We have shown that over-sulfated chondroitin sulfate/ dermatan sulfate (CS/DS) chains from various marine organisms exhibit growth factor binding activities and neurite outgrowth-promoting activities in embryonic mouse hippocampal neurons in vitro. In this study we demonstrated that CS/DS hybrid chains purified from embryonic pig brain displayed marked neuritogenic activity and growth factor binding activities toward fibroblast growth factor 2 (FGF2), FGF10, FGF18, pleiotrophin, and midkine, all of which exhibit neuroregulatory activities in the brain. In contrast, the CS/DS preparation from adult pig brain showed considerably less activity to bind these growth factors and no neuritogenic activity. Structural analysis indicated that the average size of the CS/DS chains was similar (40 kDa) between these two preparations, but the disaccharide compositions differed considerably, with a significant proportion of L-iduronic acid (IdoUA)-containing disaccharides (8ϳ9%) in the CS/DS chains from embryos but not in those from adults (<1%). Interestingly, both neurite outgrowth-promoting activity and growth factor binding activities of the CS/DS chains from embryos were abolished by digestion not only with chondroitinase ABC but also with chondroitinase B, suggesting that the IdoUAcontaining motifs are essential for these activities. These findings imply that the temporal expression of CS/DS hybrid structures containing both GlcUA and IdoUA and binding activities toward various growth factors play important roles in neurogenesis in the early stages of the development of the brain. Chondroitin sulfate proteoglycans (CS-PGs)1 are complex macromolecules consisting of a protein core and at least one covalently linked CS glycosaminoglycan (GAG) chain and are ubiquitous components in the extracellular matrices of connective tissues and at the surfaces of many cell types (for reviews, see Refs. 1-3). In the mammalian brain CS-PGs are common extracellular matrix components with a highly regulated spatiotemporal expression (4 -8). Although the CS chains attached to CS-PGs have attracted little attention until recently compared with heparan sulfate (9), recent advances in the structural biology of CS chains have suggested important biological functions in the development of the brain (10). Several studies have demonstrated that the composition of CS chains changes with aging and normal brain maturation and that some CS epitopes are only found in specific sections of the avian and mammalian brain (7,8,11). The developmentally regulated expression and tissue-specific distribution of CS variants suggest that CS chains differing in the degree and profile of sulfation exhibit distinct functions during the development of the brain.The functions of CS-PGs and CS chains in the central nervous system can be categorized as the regulation of cell adhesion and migration, neurite formation, polarization of neurons, synaptic plasticity, survival of neurons, etc. (for reviews, see Ref. 1 and 4). Concerning the neurite formation, studies have sh...

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-

To investigate the incidence, the specificity and clinical significance of positivity for serum anti-neutrophil cytoplasmic antibody (ANCA) in 31 patients with systemic lupus erythematosus (SLE), the indirect immunofluorescence (IIF) technique and enzyme-linked immunosorbent assay (ELISA) were used to measure ANCA. Purified myeloperoxidase (MPO), lactoferrin (LF), cathepsin-G (CTG) and elastase (HLE) served as ANCA antigens for ELISA. Thirteen (42%) of the 31 SLE patients showed positivity for perinuclear, but not cytoplasmic, ANCA by IIF. Five of 31 sera were positive for MPO, 10 for LF, 1 for CTG and 0 for HLE by ELISA. Patients positive for ANCA had a higher score of SLE disease activity index (SLEDAI) than those without ANCA. There was no correlation between ANCA positivity, clinical manifestations, or organic involvement. While the ANCA in patients with SLE reflected disease activity, it was unrelated to organic involvement.

Appican produced by rat C6 glioma cells, the chondroitin sulfate (CS) proteoglycan form of the amyloid precursor protein, contains an E disaccharide,^GlcUA-GalNAc(4,6-O-disulfate)^, in its CS chain. In this study, the appican CS chain from rat C6 glioma cells was shown to speci¢cally bind several growth/di¡erentiation factors including midkine (MK) and pleiotrophin (PTN). In contrast, the appican CS from SH-SY5Y neuroblastoma cells contained no E disaccharide and showed no binding to either MK or PTN. These ¢ndings indicate that the E motif is essential in the interaction of the appican CS chain with growth/di¡erentiation factors, and suggest that glial appican may mediate the regulation of neuronal cell adhesion and migration and/or neurite outgrowth. ß

Many cytokines are involved in the repair of damaged tissue, and one of these, hepatocyte growth factor (HGF), is involved not only with liver regeneration but also in the repair of other tissues. To investigate the importance of HGF in the repair of the small intestine, we evaluated its effect and that of other growth factors in IEC-6 cells, an intestinal epithelial cell line derived from normal rat small intestine. Round "wounds" were made in confluent monolayers of IEC-6 by silicon rubber-tipped steel rods and various cytokines; transforming growth factor alpha (TGF-alpha), transforming growth factor beta1 (TGF-beta1), keratinocyte growth factor (KGF), and HGF, were added. We photographed the repaired monolayers every 24 h and calculated the ratios of areas not covered by cells to initial areas. Cell proliferation with TGF-alpha, TGF-beta, KGF, or HGF was examined in terms of [3H]-thymidine uptake. Finally, we determined c-met (the HGF receptor) mRNA in the IEC-6 cells by Northern blot hybridization. HGF was the most potent of the cytokines in accelerating repair of the damaged monolayer of IEC-6. HGF was also 1.34 times more effective than control the medium for inducing cell proliferation of IEC-6. By Northern blot hybridization, three bands of mRNA bound to c-met cDNA. These results suggest that HGF is important in the repair of the small intestine.

Mesenchymal-epithelial interactions are important in the gastric mucosal repair. However, specific factors responsible for such interactions have not been established. In the present study, keratinocyte growth factor (KGF) significantly stimulated proliferation of gastric epithelial cells dose dependently and synergistically with hepatocyte growth factor (HGF), epidermal growth factor (EGF) and insulin. Restitution of gastric epithelial monolayers was also assessed, using a round wound restitution model. Keratinocyte growth factor facilitated the restitution of gastric epithelial cells significantly but did not have any effects on gastric fibroblasts. Keratinocyte growth factor receptor mRNA was expressed by gastric epithelial cells, indicating that these effects were elicited by the specific receptor mediated pathway. Northern blot analysis revealed the expression of KGF mRNA in gastric fibroblasts but not in gastric epithelial cells, indicating the production of KGF. These results suggest that KGF might be involved in gastric mucosal repair, through mesenchymal-epithelial interaction.

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