Sup35NMp morphology evaluation on Au, Si, formvar and mica surfaces using AFM, SEM and TEM.

Prion and some other incurable human neurodegenerative diseases are associated with misfolding of specific proteins, followed by the formation of amyloids. Despite the widespread usage of the transmission electron and of the atomic force microscopy for studing such amyloids, many related methodological issues still have not been studied until now. Here, we consider one of the first amyloids found in Saccharomyces cerevisiae yeast, i.e. Sup35NMp, to study the adsorption of monomeric protein and its fibrils on the surface of mica, silica, gold and on formvar film. Comparison of linear characteristics of these units calculated by processing of images obtained by the atomic force, transmission and scanning electron microscopy was carried out. The minimal number of measurements of fibril diameters to obtain the values in a given confidence interval were determined. We investigated the film formed by monomeric protein on mica surface, which veiled some morphology features of fibrils. Besides, we revealed that parts of the Sup35NMp excluded from the fibril core can form a wide "coat". The length of the protein forming the core of the fibrils was estimated.

Peptide regulation of gene expression and protein synthesis in bronchial epithelium.

INTRODUCTION: Some studies have shown that peptides have high treatment potential due to their biological activity, harmlessness, and tissue-specific action. Tetrapeptide Ala-Asp-Glu-Leu (ADEL) was effective on models of acute bacterial lung inflammation, fibrosis, and toxic lung damage in several studies. METHODS: We measured Ki67, Mcl-1, p53, CD79, and NOS-3 protein levels in the 1st, 7th, and 14th passages of bronchoepithelial human embryonic cell cultures. Gene expression of NKX2-1, SCGB1A1, SCGB3A2, FOXA1, FOXA2, MUC4, MUC5AC, and SFTPA1 was measured by real-time polymerase chain reaction. Using the methods of spectrophotometry, viscometry, and circular dichroism, we studied the ADEL-DNA interaction in vitro. RESULTS: Peptide ADEL regulates the levels of Ki67, Mcl-1, p53, CD79, and NOS-3 proteins in cell cultures of human bronchial epithelium in various passages. The strongest activating effect of peptide ADEL on bronchial epithelial cell proliferation through Ki67 and Mcl-1 was observed in "old" cell cultures. ADEL regulates the expression of genes involved in bronchial epithelium differentiation: NKX2-1, SCGB1A1, SCGB3A2, FOXA1, and FOXA2. ADEL also activates several genes, which reduced expression correlated with pathological lung development: MUC4, MUC5AC, and SFTPA1. Spectrophotometry, viscometry, and circular dichroism showed ADEL-DNA interaction, with a binding region in the major groove (N7 guanine). CONCLUSIONS: ADEL can bind to specific DNA regions and regulate gene expression and synthesis of proteins involved in the differentiation and maintenance of functional activity of the bronchial epithelium. Through activation of some specific gene expression, peptide ADEL may protect the bronchial epithelium from pulmonary pathology. ADEL also may have a geroprotective effect on bronchial tissue.

DNA-polycation complexes: effect of polycation structure on physico-chemical and biological properties.

The purpose of the study was to investigate the influence of cationic polymer structure on the formation of DNA-polycation complexes and their transfection activity. Primary, tertiary, and quaternary polyamines with molecular masses ranging from 8000 to 200,000 were investigated. DNA-cationic polymer interaction was characterized by low gradient viscometry, dynamic light scattering, circular dichroism, UV spectrometry, flow birefringence, DNA electrophoresis, and electron microscopy. Transfection activity of the complexes was evaluated by the expression of reporter gene (beta-galactosidase) and using synthetic FITC-labelled oligonucleotides. Complex formation was found to be dependent on the structure and molecular weight of the polymer and the ionic strength of the solution. Secondary DNA structure in complexes was not disrupted, and DNA was protected from protonation. Cell lines of different origin were used for testing of transfection activity of the complexes. The sensitivity of the cells to transfection was established to be highly dependent on the cell line. DNA-polycation complexes are non-toxic according to MTT. Polyallylamine, and polydimethylaminoethylmethacrylate were found to be the most promising polycations for gene delivery. Transfection efficacy of their complexes with DNA to T-98G cells reaches up to 90-100%. It was found that optimal molecular mass of polydimethylaminoethylmethacrylate is in the range of 8000-50,000 Da.