Нелинейные локальные деформации мембран эритроцитов: действие токсинов и фармпрепаратов (Часть 2)

Modifiers of membranes cause local defects on the cell surface. Measurement of the rigidity at the sites of local defects can provide further information about the structure of defects and mechanical properties of altered membranes.The purpose of the study: a step-by-step study of the process of a nonlinear deformation of red blood cells membranes under the effect of modifiers of different physico-chemical nature.Materials and methods. The membrane deformation of a viscoelastic composite erythrocyte construction inside a cell was studied by the atomic force spectroscopy. Nonlinear deformations formed under the effect of hemin, Zn2+ ions, and verapamil were studied.Results. The process of elastic deformation of the membrane with the indentation of a probe at the sites of local defects caused by modifiers was demonstrated. The probe was inserted during the same step of the piezo scanner z displacement; the probe indentation occured at the different discrete values of h, which are the functions of the membrane structure. At the sites of domains, under the effect of the hemin, tension areas and plasticity areas appeared. A mathematical model of probe indentation at the site of membrane defects is presented.Conclusion. The molecular mechanisms of various types of nonlinear deformations occurring under the effect of toxins are discussed. The results of the study may be of interest both for fundamental researchers of the blood cell properties and for practical reanimatology and rehabilitology. Модификаторы мембран порождают локальные дефекты на поверхности клеток. Измерение жесткости в зонах локальных дефектов может дать дополнительную информацию о структуре дефектов и механических свойствах мембран в целом.Цель работы: пошаговое исследование процесса нелинейной деформации мембран эритроцитов при действии на них модификаторов различной физико-химической природы.Материалы и методы. Методом атомно-силовой спектроскопии провели исследование деформации мембран внутрь клетки вязко-упругой композитной конструкции эритроцита. Исследовали нелинейные деформации при действии гемина, ионов Zn2+, фармпрепарата верапамила.Результаты. Показали процесс упруго-эластичной деформации мембраны по мере индентации зонда в областях локальных дефектов, вызванных модификаторами. На один и тот же шаг смещения z пьезосканера зонд погружался на разные дискретные величины h, которые являются функциями структуры мембраны. В областях доменов при действии гемина возникали зоны натяжения и зоны пластичности. Привели математическая модель индентации зонда в области дефектов мембран.Заключение. Обсудили молекулярные механизмы различных видов нелинейных деформаций, возникающих при действии токсинов. Результаты работы могут представлять интерес как для фундаментальных исследований свойств клеток крови, так и для практической реаниматологии и реабилитологии

Real-Time Imaging of HIF-1α Stabilization and Degradation

HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α

Vibration Diagnostics of Power Equipment Before Commissioning

The article describes how to use vibration diagnostics to detect defects in the installation of new equipment and to determine the quality of repairs based on vibroacoustic characteristics. Especially relevant is the vibration diagnostics of rolling bearings before installation on the mechanism. If there are errors in the calculations of the magnetic system of synchronous generators, vibration occurs, which is not eliminated during operation. Design errors, poor assembly of pumping units, asynchronous motors, power transformers cause increased vibration, which leads to damage to the specified equipment. The main reasons causing the vibration of the transformer tank are considered and the frequency range from 50 to 6000 Hz is determined on which these defects can occur. Full-scale experiments of the vibration of the transformer tank have shown that certain defects can occur in a narrow frequency band. The practical application vibration diagnostics of the pump unit describes in this article. Technical diagnosis of pumping units of the sewer station was carried out for various included composition. The results obtained made it possible to exclude reverse hydraulic impacts as a possible cause of the failure of the pump unit

Nonlinear Biomechanical Characteristics of Deep Deformation of Native RBC Membranes in Normal State and under Modifier Action

The ability of membranes of native human red blood cells (RBCs) to bend into the cell to a depth comparable in size with physiological deformations was evaluated. For this, the methods of atomic force microscopy and atomic force spectroscopy were used. Nonlinear patterns of deep deformation (up to 600 nm) of RBC membranes were studied in normal state and under the action of modifiers: fixator (glutaraldehyde), natural oxidant (hemin), and exogenous intoxicator (zinc ions), in vitro. The experimental dependences of membrane bending for control RBC (normal) were approximated by the Hertz model to a depth up to 600 nm. The glutaraldehyde fixator and modifiers increased the absolute value of Young’s modulus of membranes and changed the experimental dependences of probe indentation into the cells. Up to some depth hHz, the force curves were approximated by the Hertz model, and for deeper indentations h>hHz, the degree of the polynomial function was changed, the membrane stiffness increased, and the pattern of indentation became another and did not obey the Hertz model. Quantitative characteristics of nonlinear experimental dependences were calculated for deep bending of RBC membranes by approximating them by the degree polynomial function

Conformational Distortions of the Red Blood Cell Spectrin Matrix Nanostructure in Response to Temperature Changes In Vitro

The spectrin matrix is a structural element of red blood cells (RBCs). As such, it affects RBC morphology, membrane deformability, nanostructure, stiffness, and, ultimately, the rheological properties of blood. However, little is known about how temperature affects the spectrin matrix. In this study, the nanostructure of the spectrin network was recorded by atomic force microscopy. We describe how the nanostructure of the RBC spectrin matrix changes from a regular network to a chaotic pattern following an increase in temperature from 20 to 50°C. At 20–37°С, the spectrin network formed a regular structure with dimensions of typically 150±60 nm. At 42–43°С, 83% of the spectrin network assumed an irregular structure. Finally, at 49–50°С the chaotic pattern was observed, and no quantitative estimates of the spectrin structure’s parameters could be made. These results can be useful for biophysical studies on the destruction of the spectrin network under pathological conditions, as well as for investigating cell morphology and blood rheology in different diseases

Partial Fluxes of Phosphoric Acid Anions through Anion-Exchange Membranes in the Course of NaH2PO4 Solution Electrodialysis

Electrodialysis (ED) with ion-exchange membranes is a promising method for the extraction of phosphates from municipal and other wastewater in order to obtain cheap mineral fertilizers. Phosphorus is transported through an anion-exchange membrane (AEM) by anions of phosphoric acid. However, which phosphoric acid anions carry the phosphorus in the membrane and the boundary solution, that is, the mechanism of phosphorus transport, is not yet clear. Some authors report an unexpectedly low current efficiency of this process and high energy consumption. In this paper, we report the partial currents of H2PO4−, HPO42−, and PO43− through Neosepta AMX and Fujifilm AEM Type X membranes, as well as the partial currents of H2PO4− and H+ ions through a depleted diffusion layer of a 0.02 M NaH2PO4 feed solution measured as functions of the applied potential difference across the membrane under study. It was shown that the fraction of the current transported by anions through AEMs depend on the total current density/potential difference. This was due to the fact that the pH of the internal solution in the membrane increases with the growing current due to the increasing concentration polarization (a lower electrolyte concentration at the membrane surface leads to higher pH shift in the membrane). The HPO42− ions contributed to the charge transfer even when a low current passed through the membrane; with an increasing current, the contribution of the HPO42− ions grew, and when the current was about 2.5 ilimLev (ilimLev was the theoretical limiting current density), the PO43− ions started to carry the charge through the membrane. However, in the feed solution, the pH was 4.6 and only H2PO4− ions were present. When H2PO4− ions entered the membrane, a part of them transformed into doubly and triply charged anions; the H+ ions were released in this transformation and returned to the depleted diffusion layer. Thus, the phosphorus total flux, jP (equal to the sum of the fluxes of all phosphorus-bearing species) was limited by the H2PO4− transport from the bulk of feed solution to the membrane surface. The value of jP was close to ilimLev/F (F is the Faraday constant). A slight excess of jP over ilimLev/F was observed, which is due to the electroconvection and exaltation effects. The visualization showed that electroconvection in the studied systems was essentially weaker than in systems with strong electrolytes, such as NaCl

Introducing a new reporter gene, membrane-anchored Cypridina luciferase, for multiplex bioluminescence imaging

Bioluminescence reporter gene imaging is a robust, high-throughput imaging modality that is useful for tracking cells and monitoring biological processes, both in cell culture and in small animals. We introduced and characterized a novel bioluminescence reporter—membrane-anchored Cypridina luciferase (maCLuc)—paired with a unique vargulin substrate. This luciferase-substrate pair has no cross-reactivity with established d-luciferin- or coelenterazine-based luciferase reporters. We compare maCLuc with several established luciferase-based reporter systems (firefly, click beetle, Renilla, and Gaussia luciferases), using both in vitro and in vivo models. We demonstrate the different imaging characteristics of these reporter systems, which allow for multiplexed-luciferase imaging of 3 and 4 separate targets concurrently in the same animal within 24 h. The imaging paradigms described here can be directly applied for simultaneous in vivo monitoring of multiple cell populations, the activity of selected signal transduction pathways, or a combination of both constitutive and inducible reporter imaging