Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology最新文献

Blood coagulation and fibrinolysis systems are enzymatic cascades in blood plasma that control formation and dissolution of a fibrin clot, respectively. However, critical processes in both systems occur on specialized scaffolds but not in the liquid phase. These scaffolds are two- or three-dimensional matrices that provide special conditions for biochemical reactions. The following fundamental categories of scaffolds can be distinguished: (a) phospholipid membranes enriched with phosphatidylserine provided by a procoagulant subpopulation of activated platelets, as well as damaged endothelium; membranes of apoptotic bodies in atherosclerotic plaque; lipoproteins and plasma microvesicles; (b) complex of fibrin and extracellular matrix proteins, which is associated with platelets and is the leading scaffold for pro- and anti-fibrinolytic processes; (c) polymers containing phosphate groups, including platelet polyphosphates and neutrophil extracellular traps. For some of these scaffolds, there are speculations about their physiological significance and physical meaning, while the role of others seems mysterious or at least pathophysiological. Herein we consider existing ideas about the roles and mechanisms of the involvement of these scaffolds in haemostasis and thrombosis.

Using the method of protein reconstitution into planar bilayer lipid membranes, the electrophysiological properties of a novel porin channel from the marine bacterium Marinomonas primoryensis (MpOmp) were characterized. The main characteristics were determined: the conductivity value of the single MpOmp channel, its selectivity, and the values of the critical closing potential in various media (neutral, weakly acidic, alkaline). Using an in silico approach, the geometric characteristics of the MpOmp pore and the distribution of charges at the mouth and inside the porin channel were predicted.

The cytochrome c–cardiolipin complex plays a key role in triggering apoptosis via the mitochondrial pathway due to lipoperoxidase and quasi-lipoxygenase activities of cytochrome c. As a result of the formation of this complex, the conformation of cytochrome c changes, which acquires the properties of peroxidase enzymes capable of triggering lipid peroxidation reactions. The functions of the cytochrome c–cardiolipin complex are usually studied using the recording of enhanced (activated) chemiluminescence. The chemiluminescence enhancer or activator increases the luminescence intensity due to the migration of the electron excitation energy from the excited lipid peroxidation products to the activator molecules, followed by its chemiluminescence with a high quantum yield. It is advisable to use in such studies activators that enhance luminescence without a chemical reaction with the components of the system under study and keep their concentration unchanged during the reaction time. At the end of the last century, it was shown on the Fe²⁺-induced lipid peroxidation system that quinolizidine derivatives of coumarin are such compounds. The ideas about the immutability of their concentration were transferred without additional studies to systems in which lipid peroxidation is triggered by peroxidase. However, it was found in this study by spectrophotometry using a reaction catalyzed by the cytochrome c–cardiolipin complex as an example that quinolizidine derivatives of coumarin, coumarin-314 (quinolizidine[5,6,7-gh]3-ethoxycarbonylcoumarin) and coumarin-334 (quinolizidine[5,6,7-gh]3-acetylcoumarin), are direct participants in the enzymatic lipoperoxidase reaction. Based on a comparison of changes in the concentration of coumarin derivatives in the presence and absence of phosphatidic acid, we found that coumarin derivatives are predominantly substrates of the second reaction of the peroxidase catalytic cycle, that is, the reduction of a peroxidase ferriform with two oxidized equivalents (compound 1) to a peroxidase ferriform with one oxidized equivalent (compound 2). It was also shown that during the catalysis of a quasi-hypoxygenase reaction (in the absence of H₂O₂ in the system), peroxidase passes through a catalytic cycle by the mechanism of one-electron oxidation followed by reduction, while there is no ferriform stage of peroxidase with two oxidized equivalents (compound 1). The rate constants of the first-order reaction of the decomposition of coumarin derivatives during the enzymatic lipoperoxidase reaction were determined, and based on them, functions were derived for calculating correction coefficients that take into account the decomposition of coumarin derivatives for correcting chemiluminograms obtained in the study of the cytochrome c–cardiolipin complex.

Propagation of action potentials and the transfer of metabolites with the streaming cytoplasm represent two main pathways of long-distance signaling in giant cells of characean algae. This work shows that the excitation of plasma membranes and the arrest of cytoplasmic streaming in Chara cells at low light (in the absence of large pH changes on the cell surface) is accompanied by a transient increase or a series of oscillations of chlorophyll F ' fluorescence in chloroplasts located near zones of external calcification but has no effect on fluorescence of chloroplasts in the predominant non-calcified cell areas. The different sensitivity to the action potential generation in chloroplasts located near and far from the incrustation zones is probably due to structural and biochemical differences of cellular domains and to the influence of cytoplasmic streaming on chloroplast environment in the area of fluorescence measurements. The kinetic curves of F ' changes upon cell excitation and the number of individual peaks in the F ' response depended on the illuminated area of plant sample. The number of peaks diminished when the overall illumination was replaced with illumination of a narrow cell region using flexible light guides with diameters of 2.0 and 0.4 mm. The results indicate that the initial stages of F ' changes induced by the action potential generation are caused by internal processes in chloroplasts, while the subsequent changes in F ' are due to the influx of metabolites from the recovering cytoplasmic flow into the chloroplasts of the studied zone. The amplitude of excitation-induced F ' changes decreased transiently after the transition from general to local illumination, which is explained by the depletion of reducing equivalents in the cytoplasm during postillumination CO₂ fixation under cessation of NADPH photoproduction outside the narrow illuminated zone.

Localized thrombin production appears to be a key event in the hemostatic response to the vascular injury. This protein causes irreversible activation of platelets and is responsible for the formation of a fibrin mesh that stabilizes the hemostatic plug. It is generally accepted that flow has a strong inhibitory effect on the kinetics of plasma coagulation reactions, so that thrombin generation and fibrin formation are restricted to the areas, which are protected from the diluting effect of the blood flow, for example, inside the platelet aggregate or in the subendothelial matrix. However, experimental evidence indicates the possibility of in vitro fibrin polymerization at arterial shear rates in the absence of platelets. Here, using in vitro experiments and in silico models, we show that the initiation of plasma coagulation under arterial shear rates can occur due to the presence of an immobilized phospholipid fraction in the area mimicking the damaged vascular wall. Our results suggest that binding of coagulation factors to these phospholipids allows the initial stages of plasma coagulation to be protected from the flow and leads to a rapid thrombin production even under conditions of arterial blood shear rates. Thus, the obtained data suggest that under certain conditions activation of secondary hemostasis may precede and promote platelet activation and aggregation.

The pregnane X receptor (PXR) is a nuclear receptor that plays an important role in regulating the expression of biotransformation and metabolism enzymes, as well as transporter proteins. There are contradictory data in the literature on the effect of oxidative stress on the amount of PXR. The purpose of this study was to evaluate the effect of oxidative stress on the functioning of PXR. The work was performed on the Caco-2 cell line. Oxidative stress was modeled with hydrogen peroxide at concentrations of 0.1, 0.5, 1, 5, 10, 50, and 100 μM and incubation duration of 3, 24, and 72 h. The amount of PXR was estimated by Western blot method. H₂O₂ at all concentrations during incubation for 3 h did not significantly affect the amount of PXR. An increase in the exposure up to 24 h at prooxidant concentrations of 10, 50, and 100 μM led to an increase in the amount of PXR, which was combined with an increase in the content of lipid peroxidation products (LPPs). Continued exposure to hydrogen peroxide for up to 72 h was accompanied by an increase in the concentration of LPPs and a decrease in the amount of PXR to control values (at the H₂O₂ concentration of 10?μM) or below it (at H₂O₂ concentrations of 50 and 100 μM). Incubation of the cells with malonic dialdehyde, the final product of lipid peroxidation, at a concentration of 10 μM for 24 h led to an increase in the amount of PXR. Thus, exposure to hydrogen peroxide for 24 h led to an increase in the amount of PXR and was associated with the inducing effect of LPPs. An increase in the exposure to 72 h leveled this inducing effect.