Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues¹. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 Å. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.     

Diversity of cultured aerobic organisms in the areas of natural oil seepage on Lake Baikal

The diversity of cultured aerobic organisms collected from water samples and bottom sediment from two areas of natural oil seepage on Lake Baikal has been researched. Representatives of Alpha-, Beta-, and Gammaproteobacteria have been found in samples collected near the Bol’shaya Zelenovskaya River mouth, while near Cape Gorevoi Utes Betaproteobacteria were absent. Most cultures are characterized by a sufficiently high homology level (96–100%) with nucleotide sequences from the international database.     

Effect of intestinal microflora on the pathogenesis of cholera intoxication

Germ-free minipigs, previously treated with bacteroids, develop cholinergic reaction after the intragastric administration of Vibrio cholerae exotoxin. The intensity of this reaction, disturbances in homeostasis, and the character of morphological changes depend on the dose of choleragen, the bacteroid strain, and the presence of the concomitant (Escherichia coli) and residual microflora in the intestine.     

Reactogenicity of a group A and C meningococcal polysaccharide bivalent vaccine

A total of 1 968 persons aged 18-20 years belonging to organized groups were immunized with different doses of polysaccharide meningococcal divaccine, groups A and C, by means of syringes and jet injectors under the conditions of a controlled epidemiological trial. Systemic reactions to the injection of the preparation were found to be absent. Local reactions manifested by inflammation, swelling and tenderness at the site of injection were faintly pronounced and disappeared in 48 hours, which is indicative of low reactogenicity of the preparation under study.     

The protective properties of a porin from the outer membrane of Yersinia pseudotuberculosis

The protective properties of the species-specific pore-forming polypeptide of Y. pseudotuberculosis outer membrane (porin) were studied. The study showed that 80-90% of mice immunized with porin survived after challenge with 10-30 LD50 of Y. pseudotuberculosis, serovars 1 and 3. The LD50 of the preparation exceeded its ED50 more than 100-fold. Immunization made in two injections was more effective than immunization in one injection. The immunization of the animals with porin led to an increase in the activity of peritoneal exudate macrophages with respect to Y. pseudotuberculosis, serovars 1 and 3.     

Changes in the cerebral cortex in the progeny of DOCA-dependent females]

Methods of light microscopy and morphometrical analysis were used for studying semithin sections of the antero-parietal portions of the cortex of neonatal rats from mothers treated by desoxycorticosterone acetate (DOCA) during the last 7 (II group) of last 14 days (III group) of gestation. Animals of the III group were given greater doses of DOCA. Experimental neonatal rats had greater absolute and relative masses of the cerebrum and the thicker cortex: in the II group at the expense of enlargement of neurons, greater amount of glioblasts and the volume of neuropile; in the III group--at the expense of still greater neuron sizes, enlargement of glioblasts and their greater number, as well as the growth of the neuropile volume. In the neocortex of the II group of animals processes of proliferation of glioblasts were more pronounced, greater doses and amount of actions of DOCA (III group) being followed by processes of reinforced differentiation of nerve and glial cells. Experimental animals were found to have greater amount of microgliocytes, hypertrophy of endotheliocyte and pericyte nuclei. Symptoms of blood stagnation and perivascular edema were very rarely noted in neonatal rats of the III group.