A new method for the histochemical demonstration of O-acyl sugars in human colonic epithelial glycoproteins

Summary A new general method has been developed for the specific histochemical identification ofO-acyl sugars in any epithelial glycoprotein. These sugars include hexose, 6-deoxyhexose andN-acetylhexosamine with an ester substituenent(s) located on a potentialvicinal diol(s). In the procedure reported [the periodic acid-borohydride reduction-saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA-Bh*-Bh-PAS) method] the initial PA-Bh treatment rendersvicinal diols located on either sialic acid or neutral sugars PAS unreactive. In the subsequent steps ester substituents are removed from bothO-acyl sugars andO-acyl sialic acids by saponification (KOH), sialic acidvicinal diols are selectively removed by the PA^*-Bh sequence andO-acyl sugars are stained with the PAS technique. This method has the advantage that the results are obtained with a single section and the results are either positive or negative. Consequently, it is superior to the three indirect methods investigated because it does not require an observer to compare the intensity or the shade of the staining obtained with serial sections.Using the PA-Bh-KOH-PA^*-Bh-PAS method we have demonstrated, for the first time, thatO-acyl sugars occur in the epithelial goblet cell glycoproteins of adult human colon. The effect of the presence ofO-acyl sugars on the interpretation of a number of other methods for the histochemical investigation of glycoproteins is discussed. It is recommended that the results obtained with the PA-Bh-KOH-PA^*-Bh-PAS method be evaluated before histochemical procedures for the investigation of neutral sugars andO-acyl sialic acids are selected.     

Histochemical studies of epithelial cell glycoproteins in normal rat colon

Two general classes of glycoproteins have been identified in the colonic epithelial cells of the Sprague Dawley rat. Glycoproteins belonging to the first of these classes contain sialic acids both with and without side chain o-acyl substituents, abundant o-sulphate ester and 'neutral sugars' (hexose, 6-deoxyhexose or N-acetyl hexosamine residues) with oxidisable vicinal diols and are located in the goblet cells of the descending colon and in goblet cells populating the upper halves of the crypts of the ascending colon. In the descending colon, the sulphosialoglycoproteins in the goblet cells in the base of the crypts contain sialic acids without side chain o-acyl substituents. It appears that as these cells migrate up the crypts, there is o-acylation of the side chains of the sialic acids of the glycoproteins and an increase in the quantity of 'neutral sugars' without a corresponding increase in sialic acid. Glycoproteins with similar properties to those of the goblet cells of the upper halves of the crypts of the descending colon, but containing less sulphate, are found in the goblet cells of the upper half of the crypts of the ascending colon. The second general class of glycoproteins contain sialic acids all, or almost all of which, are substituted at position C8 and only relatively small quantities of sulphate. They are located in the mucous cells of the descending colon, the deep crypt secretory cells of the ascending colon and the columnar absorptive cell brush border.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its O-acyl variants or O-sulphate ester. II. Methods based upon the periodic acid-phenylhydrazine-Schiff reaction

Summary Four methods based upon the periodic acid—phenylhydrazine—Schiff reaction have been developed for the simultaneous visualization of neutral sugars with periodate oxidizablevicinal diols (hexose, 6-deoxyhexose,N-acetylhexosamine) and either sialic acids or side chainO-acyl sialic acids. In the first of these procedures, the saponification—periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification (KOH—PA—DNPH—Az—KOH) method, all sialic acids stain Azure blue, neutral sugars with oxidizablevicinal diols stain yellow and mixtures of such components stain in various shades of green. In the second technique, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A Schiff—saponification (PA—DNPH—Az—KOH), Azure Blue staining is confined to sialic acids without side chain substituents or which have anO-acyl substituent at position C7, while in the third method, the selective periodate oxidation—borohydride reduction—saponification—periodic acid oxidation—2,4-dinitrophenyl hydrazine—Azure A—Schiff—saponification (PA^*—Bh—KOH—PA—DNPH—Az—KOH) technique, only sialic acids withO-acyl substituents at positions C7, C8 or C9 (or which have two or threeO-acyl side chain substituents) stain Azure blue. Finally in the fourth procedure, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification—borohydride reduction—periodic acid oxidation—Schiff (PA—DNPH—Az—KOH—Bh—PAS), sialic acids without side chain substituents or which haveO-acyl substituents at C7 stain Azure blue, sialic acids substituted at position C8 or C9 (or which are di- or tri-substituted) stain magenta and neutral sugars stain yellow. Where mixtures of these components are present, a wide range of colours is obtained.     

Histochemical studies of intestinal epithelial goblet cell glycoproteins during the development of the human foetus

Summary Histochemical studies performed on specimens of intestine from 12 to 37-week human foetuses showed that the epithelial glycoproteins of the goblet cells of the small intestine are non-sulphated sialoglycoproteins containing neutral sugar (hexose, 6-deoxy hexose or N-acetyl hexosamine residues with Periodic acid-Schiff (PAS) reactive vicinal diols), sialic acids without O-acyl substituents, smaller and variable quantities of sialic acids with O-acyl substituents at positions C8 or C9 (or with two or three side chain substituents) and O-acyl sugars (neutral sugars with an ester substituent blocking PAS reactivity). In the lower small intestine glycoproteins containing 8 (or 9)-O-acyl sialic acids are first observed in goblet cells at the tips of the villi. As the foetus matures their quantity increases and they are found in goblet cells located along the length of the villi. Smaller quantities of O-acyl sialic acids and traces of O-acyl sugars occur in the goblet cells of the upper small intestine. The colonic goblet cells contain sulphosialoglycoproteins of two types. The first type, found in the majority of specimens, contains O-sulphate ester, neutral sugar, O-acyl sugars and 8 (or 9)-O-acyl sialic acids. The second type contains O-sulphate ester, neutral sugars, and sialic acids which are either without side chain O-acyl substituents or are a mixture of such acids and 8 (or 9)-O-acyl sialic acids; O-acyl sugars are reduced or absent. The degree of sulphation of the foetal colonic goblet cell epithelial glycoproteins differs with the region of the colon, the level of the crypt and the gestational age of the foetus in a manner consistent with that described by Lev & Orlic (1974). The detection of O-acyl sugars in foetal intestinal glycoproteins adds to the known examples of such sugars and strengthens the suggestion that they are a normal constituent of colonic epithelial glycoproteins.Part of this work was presented at the 32nd meeting of the Canadian Federation of Biological Sciences, Calgary, Alberta, June 1989 (abstract # 336).     

Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its side chain O-acyl variants or O-sulphate ester. I. Methods based upon the selective periodate oxidation of sialic acids

Summary Five new methods, based upon the selective oxidation of sialic acid residues with 0.4mm periodic acid in approximately 1m hydrochloric acid at 4°C for 1 h (PA^*), have been devised for the simultaneous visualization of neutral sugars and either sialic acid and its side chainO-acyl variants orO-sulphate ester. In the first of these, the selective periodate oxidation—borohydride reduction—saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (PA^*—Bh—KOH—PA^*—T—KOH—Bh—PAS) technique, sialic acids withO-acyl substituents at C7, C8 or C9 (or which have two of three side chainO-acyl substituents) stain blue while neutral sugars with periodate-sensitivevicinal diols (hexose, 6-deoxyhexose, andN-acetylhexosamine) stain magenta. The second method, the saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (KOH—PA^*—T—KOH—Bh—PAS), stains all sialic acids blue and neutral sugars magenta. In the third procedure, the selective periodate oxidation—Thionin Schiff—borohydride reduction—periodic acid—Schiff—saponification (PA^*—T—Bh—PAS—KOH) method, sialic acids without side chain substituents (or which have anO-acyl substituent at C7) stain blue and neutral sugars stain magenta. In the fourth method, the saponification-selective periodate oxidation—borohydride reduction—Alcian Blue pH 1.0—periodic acid—Schiff (KOH—PA^*—Bh—AB1.0—PAS) technique,O-sulphate esters stain aquamarine blue and neutral sugars stain magenta. In all of these techniques mixtures of the components stain in various shades of purple. Performance of the KOH—PA^*—Bh—AB1.0—PAS technique without the Alcian Blue pH 1.0 step provides a method for the selective identification of neutral sugars in macromolecules that also contain sialic acids.     

Histochemical analysis of glycoproteins in the unicellular glands in the epidermis of an Indian freshwater fish Mastacembelus pancalus (Hamilton)

Summary The unicellular glands in the epidermis of the Indian freshwater fish Mastacembelus pancalus consist of three types of mucous cells and sacciform cells. The histochemical properties of their secretory glycoproteins have been analysed by means of a battery of histochemical methods. These included methods for the identification and simultaneous visualization of oxidizable vicinal diols, O-acyl sugars, O-sulphate esters and sialic acid residues with or without side-chain O-acyl variants. Four general classes of glycoproteins (GPs) were identified. These included (i) GPs with O-sulphate esters and oxidizable vicinal diols, (ii) GPs with oxidizable vicinal diols and sialic acid residues with or without O-acyl substitution at C7, (iii) GPs mainly with O-sulphate esters, low moieties of GPs with oxidizable vicinal diols, O-acyl sugars and sialic acid residues with side-chain O-acyl variant predominantly at C8 (or which are di- or tri-substituted) or C9 and in traces of sialic acid residues without O-acyl substitution or with O-acyl substitution at C7, and (iv) GPs with traces of oxidizable vicinal diols, O-acyl sugars and sialic acid residues with O-acyl substitution at C8 (or which are di- or tri-substituted) or C9. The physiological significances of these GP classes and their release on the surface of the epidermis are discussed with special reference to their role in lubrication, protection and inhibition of the invasion and proliferation of pathogenic microorganisms in the epidermis, as adapted to the peculiar mode of life of the fish.     

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.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. I. A comparison between histologically normal colon, colonic tumours, ulcerative colitis and diverticular disease of the colon

Summary Chemical and histochemical methods were used to compare the epithelial glycoproteins from formalin-fixed surgical specimens of normal human large intestine, colonic tumours, ulcerative colitis and diverticular disease. All the epithelial glycoproteins contained fucose, galactose, glucosamine, galactosamine and, in addition, sialic acids both with and withoutO-acyl substituents in the side chain and/or at position C₄. The glycoproteins of the normal ascending and descending colons differed significantly with respect to the percentage of the sialic acids released following digestion of the de-O-acylated glycoprotein withVibrio cholera neuraminidase and to the molar fucose-sialic acid ratio. Statistical analysis of the chemical data showed that (a) compared to normal, the sialic acids of the tumour and ulcerative colitis glycoproteins from the descending colon were significantly less substituted in the side chain and at position C₄; (b) theO-acetyl substitution pattern of the sialic acids of the ulcerative colitis glycoproteins from the ascending colon and the quantitative composition of the carbohydrate prosthetic groups of the ulcerative colitis glycoproteins from both ascending and descending colons differed from normal; (c) it was not always possible to distinguish between the ulcerative colitis and tumour glycoproteins on the basis of theO-acetyl substitution pattern of their sialic acids; and (d), there were minor differences between normal glycoproteins and those from cases of diverticular disease.     

Histochemical identification of 9-O-acyl sialic acids: studies of bovine submandibular and rat sublingual gland and human colon

Summary Formalin-fixed tissue specimens containing glycoproteins with side chain O-acylated sialic acids were used to re-examine, compare and evaluate the usefulness of three methods based on the periodic acid-borohydride reduction-saponification-periodic acid-Schiff sequence (PA-Bh-KOH-PAS) for the histochemical identification of 9-O-acyl sialic acids (9-O-AcSA). Method I, modified from Vehet al. (1979), involved a comparison of the staining intensely obtained when both oxidation steps of the PA-Bh-KOH-PAS sequence were carried out with the selective oxidation technique of Volzet al. (1987) with that obtained when the initial oxidation step was carried out with 0.5m periodic acid for 4h at room temperature. Methods II and III, modified from Reidet al. (1978), involved an initial PA-Bh step under oxidation conditions that cleaved all the vicinal diols associated with neutral sugars and side chain unsubstituted and 7-O-acyl sialic acids. The Schiff staining obtained following subsequent re-oxidation with either 0.5m (method II) or 1% periodic acid (method III) for 4h at room temperature (PA-Bh-PAS procedure) identifies 9-O-AcSa.The results of this study indicate that (a) bovine submandibular gland acinar cell glycoproteins contain 9-O-AcSA as well as sialic acids which have ester substituents at C7 or C8, or which are di-(C7C8, C7C9, C8C9) or tri-(C7C8C9) substituted, (b) the side chain O-acyl sialic acids of the glycoproteins of Sprague Dawley rat sublingual gland acinar cells are entirely or almost entirely 9-O-AcSA and (c) it is likely that the majority of the human adult and foetal glycoproteins studied contain small quantities of 9-O-AcSA mixed with sialic acids which are substituted at C7 or C8 or which have two or three side chain O-acyl substituents. However, the interpretation of the results are complicated by observations that indicate that (a) treatment with 0.5m periodic acid either extracts or removes sialic acids from bovine submandibular gland glycoproteins, (b) some human colonic epithelial glycoproteins apparently contain a component other than 9-O-AcSA that oxidises slowly with periodic acid and (c) 1% periodic acid for 2h at room temperature oxidises a small but significant quantity of 9-O-AcSA, thus reducing the intensity of staining in methods II and III. It is concluded that when adequately controlled, methods I, II and III are capable of detecting 9-O-AcSA in glycoproteins containing large quantities of the sialic acid. However, these methods may not detect small quantities of 9-O-AcSA in the presence of large quantities of sialic acids which have O-acyl substitutents at positions C7 or C8 or which have two (C7C8, C7C9, C8C9) or three (C7C8C9) side chain O-acyl substituents. Thus, caution should be used when interpreting data that indicates the absence of 9-O-AcSA.     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, alpha-D-mannose and alpha-D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; alpha-D-mannose and alpha-D-glucose residues and terminal sialic acid-galactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae)

Glycoproteins (GPs) were visualised histochemically in the secretory cells – the mucous goblet cells (the type A and the type B), the serous goblet cells, the club cells and the epithelial cells in the gill epithelium of Rita rita. The type A mucous goblet cells, the type B mucous goblet cells and the epithelial cells elaborate GPs with oxidizable vicinal diols and GPs with sialic acid residue without O-acyl substitution. In addition, GPs with O-sulphate esters are elaborated by the type A and GPs with O-acyl sugars by the type B mucous goblet cells. GPs are absent in the serous goblet cells and are with oxidizable vicinal diols in low moieties in the club cells. The analysis of the results elucidates interesting differences in the composition and concentration of GPs in the mucus elaborated by the epithelium of the gill arches and the gill rakers; and the gill filaments and the secondary lamellae indicating the potential importance of the glycoproteins at these locations. GPs elaborated on the surfaces of the gill arches and the gill rakers could be associated to assist in feeding activities and on the surfaces of the gill filaments and the secondary lamellae in the respiratory activity.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. II. A comparison between histologically normal small intestine and Crohn's disease of the small intestine

Summary Comparative chemical and histochemical studies were performed on formalin-fixed, surgical specimens of human small intestine from cases of Crohn's disease and normal controls. The sialic acids of the crude glycoproteins isolated from normal ileum were significantly less neuraminidase-susceptible and more C₄ substituted (P<0.01) than those of the glycoproteins isolated either from normal upper small intestine (duodenum and jejunum) or from cases of Crohn's disease of the ileum. Fractionation yielded two major sialic acid-containing fractions, eluting from DEAE-cellulose with 0.2m or 0.3m sodium chloride. Both fractions contained fucose, galactose, glucosamine and galactosamine in addition to sialic acids both with and withoutO-acyl substituents at position C₄ and/or in the side-chain (side-chainO-acylated sialic acids were also detected by histochemical procedures). The fractions differed significantly from one another with respect to the neuraminidase susceptibility of their sialic acids (P<0.01), the percentage of C₄ (P<0.01) and side-chain substituted sialic acids (P<0.05), and the molar fucose-sialic acid ratio (P<0.05). TheO-acyl substitution patterns of the sialic acids of both the 0.2m and 0.3m fractions of the upper small intestine glycoproteins differed significantly from those of the corresponding fractions from normal ileum, while the sialic acids of the 0.2m fractions from Crohn's disease of the ileum differed significantly from normal with respect to neuraminidase susceptibility (P<0.01) and percentage C₄ substitution (P<0.01); the 0.3m fractions differed only in the percentage of sialic acids substituted at C₄. The differences between the sialic acids from the normal and Crohn's disease specimens were shown to be independent of either the anatomical origin of the specimen or the histopathological sub-group of the Crohn's disease specimens; no significant differences were noted between the sub-groups but all the sub-groups differed from normal.     

The detection by X-ray microanalysis of noradrenaline in sympathetic nerve terminals of the rat vas deferens

Summary The epidermis of Mystus (Mystus) vittatus contains two well differentiated mucous cells which secrete different mucosubstances. The goblet cells contain periodate reactive neutral mucosubstances, glycogen, testicular hyaluronidase resistant sulphated mucosubstances, and sialic acid rich glycoproteins. The clavate cells contain small amounts of neutral and sulphated mucosubstances and no glycoproteins. The difference in the histochemical nature of the two types of mucous cells is discussed in relation to their physiological activities.This investigation was supported by research Fellowship No. 7/176(138)77 from the Council of Scientific & Industrial Research, New Delhi to the first author     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

Summary In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, -D-mannose and -D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; -D-mannose and -D-glucose residues and terminal sialic acidgalactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

A new histochemical method for the selective periodate oxidation of total tissue sialic acids

Summary Evaluation of the intensity of the periodic acid—Schiff (PAS) staining produced following oxidation for 1 h at 4°C with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that this reagent completely oxidized all available sialic acid residues of either the sialo- or sialosulphoglycoproteins of human and rat colon or the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules orvicinal diols located on hexose, 6-deoxyhexose orN-acetylhexosamine residues (neutral sugars) of sialo- and sialosulphoglycoproteins. Furthermore, there was no extraction of epithelial glycoproteins or de-O-acylation of side chain substituted sialic acid residues. These data demonstrate that 0.4mm periodic acid in approximately 1m hydrochloric acid can be used as a specific reagent for the selective visualization of sialic acids in the PAS procedure.Studies of the mechanism of the oxidation of neutral sugars with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that their lack of PAS reactivity was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is suggested that the selectivity of 0.4mm periodic acid in approximately 1m hydrochloric acid is a result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line

Summary Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheat-germ agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(1–3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosac-charide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

Scanning electron microscopy of the disruption of tobacco hornworm, Manduca sexta, midgut by Bacillus thuringiensis endotoxin

The ingestion of Bacillus thuringiensis crystal endotoxin by Manduca sexta causes the destruction of both goblet and columnar cells of the midgut. One hour after ingestion, the microvilli show pathological effects. Nearly complete destruction of the goblet and columnar cells has taken place after 4 hr exposure to the toxin.     

Correlative histochemical study providing evidence for the dual nature of human colorectal cancer mucin

Summary Luminal secretions within colorectal cancers have been assumed to be the counterpart of normal goblet cell mucin. The aim of this study was to establish whether secretory material within colorectal cancers may in fact be traced to different lineages: goblet cells and columnar cells. The distribution of the apomucins MUC1 and MUC2, non-O-acetylated sialic acid and the carbohydrate structures sialosyl Tn, Tn, Lewis^x, sialosyl Lewis^x and Lewis^y was studied in normal colorectal mucosa and colorectal cancer specimens using standard histochemical techniques. Unmasking of MUC1 and MUC2 was achieved using periodic acid and saponification-neuraminidase-periodic acid pretreatment respectively. Within normal and malignant epithelium, correlations and/or co-localization could be demonstrated for goblet cells with MUC2, non-O-acetylated sialic acid, sialosyl Tn, Tn (Golgi region) and sialosyl Lewis^x, and for columnar cells with MUC1, Lewis^x, sialosyl Le^x, Tn (cytoplasm) and Lewis^y (UEA-1). The goblet cell spectrum was associated with mucin-like (type I) luminal secretions within cancers, whereas the columnar cell spectrum characterized non-mucin-like (type II) secretions and intracytoplasmic lumina. These data indicate that colorectal cancer mucin can be broadly separated into two types: secretory mucin linked to cells of goblet lineage and up-regulated membrane-associated mucin of presumed columnar cell origin. This revised version was published online in November 2006 with corrections to the Cover Date.     

Histomorphological and histochemical characteristics of the intestine of the Senegal sole, Solea senegalensis.

The Senegal sole, Solea senegalensis has been exploited extensively in aquaculture from different countries; at present an intensive production of larvae and adults is being achieved with some nutritional problems. Since this species displays very different life styles and feeding habits at different stages of its life history (larvae, metamorphosis, adults), and because digestive mucins are implicated in different physiological processes including: increase of digestive efficiency, promotion of macromolecules-absorption, buffering of intestinal fluid, prevention of proteolytic damage to the epithelium and defence against bacteria, etc., we studied the histomorphological aspects, as well as the histochemical distribution of carbohydrates, (PAS, Alcian Blue), proteins (Bromophenol Blue), lipids (Oil Red O, Black Sudan B) and glycoproteins (Horseradish peroxidase-conjugated lectins) in the intestinal epithelium of adult Solea senegalensis specimens. Our data are compared with those obtained in larvae and adults of this and other fish species. Primary and secondary folds, microvilli of the intestinal enterocytes, as well as mucous or goblet cells were observed with a scanning electron microscope. Enterocytes and mucocytes of the intestine of adult Solea senegalensis were characterized by a rich supply of sugar and oligosaccharides. Carbohydrates (glycogen and mucins), proteins and lipids were present in cytoplasm and microvilli--brush border--of the enterocytes, which contain GalNAc, GlcNAc, Man, Glc and sialic acid-N-acetyl-D-galactosamine glycoconjugates. Intestinal mucous cells were strongly or weakly stained with Alcian Blue (pH 2.5 and 1). PAS reactivity was intense in numerous goblet cells, but some cells were PAS unreactive or weakly stained. Some goblet cells were positive for Bromophenol Blue but numerous cells were unstained; thus many proteins and possibly lipids may be conjugated with sugars. A similar reactivity to WGA and to neuraminidase-WGA was identified in some intestinal goblet, which were Con A unreactive, indicating the absence of Man and/or Glc and NANA glycoconjugates. GalNAc residues were only scarcely present in glycoproteins of some goblet cells.     

Histochemical identification of side chain substituted O-acylated sialic acids: the PAT-KOH-Bh-PAS and the PAPT-KOH-Bh-PAS procedures

Summary Two new methods, based on the original periodic acid-Thionin Schiff-saponification-periodic acid-Basic Fuchsin Schiff (PAT-KOH-PAS) technique of Cullinget al. (1976), have been devised for the histochemical identificantion of side-chainO-acylated sialic acids. In the first of these, the periodic acid-Thionin Schiff-saponification-borohydride reduction-periodic acid-Basic Fuchsin Schiff (PAT-KOH-Bh-PAS) procedure, the specificity of the original PAT-KOH-PAS technique was improved by: (a) extending, when necessary, the initial period of periodate oxidation, (b) increasing the period of exposure to Thionin Schiff reagent from 30 min to 4 h, (c) using a Thionin Schiff reagent prepared by a different method, (d) interposing a borohydride reduction step between the saponification and PAS steps and, (e) extending the period of oxidation in the final PAS step from 10 to 30 min. In the second procedure, the periodic acid-phenylhydrazine-Thionin Schiffborohydride reduction-periodic acid-Basic Fuchsin Schiff (PAPT-KOH-Bh-PAS), based on the periodic acid-phenylhydrazine-Schiff (PAPS) technique of Spicer (1961), blue Thionin Schiff staining was confined to sialic acid residues with oxidizable side chainvicinal diols by interposing a treatment with 0.5% (w/v) aqueous phenylhydrazine hydrochloride for 2 h at room temperature between the initial periodic acid oxidation and the Thionin Schiff steps of the PAT-KOH-Bh-PAS procedure. These procedures are discussed within the general framework of the methods available for the histochemical identification of side-chainO-acylated sialic acids.