Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells

Guluronate and mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polymannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)-alpha secretion from RAW264.7 cells in a structure-depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end-structure of alginate oligomers was important for the TNF-alpha-inducing activity. Among the unsaturated guluronate (G3-G9) and mannuronate (M3-M9) oligomers, G8 and M7 showed the most potent activity, respectively. Bio-Plex assay revealed that interleukin (IL)-1alpha, IL-1beta, and IL-6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure-activity relationship profiles as seen in TNF-alpha, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll-like receptor (TLR)2 and TLR4 effectively inhibited the G8- and M7-induced production of TNF-alpha, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.     

Stromal Fibroblasts Mediate Extracellular Matrix Remodeling and Invasion of Scirrhous Gastric Carcinoma Cells

Scirrhous gastric carcinoma (SGC) has the worst prognosis of all gastric cancers, owing to its rapid expansion by invasion and frequent peritoneal dissemination. Due to the increased proliferation of stromal fibroblasts (SFs) that occurs within SGC lesions and the peritoneal metastatic sites, SFs have been proposed to support the progression of this disease. However, the biological and molecular basis and the pathological role of the intercellular interaction between SGC cells and SFs remain largely unknown. In this study, we investigated the role of SFs in the invasion of the extracellular matrix (ECM) by SGC cells. When SGC cells were cocultured with SFs derived from SGC tissue on three-dimensional (3D) Matrigel, they were attracted together to form large cellular aggregates that invaded within the Matrigel. Time-lapse imaging revealed that this process was associated with extensive contraction and remodeling of the ECM. Immunofluorescence and biochemical analysis showed that SGC cells stimulate phosphorylation of myosin light chain and actomyosin-mediated mechanical remodeling of the ECM by SFs. By utilizing this assay system for inhibitor library screening, we have identified several inhibitors that potently suppress the cooperation between SGC cells and SFs to form the invasive structures. Among them, a Src inhibitor dasatinib impaired the interaction between SGC cells and SFs both in vitro and in vivo and effectively blocked peritoneal dissemination of SGC cells. These results indicate that SFs mediate mechanical remodeling of the ECM by SGC cells, thereby promoting invasion and peritoneal dissemination of SGC.     

Poxviral Protein A52 Stimulates p38 Mitogen-activated Protein Kinase (MAPK) Activation by Causing Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Self-association Leading to Transforming Growth Factor β-activated Kinase 1 (TAK1) Recruitment

Vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. These include A52, a protein shown to inhibit IL-1- and Toll-like receptor-stimulated NFκB activation, via interaction with interleukin-1 receptor-associated kinase 2 (IRAK2). Interestingly, A52 was also found to activate p38 MAPK and thus enhance Toll-like receptor-dependent IL-10 induction, which was TRAF6-dependent, but the manner in which A52 manipulates TRAF6 to stimulate p38 activation was unclear. Here, we show that A52 has a non-canonical TRAF6-binding motif that is essential for TRAF6 binding and p38 activation but dispensable for NFκB inhibition and IRAK2 interaction. Wild-type A52, but not a mutant defective in p38 activation and TRAF6 binding (F154A), caused TRAF6 oligomerization and subsequent TRAF6-TAK1 association. The crystal structure of A52 shows that it adopts a Bcl2-like fold and exists as a dimer in solution. Residue Met-65 was identified as being located in the A52 dimer interface, and consistent with that, A52-M65E was impaired in its ability to dimerize. A52-M65E although capable of interacting with TRAF6, was unable to cause either TRAF6 self-association, induce the TRAF6-TAK1 association, or activate p38 MAPK. The results suggest that an A52 dimer causes TRAF6 self-association, leading to TAK1 recruitment and p38 activation. This reveals a molecular mechanism whereby poxviruses manipulate TRAF6 to activate MAPKs (which can be proviral) without stimulating antiviral NFκB activation.     

Asymmetric Reduction by an NADH Model Compound with l-Prolinamide in the N1-Substituent

An intracellular uricase from Bacillus fastidiosus with high catalytic capacity and strong resistance to xanthine was inactivated in water but could be essentially re-activated in solutions of high ionic strength. By polyacrylamide gel electrophoresis (PAGE), gradient PAGE, sodium-dodecyl-sulfate-PAGE, gel-filtration through Sephadex G200, and activity staining with peroxidase and its chromatogenic substrate, this homotetrameric uricase in water was found to dissociate into inactive homodimers that could form active homotetramers again in solutions of high ionic strength. Sensitivity to low ionic strength of solutions complicates formulation of this uricase as a drug and its elimination requires protein engineering.     

The role of interactions between Prorocentrum minimum and Heterosigma akashiwo in bloom formation

We examined the growth and interactions between the bloom-forming flagellates Prorocentrum minimum and Heterosigma akashiwo using bi-algal culture experiments. When both species were inoculated at high cell densities, growth of H. akashiwo was inhibited by P. minimum. In other combinations of inoculation densities, the species first reaching the stationary phase substantially suppressed maximum cell densities of the other species, but the growth inhibition effect of P. minimum was stronger than that of H. akashiwo. We used a mathematical model to simulate growth and interactions of P. minimum and H. akashiwo in bi-algal cultures. The model indicated that P. minimum always out-competed H. akashiwo over time. Additional experiments showed that crude extracts from P. minimum and H. akashiwo cultures did not affect the growth of either species, but both strongly inhibited the growth of the bloom-forming diatom Skeletonema costatum. Further experiments showed that it was unlikely that reactive oxygen species produced by H. akashiwo were responsible for the inhibition of P. minimum growth.     

Synthesis of Aromatic Carbonyl Compounds by Friedel-Crafts Reaction Using BF3 Catalyst

Friedel-Crafts reaction using BF₃ catalyst was investigated in order to synthesize various carbonyl compounds.     

A facile method for preparation of t-butyloxycarbonylamino acid p-nitroanilides

A series of p-nitroanilides of t-butyloxycarbonylamino acids, including Boc-Trp, Boc-Asn, Boc-Gln, Boc-Ser(But), Boc-Thr(But), Boc-Asp(OBut), Boc-Lys(TFA), and Boc-His(Boc), were prepared conveniently by the mixed anhydride method using 2,2-dimethylpropanoic chloride (pivaloyl chloride). The products were obtained in 40-60% yields after purification by column chromatography on Sephadex LH-20. p-Nitroanilide of histidine was purified after deprotection of Boc-His(Boc)-pNA and obtained as H-His-pNA.2HCl.