Dynamics of the Multipathway Regulation of the Vasodilator Bold Effect Induced by a Nerve Impulse: A Kinetic Model of the Neurovascular Coupling Process.

A kinetic model of the dynamics of a multipathway mechanism of neurovascular coupling induced by nerve impulses was constructed. The model calculations were compared with experimental data on the changes in the blood oxygen level dependent signal during sensory-motor and visual excitation before and after the use of the nonsteroidal anti-inflammatory drug indomethacin. The influence of the catalytic activity of key enzymes on the dynamics of the neurovascular response in the proposed model is shown. The multipathway mechanism of the biochemical reactions provides stability of the neurovascular coupling during various possible catalytic activities of the key enzymes in the process.

Thermovaccination - thermoheliox as a stimulator of the immune response. Kinetics of the synthesis of antibodies and C-reactive protein in coronavirus infection.

Clinical trials of thermoheliox application (inhalation with a high-temperature mixture of oxygen and helium, 90 °C) in the treatment of the acute phase of coronavirus infection were conducted. Dynamics of disease development in infected patients (PCR test for the virus) and, dynamics of changes in blood concentration of C-reactive protein, immunoglobulin M, specific immunoglobulin G were studied. High efficiency of thermoheliox in releasing the organism from the virus and stimulating the immune response (thermovaccination effect) was shown. The kinetic model of the process is proposed and analyzed.

Chemical kinetics of the development of coronaviral infection in the human body: Critical conditions, toxicity mechanisms, "thermoheliox", and "thermovaccination".

Kinetic modeling of the behavior of complex chemical and biochemical systems is an effective approach to study of the mechanisms of the process. A kinetic model of coronaviral infection development with a description of the dynamic behavior of the main variables, including the concentration of viral particles, affected cells, and pathogenic microflora, is proposed. Changes in the concentration of hydrogen ions in the lungs and the pH -dependence of carbonic anhydrase activity (a key breathing enzyme) are critical. A significant result is the demonstration of an acute bifurcation transition that determines life or system collapse. This transition is connected with exponential growth of concentrations of the process participants and with functioning of the key enzyme carbonic anhydrase in development of toxic effects. Physical and chemical interpretations of the therapeutic effects of the body temperature rise and the potential therapeutic effect of "thermoheliox" (respiration with a thermolized mixture of helium and oxygen) are given. The phenomenon of "thermovaccination" is predicted, which involves stimulation of the immune response by "thermoheliox".

Kinetic Modeling of the Blood Oxygenation Level Dependent (BOLD) Signals and Biocatalytic Reactions Observed in the Human Brain Using MRI: An Analysis of Normal and Pathological Conditions.

A kinetic model describing the pulse of increased oxygen concentrations and the subsequent changes in the concentration of N-acetylaspartate in the excited nervous tissue of the human brain in response to an external signal is presented. The model is based on biochemical data, a multistage and nonlinear dynamic process the BOLD signal and N-acetylaspartate. The existence of multiple steady states explains the triggering effect of the system. The inhibitory effect of the substrate is a necessary factor for the autostabilization of N-acetylaspartate. The kinetic model allows the dynamic behavior of previously unmeasurable metabolites, namely, products of the hydrolysis of N-acetylaspartate, such as acetic and aspartic acid, and glutamic acid to be predicted. Kinetic modeling of the BOLD signal and the subsequent hydrolysis of N-acetylaspartate provides information about the biochemical and dynamic characteristics of some pathological conditions (schizophrenia, Canavan disease, and the superexcitation of the neural network).

Proteome Profiling of the Exhaled Breath Condensate after Long-Term Spaceflights.

Comprehensive studies of the effects of prolonged exposure to space conditions and the overload experienced during landing on physiological and biochemical changes in the human body are extremely important in the context of planning long-distance space flights, which can be associated with constant overloads and various risk factors for significant physiological changes. Exhaled breath condensate (EBC) can be considered as a valuable subject for monitoring physiological changes and is more suitable for long-term storage than traditional monitoring subjects such as blood and urine. Herein, the EBC proteome changes due to the effects of spaceflight factors are analyzed. Thirteen EBC samples were collected from five Russian cosmonauts (i) one month before flight (background), (ii) immediately upon landing modules in the field (R0) after 169-199 days spaceflights, and (iii) on the seventh day after landing (R+7). Semi-quantitative label-free EBC proteomic analysis resulted in 164 proteins, the highest number of which was detected in EBC after landing (R0). Pathways enrichment analysis using the GO database reveals a large group of proteins which take part in keratinization processes (CASP14, DSG1, DSP, JUP, and so on). Nine proteins (including KRT2, KRT9, KRT1, KRT10, KRT14, DCD, KRT6C, KRT6A, and KRT5) were detected in all three groups. A two-sample Welch's t-test identified a significant change in KRT2 and KRT9 levels after landing. Enrichment analysis using the KEGG database revealed the significant participation of detected proteins in pathogenic E. coli infection (ACTG1, TUBA1C, TUBA4A, TUBB, TUBB8, and YWHAZ), which may indicate microbiota changes associated with being in space. This assumption is confirmed by microbial composition analysis. In general, the results suggest that EBC can be used for noninvasive monitoring of health status and respiratory tract pathologies during spaceflights, and that the obtained data are important for the development of medicine for use in extreme situations. Data are available from ProteomeXchange using the identifier PXD014191.

Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad.

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 µs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

Three Faces of N-Acetylaspartate: Activator, Substrate, and Inhibitor of Human Aspartoacylase.

Hydrolysis of N-acetylaspartate (NAA), one of the most concentrated metabolites in brain, catalyzed by human aspartoacylase (hAsp) shows a remarkable dependence of the reaction rate on substrate concentration. At low NAA concentrations, sigmoidal shape of kinetic curve is observed, followed by typical rate growth of the enzyme-catalyzed reaction, whereas at high NAA concentrations self-inhibition takes place. We show that this rate dependence is consistent with a molecular model, in which N-acetylaspartate appears to have three faces in the enzyme reaction, acting as activator at low concentrations, substrate at moderate concentrations, and inhibitor at high concentrations. To support this conclusion we identify binding sites of NAA at the hAsp dimer including those on the protein surface (activating sites) and at the dimer interface (inhibiting site). Using the Markov state model approach we demonstrate that population of either activating or inhibiting site shifts the equilibrium between the hAsp dimer conformations with the open and closed gates leading to the enzyme active site buried inside the protein. These conclusions are in accord with the calculated values of binding constants of NAA at the hAsp dimer, indicating that the activating site with a higher affinity to NAA should be occupied first, whereas the inhibiting site with a lower affinity to NAA should be occupied later. Application of the dynamical network analysis shows that communication pathways between the regulatory sites (activating or inhibiting) and the gates to the active site do not interfere. These considerations allow us to develop a kinetic mechanism and to derive the equation for the reaction rate covering the entire NAA concentration range. Perfect agreement between theoretical and experimental kinetic data provides strong support to the proposed catalytic model.

Role of Protein Dimeric Interface in Allosteric Inhibition of N-Acetyl-Aspartate Hydrolysis by Human Aspartoacylase.

The results of molecular modeling suggest a mechanism of allosteric inhibition upon hydrolysis of N-acetyl-aspartate (NAA), one of the most abundant amino acid derivatives in brain, by human aspartoacylase (hAsp). Details of this reaction are important to suggest the practical ways to control the enzyme activity. Search for allosteric sites using the Allosite web server and SiteMap analysis allowed us to identify substrate binding pockets located at the interface between the subunits of the hAsp dimer molecule. Molecular docking of NAA to the pointed areas at the dimer interface predicted a specific site, in which the substrate molecule interacts with the Gly237, Arg233, Glu290, and Lys292 residues. Analysis of multiple long-scaled molecular dynamics trajectories (the total simulation time exceeded 1.5 µs) showed that binding of NAA to the identified allosteric site induced significant rigidity to the protein loops with the amino acid side chains forming gates to the enzyme active site. Application of the protein dynamical network algorithms showed that substantial reorganization of the signal propagation pathways of intersubunit communication in the dimer occurred upon allosteric NAA binding to the remote site. The modeling approaches provide an explanation to the observed decrease of the reaction rate of NAA hydrolysis by hAsp at high substrate concentrations.

Proteomics of exhaled breath: methodological nuances and pitfalls.

BACKGROUND: The analysis of exhaled breath condensate (EBC) can be an alternative to traditional endoscopic sampling of lower respiratory tract secretions. This is a simple non-invasive method of diagnosing respiratory diseases, in particular, respiratory inflammatory processes. METHODS: Samples were collected with a special device-condenser (ECoScreen, VIASYS Healthcare, Germany), then treated with trypsin according to the proteomics protocol for standard protein mixtures and analyzed by nanoflow high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) with a 7-Tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Germany). Mascot software (Matrixscience) was used for screening the database NCBInr for proteins corresponding to the peptide maps that were obtained. RESULTS: EBCs from 17 young healthy non-smoking donors were collected. Different methods for concentrating protein were compared in order to optimize EBC preparations for proteomic analysis. The procedure that was chosen allowed identification of proteins exhaled by healthy people. The major proteins in the condensates were cytoskeletal keratins. Another 12 proteins were identified in EBC from healthy non-smokers. Some keratins were found in the ambient air and may be considered exogenous components of exhaled air. CONCLUSIONS: Knowledge of the normal proteome of exhaled breath allows one to look for biomarkers of different disease states in EBC. Proteins in ambient air can be identified in the respiratory tract and should be excluded from the analysis of the proteome of EBC. The results obtained allowed us to choose the most effective procedure of sample preparation when working with samples containing very low protein concentrations.

Hierarchical classification of hydrolases catalytic sites.

UNLABELLED: Universal ontology of catalytic sites is required to systematize enzyme catalytic sites, their evolution as well as relations between catalytic sites and protein families, organisms and chemical reactions. Here we present a classification of hydrolases catalytic sites based on hierarchical organization. The web-accessible database provides information on the catalytic sites, protein folds, EC numbers and source organisms of the enzymes and includes software allowing for analysis and visualization of the relations between them. AVAILABILITY: http://www.enzyme.chem.msu.ru/hcs/