The structural analogue of apelin-12 prevents energy disorders in the heart in experimental type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is the most severe form of diabetes, which is characterized by absolute insulin deficiency induced by the destruction of pancreatic beta cells. The aim of this study was to evaluate the effect of a structural analogue of apelin-12 ((NαMe)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH, metilin) on hyperglycemia, mitochondrial (MCh) respiration in permeabilized cardiac left ventricular (LV) fibers, the myocardial energy state, and cardiomyocyte membranes damage in a model of streptozotocin (STZ) diabetes in rats. Metilin was prepared by solid-phase synthesis using the Fmoc strategy and purified using HPLC. Four groups of animals were used: initial state (IS); control (C), diabetic control (D) and diabetic animals additionally treated with metilin (DM). The following parameters have been studied: blood glucose, MCh respiration in LV fibers, the content of cardiac ATP, ADP, AMP, phosphocreatine (PCr) and creatine (Cr), the activity of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in blood plasma. Administration of metilin to STZ-treated rats decreased blood glucose, increased state 3 oxygen consumption, the respiratory control ratio in MCh of permeabilized LV fibers, and increased the functional coupling of mitochondrial CK (mt-CK) to oxidative phosphorylation compared with these parameters in group D. In STZ-treated animals metilin administration caused an increase in the PCr content and prevention of the loss of total creatine (ΣCr=PCr+Cr) in the diabetic hearts, as well as restoration of the PCr/ATP ratio in the myocardium and a decrease in the activity of CK-MB and LDH in plasma to initial values. Thus, metilin prevented energy disorders disturbances in cardiomyocytes of animals with experimental T1DM.

[The anti-ischemic and antioxidant activity of the pharmacological agonist of galanin receptor GalR2 and carnosine in in vitro and in vivo model systems]. / Protivoishemicheskaia i antioksidantnaia aktivnost' farmakologicheskogo agonista retseptora galanina GalR2 i karnozina v model'nykh sistemakh in vitro i in vivo.

Antioxidant and anti-ischemic properties of the pharmacological agonist of galanin receptor GalR2 WTLNSAGYLLGPßAH (Gal) and its C-terminal fragment, dipeptide carnosine (ßAH), were studied in the model of regional ischemia and reperfusion of the rat heart in vivo in the dose range of 0.5-5.0 mg/kg and Cu²âº-induced free radical oxidation of low density lipoproteins (LDL) of human plasma in vitro for peptide concentrations of 0.01 mM and 0.1 mM. Gal was obtained by automatic solid phase synthesis using the Fmoc methodology; its structure was characterized by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry. Intravenous administration of the optimal dose of Gal (1 mg/kg) to rats after ischemia was more effective than carnosine in reducing of the myocardial infarct size and the activity of creatine kinase-MB and lactate dehydrogenase in blood plasma at the end of reperfusion. It also improved the metabolic state of the reperfused myocardium and reduced the formation of peroxidation products during reperfusion. Gal reduced more effectively the formation of adducts of hydroxyl radicals in the interstitium of the area at risk (AAR) of the rat heart than carnosine. Carnosine at a dose of 1 mg/kg more effectively increased the activity of catalase and glutathione peroxidase in the AAR by the end of reperfusion compared to Gal. In a model of Cu²âº-initiated oxidation of human plasma LDL 0.1 mM carnosine demonstrated a significantly more pronounced reduction in the formation of lipid radicals compared to Gal. The results show that Gal can be considered as a promising agent that reduces myocardial injury during reperfusion and oxidative stress.

[Analysis of Urine Proteome in Patients With Postinfarction Cardiosclerosis Combined With Hypertensive Disease for Assessing Endothelial Dysfunction].

In our study urine protein composition of 18 healthy volunteers was compared with that of 18 patients with ischemic heart disease and concomitant hypertension. Liquid chromatography-mass-spectrometry (LC-MS) analysis of the second fraction of morning urine was carried out using nano-line high performance liquid chromatograph and hybrid mass spectrometer. The analysis revealed 23 proteins expressed in the endothelium, according to the information contained in the database Bgee, and 49 proteins, with direct functional link with the processes in the endothelium in the reconstruction of associative networks using ANDSystem program. Comparison of urine proteome of healthy people and patients with postinfarction cardiosclerosis revealed proteins specific for patients with cardiovascular disease. Thus, proteins vitronectin, syndecan-4, a histidine rich glycoprotein, endothelial protein C receptor, colony stimulating factor, cathepsin D and sekretogranin-1 may be considered as potential markers for cardiovascular diseases. Further research in this area should be conducted for clinical and experimental verification of these hypotheses.

PROTEOMIC ANALYSIS OF URINE UNDER CONTROLLED SALT INTAKE IN PROJECT <>.

Specifics of urine proteome is sensitive to a multitude of factors. One is nutrition or entrance in organism of main nutrients including salt (NaCI). Purpose of the investigation was to study the proteomic composition of healthy human urine in the controlled environment of a 105-day isolation experiment (project <>) with various levels of salt intake. Analysis was performed using the present-day proteomics techniques based on chromatography-mass spectrometry and bio-informatics options. An attempt was made to correlate changes in processes and physiological systems with the controlled salt intake. As a result, a list of proteins directly responsible for different salt intake during the experiment and then a list of tissues where these proteins express predominantly were compiled; besides, analysis of the processes these proteins are involved in was performed.

[SHIFTS IN URINE PROTEIN PROFILE DURING DRY IMMERSION].

The study was aimed at tracking the proteomic profile of urine in 8 normal volunteers to 5-day dry immersion (DI). The proteome composition was determined by chromatography-mass spectrometry on high-efficient on-line liquid nano chromatograph Agilent 1100; complementary information about the protein spectra was obtained by dint of mass-spectrometer MaXis Impact 4G and hybrid mass-spectrometer LTQ-FT. Functional associations between proteins and biological functions were analyzed using computer system ANDCell (Associative Networks Discovery in Cells). A total of 256 proteins were identified; for 43 proteins difference in the detection rate during the baseline data collection and on DI day 4 exceeded 20%.

[Identification of biological processes on the composition of the urine proteome in cosmonauts on the first day after long space flights].

There are several difficulties that impede medical support of space missions. We can assume that the long-term space missions will be accompanied by functional changes in the organism. These changes will be a natural reaction to the factors of space flight. Complex action of factors can lead to the development of both non-specific changes (general adaptation syndrome), and specific changes. We analyzed the physiological changes after long space flights and carried out the correlation of these changes with previously identified proteins in the urine. It is possible to determine the mechanisms of adaptation of the human organism to the conditions of life on Earth after a long stay in Earth orbit.

[MODIFICATION OF URINE PROTEOME IN HEALTHY HUMAN DURING 21-DAY BED REST].

Mass spectromy-based proteomics was employed to analyze urine from 8 normal volunteers for a 21-day bedrest study (BR). The analysis included trypsinolysis in solution prior to liquid chromatography and tandem mass spectrometry (LC-MS/MS), and spectra processing using the bioinformatics tools. Relying on 221 IPI-indices with Score from 24 to 1700, 169 different proteins were identified. Molecular functions, biological processes and cell components as the loci of certain protein functioning were determined with the help of UniProt-GOA. Associative interactions networks were constructed using BiNGO. There were 14 proteins identified that are functional in the cardiovascular system mostly. They were annotated and -dynamics of their occurrence throughout the experiment was considered. Grounding on the biological functions of these proteins, an assumption of eligible activation of different biological processes during BR was made.

[Urine proteome study for the evaluation of age dynamics in healthy men].

We investigated the age dynamics of proteomic profile of urine in 52 healthy men aged 18 to 51 years. A special sample preparation was performed, followed by liquid chromatography-mass spectrometry. Liquid chromatography-mass spectrometry of the minor proteins was performed on a nano-HPLC Agilent 1100 system («Agilent Technologies Inc.¼, USA) in combination with a LTQ-FT Ultra mass spectrometer («Thermo Electron¼, Germany). A total of 259 proteins were identified. According to the TiGER database, a tissue origin was established for 141 proteins and identified 715 processes in which they participate. We found a significant positive correlation with age, the number of proteins (R=0,566; p-value=1,24E-05) and the weight of proteins (R=0,45; p value=8,17E-04). Identified 23 proteins were significantly more frequent in the urine of subjects with increasing age (p<0,05), and only one protein - RGSL, Regulator of G protein signaling protein-like (MW 125.69) - less frequently.

[Permanent proteins in healthy human's urine in the experiment with 520-day isolation].

Purpose of the study was to track permanent proteins of urine proteome in the 520-day isolation experiment at the IBMP Ground-Based Test Facility with controlled environmental parameters. Object of the investigation was urine sampled from 6 normal male subjects at the age of 25 to 37 years. Second morning aliquots were gathered during baseline data collection, on days 50, 93, 124, 153, 180, 251, 274, 303, 330, 371, 400 and 427 of isolation, and in 7 days after its completion. Samples were subject to chromatography-mass spectrometry; results were analyzed with the help of bioinformatics resources. The following 7 permanent proteins were observed in urine over the entire length of the investigation: epidermal growth factor, polymer immunoglobulin receptor, plasma serine protease inhibitor, protein AMBP, keratin, type II cytoskeletal 1, collagen alpha-1 (vi) chain, serum albumin.

[Identification proteins cardiovascular system in urine healthy subjects during "dry" immersion].

We analyzed the urine proteome in 14 healthy volunteers who underwent a 5-day dry immersion using proteomic analysis methods and bioinformatics approach. We identified 9 proteins related to cardiovascular system. It was shown that 5-day dry immersion modifies urine proteomic profile indicating renal, endocrine, circulatory and metabolic changes. Most of these changes are characterized by both a very rapid onset and very rapid restoration within return to normal conditions.

Changes of protein profile of human urine after long-term orbital flights.

We analyzed protein profile of urine samples obtained from 7 Russian cosmonauts (age 35-51 years), participants of space flights on the International Space Station lasting for 169-199 days. Gradient chromatography with linear increase of eluent proportion was carried out in a system consisting of an Agilent 1100 chromatograph (Agilent Technologies Inc.) and a hybrid mass-spectrometer LTQ-FT Ultra (Thermo). The obtained results help to understand changes in the human body induced by space flight factors.

[Urine proteome study for the evaluation of cardiovascular system state after spaceflight in human].

In order to find markers to assess the functional state of the cardiovascular system before and after spaceflight (first and seventh day after landing), we analyzed the urine proteome in ten cosmonauts aged of 35 to 51 years who have completed 169 to 199-day spaceflight onboard the ISS. A special sample preparation was performed, followed by liquid chromatography-mass spectrometry. Liquid chromatography-mass spectrometry of the minor proteins was performed on a nano-HPLC Agilent 1100 system (Agilent Technologies Inc., USA) in combination with a LTQ-FT Ultra mass spectrometer (Thermo, Germany). A total of 238 proteins was identified. According to the TIGER database, a tissue origin was established for 129 proteins. We identified 20 proteins related to the cardiovascular system. It was found that changes in cosmonauts' urine proteome comprehensively reflect the adaptive responses of cardiovascular, renal and neuroendocrine systems to long-term microgravity conditions.

[Detection of renal and urinary tract proteins in the urine before and after space flight].

The urine protein composition (proteome) of healthy human was analyzed using proteomic techniques to obtain data in physiological condition and after six months space flights. It was shown that after long duration space flights in cosmonaut's urine reveals specific minor proteins which can be identified as proteins came from kidney and urinary tract.

[Reconstruction of associative protein networks connected with processes of sodium exchange' regulation and sodium deposition in healthy volunteers by urine proteome analysis].

The study was conducted during the experiment with 105-day isolation in experimental complex. In the present investigation we collected urine samples from 6 healthy volunteers. The physical activity, diurnal rhythm, temperature parameters and level of oxygen and carbon dioxide were controlled during the experiment. According to the program, food intake (electrolytes, water, calories, fat, carbohydrates, protein, vitamins, etc.) on each stage of experiment was normalized. All samples were analyzed using mass spectrometer of an ionic cyclotron resonance with transformation of Fure LTQ FT MS (Thermo) on the basis of the AMT-tags (accurate mass and time tags) approach. Among more than 20 000 we found out 690 proteotypical proteins and we identified about 600 urine proteins. For physiological interpretation of the proteome data we used computer systems ANDCell and ANDVisio. Clustering of proteins and application of these systems revealed proteins that are most closely associated with the regime of sodium intake, as well as build the network of their interactions.

[Variability of urine proteome of healthy persons during 105-daily isolation].

Human proteome is very plastic, it changes under the influence of various biological factors. It is of big interest to find out how specific factors of an environment, including a long-term isolation affect on urine proteome. The study was conducted during the experiment with 105-day isolation. In the present investigation we collected urine samples from 6 healthy volunteers (26-41 years old). The physical activity, daily rhythms and diet were controlled. Urine samples were fractionated on magnetic beads MB-HIC C8 (MB - hydrophobic-interaction chromatography) with ClinProt robot (Bruker Daltonics) prior to MALDI-TOF mass spectrometer analysis with Autoflex III TOF/TOF (Bruker Daltonics), working in a positive linear mode. 117 peaks have been obtained in each spectrum of urine. We have shown that even during isolation and under controlled conditions of life a high variability of urine proteome of healthy personas (36 protein MC-peaks in the urine, on average) are revealed.

[Direct proteome profiling of human blood serum in the experiment with 5-day dry immersion].

Purpose of the investigation was to determine changes in blood plasma proteome in healthy human subjects (n = 14, 19 to 26 y.o.) in an experiment with dry immersion (DI). Plasma samples were drawn 7 and 2 days before the exposure, on DI days 2, 3 and 5, and on days 1, 3, 7 and 15 after the experiment. Previous to direct MALDI-TOF mass-spectrometric profiling, serum samples were pre-fractionated and enriched with magnetic particles MB WCX (WCX--a weak cation exchanger) on ClinProt (Bruker Daltonics). In each spectrum, 175 MS-peaks were detected on average within the mass range from 1000 to 17,000 Da with the signal/noise ratio = 5. Student's criterion (p < 0.05) was used to define reliable differences between DI and baseline samples from 48 peaks (27.4 % of all the proteome profile peaks). On DI days 2 and 3, growth of peak areas was observed in fragments of complement system proteins C3 and C4, high-molecular kininogen and fibrinogen that can be attributed to organism adaptation to conditions of the experiment. Significant increases of the peak area of apolipoprotein CI (reduced form with segregated threonine and proline) and C4 enzymes of the complement system, and fibrinogen on the first day after the experiment can be related to changes in motor activities of the subjects.

[Renal functioning and human body liquids during 5-day dry immersion].

Renal function and body composition, including liquids, were investigated in 14 essentially healthy male subjects during simulation of some spaceflight effects by 5-d dry immersion (DI). Noninvasive measurement of water spaces of organism was performed with the methods of bio-impedance analysis (BIA). Increase of renal excretion of liquids and appearance of negative water balance were observed. BIA revealed reductions of total body and extracellular liquids and a decrease of circulating plasma volume. This means, that DI induced hypohydration of organism. In the post-DI period, the hydration status regained its baseline level fairly soon. Lean body mass slightly decreased; adipose mass, on the contrary, exceeded baseline values. Moreover, the experiment evidenced technical impossibility to obtain valid impedansimetry data in DI.

[Investigation of urine proteome in normal humans].

Recent advances in genomics and informatics have given birth to such area in molecular biology as proteomics. Proteomics is currently tasked with the full proteoma mapping of various biological substrates (e.g. body liquids, cells and tissues) both in the norm and pathology, and discovery of biomarkers of pathologies including tumors. The review presents analysis of the urine proteoma literature over the last five years covering the methods, strategy and results of the investigations.

Analysis of antidiuretic effect of arginine-vasotocin and its analogs in primates.

Intramuscular injection of synthetic analogs of arginine-vasotocin to monkeys after 3% water loading caused antidiuretic reaction, manifesting by reabsorption of osmotically free water and paralleled by significant changes in excretion of sodium or potassium ions. It is shown that synthetic analogs can be more effective and selective than natural agonists.