Changes In Electrophoretic Mobility Research Articles

KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells.

Connexins (Cxs) are proteins that help cells to communicate with the extracellular media and with the cytoplasm of neighboring cells. Despite their importance in several human physiological and pathological conditions, their pharmacology is very poor. In the last decade, some molecules derived from aminoglycosides have been developed as inhibitors of Cxs hemichannels. However, these studies have been performed in E. coli, which is a very simple model. Therefore, our main goal is to test whether these molecules have similar effects in mammalian cells. We transfected HeLa cells with the human Cx46tGFP and characterized the effect of a kanamycin-derived molecule (KI04) on Cx46 hemichannel activity by time-lapse recordings, changes in phosphorylation by Western blot, localization by epifluorescence, and possible binding sites by molecular dynamics (MD). We observed that kanamycin and KI04 were the most potent inhibitors of Cx46 hemichannels among several aminoglycosides, presenting an IC50 close to 10 μM. The inhibitory effect was not associated with changes in Cx46 electrophoretic mobility or its intracellular localization. Interestingly, 5 mM DTT did not reverse KI04 inhibition, but the KI04 effect completely disappeared after washing out KI04 from the recording media. MD analysis revealed two putative binding sites of KI04 in the Cx46 hemichannel. These results demonstrate that KI04 could be used as a Cx46 inhibitor and could help to develop future selective Cx46 inhibitors.

Single-particle electrophoresis for studying the adsorption of cationic polymers onto anionic particles.

Understanding the adsorption of polymers onto particles is crucial for many technological and biomedical applications. Even though polymer adsorption on particles is a dynamic process, most experimental techniques can only study the adsorption indirectly, in equilibrium and on the ensemble level. New analysis methods are required to overcome these limitations. We investigated the use of single-particle electrophoresis to study the adsorption kinetics of cationic polymers onto anionic particles and compared the resulting data to a theoretical model. In this approach, the electrophoretic mobility of single polystyrene (PS) particles, exposed to different concentrations of poly(2-guanidinoethyl methacrylate), was measured as a function of time. The polymer adsorption leads to an electrophoretic mobility change of the PS particle over time, from the initial negative value to a positive value at equilibrium. By fitting the kinetics data to the Langmuir model, the adsorption rate, desorption rate and equilibrium constant were determined. Finally, the adsorption kinetics of several other polymers was investigated. This showed that the presented technique enables direct analysis and comparison of the kinetics of polymer adsorption on the single-particlelevel.

Pathophysiological Role of Variants of the Promoter Region of CITED2 Gene in Sporadic Tetralogy of Fallot Patients with Cellular Function Verification.

Tetralogy of Fallot (TOF) is a common congenital heart malformation. Genetic variants in the CITED2 coding region are known to be significantly associated with cardiac malformation, but the role of variants in the CITED2 promoter region in the development of TOF remains unclear. In this study, we investigated CITED2 promoter variants in the DNA of 605 subjects, including 312 TOF patients and 293 unrelated healthy controls, by Sanger sequencing. We identified nine CITED2 gene promoter variants (including one novel heterozygous variant). Six were found only in patients with TOF and none in the control group. The transcriptional activity of the CITED2 gene promoter in mouse cardiomyocyte (HL-1) cells was significantly altered by the six variants (p < 0.05). The results of the electrophoretic mobility change assay and JASPAR database analysis showed that these variants generated or destroyed a series of possible transcription factor binding sites, resulting in changes in the CITED2 protein expression. We conclude that CITED2 promoter variants in TOF patients affect transcriptional activity and may be involved in the occurrence and progression of TOF. These findings may provide new insights into molecular pathogenesis and potential therapeutic insights in patients with TOF.

Genetic Variants of CITED2 Gene Promoter in Human Atrial Septal Defects: Case-Control Study and Cellular Functional Verification.

Atrial septal defect (ASD) is one of the most common forms of congenital heart disease (CHD). Genetic variants in the coding region of the CITED2 gene are known to be significantly correlated with CHD, but the role of variants in the promoter region of CITED2 is unknown. We investigated variants in the promoter of the CITED2 gene in 625 subjects (332 ASD and 293 healthy controls) through Sanger sequencing. Four variants in the CITED2 gene promoter were found only in eight ASD patients with zero occurrence in the control subjects (one case of g.4078A>C(rs1165649373), one case of g.4240C>A(rs1235857801), four cases of g.4935C>T(rs111470468), two cases of g.5027C>T(rs112831934)). Cellular functional analysis showed that these four variants significantly changed the transcriptional activity of the CITED2 gene promoter in HEK-293 and HL-1 cells. Electrophoretic mobility change assay results and JASPAR database analysis demonstrated that these variants created or destroyed a series of possible transcription factor binding sites, resulting in changes in the expression of CITED2 protein. We conclude that the variants of CITED2 promoter in ASD patients affect the transcriptional activity and are likely involved in the occurrence and development of ASD. These findings provide new perspectives on the pathogenesis and potential therapeutic insights of ASD.

A phosphorylation network controls the stability of alpha‐arrestins Aly1/Art6 and Aly2/Art3

Selective protein trafficking controls the repertoire of membrane proteins at the cell surface. This contingent of surface proteins regulates nutrient/metabolite balance and response to extracellular cues. Environmental changes trigger cellular signaling that drives transitions in the plasma membrane proteome. These alterations are achieved in no small part through regulation of the α‐arrestins, an emergent and powerful class of protein trafficking adaptors. The α‐arrestins are largely cytosolic proteins that are transiently recruited to membranes via binding to membrane protein motifs. α‐Arrestins bring with them a ubiquitin ligase, which stimulates ubiquitination and subsequent endocytosis of membrane proteins. Phosphorylation of α‐arrestins is key to regulating their trafficking function; dephosphorylated α‐arrestins are generally ‘active’ endocytic adaptors. While some kinases and phosphatases for α‐arrestins are known, the degree of α‐arrestins phosphorylation is staggering, with &gt;40 phosphorylation sites identified for a single α‐arrestin, and suggests we still have much to learn about α‐arrestins regulation. We sought to determine what kinases and phosphates regulate paralogous α‐arrestins Aly1 and Aly2 using a genetic screen in yeast. Using an array of all known non‐essential kinase and phosphatase deletions, we determined which of these influenced α‐arrestin‐mediated resistance to rapamycin, an inhibitor of the TORC1 nutrient‐sensing kinase. We identified a large cohort of kinases and phosphates that influenced Aly‐dependent phenotypes, causing electrophoretic mobility changes and, in many cases, diminishing the abundance of these α‐arrestins. We focused our studies on the Sit4 protein phosphatase, a key regulator downstream of TORC1 signaling able to influence other α‐arrestins, identified in our screen. Strikingly, Aly1 and Aly2 were hyperphosphorylated and destabilized in the absence of Sit4, suggesting that excessive phosphorylation may promote degradation of these α‐arrestins. For Aly2, but not Aly1, degradation in the sit4∆ cells could be reversed by loss of the vacuolar protease Pep4, indicating that vacuolar degradation predominates for Aly2 under these conditions. Loss of the Npr1 kinase in sit4∆ cells restored Aly protein abundances and reversed the hyperphosphorylation, demonstrating that this kinase is responsible for the excess Aly phosphorylation in sit4∆ cells. This is a remarkable finding as typically Npr1 is considered inactive in sit4∆, however, we suggest that Npr1 is selectively active in the absence of Sit4, able to modify some substrates but not others. We define new features of TORC1 signaling in regulating α‐arrestin phosphorylation and stability.

Variants and Cellular Functional Verification of CITED2 Gene Promoter Region in Patients with Atrial Septal Defect

ObjectivesCongenital heart disease (CHD) is a common disease of human birth defects, and atrial septal defect (ASD) is the most common form of ASD. Genetic variants in the coding region of the CITED2 gene is known to be significantly correlated with cardiac malformations, but variants of the CITED2 promoter region and its relationship with the formation of ASD are still unclear. We hypothesize that the variants of the CITED2 promoter may be related to pathogenesis of ASD. The purpose of this study was to screen variants in the promoter region of the CITED2 gene and to verify the effect of the variants on gene expression at the cellular level.MethodsThe blood samples of 332 ASD patients and 293 unrelated healthy children used as controls were studied. The total DNA of all subjects was extracted, and the CITED2 promoter variants were screened by PCR combined with Sanger sequencing. The luciferase activity of the CITED2 promoter was measured by a dual luciferase reporter at the cellular level. Electrophoretic mobility change assay (EMSA) and bioinformatics analysis were used to test the effect of CITED2 promoter changes on transcription factor binding sites.ResultsFour variants in the promoter region of the CITED2 gene were found only in the 332 ASD patients with zero occurrence in the 293 control subjects. These included one novel heterozygous variant (g.4933C&gt;A) and three SNPs [1 case of g.4078 A&gt;C(rs1165649373), 4 cases of g.4935C&gt;T(rs111470468), 2 cases of g.5027C&gt;T (rs112831934)]. The cellular functional analysis showed that these four variants significantly changed the transcriptional activity of the CITED2 gene promoter in HEK‐293 cells (P&lt;0.05). In addition, EMSA results and database analysis showed that these variants created or destroyed a series of possible transcription factor binding sites, resulting in changes of the expression of CITED2 protein.ConclusionThe variants of CITED2 promoter sequence in ASD patients affect transcriptional activity and are likely involved in the occurrence and development of ASD. These findings may provide new perspectives on the molecular pathogenesis and potential therapeutic insights of ASD patients.

The Reciprocal Relationship between LDL Metabolism and Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus and insulin resistance feature substantial modifications of the lipoprotein profile, including a higher proportion of smaller and denser low-density lipoprotein (LDL) particles. In addition, qualitative changes occur in the composition and structure of LDL, including changes in electrophoretic mobility, enrichment of LDL with triglycerides and ceramides, prolonged retention of modified LDL in plasma, increased uptake by macrophages, and the formation of foam cells. These modifications affect LDL functions and favor an increased risk of cardiovascular disease in diabetic individuals. In this review, we discuss the main findings regarding the structural and functional changes in LDL particles in diabetes pathophysiology and therapeutic strategies targeting LDL in patients with diabetes.

Rapid and comprehensive monoclonal antibody Characterization using microfluidic CE-MS

The identification and control of monoclonal antibody (mAb) critical quality attributes (CQAs) is a key component of quality by design (QbD). In this work, rapid peptide mapping and native intact charge variants analysis have been developed to comprehensively characterize and monitor mAb CQAs using a microfluidic capillary electrophoresis - mass spectrometry (CE-MS) platform.The ultrafast peptide mapping simultaneously analyzed multiple CQAs, including protein primary structure, oxidation, deamidation, succinimide, C-terminal lysine (Lys) clipping, N-terminal cyclization, and glycosylation. The microfluidic CE-MS based peptide mapping acquired results comparable to conventional but lengthy liquid chromatography – MS (LC-MS) based approach.The native intact analysis resolved mAb charge variants with a comparable resolution as commonly achieved using capillary isoelectric focusing (cIEF). Charge variants’ identities were assigned based on characteristic mass shifts, knowledge learned from peptide mapping, and changes in electrophoretic mobility. Major mAb glycoforms of each charge variants were resolved and identified in the deconvoluted mass spectra.Furthermore, a model simulation was performed to reconstruct intact deconvoluted mass spectra using peptide mapping results. The reconstructed and experimentally determined intact deconvoluted mass spectra were highly correlated, suggesting that our data collected at the peptide level and intact level were consistent and highly comparable.Overall, the microfluidic CE-MS based peptide mapping and native intact charge variants analysis are high-throughput methods that have great potential to support biopharmaceutical development.

Determination of Two-Steps Acid Dissociation Constants of L-Ascorbic Acid by Capillary Zone Electrophoresis

Two-steps acid dissociation constants of L-ascorbic acid (AA) were determined through the changes in the effective electrophoretic mobility in capillary zone electrophoresis. Although ascorbic acid is oxidatively degradable in an aqueous solution, especially in alkaline conditions, the effective electrophoretic mobility of AA was successfully measured in the pH range between 1.87 and 11.97. In the analysis of the first-step acid dissociation constant (pKa1) at weakly acidic pH conditions, a coated capillary with 1,3-propanesultone and a pressure-assist were utilized to detect anionic AA. In the analysis of the second-step acid dissociation constant (pKa2) at weakly alkaline pH conditions, AA was successfully detected at the pH range up to 11.97 with the help of Cu catalyst to remove the dissolved oxygen in the separation buffer. Acid dissociation constants were independently determined as pKa1 = 4.15±0.01 and pKa2 = 12.07±0.04 by non-linear least-squares analyses. AA did not fully dissociate at the weakly alkaline pH range, and the effective electrophoretic mobility of the dianion form of AA was extrapolated for the analysis.

Cyclodextrins as mobile phase additives in open-tubular admicellar electrochromatography for achiral and chiral separations

Native cyclodextrins (α-, β- and γ-CD) which are traditional chiral selectors in capillary electrophoresis were studied in open-tubular admicellar electrochromatography (OT-AMEC) using cetyltrimethylammonium bromide (CTAB) as dynamic stationary pseudophase. The initial mobile phase was 0.2 mM CTAB in 25 mM sodium tetraborate (pH 9.2), which forms admicelles (bilayer) at the solid surface/liquid interface inside the capillary. Different molar ratios of [CD]:[CTAB], i.e., 0:1 to 2:1 for [α-CD]:[CTAB] and [β-CD]:[CTAB] and 0:1 to 20:1 for [γ-CD]:[CTAB] in the mobile phase were investigated. An increase in the CD concentration decreased the magnitude of the electroosmotic flow (EOF). This suggests that CDs increase the critical surface aggregation concentration of CTAB, reducing the amount of free CTAB available to aggregate at the solid surface/liquid interface. At a molar ratio of 1.5:1 for α-CD, 2:1 for β-CD and 20:1 for γ-CD, the EOF changed from anodic to cathodic in direction. This suggests that no admicelles were formed under these conditions. Consequently, the retention factors (k) of the tested analytes (dichlorprop, fenoprop, ibuprofen, ketoprofen, propranolol, verapamil, hexanophenone, verapamil and valerophenone) decreased until k = 0 (no admicelles or stationary pseudophase). We observed a change in the migration orders of the analytes because of the resulting change in the effective electrophoretic mobility. Finally, we achieved chiral separation of mecoprop and dichlorprop in OT-AMEC with α- or β-CD in the mobile phase, which was not possible in electrokinetic chromatography using the same α- or β-CD concentration in the background solution. The chiral separation was aided by the retention caused by the dynamic stationary pseudophase.

Phos-tag diagonal electrophoresis precisely detects the mobility change of phosphoproteins in Phos-tag SDS-PAGE

Phos-tag diagonal electrophoresis was developed to identify precisely a change in electrophoretic mobility of phosphoproteins in Phos-tag SDS-PAGE. Previously, if a single protein band was detected, it was impossible to determine whether mobility of the protein altered by Mn2+ Phos-tag in Phos-tag SDS-PAGE gels because SDS-PAGE and Phos-tag SDS-PAGE were performed on different gels. Moreover, when multiple protein bands were detected, it was difficult to determine whether the band with the highest mobility was altered mobility by Mn2+ Phos-tag. However, these problems were resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns were provided on a single gel. Using this technique we identified phosphorylation states of various proteins such as α-lactalbumin, α- and β-casein, ovalbumin, basic 7S globulin, and 26S proteasome subunits. In the analyses of 26S proteasome subunits from humans and yeast, we could confirm that all subunits are phosphorylated, and find that the number of major proteins with different phosphorylation states is a few in each of the subunits despite having many phosphorylation sites. SignificancePreviously, Phos-tag SDS-PAGE has been developed to identify a change in electrophoretic mobility of phosphoproteins. However, we had a problem in this technique; it was often difficult to recognize the mobility shift by Mn2+ Phos-tag when we used separately SDS-PAGE and Phos-tag SDS-PAGE. Such a problem was resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns are provided on a single gel. This technique was useful to identify phosphorylation states of various proteins.: Phos-tag diagonal electrophoresis, mass spectrometry, phosphoproteins, basic 7S globulin, proteasome

Assignment of complex species by affinity capillary electrophoresis: The case of Th(IV)-desferrioxamine B.

The electrophoretic mobility change of desferrioxamine B (DFO) was monitored by UV absorption spectrophotometry upon increasing the thorium(IV) concentration in the background electrolyte at two acidities ([HClO4 ]Tot = 0.0316 and 0.0100 M). These data enabled to assess the speciation model and to determine the equilibrium constant of [Th(DFO)H2 ]3+ at fixed ionic strength (I = 0.1 M (H,Na)ClO4 ). Affinity capillary electrophoresis (ACE) turned out to be most helpful in identifying the complexed species by ascertaining its charge and protonation state. The assignment of the correct stoichiometry relied on the reliable estimation of the electrophoretic mobility by assuming similar hydrodynamic radii for (DFO)H4+ and the chelate. The value of the apparent equilibrium constant (log β112 = 38.7±0.4) obtained by ACE compares favorably well with those reported in the literature for thorium and a range of other metal ions, according to a linear free-energy relationship. This method is useful for studying metal-ligand binding equilibria and provides valuable information for further modelling the behavior of tetravalent actinides under environmental conditions. Structural information about the prevalent solution species in acidic conditions was gained by DFT calculations, confirming the bishydroxamato coordination mode of Th4+ by the diprotonated ligand.

Relaxation of adsorbed layer thickness and electrophoresis of polystyrene latex particles after overshooting of polyelectrolytes with different charge density

Temporal change of hydrodynamic layer thickness (δH) and electrophoretic mobility (EPM) of polystyrene sulfate latex (PSL) particles adsorbed by oppositely charged polyelectrolytes were monitored herein to clarify the relaxation behavior after overshooting. Two linear polyelectrolytes with nominal molecular weight of 5 million differing in chain charge density, σ = 100 % (PTMA5M) and σ = 4 % (PTMC5M), and PSL particles of different diameters were used in the experiment. With the former polyelectrolyte, a remarkably thick layer that is formed at the incubation decays with the time scale of several tens of minutes and the EPM remains constant. The value of δH decreases with the increase of particle size, whereas with the latter polyelectrolyte, the initial value of δH is several times smaller than that of the former. There is a case wherein the EPM decreases with time, although δH remains constant. The initial δH is independent of the particle size. Ascribing the reason for the relaxation phenomena to desorption of overshot polyelectrolytes attached with meta-stable conformation in the initial stage, we have obtained an approximate picture of the conformational change for the chain of different charge density.

Determination of acid dissociation constants of flavin analogues by capillary zone electrophoresis.

Acid dissociation constants (pKa ) of nine kinds of flavin analogues as molecular catalyst candidates were determined by CZE. Although some of the analogues are instable and degradable under the light exposure or in alkaline aqueous solutions, the effective electrophoretic mobility of the flavin analogue of interest has been measured with the residual substance. The pKa values of the flavin analogues were analyzed through the changes in the effective electrophoretic mobility with varying pH of the separation buffer. One or two steps pKa values were determined by the analysis. One of the degraded species from the flavin analogues, lumichrome, was also detected in the CZE analysis, and its pKa values were also determined. While coexisting impurities generated over the storage conditions were found in some analogues, the pKa values of the target analogues were successfully determined with the help of the CZE separations. A pressure-assisted CZE was utilized for the determination or the estimation of the pKa values of such analogues as possessing carboxylic acid moiety.

Incorporating peptide aptamers into resistive pulse sensing

AbstractThe use of nanocarriers within resistive pulse sensing, RPS, aids the detection and quantification of analytes. In the absence of convection, the signal strength and frequency can dependent upon the electrophoretic mobility of the nanocarrier/analyte. Here, we have developed a simple strategy to incorporate peptide aptamers onto RPS assays with enhanced electrophoretic signals. Using a hybrid DNA–Peptide nanocarrier, an existing peptide was incorporated into a rapid assay without having to engineer or modify the peptide sequence. The surface of a nanocarrier is coated with a mixture of peptide aptamers and a non‐binding DNA. The binding of the target to the peptide creates an “analyte corona” which shields the phosphate groups of the underlying DNA. This results in a change in electrophoretic mobility of the nanocarrier. The signal is concentration‐dependent and is illustrated using a peptide to a key biomarker of infection, C‐reactive protein, CRP. As a comparison, we also show the binding of the CRP to a DNA aptamer. This universal approach can be easily adapted to other peptides without the peptide itself to undergo any chemical modifications opening new opportunities and applications in RPS strategies.

New Platinum(II) Triazole Thiosemicarbazone Complexes: Analysis of Their Reactivity and Potential Antitumoral Action.

The synthesis and characterization of three new platinum complexes, with 3,5-diacetyl-1,2,4-triazole bis(4-N-isopropylthiosemicarbazone) as a ligand, are reported. The specific conditions under which solvent coordination takes place are reported and the X-ray structure of the complex with one solvent molecule of dimethyl sulfoxide is resolved. Analysis of the reactivity of these platinum compounds aids in finding the best solution profile for biological investigations. Then, the interactions of the complexes with biological models, such as calf-thymus DNA, are studied by using UV spectroscopy and tracking the changes in electrophoretic mobility produced in the supercoiled plasmid DNA model. Initial screening of these potential antitumoral compounds indicates possible selective antitumoral action.

Epimedium koreanum Extract and Its Flavonoids Reduced Atherosclerotic Risk via Suppressing Modification of Human HDL.

Atherosclerosis is the key factor responsible for cardiovascular events, which is a major cause of morbidities and mortalities worldwide. It is well known that high-density lipoprotein (HDL) oxidation and glycation increases the risk for atherosclerosis. Epimedium koreanum has been used as a traditional oriental medicine for treating erectile dysfunction, kidney diseases, osteoporosis, and breast cancer. However, no reports on the effects of E. koreanum on HDL modification exist. In this study, we investigated the inhibitory effects of E. koreanum extract and its eight flavonoids, which are: (1) anhydroicaritin 3-O-rhamnoside, (2) β-anhydroicaritin, (3–5) epimedins A-C, (6) epimedoside A, (7) icariin, and (8) des-O-methyl-β-anhydroicaritin, against HDL modification. HDLs obtained from pooled human plasma samples were incubated in vitro with E. koreanum extract or each compound in the presence of copper sulfate or fructose. The HDL modifications were evaluated by measuring generation of conjugated dienes, production of thiobarbituric acid reactive substances, change in electrophoretic mobility of apoA-I, advanced glycation end products formation, and apoA-I aggregation. Consequently, E. koreanum extract and compound 8 suppressed HDL modification through inhibition of lipid peroxidation, apoA-I aggregation, negative charge increase, and AGEs formation. In particular, compound 8 showed more potent inhibitory effect on HDL modification than the extracts, suggesting its protective role against atherosclerosis via inhibition of HDL oxidation and glycation.

Диагностические возможности электрофоретической подвижности эритроцитов и клеток буккального эпителия при стрессе

Диагностика стресс-реактивности по анализу гормонального состояния организма, является сложной, экономически затратной и трудоемкой процедурой исследования. В связи с этим актуален поиск информационных технологий, которые достаточно легко реализуются в любой медицинской лаборатории и позволяют оценить стресс-реакцию организма. Цель работы - оценка диагностических возможностей электрофоретической подвижности эритроцитов и клеток буккального эпителия в процессе развития стрессовой реакции. Методика. Для моделирования стресса использовали внутрибрюшинное введение крысам раствора адреналина-гидрохлорида (0,1 мг/кг) однократно или ежедневно в течение 3 сут эксперимента соответственно. Контролем служили крысы, получавшие физиологический раствор (в/б) в том же объеме. Буккальный эпителий исследовали на 7-е сут после введения адреналина. Мазки клеток эпителия окрашивали по Романовскому-Гимза, на препаратах подсчитывали количество клеток с микроядрами на 1000 клеток. Электрофоретическую подвижность эритроцитов оценивали методом микроэлектрофореза. Результаты. Наблюдалась качественно однотипная динамика показателя электрофоретической подвижности эритроцитов, количественно зависящая от силы воздействия. При слабых стимулах скорость изменений электрофоретической подвижности эритроцитов незначительна и сочетается с развитием адаптивных процессов, связанных с репарацией генетических повреждений. Увеличение степени стрессового воздействия путем трехкратного введения адреналина приводит к резкому изменению электрофоретической подвижности эритроцитов и росту числа эпителиоцитов с различными патологическими изменениями. Заключение. Показана возможность использования данного метода в качестве маркерной характеристики интенсивности стрессовой реакции с выявлением ее генотоксичности. Diagnostics of stress reaction using an analysis of hormonal state is a difficult, costly, and labor-consuming research. Therefore, searching for information technologies, which could be easily used in any medical laboratory and would allow evaluating the stress reaction is relevant. We studied the electrophoretic mobility of red blood cells (RBC) and buccal epithelial cells of rats exposed to stress induced by a single or repeated intraperitoneal epinephrine injection. This study showed that these methods could be used as the marker characteristics of stress reaction intensity and genotoxicity. Qualitative changes in the electrophoretic mobility of RBC were similar but quantitative characteristics depended on the acting force. Under a weak stimulus, changes in the electrophoretic mobility of RBC were slight and associated with development of adaptive processes related with repair of genetic damage. A further increase in the acting force led to a sharp change in the electrophoretic mobility of RBC and an increase in the number of epithelial cells with pathological alterations.

Динамика морфофункциональных показателей эритроцитов при действии сульфгидрильного ингибитора ангеотензин-превращающего фермента фентиаприла в экспериментальном моделировании артериальной гипертензии

Here, we studied the effect of fentiapril on the morpho-functional parameters of erythrocytes in the experimental model of arterial hypertension (AH). The drug was administered once, intraperitoneally, at the dose of 20 mg/kg. The dynamics of the changes in the electrophoretic mobility of erythrocytes (EME), their aggregation ability, ATP and 2,3 DFG concentration in the erythrocytes, the lipid peroxidation activity (by MDA content), the number of erythrocytes and gross hemoglobin amount, the volume of erythrocytes, the single-erythrocyte hemoglobin content, and the hematocrit, were assessed 30 and 60 min, as well as 24 hours after the drug administration. An increased erythrocyte aggregation, decreased EME, increased MDA and 2,3 DFG amid concentration, decreased ATP concentration, increased erythrocyte and hemoglobin amount, as well as increased hematocrit were demonstrated in the AH model. Application of fentiapril led to a decrease in the erythrocyte aggregation, as well as their number, decrease in the hemoglobin content and hematocrit, an increase in the MDA and 2,3DFG concentration accompanied byt the decrease in the ATP concentration, compared to the animals with hypertension during 1 day of registration, and an increase in the electronegativity of erythrocytes during the 30–60 min of the experiment, with maximal decrease in blood pressure observed at 60 min of registration. The decreased blood pressure under the fentiapril action was accompanied by the restoration of the studied erythrocyte characteristics to the values ​​similar to those observed in intact animals, whereas the increased blood pressure led to a decrease in the erythrocyte membrane electronegativity and an increase in lipid peroxidation.

Exosome Isolation: Cyclical Electrical Field Flow Fractionation in Low-Ionic-Strength Fluids.

The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at ∼1.3 V AC. A375 melanoma exosome recovery was approximately 70-80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from ∼-1.6 to -0.1 μm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from ∼-5.5 to -0.1 μm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.