PAGE Electrophoresis Research Articles

Isolation of donor gamma globulin obtained from multiparous women and its effects upon expression of HLA-G and HLA-DR molecules on lymphocytes from mothers of children with septal congenital heart defects

A common pathogenetic mechanism of reproductive losses and congenital heart disease (CHD) is associated with immune inflammation in the “mother-embryo” system which affects differentiation and proliferation of cardiovascular progenitor cells. It is hypothesized that this link may be blocked by regulatory auto- and alloimmune antibodies to HLA-G and HLA-DR molecules. Moreover, these antibodies may be present at sufficient amounts in donor immunoglobulins, especially those obtained from the blood of multiparous women. Based on this suggestion, the aim of our study was to obtain enriched gamma globulin fraction from the blood of multiparous women and evaluate its functional effects towards HLA-DR and HLA-G molecules. Isolation of the gammaglobulin fraction (GGF) from the blood plasma of multiparous women was performed using affinity chromatography in several sessions. Purity grade of the resulting protein was analyzed by immunoelectrophoresis, electrophoretic separation of the protein fraction of blood serum and electrophoresis in 4.12% polyacrylamide gel with the addition of SDS (PAGE electrophoresis). PAAG electrophoresis showed that this GGF did not differ from commercial therapeutic intravenous immunoglobulin (IVIG). Assessment of the functional activity of GGF upon HLA-DR and HLA-G molecules was performed in the main group of women and their children with congenital heart disease (n = 38), and control group of women who gave birth to conditionally healthy children (n = 21). To determine the specificity of GGF with respect to HLA-G, HLA-DR molecules, as well as to compare its effect with autologous and allogeneic sera and IVIG, we developed an immunological testing protocol using flow cytometry. The protocol was arranged on the basis of the methodology of “cross-match” approach and Russian patent “Method for determining antibodies to HLA-G”. It was found that the blocking activity of female serum towards autologous (intrinsic) and allogeneic (embryo/fetus/child) HLA-G and HLA-DR molecules may determine the protective effect on development of congenital heart defects in the next generation. Donor human immunoglobulin showed a similar blocking effects to these molecules, possibly due to the presence of alloimmune antibodies to HLA classes I and II. The gammaglobulin fraction obtained from the donor blood of multiparous women has a more pronounced blocking effect on the HLA-G and HLA-DR expression. Hence, this immunobiological preparation can be considered a prototype of therapeutic and prophylactic agent blocking the genesis of congenital heart defects.

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes.

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

Swelling, Rupture and Endosomal Escape of Biological Nanoparticles Per Se and Those Fused with Liposomes in Acidic Environment.

Biological nanoparticles (NPs), such as extracellular vesicles (EVs), exosome-mimetic nanovesicles (EMNVs) and nanoghosts (NGs), are perspective non-viral delivery vehicles for all types of therapeutic cargo. Biological NPs are renowned for their exceptional biocompatibility and safety, alongside their ease of functionalization, but a significant challenge arises when attempting to load therapeutic payloads, such as nucleic acids (NAs). One effective strategy involves fusing biological NPs with liposomes loaded with NAs, resulting in hybrid carriers that offer the benefits of both biological NPs and the capacity for high cargo loads. Despite their unique parameters, one of the major issues of virtually any nanoformulation is the ability to escape degradation in the compartment of endosomes and lysosomes which determines the overall efficiency of nanotherapeutics. In this study, we fabricated all major types of biological and hybrid NPs and studied their response to the acidic environment observed in the endolysosomal compartment. In this study, we show that EMNVs display increased protonation and swelling relative to EVs and NGs in an acidic environment. Furthermore, the hybrid NPs exhibit an even greater response compared to EMNVs. Short-term incubation of EMNVs in acidic pH corresponding to late endosomes and lysosomes again induces protonation and swelling, whereas hybrid NPs are ruptured, resulting in the decline in their quantities. Our findings demonstrate that in an acidic environment, there is enhanced rupture and release of vesicular cargo observed in hybrid EMNVs that are fused with liposomes compared to EMNVs alone. This was confirmed through PAGE electrophoresis analysis of mCherry protein loaded into nanoparticles. In vitro analysis of NPs colocalization with lysosomes in HepG2 cells demonstrated that EMNVs mostly avoid the endolysosomal compartment, whereas hybrid NPs escape it over time. To conclude, (1) hybrid biological NPs fused with liposomes appear more efficient in the endolysosomal escape via the mechanism of proton sponge-associated scavenging of protons by NPs, influx of counterions and water, and rupture of endo/lysosomes, but (2) EMNVs are much more efficient than hybrid NPs in actually avoiding the endolysosomal compartment in human cells. These results reveal biochemical differences across four major types of biological and hybrid NPs and indicate that EMNVs are more efficient in escaping or avoiding the endolysosomal compartment.

Analysis of the electrophoretic profiles of the protein extracts of the species Manihot esculenta (Crantz, 1975), Persea Americana (Mill., 1768) and Actinidia delicious (A. Chevalier., 1984), and their association with the latex-fruit syndrome for searching for potential allergens

Determine the electrophoretic profiles of the extracts of Manihot esculenta, Actinidia Deliciosa and Persea Americana and their possible relationship with Latex-Fruit Syndrome. Protein extracts of M. esculenta, P. Americana and A. Deliciosa were prepared through the processes of maceration and solvent extraction from plant samples. In the case of the avocado, a prior extraction by soxhlet was carried out to eliminate the fat. The extracts were vacuum filtered, dialyzed and finally lyophilized. Separation of proteins based on molecular weight was performed by SDS PAGE electrophoresis. The electrophoretic profiles obtained were compared with the allergenic proteins previously identified in the latex extract, in order to determine a possible relationship with Latex-Fruit Syndrome, depending on the molecular weight. The extracts of M. esculenta and P. Americana showed a wide range of protein fractions with molecular weights varying from 10 to 250 KD, finding that the region with the highest concentration of bands was between 20 and 89 KD, (60 and 65%), respectively. A 20-band profile was obtained for the M. esculenta extract (Figure 1), with seven bands sharing similar weights with the latex allergens (Hev b 1, Hev b 2, Hev b3, Hev b 4, Hev b 5, Hev b 6.03, Hev b 8 and Hev b 10) (3-5). For the P. Americana extract, 20 bands were also observed (Figure 2), seven of which presented approximate weights to the Latex allergens (Hev b 1, Hev b 2 Hev b 4 Hev b 6.01 Hev b 6.03 Hev b 8 , Hev b 10 Hev b 11 Hev b 14). The Kiwi extract showed two bands of 19.1 and 22.9 KD, with weights close to latex proteins (figure 3), (Hev b 3 and Hev b 6.01), and allergens (Act d 2 and Act d 6), reported in the literature for this fruit. When analyzing the relationship between the separated protein fractions and the latex allergens described in the literature, a possible association of 35% was found for the extracts of M. esculenta and P. Americana, and 10% for A. Delicious, with great relevance being the association found with the allergens Hev b 4, Hev b 2, Hev 8 and Hev b 11, which are involved in Latex-Fruit Syndrome. The electrophoretic profiles of the prepared extracts were determined and compared with the Latex allergens. This information generates a contribution for the development of new research and advances in the standardization of these extracts on a large scale and for their future use in diagnostic tests.

Conformational Modulation of Tissue Transglutaminase via Active Site Thiol Alkylating Agents: Size Does Not Matter.

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme's dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2's conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme's ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity.

Improving CEA detection Sensitivity: Carboxyfluorescein-Loaded liposomes in aptamer sandwich assay

This study aimed to develop an effective method for highly sensitive detection of carcinoembryonic antigen (CEA), a tumor biomarker, using an aptasensor sandwich assay based on a liposome loaded with carboxyfluorescein. In this approach, liposomes loaded with carboxyfluorescein (CF) were anchored with a cholesterol-labeled anti-CEA aptamer (Apt1-Lip) using a post-insertion technique. Furthermore, the successful insertion of the aptamer into the liposome outer layer was confirmed through UV absorption, PAGE electrophoresis, dynamic light scattering (DLS), and zeta potential measurements. Additionally, a secondary aptamer was conjugated with magnetic beads via biotin-streptavidin interactions and used as a capture probe (MBs/Apt2). Aptamer-decorated liposomes successfully bound to the CEA antigen in the presence of the target, leading to the formation of a sandwich assay when combined with MBs/Apt2. The specific binding signal was further increased by dissolving the lipid nanospheres with Triton X-100. This liposome-based aptasensor demonstrated a detection limit of 35.48 pg/mL over a wide linear range from 0.1 ng/mL to 100 ng/mL, with high specificity and sensitivity at CEA. Additionally, the proposed approach exhibited notable promise for on-site testing in early clinical diagnosis, as it showed excellent selectivity, satisfactory reproducibility, and good recovery in CEA detection from human serum samples.

Misacylation of tRNA with Ser-Pro Dipeptide for In Vitro Transcription-Translation.

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami etal. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNASerPro.

Sustainable production and preparative purification of thermostable alkaline α-amylase by Bacillus simplex (ON754233) employing natural deep eutectic solvent-based extractive fermentation

Using PEG-based deep eutectic solvents (PDES), the current study proposes extractive fermentation as a sustainable process integration for the production and purification of α-amylase from Bacillus simplex (ON754233). Glucose: PEG 400 outperformed five PDES in terms of tie lie length (58) and slope value (1.23) against sodium sulphatt. Apple cider pomace was used as a low-cost, sustainable carbon source to produce-amylase, with a maximum enzyme production of 2200.13 U/mL. PDES concentration (20% w/v), salt (12.75 w/v), and apple waste (2.75 g/mL) were all optimized using response surface methodology. When scaled upto 3 L benchtop bioreactor, extractive fermentation was proved to be better technology with maximum recovery of 92.4% with highest partition coefficient (3.59). The partially purified enzyme was further purified using a Sephadex G 100 followed by DEAE-Sephadex anion exchange chromatography with a purity fold of 33. The enzyme was found to be thermostable at the temperature (60 °C), remains alkaline (pH 8), and the activity was stimulated in the presence of Mg2+ ions. With SDS PAGE electrophoresis, the molecular weight was found to be around 140 kDa. Finally, the enzyme kinetics parameters were evaluated with observed Km (0.00396 mM) and Vmax (37.87 U/mL). Thus scaling up extractive fermentation entails increasing production capacity with improved extraction efficiency using green solvents.

Получение рекомбинантного ингибитора рибонуклеаз в E. coli для использования в синтезе мРНК in vitro

Technologies underlying the production of synthetic mRNAs in vitro have significantly expanded the possibilities for research and therapeutic use of this class of molecules. The flagship application area has been the niche of mRNA vaccines, but this class of therapeutic molecules has the potential to be applied in a much broader range of situations. The process of in vitro production of artificial RNA molecules is based on an enzymatic synthesis reaction, one of the components of which is a ribonuclease inhibitor. This protein protects synthesized RNA from attacks by ribonucleases and prevents degradation of the molecules, which is critically important for RNA. Eukaryotic ribonuclease inhibitor synthesized as a recombinant protein in the cells of E. coli bacteria is the most common choice. However, the structure of this protein makes it a difficult product to make in bacteria. This study aimed to test the production of a recombinant ribonuclease inhibitor in various strains of E. coli, and to show the effect that helper polypeptides and cellular chaperones have on this process. Using genetic engineering approaches, we constructed plasmids, from which chimeric ribonuclease inhibitor molecules and helper polypeptides were produced. The influence of various components on solubility of the target recombinant protein was assessed with the help of densitometry, to which we have subjected products of the PAGE electrophoresis. It was determined that combinations of a vector with a strong promoter for the expression of the RNH1 ribonuclease inhibitor gene and helper polypeptides MBP and TIG against the background of increased expression of cellular chaperones dnaK, dnaJ, grpE give the target product yield of 45 mg/l and 60 mg/l, respectively. The selected conditions allow large-scale production of this protein for further use in in vitro RNA synthesis in the context of production of medicines.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis of toxin proteins of Bacillus thuringiensis isolated from soil at the Liwa Botanical Garden

Bacillus thuringiensis is a spore-forming bacterium known to have the ability to synthesize proteins that are toxic against insect pests. Currently, the bacterial protein toxins are widely used as pesticides because they are environmentally friendly and safe for health. As a cosmopolitan microbe B. thuringiensis is widely distributed in nature with soil as its main habitat. Liwa Botanical Gardens is one among the conservation areas in Lampung Province of Indonesia where the soil type and conditions expected to good for the bacterial life of B. thuringiensis. This study aims to determine toxin proteins extracted from B. thuringiensis isolated from the soil of the Liwa Botanical Garden by their molecular weights. The proteins were quantified using Lowry method and the protein profiling performed using SDS PAGE electrophoresis. The results showed that there were two isolates, Bt 3 and Bt 5 which showed close protein concentrations, namely 0.679 mg/ml and Bt 5 with 1.313 mg/ml respectively. Both isolates also exhibit different molecular weights. Bt 3 isolate has a molecular weight of 110.59 kDa, while Bt 5 has a molecular weight of 76.97 kDa.

Isolation and purification of isocitrate lyase by chromatographic meth-ods from the liver of rats under conditions of alloxan-induced diabetes

The homogeneous preparation with isocitrate lyase activity was obtained from the peroxisomal fraction of rat hepatocytes using chromatographic methods. Male white inbred laboratory Wistar rats (Rattus norvegicus) were used as the object of the study. Experimental diabetes was induced by a single injection of a 5% alloxan solution. The development of diabetes was monitored by measuring blood glucose levels using a glucometer (SatellitePlus PKG-02.4). Blood sampling was carried out in the morning from the tail vein. On the 10th day of the experiment, laboratory animals were subjected to decapitation, previously euthanized with ether anaesthesia, to obtain liver tissue samples. For the isolation of a homogeneous preparation of isocitrate lyase (ICL, EC 4.1.3.1.), chromatographic methods were used. Purification included several stages: fractionation of the homogenate with ammonium sulphate, gel filtration on columns filled with Sephadex G-25 and ion exchange chromatography, which was the determining stage. The anion exchanger DEAE-Sephacel was used as a sorbent. This allowed to achieve a degree of purification of 12.12 for the first ICL isoform and 24.24 for the second ICL isoform. The specific activity for the first sample was 0.04 U/mg protein, and the yield was 26.3%. The specific activity value for the second form of ICL was 0.08 U/mg protein, and the yield was 47.4%. For elution from a column filled with DEAE-Sephacel was performed using step KCl gradient (60 mM – 200 mM). Identification of the resulting preparations was carried out spectrophotometrically, following the increase in the optical density, according to the Kornberg method (λ-324 nm). The presence of isoforms was determined by specific staining of the gel after PAGE electrophoresis. As a result of four-step purification, two isoforms with different from ICL (EC 4.1.3.1) from other sources electrophoretic mobility were obtained.

Nanoparticles fabricated from the bioactive tilapia scale collagen for wound healing: Experimental approach.

The creation of innovative wound-healing nanomaterials based on natural compounds emerges as a top research goal. This research aimed to create a gel containing collagen nanoparticles and evaluate its therapeutic potential for skin lesions. Collagen nanoparticles were produced from fish scales using desolvation techniques. Using SDS PAGE electrophoresis, Fourier transform infrared spectroscopy (FTIR) as well as the structure of the isolated collagen and its similarities to collagen type 1 were identified. The surface morphology of the isolated collagen and its reformulation into nanoparticles were examined using transmission and scanning electron microscopy. A Zeta sizer was used to examine the size, zeta potential, and distribution of the synthesized collagen nanoparticles. The cytotoxicity of the nanomaterials was investigated and an experimental model was used to evaluate the wound healing capability. The overall collagen output from Tilapia fish scales was 42%. Electrophoretic patterns revealed that the isolated collagen included a unique protein with chain bands of 126-132 kDa and an elevated beta band of 255 kDa. When compared to the isolated collagen, the collagen nanoparticles' FTIR results revealed a significant drop in the amide II (42% decrease) and amide III (32% decrease) band intensities. According to SEM analysis, the generated collagen nanoparticles ranged in size from 100 to 350 nm, with an average diameter of 182 nm determined by the zeta sizer. The produced collagen nanoparticles were polydispersed in nature and had an equivalent average zeta potential of -17.7 mV. Cytotoxicity study showed that, when treating fibroblast cells with collagen nanoparticle concentrations, very mild morphological alterations were detected after human skin fibroblasts were treated with collagen nanoparticles 32 μg/ml for 24 hours, as higher concentrations of collagen nanoparticles caused cell detachment. Macroscopical and histological investigations proved that the fabricated fish scale collagen nanoparticles promoted the healing process in comparison to the saline group.

SDS-Page Analysis Of Cysticercus tenuicollis In Vitro And In Vivo In Basrah Province/ Southern Iraq

The larval stage Cysticercus tenuicollis was isolated from slaughtered sheep and goats at the Basrah slaughterhouse with 30 samples. In vitro 16 samples, 2 control, and 14 were treated with plant extracts and novel chemical compounds. In vivo 14 sample,1 control and 13 treated with plant extracts and novel chemical compounds.. SDS- page electrophoresis using the gel as separation gel of larval stage Cysticercus tenuicollis. Lanes (C) control, (A) Syzygium aromaticum, (B) Capparis spinosa, novel acylselenourea, i.e., 4-Nitro-N-((4-(N-(pyrimidin-2-yl) sulfamoyl) phenyl) carbamoselenoyl) Benz amide (1), 4-Methyl-N-((4-(N-(pyrimidin-2-yl) sulfamoyl) phenyl) carbamoselenoyl) Benz amide (2), 4-Nitro-N-((4-nitrophenyl) carbamo selenoyl) Benz amide (4), N-(2,6-dioxo-1,2,3,6-tetrahydropyrimidine-1-carbonoselenoyl)-4-nitrobenzamide (5) and novel acylthiourea 4-Methyl-N-((4-(N-(pyrimidin-2-yl) sulfamoyl phenyl) carbamothioyl) Benz amide (3). The rats were administered plant extracts and synthesis compounds orally via mouth for 15 days, Then the rats were dissected, the cysts were isolated, and their SDS page was measured.The results found in in vitro, all plant extracts, and synthesized compounds bands ranged from 67 to 17KD. In contrast, the protein bands of cysts varied from 67 to 17KD in vivo whereas Capparis spinosa and the synthetic chemical (5) showed no bands. This study recorded a clear effect on protein cotenants of C. tenuicollis which was treated with two plant extracts C. spinosa and S. aromaticum, and five novel chemical synthesized compounds in vitro and in vivo by SDS- PAGE as compared with untreated.

Quantification of C1 esterase inhibitor in human serum by enzyme-linked immunosorbent assay: Correlation with turbidimetric immunoassay

C1 inhibitor of serine proteases (C1-INH) performs a regulatory function in the complement system and vascular permeability. Deficiency of C1-INH leads to various forms of angioedema, including hereditary angioedema (HAE). The cause of HAE is a genetically determined violation of the synthesis of C1-INH. A decrease in the level of C1-INH to 50% relative to the norm leads to an increase in the production of bradykinin, which is the basis for the diagnosis of HAE. The development of affordable ELISA for the quantitative determination of C1-INH is a popular direction for clinicians. During the development of a new kit for quantitative determination of C1-INH, two mouse monoclonal antibodies (mAb) with different epitope specificities were obtained. On their basis, a sandwich-type ELISA was developed. The specificity of the obtained mAb's was confirmed using the medical device “Berinert”. To prepare calibrators, C1-INH was affinity purified from human blood plasma using a sorbent with immobilized mAbs. The identity of the C1-INH protein was confirmed by PAGE electrophoresis, immunoblotting, and mass spectrometry on MALDI-TOF/TOF UltrafleXtreme mass spectrometer. To assess the quality indicators of developed reagents kit, studies were carried out in accordance with GOST R 51352-2013 and TU 21.20.23-041-01967164-2022. Values of quality indicators: accuracy — 93.53%; measurement linearity interval — 22.00-176.07 ng/mL. Using the developed ELISA test system, we examined 28 blood sera from healthy donors and 7 blood sera from patients with confirmed HAE. In the same samples, the content of C1-INH was determined by turbidimetric method, using the "Diagnostic reagents for in vitro immunochemical studies of specific blood proteins. Model: C1-esterase inhibitor (C1 EsteraseInhibitor)" (Aptec, Belgium). The correlation coefficient was 0.94 (p < 0.05). It was found that the diagnostic sensitivity and specificity of the developed ELISA is 100%. As a result of the study, an original ELISA test system for the quantitative determination of C1-INH was developed "Reagent kit for enzyme-linked immunosorbent assay of human C1-inhibitor (C1-inh PS)".

Evaluation of serum protein electrophoresis in patients with renal disorders

One of the cardinal findings of the renal disease is proteinuria, which appears in the early phase of kidney disease and is very important in diagnosis, prognosis and decision-making in treatment process and results of treatment. Besides this abnormal protein fractions are commonly observed in renal disorder. Serum will be collected for protein electrophoresis and Quantization of each fraction was done. So the present study is designed to find the role of serum protein electrophoresis in renal disorder patients.The aim of the present study was to study protein electrophoresis pattern in various renal disorders.The present study was a hospital-based observational study. The study was conducted over a period of 1 year on 50 patients with renal disorder of either sex visiting the OPD/IPD of Medicine department of Guru Gobind Singh Medical College and Hospital Faridkot. SDS Page electrophoresis was done to evaluate serum proteins in the patients with renal disorders.The results of the study showed the comparison of protein content in patients of NS and ESRD at p<0 Albumin(t=3.86), α1 Globulin(t=6.18), t=-2.76)>0.05). The Comparison of protein content in patients of NS and CKD at p<0 Albumin(t=5.42), α1 Globulin(t=3.38)>0.05).The present study concluded that serum protein fractions are altered in various renal disorders. Protein electrophoresis should be used in addition to other renal markers for better monitoring of the disease progression.

A Novel Hyperthermostable Recombinant Protein Nanocage

Ferritin has an important role in iron storage in the cells, and due to its nanocage structure and self-assembly properties, it has wide application prospects in nanobiotechnology. The maize (Zea mays) ferritin gene ZmFer1 was cloned and expressed in Escherichia coli BL21 (DE3) for the first time. Change in macromolecular structure of ZmFer1 ferritin due to heat treatment was investigated using native PAGE electrophoresis, DLS, and TEM. Change in the secondary structures of the protein was evaluated using CD spectroscopy. Moreover, alteration in the conformation of the protein was evaluated using UV-absorption spectra and intrinsic fluorescence spectra. The Tm of ZmFer1 was obtained using DSC. Finally, the effect of heat on the function of ZmFer1 was assessed by iron loading ability. The purified ZmFer1 protein showed a homopolymer nanocage structure. The results of native PAGE electrophoresis, DLS, and TEM techniques showed that ZmFer1 protein nanocage is stable to heat treatment up to 90 °C, and some of the protein nanocages retain their macromolecular structures even at 100 °C in liquid aqueous solution. Based on the DSC results, ZmFer1 protein nanocage had a Tm of 81.9 °C. After treatment at 100 °C, stable ZmFer1 protein nanocages were able to store iron atoms. Recombinant ZmFer1 ferritin with a Tm > 80°C is a hyperthermostable protein nanocage. The results of this study are beneficial for the development of protein nanocages that are stable under extreme temperature conditions, as well as application of ZmFer1 in nanobiotechnology, biomaterials, and biomedical fields.

VNTR (CAG)n polymorphism of the ATXN2 gene and metabolic parameters of cardiovascular risk associated with the degree of obesity in the Amerindian population of Oaxaca

IntroductionThe ATXN2 gene has a VNTR (CAG)n with locus in exon1. Long alleles within the normal range (22–29 repeats) are associated with severe obesity in people from the United Kingdom, Indonesia and the Caribbean. ObjectiveTo analyse the influence of VNTR (CAG)n on metabolic profile in adults with obesity and pre-obesity, as well as to estimate its effect on the risk of developing diabetes. Methods and material255 adults of Chinantec Amerindian ethnic origin were included, who underwent anthropometric and biochemical evaluation. The VNTR was amplified by end-point PCR and by 8% PAGE electrophoresis. ResultsDifferences were found in the waist/hip circumference index and body mass index in the carriers of genotypes different to the one homozygous for 22 repeats with a Student's t-test value of 0.0041 and 0.0334, respectively. We also found an association with a family history of chronic disease. ConclusionThe VNTR of ATXN2 is associated with obesity in Mexican adults of Chinantec ancestry.

Assessing Oligomerization Status of Mitochondrial OXPHOS Complexes Via Blue Native Page.

Mitochondrial metabolism plays key roles in pathologies such as cancer. The five complexes of the oxidative phosphorylation (OXPHOS) system are crucial for producing ATP and maintaining cellular functions and are particularly exploited in cancer cells. Understanding the oligomeric state of these OXPHOS complexes will help elucidate their function (or dysfunction) in cancer cells and can be used as a mechanistic tool for anticancer agents that target mitochondria. Here we describe a protocol to observe the oligomeric state of the five OXPHOS complexes by isolating mitochondrial-enriched fractions followed by assessing their oligomeric state by nondenaturing blue native page electrophoresis.

POLYMORPHISM OF TAGLGAP MICROSATELITE LOCUS AND ITS CONNECTION WITH ALLELELIC VARIETIES OF GLIADINS OF BREAD WHEAT

Introduction. Gliadins are monomeric and highly polymorphic storage proteins of wheat endosperm, which together with glutenins form a gluten complex that determines the breadmaking properties of wheat. Allelic variants of gliadins are an important feature in the selection of material for breeding, but their determination by electrophoresis in acid PAGE is quite difficult. Aim. The aim of this study was to investigate the polymorphism of the Taglgap microsatellite locus and to analyze its correspondence to the polymorphism of allelic variants of gliadins that have been revealed by acid PAGE electrophoresis. Methods. 140 cultivars and lines of bread wheat of Ukrainian and foreign selection were analyzed. Electrophoresis of storage proteins was performed in an acid PAGE according to the method of F. O. Poperellia (1989), allelic variants were designated according to the international nomenclature (Metakovsky et al., 2018). DNA was isolated by CTAB method and PCR was performed with primers to the Taglgap microsatellite (Devos et al., 1995). PCR products were fractionated in 7% PAGE and stained with silver staining method. Nucleotide sequences were searched by BLAST and aligned by MAFT methods. The main results. 19 allelic variants of gliadins and 11 alleles of the Taglgap locus were identified. In the collection of Ukrainian varieties there were Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1l and Gli-B1o allelic variants and alleles of Taglgap 216 bp, 237 bp, 246 bp, 248 bp, 252 bp, 267 bp, 270 bp and null. In the foreign collection of varieties − Gli-B1a, Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1i, Gli-B1j, Gli-B1k, Gli -B1l, Gli-B1m, Gli-B1n, Gli-B1o, Gli-B1p, Gli-B1q, Gli-B1r, Gli-B1s and 213 bp, 216 bp, 237 bp, 246 bp, 248 bp, 250 bp, 252 bp, 270 bp, 285 bp and null. Nucleotide sequence analysis in the NCBI database showed the presence of a number of other alleles of the Taglgap microsatellite not only in bread wheat but also in some species of the Triticum L. and Aegilops L. genus. Conclusions. The detected polymorphism correlates with the polymorphism of allelic variants of gliadins of Gli-B1 locus and makes it possible to identify Gli-B1a, Gli-B1d, Gli-B1h and Gli-B1l allelic variants, and for Ukrainian varieties with high probability also Gli-B1b allelic variant. However, this marker does not allow identifying Gli-B1c, which is important for selection.

ОПРЕДЕЛЕНИЕ РАЗМЕРА НУКЛЕИНОВЫХ КИСЛОТ БАКТЕРИОФАГОВ PS.S-7 УЛГАУ И PS.S-27 УЛГАУ

The article presents results of studies on specification of the size of nucleic acids of Ps.s-7 UlGAU and Ps.s-27 UlGAU bacteriophages. The relevance of the research on classification of bacteriophages by type and size of nucleic acid is substantiated in the introduction of the article. It was established, by the method of extraction on magnetic particles and phenol-chloroform extraction, that the maximum calculated size for the DNA of Ps.s-7 UlGAU bacteriophage (isolated from a soil sample, characteristics: lytic activity, determined by the Gracio method, - 2.0 ± 0.1x109 pfu / ml, by Appelman's method - 10-8, drip test - n9; spectrum of lytic action - 85.7%) was 38137 bp, for Ps.s-27 UlGAU bacteriophage (isolated from a soil sample, characteristics: 1.0 ± 0.1x109 pfu / ml according to Grazio, according to Appelman - 10-8 , drop test - n9; spectrum of lytic action was 85.7%) DNA size was 23744 bp. The studies used a DNA molecular weight marker Quick-Load Extend DNA 500-48500 bp, a Nanodrop 2000 / 2000c spectrophotometer (ThermoFisher), and a PAGE electrophoresis technique. The obtained data will make it possible to specify the phylogenetic relationship of Ps.s-7 UlGAU and Ps.s-27 UlGAU bacteriophages with bacteriophages annotated in the NCBI database, using Protein BLAST, active against phytopathogenic Pseudomonas syringae bacteria, causing tumor neoplasms, rotting, growth cessation and death of a part of plants cultivated by humans without decay, chlorosis, necrosis, etc.