Adhesion-clutch between DCC and netrin-1 mediates netrin-1-induced axonal haptotaxis.

The growth cone, a motile structure located at the tip of growing axons, senses extracellular guidance cues and translates them into directional forces that drive axon outgrowth and guidance. Axon guidance directed by chemical cues on the extracellular adhesive substrate is termed haptotaxis. Recent studies reported that netrin-1 on the substrate functions as a haptotactic axon guidance cue. However, the mechanism mediating netrin-1-induced axonal haptotaxis remains unclear. Here, we demonstrate that substrate-bound netrin-1 induces axonal haptotaxis by facilitating physical interactions between the netrin-1 receptor, DCC, and the adhesive substrates. DCC serves as an adhesion receptor for netrin-1. The clutch-linker molecule shootin1a interacted with DCC, linking it to actin filament retrograde flow at the growth cone. Speckle imaging analyses showed that DCC underwent either grip (stop) or retrograde slip on the adhesive substrate. The grip state was more prevalent on netrin-1-coated substrate compared to the control substrate polylysine, thereby transmitting larger traction force on the netrin-1-coated substrate. Furthermore, disruption of the linkage between actin filament retrograde flow and DCC by shootin1 knockout impaired netrin-1-induced axonal haptotaxis. These results suggest that the directional force for netrin-1-induced haptotaxis is exerted on the substrates through the adhesion-clutch between DCC and netrin-1 which occurs asymmetrically within the growth cone.

Melibiosamine, a novel oligosaccharide, suppresses mitogen-induced IL-2 production via inactivation of NFAT and NFκB in Jurkat cells.

d-Glucosamine (GlcNH2) and several of its derivatives are known to possess immunosuppressive activities in various immune cell lines. The novel GlcNH2-containing oligosaccharide Galα1-6GlcNH2 (designated melibiosamine; MelNH2) is expected to be immunosuppressive also. In Jurkat cells (immortalized human T lymphocytes), interleukin 2 (IL-2) production (an index of the T-cell immune response) can be induced by stimulation with a mitogen, such as concanavalin A. Here, we compared the effects of GlcNH2 and MelNH2 on concanavalin A-induced IL-2 production (CIIP) in Jurkat cells and found that GlcNH2 and MelNH2 at millimolar levels both significantly suppressed CIIP without affecting cell viability. When we examined the effects of GlcNH2 and MelNH2 on the activation of the three transcription factors required for CIIP-NFAT (nuclear factor of activated T-cells), NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells), and AP-1 (activator protein 1)-we found that GlcNH2 and MelNH2 both suppressed CIIP by inhibiting the activation of NFAT and NFκB, but, unlike GlcNH2, MelNH2 also promoted the activation of AP-1. These results suggest that MelNH2 may be a potentially useful lead compound for development as an immunosuppressive or anti-inflammatory drug.

Structural investigation of α-l-fucosidase from the pancreas of Patiria pectinifera, based on molecular cloning.

An α-l-fucosidase (Pap-Alf) was purified from the pancreas of a starfish Patiria pectinifera by ammonium sulfate precipitation followed by several column chromatographies. The molecular mass of the purified enzyme was estimated to be 52.6 kDa by SDS-PAGE, although gel filtration analysis of the native enzyme suggests it exists as a homodimer in solution. The purified enzyme showed maximal activity at pH 5.0 and 70 °C. The enzyme was highly specific toward a fucosyl-monosaccharide (Fuc-α-pNP), but it also showed activity toward 2-sulfo-Fuc-α-pNP and fucosyl-α-lactosides (Fuc-α-Galß1→4Glc-ß-pNP). We determined the primary structure of the α-l-fucosidase and validated its expression level in starfish tissue. Whole genome sequence analysis of P. pectinifera was also performed in the present study. Detailed primary structural analysis using bioinformatics tools revealed Pap-Alf lacks the C-terminal region that is otherwise conserved in all previously described α-l-fucosidases. Quantitative gene expression analysis of Pap-Alf in each tissue indicated that the expression of Pap-Alf gene in pancreas was 5-fold higher than in ovary.

Decoupled Thermo- and pH-Responsive Hydrogel Microspheres Cross-Linked by Rotaxane Networks.

Rotaxane cross-linked (RC) microgels that exhibit a decoupled thermo- and pH-responsive volume transition were developed. The pH-induced changes of the aggregation/disaggregation states of cyclodextrin in the RC networks were used to control the swelling capacity of the entire microgels. Different from conventional thermo- and pH-responsive microgels, which are usually obtained from copolymerizations involving charged monomers, the RC microgels respond to temperature as intended, even in the presence of charged functional molecules such as dyes in the microgel dispersion. The results of this study should lead to new applications, including drug delivery systems that require a retention of their smart functions even in environments that may contain foreign ions, for example, in in vivo experiments.

Formation of Tough Films by Evaporation of Water from Dispersions of Elastomer Microspheres Crosslinked with Rotaxane Supramolecules.

Compared to rigid microspheres that consist, for example, of polystyrene or silica, soft and deformable elastomer microspheres can be used to generate colorless transparent films upon evaporating the solvent from microsphere-containing dispersions. To obtain tough films, a post-polymerization reaction to crosslink the microspheres is usually necessary, which requires extra additives during the drying process. This restriction renders this film-formation technology complex and rather unsuitable for applications in which impurities are undesirable. In the present study, it is demonstrated that tough elastomer microspheres that are crosslinked with rotaxanes can form tough bulk films upon evaporation of water from microsphere dispersions, so that post-polymerization reactions are not required. The results of this study should thus lead to new applications including coatings for biomaterials that need complete removal of all impurities from the materials prior to use.