Detection of microwave radiation of cytochrome CYP102 A1 solution during the enzyme reaction

Microwave radiation at 3.4–4.2 GHz frequency of the cytochrome P450 CYP102 A1 (BM3) solution was registered during the lauric acid hydroxylation reaction. The microwave radiation generation was shown to occur following the addition of electron donor NADPH to a system containing an enzyme and a substrate. The radiation occurs for the enzyme solutions with enzyme concentrations of 10^?8 and 10^?9 М. The microwave radiation effect elicited by the aqueous enzyme solution was observed for the first time. The results obtained can be used to elaborate a new approach to enzyme systems research, including studying of the mechanism of interaction of a functioning enzyme system with microenvironment.     

Formation of protodioscin and deltoside isomers in suspension cultures of Nepal yam (<Emphasis Type="Italic">Dioscorea deltoidea</Emphasis> Wall.) cells

Changes in the content of the furostanol glycosides protodioscin and deltoside, particularly that of the (25S)-isomers of the glycosides, during suspension cultivation of different lines of Nepal yam (Dioscorea deltoidea Wall.) cells of the strain IFR-DM-0.5 has been investigated. The composition of furostanol glycosides has been characterized, and the dynamics of the accumulation of individual glycosides during lengthy subcultivation of cells maintained in flasks or in a barbotage bioreactor has been analyzed. A positive correlation between the growth and accumulation of substances that belonged to the class of furostanol glycosides has been demonstrated for cultured dioscorea cells, whereas the content of some of the individual glycosides varied considerably between the lines of the strain, cultures maintained under different conditions, and even between cells in different phases of the growth cycle. The increased content of (25R)-forms of the glycosides (protodioscin and deltoside) was correlated with a decrease in the cellular growth rate, whereas an increase in culture growth intensity occurred concomitantly to an increase of the amount of (25S)-isomers. This may be indicative of the specific stimulatory effect of (25S)-glycosides, but not the (25R)-forms, on cell proliferation in vitro. Thus, the concentration of (25S)-forms may increase due to the autoselection of cells capable of intensive division during prolonged cultivation.     

The role of proteins of the outer membrane of <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1 in the formation and stabilization of silver sulfide nanoparticles

Silver sulfide nanoparticles stable in aqueous solutions were obtained in presence of the cells of the bacterium Shewanella oneidensis MR-1 in aqueous solution containing an equimolar mixture of AgNO₃ and Na₂S₂O₃. Proteins absorbed on the surface of Ag₂S nanoparticles were identified for the first time by MALDITOF/TOF. Among these proteins, multiheme cytochromes MtrC and OmcA, as well as the MtrB membrane porin, which forms a complex on the outer cell membrane, were detected. It was shown that an insoluble precipitate consisting of agglomerated Ag₂S nanoparticles with a wide size distribution was formed in the absence of the cells. The role of the detected proteins in the mechanism of the formation and stabilization of the Ag₂S nanoparticles in the studied system is discussed.     

Non-photochemical fluorescence quenching in photosystem II antenna complexes by the reaction center cation radical

In direct experiments, rate constants of photochemical (k_P) and non-photochemical (k_P⁺) fluorescence quenching were determined in membrane fragments of photosystem II (PSII), in oxygen-evolving PSII core particles, as well as in core particles deprived of the oxygen-evolving complex. For this purpose, a new approach to the pulse fluorometry method was implemented. In the “dark” reaction center (RC) state, antenna fluorescence decay kinetics were measured under lowintensity excitation (532 nm, pulse repetition rate 1 Hz), and the emission was registered by a streak camera. To create a “closed” [P680⁺Q_A^–] RC state, a high-intensity pre-excitation pulse (pump pulse, 532 nm) of the sample was used. The time advance of the pump pulse against the measuring pulse was 8 ns. In this experimental configuration, under the pump pulse, the [P680⁺Q_A^–] state was formed in RC, whereupon antenna fluorescence kinetics was measured using a weak testing picosecond pulsed excitation light applied to the sample 8 ns after the pump pulse. The data were fitted by a two-exponential approximation. Efficiency of antenna fluorescence quenching by the photoactive RC pigment in its oxidized (P680⁺) state was found to be ～1.5 times higher than that of the neutral (P680) RC state. To verify the data obtained with a streak camera, control measurements of PSII complex fluorescence decay kinetics by the single-photon counting technique were carried out. The results support the conclusions drawn from the measurements registered with the streak camera. In this case, the fitting of fluorescence kinetics was performed in three-exponential approximation, using the value of τ₁ obtained by analyzing data registered by the streak camera. An additional third component obtained by modeling the data of single photon counting describes the P680⁺Pheo^– charge recombination. Thus, for the first time the ratio of k_P⁺/k_P = 1.5 was determined in a direct experiment. The mechanisms of higher efficiency for non-photochemical antenna fluorescence quenching by RC cation radical in comparison to that of photochemical quenching are discussed.     

Characterization of V. cholerae T3SS‐dependent cytotoxicity in cultured intestinal epithelial cells

AM‐19226 is a pathogenic, non‐O1/non‐O139 serogroup strain of Vibrio cholerae that uses a Type 3 Secretion System (T3SS) mediated mechanism to colonize host tissues and disrupt homeostasis, causing cholera. Co‐culturing the Caco2‐BBE human intestinal epithelial cell line with AM‐19226 in the presence of bile results in rapid mammalian cell death that requires a functional T3SS. We examined the role of bile, sought to identify the mechanism, and evaluated the contributions of T3SS translocated effectors in in vitro cell death. Our results suggest that Caco2‐BBE cytotoxicity does not proceed by apoptotic or necrotic mechanisms, but rather displays characteristics consistent with osmotic lysis. Cell death was preceded by disassembly of epithelial junctions and reorganization of the cortical membrane skeleton, although neither cell death nor cell‐cell disruption required VopM or VopF, two effectors known to alter actin dynamics. Using deletion strains, we identified a subset of AM‐19226 Vops that are required for host cell death, which were previously assigned roles in protein translocation and colonization, suggesting that they function other than to promote cytotoxicity. The collective results therefore suggest that cooperative Vop activities are required to achieve cytotoxicity in vitro, or alternatively, that translocon pores destabilize the membrane in a bile dependent manner.     

Formation of a strong electric field resulting in the excitation of microplasma discharges at the edge of a dielectric film on a metal in a plasma flow

Results are presented from experimental and analytical studies of the processes resulting in the excitation of microplasma discharges (MPDs) on a metal surface partially covered with a thin dielectric film under the action of an external plasma flow in vacuum. It is shown experimentally that MPDs are excited at the interface between the open metal surface and the region covered by the dielectric film. The probability of MPD excitation is investigated as a function of the thickness of the dielectric film deposited on the metal. It is found that, for a film thickness of 1 μm, the probability of MPD excitation is close to unity. As the film thickness decreases below ~10 nm or increases above ~10 μm, the probability of MPD excitation is reduced by more than two orders of magnitude. A two-dimensional kinetic numerical code is developed that allows one to model the processes of Debye sheath formation and generation of a strong electric field near the edge of a finite-thickness dielectric film on a metal surface in a plasma flow for different configurations of the film edge. It is shown that the maximum value of the tangential component of the electric field is reached at the film edge and amounts to E _max ≈ |φ₀|/2d (where φ₀ < 0 is the electric potential applied to the metal and d is the film thickness), which for typical conditions of experiments on the excitation of MPDs on metal surfaces (φ₀ ≈–400 V, d ≈ 1 μm) yields E _max ≈ 2 MV/cm. The results of kinetic simulations confirm the qualitative idea about the mechanism of the formation of a strong electric field resulting in the excitation of MPDs at the edge of a dielectric film on a metal surface in a plasma flow and agree with experimental data.     

Radiation Spectra of the Plasma Relativistic Microwave Oscillator

Plasma Physics Reports - The spectral composition of high-power microwave radiation of the plasma relativistic oscillator in the range of 1−4 GHz was studied experimentally as a function of...