Keratin Biomembranes as a Model for Studying Onychomycosis

Difficulties in obtaining human nails that are large enough for examining the penetration of drug formulations led us to produce keratin films regenerated from human hair. We assume that these films can simulate human nail plates in drug penetration and permeation tests and can serve as a biological model for studying onychomycosis. The films were formed from keratin extracted from human hair using dithiothreitol, urea and thiourea. The obtained keratin extract was dispensed into Teflon rings and dried at 40 °C and then cured at 110 °C. The structure, surface morphology, chemical characterization and thermal stability of the films were characterized and were compared to those of human nail, hair and bovine hoof samples using SDS-electrophoresis, scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The structure of the obtained films was found to be closer to human nails than to hair or bovine hooves. The keratin films were infected with Trichophyton rubrum and were proven to be appropriate for serving as a model for studying onychomycosis.

Molecular characterization of high-molecular weight glutenin subunits 1Bx6.1 and 1By22.1 from Triticum spelta К1731 accession

Aim. Some spelt varieties, along with alleles of gliadins and high-molecular glutenin subunits (HMW-GS), identical to common wheat, contain specific alleles, that are source of Triticum aestivum gene pool enrichment. The aim of this work is the identification, molecular analysis of HMW – GS from T. spelta K1731 and evaluation of their effect on the elastic properties of gluten. Methods. Identification of HMW-GS was carried out by SDS-electrophoresis and PCR analysis. Nucleotide gene sequences were determined by Sanger sequencing. The secondary structure of proteins was predicted on the on-line CFSSP server. Results. Subunits 6.1 + 22.1 of the Glu-B1 locus encoded by the Glu-B1be allele were detected in the T. spelta K1731. The nucleotide sequences of the 1Bx6.1, 1By22.1 genes from spelt were determined, the amino acid sequence and the protein secondary structure of 6.1 + 22.1 subunits were analyzed. Conclusions. Molecular analysis of HMW-GS 1Bх6.1 and 1By22.1 from T. spelta К1731 established a low contribution to the bread-making quality of these subunits. Keywords: Triticum spelta K1731, HMW-GS, SDS electrophoresis, sequencing, secondary protein structure, gluten quality.

Mutants at gliadin loci on the basis of the common wheat cultivar Bezostaya 1

Aim. The aim of this study was to isolate and propagate mutants at gliadin loci developed on the basis of the common wheat cultivar Bezostaya 1. Methods. We searched for spontaneous and gamma-irradiation induced mutations at gliadin loci among the progeny of F1 and F2 plants from crosses between near-isogenic lines by gliadin loci on the basis of the cultivar Bezostaya 1, including lines with the wheat-rye 1BL.1RS translocation. To identify mutations, we performed acid polyacrylamide gel electrophoresis and SDS-electrophoresis of storage proteins. Results. On the basis of the common wheat cultivar Bezostaya 1, five mutants (six mutations) at gliadin loci were isolated and propagated, four of which were described for the first time. Three mutations occurred at the Gli-R1 locus involved in the wheat-rye 1BL.1RS translocation (the loss of secalins, intensification of a secalin component, and increased mobility of a secalin component). Two mutations were identified in the allele Gli-B1b, one caused the null-allele at the Gli-A2 locus. Conclusions. The material of mutants is of importance for studying the role of certain groups of storage proteins and their components in quality determination, as well as mechanisms of regulation of storage protein synthesis. Keywords: Triticum aestivum, gliadin, secalin, mutation, 1BL.1RS translocation.

PROPERTIES OF NATIVE PROTEIN-CONTAINING ANTIGENS OF STREPTOCOCCUS PNEUMONIAE

Aim. The study of immunochemical and immunobiological properties of native protein-containing antigens of pneumococcus. Materials and methods. The study was carried out on the strains of the Collective Usage Center «Collection of Mechnikov Res. Inst. for Vaccine and Sera». In the work studied the chemical composition, the molecular weight of the obtained antigens in SDS-electrophoresis and antibody titers in ELISA. Protective activity of protein-containing antigens of pneumococcus was determined in experiments of active protection of mice. Results. Protein-containing antigens of pneumococcus were isolated from S. pneumoniae serotypes 3, 6B, 10A, 14, 19F, 23F and 36. The chemical composition of the preparations contained from 16 to 35% protein. In SDS-electrophoresis in polyacrylamide gel it was established that the molecular weight of protein-containing antigens of pneumococcus ranged from 14 to 116 kDa. Using ELISA shows the cross-activity of native antigens. Virtually all drugs reacted with antimicrobial rabbit serum obtained to serotype 19F (p≤0.05). Serotype serum 14 was less active and only protein-containing pneumococcal antigens obtained from 14 and 19F serotypes (p≤0.05) interacted with it. In the precipitation test according to Ouchterlony it was confirmed that preparations of serotypes 3, 6B, 14, 19F and 36 reacted with rabbit immune serum obtained for S. pneumoniae 19F serotype. In immunoblotting it was found that protein-containing antigens of pneumococcus isolated from serotypes 3, 6B, 10A, 14, 19F and 36 were associated with monoclonal antibodies to pneumococcal protein — pneumolysin. In vivo experiments it was shown that protein-containing antigens of pneumococcus protected animals from intraperitoneal infection of S. pneumoniae in homologous and heterologous systems (p≤0.05). Conclusion. The revealed immunochemical and cross-protective activity of protein-containing antigens of pneumococcus in vitro and in vivo experiments allows to select drugs derived from serotypes 6B, 10A, 19F and 36, as the most promising for further study of the intraspecific protective activity of individual native proteins of pneumococcus.

Description of biologically active protein hydrolysates of whey and colostrum

Antioxidant, antimutagenic and antigenic properties of partial hydrolysates of whey and colostrum obtained using bacterial endopeptidase (alcalase) have been investigated. It was found that the depth of proteolysis, qualitative and quantitative composition of protein component of samples determined the level of their antiradical and antimutagenic activity. According to SDS-electrophoresis whey hydrolysate contains cleaved allergen proteins, whereas colostrum hydrolysate possesses a high molecular weight fraction (>10 kDa) of partially digested immunoglobulins. Proteolysis of β-lactoglobulin, which has a high allergenic potential, is confirmed by results of immunoprecipitation reaction. In accordance with the ORAC method antioxidant action of hydrolysed whey and colostrum increased by 2.8 and 5.0 times, respectively. Antimutagenic effect for whey hydrolysate was 15.7–49.2 % when tested on the strain Salmonella typhimurium TA 98 and 18.8–52.1 % for strain TA 100. It exceeded values of colostrum hydrolysate. Samples of whey and colostrum hypoallergenic hydrolysates with confirmed antioxidant and antimutagenic properties have been obtained.

Investigation of peroxidase preparation composition and properties isola ted from horseradish roots

<p>Using the precipitation of proteins by zinc and ammonium sulfates, then by polymer and subsequent dialysis,<br />the peroxidase preparation from horseradish roots was isolated (RZ=0.7), with yield by protein 1.7 % and specific<br />peroxidative activity 7.6 U. By SDS -electrophoresis method it was verified the obtaining of partially purified peroxidase<br />preparation with molecular mass (43±0.5) kDa. By the native electrophoresis method it was determined, that 80.8 %<br />of the total preparation protein possesses expressed peroxidase activity.</p>