Chemical and histochemical studies of normal and diseased human gastrointestinal tract. III. Changes in the histochemical and chemical properties of the epithelial glycoproteins in the mucosa close to colonic tumours

Summary Histochemical, chemical and histological studies were performed on 26 specimens of human colonic tumours and 62 specimens of mucosa taken at distances of 0.5–5.0 cm from the tumour. The tumour glycoproteins were divided almost equally between three anionic types, sulphomucin, sialomucin and mixed sialomucin and sulphomucin. All showed a reduction in staining for side chainO-acylated sialic acid. In 56% of the tumours, this was accompanied by loss of glycoprotein while, in 44%, abundant mucin was still present.Histochemical examination of the mucosal specimens indicated that in 24.2% the side chainO-acylated sialic acids did not differ from normal. In 41.9% there was a focal change and in 33.9% there was a generalized field reduction in the proportion of side chainO-acyl sialic acids. The latter were subdivided into moderate and severe. Chemical analyses correlated well with the histochemical classification of the mucosal specimens and showed that, on average, the classifications focal and severe field change were not due to sampling error. Forty-five per cent of the cases showed only focal change and 40% only field change. Mucosal specimens associated with 60% of the moderately differentiated tumours showed only focal change while those associated with 75% of well-differentiated tumours showed only field change. Abnormal patterns of staining for side chainO-acylated sialic acids (a) were largely independent of the distance from the tumour, (b) occurred in the presence of a normal pattern of staining for sialomucins and sulphomucins and (c) were associated with 61.4% of the specimens that showed no discernible evidence of histological abnormality. In contrast, only one specimen showed evidence of histological change without a corresponding change inO-acylated sialic acids. The data suggest that abnormal patterns of staining forO-acylated sialic acids may represent premalignant change but their precise significance and specificity requires further studies of non-neoplastic diseases of the colon.     

Chemical and histochemical studies of normal and diseased gastrointestinal tract. IV. A comparison of histochemical and chemical methods for the estimation of side chain O-acylated sialic acids

Summary Statistically significant correlations were obtained between a chemical assay for the proportion of colonic epithelial glycoprotein sialic acids with side chainO-acyl substituents and two histochemical methods, the PBT-KOH-PAS sequence (r _s=0.7485 forN=31,P=0.01, one-sided test) and the PAPT-KOH-Bh-PAS procedure (r _s=0.7024 forN=34). A positive correlation (r _s=0.8654 forN=30,P=0.01) was also obtained between the results of the two histochemical procedures. It is concluded that, on average, histochemical observations are a reliable semiquantitative comparative method for the estimation of side chainO-acetylated sialic acids.     

Histochemical studies of the colonic epithelial glycoproteins of the normal rabbit

Summary Two general classes of glycoproteins have been identified in the colonic epithelial cells of New Zealand white rabbits. Each is associated with an ultrastructurally distinct secretory cell. The first of these classes is found in cells, termed vesiculated columnar cells, characterized by electron-translucent vesicles, a small rough endoplasmic reticulum-Golgi complex and prominent microvilli. The glycoproteins of the vesiculated cells contain abundantO-sulphate ester, sialic acids with ester substituents at positions C-8 or C-9 (or with two or three side chain substituents) and neutral sugars withvicinal diols whose periodate oxidation is prevented by anO-acyl ester substituent(s). The second class of glycoproteins occurs in goblet cells characterized by electron-dense vesicles, an abundant rough endoplasmic reticulum, a well-developed Golgi apparatus and few, if any, microvilli. Goblet cells along the entire length of the crypts contain neutral sugars with periodate-oxidisablevicinal diols and a ferriferricyanide-reactive component. Cells in the upper halves of the crypts also contain components that are sulphated, Schiff-reactive and acid-fast. In the lower halves of the crypts, the goblet cells contain smaller quantities of the above components plus sialic acids, some of which possibly have anO-acyl substituent located at position C-8 or C-9 (or which have two or three side chainO-acyl substituents). It is suggested that the function of the glycoproteins from the vesiculated columnar cells is protective and that from the goblet cells is lubricative.     

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 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.     

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.     

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.     

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.     

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 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.     

Binding specificity of influenza C-virus to variablyO-acetylated glycoconjugates and its use for histochemical detection ofN-acetyl-9-O-acetylneuraminic acid in mammalian tissues

The specificity of influenza C-virus binding to sialoglycoconjugates was tested with various naturallyO-acetylated gangliosides or syntheticallyO-acetylated sialic acid thioketosides, which revealed binding to 9-O-acetylatedN-acetylneuraminic acid. Binding was also observed with a sample of Neu5,7Ac₂-GD3, however at a lower degree. Sialic acids with two or threeO-acetyl groups in the side chain of synthetic sialic acid derivatives are not recognized by the virus. In these experiments, bound viruses were detected with esterase substrates. Influenza C-virus was also used for the histological identification of mono-O-acetylated sialic acids in combination with an immunological visualization of the virus bound to thin-sections. The occurrence of these sialic acids was demonstrated in bovine submandibular gland, rat liver, human normal adult and fetal colon and diseased colon, as well as in human sweat gland. Submandibular gland and colon also contain significant amounts of glycoconjugates with two or three acetyl esters in the sialic acid side chain, demonstrating the value of the virus in discriminating between mono- and higherO-acetylation at the same site. The patterns of staining showed differences between healthy persons and patients with colon carcinoma, ulcerative colitis or Crohn's disease. Remarkably, some human colon samples did not showO-acetyl sialic acid-specific staining. The histochemical observations were controlled by chemical analysis of tissue sialic acids.Abbreviations BSA bovine serum albumin - BSM bovine submandibular gland mucin - HAU haemagglutination units - HPLC high-performance liquid chromatography - HPTLC high-performance thin-layer chromatography - Neu5Ac N-acetylneuraminic acid - Neu5,9Ac₂ N-acetyl-9-O-acetylneuraminic acid - Neu5,7,9Ac₃ N-acetyl-7,9-di-O-acetylneuraminic acid - Neu5,7,8,9Ac₄ N-acetyl-7,8,9-tri-O-acetylneuraminic acid - PBS phosphate-buffered saline - TLC thin-layer chromatography Dedicated to Prof. Dr Nathan Sharon on the occasion of his 70th birthday.     

Studies of the degraded carrageenan-induced colitis of rabbits. II. Changes in the epithelial glycoprotein O-acylated sialic acids associated with the induction and healing phases

Summary Rabbits fed freely with a 1% aqueous solution of degraded carrageenan developed a progressive colitis characterized after five days by severe inflammation and mucosal ulceration of the caecum. Histochemical and chemical studies indicated that there was a marked reduction in intracellular mucin and in the proportion of the epithelial glycoprotein sialic acids with substituents in the side chain and at position C₄. Changes in theO-acylated sialic acids occurred rapidly and, apparently, prior to either mucosal ulceration or a significant inflammatory response.Following the removal of carrageenan from the diet, there was evidence of progressive healing characterized by re-epitheliazation and a reduction in the inflammatory response until, at 12 days, the mucosa was comparatively normal. Healing was accompanied by an apparent increase in intracellular mucin and in the proportion of the epithelial glycoprotein sialic acids with substituents in the side chain and at position C₄. Animals sacrificed 20 days after withdrawal of the carrageenan showed a renewal of ulceration characterized by an active inflammatory process, congestion, haemorrhage, and an inflammatory exudate consiting of a massive aggregation of cosinophils together with lymphocytes and plasma cells. This was accompanied by a reduction in the proportion of side chain and C₄ substituted sialic acids.     

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.     

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.     

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)     

Identification of 9-O-acetyl-N-acetylneuraminic acid in normal canine pre-ocular tear film secreted mucins and its depletion in Keratoconjunctivitis sicca

O-Acetylated sialic acids have been reported in many sialoglycoproteins where they mediate a variety of immune and other biological events. We have previously demonstrated that the protective mucus barrier on the surface of the canine eye contains sialoglycoproteins. We have also investigated the occurrence of O-Acetylated sialic acids in these ocular mucins. Mucus aspirated from the surface of normal dog eyes and those with keratoconjunctivitis sicca (KCS) was fractionated into three pools by density gradient centrifugation. Sialic acids comprised 0.6–0.9% of the dry weight of the mucins isolated. The sialic acid profile in these pools was examined using HPLC. O-Acetylated sialic acids, mainly Neu5,9Ac₂, were detected in normal animals and made up 10–30% of the total sialic acids detected. A doubling of the sialic acid content was found in KCS mucins, but the level of 9-O-Acetylated sialic acid was reduced below 4% of total. Histological analysis of conjunctival tissue from normal and KCS dogs showed the presence of sialic acids, detected with the α(2–6) sialic acid-specific lectin Sambucus nigra, in the goblet cells and corresponding to the staining pattern for MUC5AC, the major ocular-secreted mucin gene product. In KCS animals a disruption of the normal pattern of conjunctival goblet cells was seen with preservation of the pattern of lectin binding observed in normal animals. Thus the data demonstrate the presence of mono-O-Acetylated sialic acids in normal canine ocular mucins and a loss of this population of sialic acids in dry eye disease in spite of a significant increase in total sialic acids in KCS mucin.     

Sialic acid in colonic mucin: An evaluation of modified PAS reactions in single and combination histochemical procedures

Summary Two new histochemical procedures for detecting sulphated and non-sulphated sialomucin in colonic mucosa were assessed: the saponification—Alcian Blue pH 1—periodic acid—phenylhydrazine—Schiff method (KOH—AB pH 1—PAPS) and the mild periodic acid modification of this (KOH—AB pH 1—mPAS). Using normal colonic mucosa obtained from 11 non-cancer patients, the mPAS and PAPS techniques were tested for specificity and reproducibility for staining sialic acid, either alone or in combination with Alcian Blue. A spectrophotometric method was devised to quantify the uptake of both Schiff and Alcian Blue stain by sections. At low temperature and pH5.5, the mPAS procedure had improved specificity over the PAPS procedure, and after saponification it could be used to stainO-acetyl-substituted sialic acid. When used in combination with Alcian Blue at pH 1, however, underestimation of the sialic acid content occurred owing to interference between Alcian Blue and Schiff dyes. Interference was even greater with KOH—AB pH1—PAPS procedure for both sialic acid and sulphate components. We conclude that caution must be exercised in interpretation of the staining results obtained with these new combination methods and that more accurate information on the sialic acid and sulphate content of colonic mucin is obtained by staining serial sections with the mPAS technique and Alcian Blue pH 1 alone.     

Modification of sialic acids by 9-O-acetylation is detected in human leucocytes using the lectin property of influenza C virus

Influenza C virus spike glycoprotein HEF specifically recognizesglycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid.The same protein also contains an esterase activity. Takingadvantage of these two properties, influenza C virus was usedas a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminicacid in human leucocytes. The binding of influenza C virus toleucocyte glycoproteins and gangliosides separated by sodiumdodecyl sulphate–polyacrylamide gel electrophoresis andthin-layer chromatography, respectively, was assayed using achromogenic esterase substrate. In this way, glycoproteins ofB-lymphocytes and T-lymphocytes were found to contain 9-O-acetylatedsialic acids. Of the various 9-O-acetylated gangliosides detected,one had the characteristics of 9-O-acetylated G^D3. The identificationof 9-O-acetylated sialic acids on distinct glycoproteins andglycolipids should be helpful in assigning a physiological roleto this sugar. O-acetylation gangliosides influenza C virus lymphocytes sialic acids     

Cell wall glycoproteins from Chlamydomonas reinhardii are sulphated

Abstract Both the two major structural cell wall glycoproteins and the soluble excreted glycoproteins of Chlamydomonas reinhardii Levine WT II/32 contain low levels (approx. 1–4%) of sugar O-sulphate esters, asymmetrically distributed within the molecules. Preliminary characterization of their structure is described through [³⁵S] sulphate labelling experiments. The function of the sulphated glycoproteins is discussed in terms of their structural role and their water retaining properties.     

Sialylglycoconjugates and sialyltransferase activity in the fungus Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen associated with systemic mycoses in up to 10% of AIDS patients. C. neoformans yeasts express sialic acids on the cell wall, where they play an anti-phagocytic role, and may represent a virulence factor at the initial phase of infection. Since the nature of the sialic acid-carrying components is undefined in C. neoformans, our aim in the present work was to identify sialylated molecules in this fungus and study the sialylation process. C. neoformans yeast forms were cultivated in a chemically defined medium free of sialic acids, to search for autologous sialylglycoconjugates. Sialylated glycolipids were not detected. Two glycoproteins with molecular masses of 38 and 67 kDa were recognized by Sambucus nigra agglutinin, an 2,6-sialic acid-specific lectin. The 67 kDa glycoprotein also interacted with Influenza C virus, but not with Limax flavus agglutinin, suggesting the presence of the 9-O-acetylated sialic acid derivative as a constituent of the oligosaccharide chains. A partially purified protein fraction from cryptococcal yeast forms was able to transfer sialic acid from CMP-Neu5Ac to both N-(acetyl-1-¹⁴C)-lactosamine and asialofetuin. Additional evidence for a sialyltransferase in C. neoformans was obtained through the reactivity of fungal proteins with rabbit anti-rat 2,6 sialyltransferase polyclonal antibody. Our results indicate that sialic acids in C. neoformans are linked to glycoproteins, which are sialylated by the action of a fungal sialyltransferase. This is the first demonstration of this biosynthetic step in pathogenic fungi. Published in 2003.