Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.     

Ecological description of ground beetle communities (Coleoptera, Carabidae) in northern taiga meadows of Arkhangelsk Region

Species composition and ecological structure of ground beetle population was studied in northern taiga meadows of the Arkhangelsk Region. Meadows in the northern forest zone proved to harbor 91 ground beetle species. Carabid complexes formed in the intrazonal biocenoses of the northern forest zone can be as rich as the family topical groups in the southern forest zone by the number of species and ecological diversity. Ecological properties of the fauna and ground beetle population proved similar in different parts of the forest zone. The proportion of stenobiotic meadow species proved to decrease while that of ecologically plastic ones increased from south to north. The proportion of the genus Harpalus decreased in the ground beetle population while the number of Amara species remained unaltered and their abundance increased. The changes in the species composition caused no transformation of the ecological structure of ground beetle population since they were limited to a single life form or guild.     

An Immunohistochemical Study of Localization of the Calcium-Binding Protein Recoverin in Retina of the Newt Pleurodeles waltl

The presence and localization of the calcium-binding protein recoverin, initially found in photoreceptors of the bovine eye, were immunochemically studied in retina of the new Pleurodeles waltl. Polyclonal monospecific antibodies against recoverin were raised and the methods of immunoblotting and indirect immunofluorescence were used. A protein with an apparent molecular mass of 26 kDa was found in the retina extract, which was specifically stained by the antibodies against recoverin. Localization of recoverin was studied on the retina sections: an intense reaction was found in the inner segments and a weak reaction was found in the basal part of the outer segments of photoreceptors and in Landolt's clubs of displaced bipolars. The results we obtained suggest for the first time the presence of recoverin in the retina of a representative of the Urodeles and indicate to interspecific conservativeness of this protein and differences of its localization in the retina photoreceptors in different species. The data obtained open a possibility of using recoverin as a marker protein of photoreceptors and displaced bipolars in studies of retina regeneration in newts.     

Incompatibility of the Cad plasmid from Yersinia pestis with plasmids of the IncFI group

The relation of Yersinia pestis calcium dependence plasmid (pCad) to known Inc FI (F'lac, R386, pOX38) and IncFV (F0lac) plasmids has been studied. Evidence that plasmid pCad of Yersinia pestis belongs to FI incompatibility group is presented.     

Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions.

We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.     

Plasmid content in Yersinia pestis strains of different origin

Plasmid content in 242 Yersinia pestis strains from various natural plague foci of the U.S.S.R. and other countries was studied. Of these strains, 172 (71%) were shown to carry three plasmids described previously of about 6, 45-50 and 60 MDa, respectively. Twenty strains (8%) from different foci harboured additional cryptic plasmids, most often of about 20 mDa in size. Plasmid pPst displayed considerable constancy of its molecular mass. On the contrary, size variations of pCad (45-49 MDa) and, especially, pFra (60-190 MDa) were found. Molecular mass of these plasmids correlated with the host strain origin.     

Synthesis, cloning, and expression of artificial genes, coding antigenic determinants of the foot-and-mouth virus substrain A22]

Chemical-enzymatic synthesis and cloning in Escherichia coli of double-stranded DNAs, coding for simple and complex antigenic determinants of foot-and-mouth disease virus (FMDV) strain A22, have been carried out. The simple antigenic determinants are a part of the viral coat protein VP1 (amino acid sequence 131-152 or 131-160) whereas the complex antigenic determinants comprise additionally the amino acid sequence 200-213 of VP1 linked to N-terminus of simple antigenic determinants through a tetrapeptide spacer Pro-Pro-Ser-Pro. Recombinant DNAs containing genes for antigenic determinants of FMDV fused with C-terminus of gene for human tumor necrosis factor (hrTNF) have been constructed. Expression of the hybrid genes and properties of the proteins coded were studied. All recombinant proteins were shown to interact specifically with polyclonal antibodies both against hrTNF and FMDV strain A22. The recombinant proteins produced by bacteria are perspective for study as a vaccine against FMDV.     

Determination of the initial rate of antimutagenic repair in UV-irradiated WP2 Escherichia coli cells]

The initial rates of antimutagenic dark repair were measured in Escherichia coli WP2 trpE65 cells irradiated by UV-light (11 J/m2) and then incubated in liquid media of various compositions. Samples were taken from suspension of incubated bacteria every 5 min following irradiation, mixed with acriflavine to block further repair and plated onto the selective medium containing acriflavine (1 micrograms/ml) to score the Trp+ mutations. The initial rate of antimutagenic repair was estimated from the kinetics of disappearance of mutations in several successive probes. It appeared to depend on the composition of a medium, to establish just after placing irradiated bacteria onto the medium and to decrease significantly in irradiated cells incubated under conditions favourable for growth. The decrease was not due to inhibition of postreplicative repair and was not caused by casaminoacids as such, but by combination of growth factors that provided the intensive protein synthesis. The decrease could be responsible for a strong mutational response of bacteria to irradiation because it secures the survival of premutagenic lesions in DNA till mutation fixation. It is suggested that metabolic regulation of the antimutagenic repair activity exists, based on an active switch of the energy flows required for several parallel metabolic pathways that proceed in irradiated cells.     

Localization of glycoconjugates in tissues of mammals revealed by means of labeled lectins

Histochemical peculiarities on binding of castor-oil plant, soybean and lentil lectins with tissues of the mucous membrane in the stomach, small and large intestine have been studied in the human being, rat, mouse, as well as the lectins mentioned and the maize agglutinin with the nervous tissue of the rat cerebral tissue. The reactions are carried out with nonfixed cryostat and deparaffinized histological slices. Lectins labelled with horseradish peroxidase are used. Certain specific peculiarities and differences concerning the lectin binding with tissues of the organs studied are determined. Predominant binding is noted of the soybean lectin with parietal and mucin-producing cells of the stomach, with epitheliocytes of the duodenal glands, with the brush border of the epithelial cells of the intestinal villi. The lentil and castor-oil plant lectins make contours of the basal membrane epithelium in the stomach and intestine. The lentil lectin also reacts with the germinative centers of the stomach lymphatic nodules and the castor-oil plant agglutinin--with the brush border of the small intestine epitheliocytes. The lectins used are predominantly bound with neurons of the subcortical formations of the rat brain and cerebral cortex. By means of labelled lectins of lentil, soybean, and castor-oil plant it is possible to reveal certain modifications of the rat small intestine glycoconjugates produced by means of the immortelle extract.     

Effect of forskolin on action potential, slow inward current and tension of frog atrial fibers

The new nonhormonal activator of adenylate cyclase forskolin was studied on frog atrial trabeculae by current clamp and voltage clamp methods using a double sucrose gap technique. Forskolin (5 X 10(-6) M to 2 X 10(-5) M) dose-dependently increased action potential duration, the height of the plateau and twitch tension. The time constant for inactivation of the slow inward current and the steady state kinetic variables of calcium channels d infinity and f infinity remained uneffected. Forskolin increased the amplitude of slow inward calcium current isi and of the phasic tension related to it. The maximal conductance gsi increased. These effects were indistinguishable from those obtained earlier on cardiac fibers with hormonal and nonhormonal activators of cyclic AMP-dependent phosphorylation. The beta-adrenoreceptor antagonist propranolol 10(-6)M did not decrease the effect of forskolin. Forskolin had no effect when slow inward current was previously increased by saturating concentrations of the beta-adrenergic agonist isoproterenol (10(-4)M). Our results are in favour of the hypothesis that cyclic AMP-dependent phosphorylation of membrane proteins modulates the Ca-entry in the heart cells through the membrane slow calcium channels.