Differential expression of surface glycoconjugates on Entamoeba histolytica and Entamoeba dispar.

The human large intestine can harbor two morphologically similar amoebae; the invasive Entamoeba histolytica and the non-invasive Entamoeba dispar. Whereas E. histolytica can produce intestinal and extra-intestinal lesions, E. dispar is present in non-symptomatic carriers. Although biochemical, genetic and proteomic studies have identified clear differences between these Entamoebae, it has become clear that several molecules, once assumed to be involved in tissue destruction, exist in both the virulent and the avirulent species. As surface molecules may play a role in invasion and could therefore determine which amoebae are invasive, we analyzed the glycoconjugate composition of E. histolytica and E. dispar using lectins. There was a significant difference between E. histolytica and E. dispar in the expression of glycoconjugates containing d-mannose and N-acetyl-alpha-D-galactosamine residues, but not between virulent and avirulent strains of E. histolytica. N-glycoconjugates with terminal alpha (1-3)-linked mannose residues participate in the adhesion and subsequent cytotoxicity of E. histolytica to cultured hamster hepatocytes. One of them probably is the Gal/GalNAc lectin.

Histochemical localization of N-acetyl-galactosamine in the midgut Lutzomyia longipalpis (Diptera: Psychodidae).

The binding of lectins to the midgut of the female sand fly Lutzomyia longipalpis (Lutz & Neiva) was investigated using lectin-gold conjugates. Midguts from laboratory-reared flies provided fructose solution and/or blood fed on hamster were dissected at 6, 24, and 48 h and at 5 and 7 d after feeding. Before examination by transmission electron microscopy, each midgut was sectioned, incubated with lectins from four sources (Canavalia ensiformis [ConA], Helix pomata agglutinin [HPA], peanut agglutinin [PNA], and wheat germ agglutinin [WGA] ), then conjugated with colloidal gold. Only HPA, which is specific for N-acetyl-galactosamine (GalNAc), bound to the midgut. Binding sites were cytoplasmic secretory granules and microvilli throughout the length of the midgut epithelium. Binding occurred in sand flies fed fructose as well as in flies receiving a blood meal. The presence of GalNAc on the midgut microvilli of sand flies before, during, and after blood feeding indicates this amino-sugar is not altered by digestion. As a structural component, GalNAc may represent a terminal on a receptor molecule. The failure of the sand fly peritrophic matrix to bind WGA by N-acetylglucosamine may be caused by the complex composition of the membrane, which renders N-glycan inaccessible to the lectin-gold conjugate.

Embryos of the sea urchin Strongylocentrotus purpuratus synthesize a dermatan sulfate enriched in 4-O- and 6-O-disulfated galactosamine units.

Unfertilized eggs of the sea urchin Strongylocentrotus purpuratus are surrounded by a gelatinous layer rich in sulfated fucan. Shortly after fertilization this polysaccharide disappears, but 24 h later the embryos synthesize high amounts of dermatan sulfate concomitantly with the mesenchyme blastula-early gastrula stage when the larval gut is forming. This glycosaminoglycan has the same backbone structure [4-alpha-L-IdoA-1-->3-beta-D-GalNAc-1](n) as the mammalian counterpart but possesses a different sulfation pattern. It has a high content of 4-O- and 6-O-disulfated galactosamine units. In addition, chains of this dermatan sulfate are considerable longer than those of vertebrate tissues. Adult sea urchin tissues contain high concentrations of sulfated polysaccharides, but dermatan sulfate is restricted to the adult body wall where it accounts for approximately 20% of the total sulfated polysaccharides. In addition, sulfation at the 4-O-position decreases markedly in the dermatan sulfate from adult sea urchin when compared with the glycan from larvae. Overall, these results demonstrate the occurrence of dermatan sulfates with unique sulfation patterns in this marine invertebrate. The physiological implication of these oversulfated dermatan sulfates is unclear. One hypothesis is that interactions between components of the extracellular matrix in marine invertebrates occur at higher salt concentrations than in vertebrates and therefore require glycosaminoglycans with increased charge density.

Histochemical detection of sugar residues in lizard teeth (Liolaemus gravenhorsti): a lectin-binding study.

The structural diversity of the many oligosaccharide chains of surface glycoconjugates renders them likely candidates for modulators of cell-interactions, cellular movements, differentiation, and cellular recognition. A selection of different lectins was used to investigate the appearance of cellular distribution and changes in sugar residues during tooth development in the polyphyodont lizard, Liolaemus gravenhorsti. Lectins from three groups were used: (1) N-acetylgalactosamine specificity: BS-1, PNA, RCA-120; (2) N-acetylglucosamine specificity: ECA; and (3) fucose specificity: UEA 1 and LTA.. Digital images were processed using Scion Image. Grayscale graphics in each image were obtained. The lectins used showed a strong, wide distribution of the L-fucose and N-acetylgalactosamine at the cell surface and in the cytoplasm of multinucleate odontoclast cell, while mononuclear odontoclast cells showed no binding, suggesting some roles that the residues sugar might play in the resorption of dentine or with multinucleation of odontoclast after the attachment to the dentine surface in this polyphyodont species. Further studies must be planned to determine the specific identities of these glycoconjugates,and to elucidate the roles played by these sugar residues in the complex processes related to odontogenesis in polyphyodont species.

Histochemical detection of sugar residues in lizard teeth (Liolaemus gravenhorsti): a lectin-binding study

The structural diversity of the many oligosaccharide chains of surface glycoconjugates renders them likely candidates for modulators of cell-interactions, cellular movements, differentiation, and cellular recognition. A selection of different lectins was used to investigate the appearance of cellular distribution and changes in sugar residues during tooth development in the polyphyodont lizard, Liolaemus gravenhorsti. Lectins from three groups were used: (1) N-acetylgalactosamine specificity: BS-1, PNA, RCA-120; (2) N-acetylglucosamine specificity: ECA; and (3) fucose specificity: UEA 1 and LTA.. Digital images were processed using Scion Image. Grayscale graphics in each image were obtained. The lectins used showed a strong, wide distribution of the L-fucose and N-acetylgalactosamine at the cell surface and in the cytoplasm of multinucleate odontoclast cell, while mononuclear odontoclast cells showed no binding, suggesting some roles that the residues sugar might play in the resorption of dentine or with multinucleation of odontoclast after the attachment to the dentine surface in this polyphyodont species. Further studies must be planned to determine the specific identities of these glycoconjugates,and to elucidate the roles played by these sugar residues in the complex processes related to odontogenesis in polyphyodont species.

Microscopic and histochemical study of odontoclasts in physiologic resorption of teeth of the polyphyodont lizard, Liolaemus gravenhorsti.

Using tartrate-resistant acid phosphatase (TRAP), we examined the cytodifferentiation of odontoclast cells in resorbing areas of dental tissues during the replacement of teeth in a polyphyodont lizard, Liolaemus gravenhorsti. We also report, by means of Lectin-HRP histochemistry, the distribution pattern of some specific sugar residues of TRAPase-positive cells. For detection of TRAPase activity, the azo dye-coupling technique was used. Lectin binding sites were demonstrated by means of specific HRP-lectins. The process of tooth resorption was divided into four stages: 1) preresorption-the wall of the dental pulp is covered with an odontoblast layer, and no TRAP-positive cells are in the dental pulp; 2) early resorption-TRAP-positive multinucleate odontoclasts are present on the dental wall, but the rest of the pulp surface is still covered with an odontoblast layer; 3) later resorption-the entire surface of the pulp chamber is lined with multinucleate odontoclasts; and 4) final resorption-the tooth has been totally resorbed. Odontoclasts are usually detached from the resorbed surface, and show signs of degeneration. Of the six lectins used, PNA, ECA, and UEA-1 bind to multinucleated but not mononuclear cells. All the remaining lectins, BS-1, RCA(120), and LTA showed no binding to any cells of the teeth. The significance of saccharidic moieties such as acetyl-galactosamine, acetyl-glucosamine, and fucose sugar residues is difficult to ascertain. Perhaps these oligosaccharides might be borne on molecules associated with odontoclastic resorption or associated with multinucleation of odontoclasts after attachment to the dentine surface.

Cytochemical localization of carbohydrate residues in microfilariae of Wuchereria bancrofti and Brugia malayi.

We investigated the localization of carbohydrate residues on the surface structures of microfilariae of Wuchereria bancrofti and Brugia malayi, using a panel of 10 different gold-labeled lectins and chitinase. The sheath, a structure that encloses the microfilariae, is not a homogeneous structure, presenting two clearly distinct layers. The outer layer is more electron dense and was not labeled with the lectins. The inner layer is less dense and was intensely labeled with lectins, especially those that recognize D-galactose and N-acetyl-D-galactosamine. Small differences were observed in the lectin labeling pattern of microfilariae of W. bancrofti and B. malayi. D-galactose and fucose were observed in the cuticle of both species. Chitin, as revealed with gold-labeled chitinase, was observed in the cuticle of microfilariae of W. bancrofti but not in B. malayi.

Two types of bacteria adherent to bovine respiratory tract ciliated epithelium.

Two hundred sixty tracheas were obtained from a Philadelphia abattoir under permit from the Department of Agriculture; the tracheas were excised from predominantly Holstein calves of both sexes that weighed approximately 250 kg. Tracheas were transported in normal saline to the laboratory at Thomas Jefferson University, Philadelphia, Pennsylvania. Evidence of bacteria adherent to the tracheal epithelium was found in specimens from 20/24 of these tracheas. The epithelium from each of five tracheas was placed in glutaraldehyde fixative for transmission electron microscopic examination. Epithelium from each of 12 other tracheas was placed in formaldehyde fixative for light microscopic examination. Microscopically, 13 of these 17 bovine tracheal epithelia were observed to contain bacteria located longitudinally parallel to and between cilia and microvilli of ciliated cells. Preparations of ciliary axonemes isolated from the epithelium of seven additional bovine tracheas also contained these bacteria in sections viewed by a transmission electron microscope. These bacteria had two different ultrastructural morphologies: filamentous with a trilaminar-structured cell wall and short with a thick, homogeneously stained cell wall beneath a regularly arrayed surface layer. The short bacillus had surface carbohydrates, including mannose, galactose, and N-acetylgalactosamine, identified by lectin binding. The filamentous bacillus was apparently externally deficient in these carbohydrates. Immunogold staining revealed that the filamentous bacillus was antigenically related to cilia-associated respiratory (CAR) bacillus, which has been identified in rabbit and rodent species. Significantly decreased numbers of cilia were obtained from tracheal epithelium heavily colonized by the filamentous bacilli, suggesting a pathologic change in ciliated cells.