Yersinia pestis Surface Antigens in Reception of Specific Bacteriophages.

The significance of Yersinia pestis surface antigens in adhesiveness to specific bacteriophages has been studied with the use of two methodological approaches. It was shown that Ail protein immobilized on the surface of polystyrene microspheres (but not in the solution), can bind both the Pokrovskaya phage and pseudotuberculous diagnostic phage. YapF autotransporter interacted with both phages in a water-soluble form, but YapF bound to polystyrene microspheres interacted only with the Pokrovskaya phage. An assumption was made that Ail and YapF proteins can be the primary receptors providing non-specific reversible binding to the phages used in this work.

Selective Protective Potency of Yersinia pestis ΔnlpD Mutants.

It has recently been shown that the NlpD lipoprotein is essential to Yersinia pestis virulence and that subcutaneous administration of the nlpD mutant could protect mice against bubonic and pneumonic plague better than the EV vaccine strain [PLoS One 2009. V. 4. № 9. e7023]. In this study, similar ΔnlpD mutants were generated on the basis of other Y. pestis parent strains, including strains from the subspecies microtus, which is avirulent to guinea pigs and humans. Comparative testing confirmed that immunization of mice with ΔnlpD mutants induces immunity 105 times more potent than the one induced by the administration of the EV vaccine strain. At the same time, NlpD- bacteria failed to protect guinea pigs in the case of a subcutaneous challenge with Y. pestis, inducing a 106 times less potent protection compared with that conferred by immunization with the EV vaccine strain. The possible causes of the observed phenomena are discussed.

[A molecular basis of the plague vaccine development].

Molecular mechanisms of the Yersinia pestis pathogenicity and peculiarities of maturation of specific immunity to plague are reviewed. The history and modern state of the plague vaccine development are described. Special attention is focused on the prospects in the area of the plague vaccine development. The possible approaches to improvement of vaccine preparations are discussed.

Refolding of scFv mini-antibodies using size-exclusion chromatography via arginine solution layer.

The method for refolding of mini-antibodies using size-exclusion chromatography via arginine solution layer was developed. This method allows to refold scFv, to separate both aggregated protein and low molecular weight compounds and to isolate functionally active protein preparation in monomeric form. The comparison of various scFv preparations isolated either from inclusion bodies or from soluble fraction revealed that refolded mini-antibodies demonstrate higher antigen-binding activity. Mini-antibodies refolded in the presence of arginine also demonstrate higher electrophoretic mobility during native PAGE in comparison with soluble cytoplasmic antibodies. Both soluble as well as refolded antibodies had similar CD spectra. Refolded mini-antibodies are storage-stable.

[Structural changes in Cry3A delta-endotoxin from Bacillus thuringiensis var. Tenebrionis in alcohol solutions at pH 2.0].

Conformational changes in Cry3A delta-endotoxin caused by three different alcohols (ethanol, butanol, and isopropanol) were studied using the methods of circular dichroism, scanning microcalorimetry, and electron miscroscopy. It was shown that, in addition to the standard decrease in the native structure stability, the alcohols can cause a conformational transition that results in a sharp increase in the beta-structure content and a change in the environment of aromatic residues. The conformational transition is accompanied by intermolecular association, which leads to the appearance of oligomers in the form of short filaments. When the alcohols were removed, the oligomers dissociated again into monomers, but it is likely that the native structure either is not restored or is restored only in a small portion of molecules. The oligomer structure is rather cooperative, and its thermostability is higher than that of the initial structure. The disruption of this structure upon heating, observed as a heat absorption peak, is reversible.

[Synthesis, secretion, and proteolytic degradation of diphtheria toxin in Escherichia coli]. / Sintez, sekretsiia i proteoliticheskaia degradatsiia difteriinogo toksina v Escherichia coli.

The recombinant plasmids have been constructed encoding the synthesis of a full-sized diphtheria toxin from its own or PR, PL-promoters of bacteriophage lambda in Escherichia coli cells. The high level constitutive synthesis of toxin results in slow cell growth and plasmid elimination. The toxin was mainly detected in the periplasm, partially in the membrane and to a less extent in the cytoplasm and culturing medium. The dimeric form of toxin was found in the cytoplasm. Participation of toxin B-subunit in secreting of the toxin into culturing medium is discussed. Proteolytic degradation of the synthesized toxin in different Escherichia coli strains was demonstrated. The process takes place in cytoplasm and periplasm mainly. The main enzyme participating in the process is a La-protease. The data on proteolysis obtained by immunoprecipitation immunoblotting, affinity chromatography and in mini-cells of Escherichia coli are summarized.

[Catalase biogenesis in various subcellular fractions of regenerating rat liver cells]. / Issledovanie biogeneza katalazy v razlichnykh subkletochnykh fraktsiiakh kletok regeneriruiushchei pecheni krys.

Dynamics of the accumulation and decay of catalase in different subcellular fractions of intact and regenerating rat liver is investigated. Biological activity of cytoplasmic factors regulating the rate of enzyme synthesis at post-transcriptional stages under partial hepatectomy is also studied. Different rates of the formation and decay of different catalase isoforms in intact and regenerating rat liver are observed.