Glycosphingolipids were isolated from primary cultures of porcine endothelial cells labelled with 14C-galactose or 14C-glucosamine. They were characterized by their mobility on thin layer chromatogram, their sensitivity to exoglycosidases, and their labelling with antibodies. In addition to the major glycosphingolipids, globotetra- and globotriaosylceramide, minor ones were identified as penta- and heptaglycosylceramide of the neolactoseries terminated by either Gal alpha 1-3Gal- (xenoreactive epitope) or Fuc alpha 1-2Gal- (H determinant). Two gangliosides were found, GM3 and GD3, and N-glycolylneuraminic acid was their major sialic acid. Therefore, porcine endothelial cells differ from human endothelial cells by expression of glycosphingolipids that are absent in man: two Gal alpha 1-3Gal-terminated glycolipids recognized by human natural antibodies, and two N-glycolylneuraminic acid-terminated gangliosides which are potent immunogens.

The aim of the study was to assess the isolation of HDL by fast protein liquid chromatography (FPLC) to perform kinetics studies of apolipoprotein (apo)A-I-HDL labelled with a stable isotope. Comparison between FPLC and ultracentrifugation has been made. ApoA-I-HDL kinetics were studied by infusion of [5.5.5-2 H 3 ]leucine for 14 h in five subjects. Using FPLC, pre ␤ 1 HDL and ␣ HDL (HDL 2 and HDL 3 ) were separated from 200 l of plasma samples. Total HDL was isolated by sequential ultracentrifugation (HDL-UC). The tracer-to-tracee ratio was higher in pre ␤ 1 HDL than in total HDL-UC. The higher leucine enrichment found in total HDL-UC compared to ␣ HDL suggested the existence of a mixture of apoA-I-HDL sub-classes. From this difference in enrichments, the turnover rate of total HDL-UC, usually assumed to be ␣ HDL, was probably overestimated in previous studies. To our knowledge, this study is the first report which provides a convenient tool to distinguish enrichments of apoA-I in pre ␤ 1 HDL and ␣ HDL from total HDL previously used for kinetic measurements. Plasma HDL and apolipoprotein A-I (apoA-I) concentrations are inversely correlated with the risk of coronary heart disease (1, 2). Kinetics studies are a good tool for a better understanding of the role that HDL play in the reverse cholesterol transport. Pre ␤ 1 HDL are considered as the initial acceptor of cellular cholesterol and are transformed into spherical HDL 3 as a result of the LCAT activity. Nascent discoidal pre ␤ 1 HDL are either directly synthesized by the liver and the intestine or generated when other classes of lipoproteins including ␣ HDL are remodeled by hepatic lipase (3). In plasma, pre ␤ 1 HDL represented 5.5 Ϯ 3.3% and 7.2 Ϯ 4% of total apoA-I in women and men, respectively (4).ApoA-I, a major protein component of HDL, could be endogenously labeled using stable isotopes to study HDL metabolism in humans (5). Plasma lipoprotein and HDL are usually separated by ultracentrifugation based on differential flotation using either sequential (6) or one-step gradient methods (7). It appears that the combination of high salt concentrations and high centrifugal forces may cause structural damages to the lipoproteins (8) that could change results of kinetics studies (9). Alternatively, plasma lipoproteins may also be separated by size-exclusion chromatography on agarose. Fast Protein Liquid Chromatography (FPLC), based on the gel filtration property, is a rapid, quantitative, and non-destructive method used to separate lipoproteins. Nascent pre ␤ 1 HDL can also be separated from ␣ HDL by FPLC (10). The aim of the present study is to validate the separation of HDL subclasses by FPLC in metabolic study after an endogenous labeling with stable isotopes. In this study, we have compared the leucine enrichment of apoA-I-HDL separated by FPLC to that of HDL separated by the traditional ultracentrifugation of plasma samples.

Neutral mucin oligosaccharides from the small intestine of control rats and rats infected with the parasite Nippostrongylus brasiliensis were released and analyzed by gas chromatography-mass spectrometry. Infected animals expressed seven blood group A-like structures that were all absent in the control animals. The blood group A nature of these epitopes was confirmed by blood group A reactivity of the prepared mucins, of which Muc2 was one. Transferase assays and Northern blotting on small intestines from infected animals showed that an ␣-N-acetylgalactosaminyltransferase similar to the human blood group A glycosyltransferase had been induced. The expression was a transient event, with a maximum at day 6 of the 13-daylong infection. The rat blood group A glycosyltransferase was cloned, revealing two forms with an amino acid similarity of 95%. Both types had blood group A transferase activity and were probably allelic because none of 12 analyzed inbred strains carried both types. The second type was found in outbred rats and in one inbred strain. First generation offspring of inbred rats of each type were heterozygous, further supporting the allelic hypothesis. The transient induction and the large allelic variation could suggest that glycosyltransferases are part of a dynamic system altering mucins and other glycoconjugates as a protecting mechanism against microbial challenges.The gastrointestinal tract is covered by a mucus layer, effectively protecting the epithelial cells from the hostile milieu including microorganisms and at the same time allowing digested nutrients and other smaller molecules to traverse. Microorganisms bind to intestinal mucus and epithelial cells; some of this binding is with high specificity to glycan epitopes (1). To maintain the protective layer despite continuous damaging processes, especially by these intestinal microorganisms, a system with a continuous mucus renewal is demanded. In the intestine, a majority of the mucus is produced by the goblet cells interspersed between the enterocytes. The matrix in the mucus is made up of gel-forming mucins. These are highly glycosylated proteins, where up to 80% of the mass is from O-linked oligosaccharides. Cloning and sequencing of mucin genes have revealed that the sites for glycosylation are located in domains of long stretches of protease-resistant sequences rich in the amino acids serine and threonine together with proline. Because these domains are typical for mucins, but also found in other proteins, they have been named mucin domains. Up until now, 13 human mucin genes have been identified and fully or partly sequenced (2-4), in addition to mucins identified from other species. The major intestinal mucin in rat is the Muc2 mucin, and the two mucin domains of this mucin have previously been isolated as two highly glycosylated peptides of 650 and 335 kDa, respectively (5, 6). The rat Muc2, as well as its human homologue MUC2, belongs to a class of gel forming mucins, gaining their viscous properties by oligomerization into large mucin...

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Unlike the pig-to-primate discordant xenotransplantation model, in which preexisting anti-carbohydrate antibodies are directly responsible for hyperacute rejection, in the concordant hamster-to-rat situation, the evoked IgM anti-Forssman carbohydrate antibodies do not appear to be the main cause of the vascular rejection.

Neutral glycosphingolipids were isolated from the colon of rats between birth and adulthood. The glycolipid concentration was stable during this period. Epithelial cells of the adult colon contained three times more glycolipids than the whole organ. The distribution pattern underwent only minor modifications during development. Free ceramide contributed for 23-27% of the total neutral sphingolipids at all ages. In 6-day-old rats, it was constituted of nonhydroxylated fatty acids linked to C18-sphingenine (57.3% of the bases), C18- and C20-4D-hydroxysphinganine (24.2 and 14.0% of the bases, respectively). This composition was essentially maintained during development. Glucosylceramide was the major glycolipid at all ages (40-50% of the total neutral sphingolipid content). At birth, 40% of its fatty acids were 2-hydroxylated and 93% of the bases were C18-4D-hydroxysphinganine. In adult epithelial cells, 75% of the fatty acids were 2-hydroxylated and C18- and C20-4D-hydroxysphinganine contributed for 66 and 25% of the bases, respectively. A transient increase of the contribution of nonhydroxylated fatty acids and C18-sphingenine was observed during the first week of life. C20-4D-hydroxysphinganine, which was characterized by gas-liquid chromatography of its aldehydes after periodate oxidation and of its N-acetyl O-trimethylsilyl derivatives, appeared after birth and reached 20% of the bases after two weeks. These findings are another example of the specificity of the lipidic part of glucosylceramide during the ontogenic differentiation.

The heterophile antigens Galalpha1-->3Gal and N-glycolylneuraminic acid are the major obstacle to grafting mammal organs, especially from pig, to man. Lack of expression of these common xenoantigens by birds has raised interest in ostrich as a potential organ donor for xenotransplantation. Glycosphingolipids of ostrich liver and kidney were investigated for their carbohydrate determinants. Both organs were found similar in their glycolipid composition with three major species, mono-, di-, and pentaglycosylceramide. The pentaglycosylceramide was characterized as the Forssman antigen. In both organs, the ceramide portion was highly hydroxylated with prevalence of alpha-hydroxylated fatty acids, C18 phytosphingosine in kidney and C18 sphingosine in liver Forssman glycolipid. These data indicate that hydroxylation of kidney glycosphingolipids, which is found in mammals, has been maintained since the divergence of birds from other vertebrates. Characterization of a minor glycolipid as a Forssman tetraglycosylceramide built on the galabiosylceramide core indicates that the Forssman tetraglycosylceramide also exists in vivo. Its precursors, galactosyl- and galabiosylceramide, were characterized in kidney and liver. The Forssman antigen is the third heterophile antigen against which man raises natural antibodies. Its localization in the vascular endothelium and connective tissue makes ostrich an unpromising organ or cell donor for xenotransplantation to man.