A highly sulfated chondroitin sulfate preparation, CS-E, prevents excitatory amino acid-induced neuronal cell death.

Chondroitin sulfate (CS) is a major microenvironmental molecule in the CNS, and there have been few reports about its neuroprotective activity. As neuronal cell death by excitotoxicity is a crucial phase in many neuronal diseases, we examined the effect of various CS preparations on neuronal cell death induced by the excitotoxicity of glutamate analogs. CS preparations were added to cultured neurons before and after the administration of glutamate analogs. Then, the extents of both neuronal cell death and survival were estimated. Pre-administration of a highly sulfated CS preparation, CS-E, significantly reduced neuronal cell death induced by not only NMDA but also (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate. Neither CS preparations other than CS-E nor other highly sulfated polysaccharides such as heparin and dextran sulfate exerted any neuroprotective effects. NMDA-induced current in neurons was not changed by pre-administration of CS-E, but the pattern of protein-tyrosine phosphorylation was changed. In addition, the elevation of caspase 3 activity was significantly suppressed in CS-E-treated neurons. These results indicate that CS-E prevents neuronal cell death mediated by various glutamate receptors, and suggest that phosphorylation-related intracellular signals and the suppression of caspase 3 activation are implicated in neuroprotection by CS-E.

Cell-free synthesis of the torque-generating membrane proteins, PomA and PomB, of the Na+-driven flagellar motor in Vibrio alginolyticus.

Flagellar motor proteins, PomA and PomB, are essential for converting the sodium motive force into rotational energy in the Na(+)-driven flagella motor of Vibrio alginolyticus. PomA and PomB, which are cytoplasmic membrane proteins, together comprise the stator complex of the motor and form a Na(+) channel. We tried to synthesize PomA and PomB by using the cell-free protein synthesis system, PURESYSTEM. We succeeded in doing so in the presence of liposomes, and showed an interaction between them using the pull-down assay. It seems likely that the proteins are inserted into liposomes and assembled spontaneously. The N-terminal region of in vitro synthesized PomB appeared to be lost, but this problem was suppressed by fusing GFP to the N-terminus of PomB or by mutagenesis at Pro-11 or Pro-12. A structural change of the N-terminal region of PomB by these modifications may prevent cleavage during protein synthesis in PURESYSTEM. The mutations did not affect the functioning of the motor. Using this system, biochemical analysis of PomA and PomB can be performed easily and efficiently.

Evaluation of the single yeast cell's adhesion to ITO substrates with various surface energies via ESEM nanorobotic manipulation system.

Cell-surface adhesion force is important for cell activities and the development of bio materials. In this paper, a method for in situ single cell (W303) adhesion force measurement was proposed based on nanorobotic manipulation system inside an environment scanning electron microscope (ESEM). An end effector was fabricated from a commercial atomic force microscope (AFM) cantilever by focused ion beam (FIB) etching. The spring constant of it was calibrated by nanomanipulation approach. Three kinds of hydrophilic and hydrophobic ITO plates were prepared by using VUV-irradiation and OTS coating techniques. The shear adhesion strength of the single yeast cell to each substrate was measured based on the deflection of the end effector. The results demonstrated that the cell adhesion force was larger under the wet condition in the ESEM environment than in the aqueous condition. It also showed that the cell adhesion force to hydrophilic surface was larger than that to the hydrophobic surface. Studies of single cell's adhesion on various plate surfaces and environments could give new insights into the tissue engineering and biological field.