Ca2+ -independent sortase-A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli.

A Staphylococcus aureus transpeptidase, sortase A (SrtA), which catalyzes a peptide ligation with high substrate specificity, is a useful tool to site-specifically attach proteinaceous/peptidic functional molecules to target proteins. However, its strong Ca(2+) dependency makes SrtA difficult for use under low Ca(2+) concentrations and in the presence of Ca(2+)-binding substances. To overcome this problem, we designed a SrtA mutant that Ca(2+)-independently demonstrates a high catalytic activity. The heptamutant (P94R/E105K/E108A/D160N/D165A/K190E/K196T), which resulted from a combination of known mutations at the Ca(2+) -binding site and around the substrate-binding site, successfully catalyzed a selective protein-protein ligation in the cytoplasm of Escherichia coli. Selective protein modification in living cells is a promising approach for investigating cellular events and regulating cell functions. This SrtA mutant may prove to be a versatile tool for adding new functionalities to proteins of interest by incorporating functional proteins and chemically modified peptides in living cells, which usually retain low Ca(2+) concentrations.

Determination of an acceptable assimilable organic carbon (AOC) level for biological stability in water distribution systems with minimized chlorine residual.

There is considerable interest in minimizing the chlorine residual in Japan because of increasing complaints about a chlorinous odor in drinking water. However, minimizing the chlorine residual causes the microbiological water quality to deteriorate, and stricter control of biodegradable organics in finished water is thus needed to maintain biological stability during water distribution. In this investigation, an acceptable level of assimilable organic carbon (AOC) for biologically stable water with minimized chlorine residual was determined based on the relationship between AOC, the chlorine residual, and bacterial regrowth. In order to prepare water samples containing lower AOC, the fractions of AOC and biodegradable organic matter (BOM) in tap water samples were reduced by converting into biomass after thermal hydrolysis of BOM at alkaline conditions. The batch-mode incubations at different conditions of AOC and chlorine residual were carried out at 20 °C, and the presence or absence of bacterial regrowth was determined. The determined curve for biologically stable water indicated that the acceptable AOC was 10.9 µg C/L at a minimized chlorine residual (0.05 mg Cl(2)/L). This result indicated that AOC removal during current water treatment processes in Japan should be significantly enhanced prior to minimization of the chlorine residual in water distribution.

Design of Ca2+-independent Staphylococcus aureus sortase A mutants.

The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca(2+), because binding of Ca(2+) to Glu residues distal to the active site stabilizes the substrate binding site. To obtain Ca(2+)-independent SaSrtA, we substituted two Glu residues in the Ca(2+)-binding pocket (Glu(105) and Glu(108)). Although single mutations decreased SaSrtA activity, mutations of both Glu(105) and Glu(108) resulted in Ca(2+)-independent activity. Kinetic analysis suggested that the double mutations affect the substrate binding site, without affecting substrate specificity. This approach will allow us to develop SaSrtA variants suitable for various applications, including in vivo site-specific protein modification and labeling.

A survey on levels and seasonal changes of assimilable organic carbon (AOC) and its precursors in drinking water.

In Japan, customers' concerns about chlorinous odour in drinking water have been increasing. One promising approach for reducing chlorinous odour is the minimization of residual chlorine in water distribution, which requires stricter control of organics to maintain biological stability in water supply systems. In this investigation, the levels and seasonal changes of assimilable organic carbon (AOC) and its precursors in drinking water were surveyed to accumulate information on organics in terms of biological stability. In tap water samples purified through rapid sand filtration processes, the average AOC concentration was 174 microgC/L in winter and 60 microgC/L in summer. This difference seemed to reflect the seasonal changes of AOC in the natural aquatic environment. On the other hand, very little or no AOC could be removed after use of an ozonation-biological activated carbon (BAC) process. Especially in winter, waterworks should pay attention to BAC operating conditions to improve AOC removal. The storage of BAC effluent with residual chlorine at 0.05-0.15 mgCl2/L increased AOC drastically. This result indicated the possibility that abundant AOC precursors remaining in the finished water could contribute to newly AOC formation during water distribution with minimized residual chlorine. Combined amino acids, which remained at roughly equivalent to AOC in finished water, were identified as major AOC precursors. Prior to minimization of residual chlorine, enhancement of the removal abilities for both AOC and its precursors would be necessary.

Phenolic constituents isolated from Fragaria ananassa Duch. inhibit antigen-stimulated degranulation through direct inhibition of spleen tyrosine kinase activation.

We isolated eight phenolic constituents from Fragaria ananassa Duch. (strawberry) and determined their structures using 1D, 2D-NMR. Among the isolated compounds, linocinnamarin (LN), 1-O-trans-cinnamoyl-beta-d-glucopyranose (CG), and cinnamic acid (CA) exhibited antigen (Ag)-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells. In order to reveal the underlying mechanisms, we examined the effects of LN and CA on cellular responses induced by antigen stimulation. Treatment with both LN and CA markedly inhibited antigen-stimulated elevation of intracellular free Ca(2+) concentration and reactive oxygen species (ROS). Both LN and CA suppressed Ag-stimulated spleen tyrosine kinase (Syk) activation. These results indicate that inhibition of antigen-stimulated degranulation by LN and CA is mainly due to inactivation of Syk/phospholipase Cgamma (PLCgamma) pathways. Our findings suggest that LN and CA isolated from F. ananassa Duch. (strawberry) could be beneficial agents for alleviating symptoms of type I allergy.