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

Endothelial damage as a result of complement system activation is one of the causes of thrombotic complications in COVID-19. Factor C3 plays a key role in this process. The attachment of its proteolysis product C3b to the membrane initiates the beginning of the formation of membrane attack complex C5b-9, which forms a pore in the plasma membrane and cell death. In the present study, we investigated how histamine, secreted in the body at sites of inflammation by leukocytes and mast cells, might affect the binding of C3b to endothelial cells (ECs). FITS-conjugated antibodies against the C3c fragment were used to visualize it. These antibodies bind to intact C3 and to C3b but not to C3a. We have shown that incubation of human blood plasma with cultured ECs from human umbilical vein results in accumulation of C3/C3b as rounded local and diffuse foci on the surface of the cell monolayer. Pre-activation of ECs by histamine increases the number of C3/C3b attachment sites. These data suggest that histamine can enhance endothelial layer damage during hyperactivation of the complement system in COVID-19 and endotheliopathies caused by other diseases.

The interaction of the Yersinia pseudotuberculosis porin OmpF (YpOmpF) with the fluoroquinolone antibiotic norfloxacin (Nf) and its derivatives (mono- and dihydrochloride) was studied using the bilayer lipid membrane (BLM) method, molecular modeling, and antibacterial activity testing. An asymmetric behavior of the Nf charged molecules was found: NfH⁺¹ and Nf2H⁺² moved through the YpOmpF channel, depending on the membrane voltage and on the side where the antibiotic was added. The electrophysiological data were confirmed by computational modeling. For charged forms of the antibiotic, the presence of two peripheral high-affinity binding sites (NBS1 and NBS2), as well as an asymmetric current blocking site (NBS3) near the channel constriction zone were detected. The NBS1 site located near the channel mouth has almost the same affinity for both charged forms of Nf, while the localization of the more energetically favorable NBS2 site for the two salt forms of the antibiotic differs significantly. Nf has only one binding site near the constriction zone, which is a cluster of sites with a lower overall affinity compared to the peripheral binding sites mentioned above. Slight differences were found in the antibacterial activity of the three forms of Nf, which is likely due to their different charge states and, accordingly, different permeability and/or ability to bind within the YpOmpF channel.

Currently, foreign DNA or RNA sensor proteins, which play an important role in innate immunity, are of great interest as a new avenue for cancer immunotherapy. This review considers the functioning of cytoplasmic nucleic acid sensors such as cGAS, STING, IFI16, AIM2, DAI, DDX41, DNA-PK, MRE-11, and TREX1, involved in activating the production of various cytokines.

Anticancer peptides are of interest for cancer treatment. Nowadays, the process of apoptosis is considered a molecular target for cancer therapy. In the present study, the toxic effect of the HL-7 peptide on cervical cancer cells HeLa was investigated using the MTT assay. Also, the expression levels of Bax, Bcl-2, p53, caspase-3, PTEN, and Akt genes in HeLa cells treated with HL-7 were assessed by real-time PCR. Besides, the percentage of cells in early and late stages of apoptosis was determined using flow cytometry. The obtained results indicated that the peptide HL-7 inhibited growth of HeLa cells with IC₅₀ of 31 μM. The expression levels of Bax, p53, caspase-3, and PTEN genes were increased in HeLa cells treated with the HL-7 peptide as compared to untreated HeLa cells, while the expression levels of Bcl-2 and Akt genes was decreased (p < 0.05). The results of flow cytometry analysis indicated a high percentage of cells in the late apoptosis stage (p < 0.05). Our findings suggest that peptide HL-7 can be involved in inducing the mitochondria-dependent apoptosis pathway. However, additional studies are needed to elucidate the exact mechanism of action of the peptide on HeLa cancer cells and the prospects for its therapeutic use in the clinic.

It is known that activated derivatives of long-chain fatty acids acylcarnitines (LCAC) are considered the most toxic, which, along with calcium, can participate in the induction of the mitochondrial pore, involving various types of phospholipases. In this study, the effect of inhibitors of Ca²⁺-independent and Ca²⁺-dependent phospholipases, as well as an inhibitor of carnitine palmitoyltransferase on the induction of pores with D,L-palmitoylcarnitine (PC, C16:0) was investigated. In experiments on isolated rat liver mitochondria, the effect of PC on mitochondrial respiration rate, membrane potential (ΔΨm) and mitochondrial swelling during oxidation of glutamate and pyruvate or succinate was studied. It was shown that inhibitors of carnitine palmitoyltransferase-1 etomoxir 2, Ca²⁺-dependent phospholipase cPLA2 aristolochic acid or Ca²⁺-independent phospholipase iPLA2γ bromoenol lactone and PACOCF3 caused an increase in critical concentrations of D,L-palmitoylcarnitine (PC*), which were required to decrease the membrane potential and induce mitochondrial swelling. In the ADP activated state 3 (ADP + Mg²⁺ + hexokinase), Ethomoxir 2 and aristolochic acid promoted the inhibition of respiration and dissipation of membrane potential caused by excess of PC, while phospholipase inhibitors iPLA2γ PACOCF3 and bromoenol lactone provided a pronounced protective effect. Inhibition of iPLA2γ prevented the decrease of ΔΨm and inhibition of respiration caused by PC. Thus, the results obtained indicated the involvement of mitochondrial phospholipase iPLA2γ in the induction of the mitochondrial pore by long-chain acylcarnitines.

In the aging and the development of age-associated diseases, the trigger mechanism is the hyperactivation of the hypothalamic-pituitary-adrenal neuroendocrine axis, hypersecretion of glucocorticoids, which, under excessive and long-term stimulation, have inflammatory and degenerative effects. Chronic stress exacerbates glucocorticoid-dependent atrophic changes in the aging brain, increases neuroinflammation and neurological dysfunction, and is a key risk factor for Alzheimer’s disease. In the correction of aseptic neuroinflammation in elderly and senile patients, the use of anti-inflammatory agents that exhibit anti-glucocorticoid (pro-anabolic) and anti-glutamate (anti-excitotoxic) effects is pathogenetically justified. Succinate/SUCNR1 signalling is involved in the development of immunomodulatory, trophic, and anti-hypoxic effects; however, its role in the mechanisms of stress response remains unexplored. The aim of this study was to assay the impact of succinate/SUCNR1 signalling on the development of stress-induced neuroinflammation in the cerebral cortex of old rats. The work was performed on outbred albino male rats aged 18 months. Chronic restraint stress was modelled by immobilizing animals in individual plastic cases for 6 h daily for 5 days. Mexidol (2-ethyl-6-methyl-3-hydroxypyridine (EMHP) succinate) was used as a form of succinate that crosses the blood-brain barrier. Mexidol was administered intraperitoneally to old rats at a dose of 100 mg/kg daily for 5 days 15 min before the onset of stress. The levels of proinflammatory cytokines (IL-1β, TNF-α), anti-inflammatory cytokines (TGF-β1, IL-10), glucocorticoid receptors (GRα), transcriptional coactivator PGC-1α, succinate receptor SUCNR1/GPR91, and vascular endothelial growth factor (VEGF) were determined by immunoblotting in cerebral cortex (CC) samples. It was shown that chronic immobilization stress caused an increase in the level of IL-1β and TNF-α during stress, which was accompanied by a decrease in the content of anti-inflammatory cytokines, SUCNR1, GRα, PGC-1α. The course administration of EMHP succinate limited the development of stress-induced neuroinflammation in the CC of old rats and prevented a decrease in the levels of SUCNR1, IL-10, TGF-β1, PGC-1α, and GRα. The study reveals for the first time the stress-protective potential of succinate/SUCNR1 signalling in the brain of old rats associated with the activation of PGC-1α-dependent anti-inflammatory mechanisms under conditions of chronic stress.

Gamma-aminobutyric acid (GABA) is commonly considered as a signaling molecule in the synapses of the central nervous system, where it plays the role of the main inhibitory neurotransmitter in the mature brain and participates in the process of neurogenesis. Recently, data have been obtained indicating that GABA may also be involved in the early stages of skeletal muscle tissue development. In the present study, performed on rat myocyte culture, the effect of exogenous GABA on the process of fusion of myocytes into myotubes was investigated by analyzing such a morphometric indicator as the “fusion index”. The addition of the amino acid to the culture led to a significant concentration-dependent inhibition (up to a complete cessation) of the formation of myotubes. GABA_A receptors and GABA transporters (GAT-2) were considered among possible proteins capable of mediating the effect of amino acids. Evidence of the presence of these proteins on cultured cells was obtained using immunohistochemical methods. The blockade of GABA receptors by gabazine did not affect the fusion index in any way, and GABA continued to exert inhibitory effect in its presence. Inhibition of GABA transporters by nipecotic acid reduced the myocyte fusion index; however, the GABA effect was no longer present under the action of the transporter blocker. The data obtained are consistent with the hypothesis about the participation of the amino acid GABA at the early stages of skeletal muscle development; it suggests that the inhibitory effect of the exogenous amino acid may be due to an increase in its concentration in the sarcoplasm, since both the addition of a GABA transporter blocker and an increase in the extracellular concentration of GABA negatively affect the formation of myotubes.

Neurodegeneration is a complex progressive pathological process leading to the neuronal death, which is induced by various external and internal factors. Neurodegenerative diseases, injuries of the central and peripheral nervous system, mental disorders, and a number of other pathological conditions, accompanied by functional and structural degradation of neurons and their death, is a serious problem in the global healthcare system, as due to these diseases millions of people around the world become disabled or die every year. The situation is complicated by the lack of selective, clinically effective neuroprotective drugs. It has been shown that nitric oxide (NO) and hydrogen sulfide (H₂S) are actively involved in neurodegeneration and cell death of neurons and glia, but their role is not completely clear. This review considers NO- and H₂S-dependent signaling mechanisms underlying the pathogenesis of neurodegenerative processes. The prospects for further studies of the role of NO and H₂S in the nervous tissue under conditions of pathological conditions associated with neurodegeneration are considered.

The spectral characteristics of plant surface cells of different evolutionary levels, from unicellular (diatom algae and spores of horsetails and ferns) to multicellular (woody and herbaceous species) organisms, have been studied. It was shown that the surface layers of the cuticle and cell wall of some analyzed plants included antioxidants – blue pigments azulenes. Using histochemical methods, it has been discovered that here neurotransmitter compounds (biogenic amines) are present in the excretions on the entire surface or are released from specialized secretory structures of leaves. Under conditions of high salt concentration, dopamine and histamine are secreted, which is blocked by the addition of exogenous azulene and proazulene grosshemine. We suggest that the azulene-containing surface protects from the formed reactive oxygen species and toxic biogenic amines in high concentrations.

The effect of recombinant human chorionic gonadotropin (hCG) at pregnancy-appropriate concentrations (10 and 100 IU/mL) on differentiation and functional activity of myeloid-derived suppressor cells (MDSCs) was studied. The object of the study was isolated CD11b⁺ cells that were converted to the MDSC phenotype by two-step activation with GM-CSF cytokines, IL1β and lipopolysaccharide (LPS). After a week of cultivation, the total MDSC level was determined considering the subpopulations M-MDSC and PMN-MDSC, the expression of arginase-1 (Arg1) and indoleamn-2,3-dioxydiogenase (IDO) in these cells, as well as the cytokine profile in cell culture supernatant. It was shown that hCG increased the total number of MDSCs, and its lower concentration (10 IU/mL) contributed to the differentiation of the M-MDSC subpopulation. hCG did not affect the expression of IDO expression in MDSCs, but there was a tendency to increase IDO expression under the influence of hCG at a concentration of 10 IU/mL. CD11b⁺ cells converted to the MDSC phenotype had a low Arg 1 content, making it impossible to evaluate the effect of the hormone on the expression of this enzyme. Evaluation of the cytokine profile by multiplex analysis showed that hCG did not modulate cytokine production in the culture of CD11b⁺ cells converted the MDSC phenotype. This is the first time that hCG has been shown to induce differentiation of MDSCs.