Registration of activity of a single molecule of horseradish peroxidase using a detector based on a solid-state nanopore.

This work demonstrates the use of a solid-state nanopore detector to monitor the activity of a single molecule of a model enzyme, horseradish peroxidase (HRP). This detector includes a measuring cell, which is divided into cis- and trans- chambers by a silicon nitride chip (SiN structure) with a nanopore of 5 nm in diameter. To entrap a single HRP molecule into the nanopore, an electrode had been placed into the cis-chamber; HRP solution was added into this chamber after application of a negative voltage. The reaction of the HRP substrate, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), oxidation by the enzyme molecule was performed in the presence of hydrogen peroxide. During this reaction, the functioning of a single HRP molecule, entrapped in the nanopore, was monitored by recording the time dependence of the ion current flowing through the nanopore. The approach proposed in our work is applicable for further studies of functioning of various enzymes at the level of single molecules, and this is an important step in the development of single-molecule enzymology.

Cold and distant: structural features of the nucleoprotein complex of a cold-adapted influenza A virus strain.

Two influenza A nucleoprotein variants (wild-type: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was distance between nucleoprotein complex strands. The isolated complexes of two ribonucleoprotein variants were characterized by transmission electron microscopy and differential scanning fluorimetry (DSF). Presence of the E292G substitution was shown by DSF to affect nucleoprotein complex melting temperature. In the filament interface peptide model, it was shown that the peptide corresponding in primary structure to the wild-type NP (SGYDFEREGYS) is prone to temperature-dependent self-association, unlike the peptide corresponding to E292G substitution (SGYDFGREGYS). It was also shown that the SGYDFEREGYS peptide is capable of interacting with a monomeric nucleoprotein (wild type); this interaction's equilibrium dissociation constant is five orders of magnitude lower than for the SGYDFGREGYS peptide. Using small-angle neutron scattering (SANS), the supramolecular structures of isolated complexes of these proteins were studied at temperatures of 15, 32, and 37 °C. SANS data show that the structures of the studied complexes at elevated temperature differ from the rod-like particle model and react differently to temperature changes. The data suggest that the mechanism behind cold adaptation with E292G is associated with a weakening of the interaction between strands of the ribonucleoprotein complex and, as a result, the appearance of inter-chain interface flexibility necessary for complex function at low temperature.Communicated by Ramaswamy H. Sarma.

Induced-Charge Enhancement of the Diffusion Potential in Membranes with Polarizable Nanopores.

When a charged membrane separates two salt solutions of different concentrations, a potential difference appears due to interfacial Donnan equilibrium and the diffusion junction. Here, we report a new mechanism for the generation of a membrane potential in polarizable conductive membranes via an induced surface charge. It results from an electric field generated by the diffusion of ions with different mobilities. For uncharged membranes, this effect strongly enhances the diffusion potential and makes it highly sensitive to the ion mobilities ratio, electrolyte concentration, and pore size. Theoretical predictions on the basis of the space-charge model extended to polarizable nanopores fully agree with experimental measurements in KCl and NaCl aqueous solutions.

[Current insights into chromatin structure organization].

This review summarizes current insights into organization of chromatin structure at different levels of DNA compaction. Analysis of available experimental data allowed concluding that only nucleosomal level of structural organization was sufficiently investigated, whereas structure of a 30-nm chromatin fiber remains an open issue. The data on the chromatin structure obtained at the level of the nucleus speak in favor of a biphasic fractal organization of chromatin.

[Genetic identification and study of the ability of lactobacilli isolated from the oral cavity of healthy individuals to form biofilms].

The highly antagonistic lactobacillus strains isolated from the oral cavity of human individuals were genetically passported as L. fermentum 39, L. rhamnosus 50, and L. rhamnosus 24, by applying the RAPD-PCR technique with two types of primers (M13, MSP). These lactobacillus strains showed high degrees of autoaggregation, surface hydrophobicity, coaggregation, and adhesion. These characteristics determine the obvious capacity of lactobacilli to form biofilms, which may be used to design new probiotic agents.

[Ability of human oral cavity indigenous lactobacilli to form biofilms].

AIM: To determine ability of lactobacilli strains indigenous for oral cavity of healthy persons to adhesion, autoaggregation, surface hydrophobicity, coaggregation and formation of biofilms. MATERIALS AND METHODS: Twenty-three anatagonistically active isolates of lactobacilli, including 5 strains of Lactobacillus plantarum, 6--L. rhamnosus, 4--L. paracasei, and 8--L. fermentum, were studied. Characteristics of lactobacilli associated with adhesion, autoaggregation, surface hydrophobicity, coaggregation, and formation of biofilms were determined. RESULTS: Bacteria were characterized by high and intermediate level of adhesion, autoaggregation, surface hydrophobicity, expressed ability to form biofilms and different coaggregation activity relative to test-strains Staphylococcus aureus, Escherichia coli, Candida albicans, Pseudomonas aeruginosa, and Bacillus subtilis. CONCLUSION: Studied characteristics of lactobacilli could be used for development of new probiotics aimed at stabilization of oral cavity normoflora.

[Genetic identification of antagonistically active lactobacillus strains isolated from the oral cavity of healthy individuals].

Examination of dental deposits from 45 healthy individuals detected 3 lactobacillus strains showing a high antagonism toward test cultures. The api 50 CH "bio Merieux" test systems were employed to identify strains as Lactobacillus fermentum 39, Lactobacillus rhamnosus 24 and Lactobacillus paracasei 50. The results of analyzing the sequences of the 16S rRNA genes of the test strains confirmed this identification, except for the latter strain. The taxonomic status of the third strain L. rhamnosus 50 was determined by the bioinformative analysis of the nucleotide sequence of the 16S rRNA genes.

[Lateral mechanics of muscle fibres and its role in signaling].

Different aspects of the lateral mechanics of muscle fibres have been considered. The current data on the transverse stiffness of intact muscle cells, demenbranized muscle fibres, and isolated myofibrils under various conditions and at different stages of the differentiation of myofibers. The signaling role of cytoskeleton proteins and possible mechanisms of their involvement in the structural basis of transverse stiffness are dissussed.

[Oral cavity microbiocenose in healthy teenagers and patients with chronic gastritis and gastroduodenitis].

Microbial association distinctive features and colonization degree of different oral cavity biotope were established in healthy teenagers and patients with chronic gastritis and gastroduodenitis. There were studied mucous membrane surfaces of cheek, tongue, content of gingival crevice, oral fluid and dental plaque of 24 healthy persons of the age 12-17 years and 29 patients of the same age with gastritis and gastroduodenitis. On the mucous membranes of the ill teenagers there were found Porphiromonas type bacteria, 20% more Staphylococcus, 90% less Peptostreptococcus, 30% less Lactobacillus, 20% less Veillonella and Stomatococcus. In saliva anaerobic Peptostreptococcus and facultative anaerobic Streptococcus (7 lg COE/ml); Porphiromonas, Veillonella, Peptococcus and Staphylococcus (6 lg COE/ml); Neisseria (5 lg COE/ml); Stomatococcus, Enterobacteriaceae and Bacillus (4.6 lg COE/ml); Lactobacillus and Candida (4 lg COE/ml) predominated. In patients with chronic pathology in all tested biotopes there was found the increase of the whole microbiota spectra (representatives of opportunistic pathogenic microfiora were detected more often) as well as the bacteria number if compared with healthy teenagers.