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

Principles of resistance to physicochemical stresses in extremophile organisms are unique for the development of new protective technologies. In this work, Dsup (Damage suppressor) protein, which belongs to one of the most radiation-resistant animals—tardigrade Ramazzottius varieornatus, was studied as a perspective radioprotector for proton therapy. Dsup, known for its effective DNA-protective properties, was produced in human cells HEK293 expressing Dsup gene. To study radio resistance, human cells were irradiated by high-energy protons (150 MeV) at Medical-Technical Complex (MTC) of Phasatron (JINR). Within the actual medical protocol of proton radiation therapy, Dsup was confirmed to be the potential protector for healthy cells and tissues.

The distribution of charged cytostatics or photosensitizers (PS) in tumor cells (TC) is an important factor in anticancer therapy effectiveness. The aim of our study was to build the mathematical model of the distribution kinetics of charged PSs, namely, chlorin e6 (E6^‒) and its derivative dimethyl ether (DME⁺) in TC with variable electric transmembrane potentials (TMPs) on mitochondrial and nuclear membranes. A kinetic model is presented that includes a system of four nonlinear differential equations describing the accumulation of PS in a certain model system based on the Nernst theory. This system consists of compartments separated by membranes with different TMPs. The sum of these potentials includes negative plasma and mitochondrial TMPs, as well as an energy-dependent positive TMP on the inner nuclear membrane. Numerical solutions of the nonlinear system of equations with given modulation parameters of TMPs were obtained using a special computer program. The initial rates of PSs transfer and membrane permeability were determined using the curves of fluorescence intensity changes of E6^‒ and DME⁺ in lymphoblast cells. Comparative analysis of the data showed that the effective accumulation of the charged drugs depended on the optimal TMPs ratio in tumor cells. These TMPs can be determined using voltage sensitive fluorescent cationic probe 4-(p-di-methylaminostyryl)-1-methylpyridinium (DSM⁺).

Multifunctional nanomaterials which are active in the near infrared (NIR) region and can be physically guided (laser light, magnetic field, ultrasound, etc.) hold a great promise in several biomedical applications, such as drug delivery, cell biology, biosensing, and bioimaging. In this study, we have developed multifunctional photoluminescence coding magnetic microspheres (PCMMs), studied their physical and chemical properties, and explored the possibility of using PCMMs in living biological organisms. To reach this goal, we investigated the possibility of PCMM imaging in the C. elegance animal model. We performed extensive toxicity screening of 10 types of luminescent magnetic microspheres (LMMs) and 5 types of carbon dots (CDs), which were embedded into LMM. We also explored the delivery and localization of tested nanoparticles inside the nematode body. It was found that the particles we studied are not toxic to living C. elegance tissue in the optimized concentration range and tend to extend the lifespan of nematodes. Fluorescent microscopy studies revealed the localization of CDs and LMMs in the intestinal part of the nematode body. Our results demonstrated the possibility of detecting photoluminescent PCMM magnetic microspheres in living organisms, implying the potential of PCMM for the development of this nanoscale drug delivery system for future human studies.

This study explores the interaction of bioactive AED tripeptide molecules with new lysine-based dendrimer KHR containing spacers consisting of two different amino acid residues (histidine-arginine, HR). Previous research has examined second-generation lysine dendrimers containing double charged lysine-lysine and arginine-arginine spacers as well as double hydrophobic alanine-alanine and leucine-leucine, and double pH-sensitive histidine-histidine spacers. Interactions of some of these dendrimers with several bioactive peptide, for example, AEDG were studied also. In this work, similar molecular dynamics simulations were conducted to study the complexation of 16 AED molecules with KHR dendrimer at two pH values: (a) pH > 7 with uncharged histidines and (b) pH < 5 with fully protonated histidines. Results indicate that dendrimer at both pH make complex with AED but dendrimer at smaller pH can accommodate more AED molecules.

Hypervirulent mucoviscus Klebsiella pneumoniae (hvKp) is rapidly emerging as opportunistic pathogens that have a global impact leading to a significant increase in mortality rates among clinical patients. Anti-virulence strategies that target bacterial adhesion and biofilm formation, proposed as alternatives to antibiotic treatments for reducing the rapid emergence of bacterial resistance. The main objective of this study was to examine the efficacy of fatty acid-enriched extract (AWME3) derived from the fat of Hermetia illucens fly larvae to combat biofilms formed by multidrug-resistant (MDR) and highly virulent hvKp pathogens. To reach these goals we used crystal violet (CV) and ethidium bromide (EtBr) assays on the mature biofilms of K. pneumoniae KPi1627, KPM9 and ATCC BAA-2473 strains. We found that exposure of hvKp strains to AWME3 at a concentration of 500 µg/mL (corresponding to 2× minimum inhibitory concentration, MIC) significantly affects the membrane permeability (p < 0.0001), causing serious disturbances and changes in the composition of membrane phospholipids, as confirmed by the detected increase in CV and EtBr uptake by 70 and 80%, respectively, compared to the control group. We used scanning electron microscopy (SEM) as a direct microscopic method to collect evidence of bactericidal action of AWME3 extract and the destruction of hvKp biofilms. In conclusion, our study demonstrates the exceptional capability of natural AWME3 extract, enriched with a unique combination of fatty acids, to effectively eliminate biofilms formed by highly drug-resistant and highly virulent pathogens K. pneumoniae (hvKp). Our results highlight the potential to control and minimize the rapid emergence of bacterial resistance by treating biofilm-associated infections caused by hvK pathogens with AWME3.

Hibernation of small mammals is unique in that it consists of bouts—alternating periods of deep hibernation and inter-bout arousals, during which the animals warm up and many body parameters return to the euthermic level. Significant fluctuations in body temperature during the transition of an animal from a torpid state to a euthermic one can affect membrane-bound enzymes. In this work, the activity and kinetic parameters of acetylcholinesterase (AChE) in erythrocyte membranes of small ground squirrels (Spermophilus pygmaeus Pall.) during deep torpor and in the dynamics of exit from it was studied. The activity of AChE in the membranes of ground squirrel erythrocytes was determined by Ellman’s method. The kinetic characteristics of AChE (maximum velocity (V_m), Michaelis constant (K_m) and substrate inhibition constant (K_i)) were found by the least squares method in accordance with the Haldane model. The study showed that in the torpid state, the activity and V_m of AChE do not change significantly. At the same time, K_m decreases and K_i increases, which contributes to a significant change in the character of the concentration dependence of AChE and a decrease in the degree of substrate inhibition. During arousal of ground squirrels from hibernation at Tb 25°C, V_m increases significantly, which sharply increases the efficiency of AChE catalysis, despite the fact that the K_m value also increases. After the final warming of the ground squirrels from hibernation (Tb 37°C), the values of V_m and K_m of AChE and, accordingly, the efficiency of catalysis do not normalize but remain at the level achieved at Tb 25°C. In this case, K_i values decrease relative to hibernation, which increases the degree of substrate inhibition and narrows the range of effective acetylcholine concentrations. The detected sharp changes in the activity of AChE of ground squirrel erythrocytes AChE during awakening can play an important role in the rapid decrease in the level of circulating acetylcholine and the improvement of peripheral microcirculation.

The changes of electrical parameters of azolectin membranes in a static inhomogeneous magnetic field at the one-sided addition of positively charged quasi-spherical superparamagnetic magnetite nanoparticles (MNPs) with a diameter of about 4 nm were studied. The magnet was located at different distances from the membrane, and nanoparticles were attracted to the membrane surface with different strengths by the magnetic field. After the addition of MNPs, a decrease in capacitance, polarization, and further depolarization of the membranes was observed. Zero current appeared on the bilayer lipid membranes (BLMs). There was no unambiguous dependence of the zero current on the membrane depolarization and there were no dependences of the zero current on the strength of the magnetic field and on the concentration of added MNPs. The membrane conductivity increased on average with an increase in the external magnetic field and depended on the ionic strength. Magnetic field caused a rapid accumulation of MNPs on the surface of the BLMs. As a result, the membrane was polarized. Depolarization of the membranes occurred when MNPs passed through the BLMs. The increase in conductivity reflected the processes of lipid bilayer disordering.

Members of the insulin receptor family (InsR, IGF1R, and IRR) play a key role in metabolic regulation, yet the structural mechanisms governing their activation remain incompletely understood. While extracellular and kinase domain structures are well characterized, the transmembrane domains (TMDs), responsible for transmitting conformational changes, lack high-resolution structural data, particularly in their physiologically relevant dimeric states. This limitation impedes the development of targeted therapies for diseases like diabetes and cancer, where aberrant receptor signaling is a key driver. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for studying membrane proteins in near-native environments, offering insights into their dynamics and interactions. However, production of isotopically labeled TMDs for NMR studies remains challenging due to low expression yields and solubility issues. Here, we present an optimized cell-free continuous-exchange expression system for high-yield production of InsR, IGF1R, and IRR TMDs, coupled with efficient purification proved by heteronuclear NMR spectroscopy in a membrane mimicking environment.

The effect of the geometric configuration and charge of membrane opioid receptor (OR) agonists and antagonists on binding to μ, δ, and κ opioid receptors has been studied by molecular docking. For the docking procedure, three-dimensional structures of pharmaceutical preparations obtained by X-ray diffraction analysis (XRD) and available in the Cambridge Crystal Structures Database (CCDC), as well as their three-dimensional models constructed in a molecular editor, were used. The three-dimensional crystal structure of nalmefene, which is absent in the CCDC database, was first obtained in the presented study by XRD. Protonated and deprotonated forms of ligands were considered. The results of the study using morphine, codeine, naloxone, naltrexone, and nalmefene as examples showed that the method of obtaining three-dimensional geometric structures of opioid receptor ligands practically did not affect the calculated free energy of binding ΔG, which indicates the possibility of using ligand models constructed in silico in computational experiments. The protonation state of the ligand molecule, on the contrary, had a significant effect on free energy of binding to OR, which might affect the properties of this group of drugs with pH changes in the body. When considering the binding features of opioid enantiomers to the ligand-binding site of μ-opioid receptors, using morphine as an example, it was shown that (–)-morphine and (+)-morphine share a common site for the cationic group, and not for phenolic hydroxyl, as previously assumed. At the same time, studies have shown that molecular docking only partially made it possible to describe the pharmacological effect of analgesics and their antagonists. For some substances, such as codeine and synthetic (+)-morphine, the experiment in silico overestimated the effectiveness of the drug’s interaction with OR, and this requires continued improvement of the corresponding calculation methods and models.

Irritable bowel syndrome (IBS) is a multifactorial disorder, with a high socioeconomic impact, characterized by chronic abdominal pain, bloating, and alterations in bowel habits. The aim of our study was to study the role of voltage gated and large conductance calcium-activated potassium channels in the effect of sodium butyrate on spontaneous and induced by agonists of cholinoreceptors contractions of proximal colon in a mouse model of IBS. IBS was induced by intracolonic infusion of acetic acid in the early postnatal period. The contractile activity of proximal colonic segments was then studied in isometric conditions. The amplitude and frequency of colon contractions were higher in the IBS group. Sodium butyrate demonstrated an inhibitory effect on the amplitude of spontaneous and cholinoreceptor agonist-induced contractions of colon, with lower efficiency in the IBS group. Application of the inhibitors of voltage gated and large conductance calcium-activated potassium channels (BK channels) revealed that BK channels are involved in the inhibitory effects of sodium butyrate on spontaneous and evoked contractile activity in the control group. In the IBS group, BK inhibitor was not effective to prevent sodium butyrate effects on the amplitude of spontaneous contractions; however, it prevented the inhibitory effects of sodium butyrate on the amplitude of contractions induced by epibatidine. We conclude that the inhibitory effects of sodium butyrate on the amplitude of spontaneous and carbachol-/epibatidine-induced contractions are mediated by activation of BK channels of smooth muscle cells or cholinergic terminals. Activation of these channels results in membrane hyperpolarization and a reduction in acetylcholine release with further decreased contractile activity.