Significance of Na+ in the fish pathogen, Vibrio anguillarum, under energy depleted condition

Vibrio anguillarum kills various kinds of fish over salinities ranging from seawater to freshwater. In this study, we investigated the role of Na(+) in V. anguillarum, especially under energy-depleted conditions such as in natural seawater. V. angustum S14, which is a typical marine vibrio, was used for comparison. V. anguillarum only required Na(+) for starvation-survival, but in contrast, V. angustum S14 always required Na(+) for both growth and starvation-survival. In marine vibrios, Na(+) is used in the Na(+)-dependent respiratory chain that produces the sodium motive force (SMF) across the cell membrane. It has been considered that marine vibrios always need a SMF produced by Na(+), however in the case of V. anguillarum, the SMF is not required for growth, but becomes more important for starvation-survival.     

Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin

HP1 family proteins are adaptor molecules, containing two related chromo domains that are required for chromatin packaging and gene silencing. Here we present the structure of the chromo shadow domain from mouse HP1beta bound to a peptide containing a consensus PXVXL motif found in many HP1 binding partners. The shadow domain exhibits a novel mode of peptide recognition, where the peptide binds across the dimer interface, sandwiched in a beta-sheet between strands from each monomer. The structure allows us to predict which other shadow domains bind similar PXVXL motif-containing peptides and provides a framework for predicting the sequence specificity of the others. We show that targeting of HP1beta to heterochromatin requires shadow domain interactions with PXVXL-containing proteins in addition to chromo domain recognition of Lys-9-methylated histone H3. Interestingly, it also appears to require the simultaneous recognition of two Lys-9-methylated histone H3 molecules. This finding implies a further complexity to the histone code for regulation of chromatin structure and suggests how binding of HP1 family proteins may lead to its condensation.     

Adhesion behavior of peritoneal cells on the surface of self-assembled triblock copolymer hydrogels

Adhesion behavior of cells to the surface of physical hydrogel membranes prepared by water-induced self-organization of precisely synthesized ABA-triblock copolymers comprised of poly(beta-benzyl L-aspartate) (PBLA) as A segment and poly(ethylene oxide) (PEO, molecular weight = 20 000) as the B segment were investigated. The cast film from the methylenechloride solution of these copolymers swelled in water very rapidly forming hydrogels (100-400% water content of total weight). The content of PBLA affected the strength, the hydrophobicity, and the amount of water involved in the hydrogel surface. During the early stage of cultivation with murine peritoneal cells, cell adhesion on the hydrogels of PEO and PBLA with 18 (20K18) and 25 (20K25) monomeric units was not observed, while adhesion on the hydrogels of PEO and PBLA with 32 (20K32) and 55 (20K55) monomeric units was successful, suggesting more than 12 mol % in PBLA content is necessary for adhesion of these cells. Although cell spreading on the hydrogels of 20K18, 20K25, and 20K32 was not sufficient, the hydrogel of 20K55 allowed cell adhesion and spreading to be bipolar with leading edge whose raffling is active with pseudopodium and lamellipodium as well as PBLA homopolymer, suggesting active motility of these cells. Remarkably, prolonged incubation restored adhesiveness onto the films at 20K18 in contrast to adhesion with 20K25 despite low hydrophobicity. It is conceivable that adaptation of proteins and chemical changes to the surface during the culture period may participate in these phenomena. Mechanical properties and interaction between cell and these copolymer hydrogels could be controlled by composition of block segments, and optimization for implants could also be attainable.     

Protein region important for regulation of lipid metabolism in angiopoietin-like 3 (ANGPTL3): ANGPTL3 is cleaved and activated in vivo

Angiopoietin-like 3 (ANGPTL3) is a secreted protein that is mainly expressed in the liver and regulates lipid metabolism by inhibiting the lipolysis of triglyceriderich lipoproteins. Using deletion mutants of human ANGPTL3, we demonstrated that the N-terminal coiled-coil domain-containing fragment-(17-207) and not the C-terminal fibrinogen-like domain-containing fragment-(207-460) increased the plasma triglyceride levels in mice. We also found that the N-terminal region 17-165 was required to increase plasma triglyceride levels in mice and that a substitution of basic amino acid residues in the region 61-66 of the fragment showed no increase in the plasma triglyceride levels and no inhibition of lipolysis by lipoprotein lipase. In addition, when we analyzed ANGPTL3 in human plasma, we detected cleaved fragments of ANGPTL3. By analyzing recombinant ANGPTL3 in mouse plasma, we found that it was cleaved at two sites, Arg221 downward arrow Ala222 and Arg224 downward arrow Thr225, which are located in the linker region between the coiled-coil domain and the fibrinogen-like domain. Furthermore, a cleavage-resistant mutant of ANGPTL3 was determined to be less active than wild-type ANGPTL3 in increasing mouse plasma triglyceride levels but not in inhibiting lipoprotein lipase activity. These findings suggest that the cleavage of ANGPTL3 is important for the activation of ANGPTL3 in vivo.     

Stability of the I-motif structure is related to the interactions between phosphodiester backbones

The i-motif DNA tetrameric structure is formed of two parallel duplexes intercalated in a head-to-tail orientation, and held together by hemiprotonated cytosine pairs. The four phosphodiester backbones forming the structure define two narrow and wide grooves. The short interphosphate distances across the narrow groove induce a strong repulsion which should destabilize the tetramer. To investigate this point, molecular dynamics simulations were run on the [d(C2)]4 and [d(C4)]4 tetramers in 3'E and 5'E topologies, for which the interaction of the phosphodiester backbones through the narrow groove is different. The analysis of the simulations, using the Molecular Mechanics Generalized Born Solvation Area and Molecular Mechanics Poisson-Boltzmann Solvation Area approaches, shows that it is the van der Waals energy contribution which displays the largest relative difference between the two topologies. The comparison of the solvent-accessible area of each topology reveals that the sugar-sugar interactions account for the greater stability of the 3'E topology. This stresses the importance of the sugar-sugar contacts across the narrow groove which, enforcing the optimal backbone twisting, are essential to the base stacking and the i-motif stability. Tighter interactions between the sugars are observed in the case of N-type sugar puckers.     

Crystal structure of rice alpha-galactosidase complexed with D-galactose

alpha-Galactosidases catalyze the hydrolysis of alpha-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. The crystal structure of rice alpha-galactosidase has been determined at 1.5A resolution using the multiple isomorphous replacement method. The structure consisted of a catalytic domain and a C-terminal domain and was essentially the same as that of alpha-N-acetylgalactosaminidase, which is the same member of glycosyl hydrolase family 27. The catalytic domain had a (beta/alpha)8-barrel structure, and the C-terminal domain was made up of eight beta-strands containing a Greek key motif. The structure was solved as a complex with d-galactose, providing a mode of substrate binding in detail. The d-galactose molecule was found bound in the active site pocket on the C-terminal side of the central beta-barrel of the catalytic domain. The d-galactose molecule consisted of a mixture of two anomers present in a ratio equal to their natural abundance. Structural comparisons of rice alpha-galactosidase with chicken alpha-N-acetylgalactosaminidase provided further understanding of the substrate recognition mechanism in these enzymes.