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

ABCG2 protein (breast cancer resistance protein, BCRP) is an efflux transmembrane protein involved in the transport of endogenous and exogenous substances, as well as in the development of tumor resistance to chemotherapy. In this work, the effects of sex hormones progesterone, estradiol, and testosterone on the relative content of ABCG2 in Caco-2 cells was evaluated. The role of orphan receptors (farnasoid X receptor (FXR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and liver X receptor subtype alpha (LXRα) in the effects of sex hormones was also studied. The content of ABCG2 was estimated by the Western blot technique. Hormones were used at concentrations of 1, 10, and 100 μM; exposure duration was 24 h. All hormones at all concentrations caused an increase in the content of ABCG2. Inhibition of PXR and FXR prevented an increase in ABCG2 levels induced by progesterone. Suppression of CAR and PXR partially reduced the expression of ABCG2 caused by estradiol, as compared to exposure to estrogen alone, but still the level of the transporter exceeded the control. Inhibition of PXR and FXR attenuated the inducing effect of testosterone; however, the level of the transporter exceeded the control. Thus, it was shown that all sex hormones at concentrations 1, 10, and 100 μM increased the content of ABCG2. CAR and PXR participated in the action of estradiol, while FXR and PXR participated in the action of testosterone and progesterone.

In this study we investigated the role of voltage dependent (K_V), Ca²⁺-activated (K_Ca), and inward rectifier (K_ir) potassium channels in the effects of hydrogen sulfide donor (H₂S) sodium hydrosulfide (NaHS) on spontaneous contractile activity of rat jejunum. It was shown that NaHS dose-dependently (10–500 μM) reduced the tonus of the preparation, as well as the amplitude and frequency of spontaneous contractions of jejunum preparations under isometric conditions; the half-maximal effective concentration (EC₅₀) of the inhibitory effect of NaHS on the amplitude of contractions was 165 μM. The blocker of K_V channels 4-AP (200 μM) caused an increase in the amplitude of spontaneous contractions. NaHS (200 μM) decreased the amplitude and frequency of spontaneous activity of the preparation in the presence of 4-AP as well as in the control, and the effect on basal tonus was less pronounced. Blockers of large conductance K_Ca channels (BK), non-specific TEA (3 mM) and specific paxillin (1 μM), increased the amplitude of spontaneous contractions, while the depressing effect of NaHS was completely preserved. The selective blocker of small conductance K_Ca channels (SK) NS8593 (4 μM) did not affect the tonus of the preparation and the parameters of spontaneous contractions; it did not prevent the effect of NaHS. The activator of K_ATP channels diazoxide (100 μM) caused a decrease in the basal tonus of the preparation, as well as the amplitude and frequency of spontaneous contractions. Diazoxide and the K_ATP channel blocker glibenclamide (50 μM) prevented the effect of NaHS on the tonus of the preparation. BaCl₂, the K_ir channel blocker (30 μM), caused an increase in the amplitude of spontaneous contractions and prevented the development of the NaHS inhibitory effects on the frequency and amplitude of spontaneous contractions; the decrease in tonus was less pronounced than in the control. Thus, a decrease in the basal tonus of the rat jejunum preparation under the action of the H₂S donor was associated with activation of K_ir channels, including K_ATP channels, whereas the effect of H₂S on amplitude and frequency was mediated by an increase in Ba²⁺-sensitive conductivity.

Neurotrauma is among the main causes of human disability and mortality. However, the mechanisms that mediate the survival and death of cells in the peripheral nervous system are still not fully understood. The transcription factors p53 and E2F1 are the master regulators of basic cellular functions, including DNA repair, cell cycle, metabolism, and apoptosis. Overexpression of p53 and E2F1, shown in a number of experimental models of peripheral nerve injury, suggests an important role of these proteins in the pathogenesis of neurotrauma. This review discusses the epigenetic mechanisms of p53 and E2F1 activation and regulation, which may contribute to the survival or death of neurons and glial cells after traumatic injury. Prospects for further studies of the mechanisms of regulation of the p53 and E2F1 proteins, including those involving histone deacetylases, for the development of neuroprotectors are considered.

Sperm cryopreservation is an important part of preserving the germ cells of various organisms. However, when gametes are frozen, various damage often occur, which have a significant impact in artificial insemination. After thawing, spermatozoa usually have ultrastructural, biochemical, and functional changes such as damage of cell membrane and chromatin and oxidative stress. Since spermatozoa have a limited capacity for biosynthetic activity, they have a low capacity for regeneration. The current trend is to improve sperm cryopreservation using natural extracellular vesicles and stem cells. Extracellular vesicles and stem cells have potential regenerative effects because they contain various bioactive molecules to affect sperm repair. The present review focuses on current strategies to improve sperm health after cryopreservation. In particular, this review describes the results of studies on the use of extracellular vesicles and stem cells as cryoprotectants during sperm freezing and thawing.

A method for evaluation of metabolic characteristics of intact cells based on electrochemical registration of their respiratory activity was used to monitor a reaction of lymphocytes to a potential pharmacological agent, 2-(chlorodinitromethyl)-4-methoxy-6-(4-methylpiperazin-1-yl)-1,3,5-triazine. The method ensured an estimation of cytotoxicity of the test compound and made it possible to determine its minimum toxic concentrations for human lymphocytes. It was shown that the obtained results agree with the data of the reference method, MTT-based cell viability assay.

It is known that activation of protein synthesis and hypertrophy of muscle fibers in response to mechanical stress is realized through an anabolic mTORC1-dependent signaling pathway. However, mechanosensors through which a mechanical signal can be perceived and further transmitted to the mTORC1-dependent signaling pathway (mechanotransduction) are poorly identified. Mechanically activated (MA) ion channels are candidates for the role of such sarcolemmal mechanosensors. In this regard, the aim of this study was to investigate the potential role of MA channels (Piezo1) in the activation of the mTORC1-dependent pathway in the isolated rat soleus muscle in response to mechanical stress. Wistar rats were divided into 3 groups: (1) “Control” (animal muscles were not exposed to MA channel inhibitor or Piezo1 channel activator); (2) “Gadolinium” (animal muscles were incubated with gadolinium chloride, MA channel inhibitor), and (3) “Yoda” (animal muscles were incubated with Yoda1, Piezo1 MA channel activator). In rats from each group, m. soleus was isolated from the left limb and incubated in the appropriate solution without mechanical stress in the form of a series of stretching (“Rest”); m. soleus from the right limb was subjected to a series of stretching (“Stretch”) and then incubated in the appropriate solution. Phosphorylation of mTORC1 targets (p70S6K, rpS6, and 4E-BP1) in rat m. soleus was determined by PAAG electrophoresis and immunoblotting. A series of passive stretches of isolated m. soleus led to an increase in the phosphorylation of p70S6K, its substrate rpS6, and 4E-BP1 by 38.5, 168 and 112%, respectively, compared to the muscle that was not subjected to mechanical stress. Incubation of the muscles with gadolinium completely prevented the activation of mTORC1 markers caused by a series of stretches. Incubation of m. soleus in a solution with Yoda1 resulted in a decrease in the mechano-dependent phosphorylation of p70S6K, rpS6 and 4E-BP1 compared to the muscle that was not exposed to Yoda1. Thus, the methodological approach used in this work did not reveal the participation of Piezo1 in mechano-anabolic coupling in rat m. soleus.

A quantitative study of subthreshold excitatory postsynaptic potentials (EPSP) recorded intracellularly in giant premotor interneurons of the terrestrial snail was carried out after the formation of a conditioned defensive reflex of food aversion in snails with increased level of serotonin. The results showed a significant increase in the number of low-amplitude EPSP with an amplitude from 0.3 to 0.5 mV in the giant premotor interneurons of defensive behavior after learning and increasing the level of serotonin. The observed increase in the number of EPSP may indicate either an increase in the number of action potentials in the corresponding presynaptic neurons or an increase in the amplitudes of the EPSP that were previously undetectable.

We have studied the process of electroporation of bilayer lipid membranes (BLMs) made of dioleoylphosphatidylcholine (DOPC). We obtained experimental data on the average lifetime of the membrane as a function of applied transmembrane voltage in the range of 200–375 mV. The analysis of the obtained data showed that the dependence is nonmonotonic and cannot be described in terms of the classical theory of electroporation. These results are consistent with modern models of the process of formation of through conductive pores in a membrane. The above models imply a complex pore energy profile and its dependence on a membrane tension and an external electric field. Thus, we have shown that the classical theory of electroporation does not satisfy the experimentally observed dependencies of the average membrane lifetime on the applied potential difference and requires further refinement.

One of the main stages of the infectious process, which largely determines the course and outcome of the disease, is the primary contact of the pathogen with the host cells. A key role in this interaction of gram-negative bacteria with immunocompetent cells of the macroorganism is played by lipopolysaccharide of the outer membrane, which initiates the launch and development of immune reactions by interacting with a number of specific receptors, primarily CD14 and TLR4. The aim of this study was to quantify by atomic force microscopy the force characteristics of the interaction of Yersinia pestis lipopolysaccharide of the EV vaccine strain with CD14 and TLR4 receptors on the surface of murine J774 macrophages. The lipopolysaccharide was isolated from Y. pestis cells of the EV vaccine strain grown at 27°C. Fluorescence and confocal microscopy were used to evaluate the expression of receptors on the cell surface. Using monoclonal antibodies to CD14 and TLR4 receptors, the force characteristics of the interaction of lipopolysaccharide on the surface of the cantilever probe (tip) with J774 macrophages were evaluated by force spectroscopy. The conditions of immobilization of J774 macrophages on glass made it possible to scan their surface and assess the force of adhesion to the cells of target antigens by atomic force microscopy. Incubation of immobilized macrophages in solutions with monoclonal antibodies to CD14 and TLR4 receptors caused a decrease in the main force characteristics of interaction in the J774 macrophage–Y. pestis lipopolysaccharide system compared with intact, untreated cells. A similar effect was observed after pretreatment of cells with a solution of the same lipopolysaccharide without monoclonal antibodies. The results obtained indicate the ability of the lipopolysaccharide chemically bound to the probe to interact with CD14 and TLR4 receptors on the surface of macrophages.