Sendai virus receptor: proposed recognition structure based on binding to plastic-adsorbed gangliosides.

The binding of Sendai virus to polystyrene petri dishes to which various gangliosides of defined structures had been adsorbed was determined. The ganglioside-bound virus was visualized either by a water vapor condensation method or by a hemadsorption method. By either assay, specific virus binding of high affinity was demonstrated to the gangliosides GT1a, GQ1b, and GPlc which have a common end sequence in the oligosaccharide moiety: NeuAc alpha 2 leads to 8NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc leads to. Binding also occurred to the GD1a and GT1b gangliosides, which have the same end carbohydrate sequence except for the terminal N-acetylneuraminic acid, but the affinity was only 1-9% of that of the gangliosides with a terminal disialosyl linkage. It is proposed that the structure NeuAc alpha 2 leads to 8NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc is the recognition-specific structure of the receptor for Sendai virus that is present on cell membrane gangliosides and possibly also glycoproteins. Binding tests to plastic-adsorbed glycolipids are suggested to be a useful tool for identification of the receptor recognition structure.     

Use of brefeldin A to define sites of glycosphingolipid synthesis: GA2/GM2/GD2 synthase is trans to the brefeldin A block.

Brefeldin A (BFA) induces the rapid redistribution of the Golgi complex into the endoplasmic reticulum (ER), causing the glycoproteins that are retained in the ER to be processed by Golgi enzymes. We have examined the effects of BFA on the synthesis of glycosphingolipids (GSL) to map the intracellular sites of GSL synthesis. In several cultured cell types, BFA inhibited the synthesis of the neutral GSL gangliotriaosylceramide (GA2) and monosialoganglioside GM2 and disialoganglioside GD2, where GD2 is GalNAc(beta 1----4)- [NeuAc(alpha 2----8)NeuAc(alpha 2----3)]Gal(beta 1----4)GlcCer, GM2 lacks the NeuAc(alpha 2----8) unit, and GA2 lacks both NeuAc(alpha 2----8) and NeuAc(alpha 2----3) units. The observed decrease in labeling of GA2, GM2, and GD2 in the presence of BFA was not due either to enhanced degradation of these glycolipids or to shedding of these glycolipids from the cells. In rat liver all three of these glycolipids have been shown by others to be synthesized by the same enzyme, GA2/GM2/GD2 synthase, which catalyzes the addition of N-acetylgalactosamine to lactosylceramide (Lac-Cer), GM3 [NeuAc(alpha 2----3)Gal(beta 1----4)GlcCer], and GD3 [NeuAc(alpha 2----8)NeuAc-(alpha 2----3)Gal(beta 1----4)GlcCer], respectively. Studies with a fluorescent glycolipid analog indicated that BFA redistributed the trans-Golgi stacks into a reticular pattern characteristic of the ER. These studies localize GA2/GM2/GD2 synthase, a key enzyme involved in the synthesis of complex gangliosides, to a compartment late in the intracellular trafficking pathway, which remains functionally distinct from the ER in the presence of BFA.     

A granulocyte reactive monoclonal antibody, 1G10, identifies the Gal beta 1-4 (Fuc alpha 1-3)GlcNAc (X determinant) expressed in HL-60 cells on both glycolipid and glycoprotein molecules

1G10, a monoclonal IgM antibody that identifies a differentiation antigen on human granulocytes and a subpopulation of monocytes, was found to react specifically with glycosphingolipids bearing the Gal beta 1-4(Fuc alpha 1-3)GlcNAc hapten (X determinant). This carbohydrate determinant was found on both glycolipid and glycoprotein molecules isolated from HL-60 cells (a promyelocytic leukemia cell line). Thus, this highly conserved carbohydrate-defined determinant previously described on mouse embryonic and mouse and human carcinoma cells is also expressed as a tissue-specific differentiation antigen on normal human granulocytes.     

Ganglioside GM2 as a human tumor antigen (OFA-I-1).

A monospecific antibody produced in vitro by a B-lymphoblastoid cell line transformed with Epstein-Barr virus has been shown to recognize a membrane antigen (OFA-I-1) on human tumors and fetal brain. This study identifies the chemical nature of OFA-I-1. The glycolipid fraction of antigen-rich spent medium of an OFA-I-1-positive melanoma cell line, M14, was extracted by chloroform/methanol/water, 4:8:3 (vol/vol), and was separated into fractions of neutral glycolipids and gangliosides by DEAE-Sephadex followed by base treatment and Bio-sil A column elution. OFA-I-1 antigens were found exclusively in the ganglioside fraction when assayed with monospecific anti-OFA-I-1 by an immune adherence inhibition test. The results obtained from thin-layer chromatography of the antigenic M14 ganglioside and sequential glycosidase digestion suggested that the antigen was a ganglioside GM2, GalNAc beta 1 leads to 4(NeuAc alpha 2 leads to 3)Gal beta 1 leads to 4Glc leads to Cer. These results were further supported by testing various authentic gangliosides and neutral glycolipids for OFA-I-1 antigenicity. Only GM2 showed positive reactivity.     

Identification of a human neuroectodermal tumor antigen (OFA-I-2) as ganglioside GD2.

Two monospecific human antibodies (anti-OFA-I-1 and anti-OFA-I-2) produced in vitro by lymphoblast cell lines originating from melanoma patients have been shown previously to recognize cell surface antigens (OFA-I-1 and OFA-I-2) on human tumors and fetal brain: OFA-I-1 is expressed on a variety of human tumors, while OFA-I-2 has been detected only on tumors of neuroectodermal origin. Evidence presented in this report suggests that the two antigens expressed by a cultured human melanoma cell line (M14) are chemically distinct and that OFA-I-2 is a cell surface glycolipid, ganglioside GD2: GalNAc beta 1 leads to 4 NeuAc alpha 2 leads to 8NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4Glc-ceramide.     

Sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants.

Purified sialyltransferases (CMP-N-acetyl-neuraminate:D-galactosyl-glycoprotein N-acetylneuraminyl-transferase, EC 2.4.99.1) in conjunction with neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) were used to produce cell surface sialyloligosaccharides of defined sequence to investigate their role in paramyxovirus infection of host cells. Infection of Madin-Darby bovine kidney cells by Sendai virus was monitored by hemagglutination titer of the virus produced and by changes in morphological characteristics. By either criterion, treatment of the cells with Vibrio cholerae neuraminidase to remove cell surface sialic acids rendered them resistant to infection by Sendai virus. Endogenous replacement of receptors by the cell occurred slowly but supported maximal levels of infection within 6 hr. In contrast, sialylation during a 20-min incubation with CMP-sialic acid and beta-galactoside alpha 2,3-sialytransferase restored full susceptibility to infection. This enzyme elaborates the NeuAc alpha 2,3Gal beta 1,3GalNAc (NeuAc, N-acetylneuraminic acid) sequence on glycoproteins and glycolipids. No restoration of infectivity was observed when neuraminidase-treated cells were sialylated by using beta-galactoside alpha 2,6-sialytransferase, which elaborates the NeuAc-alpha 2,6Gal beta 1,4GlcNAc sequence. These results suggest that sialyloligosaccharide receptor determinants of defined sequence are required for Sendai virus infection of host cells.     

Age-acquired immunity to a Plasmodium vivax invasion ligand,the duffy binding protein

To investigate the interactions of glycoconjugates with the innate immune system, peripheral blood mononuclear cells were stimulated with glycolipids derived from Schistosoma mansoni eggs and worms and with biochemically synthesized neoglycoconjugates. Egg glycolipids stimulated the production of interleukin (IL)--10, IL-6, and tumor necrosis factor--alpha in monocytes, whereas worm glycolipids failed to do so. When monoclonal antibodies that specifically recognize defined carbohydrate epitopes were used, the binding of a GalNAc beta 1-4(Fuc alpha 1-2Fuc alpha 1-3)GlcNAc (LDN-DF) reactive antibody was pronounced on egg glycolipids but was absent on worm glycolipids. The binding of antibodies that recognize Gal beta 1-4(Fuc alpha 1-3)GlcNAc (LewisX), GalNAc beta 1-4GlcNAc (LDN), and GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc (LDN-F) was comparable for both preparations. Cytokine production in response to neoglycoconjugates containing enzymatically synthesized glycans also was measured. The LDN-DF neoglycoconjugate was the most potent cytokine inducer, which indicates that this difucosylated glycan can act at the host-parasite interface and can trigger innate immune responses.     

Differences in the carbohydrate moieties of the common alpha-subunits of human chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, and thyrotropin: preliminary structural inferences from direct methylation analysis

The carbohydrate components of combined alpha-subunits of urinary hCG and human pituitary LH (hLH), FSH (hFSH), and TSH (hTSH), each derived from the intact hormone, were studied by direct sugar analysis and methylation analysis. The methods provide a complete survey of the structural elements contained in the complex sugars associated with these glycoproteins, but do not establish the sugar sequences or anomeric configurations of glycosidic bonds. By analogy to N-linked oligosaccharides that occur in many glycoproteins, the data suggest distinct structural features for carbohydrates of alpha-subunits combined with beta-subunits. hCG alpha contains biantennary asparagine-linked chains terminated by either NeuAc alpha 2-3Gal beta 1- or GlcNAc beta 1-2 Man alpha 1- and lacks fucose. hTSH alpha contains biantennary chains with the same termini as hCG alpha plus terminal R-O-4GalNAc and a fucosyl residue linked alpha 1-6 to the inner GlcNAc residue of the N-linked chitobiosyl core. hLH alpha may contain some high mannose chains, but primarily contains biantennary chains terminated by NeuAc alpha 2-3(6)Gal beta 1-, GlcNAc beta 1-, GalNac-1-, R'-O-6GlcNAc-1-, and R"-0-2Man-1-plus a fucosyl residue linked alpha 1-6 to the inner GlcNAc residue of the N-linked chitobiosyl core. hFSH alpha contains more complicated structures that probably include a bisecting GlcNAc residue linked beta 1-4 to a 3,6-di-O-substituted core mannosyl residue, and terminal NeuAc alpha 2-3Gal beta 1-4(+/- Fuc alpha 1-3)GlcNAc-1, Gal beta 1-4(+/- Fuc alpha 1-3)GlcNAc-1-, R"'-O-GalNAc-1-, and GalNAc-1. In addition, the presence of 2,4-di-O-substituted mannose in hFSH alpha indicates that it contains triantennary chains. The identities of the R; R', R", and R"' groups were not determined, but recent studies of glycoprotein hormones suggest that they may be sulfate groups. Our results demonstrate differential glycosylation of virtually identical polypeptide hormone alpha-subunits produced in the same organ or perhaps even in the same cell.     

Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase.

Hyperacute rejection of a porcine organ by higher primates is initiated by the binding of xenoreactive natural antibodies of the recipient to blood vessels in the graft leading to complement activation. The majority of these antibodies recognize the carbohydrate structure Gal(alphal,3)Gal (gal epitope) present on cells of pigs. It is possible that the removal or lowering of the number of gal epitopes on the graft endothelium could prevent hyperacute rejection. The Gal(alpha1,3) Gal structure is formed by the enzyme Galbeta1,4GlcNAc3-alpha-D-galactosyltransferase [alpha(1,3)GT; EC 2.4.1.51], which transfers a galactose molecule to terminal N-acetyllactosamine (N-lac) present on various glycoproteins and glycolipids. The N-lac structure might be utilized as an acceptor by other glycosyltransferases such as Galbeta1,4GlcNAc 6-alpha-D-sialyltransferase [alpha(2,6)ST], Galbeta1,4GlcNAc 3-alpha-D-Sialyltransferase [alpha(2,3)ST], or Galbeta 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.691, etc. In this report we describe the competition between alpha(1,2)FT and alpha(1,3)GT in cells in culture and the generation of transgenic mice and transgenic pigs that express alpha(1,2)Fr leading to synthesis of Fucalpha,2Galbeta- (H antigen) and a concomitant decrease in the level of Gal(alpha1,3)Gal. As predicted, this resulted in reduced binding of xenoreactive natural antibodies to endothelial cells of transgenic mice and protection from complement mediated lysis.     

The O-linked oligosaccharide structures are striking different on pregnancy and choriocarcinoma HCG

hCG is a glycoprotein hormone which is detected in the serum and urine of pregnant women and of patients with hydatidiform mole and choriocarcinoma. The molecule contains 4 O-linked sugar chains. In an effort to identify cancer markers, the structures of these sugar units on the hCG produced in pregnancy and choriocarcinoma were compared. hCG molecules in patient urines were purified by immuno-affinity chromatography and gel filtration. beta-elimination was used to cleave the O-linked sugar units, radioactive sodium borohydride to label them, and gel filtration on Bio-Gel P4 to size them and compare their elution volumes with those of standard oligosaccharides of known structure. A trisaccharide, NeuAc alpha 2-3Gal beta 1-3GalNAc-, was found to be the principal unit attached to urinary hCG from pregnant women (10 samples). A hexasaccharide, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-6)GalNAc-, which accounted for just 6% (mean, range 0-14%) of the O-linked sugar units on pregnancy hCG, was the principal unit (mean 52% of total, range 50-56%) attached to the hCG from choriocarcinoma patient urines (3 samples). These results indicate that hexasaccharide-abundant hCG is an indicator of choriocarcinoma.     

Distribution of O-linked sugar units on hCG and its free alpha-subunit

hCG, a glycoprotein hormone produced by the trophoblast in pregnancy, is composed of two dissimilar subunits, alpha and beta, joined non-covalently. hCG has four O-linked sugar units, all attached to the beta-subunit. The trophoblast also produces a free form of the alpha-subunit, which unlike the alpha-component of hCG, can contain an O-linked sugar unit. The structures of the O-linked sugar units were examined. Four structures were identified on urinary hCG. A hexasaccharide, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-6)GalNAc- accounting for 13%, a tetrasaccharide, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc-, for 34%, a trisaccharide, NeuAc alpha 2-3Gal beta 1-3GalNAc-, for 43% and a disaccharide, NeuAc alpha 2-6GalNAc- for 10% of the total O-linked sugar structures. Similar mixtures were found on peptides containing one, three or four sugar units suggesting a random distribution among attachment sites. The distribution of O-linked sugar structures on hCG and free alpha-subunit from trophoblast explant cultures was compared. The mixture of structures attached at the single site on the free alpha-subunit paralleled that at the four sites on the hCG.     

Glycosphingolipids in human pancreatic juice

Summary Glycosphingolipids in human pancreatic juice were isolated and purified by DEAE and silica gel column chromatographs, and further by HPLC on silica gel and reversed-phase columns. The structures of the glycosphingolipids were determined to be glucosylceramides and lactosylceramides by means of fast atom bombardment mass spectrometry,¹H-NMR spectroscopy, and component analysis involving GLC-mass spectrometry. The ceramide portions of the glucosylceramides consist of palmitic, tetracosanoic, and α-hydroxy tetracosanoic fatty acids, and d18:1 and t18:0 sphingosines. The ceramide portions of the lactosylceramides consist of palmitic, tetracosanoic, tetracosenoic, and tetracosadienoic fatty acids, and d18:1 sphingosine. The fatty acid compositions are different from the free fatty acid compositions of serum and pancreatic juice. The predominance of saturated, unsaturated, and hydroxy tetracosanoic fatty acids is quite unique, and the possibility that these glycosphingolipids may play a specific role in pancreatic juice deserves consideration. These glycosphingolipids were documented to be major components of the pancreatic juice from 15 patients with a variety of pancreatic diseases.     

Ceramide structure predicts tumor ganglioside immunosuppressive activity.

Molecular determinants of biological activity ofgangliosides are generally believed to be carbohydrate in nature. However, ourstudies of immunomodulation by highly purified naturally occurring tumorgangliosides provide another perspective: while the immunosuppressive activityof gangliosides requires the intact molecule (both carbohydrate and ceramidemoieties), ceramide structure strikingly influences gangliosideimmunosuppressive activity. Molecular species of human neuroblastoma GD2ganglioside in which the ceramide contains a shorter fatty acyl chain (C16:0,C18:0) were 6- to 10-fold more active than those with a longer fatty acyl chain(C22:0/C24:1, C24:0). These findings were confirmed in studies of ceramidespecies of human leukemia sialosylparagloboside and murine lymphoma GalNAcGM1b.Gangliosides that contain shorter-chain fatty acids (and are mostimmunosuppressive) are known to be preferentially shed by tumor cells.Therefore, the results suggest that the tumor cell is optimized to protectitself from host immune destruction by selective shedding of highly activeceramide species of gangliosides.     

Biosynthesis in vitro of fucose-containing glycosphingolipids in human neuroblastoma IMR-32 cells.

Two different glycolipid:fucosyltransferase activities involved in the biosynthesis in vitro of blood group-related glycosphingolipids have been detected in a membrane preparation isolated from a human neuroblastoma-derived clonal cell line, IMR-32. The membrane preparation contains an alpha (1,2)-fucosyltransferase (EC 2.4.1.89) that catalyzed the transfer of vucose from GDP--[14C]fucose to neolactotetraosylceramide or neolactopentaosylceramide to form types H-I and B-I glycolipids, respectively. The second fucosyltransferase catalyzes the transfer of fucose to lactotriaosylceramide [GlcNAc(beta1-3)Gal(beta1-4)Glc-Cer] to form a tetraglycosylceramide intermediate of the novel Lea-type glycolipid. UDP-galactose:lactotriaosylceramide beta-galactosyltransferase (EC 2.4.1.86) had 4 times the activity of UDP-galactose:alpha-galactosyltransferase (EC 2.4.1.87) when tested under similar conditions. alpha-Fucosyltransferase activities and the incorporation of [14C]fucose into glycoproteins and glycolipids were also compared in cells differentiated in the presence of 4 micron BrdUrd and 6-mercaptoguanosine.     

Expression cloning of a CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1''Cer alpha 2,8-sialyltransferase (GD3 synthase) from human melanoma cells.

Using an expression cloning approach, we have isolated a cDNA encoding GD3 synthase (CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase, EC 2.4.99.8), which is a key regulatory enzyme determining the prominence of the ganglioside biosynthesis pathway. The cloned cDNA encodes a 341-amino acid protein containing a single transmembrane domain at its N-terminal region, suggesting that the protein has a type II transmembrane topology. The sequence of alpha 2,8-sialyltransferase showed a high level of similarity with other sialyltransferases at two conserved regions typical in the sialyltransferase family. Transfected cells containing the cloned cDNA expressed GD3 ganglioside on the cell surface, which was detectable with specific anti-GD3 antibody by immunofluorescence and immunostaining after separation of isolated glycolipids on thin-layer chromatography. The cDNA hybridized to a single mRNA species of 2.4 kb in melanoma cells. This sialyltransferase is distinctive in catalyzing the formation of the alpha 2-8 linkage of sialic acids.     

Human myeloid alpha 3-fucosyltransferase is involved in the expression of the sialyl-Lewis(x) determinant, a ligand for E- and P-selectin

The sialyl-Lex determinant (NeuAc alpha 2-->3Gal beta 1-->4[Fuc alpha- 1-->3]GlcNAc) has been identified as a major ligand in the selectin- mediated adhesion of neutrophils and monocytes to activated endothelium or platelets. This carbohydrate epitope is formed by the sequential action of alpha 3-sialyltransferase and alpha 3-fucosyltransferase on N- acetyllactosamine (Gal beta 1-->4GlcNAc) disaccharide termini of glycoconjugates. We have addressed the role of the human myeloid alpha 3-fucosyltransferase in the expression of this epitope at the leucocyte surface by determining its activity in human-mouse leukemic cell hybrids (WEGLI), normal human granulocytes and chronic myeloid leukemia (CML) cells using sialylated and desialylated glycoproteins and oligosaccharides as acceptor substrates. In contrast to what has been reported for the myeloid-type enzyme, we found that the alpha 3- fucosyltransferase of the cells studied can use sialylated acceptors be it that the activity is several times lower than with asialo- substrates. Characterization of the product obtained with a sialylated oligosaccharide indicated that the enzyme can catalyze the formation of the sialyl-Le(x) structure. Flow cytometry of the WEGLI cells using a sialyl-Le(x)-specific monoclonal antibody (MoAb) showed that these cells indeed express sialyl-Lex at their surface, provided that they contain human chromosome 11. Earlier the presence of this chromosome had been correlated with the expression of alpha 3-fucosyltransferase activity. In addition to sialyl-Le(x), WEGLI cells containing chromosome 11 showed high-expression levels of related structures recognized by antibodies VIM-2 and VIM-8, suggesting that fucose addition can occur at both distal and proximal GlcNAc residues in poly- N-acetyl-lactosaminoglycan sequences. Based on the human chromosome contents it could be ruled out that the alpha 3-fucosyltransferase of WEGLI cells is a Lewis-type alpha 3/4- or plasma-type alpha 3- fucosyltransferase, the genes of which have been mapped to chromosome 19. It is concluded that the enzyme studied is of the myeloid-type and indeed is involved in the synthesis of sialyl-Le(x) (and also VIM-2 and VIM-8 structures) in leukocytes provided that its expression is at a sufficiently high level.     

Amino acid and carbohydrate structural variants of glycoprotein products (M-N glycoproteins) of the M-N allelic locus.

Major glycoprotein of MgM, MM Miltenberger III (MiIII), and M-N erythrocyte membranes from individual donors were cleaved with CNBr and their amino-terminal octapeptides were examined with respect to amino acid and carbohydrate composition. The amino-terminal octapeptides from the heterozygous MgM donor were resolved into two types, A and A'. MgM A was identical to octapeptide A from MM glycoproteins in carbohydrate and amino acid compositions. MgM A' exhibited amino acid composition similar to NN peptide A except for a single substitution of an Asx for a Thr and, as a result, was not glycosylated. MM(MiIII) octapeptide A was identical to M peptide A in amino acid composition, but differed in carbohydrate content. This glycopeptide contained three O-glycosidically linked carbohydrate units, one of which contained GlcNAc bound to a core of NeuAc, Gal, and GalNAc. About two such units were also present in the CNBr glycopeptide B of the glycoprotein, and on the basis of studies with alkaline borohydride and alkaline sulfite degradations, these units are believed to have the following structure: (formula see text) The Mg is an allelomorph of the M-N locus, likely evolved from a single base substitution in the N gene. The resulting single amino acid substitution effects the posttranslational carbohydration of neighboring Ser and Thr residues. The MM(MiIII) appears to be a product of the M gene that undergoes sequences of posttranslational glycosylations different from those of the M-N glycoproteins.     

Chronic treatment with pioglitazone does not protect obese patients with diabetes mellitus type II from free fatty acid-induced insulin resistance

CONTEXT: Thiazolidinediones increase peripheral insulin sensitivity and decrease plasma free fatty acids (FFA). However, their exact mechanism of action has not been fully elucidated. OBJECTIVE: We studied the protective effect of pioglitazone on FFA-induced insulin resistance and the effects on intramyocellular glycosphingolipids. DESIGN: We studied glucose metabolism in the basal state and during a hyperinsulinemic euglycemic clamp by using stable isotopes. Studies were performed at baseline and after 4 months of treatment with pioglitazone. Patients were then studied on a third occasion during infusion of a lipid emulsion to increase plasma FFA to pretreatment levels. All studies were combined with muscle biopsies to measure intramyocellular ceramide and glycosphingolipids. PATIENTS: Patients were obese with poorly controlled type 2 diabetes mellitus. INTERVENTION: Patients were treated with 30 mg pioglitazone once daily. Main Outcome Measure: The change in peripheral insulin sensitivity after treatment with pioglitazone and during the infusion of the lipid emulsion was the main outcome measure. RESULTS: Peripheral glucose uptake (Rd) increased significantly, but returned to baseline levels after increasing plasma FFA to pretreatment levels. Insulin-mediated suppression of FFA was increased significantly. Intramyocellular ceramide concentrations were higher during the hyperinsulinemic clamp after treatment with pioglitazone, but not in the basal state. The intramyocellular content of glycosphingolipids and plasma concentrations of ceramide and glycosphingolipids did not change. CONCLUSIONS: Pioglitazone increases Rd and insulin-mediated suppression of plasma FFA, but does not protect patients with type 2 diabetes mellitus from FFA-induced insulin resistance. This effect of pioglitazone is not attained via a decrease in intramyocellular concentrations of ceramide or glycosphingolipids.     

Abnormalities in the glycosphingolipid content of human Pk and p erythrocytes.

Erythrocytes of the rare Pk phenotype lack the blood group P antigen, and p erythrocytes lack both P and Pk antigens. On the basis of immunological data we suggested previously that the P and Pk antigens are the glycosphingolipids globoside and trihexosyl ceramide, respectively, and we have now confirmed these designations by chemical analysis of erythrocytes lacking these antigens. The Pk erythrocytes contain only traces of globoside and have a marked excess of trihexosyl ceramide in comparison with normal erythrocytes. The p erythrocytes lack globoside and trihexosyl ceramide and contain an excess of lactosyl ceramide and other complex glycolipids. Our analyses of normal erythrocytes also revealed complex gangliosides with the approximate chromatographic mobilities of GD1b and GT1, and several gangliosides containing N-acetylglucosamine.     

Specific gangliosides function as host cell receptors for Sendai virus.

The ability of specific gangliosides to function as host cell receptors for Sendai virus was investigated by using Madin-Darby bovine kidney cells which become resistant to infection upon treatment with Vibrio cholerae sialidase. Sialidase-treated cells were incubated for 20 min at 37 degrees C with individual, highly purified gangliosides containing homogeneous carbohydrate moieties and then inoculated with virus for 10 min. Susceptibility of the cells to infection was monitored by hemagglutination titer of the virus produced 48 hr after inoculation. Incubation of the cells with gangliosides containing the sequence NeuAc alpha 2,3Gal beta 1,3GalNAc (i.e., GD1a, GT1b, and GQ1b) fully restored susceptibility to infection to the cells. However, the ganglioside GQ1b in which the sequence ends with two sialic acids in a NeuAc alpha 2,8NeuAc linkage instead of a single sialic acid as in GD1a and GT1b, was effective as a receptor at a concentration 1/100th that of any of the other gangliosides tested. Incubation with gangliosides similar in structure to GD1a, GT1b, and GQ1b but lacking the sialic acid attached to the terminal galactose (i.e., GM1 and GD1b) had no effect. The results from control experiments in which gangliosides were incubated at 0 degrees C with cells or in which trypsin was used to remove gangliosides adsorbed to cells were consistent with the premise that the gangliosides must actually insert into the cellular membrane to function as Sendai virus receptors. Addition of 4 X 10(6) molecules of 14C-labeled GD1a per cell made the cells fully susceptible to infection. Analysis of the ganglioside content of cell membranes showed that gangliosides GD1a, GT1b, and GQ1b are natural components of these cells and are present in quantities sufficient to act as receptors. These results demonstrate that gangliosides with the proper carbohydrate sequence, such as GD1a, GT1b, and GQ1b, function as natural receptors for Sendai virus in host cells.