Lysozyme binding with amikacin and levofloxacin studied by tritium probe, fluorescence spectroscopy and molecular docking.

Lysozyme complexes with amikacin and levofloxacin were studied by spectroscopy approaches as well as using a tritium probe. Tritium was used as a labeling agent to trace labeled compound concentration in a system of two immiscible liquids and in the atomic form to determine the possible position of the binding site. Co-adsorption of protein and drug at the liquid-liquid interface was analyzed by scintillation phase method that allowed us to directly determine the amount of protein and drug in the mixed adsorption layer. Also, tensiometric measuring of the interfacial tension was used for calculation of binding parameters accordingly to Fainerman model. The treatment of complexes with atomic tritium followed by trypsinolysis and analysis of tritium distribution in the lysozyme peptides reveals the binding sites, binding energies in which were analyzed using molecular docking. Formation of complexes with amikacin and levofloxacin preserves secondar structure of protein. However, the formation of complex with amikacin leads to the almost total loss of the enzymatic activity of lysozyme and the redshift of the maximum on the lysozyme fluorescence band. A slight decrease in the distribution coefficient of lysozyme in the presence of amikacin assumes that the complex has higher hydrophilicity in comparison to lysozyme without additives. The most favorable for binding were the positions of the active centers that included amino acids Asp52 and Glu35, as well as in the vicinity of peptide His15-Arg21, with the participation of amino acids Tyr20, Arg14. In the case of levofloxacin, the formation of lysozyme-ligand complex in aqueous solution is possible without changing the microenvironment of the active center of the protein. Binding of levofloxacin to the active center of the enzyme was the most favorable, but Asp52 and Glu35 that are responsible for the enzymatic activity of lysozyme, were not affected.

Pentylenetetrazole-Induced Seizures Are Increased after Kindling, Exhibiting Vitamin-Responsive Correlations to the Post-Seizures Behavior, Amino Acids Metabolism and Key Metabolic Regulators in the Rat Brain.

Epilepsy is characterized by recurrent seizures due to a perturbed balance between glutamate and GABA neurotransmission. Our goal is to reveal the molecular mechanisms of the changes upon repeated challenges of this balance, suggesting knowledge-based neuroprotection. To address this goal, a set of metabolic indicators in the post-seizure rat brain cortex is compared before and after pharmacological kindling with pentylenetetrazole (PTZ). Vitamins B1 and B6 supporting energy and neurotransmitter metabolism are studied as neuroprotectors. PTZ kindling increases the seizure severity (1.3 fold, p < 0.01), elevating post-seizure rearings (1.5 fold, p = 0.03) and steps out of the walls (2 fold, p = 0.01). In the kindled vs. non-kindled rats, the post-seizure p53 level is increased 1.3 fold (p = 0.03), reciprocating a 1.4-fold (p = 0.02) decrease in the activity of 2-oxoglutarate dehydrogenase complex (OGDHC) controlling the glutamate degradation. Further, decreased expression of deacylases SIRT3 (1.4 fold, p = 0.01) and SIRT5 (1.5 fold, p = 0.01) reciprocates increased acetylation of 15 kDa proteins 1.5 fold (p < 0.01). Finally, the kindling abrogates the stress response to multiple saline injections in the control animals, manifested in the increased activities of the pyruvate dehydrogenase complex, malic enzyme, glutamine synthetase and decreased malate dehydrogenase activity. Post-seizure animals demonstrate correlations of p53 expression to the levels of glutamate (r = 0.79, p = 0.05). The correlations of the seizure severity and duration to the levels of GABA (r = 0.59, p = 0.05) and glutamate dehydrogenase activity (r = 0.58, p = 0.02), respectively, are substituted by the correlation of the seizure latency with the OGDHC activity (r = 0.69, p < 0.01) after the vitamins administration, testifying to the vitamins-dependent impact of the kindling on glutamate/GABA metabolism. The vitamins also abrogate the correlations of behavioral parameters with seizure duration (r 0.53-0.59, p < 0.03). Thus, increased seizures and modified post-seizure behavior in rats after PTZ kindling are associated with multiple changes in the vitamin-dependent brain metabolism of amino acids, linked to key metabolic regulators: p53, OGDHC, SIRT3 and SIRT5.

Changes in the Structure of Potato Virus A Virions after Limited in situ Proteolysis According to Tritium Labeling Data and Computer Simulation.

Coat proteins (CP) of the potato virus A virions (PVA) contain partially disordered N-terminal domains, which are necessary for performing vital functions of the virus. Comparative analysis of the structures of coat proteins (CPs) in the intact PVA virions and in the virus particles lacking N-terminal 32 amino acids (PVAΔ32) was carried out in this work based on the tritium planigraphy data. Using atomic-resolution structure of the potato virus Y potyvirus (PVY) protein, which is a homolog of the CP PVA, the available CP surfaces in the PVY virion were calculated and the areas of intersubunit/interhelix contacts were determined. For this purpose, the approach of Lee and Richards [Lee, B., and Richards, F. M. (1971) J. Mol. Biol., 55, 379-400] was used. Comparison of incorporation profiles of the tritium label in the intact and trypsin-degraded PVAΔ32 revealed position of the ΔN-peptide shielding the surface domain (a.a. 66-73, 141-146) and the interhelix zone (a.a. 161-175) of the PVA CP. Presence of the channels/cavities was found in the virion, which turned out to be partially permeable to tritium atoms. Upon removal of the ΔN-peptide, decrease in the label incorporation within the virion (a.a. 184-200) was also observed, indicating possible structural transition leading to the virion compactization. Based on the obtained data, we can conclude that part of the surface ΔN-peptide is inserted between the coils of the virion helix thus increasing the helix pitch and providing greater flexibility of the virion, which is important for intercellular transport of the viruses in the plants.

Ivermectin Affects Neutrophil-Induced Inflammation through Inhibition of Hydroxylysine but Stimulation of Cathepsin G and Phenylalanine Secretion.

The invasion and integrin-dependent adhesion of neutrophils to lung tissues and their secretion lead to the development of pneumonia in various pulmonary pathologies, including acute respiratory distress syndrome in coronavirus disease. We studied the effect of ivermectin, a possible therapeutic agent for inflammation and cancer, on integrin-dependent neutrophil adhesion to fibronectin and the concomitant secretion. Ivermectin did not affect the attachment of neutrophils to the substrate and the reactive oxygen species production but sharply inhibited the adhesion-induced release of hydroxylysine and stimulated the release of phenylalanine and cathepsin G. Hydroxylysine is a product of lysyl hydroxylase, which is overexpressed in tumor cells with an increased ability to invade and metastasize. The inhibition of hydroxylysine release by ivermectin, by analogy, may indicate the suppression of neutrophil invasion into tissue. The increase in the release of phenylalanine in our experiments coincided with the secretion of cathepsin G, which indicates the possible role of this enzyme in the cleavage of phenylalanine. What is the substrate in such a reaction is unknown. We demonstrated that exogenously added angiotensin II (1-8) can serve as a substrate for phenylalanine cleavage. Mass spectrometry revealed the formation of angiotensin II (1-7) in the secretion of neutrophils, which attached to fibronectin in the presence of ivermectin and exogenous angiotensin II (1-8), indicating a possible involvement of ivermectin in the inactivation of angiotensin II.

Genes Responsible for H2S Production and Metabolism Are Involved in Learning and Memory in Drosophila melanogaster.

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-ß-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.

Inhibitor of Hyaluronic Acid Synthesis 4-Methylumbelliferone Suppresses the Secretory Processes That Ensure the Invasion of Neutrophils into Tissues and Induce Inflammation.

Integrin-dependent adhesion of neutrophils to tissue, accompanied by the development of neutrophil-induced inflammation, occurs both in the focus of infection and in the absence of infection in metabolic disorders such as reperfusion after ischemia, diabetes mellitus, or the development of pneumonia in patients with cystic fibrosis or viral diseases. Hyaluronic acid (HA) plays an important role in the recruitment of neutrophils to tissues. 4-methylumbilliferon (4-MU), an inhibitor of HA synthesis, is used to treat inflammation, but its mechanism of action is unknown. We studied the effect of 4-MU on neutrophil adhesion and concomitant secretion using adhesion to fibronectin as a model for integrin-dependent adhesion. 4-MU reduced the spreading of neutrophils on the substrate and the concomitant secretion of granule proteins, including pro-inflammatory components. 4-MU also selectively blocked adhesion-induced release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, which can influence cell invasion by modifying the extracellular matrix. Finally, 4-MU inhibited the formation of cytonemes, the extracellular membrane secretory structures containing the pro-inflammatory bactericides of the primary granules. The anti-inflammatory effect of 4-MU may be associated with the suppression of secretory processes that ensure the neutrophil invasion and initiate inflammation. We suggest that HA, due to the peculiarities of its synthesis, can promote the release of secretory carriers from the cell and 4-MU can block this process.

Thermal remodelling of Alternanthera mosaic virus virions and virus-like particles into protein spherical particles.

The present work addresses the thermal remodelling of flexible plant viruses with a helical structure and virus-like particles (VLPs). Here, for the first time, the possibility of filamentous Alternanthera mosaic virus (AltMV) virions' thermal transition into structurally modified spherical particles (SP) has been demonstrated. The work has established differences in formation conditions of SP from virions (SPV) and VLPs (SPVLP) that are in accordance with structural data (on AltMV virions and VLPs). SP originate from AltMV virions through an intermediate stage. However, the same intermediate stage was not detected during AltMV VLPs' structural remodelling. According to the biochemical analysis, AltMV SPV consist of protein and do not include RNA. The structural characterisation of AltMV SPV/VLP by circular dichroism, intrinsic fluorescence spectroscopy and thioflavin T fluorescence assay has been performed. AltMV SPV/VLP adsorption properties and the availability of chemically reactive surface amino acids have been analysed. The revealed characteristics of AltMV SPV/VLP indicate that they could be applied as protein platforms for target molecules presentation and for the design of functionally active complexes.

Binding of chloroaurate to polytyrosine-PEG micelles leads to an anti-Turkevich pattern of reduction.

Here we report formation of gold nanoparticles (GNPs) in micelles of polytyrosine-PEG copolymers that combine the properties of a reducer and a stabilizer. The size and properties of the GNPs were tailored by the excess chloroaurate over the copolymer. The latter quickly formed non-covalent complexes with HAuCl4 and then slowly reduced it to form GNPs. 3 Tyr residues are consumed by reduction of one mole of chloroaurate. The size of the GNPs was controlled by the [Tyr]/[Au(iii)] molar ratio. Small GNPs with D ≅ 8 nm were formed at [Tyr]/[Au(iii)] = 0.5-1.5. 90% of these small GNPs remained bound to the copolymer and could be stored in a lyophilized state. Such polypeptide-gold hybrid materials produced at [Tyr]/[Au(iii)] = 0.5 demonstrated high activity in the catalytic reduction of 4-nitrophenol by sodium borohydride. [Tyr]/[Au(iii)] = 5 led to the formation of large nanoplates (D ≅ 30-60 nm). Thus, in the polymer-based system the GNP size grew in line with the excess of the reducing agent in contrast to Turkevich synthesis of GNPs with citric acid, which also combines the functions of a stabilizer and a reducer. The difference results from the reduction of HAuCl4 in solution according to the Turkevich method and in the micelles of the amphiphilic polymer where the seed growth is limited by the amount of neighboring reducer.

Surface characterization of the thermal remodeling helical plant virus.

Previously, we have reported that spherical particles (SPs) are formed by the thermal remodeling of rigid helical virions of native tobacco mosaic virus (TMV) at 94°C. SPs have remarkable features: stability, unique adsorption properties and immunostimulation potential. Here we performed a comparative study of the amino acid composition of the SPs and virions surface to characterize their properties and take an important step to understanding the structure of SPs. The results of tritium planigraphy showed that thermal transformation of TMV leads to a significant increase in tritium label incorporation into the following sites of SPs protein: 41-71 а.a. and 93-122 a.a. At the same time, there was a decrease in tritium label incorporation into the N- and C- terminal region (1-15 a.a., 142-158 a.a). The use of complementary physico-chemical methods allowed us to carry out a detailed structural analysis of the surface and to determine the most likely surface areas of SPs. The obtained data make it possible to consider viral protein thermal rearrangements, and to open new opportunities for biologically active complex design using information about SPs surface amino acid composition and methods of non-specific adsorption and bioconjugation.

Neutrophils Release Metalloproteinases during Adhesion in the Presence of Insulin, but Cathepsin G in the Presence of Glucagon.

In patients with reperfusion after ischemia and early development of diabetes, neutrophils can attach to blood vessel walls and release their aggressive bactericide agents, which damage the vascular walls. Insulin and 17ß-estradiol (E2) relieve the vascular complications observed in metabolic disorders. In contrast, glucagon plays an essential role in the pathophysiology of diabetes. We studied the effect of hormones on neutrophil secretion during adhesion to fibronectin. Amino acid analysis revealed that proteins secreted by neutrophils are characterized by a stable amino acid profile enriched with glutamate, leucine, lysine, and arginine. The total amount of secreted proteins defined as the sum of detected amino acids was increased in the presence of insulin and reduced in the presence of glucagon. E2 did not affect the amount of protein secretion. Proteome analysis showed that in the presence of insulin and E2, neutrophils secreted metalloproteinases MMP-9 and MMP-8 playing a key role in modulation of the extracellular matrix. In contrast, glucagon induced the secretion of cathepsin G, a key bactericide protease of neutrophils. Cathepsin G can promote the development of vascular complications because of its proinflammatory activity and ability to stimulate neutrophil adhesion via the proteolysis of surface receptors.

Isolation of influenza virus A hemagglutinin C-terminal domain by hemagglutinin proteolysis in octylglucoside micelles.

A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.

Pluronic L61 accelerates flip-flop and transbilayer doxorubicin permeation.

It has recently been found that Pluronics (block copolymers of ethylene oxide, EO, and propylene oxide, PO) favor the permeability and accumulation of anthracycline antibiotics, for example doxorubicin (Dox), in tumor cells. In an effort to understand these results, the interaction of EO(2)/PO(32)/EO(2) (Pluronic L61) with unilamellar egg yolk vesicles (80-100 nm in diameter) was examined. A partition coefficient K(p)=[Pl](membrane)/[Pl](water)=45 was determined. This corresponds to adsorption of about 20 polymer molecules to the surface of each vesicle in a 20 microM polymer solution. Despite this rather weak adsorption, Pluronic has a substantial effect upon the transmembrane permeation rate of Dox and upon the phospholipid flip-flop rate within the bilayers. Thus, the Dox permeation rate increases threefold and the flip-flop rate increases sixfold in 20 microM Pluronic. The two rates increase linearly with the amount of adsorbed polymer. The obvious ability of Pluronics to increase the mobility of membrane components may have important biomedical consequences.

Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity.

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21-->Thr and Asp66-->Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N' (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N' hypersensitive response suppression is discussed.