Accumulation of neurons differentiated from mouse embryonic stem cells in particular areas of culture plate surface.

Nanoscale magnetic beads coated with nerve growth factor (NGF) allow us to accumulate neurons differentiated from mouse ES cells in a selected area of the culture plate surface using a magnet. Neurons with neurite outgrowths within a particular area expressed TrkA and incorporated beads in the soma.

Quantitative analysis of plant polyamines including thermospermine during growth and salinity stress.

Arabidopsis thaliana was thought to contain two spermine synthase genes, ACAULIS 5 (ACL5) and SPMS. Recent investigations, however, revealed that the ACL5 gene encodes thermospermine synthase. In this study, we have established a simple method to separate two isomers of tetraamine, spermine and thermospermine, in extracts from plant tissues of less than 500 mg. Polyamines (PAs) extracted from plant tissues were benzoylated, and the derivatives were completely resolved by high-performance liquid chromatography on a C18 reverse-phase column, by eluting with 42% (v/v) acetonitrile in water in an isocratic manner at 30 degrees C and monitoring at 254 nm. The relevance of the method was confirmed by co-chromatography with respective PAs and by the PA analysis of the single- and double-mutants of acl5 and spms, which could not synthesize thermospermine and/or spermine, respectively. Furthermore, with this method, we monitored the thermospermine contents in various tissues of A. thaliana and found that stems and flowers contain two- to three-fold more thermospermine compared to whole seedlings and mature leaves. The presence of thermospermine was confirmed in Oryza sativa and Lycopersicon pesculentum. Finally we addressed whether salinity stress changes the contents of PAs including thermospermine in Arabidopsis.

Neurite outgrowths of neurons with neurotrophin-coated carbon nanotubes.

Multiwalled carbon nanotubes (CNTs) coated with neurotrophin were used to regulate the differentiation and survival of neurons. Neurotrophin (nerve growth factor [NGF] or brain-derived neurotrophic factor [BDNF]) was covalently bound to CNTs modified by amino groups using a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) reagent. The CNTs coated with NGF or BDNF promoted the neurite outgrowths of neurons in the same manner as soluble NGF and soluble BDNF. By enzyme-linked immunosorbent assay (ELISA), we demonstrated that neurotrophin-coated CNTs carry neurotrophin. These results suggest that neurotrophin-coated CNTs have biological activity and stimulate the neurite outgrowths of neurons.

Neurite outgrowths of neurons using neurotrophin-coated nanoscale magnetic beads.

Neurotrophin-coated nanoscale magnetic beads were used to regulate the differentiation and survival of neurons. The beads coated with nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) promoted neurite outgrowths of neurons in the same manner as soluble NGF or soluble BDNF, but beads coated with bovine serum albumin did not promote neurite outgrowths. When the volume of NGF-coated bead solution was increased, the number of neurons with neurite outgrowths increased. The addition of anti-NGF antibodies decreased the numbers of neurons with neurite outgrowths in proportion to the volume of anti-NGF antibodies added. NGF-coated beads appeared to bind to soma with neurite outgrowths as determined using fluorescence. In addition, hybrid beads coated with both NGF and BDNF promoted neurite outgrowths of PC12h cells, although the cells did not produce neurite outgrowths in response to BDNF. Neurons with neurite outgrowths could be concentrated within a particular area when NGF-coated beads were immobilized in a particular area of the culture plate surface using a magnet. The results demonstrate that neurotrophin-coated nanoscale magnetic beads allow us to cultivate neurons in a selected area of the culture plate surface by using a magnet. Thus, neurotrophin-coated nanoscale magnetic beads are applicable to micro-integrated systems and biosensors.

Neurite outgrowths of neurons on patterned self-assembled monolayers.

The effects of specific chemical functionalities on the neurite outgrowths of embryonic chick dorsal root ganglia (DRG) neurons and PC12h cells were investigated using a set of chemically functionalized surfaces prepared by self-assembled monolayers (SAMs) of alkanethiolates with R = NH2, COOH, and CH3 on patterned gold surfaces. The numbers of neurons with neurite outgrowths were compared in the course of a two-week cultivation period. Neurons with neurite outgrowths were observed predominantly on a patterned SAM of long-chain alkanethiolates with amino groups. After about two weeks, the neurons detached from the patterned SAM. However, the activity of beta-galactosidase immobilized via a patterned SAM did not decrease over a 13-d period, reflecting the long-term stability of the SAM. Therefore, the neurons became detached upon cell death. These results demonstrate that the patterned SAM of 11-amino-1-undecanethiolate is a scaffold suitable for making cell chips.