Oligosaccharides of human milk. Isolation and characterization of three new disialyfucosyl hexasaccharides.

Renal mitochondrial 25-hydroxyvitamin D₃-1-hydroxylase (1-hydroxylase) is sensitive to inhibition by 2 × 10^?5m calcium and 5 × 10^?3m phosphate when hydroxylation is supported by either malate or NADPH. This sensitivity to ion inhibition is observed in mitochondria from both vitamin D-deficient and repleted chicks and remains when mitochondria are frozen and thawed or are incubated in a hypotonic medium. The ionophore A23187 inhibits the 1-hydroxylase but partially reverses the inhibition exerted by 2, 5, or 7.5 × 10^?5m calcium. Addition of a kidney soluble cell fraction (37,000g supernatant) to isolated mitochondria did not enhance the 1-hydroxylase activity under conditions of varied substrate concentration, osmolarity of the incubation medium, or mitochondrial washes. It is concluded that a soluble cellular component is not involved in the regulation of the 1-hydroxylase but that intramitochondrial calcium and phosphate may well play a role in its regulation.     

Comparative Study between Gas Chromatography and Ion-exchange Chromatography on Amino Acid Analysis of Foods

Lactobacillus brevis and Saccharomyces cerevisiae were completely sterilized by the supercritical (SC) CO2 micro-bubble method. Gaseous (G) and liquid (LQ) CO2 were used in a similar manner to compare the sterilizing effect. Among the three treatments, the microorganisms were only effectively sterilized by the SC CO2 treatment at 25 MPa and 35°C.     

Formation of long-chain N-vanillyl-acylamides from plant oils

Long-chain N-vanillyl-acylamides (LCNVAs) were generated from plant oils and vanillylamine (VA) by nucleophilic amidation without any catalytic reagents. The resulting LCNVAs varied according to the fatty acid composition of the plant oil used. Therefore, the LCNVAs contained in Capsicum oleoresins were products that were spontaneously generated from the oleoresin during storage.     

Fucosyl-GM1a, an endoglycoceramidase-resistant ganglioside of porcine brain

The use of bovine brain has been prohibited in many countries because of the world-wide prevalence of mad cow disease, and thus porcine brain is expected to be a new source for the preparation of gangliosides. Here, we report the presence of a ganglioside in porcine brain which is strongly resistant to hydrolysis by endoglycoceramidase, an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. Five major gangliosides (designated PBG-1, 2, 3, 4, 5) were extracted from porcine brain by Folch's partition, followed by mild alkaline hydrolysis and PBA column chromatography. We found that PBG-2, but not the others, was strongly resistant to hydrolysis by the enzyme. After the purification of PBG-2 with Q-Sepharose, Silica gel 60 and Prosep-PB chromatographies, the structure of PBG-2 was determined by GC, GC-MS, FAB-MS and NMR spectroscopy as Fucalpha1-2Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer (fucosyl-GM1a). The ceramide was mainly composed of C18:0 and C20:0 fatty acids and d18:1 and d20:1 sphingoid bases. The apparent kcat/Km for fucosyl-GM1a was found to be 30 times lower than that for GM1a, indicating that terminal fucosylation makes GM1a resistant to hydrolysis by the enzyme. This report indicates the usefulness of endoglycoceramidase to prepare fucosyl-GM1a from porcine brain.     

Study of the sugar chains of recombinant human amyloid precursor protein produced by Chinese hamster ovary cells

The N- and O-glycans of recombinant amyloid precursor protein (APP), purified from Chinese hamster ovary cells transfected with the human 695-amino acid form of APP, were separately released by hydrazinolysis under different conditions. The reducing ends of the released N- and O-glycans were reduced with NaB3H4 and derivatized with 2-aminobenzamide (2AB), respectively. After acidic N-glycans were obtained by anion-exchange column chromatography, these were converted to neutral oligosaccharides by sialidase digestion, demonstrating that their acidic nature was entirely due to sialylation. The sialidase-treated N-glycans were then fractionated by lectin column chromatography and their structures were determined by linkage-specific sequential exoglycosidase digestion. These results demonstrated that recombinant APP has bi- and triantennary complex type N-glycans with fucosylated and nonfucosylated trimannosyl cores. In a similar fashion, the 2AB-labeled O-glycans derived from APP were determined to be mono- and disialylated core type 1 structures. Taken together, these results indicate that recombinant APP has sialylated bi- and triantennary N-glycans with fucosylated and nonfucosylated cores and sialylated O-glycans with core type 1 structures.     

Molecular evidence of the existence of two sibling species within the echinothurioid echinoid Asthenosoma ijimai from Japanese waters

The echinoid, Asthenosoma ijimai belonging to the order Echinothurioida from Japanese waters shows the geographical variation in morphological and ecological characters. The echinothurioid from Ryukyu Islands in southern Japan is cleary different from that of Sagami Bay and Suruga Bay in the middle part of Japan at non-molecular level.Their phylogenetic and taxonomic relationships were studied at the molecular level by allozyme analysis. The results demonstrated that the two echinothurioids from Ryukyu Islands and Sagami Bay do not share gene pools with each other, and they were fixed for different alleles at five genetic loci (Mdh, G6pd, Po, Alk-3 and Est-7) in a total of 23 enzyme genetic loci scored. This indicates no gene flow between the two echinothurioids, and is a molecular evidence for that they are reproductively isolated and genetically distinct species. The Nei's genetic distance (D=0.181) between the two were significantly higher than those between conspecific local populations, and comparable to those between closely related species in many other animals containing echinoderms. The present molecular data are well consistent with the non-molecular evidence from morphology, developmental biology and ecology. Putting these data together, we propose that the two echinothurioids should be classified as two sibling species of the genus Asthenosoma and would like to give the following scientific names: the echinothurioid species from Sagami Bay is Asthenosoma ijimai and that from Ryukyu Islands is A. ijimai R.