Histochemical demonstration of sulphate groups by means of diaminobenzidine

Synopsis A method is presented for the histochemical demonstration of native and induced sulphate groups. This method is similar in principle to the Bracco-Curti technique, and is based on the formation, at an acid pH, of insoluble salts formed between sulphate groups and diaminobenzidine. The salt complex is subsequently oxidized by means of osmium tetroxide and, in addition to the brown oxidized diaminobenzidine, osmium black is precipitated.This new method presents some advantages over the Bracco-Curti technique, and it may be useful in the development of a technique for the electron microscopical localization of sulphated mucopolysaccharides.On leave from the Istituto di Anatomia ed Istologia Patologica of the University of Turin and supported by a Fellowship from the Accademia Nazionale dei Lincei.     

Regulated expression of keratan sulphate and peanut agglutinin binding sites during organogenesis in the developing chick

 Keratan sulphate proteoglycans are potentially important during development and are possible binding molecules for the lectin, peanut agglutinin, a marker for areas that are inhibitory for axonal growth in early embryos. The present study describes the spatiotemporal distributions of keratan sulphate epitopes and peanut agglutinin binding sites during organogenesis in the developing chick from E5 to hatching. The widespread distributions of these molecules did not often overlap but clearly delimited different carbohydrate compartments demonstrating that peanut agglutinin does not necessarily bind to keratan sulphate proteoglycans. These markers were mostly extracellular but keratan sulphate, in particular, was found within certain specific cells in cartilage, gonad, heart and pancreas, at certain ages. The presence of keratan sulphate in putative germ cells during their migrations and in the gonads may be of particular importance. Their distributions generally evoke modulation of adhesion allowing cell migrations or morphogenetic movements related to epitheliomesenchymal interactions, but may also suggest an involvement in axonal guidance in skin, cartilage, gut and possibly heart. Furthermore, in the kidney, peanut agglutinin binding sites seem to be related to the functional differentiation of the nephrons. Accepted: 23 February 1998     

Specific binding of peanut agglutinin and soybean agglutinin to chondroitinase ABC-digested cartilage proteoglycans: Histochemical, ultrastructural cytochemical, and biochemical characterization

Summary The binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) to cartilage proteoglycans was investigated by histochemical, ultrastructural cytochemical, and biochemical methods. Following aldehyde fixation, specimens of rat epiphyseal cartilage were examined by horseradish peroxidase-labelled lectin cytochemistry with and without prior digestion in chondroitinase ABC. At the light microscope level neither PNA nor SBA exhibited any affinity for cartilage matrix, but became strongly bound following chondroitinase treatment. Similarly, at the ultrastructural level, extracellular matrix granules, presumed to be proteoglycan monomer(s), lacked PNA affinity in undigested specimens, and stained very weakly with SBA. Both PNA and SBA weakly to moderately stained thetrans cisternae of the Golgi-flattened cisternae in chondrocytes. The chondrocyte plasmalemma lacked PNA staining, but reacted weakly with SBA. Following chondroitinase digestion, PNA and SBA stained matrix granules, and the cell surface of chondrocytes intensely, whereas the Golgitrans cisternae, the Golgi-derived vacuoles, and multivesicular bodies demonstrated weak to moderate reactivity. Proteoglycan aggregates purified from rat chondrosarcoma and bovine nasal cartilage bound PNA and SBA avidly after digestion with chondroitinase. Undigested proteoglycans lacked affinity for PNA and reacted very weakly with SBA. These results indicate that both PNA and SBA specifically react with chondroitinase-modified oligosaccharide(s) bound to core proteins of cartilage proteoglycans. This provided a specific histochemical and ultrastructural cytochemical procedure for localizing chondroitin sulphate-containing proteoglycans.     

Histochemical demonstration of prolactin binding sites

We have developed a new probe for histochemical demonstration of prolactin binding sites. Ovine prolactin (oPRL) was conjugated with the N-hydroxy-succinimide ester derivative of the fluorochrome 7-amino-4-methylcoumarin-3-acetic acid. Under mild reaction conditions the ester derivative reacted with available NH2 groups of the prolactin molecule to form stable bonds. The coupling reaction yielded products that co-migrated with oPRL but had slightly decreased isoelectric points. The receptor binding and bioactivity of the flurochrome-hormone derivatives were decreased essentially proportional to the extent of conjugation. The derivatives were further tested for their ability to label PRL binding sites, using frozen sections of mammary gland and brain tissue of lactating rats. The results presented in this report describe the validation of this probe with regard to labeling of PRL binding sites at the light microscopic level in known target organs (mammary gland and choroid plexus). In addition, this fluorescent probe was used to demonstrate the presence of PRL binding sites at a novel site, the ependymal lining of the third ventricle.     

Differential binding of peanut agglutinin with lipopolysaccharide of homologous and heterologous Rhizobium

Abstract To establish the crucial role of lipopolysaccharide in the initial recognition event of symbiotic peanut-Rhizobium system the ability of various surface polysaccharides isolated from Bradyrhizobium arachis to inhibit the precipitin reaction between peanut agglutinin and asialoganglioside: deoxycholate (1:1) micelles was estimated. It was compared with that of nonsymbiotic systems e.g. Bradyrhizobium japonicum, Bradyrhizobium ciceris and Escherichia coli . Peanut agglutinin was found to interact more strongly with the lipopolysaccharide of Bradyrhizobium arachis than the exopolysaccharide or capsular polysaccharide. The inhibitory capacity of lipopolysaccharides from homologous and heterologous Bradyrhizobium as measured in terms of the concentration necessary for 50 percent inhibition of precipitin reaction were 1428, 500, 410, and 277 times less than that of lactose for Bradyrhizobium arachis, B. japonicum, B. ciceris and Escherichia coli , respectively. These results support that host lectin peanut agglutinin can recognize homologous Bradyrhizobium lipopolysaccharide by virtue of its binding specificity of higher magnitude.     

Ultrastructural visualization of selective peanut agglutinin binding sites in rat osteoclasts

We investigated the distribution of concanavalin A (ConA)-reactive alpha-D-mannosyl and alpha-D-glucosyl groups and peanut agglutinin (PNA)-reactive beta-D-galactose-(1----3)-N-acetyl-D-galactosamine residues on the surface of osteoclasts with pre-embedment ultrastructural lectin cytochemistry after aldehyde fixation of the metaphyses of the rat tibiae. By routine morphology, the plasma membrane of the ruffled border of the osteoclast was distinguished from the rest of the cell membrane, with the exception of the membrane of coated pits, by its characteristic thick coat at its cytoplasmic surface. Cytochemistry, using ConA in combination with horseradish peroxidase (ConA-HRP) and PNA conjugated to HRP, showed that binding of ConA was distributed over the entire cell surface of osteoclasts. In contrast, intense binding of PNA was limited to the membranes of the ruffled border and coated pits, whereas the remainder of the cell membrane stained weakly or not at all. These results demonstrate that preferential PNA binding sites of the cell surface correspond to coated membranes associated with osteoclastic endocytosis.     

Cell surface binding sites for peanut agglutinin in the differentiating eye disc of Drosophila

The distribution of peanut agglutinin (PNA) binding sites in imaginal discs is described using fluorescence and electron microscopy. PNA binds preferentially to the photoreceptor cell precursors in eye discs resulting in a rectilinear array of fluorescent spots that reflects that lattice-like arrangement of the presumptive ommatidia. The lectin binds to the apical surface of fixed disc cells and is taken up in presumed endocytotic vesicles in living discs. Photoreceptor precursors can be visualized with fluorescein isothyocyanate-PNA from the time they first form preclusters in the morphogenetic furrow and this technique is used to demonstrate a temperature-sensitive defect in precluster formation in the mutant shibire. PNA is localized along the sides of microvilli of disc cells, in general. The preferential binding of PNA to photoreceptor precursors is related in part to the high density of apical microvilli on these cells.     

Mutational analysis at Asn-41 in peanut agglutinin. A residue critical for the binding of the tumor-associated Thomsen-Friedenreich antigen.

Peanut agglutinin is a clinically important lectin due to its application in the screening of mature and immature thymocytes as well as in the detection of cancerous malignancies. The basis for these applications is the remarkably strong affinity of the lectin for the tumor-associated Thomsen-Friedenreich antigen (T-antigen) and more so due to its ability to distinguish T-antigen from its cryptic forms. The crystal structure of the complex of peanut agglutinin with T-antigen reveals the basis of this specificity. Among the contacts involved in providing this specificity toward T-antigen is the water-mediated interaction between the side chain of Asn-41 and the carbonyl oxygen of the acetamido group of the second hexopyranose ring of the sugar molecule. Site-directed mutational changes were introduced at this residue with the objective of probing the role of this residue in T-antigen binding and possibly engineering an altered species with increased specificity for T-antigen. Of the three mutants tested, i.e. N41A, N41D, and N41Q, the last one shows improved potency for recognition of T-antigen. The affinities of the mutants can be readily explained on the basis of the crystal structure of the complex and simple modeling. In particular, the change of asparagine to glutamine could lead to a direct interaction of the side chain with the sugar while at the same time retaining the water bridge. This study strengthens the theory that in lectins the nonprimary contacts generally made through water bridges are involved in imparting exquisite specificity.     

Histochemical demonstration of unsaturated hydrophilic lipids with palladium chloride.

Compounds in which olefinic linkages are accessible to aqueous reagents reduce the chloropalladite ion [PdCl4]2-, to metallic palladium. This reaction is used in a histochemical method whereby hydrophilic unsaturated lipids are stained dark brown or black. The specificity of the new method has been confirmed by means of solvent-extraction and chemical blocking procedures and by comparison with other histochemical techniques. Yellow staining of collagen, keratin and cytoplasm is probably due to attachment of the chloropalladite anion to proteins. The yellow background can be largely decolorized by treating the sections with aqueous pyridine, which forms colorless complexes with divalent palladium. A standard technique for staining with palladium is presented and the method is discussed in relation to other histochemical procedures that demonstrate unsaturated lipids.     

Characterization of embryonic liver suppressor cells by peanut agglutinin.

Liver cells of 19-day-old mouse embryos were separated by peanut agglutinin (PNA) into two fractions. The fraction agglutinated with the PNA was found to be enriched for cells capable of suppressing the MLC reaction and the response to the mitogens Con A, PHA, and LPS. The fraction not agglutinated by PNA was significantly less suppressive. The response to DxS was not suppressed by any of these fractions. On the other hand, the response to LPS and DxS, but not to Con A or PHA, was expressed by the nonagglutinated fraction. It is thus inferred that the suppressor cells in the embryonic liver are separable from the potentially reactive cells.     

The surface glycoproteins of human skin fibroblasts detected after electrophoresis by the binding of peanut (Arachis hypogaea) agglutinin and Ricinus communis (castor-bean) agglutinin I.

A new methodology was developed to study the cell-surface glycoproteins of cultured human skin fibroblasts. This was based on the binding of a variety of biotinyl-lectins to nitrocellulose electrophoretic transfers of total fibroblast lysates after separation in sodium dodecyl sulphate/polyacrylamide gels, followed by reaction with avidin-biotinyl-peroxidase complexes and detection with 3,3'-diaminobenzidine. The technique proved to be very sensitive and a large number of glycoproteins were detected by binding of concanavalin A and wheat-germ agglutinin. Binding of peanut agglutinin and to a lesser extent of Ricinus communis agglutinin I were found to be dependent on prior removal of sialic acid residues from the glycoproteins. Since by treatment of intact viable cells with neuraminidase only external sialic acid residues were removed, peanut agglutinin and Ricinus communis agglutinin I could thus be utilized for selective detection of cell-surface glycoproteins. Also, because peanut agglutinin was known to bind preferentially to oligosaccharides of the O-glycosidic type, and Ricinus communis agglutinin I to those of the N-glycosidic type, the two lectins were complementary in displaying the surface glycoproteins and in providing information about their oligosaccharide composition.     

In vitro effect of thymosin alpha 1 on the expression of peanut agglutinin binding by murine thymocytes

Thymosin alpha 1 induces the loss of PNA binding ability by subpopulation of thymic cells. This loss is probably due to an endocytic process. Nevertheless this disappearance is not a permanent one, suggesting a recycling of the PNA binding molecule. The cells that modulate their PNA binding sites after exposure to Thymosin alpha 1 are a small proportion of the total PNA+ thymocytes, indicating that not all thymocytes are susceptible to the thymic hormone Thymosin alpha 1. Conversely the exposure of thymocytes to Thymosin alpha 1 induces the disappearance of the binding sites for this ligand without further recycling, behavior expected for the receptor of a regulatory ligand. These results also indicate that the Thymosin alpha 1 and the PNA binding sites are on different molecules on the surface of the PNA+ thymocytes.     

Continuous titration of difference absorption upon binding of 4-methylumbelliferyl glycosides to concanavalin A and peanut agglutinin

A continuous titration of absorption differences is described. Equal volumes of the titration fluid are dispensed from two micrometer-driven Hamilton gas-tight syringes into two 1 × 1 × 4.5-cm cuvettes. These are placed in the reference and sample beam. Each cuvette stopper is equipped with a capillary inlet connected to a syringe and with a minimotor for continuous stirring. Details of the stirring device are given. The delivered volumes of titration fluid are sufficiently reproducible to allow titration of absorption differences as a function of chromophore concentration. The usefulness of this approach is tested with the binding of 4-methylumbelliferyl α-d-mannopyranoside and concanavalin A as a well-characterized system. It is applied to the binding of similarly labeled anti-t disaccharide with the lectin from peanuts. With both lectins, the change in molecular extinction coefficient of the ligand and the association constant, valid for the entire protein saturation range, were obtained. The results are identical to those from other methods, including equilibrium dialysis.