Lyudmila A. Baratova

The gasotransmitter hydrogen sulfide (H₂S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H₂S production and metabolism. Here, we investigated the role of H₂S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. We monitored the learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation–reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes.

2-Oxoacids are involved in a number of important metabolic processes and can be used as biomarkers in some human diseases. A new optimized method for quantification of 2,4-dinitrophenylhydrazine derivatives of 2-oxoacids using high-performance liquid chromatography was developed based on available techniques for quantification of 2-oxoacids in mammalian brain. The use of the 2,4-dinitrophenylhydrazine derivatives of 2-oxoacids was shown to be more advantageous in comparison with the previously used phenylhydrazine derivatives, due to a high chemical stability of the former. Here, we determined the concentrations of pyruvate, glyoxylate, 2-oxoglutarate, 2-oxomalonate, and 4-methylthio-2-oxobutyrate in the methanol/acetic acid extracts of the rat brain using the developed method, as well discussed the procedures for the sample preparation in analysis of mammalian brain extracts. The validation parameters of the method demonstrated that the quantification limits for each of the analyzed of 2-oxoacids was 2 nmol/mg tissue. The developed method facilitates identification of subtle changes in the tissue and cellular content of 2-oxoacids as (patho)physiological biomarkers of metabolism in mammalian tissues.

Influenza A virus envelope contains lipid molecules of the host cell and three integral viral proteins: major hemagglutinin, neuraminidase, and minor M2 protein. Membrane-associated M1 matrix protein is thought to interact with the lipid bilayer and cytoplasmic domains of integral viral proteins to form infectious virus progeny. We used small-angle X-ray scattering (SAXS) and complementary techniques to analyze the interactions of different components of the viral envelope with M1 matrix protein. Small unilamellar liposomes composed of various mixtures of synthetic or “native” lipids extracted from Influenza A/Puerto Rico/8/34 (H1N1) virions as well as proteoliposomes built from the viral lipids and anchored peptides of integral viral proteins (mainly, hemagglutinin) were incubated with isolated M1 and measured using SAXS. The results imply that M1 interaction with phosphatidylserine leads to condensation of the lipid in the protein-contacting monolayer, thus resulting in formation of lipid tubules. This effect vanishes in the presence of the liquid-ordered (raft-forming) constituents (sphingomyelin and cholesterol) regardless of their proportion in the lipid bilayer. We also detected a specific role of the hemagglutinin anchoring peptides in ordering of viral lipid membrane into the raft-like one. These peptides stimulate the oligomerization of M1 on the membrane to form a viral scaffold for subsequent budding of the virion from the plasma membrane of the infected cell.

An optimized method for analysis of free amino acids using a modified lithium-citrate buffer system with a Hitachi L-8800 amino acid analyzer is described. It demonstrates clear advantages over the sodium-citrate buffer system commonly used for the analysis of protein hydrolysates. A sample pretreatment technique for amino acid analysis of brain extracts is also discussed. The focus has been placed on the possibility of quantitative determination of the reduced form of glutathione (GSH) with simultaneous analysis of all other amino acids in brain extracts. The method was validated and calibration coefficient (K_GSH) was determined. Examples of chromatographic separation of free amino acids in extracts derived from different parts of the brain are presented.

The structure of the C-terminal domain of the influenza virus A matrix M1 protein, for which X-ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally-activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C-terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C-terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C-terminal domain is an almost flat layer with a three-α-helical structure. To explain the high level of tritium label incorporation into the C-terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI-TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C-terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell.

Influenza virus matrix M1 protein is one of the main structural components of the virion performing also many different functions in infected cell. X-ray analysis data with 2.08 angstrom resolution were obtained only for the N-terminal part of M1 protein molecule (residues 2-158) but not for its C-terminal domain (159-252). In the present work M1 protein of A/Puerto Rico/8/34 (H1N1) virus strain in acidic solution was investigated with the help of tritium bombardment. Tritium label incorporation into M1 protein domains preferentially labeled the C-domain and inter-domain loops. Analytical centrifugation and dynamic light scattering experiments demonstrated increased hydrodynamic parameters (diameter) that may be explained by low degree of M1 structural organization. Computational analysis of M1 protein by intrinsic disorder predictions methods also demonstrated the presence of unfolded regions mostly in the C-domain and inter-domain loops. It is suggested, that influenza virus M1 polyfunctionality in infected cell is determined by its tertiary structure plasticity which in its turn results from the presence of unstructured regions.

The investigation demonstrated that influenza A and B viruses actively interacted with a sorbent obtained from modified oxygen-containing graphite via hydrothermal treatment irrespective of the antigenic structure of surface proteins. Virionic sorption occurred in a wide range of temperatures from 8 to 34 degrees C for 15 min or more. After interaction with the sorbent, the titer of a virus decreased 4- to 256-fold. The immobilized viruses were able to interact with homologous antibodies and immune sera. Desorption of viruses with the sorbent was extremely slight. In addition to viruses, the proteins of nonviral nature--those of allantoic hen embryo liquid, immune serum, and 1% bovine serum albumin--could be immobilized to the sorbent.

We propose the modified model of the structure of coat protein (CP) subunits in filamentous virions of potato virus X (PVX). The model is similar to the one proposed by us in 2001 for the CP of another helical plant virus (potato virus A) belonging to other (potyvirus) group. In this model the PVX CP molecule consist of two main domains--a bundle of four alpha-helices located close to the virion long axis and a so-called RNP-fold (or abCd-fold) located near the virion surface. Basing on this model we suggest possible mechanism of described by J.G. Atabekov and colleagues structural transition ("remodeling") of the PVX virions resulting from their interaction with virus-specific TGB-1 protein.

It has been shown by biochemical analysis and differential scanning calorimetry that the connective tissue formed around a transplant as a result of sclero-reinforcing interference (capsula) is similar to intact sclera. The main component of newly formed capsules is collagen I whose fibers have a perfect structure and the amount of cross-links sufficient to provide normal thermomechanical properties. A fraction of collagen having thermally labile "immature" cross-links in capsules formed around the transplant impregraned with Panaxal has been detected by differential scanning calorimetry. It was suggested that fibroblasts in tissues of these capsules have a high synthetic activity.

The results of the studies of helical plant virus structures by tritium planigraphy (TP) method are discussed. TP method is based on bombardment of macromolecular objects with a stream of tritium atoms, followed by analysis of tritium label distribution along the macromolecule. By combining the TP data with the results of theoretical predictions of the protein structure, it turned out to be possible to propose a model of the coat protein structure in the virions of potato virus X (the type member of potexvirus group) and potato virus A (one of the members of potyvirus group). With the help of TP it also managed to find subtle differences in the coat protein structure between wildtype tobacco mosaic virus (strain U1) and its mutant with two amino acid substitutions in the coat protein and alter host specificity.