[Monoclonal antibodies to type A, B, E and F botulinum neurotoxins].

Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.

[Signaling function of phagocytic NADPH oxidase: activation of MAP kinase cascades in phagocytosis].

Until recently, the production of reactive oxygen species by NADPH oxidase has been considered only in the context of the oxidative damage to pathogens inside the phagosome. However, homologues of phagocytic NADPH oxidase have been found in almost all cell types, where they produce hydrogen peroxide and thereby regulate the initial intracellular stages of MAP kinase cascades. In the present work, the activation of two MAP kinase cascades, p38 and Erk1/2, during phagocytosis has been studied. It was found that phagocytosis activates both cascades. The activation of Erkl/2 is dependent, and the activation of p38 is not dependent, on the activity of NADPH oxidase. Thus, it can be stated that the activation of MAP kinases in phagocytes during phagocytosis occurs by a mechanism similar to that operating in nonphagocytizing cells, indicating the universality of the function of NADPH oxidases in different cell types.

[The dependence of stability of the green fluorescent protein-obelin hybrids on the nature of their constituent modules and the structure of the amino acid linker]. / Zavisimost' stabil'nosti gibridov zelenogo fluorestsentnogo belka s obelinom ot prirody sostavliaiushchikh ikh modulei i struktury aminokislotnogo linkera.

Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoria and the photoprotein obelin from Obelia longissima linked in-frame by inserts differing in nucleotide sequences were constructed. The expression of the chimeric genes in Escherichia coli cells resulted in synthesis of the GFP-obelin hybrid proteins. These proteins were purified to homogeneity and subjected to limited trypsinolysis. It was shown that the resistance of GFP-obelin hybrid proteins to trypsin depends on the nature of their constituent modules and the amino acid sequences of linkers between the modules. The kinetics of accumulation of full-length hybrid proteins during the growth of bacterial cells does not depend on the structure of the peptide linkers. Most of the full-length product accumulates in cells in the form of inclusion bodies resistant to endogenous proteases. The soluble fraction of the protein undergoes considerable proteolysis regardless of the linker structure.

[Stabilized reaction mixture for in vitro mRNA translation]. / Stabilizirovannaia reaktsionnaia smes' dlia transliatsii mRNK in vitro.

Here we describe a method for obtaining a ready-to-use stabilized reaction mixture for in vitro translation of mRNA. We also demonstrate the stabilization of a complete translation mixture containing wheat germ extract, amino acids, ATP, GTP, creatine phosphate, creatine kinase, and the reaction buffer by lyophilization in the presence of various sugars. The greatest stabilizing effect is achieved by supplementing the mixture with 10% (mass/volume) trehalose, which is also a unique translation activator, enhancing the translation of various mRNAs. A lyophilized complete translation mixture containing trehalose can be stored at 4-8 degrees C for several months without losing its activity. The mixture can be easily reconstituted by adding an aqueous mRNA solution and retains the potential for reproducible functioning. This allows the employment of such a cell-free translation system for analytical screening of a broad spectrum of compounds inhibiting translation at various stages.