Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice,Spitzbergen

The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4°C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses and of Proteobacteria, the Bacillus–Clostridium group, the order Actinomycetales, and the Cytophaga–Flexibacter–Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25°C. Only a few strains were psychrophilic, with an optimal growth at 10–15°C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4°C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, -amylase and -galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10°C, and almost no production was detected at higher temperatures (20–30°C). Catalytic activity was detected even below the freezing point of water (at –5°C), demonstrating the unique properties of these enzymes.     

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.     

Structure, dynamics, and elasticity of free 16s rRNA helix 44 studied by molecular dynamics simulations

Molecular dynamics (MD) simulations were employed to investigate the structure, dynamics, and local base-pair step deformability of the free 16S ribosomal helix 44 from Thermus thermophilus and of a canonical A-RNA double helix. While helix 44 is bent in the crystal structure of the small ribosomal subunit, the simulated helix 44 is intrinsically straight. It shows, however, substantial instantaneous bends that are isotropic. The spontaneous motions seen in simulations achieve large degrees of bending seen in the X-ray structure and would be entirely sufficient to allow the dynamics of the upper part of helix 44 evidenced by cryo-electron microscopic studies. Analysis of local base-pair step deformability reveals a patch of flexible steps in the upper part of helix 44 and in the area proximal to the bulge bases, suggesting that the upper part of helix 44 has enhanced flexibility. The simulations identify two conformational substates of the second bulge area (bottom part of the helix) with distinct base pairing. In agreement with nuclear magnetic resonance (NMR) and X-ray studies, a flipped out conformational substate of conserved 1492A is seen in the first bulge area. Molecular dynamics (MD) simulations reveal a number of reversible alpha-gamma backbone flips that correspond to transitions between two known A-RNA backbone families. The flipped substates do not cumulate along the trajectory and lead to a modest transient reduction of helical twist with no significant influence on the overall geometry of the duplexes. Despite their considerable flexibility, the simulated structures are very stable with no indication of substantial force field inaccuracies.     

CD40 stimulation induces differentiation of acute lymphoblastic leukemia cells into dendritic cells

Despite the very high percentage of long-term remissions in acute lymphoblastic leukemia (ALL) in children, some of them suffer from recurrence of the disease. New treatment modalities, e.g. effective geno- and immunotherapy are needed. The use of neoplasmatic cells to present tumor antigens is one of the approaches in cancer vaccines. ALL cells lack the expression of costimulatory molecules and are poor antigen presenting cells (APCs) for T-cell activation. CD40/40L interaction stimulates B-cells to proliferate, differentiate, upregulate costimulatory molecules and increase antigen presentation. The aim of the study was to test the hypothesis that ALL cells can be turned into professional APCs by CD40L activation. Children with B-cell precursor ALL were enrolled into the study. Mononuclear cells from bone marrow or peripheral blood were stimulated with CD40L and interleukin 4. Results: 1) after culture we noted upregulation of all assessed costimulatory, adhesion and activatory molecules i.e. CD1a, CD11c, CD40, CD54, CD80, CD83, CD86, CD123, HLA class I and II; 2) CD40L activated ALL cells induced proliferation of allogeneic T-cells (measured by [(3)H]thymidine incorporation). These results confirm the possibility of enhancing the immunogenicity of ALL cells with the CD40L system and indicate that this approach can be used in immunotherapeutic trials.     

Influence of Lactobacillus on the migration activity of macrophages

Influence of Lactobacillus on the migration activity of intact macrophages during their cultivation with serum and supernatant of cells from Peyer's patches and spleen that were obtained from mice of CBA line orally sensibilized with lactobacteria was studied. Data about stimulation of production of factor inhibiting migration of macrophages by immunocompetent cells and development of delayed-hypersensivity reaction are obtained. Mechanisms of complex influence of lactobacteria on cell factors of innate and adaptive immunity are discussed.     

Motor and cognitive functions of the neostriatum during bilateral blocking of its dopamine receptors

In experiments on 60 Sprague-Dawley rats, effects of systemic and intrastriatal injections of se-lective blocker of D1 receptors SCH23390 on elaboration of discriminational conditioned reflex of active avoidance (CRAA) were studied in T-maze and on behavior in test of the "open field". Systemic administration of this inhibitor at doses of 0.025 mg/kg produced a several fold decrease of percentage of correct realizations of the discriminational CRAA and of motor activity in the "open field" test. Bilateral microinjections of SCH23390 into the rat neostriatum at a dose of 0.004-1.0 mkg did not deteriorate learning of the discriminational CRAA as compared with intact control, although a marked inhibition of motor activity was observed in the open field, test. Analysis of the data has also shown a statistically significant decrease of percentage of errors in the starting maze compartment in experiments with intrastriatal injection of SCH23390 to rats. At the same time, the intrastriatal injection to rats of raclopride, a blocker of D2 dopamine receptors, at a dose of 0.004 mkg produced a sharp and prolonged deterioration of learning of the discriminational CRAA. The data obtained have allowed the following conclusions to be made: 1. Difference of effects of the systemic and intrastriatal SCH23390 injections seems to be due to that the behavioral changes observed at the systemic administration can be provided predominanantly by structures differing from neostriatal D1 receptors; 2. Effect of nigrostriatal dopaminergic system on the neostriatum through D1 receptors is complex: activation of motor activity (efferent spine cells of the direct pathway) and a poor modulation of the learning process (large aspine cholinergic interneurons); 3. The modulation of the learning process seems to occur through neostriatal D2 receptors (large aspine cholinergic interneurons).     

Characterization of antimicrobial compounds produced by Pseudomonas aurantiaca S-1.

Pseudomonas aurantiaca S-1 can serve as a natural source of pesticides towards phytopathogens like Fusarium oxysporum P1 and Pseudomonas syringae pv. glycinea BIM B-280. The largest pool of produced antimicrobial compounds was found in four days-old spent culture supernatant. At least two groups of bioactive substances were identified, one responsible for the antibacterial activity and the other for the antifungal activity. The fraction with strong antibacterial activity had the molecular mass 282.8 and formula C18H36NO, and the fraction with strong antifungal activity had molecular mass 319.3 and molecular formula C20H31O3 which could be a new fungicide. Additionally, P. aurantiaca S-1 was able to produce indoleacetic acid and siderophores.