Globo-series Glycolipids Research Articles

The amino-terminal domain of the P-pilus adhesin determines receptor specificity.

Pyelonephritic isolates of Escherichia coli commonly express P-pili, which mediate bacterial attachment to glycolipids on epithelial cell surfaces. Three classes of P-pili have been defined, based on varying specificity for galabiose-containing glycolipids. Variation in adhesive capacity is correlated with a shift in preferred host, suggesting that host tropism depends largely on detailed specificity for the globoseries glycolipids. In this study we examined the importance of the PapG adhesin in determining receptor specificity. Translational fusions were constructed between the amino-terminus of the PapG adhesin from each of the three pilus classes and a reporter protein. The binding specificity of the purified fusion proteins in vitro was identical to that seen with whole bacteria. Adherence of intact bacteria to cultured kidney cells was markedly reduced by a monoclonal antibody specific for the Class III adhesin (previously denoted PrsG), confirming the importance of the amino-terminus of PapG in mediating attachment to a receptor when presented on the eukaryotic cell surface. These results suggest that the detailed receptor specificity resides solely within the amino-terminus of the PapG adhesin and is independent of the complex pilus architecture.

Glycolipids of germ cell tumors: extended globo-series glycolipids are a hallmark of human embryonal carcinoma cells.

Glycolipids of human germ cell tumor lines were analyzed to define the most common immunohistochemical profiles of embryonal carcinoma (EC), differentiated derivatives of EC, yolk sac carcinoma (YC) and choriocarcinoma (CC). Glycolipid composition was examined by high-performance thin-layer chromatography (HPTLC) combined with immunostaining with a panel of anti-carbohydrate monoclonal antibodies (MAbs). All EC cell lines were found to contain high levels of globo-series glycolipids, including globotriosylceramide (Gb3), globoside (Gb4), Gb5 (Gal beta 1-->3Gb4) and GL7 (sialyl Gal beta 1-->3Gb4). Somatic differentiated derivatives (e.g., EC cells treated with retinoic acid) contained decreased levels of globo-series glycolipids and increased levels of lacto- and ganglio-series glycolipids, including GD3, GT3 and GD2. CC cell lines contained relatively large amounts of Gb3 but did not contain extended globo-series glycolipids Gb5 and GL7. CC cell lines also contained a macroglycolipid reactive with the antibody to SSEA-1 (Lex). Glycolipids were not detected in two YC cell lines, while other YC cell lines contained globo-series core glycolipids (Gb3 and Gb4) and gangliosides. We conclude that EC, YC and CC have distinct patterns of membrane glycolipid expression that can be identified by HPTLC and immunostaining. Our results indicate that globo-series glycolipids Gb5 and GL7, which carry stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4), are a hallmark of human EC cells. Cell lines derived from human germ cell tumors that do not express Gb5 and GL7 deserve to be re-evaluated, since they may represent different stem cells, most likely equivalent to somatic cells and their developmentally committed precursors (e.g., neuroblasts).

CD19 has a potential CD77 (globotriaosyl ceramide)-binding site with sequence similarity to verotoxin B-subunits: implications of molecular mimicry for B cell adhesion and enterohemorrhagic Escherichia coli pathogenesis.

The glycosphingolipid globotriaosyl ceramide (CD77) and other globo-series glycolipids containing terminal galactose (Gal)alpha 1-4Gal residues function as receptors for the verotoxin (Shiga-like toxin) family of Escherichia coli-elaborated toxins. CD77 is also a marker for germinal center B lymphocytes and Burkitt's lymphoma cells. The pan B cell marker CD19 is a 95-kD membrane protein that appears early in B cell differentiation and is only lost upon terminal differentiation to plasma cells. CD19 is involved in signal transduction and has a regulatory role in B cell proliferation and differentiation in response to activation in vitro. However, an endogenous ligand for CD19 has not yet been identified. We report herein that the extracellular domain of CD19 has a potential CD77-binding site with extensive sequence similarity to the verotoxin B-subunits. These B-subunit-like sequences on CD19 are in close proximity following the organization of intervening amino acids into disulfide-linked domains. Cocapping of CD19 and CD77 on Burkitt's lymphoma-derived Daudi cells with anti-CD19 antibodies indicates that CD19 and CD77 are associated on the B cell surface. Cell surface binding of anti-CD19 antibodies is decreased on CD77-deficient mutant Daudi cells, suggesting that CD77 expression influences the surface expression of CD19. Wild-type Daudi cells, but not the CD19/CD77-deficient mutants, bind to matrices expressing the carbohydrate moiety of CD77 or other Gal alpha 1-4Gal containing glycolipids. This binding can be inhibited by anti-CD77 antibodies, the CD77-binding verotoxin B-subunit or anti-CD19 antibodies. Daudi cells exhibit a degree of spontaneous homotypic adhesion in culture while the CD77/CD19-deficient Daudi mutants grow as single cells. The stronger homotypic adhesion that occurs in B cells after antibody ligation of CD19 and that involves, to some extent, the integrin system, is also dramatically lower in the mutant cells relative to the parent cell line. However, reconstitution of mutant cells with CD77 restores the anti-CD19 mAb-induced adhesion to wild-type Daudi cell levels. These studies represent the first time that CD19-mediated signaling has been reconstituted in a low-responder B cell line. These convergent observations provide compelling evidence that CD19/CD77 interactions function in adhesion and signal transduction at a specific stage in B cell development and suggest that such interactions have a role in B lymphocyte homing and germinal center formation in vivo. By targeting CD77+ B cells, verotoxins may suppress the humoral arm of the immune response during infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of Forssman antigen in human large intestine.

Forssman antigen is a commonly occurring heterophile antigen but is thought not to be present in most humans. Recent biochemical studies, however, have shown the presence of Forssman antigen in several forms of human cancer, including gastric, colon, and lung cancers. Immunohistochemical staining with both monoclonal and polyclonal antibodies has failed to demonstrate this antigen in human tissues. In this study we conclusively demonstrated the presence of Forssman antigen in cytoplasm of colon goblet cells, especially those in the so-called transitional mucosa adjacent to carcinoma. Specimens from 69 of 70 patients with colon cancer contained the antigen in goblet cells in transitional mucosae. The antigen was successfully demonstrated only after removal of sialic acid by alkaline hydrolysis-neuraminidase digestion. Localization of the antigen was quite different from that of Tn and human blood group A, B, and H antigens. This antigen was thought to be associated exclusively with globo-series glycolipids, and its existence in glycoproteins has not been conclusively demonstrated. However, the results presented here, based on proteolytic digestion and lipid extraction studies, strongly suggest that the antigen is also contained in glycoproteins.

Generation of One Set of Murine Monoclonal Antibodies Specific for Globo-Series Glycolipids: Evidence for Differential Distribution of the Glycolipids in Rat Small Intestine

We generated four murine monoclonal antibodies (MAbs) specific for globo-series glycolipids by immunizing C3H/HeN mice with these purified glycolipids adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. By use of a wide variety of glycolipids, the precise structures recognized by these MAbs were elucidated through an enzyme-linked immunosorbent assay and an immunostaining or thin-layer chromatography. These four MAbs, designated as BGR23, BGR47, BMR26, and BGR27, exhibited highly restricted binding specificities, reacting only with the globo-series glycolipids Gb3Cer, III3Galα-Gb3Cer, Gb4Cer, and IV3GalNAcα-Gb4Cer, respectively, which were used for immunization. None of the other various glycolipids or gangliosides were recognized. We determined the localization of these globo-series glycolipids in adult rat small intestine by means of an immunofluorescence technique with these MAbs. Our study revealed that there is a differential distribution of these glycolipids in the rat tissue. III3Galα-Gb3Cer was demonstrated on the cryptic cells and circular muscle, whereas Gb4Cer was localized on both the circular and longitudinal muscles. The expression of Gb3Cer was associated with the epithelium and the capillary endothelial cells in the lamina propria mucosae as well as with the tunica submucosa, whereas IV3GalNAcα-Gb4Cer was detected on the epithelium, capillary endothelial cells in the lamina propria mucosae, and both the muscle layers.

Alteration of the glycolipid binding specificity of the pig edema toxin from globotetraosyl to globotriaosyl ceramide alters in vivo tissue targetting and results in a verotoxin 1-like disease in pigs.

All members of the verotoxin (VT) family specifically recognize globo-series glycolipids on the surface of susceptible cells. Those toxins that are associated with human disease, VT1, VT2, and VT2c, bind to globotriaosyl ceramide (Gb3) while VT2e, which is associated with edema disease of swine, binds preferentially to globotetraosyl ceramide (Gb4). We were recently able to identify, using site-directed mutagenesis, amino acids in the binding subunit of these toxins that are important in defining their glycosphingolipid (GSL) binding specificity (Tyrrell, G. J., K. Ramotar, B. Boyd, B. W. Toye, C. A. Lingwood, and J. L. Brunton. 1992. Proc. Natl. Acad. Sci. USA. 89:524). The concomitant mutation of Gln64 and Lys66 in the VT2e binding subunit to the corresponding residues (Glu and Gln, respectively) found in VT2 effectively converted the GSL binding specificity of the mutant toxin from Gb4 to Gb3 in vitro. We now report that the altered carbohydrate recognition of the mutant toxin (termed GT3) has biological significance, resulting in a unique disease after intravascular injection into pigs as compared with classical VT2e-induced edema disease. The tissue localization of radiolabeled GT3 after intravascular injection was elevated in neural tissues compared with VT2e accumulation, while localization of GT3 to the gastrointestinal tract was relatively reduced. Accordingly, the pathological lesions after challenge with GT3 involved gross edema of the cerebrum, cerebellum, and brain stem, while purified VT2e caused hemorrhage and edema of the cerebellum, and submucosa of the stomach and large intestine. In addition, both radiolabeled toxins bound extensively to tissues not directly involved in the pathology of disease. VT2e, unlike GT3 or VT1, bound extensively to red cells, which have high levels of Gb4. The overall tissue distribution of VT2e was thus found to be influenced by regional blood flow to each organ and not solely by the Gb4 levels of these tissues. Conversely, the distribution of GT3 (and VT1), which cleared more rapidly from the circulation, correlated with respective tissue Gb3 levels rather than blood flow. These studies indicate the primary role of carbohydrate binding specificity in determining systemic pathology, suggest that the red cells act as a toxin carrier in edema disease, and indicate that red cell binding does not protect against the pathology of systemic verotoxemia.

Glycolipid of human pancreatic cancer; the appearance of neolacto-series (type 2 chain) glycolipid and the presence of incompatible blood group antigen in tumor tissues

Glycolipid isolated from normal and cancerous human pancreatic tissues were characterized chemically and immunologically. The major neutral glycolipids in both normal and cancerous tissues were composed of globo-series glycolipids and lacto-series glycolipids. The mole percentage of fucolipids in the total neutral glycolipids of normal tissues was 20–40%, and in general the fucolipids corresponded to blood group glycolipids related to the patient's blood group, however, in cancerous tissues the amount of these fucolipids was decreased. Immunostaining revealed that normal tissues contained only lacto-series (type 1 chain) glycolipids. In contrast, cancerous tissues contained the neolacto-series (type 2 chain) glycolipids as well as the lacto-series glycolipids. Incompatible blood group antigens, A active glycolipids in a blood type O patient and B active glycolipids in a blood type A patient, were also detectable in the neutral glycolipid fractions of the pancreatic cancer tissues.

UDP-galactose:globoside galactosyltransferase in murine kidney.

There are increased levels of stage-specific embryonic antigens-3 and -1 (SSEA-3 and SSEA-1) globo-series glycolipids in male versus female DBA/2 and C57BL/6 kidneys, respectively. To determine what enzymatic steps may be responsible for these differences, the activity and properties of UDP-galactose:globoside galactosyltransferase were studied in male and female mouse kidney microsomes. This enzyme participates in the biosynthesis of galactosylgloboside, SSEA-3 glycolipid; the reaction product was identified by high performance thin-layer chromatography (HPTLC) immunostaining. In C57BL/6 mice, the specific activity of the enzyme, in the presence of CHAPS, was 2-fold greater in the male than that in the female. Optimum pH for the enzyme from both sexes was about 5.6, and Mn2+ was essential for maximal activity. Fifty percent of the male and female enzyme activity was lost after preincubating the microsomes for 1 min at 55 degrees C; thereafter, the enzyme from female microsomes had a slower rate of denaturation. The Km for globoside in presence of sodium cholate for both male and female was 0.035 mM, but it was approximately 2-fold greater for the female in presence of CHAPS. The enzyme in male and female microsomes was differentially activated by CHAPS and cholate. The results suggest the presence of an enzyme modulator in these membranes. In DBA/2 mice, the enzyme activity was about 2-fold greater in males than that in the female. The specific activity of the enzyme in the two strains was of a similar magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)

Different patterns of glycolipid antigens are expressed following differentiation of TERA-2 human embryonal carcinoma cells induced by retinoic acid, hexamethylene bisacetamide (HMBA) or bromodeoxyuridine (BUdR)

Abstract NTERA-2 cl. Dl human embryonal carcinoma (EC) cells were induced to differentiate by either bromodeoxyuridine (BUdR) or hexamethylene bisacetamide (HMBA), and also by retinoic acid. Following exposure to each of these inducers, the globoseries glycolipid antigens stage-specific embryonic antigens -3 and -4 (SSEA-3 and -4) and the glycoprotein antigen TRA-1–60, all characteristic of the human EC cell surface, underwent a marked reduction in expression within about 7 days. At the same time, the lacto series glycolipid antigen SSEA-1, and ganglioseries antigens A2B5 (GT 3) and ME311 (9–0-acetyl GD 3) were induced in BUdR- and retinoic acid-treated cells. However, these antigens did not appear during the first 7–14 days of HMBA-induced differentiation. The observations of cell surface antigen expression were paralleled by analysis of glycolipids isolated from the cells by thin-layer chromatography. This analysis, in which the new monoclonal antibodies VIN-IS-56 and VIN-2PB-22 were included, also revealed expression of gangliosides GD 3 and GD 2 in all differentiated cultures, albeit at much lower levels following HMBA exposure than following retinoic acid or BUdR-exposure. Further, disialylparagloboside was detected in retinoic acid and BUdR-induced, but not HMBA-induced, cultures. Taken with morphological observations, the results suggest that HMBA induces differentiation of NTERA-2 cl.D1 EC cells along a pathway distinct from the pathway(s) induced by retinoic acid and BUdR.

Glycosphingolipids of human myometrium and endometrium and their changes during the menstrual cycle, pregnancy and ageing

The glycolipid composition of human myometrium and endometrium was examined at various stages of maturation and reproduction. The major neutral glycolipids of both myometrium and endometrium were identified by high-performance thin-layer chromatography as globo-series glycolipids, Gb3 and Gb4. The major acidic glycolipids (gangliosides) were identified similarly as GM3 and GD3, with lesser amounts of GM1, GD1a, and GT1b. During pregnancy, GD3 expression declined in both myometrium and endometrium, whereas GM3 expression increased. Reciprocal changes in GM3/GD3 expression were mirrored by appropriate changes in the glycosyltransferases required for their synthesis; alpha 2----3sialyltransferase activity increased approximately 3-fold during pregnancy, while alpha 2----8sialyltransferase activity declined to about 20%. The results focus attention on the glycolipids of uterine tissues, their regulation, and their possible role in reproduction and fertility.

Glycolipid glycosyltransferases in human embryonal carcinoma cells during retinoic acid induced differentiation

Retinoic acid induced differentiation of TERA-2-derived human embryonal carcinoma cells is accompanied by a dramatic reduction of extended globo-series glycolipids, including galactosyl globoside, sialylgalactosyl globoside, and globo-A antigen (each recognized by specific MoAbs). Associated with these glycolipid changes, the activities of two key enzymes, alpha 1----4 galactosyltransferase (for synthesis of globotriaosyl core structure) and beta 1----3 galactosyltransferase (for synthesis of galactosyl globoside), were found to be reduced 3- to 4-fold. The latter enzyme plays a key role in the synthesis of extended globo-series structures, and its characterization has not been reported previously. Therefore, its catalytic activity was studied in detail, including substrate specificity, detergent and phospholipid effects, pH and cation requirements, and apparent Km. During retinoic acid induced differentiation, a series of Lex glycolipid antigens (recognized by anti-SSEA-1 antibody) and their core structures (lacto-series type 2 chains) increase dramatically. In parallel with these changes in glycolipid expression, the activities of two key enzymes, beta 1----3 N-acetylglucosaminyltransferase (for extension of lacto-series type 2 chain) and alpha 1----3 fucosyltransferase (for synthesis of Lex structure), were found to increase by 4- and 2-fold, respectively. Similarly, an increase in the expression of several gangliosides (e.g., GD3 and GT3) during retinoic acid induced differentiation was mirrored by a 4-fold increase in the activity of alpha 2----3 sialyltransferase (for synthesis of ganglio core structure, GM3). The results suggest a coordinate regulation of key glycosyltransferases involved in core structure assembly and terminal chain modification.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of Unique Globo-series Glycolipids in PC 12h Pheochromocytoma Cells

In a previous paper, we reported the presence of a unique globo-series glycolipid as one of the major neutral glycolipid: Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1' Cer, in the subcloned PC 12h pheochromocytoma cells (Ariga, T., Yu, R. K., Scarsdale, J. N., Suzuki, M., Kuroda, Y., Kitagawa, H., and Miyatake, T. (1988) Biochemistry 27, 5335-5340). Recently we found that the subcloned PC 12h cells accumulated other unusual neutral glycolipids. In order to characterize these glycolipids, PC 12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated four minor neutral glycolipids, which were purified by droplet counter-current, Iatrobeads column, and preparative thin-layer chromatographies. These glycolipid structures were determined by fast atom bombardment-mass spectrometry, proton nuclear magnetic resonance spectroscopy, permethylation study, and sequential degradation with various exoglycosidases to be as follows: A, Fuc alpha 1-2Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; B, GalNAc beta 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; C, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; and D, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer. Glycolipids A and B were tentatively characterized in normal rat small intestine (Breimer, M. E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 557-568; Angstrom, J., Breimer, M. E., Falk, K.-E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 682-688). Glycolipids C and D have not been reported in the literature.

Oxidation of glycolipids in liposomes by galactose oxidase.

Small unilamellar phosphatidylcholine vesicles containing globo-series glycolipids were labeled by the galactose oxidase/NaB[3H]4 procedure. The major glycolipid of human red cells, globoside, was the best substrate for galactose oxidase both in vesicles and in tetrahydrofuran-containing buffer. The oxidation rates of membrane-bound ceramide trihexoside and Forssman glycolipid were one-fourth and one-tenth, respectively, of the oxidation rate of globoside. Membrane-bound ceramide dihexoside was not a substrate for galactose oxidase, although it was readily oxidized in tetrahydrofuran-containing buffer. Soluble sialoglycoproteins and membrane-incorporated glycophorin A stimulated the oxidation of globoside-containing vesicles, whereas membrane-bound GD1a ganglioside had no effect on globoside oxidation.

Glycolipid core structure switching from globo- to lacto- and ganglio-series during retinoic acid-induced differentiation of TERA-2-derived human embryonal carcinoma cells

We have analyzed the glycolipid markers of a recently cloned human embryonal carcinoma (EC) cell line, NTERA-2, which differentiates extensively into a variety of somatic cell types when exposed to retinoic acid. These tumor cells provide a model system that can be used to study the ontogeny of glycolipid diversity during human embryonic development. Glycolipid antigens were identified by cell surface immunofluorescence and thin-layer chromatography immunostaining using a comprehensive set of anticarbohydrate monoclonal antibodies. Undifferentiated NTERA-2 cells were found to express predominantly globo-series glycolipids, including Gb 3, Gb 5 (IV 3GalGb 4), globo-ganglioside (IV 3NeuAcα2→3GalGb 4), globo-H (IV 3Fucα1→2GalGb 4), and globo-A (IV 3GalNAcα1→3[Fucα1→2]GalGb 4). When NTERA-2 cells were induced to differentiate by culturing in the presence of 10 −5 M retinoic acid, a remarkable shift of cellular glycolipids from globo-series to lacto- and ganglio-series was observed: Globo-series structures declined, particularly during the period 7–20 days after first exposure to retinoic acid, while lacto-series structures, including fucosylα1→3 type 2 chain (Le x) and sialosyl type 2 chain, and ganglio-series structures, including GM 3, GD 3, 9- O-acetyl-GD 3, GM 2, GD 2, and GT 3, increased. The presence of globo-A and globo-H as the major ABH blood group antigens in undifferentiated NTERA-2 cells suggests that globo-series blood group antigens are embryonic antigens, synthesis of which switches to lacto-series during human development. Two-color immunofluorescence analysis indicated preferential expression of several ganglio- and lacto-series antigens on different subsets of differentiated cells and permitted the relationship of these subsets to the development of neurons in NTERA-2 cultures to be determined. The results suggest that glycosyltransferases, particularly those involved in controlling glycoconjugate core structure assembly, are key enzymes regulated during the differentiation of human EC cells and, by implication, during human embryogenesis.

Bacterial virulence versus host resistance in the urinary tracts of mice.

The relative contributions of host resistance and bacterial virulence were analyzed in a mouse model for ascending urinary tract infection. The congenic mouse strains C3H/HeJ and C3H/HeN were used in parallel. They differ in their reactivity to lipopolysaccharide (LPS) and susceptibility to experimental urinary tract infection. C3H/HeJ cells are susceptible to infection and are nonresponders to LPS (Lpsd Lpsd), whereas C3H/HeN cells respond to LPS and are resistant to infection (Lpsn Lpsn). The Escherichia coli pyelonephritis isolate GR-12, serotype O75K5, expressing adhesins specific for globoseries glycolipids (P fimbriae) and for mannosides (type-1 fimbriae), and its derivatives deficient in these factors were used, either singly or in combination, to establish experimental infections. In C3H/HeN mice, the relative persistence of E. coli was inversely proportional to its phagocytosis in vitro. Loss of the O75 and K5 antigens increased the tendency toward hydrophobic interaction, promoted phagocytosis, and reduced persistence in the kidneys. This was not the case in C3H/HeJ mice, in which O75- and K5- serotypes persisted in the same extent as did the parent strain. The total number of bacteria recovered from the kidneys of C3H/HeJ mice was about 1,000-fold higher than the number recovered from kidneys of C3H/HeN mice 24 h after infection. Previous studies have demonstrated a delayed influx of polymorphonuclear leukocytes into the urinary tracts of C3H/HeJ mice. The results are consistent with the hypothesis that phagocyte activation through LPS is a major defense mechanism against E. coli in the kidney, a property in which C3H/HeJ mice are deficient.

Receptor-specific agglutination tests for detection of bacteria that bind globoseries glycolipids.

Specific binding to the globoseries of glycolipid receptors explains the adherence of uropathogenic Escherichia coli to host cells. The minimal receptor disaccharide Gal alpha 1----4Gal beta [galactose alpha (1----4)galactose beta] is recognized by most attaching clinical isolates. However, wild-type isolates can express adhesins with several different receptor specificities. Bioassays do not permit separate analysis of each receptor specificity, since the target cells contain multiple potentially receptor-active molecules. In this study, bacterial adhesins were analyzed by using receptors immobilized into latex beads in one of two ways. In one way, di- and trisaccharides were covalently linked via a spacer arm to latex beads coupled with bovine serum albumin. In the other way, receptor-active glycolipids were coated onto the bovine serum albumin-latex beads. The latex beads were subsequently used for agglutination by using type strains with known receptor specificity. The composition was optimized regarding receptor structure and size of latex beads. Gal alpha 1----4Gal beta was as active as the trisaccharide derivative Gal alpha 1----4Gal beta 1----3glucose or Gal alpha 1----4Gal beta 1----3glucosamine. Among the natural glycolipids tested, globotetraosylceramide was the most active. Subsequently, the sensitivity and specificity of the Gal alpha 1----4Gal beta-latex and globotetraosylceramide-latex reagents were compared for 733 E. coli urinary isolates. Hemagglutination of human erythrocytes was used as the positive standard. No significant difference in the specificity or sensitivity of the latex reagents was found; the sensitivity ranged from 86%, when isolates agglutinating human erythrocytes of blood groups P1 and p were included, to 93%, when those isolates agglutinating erythrocytes of blood group p were excluded. These reagents provide tools for bacterial identification in patients with urinary tract infection.

Differentiation of TERA-2 human embryonal carcinoma cells into neurons and HCMV permissive cells: Induction by agents other than retinoic acid

Retinoic acid induces the differentiation of NTERA-2 cl. D1 human embryonal carcinoma (EC) cells into neurons, cells permissive for the replication of human cytomegalovirus (HCMV), and other cell types that cannot as yet be classified but are distinguishable from the stem cells. We tested several additional agents for their ability to induce the differentiation of these EC cells. No differentiation was induced by butyrate, cyclic AMP, cytosine arabinoside, the tumor promoter 12-0-tetradecanoylphorbol 13-acetate (TPA), or the chemotherapeutic agent cis-diaminedichloroplatinum, although morphological changes were detected at the highest concentrations of these agents that permitted cell survival. However, retinal, retinol, 5-bromouracil 2′deoxyribose (BUdR), 5-iodouracil 2′deoxyribose (IUdR), hexamethylene bisacetamide (HMBA), dimethylacetamide (DMA), and dimethylsulfoxide (DMSO) all induced some neuronal differentiation, but to a lesser extent than retinoic acid. Also, BUdR, IUdR, HMBA, and DMA induced the appearance of many cells permissive for the replication of HCMV. Differentiation was, in all cases, accompanied by the loss of SSEA-3, a globoseries glycolipid antigen characteristically expressed by human EC cells. However, another glycolipid antigen, A2B5, which appears in 60%–80% of differentiated cells 7 days following retinoic acid induction, was detected in less than 20% of the cells induced by the other agents studied. This implies that the HCMV-permissive cells induced by retinoic acid are not identical to those induced by BUdR, IUdR, and DMA.

Influence of blood group on the availability of receptors for attachment of uropathogenic Escherichia coli

Escherichia coli strains with defined receptor specificity were used as probes to analyze the individual variation in host cell receptors with respect to blood groups. The adhesins were initially characterized as mannose sensitive (MS), mannose resistant (MR), or nonagglutinating (-). The receptor specificity of the strains with MR adhesins was defined by agglutination of synthetic Gal alpha 1----4Gal beta covalently linked via a spacer arm, (CH2)2S(CH2)2CO approximately H-bovine serum albumin (BSA) to BSA-latex beads as specific for the globoseries glycolipid receptors (MR:GS). Strains with MR adhesins not reacting with Gal alpha 1----4Gal beta-BSA-latex were designated MR:nonGS. The attachment and hemagglutination of the MR:GS strains was strictly dependent on Gal alpha 1----4Gal beta-containing receptors, as shown by the absence of binding to cells from individuals of blood group P lacking these structures. Previous reports showed differences in the composition of globoseries glycolipids between erythrocytes from individuals of P1 and P2. No significant difference was found, However, in the mean adhesion to P1 and P2 epithelial cells or in the agglutination titer for P1 and P2 erythrocytes. The MR:GS receptors were equally distributed on squamous and transitional epithelial cells. In contrast, the distribution of MR:nonGS receptors was skewed. Attachment occurred mostly to squamous epithelial cells. The attachment of strains with MR:nonGS adhesins was independent of the P blood group of the cell donor. The binding ability of MR:GS and MR:nonGS adhesins appeared independent and additive. The attachment was not influenced by the ABH blood group. However, increased binding to epithelial cells from nonsecretors occurred regardless of the P blood group, suggesting a shielding of receptors by products controlled by the secretor genes. These results illustrate how individual variation in cell surface components with and without receptor activity determine the interaction of a ligand with a known receptor.

Complete structural analysis of globoseries glycolipids by two-dimensional nuclear magnetic resonance.

Combined two-dimensional proton nuclear magnetic resonance allowed the determination of complete oligosaccharide structures of glycolipids belonging to the globo series, without any other analytical methods. Although a chemical modification by peracetylation was required for the above purpose, the derivatization permitted facile assignment of the pyranose ring proton resonances of the oligosaccharide moiety. Two-dimensional chemical-shift-correlated spectroscopy of the acetylated glycolipid enabled us to elucidate the glycosidic positions from the chemical shifts of the protons at the substituted sites. The monosaccharide species were also identified from the characteristic splitting patterns of the methine protons on individual pyranose rings. The sequence of the monosaccharides was inferred from the interresidue connectivity across glycosidic linkages shown by two-dimensional nuclear Overhauser effect spectroscopy, which also gave intraresidue interaction on the pyranose rings. The linkage sites of long oligosaccharide chains having more than five monosaccharides, such as globopentaosylceramide, were analyzed by two-dimensional J-relayed coherence transfer, which yielded 1,3 interactions along with 1,2 interactions.

Structure and expression of differentiation antigens on functional subclasses of primary sensory neurons.

Subpopulations of dorsal root ganglion neurons can be distinguished on the basis of their peripheral receptive properties, spinal terminal arbors and neuropeptide content. We have used monoclonal antibodies (MAbs) to define antigenic determinants on functional populations of DRG neurons projecting to the superficial dorsal horn of the spinal cord. Three MAbs recognize defined carbohydrate epitopes associated with lacto- and globo-series glycolipids that constitute the stage-specific embryonic antigens (SSEAs) 1, 3 and 4. SSEA-3 and SSEA-4 are present in the cytoplasm of about 10% of DRG neurons in adult rat. These neurons are distinct from those that contain substance P, somatostatin or the fluoride-resistant acid phosphatase enzyme, FRAP. SSEA-1 is present in a small percentage of DRG neurons. SSEAs are present on the surface of DRG neurons maintained in dissociated cell culture: 6% are SSEA-1+, 7% are SSEA-3+ and 10-15% are SSEA-4+. MAbs LD2, KH10, TC6 and TD10 identify epitopes expressed coincidently in 25% of small DRG neurons that project to lamina II of the dorsal horn. All somatostatin- but less than 1% of substance P-immunoreactive DRG neurons express these antigens. MAb LA4 labels a distinct population of small DRG neurons that also projects to lamina II. There is extensive overlap between LA4+ neurons and those that contain FRAP. Antigens recognized by these MAbs are expressed on the surface of 10-20% of DRG neurons in culture. Preliminary biochemical studies suggest that these antigens may be glycolipids. Molecules bearing carbohydrate differentiation antigens may be involved in the development and specification of sensory connections in the dorsal horn of the spinal cord.