Francisco Hernández

Glycoconjugate distribution in the human fundic mucosa revealed by lectin- and glycoprotein-gold cytochemistry

SummaryThe glycoconjugates of the human fundic mucosa were characterized at the ultrastructural level by means of direct (Helix pomatia agglutinin-gold complex) and indirect lectin techniques (Concanavalin A and horseradish peroxidase-gold complex; wheat germ agglutinin and ovomucoid-gold complex). Surface mucous cells and mucous neck cells secreted O-glycoproteins with N-acetylgalactosamine and N-acetylglucosamine residues at the non reducing terminus of the saccharidic chain. The secretory granules of the mucous neck cells showed condensed areas slightly reactive to ConA. The results obtained in the chief cells suggest that these cells secrete N-glycoproteins rich in mannose and/or glucose residues. “Transitional cells”, presenting both morphological characteristics and lectin binding pattern intermediate to the mucous neck and chief cells have been observed. The surface of the intracellular canaliculi of the parietal cell was labelled by HPA, WGA and ConA. In the neck region of the gastric glands, immature parietal cells containing abundant mucous granules reactive to HPA, WGA and ConA were observed. The present results further corroborate the existence of a common cell precursor for surface mucous, mucous neck and parietal cells. In a further step, mucous neck cells gradually differentiate into chief cells the transitional cells being an intermediate stage.

Lectin cytochemical characterization of the N- and O-linked oligosaccharides in the human rectum.

The oligosaccharides of the mucus glycoproteins of the human rectum are important for the lubricant and protective role suggested for the rectal mucus. Changes in oligosaccharide composition are observed in several colon diseases, and some of these changes could be used as diagnostic and prognostic indicators. Thus, a previous knowledge of the normal mucus glycoproteins is necessary. The aim of the present study is the characterization of the oligosaccharides of the goblet cells and enterocytes of the human rectum. For this, a battery of 15 lectins, in combination with chemical and enzymatic deglycosylation procedures, was used. Our results suggest the presence of N-acetylglucosamine (GlcNAc), Man, Glc, N-acetylneuraminic acid (Neu5Ac)(α2–6)- and Neu5Ac(α2–3)-linked, N-acetylgalactosamine (GalNAc) and Gal(β1–3)GalNAc in the oligosaccharides of the goblet cells. Moreover, N-linked oligosaccharides specifically contained Gal(β1–4)GlcNAc, while AAA-positive Fuc was only detected in O-linked oligosaccharides. Some of these carbohydrates were only visualized after removal of N- or O-linked oligosaccharides, suggesting a high level of approximation between the oligosaccharide chains, that render the carbohydrate inaccessible to the lectins. Differences in the labelling pattern between the goblet cells of the surface epithelium and the upper half of the crypts, and those of the lower half of the crypts suggests a maturation process for the goblet cells, which modifies the oligosaccharide composition of the secreted glycoproteins, as they ascend throughout the crypts. This maturation process includes the incorporation of new carbohydrates (GlcNAc), and the masking (Neu5Ac(α2–3)-linked) or unmasking (Glc and GalNAc) of others.

N- and O-linked Oligosaccharides in the Secretory Granules of Rat Paneth Cells: An Ultrastructural Cytochemical Study

Paneth cells are located at the base of the intestinal glands. The origin, composition, and function of these cells have not been well established. The sharing of a common pathway of development with the goblet cells has been suggested. The aim of the present study was to explore the cytochemical composition of rat Paneth cells and to discuss a possible developmental relationship between goblet and Paneth cells. Lectins (WGA, LTA, UEA-I, AAA, and HPA) were used as a precise tool for the ultrastructural localization of carbohydrates. Several procedures were performed in combination with lectin cytochemistry: β-elimination, a reaction that specifically removes O-linked oligosaccharides (typical of mucin-type glycoproteins of goblet cells); and treatment with peptide N-glycosidase F, an enzyme that removes N-linked oligosaccharides from glycoproteins. Secretory granules of Paneth cells showed a biphasic nature composed of an electron-lucent peripheral halo containing O-linked oligosaccharides with GalNAc and GlcNAc residues and N-linked oligosaccharides with GlcNAc residues (only sparse Fuc residues were scarcely identified in O-linked oligosaccharides), and an electron-dense core containing N- and O-linked oligosaccharides with Fuc residues. Neither GlcNAc nor GalNAc was identified. The occurrence of O-linked oligosaccharides in the Paneth cells and the biphasic nature of the secretory granules, similar to that of transitional cells intermediate between mucous and serous cells of other tissues, favor the hypothesis of a common lineage for goblet and Paneth cells.

New arenediethynylgold(I) complexes. Crystal structures of [Ph3PAuCC(phenylendiyl-1,3)CCAuPPh3] and [Ph3PAuCC(mesitylendiyl-1,3)CCAuPPh3]

By reacting the dialkynes HCC(Ar)CCH [Ar=1,3-C6H4 (mphen), 1,3-(C6HMe3-2,4,6) (mes), 1,4-(C6Me4-2,3,5,6) (dur)] with [AuClL] [L=dimethylsulfide (dms), tetrahydrothiophene (tht)] in the presence of an excess of Et3N the polymeric complexes [AuCC(Ar)CCAu]n [Ar=mphen (1), mes (2) dur (3)] were prepared. Upon reaction of these complexes with two equivalents of phosphine or isocyanide ligands (L), complexes [LAuCC(Ar)CCAuL] [Ar=mes, L=tBuNC (4), XyNC (5); Ar=dur, L=PPh3 (6), tBuNC (7)] were prepared in good yields. The complex [tBuNCAuCC(mphen)CCAuCNtBu] (8) was prepared by reaction of the corresponding diethynylarene with 2 equiv. of [AuCl(CNtBu)] in the presence of NEt3. By reacting the appropriate diethynylarene with 2 equiv. of [Au(acac)PPh3], complexes [Ph3PAuCC(Ar)CCAuPPh3] [Ar=mphen (9), mes (10)] were prepared. The carbene complex [(tBuNH)(Et2N)CAuCC(mes)CCAuC(NHtBu)(NEt2)] (11) was obtained by reacting the corresponding isocyanide complex 4 with diethylamine. The crystal structures of complexes 9 and 10 have been determined by X-ray diffraction studies. In both cases one of the gold atoms is in an essentially linear environment [CAuP: 176.95(14) (9), 177.19(8)° (10)] while the other CAuP bond angle is appreciably bent [170.07(11) (9), 171.22(8)° (10)].

Lectin-Gold Localization of Fucose Residues in Human Gastric Mucosa

The oligosaccharides of the mucous gastric glycoproteins are involved in the protection of the gastric mucosa and are altered in different diseases. Therefore, it is important to know their composition in health, to better determine the alterations induced by the disease. Moreover, analysis of the molecular composition of the fundic gland cells has been previously used to obtain new insights into the origin of the different cell types. The aim of the present study was the localization in the subcellular structures of the fucose residues of the oligosaccharides in human fundic glands. For this, lectin cytochemical methods were used at the light and electron microscopic levels. They were combined with enzymatic and chemical treatments to characterize the nature of the oligosaccharide chains containing the fucose residues. The presence of this carbohydrate belonging to N- or O-linked oligosaccharides has been demonstrated in the secretory granules of the surface, gastric pit, mucous neck, and transitional cells of the fundic mucosa, and in the intracellular canaliculi and tubulovesicular system of the parietal cells. These fucose residues were added in the trans-Golgi regions to the elongating chains. Additional fucose linked to the innermmost N-acetylglucosamine of the N-linked oligosaccharides was found in the chief cells, being incorporated in the cis-Golgi. The findings in the transitional cells corroborate the origin of the chief cells from the mucous neck cells.

Characterization of glycoproteins in the epithelial cells of human and other mammalian gallbladder. A review.

The mammalian gallbladder mucosa is lined by a simple columnar epithelium. Typical surface epithelial cells (principal cells) contain short microvilli, secretory granules, dense bodies, mitochondria and Golgi apparatus. Dense bodies are thought to be lysosomes. Secretory granules contain mucous glycoproteins which are released to the lumen by exocytosis. Oligosaccharide side chains of mucous glycoproteins may provide a favorable environment for nucleation of cholesterol in gallstone formation; therefore they have been studied during the past decades. Histochemical techniques allow the in situ identification of carbohydrates at both the cellular and subcellular levels. The oligosaccharide chains of principal cell mucous glycoproteins have been studied by classical histochemical techniques (PAS, alcian blue, HID, etc). These techniques indicate that mammalian gallbladder mucous glycoproteins are heavily sulphated, whereas sialic acid residues are scarce. Neutral mucins have not been described in the mammalian gallbladder. Electron microscopic studies have located the oligosaccharide chains in secretory granules and Golgi apparatus. More recently, lectins (molecules which specifically recognize and bind with different saccharides or saccharide sequences) have been applied for the intracellular localization of carbohydrate residues. Lectin histochemistry has detected fucose, galactose, N‐acetylglucosamine, N‐acetylgalactosamine and N‐acetylneuraminic acid residues in mucous granules, Golgi apparatus and apical membrane of human principal cells. Mannose residues were observed only in dense bodies. The combined use of deglycosylation procedures and lectin histochemistry has revealed a variety of terminal sequences in oligosaccharide chains of gallbladder mucous glycoproteins: Neu5Ac(α2‐3)Gal(β1‐3)GalNAc, Neu5Ac(α2‐3)Gal(β1‐4)GlcNAc and Gal(β1‐4)GlcNAc. This technology also suggested the occurrence of N‐linked oligosaccharides in the dense bodies of principal cells. Mucous granules mainly contained mucin‐type O‐linked oligosaccharides although some N‐linked chains have also been detected. Gallstone formation is probably a complex process depending on multiple factors. Mucous glycoproteins are one of the factors involved in this process. Histochemical methods offer an excellent research tool for the characterization of glycoproteins in the epithelial cells of the gallbladder, thus contributing to the elucidation of the pathophysiology of gallstone formation. Microsc. Res. Tech., 38:616–630, 1997.

Subcellular characterization of glycoproteins in the principal cells of human gallbladder

Gallbladder mucus is mainly composed of glycoproteins, which seem to play a critical role in cholesterol nucleation during gallstone formation. The biosynthetic pathway and sequential processing as well as the characterization of the oligosaccharide sidechains of human gallbladder secretory glycoproteins have not been completely defined. The aim of the present study is the subcellular characterization of the glycoproteins in the principal cells of human gallbladder. Principal cells of normal human gallbladder were studied by means of a variety of cytochemical techniques, including lectin histochemistry, enzyme and chemical treatments, immunocytochemistry and lectin-gold technology. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid residues were detected in mucous granules, Golgi apparatus and apical membrane of principal cells. Mannose residues were only observed in dense bodies. Oligosaccharide side-chains of the glycoproteins contained in the biliary mucus are synthesized in the Golgi apparatus of the principal cells of the gallbladder epithelium and are also contained in the mucous granules of these cells. Terminal N-acetylneuraminic acid(α2-3)galactose(β1-3)N-acetylgalactosamine, N-acetylneuraminic acid(α2-3)galactose(β1-4)N-acetylglucosamine and galactose(β1-4)N-acetylglucosamine sequences are contained in the oligosaccharide chains of gallbladder mucus glycoproteins. The dense bodies detected in the cytoplasm of the principal cells contained N-linked glycoproteins. Mucin-type O-linked glycoproteins were the main components of the mucous granules although some N-linked chains were also detected.

Lectin Histochemical Identification of the Carbohydrate Moieties on N- and O-linked Oligosaccharides in the Duct Cells of the Testis of an Amphibian Urodele, The Spanish Newt (Pleurodeles Waltl)

The aim of the present work was to study the carbohydrate moieties present on N- and O-linked oligosaccharides of duct cells of a urodele amphibian testis, by means of lectin histochemistry. It was found that duct cells have a carbohydrate composition that includes ensuremath α(1,3)-, α(1,4)- or α(1,6)-linked Fuc and Man on N-linked oligosaccharides, Gal and GlcNAc on O-linked oligosaccharides, and DBA-positive GalNAc, α(1,2)-linked Fuc and Neu5Acα(2,3)Gal β(1,4)GlcNAc on both N- and O-linked oligosaccharides. All the duct cells showed the same lectin labelling pattern, the only exception being some sparse duct cells that showed the sequence Neu5Acα(2,6)Gal/GalNAc. The possible roles of duct cells in sperm maturation and the hypothesis for a common origin of duct and follicle (Sertoli) cells in the urodele testis are discussed.

A histochemical study of the mucins in the digestive tract of the chicken

Little is known about the distribution of glycoproteins in the digestive tube of birds. In the present study, the localization and distribution of mucins in the digestive tract of the chicken are reported. Sialo- and sulpho-mucins were widely distributed throughout the chicken digestive tube. Some of the mucous cells of the proximal segment of the proventriculus presented neutral glycoproteins; in the medial segment, surface cells containing only sialo-mucins were observed. Surface cells of the gizzard contained both sialo- and sulpho-mucins while PAS-positive material was localized in the lumen of the glands. 2 types of mucous cells were observed in the small intestine; 1 type contained only sialo-mucins and the other contained both sialo- and sulpho-mucins. In the large intestine and caecum, both types of acid mucins were present in the mucous cells. In conclusion, the distribution of glycoproteins in the chicken reported in the present study show marked differences with that reported in other avian species.

Distribution of mucins in the mucosa of the digestive tract of reptiles: a histochemical study

In the present study, the distribution and characteristics of mucins in the digestive tract of 3 reptiles (Lacerta lepida, Mauremys caspica and Testudo graeca) are investigated. In the esophagus of Testudo graeca, both the glands and the mucous cells of the surface epithelium contained sulphosialo-mucins. In Lacerta lepida, esophageal mucous cells contained either-sialo-mucins or sulphosialo-mucins. The esophageal mucous cells of Mauremys caspica contained acidic mucosubstances. The stomach of the species studied revealed a small amount of acidic mucosubstances; in Lacerta lepida and Testudo graeca, abundant neutral mucins were detected. In the intestine, the amount of acidic mucosubstances was increased in a caudal direction, the sulphosialo-mucins being predominant. In conclusion, acid mucins were more abundant in the esophagus and intestine than in the stomach. This may be related to cytoprotective roles in the esophagus and protection against potential pathogens in the intestine.