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

Attachment to the vascular endothelium is the first step of the release of mature neutrophil granulocytes from the bone marrow into the blood and subsequent migration to the inflammatory center. 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 work 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 applying 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/α3α6*β2 was negligible, possibly due to low expression of α3/α6* 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.

Proteins of the anoctamine family (ANO) form calcium-activated chloride channels (CaCC) and phospholipid scramblases. The ANO6 (TMEM16F) protein, which combines the functions of a calcium-dependent scramblase and those of an ion channel, is considered as a molecular target for the treatment of blood clotting disorders, COVID-19-associated pneumonia, neurodegenerative diseases, and other pathologies. CaCCinh-A01, which is a channel blocker of the ANO family, is studied as a potential pharmacological drug. Previously, the effect of this inhibitor was studied using methods representing the integral ion current through the membrane, which does not allow the properties of single channels to be distinguished. Therefore, it remains unknown which characteristics of single channels are sensitive to the blocker: the channel open probability, the current amplitude, or the dwelling time of the channel open state. By registration of single ANO6 channels in HEK293 cells, we showed that the action of the inhibitor is due to a decrease in both the current amplitude and the dwelling time of the single ANO6 channels open state, which, in turn, leads to a decrease in their open state probability. Thus, we have characterized the mechanism of current reduction through ANO6 channels by the inhibitor CaCCinh A01.

The thickness of the lipid membrane is its substantial characteristics. Usually, the thickness of a lipid bilayer is experimentally determined by measuring its electrical capacitance in the approximation of a plane-parallel capacitor. However, membranes formed from a mixture of lipids or containing membrane-deforming inclusions are laterally inhomogeneous, and for them the plane-parallel capacitor approximation generally does not hold. In this work, corrections to the electrical capacitance resulting from deformation of the lipid membrane were numerically calculated. It is shown that the model of a planar capacitor (or their parallel connections), in the general case, does not quantitatively describe these corrections due to the non-zero tangential component of the electric field strength. It is shown that the relative deviation of corrections to the electrical capacitance calculated in various simplified models from the exact solution can reach 50%.

The protein endophilin A, which in the mammalian genome is encoded by three genes, endophilin A1, A2, and A3, 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. Our experiments showed that deletion of endophilin genes does not change the density of SVs in the reserve pool. At the same time, the deletion of dynamin 1 and dynamin 3 genes leads to a significant increase in the vesicle density. We 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.

It is known that the molecules of innate and adaptive immunity can influence the synaptic activity of the central nervous system (CNS). Interferons (IFNs) are commonly used to treat viral and oncological diseases, although they are classified as ototoxic substances and their impact on the synaptic activity of the inner ear is not yet fully understood. In this study, the effect of interferon α2b (IFN-α2b) on the function of afferent glutamatergic synapse in the presence of drug application to the synaptic zone has been analyzed. The study was performed on the isolated vestibular apparatus of a frog (Rana temporaria) using the multiunit recording. Results showed that IFN-α2b (0.2–40 ng/mL) caused an increase in the background pulse activity of afferent fibers, followed by a decrease in the frequency of discharges, especially at high concentrations of IFN-α2b. Additionally, IFN-α2b reduced the glutamate (L-Glu) evoked response and influenced the level of afferent fiber activity restored by L-Glu when the glutamate release from hair cells was blocked in hyper-Mg²⁺ and hypo-Ca²⁺ solution. This indicates that IFN-α2b has a postsynaptic effect. Overall, the findings suggest that IFN has a neuromodulating effect on the synaptic activity of the afferent synapse of the vestibular apparatus.

Serotonin is a regulator of early embryonic development and has a complete functional system in preimplantation mammalian embryos. In the present work, the spatial distribution of serotonin, the vesicular monoamine transporter VMAT2, and the 5-HT_1D and 5-HT_2A receptors at different stages of early embryonic development was described. Serotonin, the VMAT2 transporter, and the 5-HT_1D receptor are visualized in the cortical compartment of cells, whereas the 5-HT_2A receptor has a more even distribution throughout the cytoplasm. The comparison showed that there were no statistically significant differences between the immunoreactive particle sizes of serotonin and the VMAT2 transporter, suggesting the presence of vesicles in which serotonin accumulates with the involvement of VMAT2 for further intercellular signal transduction. Moreover, the patterns of immunoreactivity of the two serotonin receptors, 5-HT_1D and 5-HT_2A, differ markedly, which may indicate that they simultaneously serve different functions in early embryogenesis.

Norepinephrine (NE), which is released by sympathetic nerve endings, causes an increase in the frequency of spontaneous action potentials in the pacemaker cardiomyocytes of the sinoatrial node (SAN) of the heart. This results in an increase in heart rate (HR). Two types of postsynaptic adrenoreceptors (ARs), α1-AR and β-AR, mediate the effects of NE. The role of α1-AR in the sympathetic control of heart rate and SAN automaticity, as well as the membrane mechanisms involved in α1-AR-mediated pacemaker control, have not yet been elucidated. In this study, we utilized immunofluorescence confocal microscopy to examine the distribution of α1A-AR in the SAN of rats. Additionally, we assessed the expression of α1A-AR mRNA in the SAN tissue using RT-PCR. Furthermore, we investigated the impact of α1-AR stimulation on key functional parameters of the pacemaker, including the corrected sinus node recovery time (SNRT/cSNRT) and the SAN accommodation, using the Langendorff perfused heart technique. We also used optical mapping of the electrical activity of perfused, isolated tissue preparations to study the effect of α1-AR stimulation on the spatiotemporal characteristics of SAN excitation. We tested the effects of chloride transmembrane conductance blockade on alteration of functional parameters and pattern of SAN excitation caused by α1-AR. Fluorescent signals corresponding to α1A-AR have been identified in SAN cardiomyocytes, indicating the presence of α1A-AR at protein level. The expression of α1A-AR in SAN has been also confirmed at the mRNA level. The stimulation of α1-AR affects SAN functioning. Phenylephrine (PHE) utilized as α1A-AR agonist caused a decrease in SNRT/cSNRT, as well as an acceleration of SAN accommodation. These effects were rate dependent and were observed in a high frequency range of pacemaker tissue stimulation. PHE induces changes in the excitation pattern of the SAN. The effects of PHE on functional parameters and SAN excitation pattern are attenuated by Ca²⁺-dependent chloride channel blocker NPPB but remains unaffected by the protein kinase C inhibitor BIM. Our results suggest that cardiac α1-ARs are important for maintaining function of SAN pacemaker at high heart rates and that α1-AR signalling cascades in the SAN by targeting Ca²⁺-dependent chloride channels are involved in the α1-adrenergic modulation of the electrophysiological properties of the heart pacemaker.

The ability of humoral agonists (and their persistent analogues) to induce contractions of denervated m. soleus and m. extensor digitorum longus of mice was investigated. Earlier, we found a change in the effectiveness of the ATP modulating effect under some non-physiological factors in the neuromuscular synapses of rodents. The aim of this study was to evaluate the effect of ATP on the contractility of isolated skeletal muscles of a mouse after traumatic denervation. It has been shown that 28-day denervation led to an increase in the strength of contractions of m. soleus and m. extensor digitorum longus caused by an acetylcholine analog. ATP application induced a contraction of denervated muscles, but not of intact ones. In the presence of a non-selective P2 receptor antagonist suramin, the effect of ATP ceased. We suggest that activation of postsynaptic P2X receptors of denervated muscles could cause their contraction. Apparently, this effect was caused by an increase in the expression of postsynaptic receptors in response to a violation of neurotrophic control and the conductive ability of the nerve fiber.

One of the urgent tasks of tissue engineering is the development of stable non-toxic materials that support cell migration during tissue regeneration. This study was aimed at obtaining new gel materials based on collagen and derivatives of taxifolin, taxifolin pentaglutarate and a conjugate of taxifolin with glyoxylic acid and investigating their properties. It was shown that an increase in the proportion of polyphenols in the gel led to a decrease in the rate of degradation of the material. The obtained materials did not negatively affect the viability of NIH/3T3 mouse fibroblasts. The cells were attached to the surface of the materials and spread out on the surface of the material containing taxifolin pentaglutarate. It was also found that fibroblasts migrated through the obtained materials. An increase in the proportion of the conjugate of taxifolin with glyoxylic acid in the material led to inhibition of migration through the material, whereas an increase in the proportion of taxifolin pentaglutarate in the material, on the contrary, led to a significant increase in cell migration through the material. The results obtained indicated the possibility of modulating cell adhesion and migration in biomaterials by including various taxifolin derivatives in their composition. Thus, materials obtained on the basis of collagen and taxifolin derivatives may be of interest for regenerative medicine.