Monoclonal Antibody HlyIIC­15 to C-End Domain HlyII B. cereus Interacts with the Trombin Recognition Site.

Among the panel of monoclonal antibodies to the recombinant protein HlyIICTD Bacillus cereus an antibody was found capable of forming an immune complex with a thrombin recognition region, the amino acid sequence of which is located inside the recombinant HlyIICTD. Localization of the epitope was carried out using peptide phage display methods, as well as enzyme immunoassay and immunoblotting for interaction with recombinant proteins, either containing or not containing individual components HlyIICTD. The identified epitope is located in the region of the thrombin site and retains the ability to interact with the antibody after the proteolyotic attack of the protein by thrombin.

[Detection of AlpA and AlpB lytic endopeptidase propeptides of Lysobacter sp. XL1 by sandwich-enzyme immunoassay based on monoclonal antibodies].

The extracellular lytic endopeptidases AlpA and AlpB of the bacterium Lysobacter sp. XL1 are highly homologous and synthesized as precursors consisting of signal peptide, propeptide and mature form. In this work, two monoclonal antibodies against propeptide endopeptidase AlpA (ProA) and eleven against propeptide endopeptidase AlpB (ProB) were obtained to study the AlpA and AlpB endopeptidases secretion. The affinity constants of the antibodies against ProA were 2.9 x 10(9) and 3.5 x 10(9) M(-1), and the affinity constants of the antibodies against ProB were from 1.5 x 10(8) to 2.2 x 10(9) M(-1). The obtained antibodies did not have cross-reactivity between themselves, as well as mature forms of the enzymes. The monoclonal antibodies based sandwich-type enzyme immunoassay has been developed for measuring the propeptide in a native form. The linear range of determination ProA was 1.5-100 ng/mL with 6% error of measurement, and for determining ProB 0.2-6.25 ng/mL with 6% error. Using the developed assay, ProA and ProB propeptides have been detected in cell lysates of Lysobacter sp. XL1 in an amount 1.18 ± 0.03 ng and 0.096 ± 0.002 ng per 1 OD540 of the bacterial culture, respectively. The immunochemical assay for detection various forms of AlpA and AlpB lytic endopeptidases can be useful when dealing with issues related to their secretion into the environment.

[Chemical composition of breast milk in females with preterm deliveries in the Primorsky Krai].

In the article there are presented data on the chemical composition of breast milk in females with preterm labor in the Primorye Territory, who were in the Department of newborns for premature babies of the Municipal Institution of Health "Children's city clinical hospital" in Vladivostok during 2010-2011 to care for their newborn infants. Laboratory studies were performed in the Federal State Institution of Health "Center of Hygiene and Epidemiology in the Primorye Territory."

[Preparation and characterization of monoclonal antibodies to Bacillus anthracis protective antigen].

Anthrax is the widespread acute infection disease, affecting animals and humans, refers to the bioterrorist threat agents of category A, because of the high resistance of Bacillus anthracis spores to adverse environmental factors and the ease of receiving them. We obtain a representative panel of 20 monoclonal antibodies against the key component of pathogenic exotoxins, anthrax protective antigen. Quantitative sandwich-ELISA for protective antigen with antibody obtained was developed. Six pairs of monoclonal antibodies showed the detection limit up to 1 ng/ml concentration of the protective antigen in blood serum.

[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.

[Anti-idiotypic monoclonal antibodies against latrotoxin interact with rat brain synaptosomes and modify latrotoxin-induced calcium entry into synaptosomes]. / Antiidiotipicheskie monokonal'nye antitela protiv latrotoksina vzaimdeistvuiut s sinaptosomami iz mozga krysy i modifitsiruiut latrotoksinindutsirovannyi vkhod kal'tsiia v sinaptosomy.

Hybridoma lines producing anti-idiotypic monoclonal antibodies (AImAbs) were prepared by fusing splenocytes of mice immunized with alpha-latrotoxin (LT) and P3-X63Ag8.653 myeloma line cells. AImAbs (1) bind to the rat brain synaptosomes, (2) do not affect the LT binding to the high-affinity receptor, and (3) do not react with LT in solution. The effect of AImAbs on the LT-induced 45Ca2+ influx into rat brain synaptosomes was studied. Some antibodies (6.6D11 and 11.7B7) were found to strongly inhibit this process. The result obtained indicate that the presynaptic membrane contains unidentified components interacting with LT. The distortion of the interaction of LT with these unknown components affects the LT-induced calcium influx into synaptosomes.

[Contacts of ribosomal proteins with tRNAPhe and 16S RNA in analogs of the 30S initiation complex]. / Kontakty ribosomnykh belkov s tRNKPhe i 16S RNK v analogakh 30S initsiatornogo kompleksa.

Direct RNA-protein contacts have been studied by means of ultraviolet-induced (254 nm) cross-links inside complexes of NAcPhe-tRNAPhe, Phe-tRNAPhe and deacylated tRNAPhe with poly(U)-charged 30S subunit of Escherichia coli ribosome. In the first two complexes tRNA directly contacts with the similar sets of proteins (S4, S5, S7, S9/S11; S6 and S8 are found only in the second complex). These sets are similar to that in the fMet-tRNAfMet X 30S X mRNA complex, evidencing similar disposition of tRNAs in these three complexes. 16S RNA contacts in free 30S subunit mainly with proteins S4, S7 and S9/S11. In both complexes, containing NAcPhe-tRNAPhe and Phe-tRNAPhe, 16S RNA contacts with essentially the same proteins (S4, S5, S7, S8, S9/S11, S10, S15, S16 and S17) and in the same ratio, evidencing similar conformation of 30S subunit in these two complexes. In the third complex deacylated tRNAPhe contacts with proteins S4, S5, S6, S8, S9/S11 and S15, 16S RNA-protein interaction differs from those in the first two complexes by a remarkable decrease of cross-linked proteins S8, and S9/S11 and by the appearance of a large amount of cross-linked proteins(s) S13/S14. Hence, this complex differs from the first two by conformation of 30S subunit and, probably, by disposition and/or conformation of tRNA.