Revised classification of native probiotic strains of Lactobacillus used in Russian Federation

Thirteen strains of industrial bacterial cultures of the genus Lactobacillus (from a collection of Gabrichevsky Research Institute of Epidemiology and Microbiology) were studied. These strains were used for decades in Russian Federation for food and drug production, as ferments for lactic acid products, for production of probiotics, biologically active and veterinary preparations. Complex analysis of data on cultures obtained using microbiological and molecular-genetic methods was conducted for the first time. Biochemical characteristics of these cultures were studied and the sequence of the proximal region of 16S ribosomal RNA gene was determined. The employment of the test system API-50CHL was shown to broaden the opportunities of a more accurate biochemical identification of bacteria belonging to the genus Lactobacillus, in comparison with the set ANAEROTEST-23. According to the results obtained in a comparative analysis of nucleotide sequences of 16S rRNA gene, all strains examined show 97-99% homology of the proximal region of this gene with that of the type representatives of studied species. These data allowed taxonomic reclassification of the species position of cultures with consideration of the more advanced level of systematics. Nucleotide sequences of gene fragments of examined lactobacilli strains were recorded in NCBI database (accession numbers of deposits GU560031, GU560032, GU560033, GU560034, GU560035, GU560036, GU560037, GU560038, GU560039, GU560040, GU560041, GU560042, GU560043).     

Comparative effects of limited tryptic hydrolysis on physicochemical and structural features of seed 11S globulins

The effect of the limited proteolysis by trypsin on selected seed storage 11S globulins (broad bean and pea legumins, glycinin and helianthinin) was studied by high-sensitive differential scanning calorimetry, fluorescence spectroscopy and analysis of proteolysis kinetics. Different behaviour of glycinin and helianthinin, on one hand, and broad bean and pea legumins, on the other, were observed: in the first group changes in the physicochemical characteristics of the proteins due to their limited proteolysis are more pronounced in comparison with the second one, in relation with the extent of primary structure modifications. The differences observed have been evaluated in relation with the amino acid sequence features of the four 11S globulin studied and agree with the literature data concerning the protein structural changes in the course of the limited proteolysis.     

Low-Molecular-Weight Glycoprotein from Cattle Blood Serum: Structure and Properties

The amino acid composition, structure, and physicochemical properties of a low-molecular-weight glycoprotein from cattle blood serum (SGP) were studied. The content of carbohydrates (represented by mannose-rich oligosaccharides) amounted to 45–50 wt %. The value of the specific partial heat of SGP, measured by differential scanning calorimetry (DSC), equaled 1.8 J/(g K), which is characteristic of unfolded proteins. Circular dichroic (CD) spectra of SGP led us to conclude that it is not highly structured and that it occurs in the shape of a statistical globule. The protein was deglycated using anhydrous trifluoromethane sulfonate (TFMS), after which its amino acid composition and the sequence of a fragment were determined. The results indicate that SGP is a protein not studied previously.     

Genetic properties of phage lambda recombinant molecules. The effect of an inserted fragment on the hybrid yield and recombination with respect to the h- and vir-markers

The yield of mature phages in lambda gt-lambda Dm34, lambda gt-lambda DM225 and lambda gt-lambda Dm234 is 2, 8 and 8 times lower (respectively) as compared to the wild type. The output of hvir-recombinants in lambda gt-lambda Dm225 is more than an order lower as compared to the other phages. As distinct from two other hybrids, the decrease in lambda gt-lamba DM225 yield cannot be explained by red-genotype and a shortening of the phage genome as well as the decrease in the output hvir-recombinants cannot be attributed to a decrease in the yield of mature particles and to a shortening of the distance between markers. The data obtained show a contribution of Drosophila DNA to the observed effects.     

Na+-K+-ATPase in frog esophagus mucociliary cell membranes: inhibition by protein kinase C activation

We examined protein kinase C (PKC)-dependentregulation ofNa⁺-K⁺-ATPasein frog mucociliary cells. Activation of PKC by12-O-tetradecanoylphorbol-13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol(diC8) either in intact cells or isolated membranes resulted in aspecific inhibition ofNa⁺-K⁺-ATPaseactivity by ~25-45%. The inhibitory effects in membranes exhibited time dependence and dose dependence [half-maximalinhibition concentration (IC₅₀) = 0.5 ± 0.1 nM and 2.4 ± 0.2 µM, respectively, for TPA anddiC8] and were not influenced byCa²⁺. Analysis of the ouabaininhibition pattern revealed the presence of twoNa⁺-K⁺-ATPaseisoforms with IC₅₀ values forcardiac glycoside of 2.6 ± 0.8 nM and 409 ± 65 nM,respectively. Most importantly, the isoform possessing a higheraffinity for ouabain was almost completely inhibited by TPA, whereasits counterpart was hardly sensitive to the PKC activator. The resultssuggest that, in frog mucociliary cells, PKC regulatesNa⁺-K⁺-ATPaseand that this action is related to the specificNa⁺-K⁺-ATPaseisoform.

     

Cholinergic regulation of the Na, K-ATPase activity of the pig kidney

Acetylcholine does not change the activity of Na, K-ATPase isolated from pig kidney. The enzyme is shown to have insignificant acetylcholinesterase activity. It is suggested that Na, K-ATPase sensitivity to acetylcholine disappears in the course of enzyme purification and that acetylcholinesterase activity is extrinsic.     

Responses of cultured rat trigeminal ganglion neurons to bitter tastants

The initial steps in taste and olfaction result from the activation by chemical stimuli of taste receptor cells (TRCs) and olfactory receptor neurons (ORNs). In parallel with these two pathways is the chemosensitive trigeminal pathway whose neurons terminate in the oral and nasal cavities and which are activated by many of the same chemical stimuli that activate TRCs and ORNs. In a recent single unit study we investigated the responses of rat chorda tympani and glossopharnygeal neurons to a variety of bitter-tasting alkaloids, including nicotine, yohimbine, quinine, strychnine and caffeine, as well as capsaicin, the pungent ingredient in hot pepper. Here we apply many of these same compounds to cultured rat trigeminal ganglion (TG) neurons and measure changes in intracellular calcium [Ca2+]i to determine whether TG neurons will respond to these same compounds. Of the 89 neurons tested, 34% responded to 1 mM nicotine, 7% to 1 mM caffeine, 5% to 1 mM denatonium benzoate, 22% to 1 mM quinine hydrochloride, 18% to 1 mM strychnine and 55% to 1 microM capsaicin. These data suggest that neurons from the TG respond to the same bitter-tasting chemical stimuli as do TRCs and are likely to contribute information sent to the higher CNS regarding the perception of bitter/irritating chemical stimuli.      

Results of phase I clinical trials of a combined vaccine against HIV-1 based on synthetic polyepitope immunogens

The CombiHIVvac candidate vaccine against HIV-1/AIDS containing two synthetic polyepitope immunogens such as TBI and TCI to stimulate the humoral and cellular response is described. The recombinant TBI protein is constructed as a polypeptide with predetermined tertiary structure and contains epitopes of Env and Gag proteins of HIV-1. TCI contains CD8⁺ CTL and CD4⁺ Th epitopes of the major viral proteins such as Env, Gag, Pol and Nef which are highly conserved among subtypes A, B and C of HIV-1. A gene encoding the polyepitope TCI immunogen is inserted into a pcDNA-3.1 plasmid vector. The CombiHIVvac vaccine was designed as virus-like particles containing the pcDNA-TCI plasmid in their cores (DNA vaccine) and the TBI protein conjugated with polyglucin on their surfaces. Immunogenicity and safety of CombiHIVvac has been shown in preclinical studies in several animal species. Phase I clinical trials of the vaccine have been completed and the results obtained in human volunteers confirmed that the CombiHIVvac candidate vaccine was safe and did not cause side effects, at the same time, inducing the HIV-specific humoral and cellular immune response. The phase II clinical trials have been approved by the Ministry of Health and Social Development of the Russian Federation.