SummaryNeuronal growth cones are essential for nerve growth and regeneration, as well as for the formation and rearrangement of the neural network. To elucidate phosphorylation-dependent signaling pathways and establish useful molecular markers for axon growth and regeneration, we performed a phosphoproteomics study of mammalian growth cones, which identified >30,000 phosphopeptides of ∼1,200 proteins. The phosphorylation sites were highly proline directed and primarily MAPK dependent, owing to the activation of JNK, suggesting that proteins that undergo proline-directed phosphorylation mediate nerve growth in the mammalian brain. Bioinformatics analysis revealed that phosphoproteins were enriched in microtubules and the cortical cytoskeleton. The most frequently phosphorylated site was S96 of GAP-43 (growth-associated protein 43-kDa), a vertebrate-specific protein involved in axon growth. This previously uncharacterized phosphorylation site was JNK dependent. S96 phosphorylation was specifically detected in growing and regenerating axons as the most frequent target of JNK signaling; thus it represents a promising new molecular marker for mammalian axonal growth and regeneration.

Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. To examine how signaling protein complexes are clustered in rafts, we focused on the functions of glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing mouse neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a GPM6a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a, such as Rufy3, Rap2, and Tiam2/STEF, accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation in neuronal development. RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of neuronal polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals. Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. We focused on glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation. RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.

Eight new labdane-type diterpenes (1-8) were isolated from the fruit of Vitex rotundifolia along with two known abietane-type diterpenoids (9, 10), and their structures were characterized on the basis of spectroscopic data and X-ray crystallographic analysis. Among them, the abietane-type diterpenoid ferruginol (9) exhibited a stronger antioxidative activity than the standard antioxidant, 3-tert-butyl-4-hydroxyanisole (BHA), using a ferric thiocyanate method.

Ten new labdane-type diterpenes were isolated from the fruit of Vitex rotundifolia L. (Verbenaceae), along with a known diterpene, vitexilactone. Their chemical structures were determined on the bases of spectroscopic data.

A new labdane-type diterpene, vitexifolin A (1), a new clerodane-type diterpene, vitexifolin B (2), a new abeoabietane-type diterpene, vitexifolin C (3), and two new norlabdane-type diterpenes, vitexifolin D (4) and vitexifolin E (5), were isolated from the fruits of Vitex rotundifolia, along with a known halimane-type diterpene, vitetrifolin D (6), two known norlabdane-type diterpenes, trisnor-gamma-lactone (7) and iso-ambreinolide (8), and three known flavonoids, casticin (9), artemetin (10), and 5,3'-dihydroxy-6,7,4'-trimethoxyflavanone (11). Their chemical structures were determined on the basis of spectroscopic data. Casticin (9) exhibited considerable growth inhibitory activity against human lung cancer cells (PC-12) and human colon cancer cells (HCT116) using the MTT assay.

Forty-three polyphenols from tea leaves were evaluated for their anti-oxidative effect against lipid peroxidation by the ferric thiocyanate method in vitro. Among these, 1,4,6-tri-O-galloyl-beta-D-glucose (hydrolyzable tannin) showed the highest anti-oxidative activity against lipid peroxidation, even stronger than that of 3-tert.-butyl-4-hydroxyanisole (BHA). The assay demonstrates that tea polyphenols, except for desgalloylated dimeric proanthocyanidins that possess a catechin structure in the upper unit and desgalloylated flavan-3-ols, and excepting theaflavin 3,3'-di-O-gallate, had more anti-oxidative activity than that of alpha-tocopherol. The chemical structure-activity relationship shows that the anti-oxidative action advanced with the condensation of two molecules of flavan-3-ols as well as with 3-O-acylation in the flavan skeleton such as that by galloyl, (3'-O-methyl)-galloyl, and p-coumaroyl groups.

Submicron patterning technologies for the PZT/Pt/Ti/TiN/Ti structure with a spin on glass (SOG) mask were demonstrated using a high-density ECR plasma and a high substrate temperature above 300° C. A 30%-Cl2/Ar gas was used to etch a lead zirconate titanate (PZT) film. No deposits remained, which resulted in an etched profile of more than 80°. A 40%-O2/Cl2 gas was used to etch a Pt film. The etching was completely stopped at the Ti layer. 30-nm-thick deposits remained on the sidewall. They were removed after dipping in hydrochloric acid. The etched profile of a Pt film was more than 80°. The Ti/TiN/Ti layer was etched with pure Cl2 gas. The size shift from the SOG mask was less than 0.1 µ m. Interdiffusion between SOG and PZT was not detected by transmission electron microscopy and energy dispersive X-ray spectroscopy (TEM-EDX) analysis.

The methanol extract of the aerial part of Tessaria integnfolia (common name: pajaro bobo), which is used as an herb for the treatment of asthnra and liver injury in Peru, showed a stronger antioxidative activity than the standard synthetic antioxidant, 3-tert-butyl-4-hydroxyanisole (BHA). From this extract, five new eudesmane-type sesquiterpenoids were isolated along with eighteen known compounds, and their structures were elucidated on the basis of MS, IH-NMR and 13C-NMR data. Among them, eleven phenolic compounds, four flavonoids, two lignans and five caffeoyl quinic acid derivatives were examined for their antioxidative activity using the ferric thiocyanate method, and nine compounds indicated a stronger antioxidative activity than the standard natural antioxidant, a-tocopherol at a concentration of 0.5 mM. Especially, two flavonoids were identified as stronger antioxidants than BHA. The scavenging effect of the phenolic compounds on the stable radical 1,1-diphenyl-2-picrylhydrazyl was examined. All tested compounds showed this scavenging effect, with the effect of five caffeoyl quinic acid derivatives and three flavonoids being almost twice or more that of a-tocopherol at a concentration of 0.01 mM.