popularity, the knowledge of its acid/base and Zn(II) coordination abilities and of its spectroscopic properties remained very limited. Also the published conditional dissociation constant ((C)Kd) values at pH7.4 are slightly discrepant, (15nM or 8.9nM). In this work we determined the (C)Kd for Zn(II) complexation by FluoZin-3 at pH7.4 with nitrilotriacetic acid (NTA) as competitor using two independent methods: fluorimetry and UV-Vis spectroscopy. For the first time, we investigated FluoZin-3 alone and complexed with Zn(II) in the wide range of pH, determining the total of eight pKa values from fluorescence spectra and from various regions of UV-Vis spectra. The validated values of (C)Kd (9.1±0.4nM; -log (C)Kd=8.04) and of the absolute (pH-independent) stability constant log ßZnL (8.16±0.05) were provided by fluorescence spectroscopy experiments performed at 1µM concentrations. Our experiments demonstrated that both of aminocarboxylate moieties of FluoZin-3 bind the Zn(II) ion synergistically.

Detection of African Swine Fever Antibodies in Experimental and Field Samples from the Russian Federation: Implications for Control.

African swine fever (ASF) re-entered in Europe in 2007 by Georgia rapidly affecting neighbouring countries. Since then, ASF has caused severe problems to the Russian Federation (RF) and spread to Northern and Western regions, including Ukraine (2012 and 2014) and Belarus (2013). At the beginning of 2014, dead wild boars were found in Lithuania and Poland. Several outbreaks have been later notified in the European Union(EU), affecting domestic pigs and wild boar of Latvia, Lithuania and Poland, and also wild boar in Estonia, causing major problems for the EU pig sector. Some studies have been performed with this ASFV isolate, revealing that it belongs to genotype II and causes an acute form of the disease. However, few data are available about the presence of antibodies in field and experimental samples from the affected area. This study analysed samples from experimental infections with ASFV isolated from the RF in 2013 (74 sera and 3 tissue exudates), and field samples from the RF from 2013 to 2014 (266 samples, including 32 and 7 tissue exudates from domestic pigs and wild boar, respectively). All samples were tested by a commercial ELISA and, some of them (79), also by immunochromatographic tests. Positive and doubtful samples were confirmed by immunoblotting test. Positive results were found in experimental and field samples, which confirm the presence of antibodies against ASFV in the RF. Antibodies were detected in animals inoculated with three different ASFV isolates, with some differences found among them. Only a small percentage of field samples was positive for ASF antibodies (3.7%), in agreement with other observations that reported a high virulence for the ASFV isolates in the area. These results confirm the potential presence of survivor animals that should be considered in affected areas to help design effective control and eradication plans against ASF.

Conservative mutation Met8 --> Leu affects the folding process and structural stability of squash trypsin inhibitor CMTI-I.

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process.

Factors improving the accuracy of determination of 15N relaxation parameters in proteins.

A number of factors at all stages of data processing which affect the accuracy of determination of 15N relaxation parameters in 15N-labeled proteins is discussed. Methods which allow to improve accuracy of the determined parameters are presented using data obtained for Cucurbita maxima trypsin inhibitor.

NMR measurements of proton exchange between solvent and peptides and proteins.

Scope and limitations of the NMR based methods, equilibration and magnetization transfer, for measuring proton exchange rates of amide protons in peptides and proteins with water protons are discussed. Equilibration is applied to very slow processes detected by hydrogen-deuterium exchange after a solute is dissolved in D2O. Magnetization transfer allows to study moderately rapid processes in H2O. A number of precautions should be undertaken in order to avoid systemic errors inherent in the magnetization transfer method.

Spectroscopic detection of photoproducts in lecithin model system after 8-methoxypsoralen plus UV-A treatment.

Photoreactions of 8-methoxypsoralen (8-MOP) in the presence of synthetic lecithins esterified at the beta-position with linoleic/oleic and gamma-palmitic acid (PCd2/d1pal) have been studied. Following UV-A (320-400 nm) irradiation, the photoproducts separated by thin-layer chromatography are analysed by UV absorption spectroscopy, nuclear magnetic resonance and mass spectroscopy. The new isolated products are lecithin double cyclobutane adducts, PC-(8-MOP)2, fatty acid-8-MOP split adducts from phosphatidylcholine and lecithin adducts with photo-oxidized 8-MOP. The photolysis performed in the presence of 8-MOP is related to the previously reported lecithin cyclobutane adducts with psoralen. A hypothetical scheme of lecithin photolysis under PUVA (psoralen + UV-A) treatment is proposed. We suggest that photolysis of lecithin may have a significant role in the chain of reactions triggered in cell membrane submitted to PUVA treatment.

[Quantitative analysis of morphogenesis of local nerve plexuses forming in tissue culture]. / Kolicherstvennyi analiz morfogeneza lokal'nykh nervnykh spletenii, formiruiushchikhsia v kul'ture tkanei.

Discrete stages of formation of nervous plexuses in the culture of isolated neurons of a mollusc were analyzed autonomically. Area of plexus expansion (S1) and area, occupied by nerve structures (neurites and neuron bodies) were determined. It was demonstrated that despite intensive radial growth of peripheral neurites and S1 growth the density of plexus (S2/S1) was not changed. Relative acceleration of plexus expansion (as compared to S2 growth) registered on 40th and 50th hrs of cultivation indicates the absence of necessary cells in the plexus and development of contacts with adjacent local plexuses. Scanning of lamellar structures revealed submembranous aggregation of cytoskeleton apart from longitudinally directed cytoskeletal bands, which prevents one of the ways of neurite ramification. Elimination of lamellae between cytoskeletal bundles leads to formation of several neurites.

[Innervation of the humerus metaepiphysis in newborn and one year old children]. / Innervatsiia metaépifiza plechevoi kosti novorozhdennykh i detei pervogo goda zhizni.

Analysis of home and foreign literature on metaepiphyseal cartilage innervation and blood supply was presented, data on these organ diseases substantiate theoretical and clinical significance of the study was performed. Using macro-microscopic preparation, histological study, automatic image analyzer branches of humerus proximal metaepiphysis were shown to originate from axillary and subscapulary nerves. Nerves and vessels penetrating in the cartilaginous canals form neurovascular complexes. Cartilaginous canals are located unevenly. Nervous structures were found in the canals and their characteristics was given.