Neuraminic acid-specific modification and tritium labelling of gangliosides.

1. A crude ganglioside mixture and pure GM1 and GD1a from bovine brain grey matter were prepared on a large scale. 2. The C7- and G8-analogues of NeuNAc were prepared from Collocalia mucoid and their structures established by gas-liquid chromatography and mass spectrometry. 3. Using model compounds in addition to various gangliosides, the conditions for the periodate oxidation and subsequent borohydride reduction of gangliosides were investigated with regard to the yield of C7- and C8-analogues of NeuNAc and the integrity of other monosaccharides in the oligosaccharide chain. These conditions were optimised to yield maximum C8-NeuNAc production and low C7-NeuNAc formation. Thus products were obtained which closely resemble the native gangliosides. 4. Using boro [3H] hydride, ganglioside derivatives with high specific radioactivity were prepared for the first time, containing either NeuNAc and labelled C8-NeuNAc or mainly labelled C7-NeuNAc depending on the prevailing conditions.

Rat colonic mucosal cell sialic acid metabolism in azoxymethane-induced tumours.

Colonic tissue was examined from normal (control) rats and azoxymethane- (carcinogen-) treated animals. Tumour-bearing colons from azoxymethane-treated rats were divided into malignant and non-malignant areas. Mucosal cells were prepared from the three types of colonic tissue and then examined for DNA and protein content and for the activities of ten enzymes involved in sialic acid metabolism. Enzyme activities were related to either the protein or the DNA content of fractions. The DNA content of cell homogenates was significantly different between tumour and non-malignant tissue and between both these tissues and normal mucosa. The protein content of the 100000 X g membrane pellet and supernatant fraction did not vary significantly between normal and non-malignant material but both these tissues differed significantly from tumour tissue. Significant variation between normal control and tumour tissue was detected at all levels of sialic acid metabolism, including N-acetylhexosamine interconversion and phosphorylation, sialic acid formation and activation, CMP-NeuAc breakdown and transfer and sialic acid release from glycoconjugates. The results indicate that major changes at all levels of sialic acid metabolism are associated with malignancy in rat colonic mucosa. Some of these changes are apparent in non-malignant mucosa and may reflect a pre-malignant state.

The specificity of viral and bacterial sialidases for alpha(2-3)- and alpha(2-6)-linked sialic acids in glycoproteins.

The anomeric specificity of six sialidases (Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, Newcastle disease virus, fowl plague virus and influenza A2 virus sialidases) was assessed with sialylated antifreeze glycoprotein, ovine submandibular gland glycoprotein and alpha 1-acid glycoprotein, resialylated specifically in alpha(2-3) or alpha(2-6) linkage with N-acetylneuraminic acid or N-glycolylneuraminic acid using highly purified sialyltransferases. The rate of release of sialic acid from these substrates was found to correlate well with the specificity observed earlier with the same sialidases using small oligosaccharide substrates, i.e., alpha(2-3) glycosidic linkages are hydrolyzed faster than alpha(2-6) linkages, with the exception of the enzyme from A. ureafaciens. Sialidase activity was higher with N-acetylneuraminic acid when compared with N-glycolylneuraminic acid. The studies also showed that the core oligosaccharide and protein structure in glycoproteins may influence the rate of release for different glycosidic linkages.

The role of mucins in host-parasite interactions: Part II - helminth parasites.

Some parasites express mucin-like molecules. These have possible roles in attachment and invasion of host cells and in the avoidance of host immune processes. Enzymes of parasite origin might also facilitate infection, either by degrading host mucus barriers or by generating binding sites on host cells. Host mucins have roles in preventing parasite establishment or in parasite expulsion. They, in turn, might be exploited by parasites, either as sources of fuel or binding sites, or as host-finding targets. Here, we describe the biochemical properties of mucins and mucin-like molecules in relation to interactions (established and putative) between helminth parasites and their hosts.

Mucins in the gastrointestinal tract in health and disease.

Mucins form part of the dynamic, interactive mucosal defensive system active at the mucosal surface of the gastrointestinal tract. They are carbohydrate rich glycoproteins with unique molecular structure and chemical properties. The family of mucin (MUC) genes has 13 members that can be divided into secreted and membrane-associated forms each with characteristic protein domains and tissue specific glycosylation. Biosynthetic pathways have been described for the secreted and membrane-associated mucins and their eventual degradation and turnover. Mucins are present at all mucosal surfaces throughout the body in typical combinations and relate to the demands of organ function. Patterns of MUC gene expression with gastrointestinal site specific glycosylation are clearly important but are not yet well defined. Mucin production during fetal development shows distinct patterns that may correlate in many cases with neoplastic expression in adult life. An increasing number of protective proteins have been identified that appear in the adherent mucus layer at the mucosal surface. These proteins are co-secreted with mucins in some cases, interact with mucins at a molecular level through peptide and carbohydrate sites or benefit from the viscoelastic, aqueous environment afforded by the mucus gel to effect their defensive roles. The mechanism of many of these interaction remains to be elucidated but is clearly part of an integrated innate and adaptive mucosal defensive system relying on the mucins as an integral component to provide a mucus gel. Recent improvements in the description of MUC gene expression and mature mucin synthesis in the healthy gastrointestinal tract has formed a basis for assessment of mucosal disease at sites throughout the tract. Pathological patterns of mucin expression in disease appear to follow tissue phenotype, so that gastric and intestinal types can be defined and appear in metaplasia in e.g. esophagus and stomach. Adaptation of previous mucin based, histochemical classification of intestinal metaplasia to assess MUC gene expression has proved helpful and promises greater value if reliably combined with mucin linked glycosylation markers. Few changes in MUC gene expression or polymorphism have been detected in inflammatory bowel diseases in contrast to malignant transformation. Glycosylation changes however, are evident in both types of disease and appear to be early events in disease pathogenesis. Review of the major mucosal diseases affecting the gastrointestinal tract in childhood reveals parallel patterns to those found in adult pathology, but with some novel conditions arising through the developmental stages at lactation and weaning. The impact of bacterial colonization and nutrition at these stages of life are important in the evaluation of mucosal responses in pediatric disease.

Membrane-associated mucins in normal human conjunctiva.

PURPOSE: To examine the presence of specific membrane-associated mucins in normal human conjunctiva. METHODS: Glycoconjugates were extracted from membranes with two detergents: octylglucoside and Triton X114. Mucins were separated by cesium chloride density gradient centrifugation. Size was assessed by gel filtration on Sepharose CL2B and charge by ion-exchange chromatography on MonoQ. Cross reaction with antibodies against mucin gene products was assessed in blots of electrophoresis gels. RESULTS: Extraction of total tissue membranes yielded material with a buoyant density typical of mucins. Gel filtration showed material reacting with antimucin antibodies in a range of molecular sizes. Agarose electrophoresis confirmed the presence of MUC1 and MUC4 and the absence of MUC2 or MUC5AC. Isolation of membrane mucins by sequential, exhaustive extraction with octylglucoside followed by Triton X114 suggested the existence of mucins in different membrane environments. Reagents to carbohydrate epitopes revealed high mobility material, comigrating with MUC1 and MUC4. Low mobility membrane-bound mucins did not cross-react with any antibodies to mucin genes known to be expressed in human conjunctiva. CONCLUSIONS: Membrane-associated mucins are distinct from secreted mucins in normal human conjunctiva. MUC1 and MUC4 mature products decorate the membranes of conjunctival epithelial cells. Their segregation between octyl glucoside and the detergent and aqueous phases of Triton X114 suggests a variety of membrane anchoring modes.

Polydispersity of normal human conjunctival mucins.

PURPOSE: To isolate all constituent mucins from human conjunctival mucus. METHODS: Mucins were extracted from human conjunctiva in guanidine hydrochloride and protease inhibitors. The mucins were isolated by density gradient centrifugation, gel filtration, and ion exchange chromatography. Throughout purification, the mucin profile was monitored by agarose electrophoresis and vacuum blotting. Blots were probed for peptide and carbohydrate epitopes. The latter included IE3 and TKH2 specific for Tn and sialyl-Tn, respectively, considered tumor-related antigens. In vivo impression cytology specimens of normal conjunctival goblet cells also were probed with the same reagents. Oligosaccharides were released from isolated mucins by alkaline beta-elimination and then size fractionated. RESULTS: Human conjunctival mucins consist of at least three size populations; the largest is excluded on Sepharose CL2B. The two largest populations are polydisperse. Their overall electrophoretic pattern is conserved between individuals. Similar charge distributions were detected in different buoyant density ranges from the density gradient centrifugation: a less charged population containing three components and a highly charged population with two components on agarose electrophoresis. Cross-reaction with IE3 and TKH2 was detected throughout purification in the largest mucins, which were presumably mature, and in impression cytology. Oligosaccharides from mucins in each buoyant density were largely in the monosaccharide and disaccharide range, consistent with Tn and sialyl-Tn standards. CONCLUSIONS: Secreted human conjunctival mucins are polydisperse, with discrete components appearing consistently in pooled and individual samples. They have a unique oligosaccharide pattern containing Tn and sialyl-Tn. This indicates normal roles in normal human ocular mucins for these antigens, which are disease markers in other tissues.

Studies on the interaction of Clostridium perfringens sialidase with sialic acid linked to the internal galactose in monosialogangliotetraosyl ceramide.

Investigation of the action of highly purified Clostridium perfringens sialidase on ganglioside II3Neu5Ac-Gg4Cer and its oligosaccharide II3Neu5Ac-Gg4, in the presence and absence of sodium cholate, extend earlier results obtained with impure enzyme fractions. Sialidase labeled with 125I was found to bind to various ganglioside substrate micelles, including II3Neu5Ac-Gg4Cer, and to mixed ganglioside-sodium cholate micelles. No binding occurred between the enzyme and the ganglioside-derived oligosaccharide II3Neu5Ac-Gg4, even when radioactive II3Neu5Ac-Gg4-[3H]ol was used. The binding of sialidase to micellar substrate is a condition for enzymic hydrolysis. Correspondingly, II3Neu5Ac-Gg4Cer and II3Neu5Ac-Gg4Cer-sodium cholate micelles were hydrolyzed by the enzyme but II3Neu5Ac-Gg4 was not. Ganglioside oligosaccharide analogues containing an amino function at the reducing terminus or between two oligosaccharide chains, II3Neu5Ac-Gg4-NH2 and (II3Neu5Ac-Gg4)2NH, were hydrolyzed in the absence of cholate. A synthetic analogue of II3Neu5Ac-Gg4Cer containing only the fatty acid moiety and not the sphingosine residue (I1-deoxy-I1-stearamido-II3-monosialo-gangliotetraitol ) behaved as the ganglioside in the presence and absence of sodium cholate.

Lack of mucin MUC5AC field change expression associated with tubulovillous and villous colorectal adenomas.

BACKGROUND: MUC5AC is a secreted mucin aberrantly expressed by polypoid colorectal adenomas. It has been hypothesised that the "normal" surrounding colorectal mucosa expresses MUC5AC as a field change phenomenon that can be used to predict adenoma recurrence following resection. AIM: To determine if there is a field change of de novo MUC5AC expression in histologically normal rectal mucosa adjacent to villous and tubulovillous adenomas, and thus whether MUC5AC expression can be used as a marker of early tumour recurrence. METHODS: In a prospective cohort study paired mucosal biopsies of adenomatous and macroscopically "normal" mucosa were obtained from 11 patients with villous and 11 patients with tubulovillous adenomas who underwent primary resection for purpose of cure. The tissues were studied to determine MUC5AC gene expression by immunohistochemistry and in situ hybridisation. Patients were followed up by flexible sigmoidoscopy to detect the presence of early local recurrence. RESULTS: 10 villous adenomas showed mature MUC5AC glycoprotein and all 11 expressed MUC5AC mRNA. Five tubulovillous adenomas showed mature MUC5AC glycoprotein and 10 expressed MUC5AC mRNA. Neoexpression of the MUC5AC mucin gene was not detected in any of the mucosal biopsies taken adjacent to either villous or tubulovillous adenomas, even in three patients with early, locally recurrent disease. CONCLUSIONS: Aberrant MUC5AC gene expression is not a "field change" in the colorectal mucosa in patients with rectal adenomas and therefore cannot be used to predict local recurrence of villous and tubulovillous adenomas.

Preliminary study pointing out a significant alteration in the biochemical composition of MUC2 in colorectal mucinous carcinoma.

OBJECTIVES: In this study, we characterized colonic MUC2 mucin from a mucinous carcinoma cell line and tried to find out carcinoma-associated alterations by comparing the results with those obtained from its benign phenotype previously. DESIGN AND METHODS: The molecular size distribution of the extracted molecules and their reactivity with two different MUC2 polypeptide antibodies indicated the presence of precursor and mature forms of the mucin in both cell lines. Isopycnic density gradient centrifugation gave good resolution of mature and precursor forms of MUC2 as assessed by agarose gel electrophoresis. Using this approach, we compared the different forms of MUC2 between benign and malign colonic cells. RESULTS: In the comparison, we detected some aberrant glycosylated MUC2 molecules in mucinous carcinoma cell line. Agarose gel electrophoretic analysis of the low-density fractions indicated that these molecules are more charged than precursors, however, they are smaller and/or less glycosylated than mature MUC2 molecules. CONCLUSION: The identification of unusual partially glycosylated forms of the major colonic mucin MUC2 is novel and unexpected. Implication of defective processes in the post translational modification/ processing of MUC2 opens a new field in the cancer mucin biology.

Human preocular mucins reflect changes in surface physiology.

BACKGROUND/AIMS: Mucin function is associated with both peptide core and glycosylation characteristics. The authors assessed whether structural alterations occurring during mucin residence in the tear film reflect changes in ocular surface physiology. METHODS: Ocular surface mucus was collected from normal volunteers as N-acetyl cysteine (NAcCys) washes or directly from the speculum after cataract surgery. To assess the influence of surface health on mucins, NAcCys washings were also obtained from patients with symptoms, but no clinical signs of dry eye (symptomatics). Mucins were extracted in guanidine hydrochloride (GuHCl) with protease inhibitors. Buoyant density of mucin species, a correlate of glycosylation density, was followed by reactivity with anti-peptide core antibodies. Mucin hydrodynamic volume was assessed by gel filtration on Sepharose CL2B. RESULTS: Surface fluid and mucus contained soluble forms of MUC1, MUC2, MUC4, and MUC5AC and also the same species requiring DTT solubilisation. Reactivity with antibodies to MUC2 and MUC5AC peaked at 1.3-1.5 g/ml in normals, while dominated by underglycosylated forms in symptomatics. Surface mucins were predominantly smaller than intracellular species. MUC2 size distributions were different in symptomatics and normals, while those of MUC5AC were similar in these two groups. CONCLUSIONS: A reduction in surface mucin size indicates post-secretory cleavage. Dissimilarities in surface mucin glycosylation and individual MUC size distributions in symptomatics suggest changes in preocular mucin that might precede dry eye signs.

Colorectal carcinogenesis: sequential steps in the in vitro immortalization and transformation of human colonic epithelial cells (review).

The development of colorectal cancer is an excellent example of the complex multistage nature of carcinogenesis and most colorectal cancers are thought to develop from adenomas. In this paper we have reviewed in vitro models developed in our laboratory for the study of human colorectal carcinogenesis. For these studies epithelial cell lines have been isolated from hereditary and sporadic colorectal adenomas representing different stages in tumour progression. Karyotypic analysis has shown specific abnormalities of chromosomes 1, 7, 14, 17, 18 and 22 to occur in these premalignant adenoma cell lines. The majority of cell cultures derived from small adenomas (less than 1 cm in diameter) senesced whereas the larger adenomas (greater than 2 cm in diameter) were more likely to give rise to immortal cell lines indicating that the acquisition of in vitro immortality occurs at a relatively late stage of colorectal carcinogenesis. Abnormalities of chromosome I have been implicated in tumour progression and in the in vitro immortalization of colorectal adenomas. Furthermore, several stages have been described in the transformation of an adenoma cell line PC/AA to a tumorigenic phenotype. Sodium butyrate and the potent carcinogen N-methyl-N-nitro-nitrosoguanidine (MNNG) were used in this transformation. Sodium butyrate is proposed to act as a possible promoter of colorectal carcinogenesis, and MNNG to cause the further genetic changes required for the conversion of the premalignant cells to a carcinoma. Markers to study the progression of an adenoma cell line to a tumorigenic phenotype in vitro include in vitro immortalization, aneuploidy, clonogenicity, resistance to the inhibitory effects of sodium butyrate, anchorage independent growth, ras gene activation, production of active proteinases and tumorigenicity in athymic nude mice. A role for a constitutively produced tumour promoter in colorectal carcinogenesis is discussed together with the possibility that different events are involved in the development of sporadic versus hereditary tumours due to the importance of the microenvironment in hereditary cancer. Our in vitro progression provides the first experimental evidence for the adenoma to carcinoma sequence and the cytogenetic evidence suggests that it is relevant to in vivo carcinogenesis.

Conjunctiva : organ and cell culture.

The conjunctiva is the mucous membrane of the eye. It is a delicate transparent epithelium with its own stroma overlying the tough white sclera, which forms the ball of the eye. Only at the circular limbus, where the sclera meets the transparent cornea, and at the eyelids, is the conjunctiva firmly attached, thus permitting free movement of the eye The loose conjunctiva between these points of attachment folds into a blind sac deepest under the upper and lower lids. Removed in one piece from the eye, the conjunctiva is a flimsy sheet (usually with adherent fascial tissue called Tenon's capsule) with an 1l-mm circular defect in the center.

Application of a novel human cervical mucin-based assay demonstrates the absence of increased mucinase activity in bacterial vaginosis.

Enzymes produced in bacterial vaginosis (BV) have been proposed as possible mediators of pre-term birth. Most studies have concentrated on mid-trimester measurements of enzyme activity, and utilize synthetic substrates to measure enzyme activity, which may not accurately represent mucinase activity in vivo. We have developed a novel ELISA mucinase assay using biotinylated human cervical mucin as a substrate. The assay is rapid, sensitive and can be used to screen large numbers of samples. The new assay has been used to assess vaginal mucinase activities in 92 women <14 weeks gestational age with and without BV. No differences in mucinase activity were detected between normal and BV groups while significant elevation of sialidase and other glycosidases was confirmed as reported before. This study shows that significant mucinase activity is a normal event in the mucus barrier, but does not reflect changes identified for individual enzyme activities associated with BV.

Mucinase and sialidase activity of the vaginal microflora: implications for the pathogenesis of preterm labour.

Evidence linking bacterial vaginosis (BV) to chorioamnionitis and spontaneous preterm birth is mounting. Successful treatment of BV could reduce the rate of late miscarriage and preterm birth. Mucinase and sialidase activity have been implicated in the pathogenesis of BV. This study extends the work of previous studies to investigate sialidase, other known mucin degrading enzymes and overall mucin degrading activity in samples of vaginal fluid from women with and without BV. Samples from 31 women were diagnosed for BV, and tested for enzyme activity using established assays. Activity was recorded in all samples. Significant increases in activity were detected in BV samples for sialidase using a mucin (BSM P<0.005) and serum type glycoprotein (AGP P<0.005) substrates, beta-galactosidase (P<0.001), and beta-N-acetylhexosaminidase (P<0.01). No significant increases in BV patients were detected in O-glycanase, proteinase, arylesterase, sulphatase or whole mucinase activities. These results support the hypothesis that certain BV-associated enzymes may detrimentally affect the mucosal barrier, permitting bacteria access to the uterus.

Distribution of MUC1 in the normal human oral cavity is localized to the ducts of minor salivary glands.

The change in expression of MUC1 from health to disease forms the basis of its use as a potential disease marker. Previous attempts at isolating MUC1 from normal, healthy human oral mucosa have, however, drawn conflicting conclusions as to its presence. Furthermore, when MUC1 was detected in the oral glycocalyx, it was not clear which cells were synthesising it. We examined human oral glycocalyx using pooled buccal smears from 50 normal individuals. Following isopycnic density centrifugation and membrane extraction with octyl glucoside and saponin, MUC1 was detected with the polyclonal antibody CT1. Immunohistochemistry using antibodies CT1 and BC2 was performed on sections from eight labial, seven palatal, four buccal, three retromolar pad, three dorsum of tongue and two ventral surface of tongue biopsies. In-situ hybridisation using MUC1 and cytoplasmic tail oligoprobes on sections from four palatal, seven labial and two retromolar pad biopsies was also carried out. MUC1 mRNA could only be detected in the minor salivary mucous glands. MUC1 has already been identified in the ducts of normal parotid and submandibular gland, and our findings demonstrate a similar distribution in minor salivary glands. We conclude that when present in the normal oral glycocalyx, the only oral source of MUC1 is from cell membranes of the minor salivary glands.

Children with Hirschsprung's disease have an abnormal colonic mucus defensive barrier independent of the bowel innervation status.

The mucus gel layer in the colon plays an important role in the defensive mechanisms against pathogenic organisms. Mucin glycoproteins or mucins are the major component of this gel. We studied the mucins in patients who had Hirschsprung's disease (HD) by colonic mucosal organ culture with radioactive mucin precursors [35S]-sulphate and [3H]-glucosamine. The secreted and cellular mucus fractions were collected after 24-hour incubation, and mucins were purified by gel filtration. The ratio of incorporation of the precursors and their turnover were quantified. Purified mucins were tested against wheat germ agglutinin for total mucin turnover. We used nine aganglionic bowel samples, 10 ganglionic bowel samples from HD patients, and 13 age-matched normal controls. There were no significant differences in the three groups in ratio of incorporation. The turnover with both radioactive precursors was reduced in both aganglionic and ganglionic bowel of HD, these differences were significant in [35S]-sulphate incorporation in the cellular fraction, ganglionic bowel being most affected. Total mucin turnover, as assessed by reactivity with wheat germ aggultinin, was reduced in both HD groups, being significant in the cellular fraction, aganglionic bowel being the most affected. These results signify an abnormal mucus defensive barrier in the colon of HD patients, even in the ganglionic bowel, which is thought to be normal and is retained at the definitive pull-through operation. This abnormality may be an etiological factor in the pathogenesis of enterocolitis of HD.

Turnover of radioactive mucin precursors in the colon of patients with Hirschsprung's disease correlates with the development of enterocolitis.

BACKGROUND/PURPOSE: Mucin glycoproteins (mucins) in the colonic mucus gel layer interact with pathogens performing protective functions by a variety of mechanisms. It is recognised that patients with Hirschsprung's disease (HD) are prone to episodes of enterocolitis even after corrective surgery, the aetiology of which is poorly understood. The authors correlated the turnover of radioactive mucin precursors in organ culture of the proximal ganglionated colon at the time of pull-through with the development of postoperative enterocolitis. METHODS: The colonic mucins in the retained proximal ganglionated colon of nine HD patients at the time of pull-through were studied. Organ culture of intact mucosa was performed with radioactive mucin precursors 35S-sulphate and 3H-glucosamine. Mucins in the secretions and epithelial cells were then purified by gel filtration. Turnover of the isotopes was determined by relating radioactivity to tissue DNA content. These patients were followed up prospectively for a mean duration of 30.8 months. The patients were assigned to one of two groups according to the criteria of requiring hospital admission for enterocolitis during this period. There were five patients in the group that remained well after corrective surgery and four in the group that developed entercolitis. The turnover values of both radioisotopes were analysed for differences in the two groups of patients. RESULTS: Patients in the enterocolitis group had a median value for turnover of 331 dpm/microg DNA, and the group that was well had a median value of 2044 dpm/microg DNA. These differences were statistically significant (Mann-Whitney, P = .037). CONCLUSIONS: A reduced turnover of mucins as shown by incorporation of radioactive precursors will give rise to a defective colonic mucus-defensive barrier. It can be inferred that the lower the turnover, the more prone a patient is to postoperative enterocolitis. It is therefore possible that organ culture with radioactive mucin precursors of the proximal ganglionated mucosa performed at the time of pull-through has a predictive value in the development of postoperative enterocolitis.

Histochemical and genetic analysis of colonic mucin glycoproteins in Hirschsprung's disease.

BACKGROUND/PURPOSE: Mucin glycoproteins (mucins) recently have been shown to be deficient in the colonic mucosa of patients with Hirschsprung's disease (HD). The authors performed a detailed histo- and immunohistochemical analysis of mucins in the colonic mucosa and studied the expression of mucin genes to characterize histologically mucin quality and gene expression in HD compared with controls. METHODS: Paraffin-embedded 4-microm thick sections from patients with HD (n = 11 ganglionic, 10 aganglionic) and controls (n = 19) were taken. Slides were stained with mild periodic acid Schiff with and without saponification with KOH (reacts with O-actylated mucins), high iron diamine/alcian blue (differentiates sulphated v nonsulphated mucins), the monoclonal antimucin antibodies, PR3A5 (against di- and tri-O-acetylated sialic acids) and 91.9H (against sulphated mucins). O-acetylation and sulphation both confer an increased resistance of mucins to bacterial degradation and are thought to be important in the defensive function of the colonic mucus gel layer. In situ hybridization was used to study expression of the mucin genes MUC 1, 2, 3, 4, 5AC, 5B, 6, 7, and 8. [35S]-sulphate-labelled antisense oligonucleotide 48mer probes designed to the known tandem repeat domains of MUC genes were used. After hybridization and washing the slides were opposed to Hyperfilm MP for 7 days. The autoradiographs were scored by three independent observers for differences in expression and by image analysis. Those with positive findings were dipped in photographic emulsion, developed, and counterstained for photomicrographs. RESULTS: There were different patterns of staining dependent on the region of the colon and especially the age of the patient with three reagents. No significant differences in the histological staining pattern was detected between HD patients and controls. The colonic mucins in HD were found to be primarily O-acetylated and sulphated. The MUC gene expression was similar in patients and controls. MUC2 and 4 were strongly expressed, MUC1, 3, and 5B had moderate to weak expression, and MUC 5AB, 6, 7, and 8 had baseline expression. CONCLUSIONS: The mucin glycoproteins in children with HD, although quantitatively deficient, show no qualitative differences on histo- and immunohistochemical staining from normal controls. The expression of all the known mucin genes, the genetic control of mucin secretion, and the quality of mucins, is similar to normal controls.

Surface mucus in the non-glandular region of the equine stomach.

In horses, ulceration of the non-glandular region of the stomach is common and has been attributed to the lack of a protective mucus covering. This study aimed to determine whether the non-glandular region is covered by a mucus layer. A mixture of antibodies raised against human gastric mucin (MUC 5 AC) showed a tissue distribution in the glandular region of the equine stomach similar to that seen in humans. Dot blots of mucus from the glandular and non-glandular regions showed cross-reactivity with these antibodies. Various histological fixation and processing techniques were compared for their ability to preserve mucus in the non-glandular region. Fixing frozen sections on-slide for 20 seconds in 20 per cent formalin/1 per cent cetylpyridinium chloride was considered the best method. In conclusion, the equine stomach expresses a gene homologous to human MUC 5 AC. Its product is expressed as a neutral mucin, which is present in the mucus that covers both the glandular and non-glandular regions. Future comparison of mucus composition in the healthy and ulcerated stomach will improve our understanding of gastric ulceration in the horse.