Normal Human Colonic Mucosa Research Articles

Expression of functional CXCR4 chemokine receptors on human colonic epithelial cells.

In addition to their role as regulators of leukocyte migration and activation, chemokines and their receptors also function in angiogenesis, growth regulation, and HIV-1 pathogenesis--effects that involve the action of chemokines on nonhematopoietic cells. To determine whether chemokine receptors are expressed in human colonic epithelium, HT-29 cells were examined by RT-PCR for the expression of the chemokine receptors for lymphotactin, fractalkine, CCR1-10, and CXCR1-5. The only receptor consistently detected was CXCR4 (fusin/LESTR), although HT-29 cells did not express mRNA for its ligand, stromal cell-derived factor (SDF-1alpha). Flow cytometric analysis with anti-CXCR4 antibody indicated that the CXCR4 protein was expressed on the surface of roughly half of HT-29 cells. CXCR4 was also expressed in colonic epithelial cells in vivo as shown by immunohistochemistry on biopsies from normal and inflamed human colonic mucosa. The mRNA for SDF-1alpha and other CC and CXC chemokines was present in normal colonic biopsies. The CXCR4 receptor in HT-29 cells was functionally coupled, as demonstrated by the elevation in [Ca2+]i, which occurred in response to 25 nM SDF-1alpha and by the SDF-1alpha-induced upregulation of ICAM-1 mRNA. Sodium butyrate downregulated CXCR4 expression and induced differentiation of HT-29 cells, suggesting a role for CXCR4 in maintenance and renewal of the colonic epithelium. This receptor, which also serves as a coreceptor for HIV, may mediate viral infection of colonic epithelial cells.

Expression of glutathione S-transferase theta class isoenzymes in human colorectal and gastric cancers.

Glutathione S-transferases (GSTs) are a superfamily of detoxification enzymes, which play an important role in the protection of tissues against potentially harmful compounds. In humans, two theta class isoenzymes, GSTT1-1 and GSTT2-2, have been described so far. Both enzymes were claimed to have an important role in human carcinogenesis. In colorectal and gastric tissues, the expression of the other isoenzymes changes after malignant transformation. No data on the expression levels of the theta isoenzymes in these tissues are available. The aim of this study was to determine the protein levels of the two theta class isoenzymes in human colorectal and gastric cancers and paired normal tissue. Cytosolic fractions of normal and matched tumor tissue samples from 20 patients with colorectal or gastric adenocarcinomas were analyzed on immunoblots using specific antibodies against GSTT1-1 and GSTT2-2, respectively. In addition paraffin-embedded sections of these tissues were examined immunohistochemically for GSTT1-1 expression. In both types of tissue, theta class isoenzymes were highly expressed. Expression of GSTT1-1 was higher in gastric than in colorectal tissues. The GSTT2-2 levels were comparable in both tissues. A great interindividual difference in expression was demonstrated. In colon, there was no change in the theta class isoenzyme levels after malignant transformation. Gastric tumors had significantly lower expression of both theta class isoenzymes compared with the normal mucosa. In colon, GSTT1-1 was expressed in the enterocytes and goblet cells. In gastric tissues, staining was seen in upper and deeper mucous cells, chief cells and, to a lesser extent, in parietal cells. In both types of tumors, staining was seen in adenomatous cells. In conclusion, in both normal human colonic and gastric mucosa, GSTT1-1 and GSTT2-2 are present at high levels, whereas after malignant degeneration, expression is not influenced or is even downregulated.

Ovarian strumal carcinoid with severe constipation: Immunohistochemical and mRNA analyses of peptide YY

Functioning ovarian carcinoid tumors are well known to cause carcinoid syndrome. Recently, strumal and trabecular ovarian carcinoid tumors are reported to cause severe constipation possibly because of tumor-producing peptide YY (PYY). We studied a case of primary ovarian strumal carcinoid who had had severe constipation until the tumor was removed by surgical operation. Immunohistochemically, many tumor cells were strongly positive for PYY. By Northern blot and reverse transcription polymerase chain reaction analyses, PYY mRNA was expressed in a complete form as detected in normal human colon mucosa. From these findings, an ovarian strumal carcinoid is strongly suggested to express complete PYY mRNA and therefore complete PYY protein that results in severe constipation.

Modulation of base hydroxylation by bile acids and salicylates in a model of human colonic mucosal DNA: putative implications in colonic cancer.

Bile acids are believed to be involved in the formation of colonic cancer, and 5-aminosalicylic acid and other salicylates may have a protective role. The precise mechanisms of both actions are not known, but modifications (stimulation or inhibition) of basal or oxygen-radical induced DNA base hydroxylation as potential early events in tumor formation by these compounds may be involved in such actions. We, therefore, investigated whether: (1) bile acids in concentrations as they occur systemically or intraluminally are able to enhance basal or OH*-radical-stimulated base hydroxylation in DNA from calf thymus; (2) 5-aminosalicylic acid, its main intestinal metabolite N-acetyl-aminosalicylic acid and salicylate, the main aspirin metabolite, are able to inhibit this hydroxylation; and (3) DNA from calf thymus can be used as a model by comparing its base composition and hydroxylation with DNA from normal human colonic mucosa. We found an enhancement of the OH*-radical-induced DNA hydroxylation especially 8-OH adenine with 214.0%. On the other hand 5-ASA, N-acetyl-ASA, and salicylate showed a concentration-dependent inhibition of OH*-stimulated hydroxylation with IC50 between 0.04 +/- 0.01 mM (X +/- SD) and 1.3 +/- 0.1 mM. No effects were observed on basal hydroxylation. Electron spin resonance spectroscopy studies showed reduction of the corresponding base signals pointing to a scavenger mechanism. In DNA isolated from normal human colonic mucosa (N = 7) a similar base distribution was found as in calf thymus; hydroxylation was < or = 1.0% in both systems. From our results we conclude that DNA from calf thymus may serve as a model for human colonic mucosal DNA and that one of the carcinogenic actions of bile acids may be enhancement of oxygen-radical-induced DNA base hydroxylation, especially 8-OH adenine. The absence of effects under unstimulated conditions supports their role as cocarcinogens. The concentration-dependent inhibition of OH*-stimulated DNA hydroxylation by 5-ASA, salicylate, and N-acetyl-ASA may be a possible mechanism of chemoprevention.

Monoclonal antibody 91.9H raised against sulfated mucins is specific for the 3'-sulfated Lewisa tetrasaccharide sequence.

The IgG1hybridoma antibody, 91.9H, was originally raised against sulfated mucins isolated from normal human colonic mucosa. Previous studies have shown that the 91.9H antigen is expressed on normal colonic epithelial cells and the sulfomucins that they produce, but not in the normal small intestine and stomach. Tissue-specific changes occur in 91.9H antigen expression in disease: the antigen diminishes in colonic carcinomas, whereas in regions of gastric mucosa showing intestinal metaplasia and in gastric carcinomas, the antigen is expressed as a "neo-antigen." This report is concerned with elucidation, by the neoglycolipid technology, of the determinant recognized by antibody 91.9H using sulfated and sialyl oligosaccharides of Lewisa(Lea) and Lextypes, and analogs that lack sulfate, sialic acid, or fucose. Binding experiments with the lipid-linked oligosaccharides immobilized on chromatograms or on microwells, and inhibition of binding experiments with free oligosaccharides based on di-, tri- and tetrasaccharide backbones, show that the 91.9H antigenic determinant is based on a trisaccharide backbone, and consists of the 3'-sulfated Leatetrasaccharide sequence, which is a potent ligand for the E- and L-selectins. The antibody gives a relatively low signal with the 3'-sulfated non-fucosylated backbone, and has no detectable cross-reaction with the 3'-sulfated Lexisomer, nor with sialyl-Leaand -Lexanalogues. Antibody 91.9H is a valuable addition, therefore, to the repertoire of reagents for mapping details of the distribution, and determining the relative importance of sulfated and sialyl oligosaccharides as ligands for the selectins, in normal and pathological epithelia and endothelia.

Expression of alpha5 (CD49e) and alpha6 (CD49f) integrin subunits on T cells in the circulation and the lamina propria of normal and inflammatory bowel disease colonic mucosa.

Intestinal lamina propria T cells are believed to be derived, via the systemic circulation, from gut-associated lymphoid tissue. After migration into the lamina propria, T cells are capable of luminally directed migration following the loss of surface epithelial cells. For adhesion and migration within the extracellular matrix, T cells are likely to utilize the integrin family of adhesion molecules. The aim of this study was to quantitatively and qualitatively investigate the expression of alpha5 and alpha6 integrin subunits on the surface of human T cells that: (a) migrated out of the lamina propria, (b) remained resident within the matrix and (c) were present in the circulation. In both subpopulations of CD4 and CD8-positive T cells, from both normal and inflamed (inflammatory bowel disease) colonic mucosa, there were significantly fewer alpha5 and alpha6-positive cells than in the peripheral blood. In addition, there were significantly fewer alpha6 integrin molecules on the surface of CD4 and CD8-positive lamina propria T-cell subpopulations, compared with those in the circulation. Our studies suggest that, following migration into the lamina propria, there is down-regulation of alpha5 and alpha6 integrin-subunit expression on the surface of T cells. Molecules other than members of very late activation antigen-5 (VLA-5) (alpha5beta1) and VLA-6 (alpha6beta1) families of adhesion molecules are likely to be important in interactions with extracellular components in the lamina propria of normal and inflamed human colonic mucosa.

Effects of Acetaldehyde on Brush Border Enzyme Activities in Human Colon Adenocarcinoma Cell Line Caco‐2

The treatment of Caco-2 cells, a human colon adenocarcinoma cell line that closely resembles normal human small intestinal epithelial cells, with acetaldehyde resulted in significantly decreased activities of brush border enzymes sucrase, maltase, lactase, and gamma-glutamyltransferase; alkaline phosphatase activity was not affected. In the case of sucrase and maltase, the activities were also decreased by a combination of acetaldehyde and ethanol, although ethanol alone markedly increased them. The possibility that intraintestinal acetaldehyde, formed by intestinal microbes, might play a role in some small intestinal enzyme deficiencies observed earlier in alcoholics should therefore be considered. The mechanism by which acetaldehyde alters these enzyme activities remains unclear. The observation that acetaldehyde also disturbed cell polarization, an initial step in the process of differentiation in Caco-2 cells, indicates that acetaldehyde might decrease these enzyme activities by interfering with cell differentiation. Because ethanol and acetaldehyde metabolizing enzymes have not been previously studied from Caco-2 cells, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities were also measured from these cells, and their ALDH isoenzyme pattern was characterized. Like many cancerous cell lines, Caco-2 cells were found to express no ADH. They, however, possessed ALDH activity that was comparable with normal colonic mucosal activity and also expressed the same ALDH classes (ALDHs 1 to 3) than normal human colonic mucosa.

Enterocyte differentiation is compatible with SV40 large T expression and loss of p53 function in human colonic Caco-2 cells: Status of the pRb1 and pRb2 tumor suppressor gene products

Transfer of the SV40 large-T (LT) oncogene into isolated human and murine intestinal epithelial cells induced alterations of the ultrastructural organization and polarization of the resulting immortalized cell lines. We now demonstrate that the functional expression of the SV40 LT antigen in Caco-2 cells did not alter phenotypic markers of differentiation, including expression of villin, sucrase–isomaltase, brush border and dome formation. As compared to parental cells, the transfected Caco-2 LT9 cells exhibited similar growth curves and no invasive properties in vitro. The major oncogenic function of the SV40 LT antigen in transfected Caco-2 cells is associated with reduced latency times necessary for the manifestation of tumors in athymic nude mice. The Caco-2 cell line contained deleted and mutant p53 alleles (stop codon in position 204) and has no detectable truncated p53 protein by Western blot. Molecular complexes between the SV40 LT antigen and the retinoblastoma-related proteins pRb1 and Rb2 were clearly identified at the different phases of the growth curve. When compared to normal human colonic crypts, Caco-2 cell differentiation is related to partial redistribution of pRb1 into hypophosphorylated, antiproliferative forms. The pRb2 protein is found elevated in a subset of human colorectal tumors and their corresponding liver metastases. We conclude that: (1) Caco-2 cells exert a dominant control against the oncogenic functions of the LT antigen; (2) loss of p53 function is not restrictive for the establishment of polarity and differentiation of the enterocyte lineage; (3) the levels and phosphorylation status of the Rb1 and Rb2 proteins may play important roles in the proliferation and differentiation of normal and neoplastic human colonic mucosa.

Colon carcinoma glycoproteins carrying alpha 2,6-linked sialic acid reactive with Sambucus nigra agglutinin are not constitutively expressed in normal human colon mucosa and are distinct from sialyl-Tn antigen.

In human colon carcinoma, increased amounts of sialic acids have been found and correlated with tumor progression. Further, the degree of O-acetylation of sialic acid residues in normal mucosa is higher than in colon carcinoma. Thus, tumor-associated sialylated antigens may be constitutively expressed in O-acetylated form in normal mucosa unreactive with the respective monoclonal antibodies. We have earlier demonstrated a colon carcinoma-associated expression of alpha 2,6-linked sialic acid residues with the Sambucus nigra agglutinin (SNA). We report now that de-acetylation of normal and transitional colonic mucosa, in contrast to sialyl-Tn antigen, does not result in SNA binding. Further, the alpha 2,6-linked sialic acid recognized by SNA is distinct from that of sialyl-Tn antigen. This is confirmed by Northern blotting detecting transcripts for alpha 2,6 sialyltransferase of N-glycoproteins and measurement of activity for this sialyltransferase. Blot analysis by SNA of colon carcinoma cells revealed few reactive glycoproteins. Quantitative differences in lectin labeling and sialyltransferase activity were found in HCT116 colon carcinoma cell sub-lines. Our data suggest that SNA binding in human colon carcinoma is due to de novo expression of a specific sialic acid present on selected glycoproteins.

Colon carcinoma glycoproteins carrying α 2,6‐linked sialic acid reactive with Sambucus Nigra agglutinin are not constitutively expressed in normal human colon mucosa and are distinct from sialyl‐tn antigen

In human colon carcinoma, increased amounts of sialic acids have been found and correlated with tumor progression. Further, the degree of O-acetylation of sialic acid residues in normal mucosa is higher than in colon carcinoma. Thus, tumor-associated sialylated antigens may be constitutively expressed in O-acetylated form in normal mucosa unreactive with the respective monoclonal antibodies. We have earlier demonstrated a colon carcinoma-associated expression of α 2,6-linked sialic acid residues with the Sambucus nigra agglutinin (SNA). We report now that de-acetylation of normal and transitional colonic mucosa, in contrast to sialyl-Tn antigen, does not result in SNA binding. Further, the α 2,6-linked sialic acid recognized by SNA is distinct from that of sialyl-Tn antigen. This is confirmed by Northern blotting detecting transcripts for α 2,6 sialyltransferase of N-glycoproteins and measurement of activity for this sialyltransferase. Blot analysis by SNA of colon carcinoma cells revealed few reactive glycoproteins. Quantitative differences in lectin labeling and sialyltransferase activity were found in HCT116 colon carcinoma cell sub-lines. Our data suggest that SNA binding in human colon carcinoma is due to de novo expression of a specific sialic acid present on selected glycoproteins.Int. J. Cancer 70:575–581. © 1997 Wiley-Liss Inc.

Novel alternative splicing predicts a secreted extracellular isoform of the human receptor-like protein tyrosine phosphatase LAR

RT-PCR was used to examine the expression of LAR (encoding the leukocyte-common antigen-related protein tyrosine phosphatase) in normal human colon mucosa, and colon polyps and tumors. Although the LAR protein was not detected in the colon in a previous immunohistochemical study, amplification of a region of LAR between the most membrane proximal (eighth) fibronectin type-III (FN-III) repeat and the transmembrane domain demonstrated LAR expression in all samples, but showed no difference in expression within matched samples from each patient examined. An additional minor fragment amplified in all reactions was consistently observed in colon and various cell line samples using this and two other LAR-specific sets of primers. Cloning and sequencing of the fragment identified it as deriving from a novel alternatively spliced form of LAR containing a retained intron of 85 bp. This intron encodes an additional 13 amino acids followed by an in-frame stop codon, thus its retention is predicted to give rise to a secreted LAR extracellular region isoform(s). LAR transcripts containing the intron were detected by RNase protection assay of colon samples and were present in most human tissues examined by Northern analysis. A protein in colon tumor extract was recognized by antiserum raised to the intron-encoded sequence. Soluble isoforms of the LAR extracellular immunoglobulin (Ig)-like/FN-III repeat-containing region could have a biological function distinct from those isoforms localized at the cell surface and/or coupled to intracellular phosphatase activity.

Characterization of the CCK-B/gastrin-like receptor in human colon cancer.

The gastrointestinal peptide, gastrin, tonically stimulates growth of human colon cancer cells in vivo and in vitro, and does so in a receptor-mediated fashion. This study defined the nature of gastrin binding in human colon cancer using [3H]L-365,260, a specific cholecystokinin B (CCK-B)/gastrin antagonist found to block gastrin's effects on growth. Following elucidation of optimal binding conditions (e.g., pH, time, and temperature) in log phase HT-29 human colon cancer cells, specific and saturable binding with a dissociation constant of 4.8 +/- 0.7 nM and a maximal binding capacity (Bmax) of 320 +/- 120 fmol/mg protein, consistent with a single binding site, was recorded. Binding was localized to the membrane fraction. Exposure to gastrin or receptor antagonist decreased and increased, respectively, the Bmax. Competition experiments indicated that L-365,260 was 25- and 200-fold more effective at displacing radiolabeled L-365,260 than gastrin and cholecystokinin, respectively. In contrast to log phase cells, the Bmax was decreased by 67 to 76% in confluent and postconfluent cultures. Binding activity was observed in other cell lines examined, as well as in xenografts and colon cancers obtained at surgery. Binding in normal human colonic mucosa was 10-fold less than in colon cancer. These results provide the first comprehensive identification and characterization of a CCK-B/gastrin-like receptor in human colon cancer.

Rapid onset of apoptosis in vitro follows disruption of beta 1- integrin/matrix interactions in human colonic crypt cells

BACKGROUND & AIMS: Apoptosis by loss of adherence is a recently described phenomenon termed "anoikis." beta 1, integrins are heterodimeric surface molecules mediating adhesion to the extracellular matrix. The aim of this study was to address whether anoikis accounts for the elimination of senescent enterocytes and, if so, whether beta 1 integrins are involved. METHODS: Whole crypts were isolated from normal human colonic mucosa and examined in vitro. RESULTS: The vast majority of cells in resuspended crypts rapidly underwent apoptosis within 4 hours. Apoptosis was partially inhibited when cells had contact with collagen I-coated membranes or when whole crypts were embedded in a collagen gel. Preincubation of crypts with an inhibiting anti-beta 1 antibody before readhesion caused a much higher apoptotic rate. Confocal microscopy of embedded crypts revealed two critical zones of high sensitivity to temporary loss of adherence: the base of the crypts where stem cells are supposed to reside and the crypt mouth including the surface epithelium. CONCLUSIONS: Survival of colonic epithelia crucially depends on matrix adhesion and is likely to be guaranteed by beta 1-integrin/ matrix interaction. The data strongly suggest that anoikis is the way senescent colon cells die. (Gastroenterology 1996 Jun;110(6):1776-84)

Platelet activating factor: release from colonic mucosa in patients with ulcerative colitis and its effect on colonic secretion.

Inflammatory mediators have been implicated in the pathophysiology of ulcerative colitis. They may stimulate intestinal secretion and contribute to the production of diarrhoea. Platelet activating factor (PAF) may be responsible for a high proportion of this secretory response. Biopsy specimens from inflamed and quiescent mucosa of patients with ulcerative colitis and normal human colonic mucosa were cultured or co-cultured. The release of PAF, prostaglandin E2, and leukotriene D4 into the culture medium was measured and the ability of this culture medium, from inflamed and normal tissues, to influence secretion in rat colonic mucosa was assessed. PAF was liberated by inflamed tissue. Its release from quiescent but not normal tissue was stimulated by medium in which inflamed mucosal biopsy tissues had been cultured and by exogenous bradykinin and 5-hydroxytryptamine, but not by histamine. PAF stimulated eicosanoid production. The rise in short circuit current produced in vitro by inflamed tissue culture medium was inhibited by the PAF receptor antagonist (CV 6209) (46%) (32.4 (2.9) v 17.5 (1.19) muA.cm-2, p < 0.005) and further by combined cyclooxygenase and lipoxygenase inhibition (indomethacin plus ICI 207968) (58%) (32.4 (2.9) v 13.6 (1.9) muA.cm-2, p < 0.005). Mepacrine and hydrocortisone attenuated considerably the electrical response evoked by medium from inflamed mucosa to a similar extent (32.4 (2.9) v 6.3 (1.2) v 5.1 (0.9) muA.cm-2, p < 0.001). These data suggest that PAF accounted for 46% of the culture medium secretory effect. Thus, any attempt to block its release in patients with ulcerative colitis may have only a partial effect on their symptoms.

Expression and characterization of CD46, membrane cofactor protein, in human colonic mucosa

CD 46, membrane cofactor protein (MCP), is a membrane regulatory glycoprotein of the complement system, and acts as a cofactor of factor I, which inactivates C3b and C4b bound on autologous cell membrane. MCP is present on human peripheral blood cells except erythrocytes, fibroblasts, epithelial and endothelial cells, and has been proved to exist in other organs including gastrointestinal tract recently. In this study the expression and characterization of MCP on normal human colonic mucosa was investigated. Immunohistochemical study showed MCP in the colonic mucosal epithelium. Western blot analysis revealed that MCP protein was expressed as a broad band of 50-65 kDa in all cases and another faint band of 43-46 kDa in 3 out of 20 cases, though the latter band was highly suspected to be originated from contaminated mononuclear cells in the colon.

Ornithine decarboxylase levels in patients with normal colonic mucosa.

We describe a systematic examination of ornithine decarboxylase activity in 120 colonic mucosal samples which were obtained from 20 subjects without colonic disease to establish the normal mean and standard deviation from proximal to distal colon. Ornithine decarboxylase activity was determined by releasing CO2 from DL-[1-14C]ornithine. The mean ornithine decarboxylase levels (CO2 liberated) ranged from 0.26 +/- 0.08 nmol/h.mg protein in the caecum to 0.44 +/- 0.16 nmol/h.mg protein in the rectum. There was no difference between sex and age. Ornithine decarboxylase was not stimulated by guanosine 5'-triphosphate. alpha-Difluoromethylornithine showed an ornithine decarboxylase inhibition of 97.1%. Ornithine decarboxylase activity can be measured with reliable precision and reproducibility. The knowledge of the normal range of ornithine decarboxylase activity in normal human colonic mucosa is necessary for the determination of ornithine decarboxylase activity in pathological findings, especially in malignant transformation.

Biosynthesis of Platelet-Activating Factor in Normal and Inflamed Human Colon Mucosa: Evidence for the Involvement of the Pathway of Platelet-Activating Factor Synthesis De Novo in Inflammatory Bowel Disease

1. Platelet-activating factor can be synthesized by two distinct biochemical pathways and is degraded by a number of enzymes, the first step of which is deacetylation by a specific acetyl hydrolase. 2. The biochemical pathway of platelet-activating factor synthesis de novo and the first step in platelet-activating factor degradation have been investigated for the first time in incubates of normal human colon mucosa and in inflamed mucosa from patients with inflammatory bowel disease. 3. In the presence of 100 mumol/l CDP-choline and 100 mumol/l hexadecyl acetyl glycerol, homogenates from inflamed mucosa synthesized significantly greater platelet-activating factor [851 +/- 574 pmol/mg of protein (mean +/- SEM) in 90 min incubation] than normal mucosa [105 +/- 61 pmol/mg of protein in 90 min incubation] (P < 0.05). 4. Under the same conditions of assay, the percentage turnover to inactive lyso-platelet-activating factor was similar in inflamed mucosa (35.5 +/- 9.4%) and normal mucosa (42.7 +/- 8.5%) in 90 min (P > 0.05). 5. The identity of platelet-activating factor was confirmed by HPLC, by its mobility on TLC and by the ability of WEB 2170, a selective platelet-activating factor receptor antagonist, to block its platelet-aggregatory action. 6. These findings confirm the presence of the pathway for the synthesis de novo of the potently proinflammatory platelet-activating factor in human colon mucosa in inflammatory bowel disease.

Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease.

Recent studies have suggested that nitric oxide (NO.), the product of nitric oxide synthase in inflammatory cells, may play a part in tissue injury and inflammation through its oxidative metabolism. In this study the colonic generation of oxides of nitrogen (NOx) and nitric oxide synthase activity was determined in ulcerative colitis and Crohn's disease. Colonic biopsy specimens were obtained from inflammatory bowel disease patients and from normal controls. Mucosal explants were cultured in vitro for 24 hours and NOx generation was determined. Nitric oxide synthase activity was monitored by the conversion of [3H]-L-arginine to citrulline. Median NOx generation by inflamed colonic mucosa of patients with active ulcerative colitis and Crohn's colitis was 4.2- and 8.1-fold respectively higher than that by normal human colonic mucosa. In ulcerative colitis and Crohn's colitis nitric oxide synthase activity was 10.0- and 3.8-fold respectively higher than in normal subjects. Colonic NOx generation is significantly decreased by methylprednisolone and ketotifen. The decrease in NOx generation by cultured colonic mucosa induced by methylprednisolone suggests that NO synthase activity is induced during the culture and the steroid effect may contribute to its therapeutic effect. Enhanced colonic NOx generation by stimulated nitric oxide synthase activity in ulcerative colitis and Crohn's disease may contribute to tissue injury.

Synthesis of O-glycan core 3: characterization of UDP-GlcNAc: GalNAc-R beta 3-N-acetyl-glucosaminyltransferase activity from colonic mucosal tissues and lack of the activity in human cancer cell lines.

UDP-GlcNAc: GalNAc-R beta 3-GlcNAc-transferase (core 3 beta 3-GlcNAc-T, where GlcNAc is N-acetyl-D-glucosamine, GalNAc is N-acetyl-D-galactosamine and T is transferase) is expressed in a tissue-specific fashion and is high in normal colonic tissue, but downregulated in colon cancer. To further study the control of this enzyme, we examined the activity in pig, rat and human colonic tissues, and several human cancer cell lines. The enzyme was difficult to solubilize by detergents and was extremely unstable in the solubilized form. Using synthetic derivatives of the GalNAc-R substrate, we showed that the specificity of the enzyme in normal rat and human colonic mucosa requires all the substituents of the GalNAc-sugar ring of substrates for maximal activity. Core 3 beta 3-GlcNAc-T was significantly influenced by the structure of the aglycon group. None of the inactive substrate derivatives could inhibit the activity. N-Iodoacetamido-galactosamine alpha-benzyl was a weak substrate and significantly inhibited the incorporation of GLcNAc into GalNAc alpha-benzyl by human colonic homogenates. Surprisingly, none of the colonic cancer cell lines or any other cancer and leukaemia cells examined exhibited detectable activity of the enzyme, although a number of other glycosyltransferase activities involved in O-glycan biosynthesis were active. Mixing experiments did not reveal an endogenous inhibitor in HL60 cells or an activator of core 3 beta 3-GlcNAc-T in human colonic mucosa. Thus, the lack of core 3 beta 3-GlcNAc-T in human colonic mucosa. Thus, the lack of core 3 beta 3-GlcNAc-T activity in cancer cell lines may be due to cell transformation or cell culturing.

The high-affinity folate receptor of normal and malignant human colonic mucosa.

The hypothesis that folate depletion is a risk factor for development of colonic neoplasia prompted us to study the presence of a putative folate receptor in human colon mucosa. Binding of 3H-folate to normal and malignant mucosa studied by equilibrium dialysis was of high-affinity type (K = 10(10) L/mol) and displayed apparent positive cooperativity. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. As compared to methotrexate, 5-formyltetrahydrofolate was a potent inhibitor of binding. Gel filtration revealed a 25 kDa and a 100 kDa peak of folate-binding activity. Immunoreactivity studies were performed with rabbit antibodies against human 25 kDa milk folate-binding protein. Immunoblotting showed a single band at 65 kDa, and tissue sections exhibited immunostaining of mucosal areas. The present folate receptor with characteristics similar to those of other high-affinity folate-binding proteins may serve as a regulator of intracellular folate concentration in colon mucosa.