Biologičeskie membrany

The first stage of mature neutrophil granulocytes leaving the bone marrow into the blood and migration to inflammatory center is attachment to vascular endothelium. Disturbance of neutrophil adhesiveness is critical for many diseases with inflammatory components. Endo- and exogenous factors modify the cell ability to adhere via different receptors, including nicotinic acetylcholine receptors (nAChRs). However, the involvement of nAChRs in the regulation of bone marrow (BM) granulocyte adhesion and the role of signaling components in the action of nicotine are poorly understood. In this work the role of different types of nAChRs in the regulation of murine BM granulocyte adhesion during acute inflammation was studied. The study was performed on BM granulocytes of the BALB/c mouse strain using static adhesion assay, confocal microscopy, inhibitor assay, and reverse transcription PCR (RT-PCR). The role of nAChR types was assessed using selective antagonists: 10 nM α-CTX (α7), 10 nM GIC and 5 nM MII (α3β2), 200 nM MII (α3β2 and α7), RgIA and Vc1.1 (α9α10). The number of attached BM granulocytes did not differ between animals with and without inflammation. Nicotine (0.01–100 µM, 30 min) significantly increased cell adhesion in both groups. Toxins (α-CTX, RgIA, Vc1.1) enhanced cell adhesion in both groups, as 200 nM MII did in controls. Fluorescence labelling assays showed expression of α7 and α10 nAChR subunits on cytoplasmic membrane of native BM granulocytes. Using inhibitors, we showed that the effect of nicotine on BM granulocyte adhesion was mediated by heterotrimeric G-proteins, PKC, PI3K, and ROCK both normally and in the presence of inflammation. α7 and α9α10 nAChRs were predominantly involved in regulation of BM granulocyte adhesion, and participation of α3β2 was negligible, possibly due to low expression of α3 subunits. In the regulation of cell adhesion by nicotine, the development of inflammation in the body enhanced the role of α7 nAChRs, which are conventionally expressed on the membrane of BM granulocytes.

Lipid droplets are organelles responsible for the accumulation and breakdown of neutral fats in the human body. Lipid droplets have a monolayer shell of phospholipids, which prevents their spontaneous fusion. The fusion of lipid droplets is carried out by specialized fusion proteins and is regulated by the lipid composition of the monolayer membrane. The efficiency of fusion is determined by the energy needed for the local approach of lipid droplets and the topological rearrangement of their monolayers. In this work, the fusion of monolayers is modeled within the framework of the theory of membrane elasticity. The energy barrier for fusion is calculated under various conditions simulating possible compositions of monolayers, as well as the possible effects of proteins. The calculation results show that the height of the barrier is most dependent on the distance between lipid droplets, which is determined by the fusion proteins. Lipid composition also affects the fusion efficiency and can change it several tens of times, which is consistent with previously obtained data on bilayer fusion.

One of the risk factors for infection caused by SARS-CoV-2 is hyperactivation of the complement system, which can lead to activation and damage to the endothelium, thrombosis and, in some severe cases, the development of multiple organ failure. Using quantitative immunohistochemistry, we studied the opsonization of human umbilical vein endothelial cells (HUVEC) by complement factors C3/C3b when exposed to blood plasma from patients with a confirmed diagnosis of COVID-19 in order to evaluate the possibility of using this approach to study the state of the complement system in COVID-19 ex vivo. For these purposes, FITC-labeled antibodies specific to the C3c domain of factors C3/C3b were used. The integral intensity of the recorded fluorescence and the number of C3/C3b-immunopositive structures were chosen as parameters for assessing the binding of C3/C3b to cells. We have shown that when HUVEC are incubated with plasma from patients, the total number of C3/C3b-immunopositive structures recorded on the membrane of these cells is 5.8 ± 2.8 times (mean ± SD, p < 0.001) higher than when exposed to plasma from healthy people. In this case, the integrated fluorescence intensity increased by 6.3 ± 3.2 times (mean ± SD, p ≤ 0.0001) compared to the control. The area of immunopositive structures recorded after exposure to plasma cells from healthy donors and patients with COVID-19 and selected for analysis ranged from 2.2 to 70 μm². Immunopositive particles with an area of 2.2–10.9 μm² after incubation with plasma of COVID-19 patients had a more elongated shape compared to controls. The average number of particles per cell was 0.49 ± 0.06 (mean ± SD, n = 6) in the control, and 2.4 ± 0.4 (mean ± SD, n = 13, p < 0.001) during incubation with patient plasma. Analysis of particle area distribution showed that the most pronounced differences in the number of C3/C3b-immunopositive structures compared to the control were observed among large particles. Thus, we showed an increase in the level of opsonization of endothelial cells by complement factors C3/C3b in the presence of plasma from COVID-19 patients in comparison with control plasma. The increase is due to an increase in the number of C3/C3b-immunopositive structures, mostly large ones. We assume that the proposed approach will allow us to study the role of the complement system in damage to vascular endothelial cells in patients with COVID-19 using an ex vivo model, as well as to evaluate the level of complement activation in the plasma of patients and the effectiveness of their treatment.

It has been established that cyclic adenosine monophosphate (cAMP), a second messenger of the adenylate cyclase signaling system, and Ca²⁺ are able to dose-dependently modulate the activity of various forms of superoxide dismutase (SOD) in the root cells of pea seedlings of the Rondo variety. The effect of cAMP on SOD activity in pea root cells was studied on intact seedlings by incubating their roots in 50 nM n-dibutyryl-cAMP, a fat-soluble analogue of cAMP, which led to an increase in the intracellular concentration of cAMP. Incubation of similar roots in 800 μM suramin, an inhibitor of transmembrane adenylate cyclase, contributed to a significant decrease in endogenous cAMP levels. In each of these variants, the SOD activity measured in the supernatant obtained from the root homogenate changed. Under the influence of n-dibutyryl-cAMP, the total SOD activity increased to 230%; SOD inhibitors, 3 mM KCN or 3 mM H₂O₂, added to the homogenate, reduced its activity (180 and 190% of the control, respectively). During incubation with suramin, the total activity decreased to 40% of the control value, while with the additional use of SOD inhibitors it decreased to 50–60%. Incubation of seedlings in 400 μM LaCl₃ solution resulted in a decrease in total SOD activity to 73% and in the presence of 3 mM KCN, to 56% of the control, and when 3 mM H₂O₂ was added to the homogenate, to 67%. A similar incubation of seedlings in 1 mM EGTA led to a decrease in total activity by 32%, and the inhibitors had no additional effect. The effect of calcium deficiency or excess on SOD activity was studied in a homogenate of pea seedling roots. When a calcium ion chelator, 100 mM EGTA, was added to the root homogenate, a decrease in the total SOD activity to 81% was observed; when inhibitors (H₂O₂ or KCN) were added, an even greater decrease in SOD activity occurred, up to 65 and 51%, respectively. The addition of 500 nM CaCl2 to the homogenate slightly increased the total SOD activity; KCN reduced SOD activity by approximately 20%, and H₂O₂ had no effect on this indicator. When a higher concentration of CaCl₂, 500 μM, was added, the total activity did not change (100%), in the variant with KCN it decreased by 30%, and when H₂O₂ was added it remained almost unchanged. We conclude that cAMP most likely has an indirect effect on SOD activity, while calcium ions probably act directly on the active site of the enzyme molecule; Moreover, each form of SOD differs in sensitivity to calcium.

Protein endophilin A regulates the synaptic vesicle cycle during exo- and endocytosis, and it is present in the reserve pool of synaptic vesicles (SVs), where its function is unknown. In vitro experiments suggest that endophilin via its SH3 domain interactions incorporates several components into the protein liquid phase that organizes SVs in the reserve pool. We investigated the effect of deletion of the genes encoding endophilin and one of its binding partners, dynamin, on the organization of SVs in living synapses formed by cortical neurons in culture. Experiments showed that deletion of endophilin genes does not change the density of SVs in the reserve pool. At the same time, deletion of the major dynamins 1 and 3 leads to a significant increase in the vesicle density. These results suggest that other SH3-domain-containing proteins, which are components of the protein liquid phase, complement the function of endophilin in the SV reserve pool.