Showing papers by "Anthony P. Corfield published in 1995"

Glycosylation patterns of mucins in colonic disease

Abstract: Glycosylation patterns of mucins in colonic disease A. P. Corfield*, N. Myerscough*, M. Gough*, I. Brockhausent, R. Schaued and C. Paraskevas *Department of Medicine Laboratories, Bristol Royal Infirmary, Bristol BS2 8HW, U K , +Department of Pathology and Microbiology, School of Medical Sciences, Bristol BS8 ITD, U K , $Biochemistry Department, Hospital for Sick Children, Toronto M5G I X8, Canada, and §Biochemisches lnstitut I0 Universitat Kiel, D-24098 Kiel, Germany 840

South Asian and European colitics show characteristic differences in colonic mucus glycoprotein type and turnover.

Abstract: South Asians in Britain have a high incidence of ulcerative colitis and a low incidence of colorectal cancer. The pattern of mucus production in 12 South Asian and 16 European colitics and a control group of 19 South Asians was studied. Three types of mucin were identified after organ culture of colonic biopsy specimens with a dual label of [3H]-glucosamine and sodium [35S]-sulphate: type A had a high [35S]:[3H] ratio and high incorporation ([3H] dpm/micrograms DNA > 500); type B had a low ratio and high incorporation; and type C had low incorporation but with either high (C1) or low (C2) ratios. European colitic mucins show a significant reduction in the level of sulphation detected by mucin histochemistry with high iron diamine/Alcian blue staining, together with predominantly type B or C2 mucins (low sulphation). South Asian colitics showed histochemically normal patterns of high sulphation and largely type A and C1 mucins (high sulphation). There was no correlation of mucin type with disease activity index in either ethnic group. The appearance of apparently normal mucin in patients with ulcerative colitis may be a useful marker for the identification of a subgroup at low risk of colorectal cancer.

Expression of mucin genes in ulcerative colitis.

Abstract: Eight different human mucin (MUC) genes have been identified. The major mucin produced and secreted by the colorectal mucosa is believed to be MUC2 11-31. We have examined the colonic mucins from normal and ulcerative colitis (UC) patients in order to determine whether the changes identified in mucin glycosylation 141 are also reflected at the level of mucin gene expression in the colonic mucosa. Amino-acid analysis of mucin glycopolypeptide and in situ hybridization with 8 MUC gene probes have been used to this end. Tissue obtained at surgery was processed within 30 min of collection. Tissue samples used to prepare mucin glycopolypeptide were irrigated with PBSprotease inhibitor buffer and scraped. The extracted mucus was separated as the Vo fraction on Sepharose CL 28 chromatography and this fraction was exhaustively digested with pronase. After chromatography on Sephadex G150 the excluded peak containing the glycopolypeptide was pooled and used for analysis 151. Amino acids, monosaccharides and sulphate were determined in these fractions as before [6,7] The expression of MUCl, 2,3,4,5B, X, 6 and 7 was examined by in situ hybridization in routine colonic tissue from a histopathology archive and specimens were obtained by pancolectomy and partial colectomy. Three disease groups were studied, those with ulcerative colitis (UC), UC with dysplasia and UC with carcinoma. In situ hybridization was performed by the method of Levy and Lightman.[S] using antisense 48mers to the tandem repeat sequences of mucin genes 121. Oligonucleotides were end labelled with a terminal deoxytransferase kit (Boehringer Mannheim) and [35S] deoxy-(a-thio) adenine triphosphate at 35 MBq/ mmol (Amersham International, Amersham). Controls included mucosal and non-mucosal tissues and hybridisation with a non-mucin gene related antisense probe TRK, to a neurotrophin receptor. Colorectal tissue was fixed in 4% paraformaldehyde in PBS pH 7.4 for up to 2 days and Paraffin embedded. Sections were deparaffinised and incubated with 9Op1 hybridisation buffer containing 1xlWcpm of [35S] labelled probe for 1620 hours at 45T, followed by stringent washing at 65% Analysis of the glycopolypeptide amino acids obtained from 24 control and 13 UC patients showed a close correlation with the predicted ratio of serine : threonine : proline expected for the MUC2 tandem repeat (Table 1). There was no significant difference in the ratio between the control and UC groups. However, a significant reduction of proline 146*14 and 103+13 (~4.04) and threonine 181k18 and 118*17 ( ~ 0 . 0 2 ) nmole/mg dry weight was detected between control and UC groups respectively. Results from in situ hybridization showed normal, significant expression of MUCZ and MUC4 with low levels of MUC3 and MUCl. Levels of the remaining MUC genes tested were at background. Table 1 Ratio of serine threonine and proline in glycopolypeptide and predicted from mucin gene MUC tandem reDeats