Flow cytometric analysis reveals the presence of asialo GM1 on the surface membrane of alloimmune cytotoxic T lymphocytes

Previous studies have demonstrated that natural killer (NK) cells express the glycolipid asialo GM1, as evidenced by the sensitivity of NK cells to treatment with anti-asialo GM1 serum and complement. Because alloimmune cytotoxic T lymphocytes (CTL) were found to be insensitive to treatment with anti-asialo GM1 serum and complement, it was concluded that asialo GM1 is expressed by NK but not by CTL. However, fluorescence studies indicated that a significant proportion of peripheral T cells did express asialo GM1. Flow cytometric studies were undertaken to determine the extent to which alloimmune CTL express asialo GM1. Affinity-purified, monospecific IgG anti-asialo GM1 antibodies were used to label cells from mixed lymphocyte cultures. Separation of asialo GM1-positive and -negative fractions by cell sorting revealed that the majority of CTL activity resides in the asialo GM1-positive population. When these studies are compared with similar studies of splenic NK activity, it is apparent that, despite the relative insensitivity of CTL to treatment with anti-asialo GM1 and complement, both CTL and NK activity are enriched in the asialo GM1-positive cell population obtained by cell sorting.     

Structural and compositional difference in the neutral glycolipids between epithelial and non-epithelial tissue of the mouse small intestine

Five major neutral glycolipids, GL-1-GL-5, were isolated from the the mouse small intestine. Their structures and distribution were determined by permethylation analysis, sequential degradation with exoglycosidases and/or immunohistochemistry. The molar ratio of GL-1, GL-2, GL-3, GL-4 and Gl-5 in the whole small intestine was 1:0.04:0.03:0.42:0.02. The structures of GL-1 and GL-4 present in epithelial cells were reported previously to be glucosyl ceramide and asialo G_M1, respectively (Umesaki, Y., Suzuki, A., Kasama, T., Tohyama, K., Mutai, M. and Yamakawa, T. (1981) J. Biochem. 90, 1731–1738). GL-5, also present in the epithelial cells, was fucosyl asialo G_M1, and fucose was shown to be linked to terminal galactose of asialo G_M1 in the manner of α(1–2) bond. GL-2 and GL-3, present in the residual tissue after scraping the mucosa, were determined to be globoside and Forssman glycolipid, respectively. Both globoside and Forssman glycolipid of the non-epithelial tissue had non-hydroxy fatty acid (C₁₆–C₂₄) in combination with sphingosine (C₁₈) as the ceramide components, in contrast with the ceramide structures of the epithelial glycolipids, which contained α-hydroxy fatty acids in combination with phytosphingosine. Immunohistochemical staining using anti-glycolipid antibodies confirmed the distribution of asialo G_M1 and fucosyl asialo G_M1, and Forssman glycolipid in the epithelial and non-epithelial tissue, respectively.     

Glycolipid-lectin interactions: reactivity of lectins from Helix pomatia, Wisteria floribunda, and Dolichos biflorus with glycolipids containing N-acetylgalactosamine

The autoradiographic detection of 125I-labeled lectins binding to glycolipids on thin-layer chromatograms can be used to rapidly analyze total glycolipid extracts of cells or tissues for specific oligosaccharide structures. The Helix pomatia lectin which binds with high affinity to terminal alpha-linked GalNAc residues did not bind to globoside (terminal beta 1-3GalNAc) but did bind the ganglioside GM2 and its asialo derivative which have terminal beta 1-4GalNAc residues. The lectin from Dolichos biflorus bound specifically to the Forssman glycolipid with relatively low affinity. The lectin from Wisteria floribunda was bound to Forssman glycolipid, globoside, and the asialo derivative of the ganglioside GM2. The interactions of these lectins with the glycolipid-derived, 3H-labeled oligosaccharides was also analyzed by affinity chromatography. The results indicated that the reactivity of multivalent carbohydrate-binding proteins with polyvalent surfaces of glycolipids is strong enough to permit detection of low-affinity interactions that may not be observed in binding assays that are based on carbohydrate-protein interactions in solution. The autoradiographic analysis of 125I-Helix pomatia lectin binding to thin-layer chromatograms of total lipid extracts from human erythrocyte membranes detected the quantitative differences in the A-active glycolipids from type A1 and A2 cells.     

Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.     

Adhesion of Bordetella pertussis to sulfatides and to the GalNAc beta 4Gal sequence found in glycosphingolipids

The adherence of the human respiratory pathogen, Bordetella pertussis, to purified glycosphingolipids was investigated using thin layer chromatography overlay assays. Both virulent and avirulent strains of B. pertussis bound to asialo GM1. The bacterium did not bind to the gangliosides GM1, GD1a, GD1b, and GT1b, nor to lactosylceramide, trihexosylceramide, globoside, or Forssman antigen. However, after treatment of the chromatography plates with sialidase, B. pertussis bound to the gangliosides GM1, GM2, GD1a, GD1b, and GT1b but not to GM3. Comparison of the oligosaccharide structures of these gangliosides suggests that the minimum sugar structure needed for avid bacterial binding is GalNAc beta 4Gal. This structure has been previously implicated as a receptor for other human respiratory pathogens (Krivan, H. C., Roberts, D. D., Ginsburg, V. (1988) Proc. Natl. Acad. Sci. U.S.A 85, 6157-6161). Virulent strains of B. pertussis also bound specifically to sulfatide. This response was dose-dependent and inhibited by the anionic polysaccharide dextran sulfate. The sulfated-sugars dextran sulfate, fucoidan, and heparin inhibited the attachment of virulent strains of B. pertussis to human WiDr cells and to hamster trachea cells indicating that sulfatides on the surface of mammalian cells may function as a receptor for B. pertussis. The occurrence of both sulfatides and asialo GM1 in human lung and trachea suggests that these glycolipids may serve as specific receptors for B. pertussis.     

Granulated metrial gland cells of pregnant mouse uterus are natural killer-like cells that contain perforin and serine esterases

The mouse uterus during pregnancy contains a large population of lymphoid cells termed granulated metrial gland (GMG) cells. Our observations suggest that these cells are highly activated cytolytic lymphocytes related to NK or lymphokine-activated killer cells. Immunostaining demonstrated asialo GM1 and Thy-1 on GMG cells, both of which are expressed by NK cells. Decidua basalis tissue and isolated GMG cells contained three proteins that are characteristic of activated cytolytic lymphocyte granules: perforin, serine esterase 1, and serine esterase 2. These mediators were demonstrated in GMG cells by Western blot analysis using polyclonal antisera and by Northern blot analysis using specific cDNA probes for their mRNA. The proteins were not detected in normal spleen or liver or in asialo GM1+ cells isolated from those organs, consistent with the absence of these mediators from resting cytolytic cells. The amount of perforin in GMG cells was similar to that present in cloned, IL-2-stimulated, CTL shown previously to contain a large amount of this protein. A large population of NK cells bearing the surface marker LGL-1 was demonstrated at the implantation site by labeling with monoclonal antibody 4D11, but T cells were not detected. Many LGL-1+ cells at the implantation site expressed the GMG cell markers asialo GM1, Thy-1, and perforin. Staining intensities were inversely correlated, with LGL-1-bright cells showing little or no staining of GMG cell markers and LGL-1-faint cells showing more obvious staining of GMG cell markers. This suggests that LGL-1+ NK cells may differentiate in situ to GMG cells, losing LGL-1 and gaining a high concentration of GMG cell markers in the process. Activated cytolytic cells related to NK or lymphokine-activated killer cells may function in the pregnant rodent uterus to intercept and kill aberrant placental or embryonic cells that might otherwise enter the female and proliferate.     

Preparation of monoclonal antibodies against a glycolipid asialo GM1

Five IgM monoclonal antibodies (MAbs), MW-1, MW-2, MW-3, MW-4, and MW-5, against a glycolipid asialo GM1 were prepared from hybridoma clones obtained by the fusion of mouse NS-1 myeloma cells with spleen cells from a mouse immunized with asialo GM1 adsorbed to naked Salmonella. All the MAbs reacted only with asialo GM1 when their reactivities were examined by enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC)-immunostaining using structurally related glycolipids. The MAbs showed a complement-dependent lysis of mouse natural killer (NK) cells, but the lytic activities were weaker than that of a rabbit polyclonal anti-asialo GM1 antibody. When they were mixed, the anti-NK activity was increased to a level almost comparable to that of the polyclonal antibody. These results suggest that all the MAbs obtained are specific for asialo GM1 and that they may be different in fine specificity for the glycolipid. Significance of the MAbs in immunological and neurochemical studies is discussed.     

Esterase XXIII

Summary The intracellular distribution of non specific esterases in various villous cells of mouse jejunum was investigated using two substrates, 8-acetoxiquinoline (Q-O-2) and 8-acetyl mercaptoquinoline (Q-S-2) respectively. With the more selectively staining Q-S-2 a uniform reaction was demonstrated in all enterocytes which was mainly located in the endoplasmic reticulum and the nuclear envelope. Possibly it is a matter of a single enzyme being hardly detachable from the membranes. With the non selectively staining Q-O-2 several esterases were demonstrated Es-2, and Es-9 among them. The main reaction was found in columnar and Goblet cells, in both at all cellular membranes and in the mitochondrial matrix, additionally in cisterns and vesicles of the endoplasmic reticulum and of the Golgi apparatus and at lipid droplets of the former, finally at mucous droplets of the latter. An extracellular reaction was found in the intercellular cleft and between the microvilli of the brush border.Supported by the Deutsche Forschungsgemeinschaft     

Polarity of the Forssman glycolipid in MDCK epithelial cells

To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.     

Evaluation of sequential glomerular eluates from rats with Heymann nephritis

This study was undertaken to characterize the antigen-antibody content of sequential glomerular eluates from rats with Heymann nephritis. Serum and renal tissue were harvested every 2 wk after immunization with renal tubular antigen (Fx1A). Circulating antibody to the tubular antigen was detectable in the circulation from days 7 to 98. Direct immunofluorescence of renal tissue demonstrated an increase in IgG deposits through day 49 with stabilization thereafter. Tubular antigen deposits peaked at day 49 and then declined. One-hour and 3-hr acid eluates of isolated glomeruli were analyzed for IgG content, antibody specificity, and antigen content. Antibody from the 1-hr eluate bound to the tubular brush border but not the glomerulus, whereas the 3-hr eluate demonstrated binding to the glomerulus and not to the tubular brush border. In addition to rat IgG, the 1-hr eluate demonstrated a 70 kD band and the 3-hr eluate demonstrated a 45 kD band by polyacrylamide gel electrophoresis. By Western blot, antibody to the brush border bound to the 70 kD band. Anti-idiotypic antibody to anti-Fx1A, which binds to the glomerulus by indirect immunofluorescence, bound to the 45 kD band. The 3-hr eluate, but not the 1-hr eluate, precipitates radiolabeled F(ab')2 fragments from anti-Fx1A antibody but not from normal rat IgG. Quantitative analysis of the sequential eluates demonstrated that the 70 kD-anti-Fx1A system predominated early in the course of disease, whereas the 45 kD-anti-idiotype antigen-antibody system predominated late in the course of the disease. These observations confirm that two antigen-antibody systems contribute to the immune deposits in Heymann nephritis.     

Apical and basal Forssman antigen in MDCK II cells: a morphological and quantitative study.

We have studied the surface distribution of a glycosphingolipid (the Forssman antigen) in MDCK II and CCL39 cells. The Forssman antigen is mobile on the surface of both these cell lines. Its surface distribution is homogenous on non-polarized cells. Under conditions where MDCK II cells are well polarized, the Forssman antigen is present in equal amounts on the apical membrane and on the basal membrane and its processes. Very little Forssman antigen can be detected on the lateral membrane. The nature of the mechanism excluding the Forssman antigen from the lateral domain remains to be determined. This surface distribution is established within hours after plating and was observed with cells grown on different types of filters. The surface density of the Forssman antigen on the apical and on the basal domain has been estimated. No involvement of the basal Forssman antigen in cell attachment could be demonstrated. However, the apical Forssman antigen appears to be essential to the establishment of the cells in culture.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Esterase. XXIII. Electron microscopical demonstration of non-specific esterases in the jejunum of the mouse (Mus musc.) with two quinoline derivatives.

The intracellular distribution of non specific esterases in various villous cells of mouse jejunum was investigated using two substrates, 8-acetoxiquinoline (Q-O-2) and 8-acetyl mercaptoquinoline (Q-S-2) respectively. With the more selectively staining Q-S-2 a uniform reaction was demonstrated in all enterocytes which was mainly located in the endoplasmic reticulum and the nuclear envelope. Possibly it is a matter of a single enzyme being hardly detachable from the membranes. With the non selectively staining Q-O-2 several esterases were demonstrated Es-2, adn Es-9 among them. The main reaction was found in columnar and Goblet cells, in both at all cellular membranes and in the mitochondrial matrix, additionally in cisterns and vesicles of the endoplasmic reticulum and of the Golgi apparatus and at lipid droplets of the former, finally at mucous droplets of the latter. An extracellular reaction was found in the intercellular cleft and between the microvilli of the brush border.     

Studies on the brush border membrane of the mouse duodenum

Summary Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

Studies on the brush border membrane of the mouse duodenum. I. Membrane isolation and analysis of protein components.

Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

Receptor-mediated uptake of asialoganglioside liposomes: sub-cellular distribution of the liposomal marker in isolated liver cell types

The use of asialo GM1-containing small unilamellar liposome preparations in vivo caused a 2.8-fold increase in the uptake by the liver as compared with the control (neutral) preparations (without asialo GM1). The uptake of negatively charged dicetylphosphate and dipalmitoyl phosphatidic acid-containing small unilamellar liposomes was found to be 1.6-and 1.8-fold respectively higher than that of the neutral preparations. In studies with isolated liver cell types, inhibition of the galactosylated liposome uptake by asialofetuin indicated a possible involvement of hepatic galactose receptors in the recognition of asialo GM1 liposomes by the hepatic parenchymal cells, which in turn were found to be mainly responsible for the enhanced incorporation of these liposomes in the liver. Sub-cellular distribution studies with isolated liver cell types indicated lysosomal localization of the liposomes both in parenchymal and nonparenchymal cells, and it has been proposed that the asialo GM1 liposomes are cointernalized with asialofetuin through a common lysosomal route of ligand internalization.     

Immunoelectron microscopic identification of asialo GM1-positive cells in adult rat liver.

For the electron microscopic identification of asialo GM1-positive cells, fresh-frozen sections fixed with cold acetone and PLP-fixed vibratome sections of adult rat livers were prepared immunocytochemically using the avidin-biotin-peroxidase complex method. Asialo GM1-positive cells were located mainly in the sinusoids, and rarely in Glisson's sheath and portal veins. In the sinusoids, most pit cells, showing the ultrastructural characteristics of large granular lymphocytes (LGL), were positive for asialo GM1 but a few pit cells were asialo GM1-negative. There were several, morphological differences between asialo GM1-positive and -negative pit cells. The asialo GM1-negative pit cells were smaller and had less-developed cell organelles and fewer dense granules, suggesting a more immature stage of development. Almost all the monocytes, segmented neutrophils and eosinophils, and small or large lymphocytes in the sinusoids also showed positive reaction for asialo GM1. In Glisson's sheath, in addition to pit cells and lymphocytes, mast cells were also positive for asialo GM1. In contrast, fixed cells such as liver parenchymal cells, endothelial cells, Kupffer cells and Ito cells within the liver lobules, as well as biliary epithelial cells, smooth muscle cells, fibroblasts, endothelial cells and pericytes in Glisson's sheath were all negative for asialo GM1. Thus, cell surface asialo GM1 expression is not specific for pit cells (LGL) in the rat liver.     

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.     

Factors regulating the expression of the neutral glycolipids in the mouse small intestinal mucosa

GDP-fucose:asialo GM1 alpha(1-2)fucosyltransferase (FT) is induced in the small intestinal mucosa after microbial contamination of germ-free mice (Umesaki, Y., Sakata, T. and Yajima, T. (1982) Biochem. Biophys. Res. Commun. 105, 439-443). As a result, asialo GM1 glycolipid, a major component of the epithelial cell membrane, drastically converted into fucosyl asialo GM1. There were many other examples in which FT was induced. One was the weaning period for conventional mice. Others included injuries of the small intestine by punctures or administration of cytosine arabinoside, and the injection of protein synthesis inhibitors, such as cycloheximide or emetine, or the soluble fraction of the small intestinal homogenate (SISF). The induction of FT was more rapid after injection of cycloheximide or SISF than after injury, mechanical puncturing or after administration of cytosine arabinoside. The changes in the neutral glycolipids of the small intestine by injection of cycloheximide or SISF were analyzed in detail. FT activity started to increase after approx. 5 h and reached the maximum 10-12 h after injection of cycloheximide or SISF, and rapidly declined thereafter. The conversion of asialo GM1 into fucosyl asialo GM1 started after about 10 h and reached the maximal value 24 h after the treatment. Fucosyl asialo GM1 persisted for a few days, although the FT activity fell to near the basal level. On the other hand, the amount of glucosyl ceramide was constant after these treatments. There was little difference in the time-courses of both the FT activity and the glycolipid conversion between these treatments. In the case of co-injection of cycloheximide and SISF, the effect of both materials on FT activity induction was synergistic. The distribution of FT activity and immunohistochemical staining using anti-fucosyl asialo GM1 antibody along the crypt-villus axis showed a stronger expression of fucosyl asialo GM1 in villus portion, the post-mitotic cell zone, than in the crypt portion. Asialo GM1 was converted into fucosyl asialo GM1 after the induction of FT by the various treatments mentioned above. Especially the effects of cycloheximide and/or SISF on FT induction suggest at least the presence of a regulatory protein(s) which controls the glycolipid expression in the small intestine.