Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Differential Binding to Glycotopes Among the Layers of Three Mammalian Retinal Neurons by Man-Containing N-linked Glycan, Tα (Galβ1–3GalNAcα1-), Tn (GalNAcα1-Ser/Thr) and Iβ/IIβ (Galβ1–3/4GlcNAcβ-) Reactive Lectins

Carbohydrate structures between retinal neurons and retinal pigment epithelium (RPE) play an important role in maintaining the integrity of retinal adhesion to underlying RPE, and in retinal detachment pathogenesis. Since relevant knowledge is still in the primary stage, glycotopes on the adult retina of mongrel canines (dog), micropigs and Sprague-Dawley rats were examined by lectino-histochemistry, using a panel of 16 different lectins. Paraffin sections of eyes were stained with biotinylated lectins, and visualized by streptavidin-peroxidase and diaminobenzidine staining. Mapping the affinity profiles, it is concluded that: (i) all sections of the retina reacted well with Morniga M, suggesting that N-linked glycans are present in all layers of the retina; (ii) no detectable human blood group ABH active glycotopes were found among retinal layers; (iii) outer and inner segments contained glycoconjugates rich in ligands reacting with T _α (Galβ1–3GalNAcα1-Ser/Thr) and Tn (GalNAcα1-Ser/Thr) specific lectins; (iv) cone cells of retina specifically bound peanut agglutinin (PNA), which recognizes T _α residues and could be used as a specific marker for these photoreceptors; (v) the retinas of rat, dog and pig, had a similar binding profile but with different intensity; (vi) each retinal layer had its own binding characteristic. This information may provide useful background knowledge for normal retinal physiology and miscellaneous retinal diseases, including retinal detachment (RD) and age-related macular degeneration (ARMD).     

Quantitative estimation of the surface carbohydrates on the infection structures of rust fungi with enzymes and lectins

Lectins of Triticum vulgaris (WGA), Concanavalia ensiformis (ConA), Phaseolus vulgaris (PHA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Griffonia simplicifolia (GSA II) and the enzymes endo-(13)--D-glucanase, exo-(13)--D-glucanase and laminarinase were tested for binding to the infection structures of Puccinia coronata and Uromyces appendiculatus. The enzymes and lectins were labeled with fluorescein and the fluorescence was measured with a microscope photometer. GSA II and ConA bound to all parts of the two rust fungi to a certain extent. The germ tubes of P. coronata bound at least two times more WGA than did the germ tubes of U. appendiculatus. The appressoria of both rust fungi additionally bound exo-(13)--glucanase, endo-(13)--glucanase and laminarinase. The substomatal vesicle and the infection hypha of both rust fungi mainly bound the glucanases. Furthermore, the substomatal vesicle of U. appendiculatus bound PHA. No obvious binding with LTA, RCA I and PNA was observed. Binding generally could be inhibited by appropriate haptens. Binding to uredospores generally appeared unspecific. The results indicate that the germ tubes have chitin on their outer surfaces, the appressoria chitin and glucans and the substomatal vesicles and infection hyphae mainly glucans. Compared to P. coronata, U. appendiculatus has more terminal linked glucose residues or the glucan has more (13)--linkages. Also, U. appendiculatus has N-acetylgalactosamine or a similar sugar on the surface of the substomatal vesicle.Abbreviations ConA Concanavalia ensiformis agglutinin - FITC fluorescein isothiocyanate - GSA II Griffonia simplicifolic agglutimin II - LTA Lotus tetragonolobus agglutinin - PBS phosphate buffered saline - PNA Peanut agglutinin - RCA I Ricinus communis agglutinin I - PHA Phaseolus vulgaris agglutinin - WGA Wheat germ agglutinin     

Molecular imaging of c-Met tyrosine kinase activity

Fluorescent flow cytometry has become the method of choice for interrogation of bacterial populations at the single-cell level. However, limitations of this technique include issues of dynamic range, spectral overlap, photobleaching, and overall low signal intensity due to the small size of bacteria. The recent development of mass cytometry allows single-cell analysis with the resolution of inductively coupled plasma mass spectrometry, facilitating multiparametric analysis. Using a combination of a metal-based membrane stain and lectins conjugated to lanthanide-chelating polymers, we demonstrate that individual Escherichia coli cells can be differentiated based on their cell surface polysaccharides using mass cytometry. The model E. coli system involves evaluation of three different surface polysaccharides using element-tagged concanavalin A and wheat germ agglutinin lectins. Finally, this technique enabled experiments designed to follow the export of O-antigen substituted lipopolysaccharide in a conditional mutant. These studies revealed that the culture responds as a uniform population and that lipopolysaccharide export is approximately 10 times faster than the logarithmic bacterial doubling time.     

Lectinochemical studies on the affinity of Anguilla anguilla agglutinin for mammalian glycotopes

Anguilla anguilla agglutinin (AAA) is a fucose-specific lectin found in the serum of the fresh water eel. It is suggested to be associated with innate immunity by recognizing disease-associated cell surface glycans, and has been widely used as a reagent in hematology and glycobiology. In order to gain a better understanding of AAA for further applications, it is necessary to elucidate its binding profile with mammalian glycotopes. We, therefore, analyzed the detailed carbohydrate specificity of AAA by enzyme-linked lectinosorbent assay (ELLSA) with our extended glycan/ligand collection and lectin-glycan inhibition assay. Among the glycans tested, AAA reacted well with nearly all human blood group Ah (GalNAcalpha1-->3[LFucalpha1-->2]Gal), Bh (Galalpha1-->3[LFucalpha1-->2]Gal), H LFucalpha1-->2Gal) and Leb (Fucalpha1-->2Galbeta1-->3[Fucalpha1-->4]GlcNAc) active glycoproteins (gps), but not with blood group Lea (Galbeta1-->3[Fucalpha1-->4]GlcNAc) substances, suggesting that residues and optimal density of alpha1-2 linked LFuc to Gal at the non-reducing end of glycoprotein ligands are essential for lectin-carbohydrate interactions. Blood group precursors, Galbeta1-3GalNAc (T), GalNAcalpha1-Ser/Thr (Tn) containing glycoproteins and N-linked plasma gps, gave only negligible affinity. Among the mammalian glycotopes tested, Ah, Bh and H determinants were the best, being about 5 to 6.7 times more active than LFuc, but were weaker than p-nitrophenylalphaFuc indicating that hydrophobic environment surrounding the LFuc moiety enhance the reactivity. The hierarchy of potency of oligo- and monosaccharides can be ranked as follows: p-nitrophenyl-alphaFuc > Ah, Bh and H > LFuc > LFucalpha1-->2Galbeta1-->4Glc (2'-FL) and Galbeta1-->4[LFucalpha1-->3]Glc (3'-FL), while LNDFH I (Leb hexa-), Lea, Lex (Galbeta1-->4[Fucalpha1-->3]GlcNAc), and LDFT (gluco-analogue of Ley) were inactive. From the present observations, it can be concluded that the combining site of AAA should be a small cavity-type capable of recognizing mainly H/crypto H and of binding to specific polyvalent ABH and Leb glycotopes.     

Characterizing the silica deposition vesicle of diatoms

Summary The present study on a centric diatom,Ditylum brightwellii, includes two parts: detection of sugars in the silica deposition vesicle (SDV) with lectins and labeling the developing siliceous cell wall in the SDV with rhodamine 123. Cells with developing valves are treated with SDS to remove all the cytoplasmic contents, then either stained with fluorescein labeled lectins or thin-sectioned and stained with colloidal gold labeled lectins. The results show that mannose is part of the organic matrix in the SDV. Rhodamine 123, a non-toxic fluorescent laser dye, enters the cell immediately and is trapped in the SDV probably by the high reducing potential of the SDV. Silica is co-deposited with rhodamine 123 in the SDV, and the resulting valves and girdle bands become fluorescent. Implications of this study for the mechanism of silicification are discussed.Abbreviation SDV Silica deposition vesicle     

Lectins and also bacteria modify the glycosylation of gut surface receptors in the rat

Oral exposure to lectins or the presence or absence of bacteria in the rat small intestine were shown by histological methods using anti-lectin antibodies or digoxigenin-labelled lectins to have major effects on the state of glycosylation of lumenal membranes and cytoplasmic glycoconjugates of epithelial cells. Taken together with the dramatic effects of exposure to lectins on gut function, metabolism and bacterial ecology, this can be used as a basis for new perspectives of biomedical manipulations to improve health. Abbreviations: DIG, digoxigenin-labelled; POD, peroxidase-labelled; Spf, specific pathogen-free; TBS, Saline (0.9% w/v NaCl solution) buffered with 0.05m Tris-glycine, pH 7.8; PAP, antibody-peroxidase-antiperoxidase. For lectins see Table 1.     

The effects of lectins on the interaction between macrophages and bone in vitro

Summary Recent studies have demonstrated that the attachment of elicited rat macrophages to bone is mediated by specific saccharides located on the cell and/or bone surfaces. We have used a macrophage-bone culture system to study the effects of two lectins, concanavalin A (con A) and soybean agglutinin (SBA), on the morphology of macrophage attachment to a devitalized bone surface and subsequent functional activity. Macrophages were obtained from 3- to 4-week-old rats by peritoneal lavage and the adherent pool was used to prepare cell suspensions. Con A-treated, SBA-treated or control cell suspensions were aliquoted onto the endocranial surface of devitalized rat calvariae. The cells were allowed to attach for 1 h at 37° C, after which, the bone samples were removed from culture and prepared for scanning electron microscopy (SEM). The morphology of con A-treated macrophages attached to bone was markedly different from that of control or SBA-treated cells. Con A altered the attachment and subsequent spreading of macrophages on bone as visualized by SEM. Furthermore, the number of con A-treated cells that attached to bone and the average surface area of cell membrane apposed to the matrix was significantly different from that of control or SBA-treated cells. A ⁴⁵Ca bone-release assay was performed to evaluate the functional significance of the morphological findings. Lectin-treated or control cell suspensions were allowed to attach to the endocranial surface of ⁴⁵Ca pre-labeled calvariae for 1 h. Following attachment, the samples were cultured for 72 h. The con A-treated cultures demonstrated a significant decrease in the release of ⁴⁵Ca after 48 and 72 h in comparison to control cultures, while the ⁴⁵Ca released from SBA-treated cultures did not differ significantly from controls. These results suggest that certain sugar residues common to membrane-associated glycoconjugates and the organic component of the bone matrix regulate the attachment of macrophages to bone and their subsequent bone-resorbing activity.