Ceramide generated from sphingomyelin has emerged as a new but conserved type of biologically active lipid. We previously found that endogenous sphingolipids are required for the normal growth of cultured cerebellar Purkinje neurons and that sphingomyelin is present abundantly in the somatodendritic region of these cells. To gain further insight into a potential role of the sphingomyelin/ceramide pathway, we investigated the effects of depletion of sphingolipids on the phenotypic growth and survival of immature Purkinje cells and the ability of ceramide or other sphingolipids to antagonize these effects. Inhibition of ceramide synthesis by ISP-i, a specific inhibitor of serine palmitoyltransferase, decreased cellular levels of sphingolipids. This treatment resulted in a decrease in cell survival accompanied by an induction of apoptotic cell death and aberrant dendritic differentiation of Purkinje cells with no detectable changes in other cerebellar neurons. Cell-permeable ceramides, sphingosine, or sphingomyelin overcame these abnormalities more effectively than other sphingolipids when added simultaneously with ISP-i. Exposure to bacterial sphingomyelinase in turn enhanced cell survival and dendritic branching complexity of Purkinje cells at different optimal concentrations. Furthermore, cell-permeable ceramide acted synergistically with the neurotrophin family, which has been previously shown to support Purkinje cell survival. These observations suggest that ceramide is a requisite for the survival and the dendritic differentiation of Purkinje cells.

Caenorhabditis elegans and Drosophila melanogaster are relevant models for studying the roles of glycosaminoglycans (GAG) during the development of multicellular organisms. The genome projects of these organisms have revealed the existence of multiple genes related to GAG-synthesizing enzymes. Although the putative genes encoding the enzymes that synthesize the GAG-protein linkage region have also been identified, there is no direct evidence that the GAG chains bind covalently to core proteins. This study aimed to clarify whether GAG chains in these organisms are linked to core proteins through the conventional linkage region tetrasaccharide sequence found in vertebrates and whether modifications by phosphorylation and sulfation reported for vertebrates are present also in invertebrates. The linkage region oligosaccharides were isolated from C. elegans chondroitin in addition to D. melanogaster heparan and chondroitin sulfate after digestion with the respective bacterial eliminases and were then derivatized with a fluorophore 2-aminobenzamide. Their structures were characterized by gel filtration and anion-exchange high performance liquid chromatography in conjunction with enzymatic digestion and matrix-assisted laser desorption ionization time-of-flight spectrometry, which demonstrated a uniform linkage tetrasaccharide structure of -GlcUA-Gal-Gal-Xyl-or -GlcUA-Gal-Gal-Xyl(2-O-phosphate)-for C. elegans chondroitin and D. melanogaster CS, respectively. In contrast, the unmodified and phosphorylated counterparts were demonstrated in heparan sulfate of adult flies at a molar ratio of 73:27, and in that of the immortalized D. melanogaster S2 cell line at a molar ratio of 7:93, which suggests that the linkage region in the fruit fly first becomes phosphorylated uniformly on the Xyl residue and then dephosphorylated. It has been established here that GAG chains in both C. elegans and D. melanogaster are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide sequence, suggesting that indispensable functions of the linkage region in the GAG synthesis have been well conserved during evolution. Mutations affecting the genes encoding putative proteins related to GAG biosynthetic enzymes have also been described for these organisms. Mutations in the tout velu (ttv) gene of D. melanogaster cause defects in Hedgehog movement in mosaic wing discs (1, 6). The ttv gene is a putative ortholog of vertebrate EXT1, which encodes a heparan polymerase and is associated with the hereditary multiple exostoses syndrome in humans (7). The pipe gene, which affects dorsal-ventral patterning of D. melanogaster development, encodes a homolog of vertebrate HS 2-O-sulfotransferase (2, 8). The sugarless and sulfateless genes, both of which affect the fibroblast growth factor signaling during the D. melanogaster development, en-

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The orbital phase continuity-discontinuity property is shown to control electron delocalization and polarization even in open chain conjugated molecules. Its chemical consequences are discussed.