Toll-like receptor 3 contributes to spinal glial activation and tactile allodynia after nerve injury

Toll-like receptors (TLRs) play an essential role in innate immune responses and in the initiation of adaptive immune responses. Microglia, the resident innate immune cells in the CNS, express TLRs. In this study, we show that TLR3 is crucial for spinal cord glial activation and tactile allodynia after peripheral nerve injury. Intrathecal administration of TLR3 antisense oligodeoxynucleotide suppressed nerve injury-induced tactile allodynia, and decreased the phosphorylation of p38 mitogen-activated protein kinase, but not extracellular signal-regulated protein kinases 1/2, in spinal glial cells. Antisense knockdown of TLR3 also attenuated the activation of spinal microglia, but not astrocytes, caused by nerve injury. Furthermore, down-regulation of TLR3 inhibited nerve injury-induced up-regulation of spinal pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α. Conversely, intrathecal injection of the TLR3 agonist polyinosine–polycytidylic acid induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Indeed, TLR3-deficient mice did not develop tactile allodynia after nerve injury or polyinosine–polycytidylic acid injection. Our results indicate that TLR3 has a substantial role in the activation of spinal glial cells and the development of tactile allodynia after nerve injury. Thus, blocking TLR3 in the spinal glial cells might provide a fruitful strategy for treating neuropathic pain.     

CD44 suppresses TLR-mediated inflammation

The cell adhesion molecule CD44, which is the major hyaluronan receptor, has been implicated in the binding, endocytosis, and metabolism of hyaluronan. Previous studies have revealed that CD44 plays crucial roles in a variety of inflammatory diseases. In recent years, TLRs, which are ancient microbial pattern recognition receptors, have been shown to initiate an innate immune response and have been linked to a variety of inflammatory diseases. The present study shows that CD44 negatively regulates in vivo inflammation mediated by TLRs via NF-kappaB activation, which leads to proinflammatory cytokine production. Furthermore, our results show that CD44 directly associates with TLR2 when stimulated by the TLR2 ligand zymosan and that the cytoplasmic domain of CD44 is crucial for its regulatory effect on TLR signaling. This study indicates that CD44 plays a protective role in TLR-mediated inflammation and is the first to demonstrate a direct association between CD44 and a TLR.     

Biochemical characterization of the Rho GTPase-regulated actin assembly by diaphanous-related formins, mDia1 and Daam1, in platelets

The diaphanous-related formins are actin nucleating and elongating factors. They are kept in an inactive state by an intramolecular interaction between the diaphanous inhibitory domain (DID) and the diaphanous-autoregulatory domain (DAD). It is considered that the dissociation of this autoinhibitory interaction upon binding of GTP-bound Rho to the GTPase binding domain next to DID induces exposure of the FH1-FH2 domains, which assemble actin filaments. Here, we isolated two diaphanous-related formins, mDia1 and Daam1, in platelet extracts by GTP-RhoA affinity column chromatography. We characterized them by a novel assay, where beads coated with the FH1-FH2-DAD domains of either mDia1 or Daam1 were incubated with platelet cytosol, and the assembled actin filaments were observed after staining with rhodamine-phalloidin. Both formins generated fluorescent filamentous structures on the beads. Quantification of the fluorescence intensity of the beads revealed that the initial velocity in the presence of mDia1 was more than 10 times faster than in the presence of Daam1. The actin assembly activities of both FH1-FH2-DADs were inhibited by adding cognate DID domains. GTP-RhoA, -RhoB, and -RhoC, but not GTP-Rac1 or -Cdc42, bound to both mDia1 and Daam1 and efficiently neutralized the inhibition by the DID domains. The association between RhoA and Daam1 was induced by thrombin stimulation in platelets, and RhoA-bound endogenous formins induced actin assembly, which was inhibited by the DID domains of Daam1 and mDia1. Thus, mDia1 and Daam1 are platelet actin assembly factors having distinct efficiencies, and they are directly regulated by Rho GTPases.     

Enzymatic properties of cellobiose 2-epimerase from <Emphasis Type="Italic">Ruminococcus albus</Emphasis> and the synthesis of rare oligosaccharides by the enzyme

The gene for cellobiose 2-epimerase (CE) from Ruminococcus albus NE1 was overexpressed in Escherichia coli cells. The recombinant CE was purified to homogeneity by a simple purification procedure with a high yield of 88%, and the molecular mass was 43.1 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and 44.0 kDa on gel chromatography. It exhibited optimal activity around at 30 degrees C and pH 7.5, and the enzyme activity was inhibited by Al3+, Fe3+, Co2+, Cu2+, Zn2+, Pb2+, Ag+, N-bromosuccinimide, iodoacetate, and 4-chloromercuribenzoate. In addition to cello-oligosaccharides, the enzyme was found to effectively 2-epimerize lactose to yield 4-O-beta-D-galactopyranosyl-D-mannose (epilactose), which occurs in cow milk as a rare oligosaccharide. The Km and kcat/Km values toward lactose were 33 mM and 1.6 s(-1) mM(-1), and those toward cellobiose were 13.8 mM and 4.6 s(-1) mM(-1), respectively. N-Acetyl-D-glucosamine, uridine 5'-diphosphate-glucose, D-glucose 6-phosphate, maltose, sophorose, laminaribiose, and gentiobiose were inert as substrates for the recombinant CE. We demonstrated that epilactose was resistant to rat intestinal enzymes, utilized by human adult bifidobacteria, and stimulated the tight junction permeability in Caco-2 cells. These results strongly suggest that this rare disaccharide is promising for use as a prebiotic.     

The expression of sialylated high-antennary N-glycans in edible bird's nest

Edible bird’s nest (EBN) is the nest made from the saliva of Collocalia swift. Recently, we have found that EBN extract could strongly inhibit infection of influenza viruses in a host-range-independent manner [Guo, C. T.; Takahashi, T.; Bukawa, W.; Takahashi, N.; Yagi, H.; Kato, K.; Hidari, K. I.; Miyamoto, D.; Suzuki, T.; Suzuki, Y. Antiviral Res. 2006, 70, 140–146]. Although this antiviral activity might be attributed to O- or N-glycoconjugates, no N-glycan structures have so far been described for EBN. Here, we report the N-glycosylation profile of EBN, in which a tri-antennary N-glycan bearing the 2,3-N-acetylneuraminic acid residues is displayed as a major component. We suggest that the sialylated high-antennary N-glycans of EBN contribute to the inhibition of influenza viral infection.     

A strategy for screening an inhibitor of viral silencing suppressors, which attenuates symptom development of plant viruses

To find out whether we can control plant virus diseases by blocking viral RNA silencing suppressors (RSSs), we developed a strategy to screen inhibitors that block the association of RSSs with siRNAs using a surface plasmon resonance assay. The screened chemicals were tested in competition with RSSs for binding to siRNAs using a mobility shift assay. We then confirmed that tested chemicals actually inhibited the RSS activity in vivo using a protoplast assay which was developed for this purpose. This entire system can be adapted to screening inhibitors of not only plant viruses but also some animal viruses possessing RSSs.     

Expression pattern of annelid <Emphasis Type="Italic">Zic</Emphasis> in embryonic development of the oligochaete <Emphasis Type="Italic">Tubifex tubifex</Emphasis>

Embryonic expression of a Zic homologue (Ttu-Zic) was examined in the oligochaete annelid Tubifex tubifex. The body plan of T. tubifex is characterized by obvious segmentation in the ectoderm and mesoderm. Ttu-Zic expression is detected in the mesodermal germ band and a subset of micromere descendants. Ttu-Zic is transiently expressed in primary m-blast cells (i.e., founder cells of mesodermal segments) as early as the time of their birth from M teloblasts. During its development, each mesodermal segment experiences two additional phases of Ttu-Zic expression. Ttu-Zic expression in micromere descendants is seen on the anterior surfaces of embryos undergoing teloblastogenesis; subsequently, these cells proliferate to form bilateral clusters, which then become internalized. Finally, clusters of Ttu-Zic-expressing cells are found in the center of the prostomium, corresponding to the cerebral ganglion. The Ttu-Zic expression profile in the early embryogenesis of T. tubifex may be homologous to those of evolutionarily distant animals.     

Endothelium-dependent vasorelaxation effect of rutin-free tartary buckwheat extract in isolated rat thoracic aorta

The aim of this study was to point out the potential of tartary buckwheat on vascular functions. A nonabsorbed fraction of hot-water extract of tartary buckwheat on a SP70 column (TBSP-T), which was free from rutin, was used for this aim. In a contractile experiment using Sprague-Dawley rat thoracic aorta rings contracted by 1.0 microM phenylephrine (PE) or 50 mM KCl, TBSP-T evoked a significant vasorelaxation [EC50 (mg/ml): PE; 2.2; KCl, 1.9]. By a further fractionation of TBSP-T by liquid-liquid partitioning into basic, neutral and acidic fractions, a marked enhancement of vasorelaxation effect was observed only for acidic fraction (EC50, 0.25 mg/ml). The action of acidic fraction was significantly attenuated in endothelium-denuded aortic rings and in the presence of nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (100 microM). The fraction also enhanced the cyclic guanosine monophosphate (cGMP) production in aortic rings contracted with PE [cGMP (pmol/mg protein): PE, 7.2+/-2.3; PE+Acidic fraction, 35+/-8]. These results indicate that acidic fraction could mediate NO/cGMP pathways, thereby exerting endothelium-dependent vasorelaxation action. In conclusion, tartary buckwheat was proven to regulate vascular tones and have latent acidic candidates except for rutin.     

Development of an amperometric flow analysis sensor for specific detection of D-psicose

The aim of this study was to develop a simple, rapid and highly sensitive sensor for measuring the rare sugar d-psicose. The proposed system adopts amperometric flow analysis and two consecutive enzyme reactions consisting of a reactor packed with d-tagatose 3-epimerase (DTE)-immobilized beads, which converts d-psicose to d-fructose, and a carbon-paste electrode containing d-fructose dehydrogenase (DFDH). In order to fabricate a robust sensor system, various experimental parameters were optimized including the buffer composition, flow rate for the two enzyme reactions and the size of micro-flow cell. The developed sensor responded linearly to d-psicose concentration in the range from 0.08 to 50mM (R(2)=0.988). The signal/noise ratio was 3.0 for the 0.08 mM d-psicose solution, and the relative standard deviations were 1.7 (n=20) and 2.6% (n=20) for the 10 and 20mM d-psicose solutions, respectively. One round of assay was completed within 8 min. Our results suggest that the sensor can be used not only for the detection of d-psicose in food samples but also for monitoring d-psicose within the environment. Moreover, the sensor system can be applied to the detection of many other rare sugars by using the same measurement principle.