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On the basis of the results outlined in our previous report, bacterial sialyltransferases (ST) from marine sources were further characterized using glycosphingolipids (GSL), especially ganglio-series GSLs, based on the enzymatic characteristics and kinetic parameters obtained by Line weaver-Burk plots. Among them, GA1 and GA2 were found to be good substrates for these unique STs. Thus, new gangliosides synthesized by α2-3 and α2-6STs were structurally characterized by several analytical procedures. The ganglioside generated by the catalytic activity of α2-3ST was identified as GM1b. On the other hand, when enzyme reactions by α2-6STs were performed using substrates GA2 and GA1, very unique gangliosides were generated. The structures were identified as NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer and NeuAcα2-6Galβ1-3GalNAcβ1-4Galβ1-4Glcβ-Cer, respectively. The synthesized ganglioside NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer showed binding activity to the influenza A virus {A/Panama/2007/99 (H3N2)} at a similar level to purified sialyl(α2-3)paragloboside (S2-3PG) and sialyl(α2-6)paragloboside (S2-6PG) from mammalian sources. The evidence suggests that these STs have unique features, including substrate specificities restricted not only to lacto-series but also to ganglio-series GSLs, as well as catalytic potentials for ganglioside synthesis. This evidence demonstrates that effective in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing sialic acid (Sia) modifications, thereby preparing large-scale gangliosides and permitting the exploration of unknown functions.

Previous studies from our laboratory indicated that pre-germinated brown rice (PR) contained certain unknown bioactive lipids that activated two enzymes related to diabetes: Na+/K+ATPase and homocysteine-thiolactonase. In this paper, we report on the isolation and structural characterization of the activator lipids from PR bran as acylated steryl glucosides (ASGs). The activator lipid was isolated by silica gel column chromatography, and its chemical structure was determined by NMR, GC-MS, and tandem mass spectrometry. We demonstrated that the bioactive component consists of a mixture of acylated steryl beta-glucosides. Delta8-cholesterol and 2-hydroxyl stearic acid were identified as constituents of ASGs. The steryl glucosides (SGs) subsequent to alkaline hydrolysis lost this enzyme activator activity. Soybean-derived ASGs were not active. This activity may be quite peculiar to PR-derived ASGs. Our findings suggest that the molecular species of ASG may play an important contributing role in the anti-diabetic properties of a PR diet.

By TLC, GM4 was found to be the major ganglioside in the liver of six shark species examined: Odontaspis taurus, Negaprion brevirostris, Sphyrna lewini, Mustelus griseus, Mustelus manazo, and Prionace glauca. A detailed analysis of the glycosphingolipids (GSLs) in the liver of O. taurus (sand tiger shark) showed that it contained approximately 110 nmol of lipid-bound sialic acid per gram of wet tissue, of which 80% was GM4. By extracting the liver of O. taurus with chloroform/methanol, followed by chromatographic separation of GSLs using DEAE-Sephadex A-25 and Iatrobeads columns, we have isolated GM4 in pure form with a yield of approximately 5 mg per 100 g of wet tissue. The structures of both the sugar chain and the ceramide moiety of this GM4 were analyzed by chemical analysis, mass spectrometry, and NMR spectroscopy. Similar to GM4 isolated from other sources, 92% of fatty acids in the ceramide of this GM4 were 2-hydroxylated. However, unlike the long-chain bases found in other GSLs, the total long-chain bases in this GM4 were found to contain 43% octadecasphingenine and 50% nonadecasphingenine. Immunohistochemical analysis using a monoclonal antibody against GM4 revealed that the hepatocytes of both M. griseus (spotless smooth hound) and M. manazo (smooth hound) were filled with lipid droplets and GM4 was primarily associated with the membrane structure surrounding lipid droplets.