Electrophoretic Separation Of Proteins Research Articles

Cellulose acetate electrophoretic separation of serum and urine proteins in Nigerian children with autism spectrum disorders

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) characterized by social communication challenges and restricted, repetitive behaviors. While genetic and environmental factors are known to contribute to ASD, the role of the immune system remains unclear. This study investigated the separation patterns of serum and urine proteins in Nigerian children with ASD compared to typically developing children and children with other NDDs.MethodsForty-seven participants aged 3–8 years were recruited, including 16 children diagnosed with ASD and 16 children with other NDDs, both according to DSM-5 criteria, along with 15 neurotypical children. Blood and urine samples were collected for protein analysis. Total protein and albumin levels were measured in both serum and urine using established methods. Protein separation in serum and urine was performed using cellulose acetate electrophoresis, followed by densitometry analysis of the electrophoretic patterns.ResultsThe results revealed no significant differences in total serum protein levels and most protein fractions between the groups. However, children with other NDDs exhibited significantly lower levels of alpha-2 globulin compared to neurotypical children. Conversely, both ASD and NDD groups showed significantly higher gamma globulin levels compared to the control group. Interestingly, spot urine protein levels were significantly higher in children with ASD compared to neurotypical children.ConclusionThe observed changes in alpha-2 and gamma globulin levels suggest potential immune system involvement in ASD and other NDDs. The higher urine protein excretion in the ASD group warrants further investigation to explore the potential of urinary protein biomarkers for ASD diagnosis.

Investigation of von Willebrand factor multimer abnormalities before and after aortic valve replacement using the Hydragel-5 assay

BackgroundSevere aortic stenosis (sAS) is associated with acquired von Willebrand syndrome (AVWS) by loss of high-molecular-weight multimers (HMWM) of von Willebrand factor (VWF), potentially resulting in perioperative bleeding. Analysis of VWF multimers remains challenging. Recently, the new, rapid Hydragel 5 assay has been developed, using electrophoretic protein separation for dividing VWF-multimers into low (LMWM), intermediate (IMWM), and HMWM, the hemostatically active part of VWF. Here, we evaluated its impact on predicting blood loss in presence of AVWS after surgical aortic valve replacement (SAVR). MethodsWe prospectively examined 52 patients (age: 68 ± 7 years; 54 % male) admitted to SAVR. They were divided in two groups (A: normal VWF, n = 28; B: abnormal VWF, n = 24, defined as VWF-activity/antigen (VWF:Ac/Ag)-ratio < 0.7 and/or HMWM loss). Blood samples and echocardiographic data were collected before, seven days and three months after SAVR. Blood loss and transfusions were recorded. ResultsBaseline characteristics and clinical data were similar in both groups. HMWM loss was present in 38.5 % of all patients. HMWM, the VWF:Ac/Ag- and HMWM/(IMWM+LMWM)-ratios were significantly decreased preoperatively in group B but normalized after SAVR. Bleeding, re-thoracotomy and transfusion rates were comparable. HMWM loss was inversely correlated with the peak aortic gradient (Pmax) and positively with the aortic valve area (AVA), while HMWM/(IMWM+LMWM)-ratio negatively correlated with the mean aortic gradient (Pmean). ConclusionHMWM and HMWM/(IMWM+LMWM)-ratio inversely correlate with severity of AS and normalize after SAVR. The Hydragel-5 assay's might be valuable for routine diagnostics to assess bleeding risk and postoperative normalization of AS and VWF abnormalities in SAVR patients.

Scaling the electrophoretic separation of rapeseed proteins and oleosomes

Gentle extraction of ingredients from raw materials is essential for high-quality food ingredients and can lead to reducing the use of water, chemicals, and energy in the extraction. For example, a simple aqueous extraction can yield a mixture of oil, in the form of a natural oil-in-water oleosome emulsion, and proteins. The oleosomes and proteins can then be further separated in a next step. We explored a continuous counter-current electrophoretic process that separates oleosomes and proteins based on their electrophoretic mobility by balancing an electric field with an opposing solvent flow. The separation is accomplished through the retention of the component with the higher electrophoretic mobility, the oleosomes, and the passage of the proteins, having lower mobility. The fluxes of oleosomes and proteins from rapeseed, after aqueous extraction, were analyzed as a function of the electric field (0–75 Vcm−1) and 1.2 ± 0.1 mLmin-1 solvent flow rate. At 50 Vcm−1, the permeation flux of proteins was 10-fold higher than that of oleosomes, as shown by the selectivity increasing to 9.84 from 1.90 at 25 Vcm−1. The difference in their flux promises to become more pronounced under an increasing treatment duration, but two main technical limitations, electrolysis-based pH alteration and membrane fouling, restrict further separation. We expect the listed challenges can be mitigated with the addition of electrode rinse chambers and the use of larger pore size membranes.

Study of variability alpha-amylase isozymes in wheat varieties of Ukrainian breeding

Aim. To investigate the variability of alpha-amylase isoenzymes in soft wheat varieties of Ukrainian selection, created by breeding institutions located in different climatic zones (Forest Steppe and Steppe). Methods. Isoenzymes of alpha-amylase were detected by electrophoretic protein separation in a polyacrylamide gel. The fall number on the «Falling Number 1700» was determined. Results. For a representative assessment of the distribution of alpha-amylase isoenzymes, we studied the zoned varieties of common wheat from two breeding centers located in different climatic zones: the Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine, Kyiv (Forest-steppe) and the Breeding and Genetics Institute of the National Academy of Sciences, Odessa (Steppe). The variability of wheat grain alpha-amylase was revealed. A search for associations between alpha-amylase variants and resistance to pre-harvest grain germination was conducted. Conclusions. The prevalence of alpha-amylase isoenzyme variants in Ukrainian common wheat varieties was estimated. Among Odessa varieties, the AbCd alpha-amylase variant predominates with a frequency of 50.0 %. AbCd and AbCD phenotypes with a frequency of 38.8 % predominate in the selection varieties of the IFRG of the National Academy of Sciences of Ukraine. It was shown that the genotypes containing the AbCD and ABсD isoenzyme variant are more resistant to pre-harvest germination, and the ABCd and AbcD variants are less resistant to pre-harvest grain germination.

Continuous counter-current electrophoretic separation of oleosomes and proteins from oilseeds

Oilseeds, with their high content of oil and protein, will play an important role in the transition to plant based foods. Currently, seed oil extraction involves elevated temperatures and the use of organic solvents, which degrade the quality of both oils and proteins. To take full advantage of the oilseeds, a gentler process is needed. We here propose a mild alkaline extraction followed by a continuous electrophoretic separation process that is based on differences in electrophoretic mobility and recovers intact oleosomes and proteins from the seeds. Rapeseed oleosomes and proteins are both negatively charged at pH ≥ 5, yet exhibit significantly different electrophoretic mobility. Therefore, separation can be achieved by imposing a counter-current hydrodynamic flow rate between their electrophoresis rate. Thus, the compounds with higher mobility, oleosomes, are retained by the electric field, and the compounds with lower mobility, proteins, go along with the flow. The separation was modeled using the Nernst-Planck equation and demonstrated using a PDMS-based microfluidic system. Both the modeling and the experimental studies confirmed that the direction and rate of migration of the compounds can be steered by the electric field strength and the convective flow velocity. The proposed electrophoretic approach is feasible and scaleable, and may be a novel path to separate differently charged components under mild conditions, thereby preserving their original native properties.

Western Blotting with Fast SDS-PAGE and Semi-Dry Protein Transfer.

Western blotting is a method widely used in cell and molecular biology for the specific detection of proteins in biological samples. It is time-consuming and normally takes up to 2 days to complete. This protocol introduces a more time-efficient method to complete western blots, covering the preparation of protein extracts (including strategies for solubilization), electrophoretic separation of proteins, transfer of proteins to the membrane, and probing with antibodies. We describe an SDS-PAGE protocol that achieves a gradient-like separation of proteins (10-400 kDa) on a single-percentage polyacrylamide gel in only 45 min. Additionally, we present a rapid (10-14 min) semi-dry transfer of proteins from standard Tris/glycine polyacrylamide gels onto a membrane using homemade Tris/HEPES- or Tris/EPPS-based buffers. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Support Protocol 1: Cell lysis and protein extraction Support Protocol 2: Protein quantification with BCA assay and sample preparation for loading on gel Basic Protocol 2: Protein transfer with a fast semi-dry transfer (FSDT) buffer Basic Protocol 3: Immunoprobing, chemiluminescent visualization, stripping, and reuse of membranes.

Protein Antioxidant Complex of Water Extract of Larvae of Black Beetles &lt;i&gt;Ulomoides dermestoides&lt;/i&gt;

An aqueous extract from the larvae of black beetles Ulomoides dermestoides grown under controlled conditions was obtained and its proteomic analysis based on one-dimensional electrophoretic separation of proteins, their trypsinolysis in a gel and subsequent chromatography-mass spectrometry was performed. Protein identification was performed using MaxQuant v.1.6.3.4 software. The Uniprot database was used to identify proteins by homology. Catalase activity was determined spectrophotometrically by hydrogen peroxide concentration decrease. Antioxidant activity was evaluated by luminol chemiluminescence quenching. The extract contained proteins that can determine the biological activity of the extract: universal regulators of cellular processes calmodulin (62%), cytochrome c-2 (13.5%), nucleoside diphosphate kinase (11.1%), the enzymatic antioxidants superoxide dismutase, catalase, glutathione S-transferase, peroxiredoxin, glutathione synthetase as well as thioredoxin, 70 and 60 kDa heat shock proteins, chitinase complex (13.4% in total). Catalase activity was 6.3 ± 1.1 µmol H2O2/min/mg protein; antioxidant activity of 1 mg protein per ml extract was equivalent to 1.36 ± 0.3 mM trolox. The prospects for practical use of the extract as a natural antioxidant complex were noted.

Biochemical, Histological Changes, Protein Electrophoretic Pattern, and Field Application of CuPb-Ferrite/TiO2 Nanocomposites for Controlling Terrestrial Gastropod Eobania vermiculata (Müller).

Eobania vermiculata is a hazardous snail that can damage ornamental plants and cause significant harm to plant sections in Egyptian areas. Herein, the molluscicidal activity of CuPb-Ferrite/TiO2 and TiO2 nanoparticles (NPs) against E. vermiculata was evaluated using the poisonous bait method. LC50 values were determined using the leaf dipping and contact methods, with values of 631.23 and 1703.49 ppm for CuPb-Ferrite/TiO2 and 193.67 and 574.97 ppm for TiO2. Exposure to both NPs resulted in a significant increase in the biochemical parameters of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), as well as a decrease in total protein (TP) percentage of E. vermiculata. Histological examinations revealed that many digestive cells had ruptured, and their contents had been lost, while the foot's epithelial layer became ruptured. The average reduction was 66.36% for CuPb-Ferrite/TiO2 NPs compared to the recommended molluscicide, Neomyl, with a 70.23% reduction in the field application. Electrophoretic separation of total protein using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment with LC50 concentrations of TiO2 and CuPb-Ferrite/TiO2 demonstrated the potency of these synthetic compounds as molluscicidal agents. Therefore, we recommend the use of CuPb-Ferrite/TiO2 NPs as a novel land snail molluscicide because it is safe to use, and the baits are arranged to not affect irrigation water, with a high molluscicidal effect.

The effect of GdYVO4:Eu3+ nanocrystals on the intercellular adhesion of mesenchymal stem cells in vitro

Adult stem cells, such as MSCs, spontaneously differentiate in vitro. This makes it difficult both to study this important cell type and to grow large numbers of MSCs for clinical use. While conventional cell cultivation methods cannot cope with this problem, nanostructured materials science offers hope. The effect of small-sized spherical nanoparticles based on orthovanadates of rare-earth elements activated by europium (GdYVO4:Eu3+ nanoparticles, diameter 1–2 nm) on cell-cell adhesion of rat bone marrow mesenchymal stem cells (rBM-MSCs) in vitro was studied using electrophoretic separation of proteins, immunofluorescence and confocal laser scanning microscopy. Our study revealed that rBM-MSCs treated with small-sized GdYVO4:Eu3+ nanoparticles had a significant impairment of intercellular adhesion in vitro. The pre-incubation of mesenchymal stem cells of rat bone marrow with GdYVO4:Eu3+ nanocrystals at a non-toxic concentration of 0.5 µg/mL during 1 hour of cultivation did not lead to significant changes in cell monolayer, the number of cells and the area of cell bodies did not change. However, the density of the monolayer and the area of the cell field decreased after the incubation. The incubation of cells with nanoparticles led to an increase in the area of the intercellular gate – a location of disruption of cell adhesion, compared to cells without nanoparticles in culture medium. The pre-incubation of rBM-MSCs with nanocrystals caused no changes in the content of total cadherins in the plasma membrane; a decrease in the content of cytoplasmic calreticulin and an increase in the content of surface calreticulin; a decrease in the content of free calcium in the cytoplasm, and an increase in protein-bound intercellular calcium and calcium in the extracellular space. The colocalization analysis revealed that the colocalization of calreticulins with cadherins on the outer surface of the plasma membrane of cells significantly increased after the incubation with GdYVO4:Eu3+ nanocrystals. The paper proposes a possible mechanism of reducing the degree of adhesion by nanocrystals. This study emphasizes the possibility of modulating MSCs adhesion using GdYVO4:Eu3+ nanoparticles. The development of new technologies capable of mitigating adhesion is crucial for the development of regenerative strategies using stem cells.

Heritability and amylose content in hybrid lines of late-generation rice with colored pericarp.

Improving grain quality in rice breeding is one of the main tasks. This concerns the creation of rice varieties with colored pericarp uncommon in the Republic of Kazakhstan, and the assessment of its quality is an important stage of breeding. Rice with colored pericarp is an important dietary crop, more useful for the human body than white rice. Regardless of the type of rice, the amount of amylose in rice grain is a crucial indicator that determines the quality of rice. The paper presents the results of electrophoretic separation of spare grain proteins of rice hybrids and dihaploids with colored pericarp and their parent forms obtained as a result of the hybridization of varieties with colored pericarp (Black Rice (China), Mavr (Russia), and Yir 5815 (Ukraine)) with white rice varieties zoned in Kazakhstan. The hybridization of the rice varieties with colored pericarp with white rice varieties was carried out to obtain rice varieties with colored pericarp oriented to the soil and climate of Kazakhstan. Analyzing the results of electrophoresis and the amount of amylose, it was found that hybrid lines differed in amylose content. One of the studied hybrids was high in amylose, four had a medium amylose content, ten had a low amylose content, three had a very low amylose content, and six were glutinous. According to the results of electrophoretic separation of spare rice grain proteins, the spectrum of the enzyme determining amylose was detected in five hybrids, which corresponds to the results of spectrophotometric determination of amylose: high amylose in one hybrid and medium amylose content in four. The results show that the hybrids obtained as a result of hybridization are true hybrids and as a result of long-term selection, the amylose content in the F7-F8 hybrids stabilized. The hybrids can be used in further breeding of rice with colored pericarp.

Acceleration of the selection process in the population of Black and Spotted cattle by using genetic markers

Genetic researches of cattle breeds in the western region of Ukraine are ecologically necessary, and they also become the most actual in connection with the formation of breed associations. In addition, the research of the genetic characteristics of various breeds for use in agricultural production is an attempt to identify the most common genetically determined characteristics in the herd. The choice of such economically valuable characteristics is determined by modern methods of research in agriculture by selecting herds with such a gene pool in which this characteristic is the most typical. The genetic structure was evaluated by genetically determined polymorphism of blood groups and genetic-biochemical systems. Experiments were carried out on erythrocytes and blood plasma. Blood from the animals was taken from the jugular vein in a test tube with heparin. Polymorphism of proteins and enzymes was evaluated using the method of electrophoretic separation of proteins in 13 % starch gel in horizontal chambers followed with subsequent histochemical staining. Group erythrocyte antigens were determined according to the generally accepted methodology based on hemolytic tests using 48 reagents of 9 genetic systems. The genetic structure of this breed is characterized by the following features: the transferrin locus is characterized by the greatest heterozygosity – 0.729, but according to the Hardy-Weinberg law, this locus is in a balanced state (Р – 0.104). The dominant frequency of occurrence is the allele Tf A – 0.438, mainly as a result of AD1 and AD2 heterozygotes. The amylase locus (AM-l) is in an unbalanced state – there are not enough heterozygotes and too many homozygotes (Р – 0.013). The frequencies of occurrence of two alleles Am-l B and Am-l C are the same and amount to 0.500. Hemoglobin is monomorphic in all investigated animals (hemoglobin AA). For purine nucleoside phosphorylase (NP), there are no significant differences within the studied group of animals.The higher frequency of occurrence of the Np L allele is 0.872, but the Np H allele occurs less often, its frequency is 0.128, the heterozygosity of this locus is 0.225. As for blood groups, the vast majority of antigens detected with the help of 48 specific antisera are found in the antigenic spectrum of the investigated Black and Spotted dairy breed. However, the frequency of some antigens (less than 0.057) is extremely low (I1, Q, T1, T2, P1, P2, Y, R, U, I', B'', H''), others (J, B ', U') is slightly higher (0.082-0.099), and some (A2, G2, G3, B2, I2, O1, O2, Y2, D', G', E'2, O', Q', G'', C1, C2, E, W, X2, L, F, S, H'', Z) was sufficiently high (from 0.11 to 0.888). The Black and Spotted dairy breed has a unique genetic structure based on the investigated loci.

The Effect of Molecular Mass of Hydroxyethyl Cellulose on the Performance of Capillary Electrophoretic Separation of Proteins

Capillary electrophoresis (CE) is a versatile analytical separation method in the field of biochemistry. Although it has been proved that the relative molecular mass (Mr) of the polymers determines the threshold concentration of the entangled polymer solution, which will affect the separation performance of DNA molecules, there is still no report on the effect of Mr on the separation performance of proteins. Herein, we have thoroughly performed the CE of proteins ranged from 14.3 kDa to 116 kDa in a mixed hydroxyethyl cellulose (HEC) solution. The mixed solution was obtained with various Mr including 90,000, 250,000, 720,000, and 1,300,000. Then, we found that the mixed polymer provided a high resolution for small protein molecules while increasing the efficiency of large ones. Results demonstrated that the migration time decreased if HEC (1,300,000) was mixed with the lower Mr one, and the mixed solution (1,300,000/250,000) offered the highest resolution. The resolution was negatively correlated with the electric field strength. Finally, we have employed the optimal electrophoretic conditions to separate proteins in human tears, and it showed that lysozyme, lipocalin, and lactoferrin from human tears were successfully resolved in the mixed HEC. Such work indicates that CE has the potential to be developed as a tool for the diagnosis of xerophthalmia, meibomian gland dysfunction, or other eye diseases.

Alterations in The Functional and Structural Properties of Erythrocytes Due to Alcohol Intoxication; Correction of Disorders

Alcohol abuse is among the leading reasons for disability and accounts for at least 5% of the global health burden. This study aims to define changes in the functional and structural properties of erythrocytes in chronic ethanol intoxication and to design methods for correction of the identified disorders. The experiments has been conducted on 93 healthy sexually mature and newborn Wistar rats. Acute and chronic alcohol intoxication has been designed by forced intragastric administration of 20% ethanol solution at a dose of 3 ml/kg (2.92 g/kg) on a daily basis for 5, 30 and 60 days. From the newborn animals we obtained allogeneic hepatocytes and their cultural fluid, from which proteins were isolated. The proteins of allogeneic hepatocyte and the combined administration of immunomodulatory, hepatoprotective and antioxidant agents were used to correct disorders in chronic intoxication. The membranes of circulating erythrocytes were obtained by the method of Dodge; electrophoretic separation of membrane proteins was carried out according to the method of Laemli. The lipid content in the erythrocyte membrane was determined by liquid thin-layer chromatography. It has been elucidated that in presence of chronic alcohol intoxication there is a change in the content of the membrane proteins in circulating erythrocytes, which are responsible for flexibility and shaping, intrinsic cellular metabolism, stabilization and structure formation, and a disruption in the lipids representativeness and ratio that form the basis for the lipid framework of the plasmalemma and play the major role in arrangement of protein macromolecules and the normal metabolism of red blood cells as well. When analyzing the corrective activity of the proteins of allogeneic hepatocytes and the combination Glutoxim + Mexidol + Heptral, we have established their high efficiency towards the disorders in the functional and structural properties of circulating erythrocytes caused by chronic alcohol intoxication.

Electrokinetic characterization of hybrid NOA 81-glass microchips: Application to protein microchip electrophoresis with indirect fluorescence detection.

A low-cost and straightforward hybrid NOA (Norland optical adhesive) 81-glass microchip electrophoresis device was designed and developed for protein separation using indirect fluorescence detection. This new microchip was first characterized in terms of surface charge density via electroosmotic mobility measurement and stability over time. A systematic determination of the electroosmotic mobility (μeo ) over a wide pH range (2-10) and at various ionic strengths (20-50mM) was developed for the first time via the neutral marker approach in an original simple frontal methodology. The evolution of μeo was proved consistent with the silanol and thiol functions arising from the glass and the NOA materials, respectively. The repeatability and reproducibility of the measurements on different microchips (RSD<14%) and within 15 days (less than 5% decrease) were successfully demonstrated. The microchip was then applied for the efficient electrophoretic separation of proteins in a zonal mode coupled with indirect fluorescence detection, which is, to our knowledge, the first proof of concept of capillary zone electrophoresis in this hybrid microsystem.

Paper-based open microfluidic platform for protein electrophoresis and immunoprobing.

Protein electrophoresis and immunoblotting are indispensable analytical tools for the characterization of proteins and posttranslational modifications in complex sample matrices. Owing to the lack of automation, commonly employed slab-gel systems suffer from high time demand, significant sample/antibody consumption, and limited reproducibility. To overcome these limitations, we developed a paper-based open microfluidic platform for electrophoretic protein separation and subsequent transfer to protein-binding membranes for immunoprobing. Electrophoresis microstructures were digitally printed into cellulose acetate membranes that provide mechanical stability while maintaining full accessibility of the microstructures for consecutive immunological analysis. As a proof-of-concept, we demonstrate separation of fluorescently labeled marker proteins in a wide molecular weight range (15-120kDa) within only 15min, reducing the time demand for the entire workflow (from sample preparation to immunoassay) to approximately one hour. Sample consumption was reduced 10- to 150-fold compared to slab-gel systems, owing to system miniaturization. Moreover, we successfully applied the paper-based approach to complex samples such as crude bacterial cell extracts. We envisage that this platform will find its use in protein analysis workflows for scarce and precious samples, providing a unique opportunity to extract profound immunological information from limited sample amounts in a fast fashion with minimal hands-on time.

Fundamentals of Capillary Electrophoretic Migration and Separation of SDS Proteins in Borate Cross-Linked Dextran Gels.

Recent progress in the development and production of new, innovative protein therapeutics require rapid and adjustable high-resolution bioseparation techniques. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using a borate (B) cross-linked dextran (D) separation matrix is widely employed today for rapid consistency analysis of therapeutic proteins in manufacturing and release testing. Transient borate cross-linking of the semirigid dextran polymer chains leads to a high-resolution separation gel for SDS-protein complexes. To understand the migration and separation basis of the D/B gel, the present work explores various gel formulations of dextran monomer (2, 5, 7.5, and 10%) and borate cross-linker (2 and 4%) concentrations. Ferguson plots were analyzed for a mixture of protein standards with molecular weights ranging from 20 to 225 kDa, and the resulting nonlinear concave curves pointed to nonclassical sieving behavior. While the 2% D/4% B gel resulted in the fastest analysis time, the 10% D/2% B gel was found to produce the greatest separation window, even higher than with the 10% D/4% B gel, due to a significant increase in the electroosmotic flow of the former composition in the direction opposite to SDS-protein complex migration. The study then focused on SDS-CGE separation of a therapeutic monoclonal antibody and its subunits. A combination of molecular weight and shape selectivity as well as, to a lesser extent, surface charge density differences (due to glycosylation on the heavy chain) influenced migration. Greater molecular weight selectivity occurred for the higher monomer concentration gels, while improved glycoselectivity was obtained using a more dilute gel, even as low as 2% D/2% B. This latter gel took advantage of the dextran-borate-glycoprotein complexation. The study revealed that by modulating the dextran (monomer) and borate (cross-linker) concentration ratios of the sieving matrix, one can optimize the separation for specific biopharmaceutical modalities with excellent column-to-column, run-to-run, and gel-to-gel migration time reproducibilities (<0.96% relative standard deviation (RSD)). The widely used 10% dextran/4% borate gel represents a good screening option, which can then be followed by a modified composition, optimized for a specific separation as necessary.

ASSESSMENT OF THE GENETIC VARIABILITY OF DIFFERENT AGE GROUPS OF SILVER AND BIGHEAD CARPS BY BIOCHEMICAL POLYMORPHISM

Goal. The study aimed to analyze and assess the genetic variability of different age groups of silver and bighead carps by the polymorphism of protein and enzyme systems.&#x0D; Methods. Blood samples were taken from silver (Hypophthalmichthys molitrix) and bighead (Aristichthys nobilis) carps of "Sunpoint Ukraine" LLC, Slobozhanske, Zmiiv district, Kharkiv region.&#x0D; Blood from the tail vein was collected from age-2 and age-3 fish in vivo and placed into tubes with an anticoagulant. Whole blood samples were separated into fractions by centrifugation and as a result plasma and erythrocytes were packed into separate tubes. The methods of vertical polyacrylamide and horizontal starch electrophoresis were used for the electrophoretic separation of proteins and enzymes of silver and bighead carps blood. Histochemical staining of the gel plates was carried out with t) and bighead he following genotyping of allelic variants of protein and enzyme systems. Biochemical systems such as loci of prealbumin (Pralb), esterase (EST, K.F. 3.1.1.1), malate dehydrogenase (MDH, K.F. 1.1.1.37), enzyme malic (ME, K.F. 1.1.1.40) and carbonic anhydrase (CA, K.F. 4.2.1.1) were investigated. Statistical processing of experimental data was performed using Biosys-1. Cluster analysis was carried out using MEGA-X.&#x0D; Results. Analysis of the peculiarities of the distribution of allele frequencies and the genotypic composition of Pralb, EST, MDH, ME, and CA loci was carried out in the age-2 and age-3 groups of silver and bighead carps. The prevalence of the frequency of the fast migrating allelic variant F was revealed in comparison with the slow migrating gene at EST locus in the age-3 group of silver carp (Est F = 0.620). The prevalence of the frequency of the fast allelic variant Pralb A in comparison with the slow Pralb B was observed in the silver carp groups (Pralb A = 0.812 in the age-2 and Pralb A = 0.726 in the age-3 groups. According to the genotypic composition of the studied loci, there was an excess of heterozygous individuals in the age-2 group of silver carp, which indicates their genetic imbalance. The formation of breeding stocks of silver and bighead carps requires control of the level of genetic variability of different age groups to monitor changes in the genetic structure and maintain the optimal level of heterozygosity in stocks. The highest level of average heterozygosity (79.4%) was found in the age-2 group of silver carp, which prevailed over the expected average heterozygosity with a value of 49.4%. In comparison with other studied groups, the age-2 group of bighead carp had the lowest level of average heterozygosity of 59.1% versus the theoretically calculated level of 45.5%. The age-2 and age-3 groups of bighead carp had the highest level of heterozygosity at Ca locus at the level of 82.8 and 78.6% respectively, in contrast to the expected heterozygosity at this locus at the level of 49.9 and 49.7%. A dendrogram of the genetic relationships of different age groups of silver carp was constructed based on the values of genetic distances. The formed clusters indicated the formation of the genetic structure of the silver and bighead carps by species affiliation.&#x0D; Conclusions. The analysis of the genetic structure of different age groups of the silver and bighead carps was performed by the polymorphism of protein and enzyme systems such as Pralb, EST, MDH, ME, CA loci. The study describes the features of the distribution of allelic frequencies and genotypic variants of the aforementioned loci. The level of average heterozygosity, observed and expected heterozygosity at loci in age-2 and age-3 groups of silver and bighead carps were determined. The high genetic variability of an age-2 group of silver carp at the level of 79.4% indicates the need to stabilize their genetic structure. As a result of the cluster analysis, it was determined that the formation of the genetic structure of the silver carp groups of the "Sunpoint Ukraine" farm in the Kharkiv region occurs by species affiliation. The study and use of biochemical markers will allow differentiating stocks of silver and bighead carps. As well as in complex analysis with other molecular genetic markers will allow them to develop a genetic passport.

ASSESSMENT OF GENETIC STRUCTURE VARIABILITY OF RAINBOW TROUT Oncorhynchus mykiss (WALBAUM, 1792) OF UKRAINIAN LOCAL STOCKS USING POLYMORPHIC BLOOD PLASMA PROTEINS

Rainbow trout is a valuable species of aquaculture, which is characterized by a high level of variability. Aim. The goal was to study the peculiarities of genetic variability of rainbow trout based on polymorphism of transferrin (TF), posttransferrin (PTF), esterase (EST) (EC 3.1.1.1.) and albumin (ALB) loci. Methods. Electrophoretic separation of plasma proteins of rainbow trout from three local stocks (Chernivtsi, Kharkiv and Transcarpathian) was carried out in the native polyacrylamide gel. Results. Peculiarities of the distribution and relative electrophoretic mobility of allelic variants of the studied loci of rainbow trout of Ukrainian local stocks were established.The results demonstrated the effectiveness of the analysis using methods of biochemical genetics to establish the characteristics of the genetic structure, the level of heterozygosity of local stocks and to carry out their differentiation. Conclusion. The use of selected markers would allow monitoring the dynamics of changes in the state of local stocks under the established conditions of cultivation in the future. Key words: rainbow trout, polymorphic blood plasma proteins, transferrin, posttransferrin, esterase, albumin.

Влияние хронической свинцовой интоксикации на функциональные свойства и изоформный состав миозина левого желудочка сердца крыс

The chronic lead intoxication was induced by the administration of intraperitoneal injections of lead acetate to outbred rats 3 times a week for 5 weeks. Using an in vitro motility assay, it was shown that lead intoxication causes changes in actin-myosin interaction in vitro, in particular, a decrease in the maximum sliding velocity of native thin filaments along myosin isolated from the myocardium of the left ventricle of the rat heart. No statistically significant changes were found in the calcium sensitivity and cooperativity of the “pCa-velocity” curve, the characteristics of the fraction of motile filaments and isometric force. Using electrophoretic separation of proteins, a shift in the ratio of myosin heavy chains towards β-chains with slower ATPase activity was found.

Immunogenicity of antigens from outer membrane vesicles of Neisseria meningitidis associated with bilayer fragment of dioctadecyldimethylammonium in Swiss adult mice.

PurposeIn the present study, meningococcal serogroup B outer membrane vesicles (OMVs) were associated with bilayer fragments of a cationic lipid, dioctadecyldimethylammonium (DDA-BF), used as adjuvant, in an antigenic preparation tested in adult female outbred mice. This adjuvant was compared to the traditional adjuvant aluminum hydroxide.Materials and MethodsThe potential in generating humoral response was evaluated by enzyme-linked immunosorbent assay (ELISA). Individual serum was collected and immunoglobulin G (IgG), IgG1, IgG2a, and IgG2b were quantified. Analyses were carried out 15 and 60 days after immunization. Antibodies avidity index were also analyzed by ELISA. Immunoblot and dot-ELISA were carried out to evaluate specific reaction for homologous strains and cross-reactive antigens present in other meningococcal strains isolated in 2011–2012 year, in Brazil. Delayed type hypersensitivity was used as indicative of cellular immunity and compared between two experimental groups, 24 hours after homologous strain challenge.ResultsThe OMVs of Neisseria meningitidis, and N. lactamica (related species) were characterized by electrophoretic separation of proteins in 13% polyacrylamide gel. The strains presented antigens in the range of 8 to 130 kDa, showing a heterogeneous protein migration pattern. In the group immunized with OMVs/DDA-BF, we found no significant production of total IgG 15 days after the first immunization. On the other hand, 60 days after first immunization both adjuvants act benefiting total IgG production similarly. The antibodies of the IgG isotype produced by animals immunized after one or two doses after first immunization, showed intermediate and high avidity, independent on the adjuvant used. In both experimental groups the swelling of the footpads was significantly higher than those of the controls, suggesting that only one dose was enough to stimulate the generation of cellular immunity.ConclusionThe use of this cationic adjuvant for N. meningitidis OMVs preparation revealed good potential for future new antigen preparation for N. meningitidis vaccine.