Glycoconjugates in normal human kidney

Summary The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Glycoconjugates of the normal human colorectum: a lectin histochemical study

Summary Previous studies of the normal human colorectum by lectin histochemistry have used a mixture of tissues, including those derived from colons harbouring neoplasia and inflammatory bowel diseases. In the current investigation, tissues from patients without either of these conditions have been examined with a wide panel of lectins, encompassing specificities directed against both N- and O-linked sequences, using an avidin peroxidase revealing system and evaluated with a semiquantitative scoring method. The results of binding of these lectins have been compared with those seen in the resection margins of (at least 5 cm away from) colorectal carcinomas. Consistent regional variations were noted between right- and left-sided colonie tissues, with more diverse glycan structures and a greater sialyl content in the distal colon. There was evidence of graduation of formation of oligosaccharide chains in developing crypts, possibly related to the maturation and expression of glycosyl transferases responsible for the incorporation of mannose residues of N-linked oligosaccharides and of N-acetylgalactosamine and N-acetylglucosamine. Comparison with previous reports has revealed some variations, possibly related to tissue fixation and processing and to lectin concentrations employed, which raises the question of standardization of methodologies in lectin histochemical investigations.     

Glycoconjugates of the human trabecular meshwork: a lectin histochemical study

Summary Twelve specimens of resin-embedded human trabecular meshwork were probed with a panel of 21 biotinylated lectins, using an avidin-biotin peroxidase revealing system, in order to determine the normal pattern of saccharide expression in this tissue. High-mannose, intermediate and hybrid N-linked glycans, and complex N-linked bisected and non-bisected bi/tri-antennate glycans, as shown by the binding ofCanavalia ensiformis (ConA),Pisum sativum (PSA),Lens culinaris (LCA) agglutinins andPhaseolus vulgaris erythroagglutinin (ePHA), were strongly expressed by the canal of Schlemm endothelium and juxtacanalicular tissue, but less so by the corneoscleral meshwork. Highly branched complex glycans were not found, as there was no binding byPhaseolus vulgaris leukoagglutinin (1PHA). Sialyl residues, especially thoseα2,6-linked as demonstrated by strongSambucus nigra (SNA) lectin staining, were also abundant in this area.N-acetyllactosamine sequences and some O-linked glycans were present in the trabecular meshwork, as shown bySolanum tuberosum (STA),Datura stramonium (DSA), andJacalin (Jac) lectin binding, while fucose residues were not detected byTetragonolobus purpureas (LTA) orUlex europaeus-1 (UEA-1) agglutinins. These results indicate similarities with renal glomerular and vascular endothelium, although the lack of binding with UEA-1 agglutinin suggests differences which may relate to the specialized function of the trabecular meshwork. This study provides a baseline for comparative analysis of the glycans of human trabecular meshwork in pathological conditions such as primary open-angle glaucoma.     

Distribution and changes of glycoconjugates in rat colonic mucosa during development. A histochemical study using lectins

A study was made of the modifications of glycoconjugates in rat colonic mucosa during development. Sections of the caecum, and proximal and distal portions of the colon from Sprague Dawley rats at different stages of development (embryos, fetuses, suckling, weaning and adult rats) were examined. The sections were incubated with a battery of eight fluoresceinated lectins: DBA, SBA, WGA, LFA, PNA, GS-I, UEA-I and Con A. Some sections were treated with neuraminidase, and others were submitted to sequential saponification-neuraminidase treatment prior to incubation with the lectin (WGA, PNA or LFA). The intensity of the fluorescence was evaluated and graded from absent (-) to very positive (4+). Gradual and progressive changes were seen in colonic glycoconjugates during development. These changes revealed a unique developmental pattern for each lectin, which was independent for each cellular compartment (goblet cells, luminal surface and supranuclear region). Local and regional differences, observed between the different colonic sections, were already present from early stages of development. Moreover, our study showed that for several glycoconjugates, the differentiation process in colonic mucosa began in the distal region and continued through to the proximal region, the former being the first to reach the adult pattern. In the caecum, some lectins maintained a fetal pattern throughout all the periods of development up to the adult stage.     

The normal juxtaglomerular apparatus in the human kidney. A morphological study.

Out of 49 serially studied juxtaglomerular apparatuses, 6 typical variants from two normal human kidneys were reconstructed graphically. The agranular Goormaghtigh cells filled the entire space between the macula densa, the afferent and the efferent arterioles and the glomerular mesangium. The Goormaghtigh cells were always in direct contact with all the other structures. They also invariably continued into the glomerular mesangium. The distal tubule regularly showed widening in the macula densa segment and, at this level, there was considerable variation in the shape of the distal tubule. Direct contact between the macula densa and the hilar arterioles was not always present, the area of contact was usually greater with the afferent than with the efferent arteriole.     

Glycoconjugates of the cecal intestinal epithelium of the chick before and after hatching: histochemical study performed with peroxidase-conjugated lectins]

A battery of seven different horseradish peroxidase-labelled lectins (PNA, ConA, DBA, SBA, LTA, WGA and UEA I) was used to study the distribution and changes of carbohydrate moieties of glycoconjugates in the caecal epithelium (proximal and distal tracts) of the chick embryo and of the 3 days old chicken. The chief results showed that: 1. The appearance of some sugar residues was earlier observed at the epithelium of the distal tract than the proximal one (Tab. 2). 2. The presence of sialic acid was detected only after hatching (Fig. 4, Tab. 2). 3. During the embryonic caecal development enterocytes and goblet cells were characterized by the presence of the same sugar residues (Tab. 2). 4. By a quantitative point of view, differences in sugar residues content between the epithelium of the proximal and distal tract were observed. The epithelial cells of the distal tract were generally characterized by an higher content of saccharide moieties (Tab. 2). 5. At the end of the incubation period and after hatching enterocytes and goblet cells showed differences in content of some sugar residues (Fig. 1-3, Fig. 5-8, Tab. 2).     

Sugar residues of glycoconjugates in the olfactory epithelium of the human fetus: histochemical study using peroxidase-conjugated lectins]

The oligosaccharide content of glycoconjugates was studied at the olfactory epithelium of human fetuses ranging from 8 to 12 weeks of gestation by mean of peroxidase labelled lectins (DBA, PNA, WGA, SBA, ConA, LTA, UEA I). The main results demonstrated that: 1-The olfactory epithelium (olfactory cells, supporting cells and basal cells) was generally characterized by different amount of a-D-mannose, a-D-galactosamine, a-D-glucose, D-galactose-(beta 1-3)-N-acetyl-galactosamine, sialic acid and a-L-fucose. 2-At the 11th-12th week of gestation the largest amount of sugar residues was detected at the olfactory cells and at some basal cells. 3-At the 12th week of gestation, UEA I may be considered a specific marker of the olfactory cells in different stages of development.     

Distribution of glycoconjugates in normal human skin using biotinyl lectins and avidin-horseradish peroxidase

Lectin binding patterns in normal human skin were studied using five different biotinyl lectins and avidin-horseradish peroxidase. The staining pattern was specific for each lectin. In the epidermis, peanut agglutinin (PNA) and soybean agglutinin (SBA) preferentially stained the cell membranes of keratinocytes in the spinous and granular cell layers, indicating changes in the saccharide residues during keratinocyte differentiation. In the secretory segment of an eccrine sweat gland, the superficial cells gave a strong granular staining with Ricinus communis agglutinin (RCA). Dolichos biflorus agglutinin (DBA) and SBA, on the other hand, strongly stained the basal cells. With these lectins, two types of cells in the secretory segment were clearly distinguished. These results show that (1) PNA and SBA binding sites increase during the course of keratinocyte differentiation, and (2) RCA, DBA, and SBA are good markers to distinguish two types of cells in the secretory segment of an eccrine sweat gland.     

A histochemical study of acid phosphatase in normal and virus-transformed cultured fibroblasts.

The distribution of acid phosphatase has been investigated in normal and virus-transformed cultured hamster and mouse fibroblasts. The enzyme was found to be present in lysosomes, autophagic vacuoles and elements of the Golgi apparatus. It was also found to be associated with a surface coat in some virus-transformed mouse cells and in the cytoplasm of both normal and transformed hamster cells.     

Detection of Y-specific repeat sequences in normal and variant human chromosomes using in situ hybridization with biotinylated probes

In situ hybridization with a cloned human Y-specific repeat, pY3.4, derived from the 3.4-kb HaeIII repetitive sequences, is useful in identifying Yq-autosome translocations. In this study nonradioactive procedures were also employed to detect the sites of hybridization. Using a biotinylated probe and either immunofluorescence or horseradish peroxidase reaction, the chromosomes of three probands and members of their families with probable Y-autosome translocations were examined. It was found that not all such translocations can be correctly diagnosed based on conventional banding analysis. The present data indicate the importance of chromosome-specific probes in studying chromosome rearrangements in man.     

A method of selective histochemical analysis of sialoglycoproteins using lectins from elder (Sambucus nigra L.)]

Good potentialities in application of elderberry (Sambucus nigra L.) bark lectin for selective histochemical identification of sialylated glycoconjugates has been demonstrated using lectin-peroxidase technique. In order to omit this lectin binding to D-galactose and N-acetyl-D-galactosamine residues, preincubation of tissue sections with non-marked PNA and SBA (or other lectins with similar carbohydrate specificity) is proposed. By means of neuraminidase digestion it has been ascertained, that oligosaccharide chains of secretory glycopolymers, synthesised in ovine submandibular gland mucocytes, contain DGal and DGalNAc residues penultimate to terminal sialic acids.     

Lectin binding sites in normal and phenobarbitale/halothane treated rat liver. A histochemical study

The content of carbohydrate residues of both normal and phenobarbitale-halothane-hypoxia exposed rat liver has been examined by means of lectin histochemistry. Eight biotinylated lectins specific to galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose and mannose were applied to paraffin sections of rat liver at light microscopic level. The most distinct binding was observed at the structures of the "perisinusoidal functional unit": Kupffer cells are bound by S-WGA, SBA and PNA. Bile canaliculi display binding sites for RCA I and WGA. Cytoplasm of hepatocytes appears lectin-negative, except for PSA. The enhanced reaction of S-WGA, PNA and SBA after the preincubation of the sections with neuraminidase indicates the occurrence of sialic acid in Kupffer cells. The phenobarbitale-halothane-hypoxia exposed rat liver shows centrolobular degeneration of hepatocytes with a diminished amount of hepatocyte and Kupffer cells as well. The lectin binding pattern of sinusoidal walls, membranes of hepatocytes and bile canaliculi remains the same compared to that of normal rat liver. This finding suggests that at least the carbohydrate content of membranes in the liver resists severe destruction under phenobarbitale-halothane-hypoxia. It is assumed that there exists a connection between intact carbohydrate residues and the regeneration of liver parenchyma.     

Saccharide distribution in human trophoblast demonstrated using fluorescein-labelled lectins

Synopsis The reactions of six Fluorescein-conjugated lectins with human trophoblast and foetal limb are described. The trophoblasi had marked membrane fluorescence affer treatment with wheatgerm andLotus tetragonolobus lectins. Concanavalin A reacted strongly with the trophoblastic cytoplasm but did not react with the nuclei. Most of the lectins had only weak reactions with foetal tissue, apart from the cartilage matrix which fluoresced strongly after treatment with several of the lectins. In addition, concanavalin A reacted with amylase-labile material, probably glycogen, in the skin epidermis and chondrocyte cytoplasm.Various substances were tested in order to determine the nature of the wheatgerm lectin receptor of trophoblast. Several secretory and binding proteins were found not to be responsible, and the identity of the receptor remains unknown.     

The outer mantle epithelium of Haliotis tuberculata (Gastropoda Haliotidae): an ultrastructural and histochemical study using lectins

The mantle of molluscs has been the subject of many studies as it is the organ that forms the shell. Microscopic studies in particular focus on the outer mantle epithelium, but few studies address this epithelium in a histochemical way. In this study, the outer mantle epithelium in adult specimens of Haliotis tuberculata is studied, that is, in specimens involved in maintaining and repairing the shell rather than in generating it. The epithelial cells are studied by scanning (SEM) and transmission electron microscopy (TEM), and by histochemical techniques, including the use of lectins for their biochemical characterization. The epithelium is composed of pigmented epidermal cells with small microvilli and junctional complexes. It furthermore contains a few ciliated cells, as well as two types of secretory cells which differ in the ultrastructural appearance of their secretory granules and their glycoconjugate content. Histochemical study shows secretory cells containing sulphated glycoconjugates such as glycosaminoglycans or mucins rich in N‐acetylgalactosamine and N‐glycoproteins rich in fucose. Furthermore, the apical regions of the epidermal cells are positive for lectins that label fucose, mannose and N‐acetylglucosamine. The role of epithelial cells in the synthesis of structural components of the shell is discussed.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. V. A differential diagnostic method for the histochemical classification of glycoproteins

Summary A differential diagnostic scheme is described for the division of colonic epithelial glycoproteins into eleven histochemically distinct classes. The scheme depends upon the use of seven histochemical techniques which, collectively, permit the differential staining ofO-sulphate ester, sialic acid and its side chainO-acyl variants and vicinal diols located on carbohydrate residues other than sialic acids. Elements of the scheme also provide a general approach to the classification of epithelial glycoproteins in anatomic sites other than the colon.Application of the scheme permitted the classification of the epithelial glycoproteins in the mucosa 0.5–5.0 cm from human colonic tumours and provided direct confirmation of previous observations that changes from normal in the relative proportions of either side chainO-acylated sialic acids or sialic acids andO-sulphate esters can occur independently of one another.     

A histochemical study of amines in palatal tissue from normal and glycosuric mice

Synopsis A fluorescence histochemical technique for localizing catecholamines was applied to palatal tissue obtained from laboratory mice and from glycosuric and nonglycosuric Australian hopping mice. Specific catecholamine fluorescence related to blood vessels was demonstrated in all the tissue samples, though there was a tendency for this to be less marked in palates taken from the glycosuric animals.Although the presence of discrete areas of bright yellow fluorescence was noted in the tissue of the laboratory mice, these areas of yellow fluorescence (probably serotonin within mast cells) were virtually absent from the palates of the hopping mice despite the fact that there was little difference in the respective mast cell populations in the palatal tissues of the laboratory and hopping mice as determined by metachromatic staining procedures.