Cytolytic peptides are a large, heterogeneous group of proteinaceous compounds capable of disrupting the integrity of biological membranes and causing cell lysis. Arthropod venom is a rich source of cytolytic peptides. Melittin from honeybee venom, as well as several peptides from the venom of the spider Lachesana tarabaevi, have previously been demonstrated to inhibit the growth of the intracellular parasitic bacterium Chlamydia trachomatis. In this study, using the Tet-On system for regulated gene expression in HEK293 cells, it was demonstrated that oxyopinins—cytolytic peptides derived from the venom of the spider Oxyopes takobius—exhibit similar anti-chlamydial activity. Oxyopinins have diverse amino acid sequences and are not homologous to previously studied peptides. This work significantly expands the range of known compounds with anti-chlamydial activity.
In this work we investigated the pore-forming activity of ShlA toxin from Serratia proteamaculans in lipid bilayers modelling the membranes of epithelial cells HeLa and Caco-2 and assessed the role of CaCl2 in its modification. It was found that the addition of conditioned S. proteamaculans media enriched with ShlA toxin to planar lipid bilayers formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sphingomyelin (SM)/cholesterol (CHOL) (33/33/33 mol
On April 16, 2026, Eugene V. Grishin would have turned 80. Eugene has been gone for ten years now, but his contribution to the science of membrane receptors is so significant that the influence of his work and ideas on our research is still felt today. In this article, I will discuss some memorable events related to Grishin in the scientific community.
One of the problems of modern neuropharmacology is the search for new structures that affect specific targets in the nervous system. Among such promising structures is diazepinobenzimidazole, which includes two privileged subclasses: diazepine and benzimidazole. One of its derivatives, DAB-19, demonstrates a wide range of activities and appears to be very promising. However, the targets of DAB-19 and the molecular mechanisms of its action remain poorly understood. DAB-19 inhibits AMPA- and NMDA-subtypes of glutamate receptors, blocks voltage-gated sodium channels, and enhances spontaneous synaptic release of glutamate. This complex action profile complicates the analysis of action mechanisms. In this study, we investigated the effects of several analogues of DAB-19 to separate the action on different targets by structure–function analysis. We found that the presence of a side chain in diazepinobenzimidazole derivatives is necessary for both stimulating spontaneous release and blocking sodium channels. The presence of a long side chain containing an aromatic ring is critical for stimulating spontaneous release. Compounds with a shorter radical with a saturated ring lose their ability to stimulate spontaneous release but retain their ability to block sodium channels. The results are important for the further targeted design of new compounds that modulate the functions of the central nervous system (CNS).
Two-pore calcium channels (TPCs) are localized in the membranes of endosomes and lysosomes and play a crucial role in the regulation of vesicular transport, autophagy, protein, nucleic acid, and lipid catabolism, recycling of plasma membrane receptors, and other processes occurring in the endolysosomal system. Humans express two types of channels, TPC1 and TPC2. Dysfunction of these channels is the cause of severe pathologies, including cancer and neurodegenerative diseases. Understanding the mechanisms of TPC channel regulation and identifying pharmacological pathways to influence their activity are essential for developing new approaches to the prevention and treatment of these diseases. This review characterizes the properties of two-pore channels and describes their regulation by phosphatidylinositol-3,5-bisphosphate and NAADP (nicotinic acid adenine dinucleotide phosphate), changes in channel activity during cell starvation, as well as the effects on TPC channels exerted by sphingosine, protein kinases P38 and JNK, and regulatory proteins mTor, Rab7a, and Tmem63a. Information on the pharmacology of TPC channels is provided. Groups of NAADP receptor site antagonists and compounds acting directly on the channel protein are highlighted. Part of the review is devoted to the description of plant-derived channel regulators and their potential use as anticancer drugs.
Potassium channels are the most diverse superfamily of ion channels, controlling many cellular functions. In this review, we focused on describing the properties of potassium channels of the Kv7 (KCNQ) and GIRK (KCNJ) families. We pay particular attention to the role of these channels in regulating neuromuscular transmission and skeletal muscle activity. Although Kv7 and GIRK channels belong to different classes of potassium channels, they perform similar functions by controlling cellular excitability. In skeletal muscles and motor nerve terminals, Kv7 and GIRK channel subtypes, located at the intersection of many signaling pathways, are involved in regulating the muscle fiber contractility and the intensity of spontaneous and evoked release of the neurotransmitter acetylcholine. Kv7 and GIRK channels are targets for a number of endogenous modulators and toxins, and their dysfunction is associated with the pathogenesis of a wide range of disorders. A detailed study of the properties and functions of Kv7 and GIRK potassium channels will provide a deeper understanding of the therapeutic potential of using their antagonists and agonists for the treatment of various neurological, including neuromuscular, diseases.
The first messengers regulate cellular functions by involving surface receptors coupled to diverse intracellular signaling pathways. Those do not function completely independently, and an increasing body of evidence argues for the existence of crosstalk between signaling systems and their coordination. Here we analyzed a potential contribution of the PI3K–Akt cascade to the regulation of cAMP signaling initiated by noradrenaline (NA) in HEK-293 cells that expressed Pink Flamindo, a genetically encoded cAMP sensor, at a constant and high level. This feature makes this cell line a sufficiently effective cellular model for the analysis of mechanisms involved in the generation of intracellular cAMP signals. As was shown, the stimulation of β‑adrenoreceptors (β-AR) by NA led to a rise in cAMP in HEK-293 cells. Being applied shortly for 100–150 s, NA initiated cAMP signals, the magnitude of which gradually increased with an agonist concentration, exhibiting the EC50 of about 1 µM. In the presence of 150 nM insulin, the magnitude of cAMP responses to 1 µM NA decreased by 31
Fluoroacetate (FA) is a metabolic poison whose toxic effect is caused by the inhibition of mitochondrial aconitase, leading to the accumulation of citrate in cells and target organs, primarily in the brain, spinal cord, heart, and kidneys. Previously, during the development of a treatment regimen for acute FA poisoning, a positive effect of methylene blue (MB) on the survival of experimental animals was revealed. The mechanism of action of MB in acute FA intoxication has not been established; however, based on the physicochemical characteristics and effects of MB described in the literature, we hypothesized that MB, as an artificial electron acceptor, promotes citrate utilization via cytoplasmic aconitase and isocitrate dehydrogenase, which leads to a decrease in citrate levels and restoration of Ca2+ ion balance. The aim of this study is to identify the effects of FA on calcium balance and mitochondrial potential in astrocytes and hippocampal neurons of rats under in vitro conditions, as well as the nature of the modulation of these effects by methylene blue, the transmembrane electron transport inhibitor 4-hydroxycoumarin (4HC), the Ca2+ chelator BAPTA, and the ryanodine receptor inhibitor dantrolene. It was found that FA causes death of astrocytes, but not neurons, in primary hippocampal cultures, which is associated with an increase in intracellular calcium ion levels ([Ca2+]i). Citrate has no toxic effect on astrocytes but potentiates the drop in mitochondrial membrane potential in response to FA. 4-Hydroxycoumarin suppresses the calcium response in neurons and has virtually no effect on astrocytes. However, 4HC suppresses changes in the mitochondrial potential of astrocytes. In response to the addition of FA in the presence of citrate, dantrolene prevents an increase in [Ca2+]i, especially in neurons. At the same time, MB neutralizes the effect of FA, preventing a decrease in the rate of mitochondrial potential change in both neurons and astrocytes. Thus, the effects of MB on astrocytes may be associated with the inhibition of transmembrane electron transport, a reduction in the level of reactive oxygen species, and the modulation of ryanodine receptor sensitivity. The observed effects support our hypothesis regarding the molecular mechanism of action of MB in astrocytes; however, further experiments are needed to obtain direct evidence.
Cholinergic neuromuscular synapses in skeletal and respiratory muscles play a crucial role in respiration and movement, transmitting excitation from the motor neuron to the muscle fibers. Key players in this process include voltage-gated ion channels, the machinery for releasing neurotransmitter acetylcholine from nerve terminals, and receptor–channel complexes on the muscle fiber membrane. Neurotoxins specific to various molecular structures involved in synaptic function are widely used to investigate the molecular mechanisms underlying the processes that ensure the plasticity and high reliability of the synaptic machinery. This review examines presynaptic and postsynaptic neurotoxins that act on ion channels, neurosecretory processes, and postsynaptic receptors and are used to study neuromuscular transmission.
Linear amphipathic peptides are components of some natural venoms exhibiting pronounced cytolytic activity. They are characterized by a low selectivity and a receptor-independent mechanism of action. Their common structural property is a spatial separation of hydrophobic and polar (charged) amino acid residues within a single molecule, as well as a relatively large positive electrical charge. Cytolytic peptides of venoms exhibit antimicrobial properties and are considered prototypes for potential antibiotics. Classical antimicrobial peptides have the same structural properties as the cytolytic peptides of natural venoms. Amphipathic peptides form pores in lipid membranes by two possible mechanisms: local and nonlocal. The local mechanism involves membrane destabilization immediately adjacent to the peptide molecule, partially embedded in the lipid monolayer. The nonlocal mechanism works only on closed membranes with one-sided addition of amphipathic peptides. In this case, the incorporation of peptides into the contact membrane monolayer induces lateral pressure in it and lateral tension in the opposite monolayer. This asymmetry in lateral tension/pressure facilitates the formation of large metastable lipid pores. In this study, we performed a theoretical analysis of the nonlocal mechanism of pore formation. Using the theory of lipid membrane elasticity, we calculated the dependences of pore energy on radius for various values of lateral tension and pressure in membrane monolayers and determined the dependences of the energy barriers to the formation and closure of a metastable pore, as well as of its radius, on lateral tension/pressure. A mechanism for the local formation of through defects in the membrane by single molecules of amphipathic peptides is proposed. Lipids and amphipathic peptides can pass through the defect walls to the opposite membrane monolayer. The structural characteristics of the peptides responsible for the efficient formation of defects are determined.
Injuries to the central and peripheral nervous systems are accompanied by complex cellular and molecular processes, including neuroinflammation, oxidative stress, and programmed cell death. Nitric oxide (NO) and hydrogen sulfide (H2S) play a key role in these processes, demonstrating dual effects. Apoptosis is a key mechanism involved in the death of neurons and glial cells in neurotrauma. NO and H2S can regulate the expression of anti- and pro-apoptotic genes through direct modification of DNA and RNA or through more complex epigenetic mechanisms involving the activation or inhibition of transcription factors. This review provides a detailed overview of NO- and H2S-dependent signaling mechanisms that regulate the expression of anti- and pro-apoptotic genes in various types of neurotrauma, as well as the dual effects of these gas transmitters in pharmacological regulation.
Early changes in the myosin genes expression pattern lead to slow-to-fast fiber type shift, functional and metabolic alterations in skeletal muscles. A detailed understanding of myosin isoform transcriptional regulation could be the key for effective countermeasures against the unloading-induced negative changes. Slow-type myosin mRNA expression changes non-linearly in postural soleus muscle during rat hindlimb suspension, with a partial re-activation of slow-type myosin expression between the 2nd and the 4th day. Calcium ions accumulation is a well-studied activator of slow-type myosin expression, and after the second day of rat hindlimb suspension calcium was shown to increase in soleus muscle myoplasm. We suggested that calcium ions accumulation could contribute to the transient re-activation in slow-type myosin gene expression after the 2nd day of rat hindlimb suspension via activation of calcineurin/NFATc1 and/or blocking of HDAC4/MRF4 signaling pathways. If this hypothesis is correct, the prevention of the unloading-induced calcium accumulation by L-type channels inhibitor nifedipine would be expected to downregulate the expression of slow-type myosin. To test our hypothesis, the experiment with 3-day rat hindlimb suspension (HS) accompanied by nifedipine (L-type calcium channels blocker) administration was conducted. After 3 days of HS, we observed an increase in slow-type myosin mRNA expression and an increase in NFATc1 MCIP1.4 reporter activity, which was not observed in the group receiving nifedipine. In the group with 3-day rat hindlimb suspension and receiving nifedipine (group HS + Nifedipine), we also observed an accumulation of HDAC4 and MRF4 in the nucleus and a decrease in NFATc1 content in the nucleus compared to 3 days of HS. These data indicate that calcium ions accumulation after 3 days of HS contributes to the reactivation of slow myosin expression and activates both HDAC4-dependent and NFATc1-dependent signaling pathways during unloading.
Myeloid-derived suppressor cells (MDSC) play a key role in the formation of immune tolerance, including during pregnancy, due to their ability to suppress the immune response using various mechanisms. One of the important regulators of the immune system during gestation is pregnancy specific glycoprotein (PSG), which has pronounced immunosuppressive properties. The aim of this study was to investigate the effect of native and recombinant PSG on the functional activity of MDSC obtained from the peripheral blood of healthy donors. For this purpose, CD11b+ cells were isolated by immunomagnetic separation and differentiated into MDSCs using GM-CSF, IL-1β, and lipopolysaccharide (LPS). Physiological concentrations of native (1, 10, 100 μg/mL) and recombinant (1, 10 μg/mL) PSG were used in the experiment. Cell phenotyping was performed by flow cytometry with determination of PD-L1 and CD73 membrane proteins, as well as measurement of inducible NO synthase (iNOS) levels and analysis of the cytokine profile (17 markers) using multiplex analysis. It was found that recombinant PSG at a concentration of 1 μg/mL significantly increased the density of PD-L1 protein on the surface of MDSC, and at a concentration of 10 μg/mL increased the level of CD73, while native PSG had no significant effect on these parameters. Both variants of PSG did not affect iNOS production, but recombinant PSG (10 μg/mL) reduced the level of MIP-1β chemokine without altering the production of other cytokines studied. The results obtained indicate that recombinant PSG can enhance the immunosuppressive potential of MDSC by increasing the membrane molecules PD-L1 and CD73, as well as suppressing MIP-1β production, which may be important for the development of new bio-pharmacological approaches to correcting the immune response in autoimmune diseases and transplantation. The structural features of recombinant PSG associated with post-translational modifications probably determine its selective effect on the functional properties of MDSC.
Piezo1 channels are mechanically activated (MA) cation channels that play an important role in skeletal muscle physiology. However, the specific mechanisms of Piezo1 regulation in skeletal muscle are not yet fully understood. At the same time, the activity of different ion channels is known to be dependent on lipid rafts, which are dynamic microdomains in the cell membrane enriched in sphingolipids and cholesterol. Therefore, the aim of this study was to evaluate the role of lipid rafts in the muscle-specific regulation of Piezo1 using a cholesterol-removing agent, methyl-beta-cyclodextrin (M beta CD), and the sphingolipid sphingosine-1-phos-phate (S1P) in a C2C12 myoblast model. Fluorescence labeling and intracellular calcium measurements demonstrated that the disruption of lipid rafts with M beta CD resulted in disassembly of the initially present actin cytoskeleton in C2C12 cells and decreased Piezo1-mediated Ca(2+)influx into the cells. In contrast, stimulation of myoblasts with S1P resulted in increased formation of lipid rafts and actin stress fibers, as well as enhanced Ca(2+)influx through Piezo1 channels. These findings suggest the involvement of lipid rafts in the regulation of Piezo1 activity in C2C12 myoblasts, which may be mediated by lipid raft components themselves and/or by lipid raft-induced rearrangements of the actin cytoskeleton.
The research described in this paper is based on a mathematical model for the proton-dependent regulation of electron and proton transport in chloroplasts. We have considered redox transformations of photosynthetic (PS) reaction centres PS1 (P700) and PS2 (P680) and of the mobile electron transport carriers ferredoxin (Fd), plastoquinone (PQ), and plastocyanin (Pc), as well as the transmembrane proton transport processes associated with electron transport and ATP synthesis by membrane ATP synthase. The simulation results are in good agreement with the literature data on pH measurements in the lumen (pHin) and stroma (pHout) of chloroplasts at different cytosol pH values (pHcyt). The steady-state pH values established in these compartments when chloroplasts are illuminated under photophosphorylation conditions satisfy the following inequalities: pHin ≈ 6.2–6.4 < pHcyt ≈ 7.2–7.4 < pHout ≈ 7.8–8.0. Variation of the pHcyt model parameter affects the electron flows along different electron transport pathways, such as non-cyclic electron transport from PS2 to PS1 and further into the Calvin–Benson cycle, cyclic electron transport around PS1, and pseudocyclic electron transport involving molecular oxygen (the ‘water–water’ cycle). It has also been shown that sufficiently strong acidification of the cytosol (pHcyt < 7) reduces electron efflux from the acceptor side of PS1 (noncyclic and pseudocyclin electron transport) and stimulates cyclic electron transport around PS1, and also decreases the rate of pH-dependent ATP synthesis.
The spectral study of the surface and isolated organelles of cells of azulene-containing plants of temperate climate, dry and humid subtropics of Russia was carried out. The presence of azulenes in leaves, flower petals, nuclei and chloroplasts was detected, which was assessed by the appearance of blue color and characteristic maxima in the region of 580–640 nm in the absorption spectra and 405–430 nm in the fluorescence spectra. These data were confirmed experimentally on extracts of these hydrophobic pigments obtained using acetone or ethanol solvents after infusion of intact leaves or isolated organelles (nuclei and chloroplasts) for 10 min (from the surface) or 24 h (also from the surface and from the inside), respectively, as well as after chromatographic purification of the azulene fraction from chlorophyll. The results obtained may be of interest for cellular monitoring of azulene-containing plants of species perspective for pharmacology.
-Microglia cells in the brain are considered resident macrophages possessing a number of functional and physiological characteristics typical of these immune cells. Microglia are involved in neuroinflammatory processes of various etiologies, during which they undergo phenotypic changes. In neuron-glial cultures, microglial cells typically have low proliferative capacity due to the absence of necessary growth factors. In this study, we evaluated the effect of a combination of compounds critical for microglial proliferation, such as transforming growth factor beta (TGF beta), macrophage colony-stimulating factor (MCSF), and cholesterol, on the number and functional activity of microglial cells in hippocampal cultures from newborn rats. We found that the combination TGF beta + MCSF + cholesterol increased the number of microglial cells in cultures by more than twofold. RT-PCR analysis showed that exposure to the pro-inflammatory agent lipopolysaccharide (LPS) in cultures grown using this combination of factors led to increased expression of genes encoding inflammation-associated proteins, such as IL-1 beta, TNF alpha, STAT3, as well as the gene encoding protein vimentin, which acts as a situational marker of reactive microglia. Additionally, incubation with LPS led to increased cell death in the cultures. In the case of hypoxic episode exposure, expression of genes encoding the mentioned pro-inflammatory proteins was suppressed, while the increase in cell death was insignificant. LPS, as well as chemotactic formylated peptide (fMLP, an immune cell activator), caused enhanced production of superoxide anion and increased intracellular Ca2+ concentration in microglial cells. Thus, the described effects of LPS may indicate that the combination TGF beta + MCSF + cholesterol added to the culture medium promotes the preservation and proliferation of functionally active microglial cells in neuron-glial cultures.
Miniature endplate potentials (MEPPs) and multiquantal endplate potentials (EPPs) caused by short rhythmic nerve stimulation were recorded in newly formed neuromuscular synapses of mice using intracellular microelectrode technique. In this work, we investigated which pathway of maturation of mature brain-derived neurotrophic factor (BDNF) from its precursor proBDNF dominates in muscle fibers during their reinnervation—extracellular or intracellular. Matrix metalloprotease 3 (MMP-3) or intracellular proconvertase furin were selectively inhibited in combination with the release of endogenous neurotrophin from muscle fibers upon stimulation of protease-activated receptors (PAR1). It was confirmed that PAR1 stimulation causes an increase in the amplitude of MEPPs due to the release of endogenous BDNF from muscle fibers and its retrograde effect aimed at increasing the quantal size of the acetylcholine (ACh). MMP-3 does not participate in the maturation of BDNF. Inhibition of furin led to a change in the synaptic effect upon stimulation of PAR1. An increase in the amplitude of MEPPs upon activation of PAR1 changes to a decrease in the frequency of MEPPs, which is characteristic of the effect of proBDNF in newly formed synapses. Thus, it has been shown that it is possible to stop the maturation of muscle BDNF by inhibiting the activity of furin at the stage of proneurotrophin in weakened regenerating synapses and eventually ensure the appearance of proBDNF in the synaptic cleft with its spectrum of effects. This may change the balance of the retrograde effect of BDNF and its proneurotrophin on the functioning of newly formed motor synapses. Moreover, a change in this balance can potentially affect not only the regulation of quantal ACh release, but also the rate and severity of reinnervation, since BDNF and proBDNF have a multidirectional effect on the elimination of excessive synaptic contacts in embryogenesis and post-traumatic muscle reinnervation.
Under conditions of insufficient muscle activity (with their functional unloading), a number of pathological processes are observed, leading to deterioration of muscle functions. Some of these processes are based on changes in gene expression, which leads to the transformation of the phenotype of muscle fibers from “slow,” with a predominantly oxidative metabolism and resistant to fatigue, to “fast,” with a predominantly glycolytic metabolism and prone to fatigue. Based on the literature data, it can be assumed that CpG methylation of promoter regions of genes may be involved in regulating the expression of genes that implement the “slow” and “fast” phenotype of muscle fibers. A decrease in the expression of genes regulating mitochondrial biogenesis and muscle fiber phenotype under conditions of mechanical muscle unloading may be determined by a lack of alpha-ketoglutarate (a coenzyme of TET translocases demethylating CpG islands). To test this hypothesis, male Wistar rats were divided into three groups of 8 animals each: (1) C, vivarium control with daily intraperitoneal administration of placebo (saline solution); (2) 7HS, 7-day hind limb suspension with daily intraperitoneal administration of placebo (saline solution), and (3) 7HSD, 7-day hind limb suspension with daily intraperitoneal administration of 200 mg/kg dimethyl-2-ketoglutarate (precursor of alpha-ketoglutarate). Based on the analysis of experimental data, it was found that the administration of dimethyl-2-ketoglutarate to the suspended animals partially prevents the decrease in the accumulation of mRNA regulators of mitochondrial biogenesis and the content of mitochondrial DNA observed during 7-day hind limb suspension. This effect can be realized through the violation of the CpG methylation by the drug; however, in the 7HSD group, an increase in the phosphorylation of AMP-activated protein kinase was also found compared to the 7HS and C groups, which may explain the effect of dimethyl-2-ketoglutarate on the accumulation of mRNA regulators of mitochondrial biogenesis and the content of mitochondrial DNA upon suspension of the hindlimbs of rats.
In arterial smooth muscle cells, Piezo1 channels are involved in the regulation of vascular tone and remodeling in various diseases. They are non-selective cation channels, the activation of which can lead to depolarization of the smooth muscle cell membrane, Ca2+ entry through voltage-gated channels and the development of contraction. This work tested the hypothesis that Piezo1 channels are involved in regulating the tone of smooth muscle cells in small cerebral arteries, and functional contribution of these channels may change in chronic carotid artery stenosis. Constricting clips were placed on both common carotid arteries in rats (reducing the volume velocity of blood flow by at least 70
