Preparation of O-Glycans from Mucins Using Hydrazine Treatment.

Mucin glycomic analysis is crucial owing to the participation of mucin O-glycans in several biological functions. Liquid chromatographic analysis of fluorescently labeled glycans is an effective tool for glycomic analysis. The first step of this analysis involves the release of O-glycans from mucins. As no enzyme is known to release all glycans, chemical methods are required for the process; therefore, hydrazine treatment is a commonly used chemical method. It enables the release of O-glycans from mucin while preserving the aldehyde group at the reducing end. This ensures that the reducing end can be modified using fluorescent reagents. However, it is also accompanied by the degradation of the glycans through a process called "peeling." Here, we describe a method for releasing glycans from mucins using hydrazine treatment with minimal "peeling."

MALDI-TOF MS/MS Analysis of Permethylated O-Glycan Alditols Released from Mucins.

Mucin glycans are associated with the function of mucin in maintaining mucosal homeostasis. Therefore, the glycomic analysis of mucins is crucial. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most suitable methods for the glycomic analysis of mucin O-glycans. In this chapter, we describe methods for analyzing permethylated O-glycan alditols released from mucins by MALDI-TOF MS and MALDI-TOF tandem mass spectrometry (MALDI-TOF MS/MS).

Analysis of A4gnt Knockout Mice Reveals an Essential Role for Gastric Sulfomucins in Preventing Gastritis Cystica Profunda.

Gastric adenocarcinoma cells secrete sulfomucins, but their role in gastric tumorigenesis remains unclear. To address that question, we generated A4gnt/Chst4 double-knockout (DKO) mice by crossing A4gnt knockout (KO) mice, which spontaneously develop gastric adenocarcinoma, with Chst4 KO mice, which are deficient in the sulfotransferase GlcNAc6ST-2. A4gnt/Chst4 DKO mice lack gastric sulfomucins but developed gastric adenocarcinoma. Unexpectedly, severe gastric erosion occurred in A4gnt/Chst4 DKO mice at as early as 3 weeks of age, and with aging these lesions were accompanied by gastritis cystica profunda (GCP). Cxcl1, Cxcl5, Ccl2, and Cxcr2 transcripts in gastric mucosa of 5-week-old A4gnt/Chst4 DKO mice exhibiting both hyperplasia and severe erosion were significantly upregulated relative to age-matched A4gnt KO mice, which showed hyperplasia alone. However, upregulation of these genes disappeared in 50-week-old A4gnt/Chst4 DKO mice exhibiting high-grade dysplasia/adenocarcinoma and GCP. Moreover, Cxcl1 and Cxcr2 were downregulated in A4gnt/Chst4 DKO mice relative to age-matched A4gnt KO mice exhibiting adenocarcinoma alone. These combined results indicate that the presence of sulfomucins prevents severe gastric erosion followed by GCP in A4gnt KO mice by transiently regulating a set of inflammation-related genes, Cxcl1, Cxcl5, Ccl2, and Cxcr2 at 5 weeks of age, although sulfomucins were not directly associated with gastric cancer development.

Interleukin-13/interleukin-4 receptor pathway is crucial for production of Sda-sialomucin in mouse small intestinal mucosa by Nippostrongylus brasiliensis infection.

Mucin is a major component of mucus in gastrointestinal mucosa. Increase of specific sialomucins having Sda blood group antigen, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß-, is considered to be associated with expulsion of the parasitic intestinal nematode Nippostrongylus brasiliensis. In this study, we examined the relationship between interleukin (IL)-13 pathway and expression of Sda-sialomucins in small intestinal mucosa with N. brasiliensis infection. Nematode infection induced marked increases in small intestinal mucins that reacted with anti-Sda antibody in wild type (wt) mice. However, this increase due to infection was supressed in IL-4 receptor α deficient (IL-4Rα-/-) mice, which lack both IL-4 and IL-13 signaling via IL-4R, and severe combined immunodeficient (SCID) mice, which have defects in B- and T-lymphocytes. Analysis using tandem mass spectroscopy showed that Sda-glycans were not expressed in small intestinal mucins in IL-4Rα-/- and SCID mice after infection despite the appearance of Sda-glycans in the infected wt mice. Inoculation of recombinant IL-13 into the infected SCID mice restored expression of Sda-glycan. Our results suggest that the IL-13/IL-4R axis is important for the production of Sda-sialomucins in the host intestinal mucosa with parasitic nematode infection.

Comparison of Methods to Release Mucin-Type O-Glycans for Glycomic Analysis.

Mucin-type O-glycans (O-glycans) are one of the most common glycans attached to proteins. To develop an optimized glycomic analysis protocol, O-glycans were released from glycoproteins using hydrazine, ammonia, or sodium hydroxide treatment, followed by hydrophilic interaction liquid chromatography to evaluate O-glycan release. We found that porcine gastric mucin or bovine fetuin treated at 60 °C for 6 h with hydrazine gas in the presence of malonic acid yielded O-glycans with only a small amount of degraded, so-called "peeled" products. Ammonia treatment also yielded intact O-glycans but with additional peeled products containing GlcNAc at the reducing end. In contrast, sodium hydroxide treatment yielded mainly peeled glycans, including those containing GlcNAc at the reducing end. Importantly, O-glycans obtained from rat gastric mucin treated with hydrazine and labeled with anthranilic acid had a nearly identical profile following hydrophilic interaction liquid chromatography as permethylated O-glycan alditols analyzed by mass spectroscopy. Taken together, the data suggest that glycan release using hydrazine treatment, followed by high-performance liquid chromatography after fluorescent labeling, is a suitable method for glycomic analysis of mucin-type O-glycans.

A cysteine protease from Spirometra erinaceieuropaei plerocercoid is a critical factor for host tissue invasion and migration.

Sparganosis in humans caused by the plerocercoid larvae of Spirometra erinaceieuropaei is found worldwide, especially in Eastern Asia and the Far East. Previous studies have suggested that dissolution of plerocercoid body, plerocercoid invasion of host tissue, and migration are important processes for sparganosis progression. However, the mechanisms underlying these processes have yet to be determined. Here, we demonstrated the enzymatic property and involvement of a native 23kDa cysteine protease (Se23kCP), purified from plerocercoids, in sparganosis pathogenesis. Se23kCP is mature protease consisting of 216 amino acids and has a high sequence similarity with cathepsin L in various organisms. Se23kCP conjugated with N-glycans, which have a core fucose residue. Both cysteine and serine protease-specific activities were determined in Se23kCP and their optimal pHs were found to be different, indicating that Se23kCP has a wide range of substrate specificity. Se23kCP was secreted from tegumental vacuoles of the plerocercoid to host subcutaneous tissues and degraded human structural proteins, such as collagen and fibronectin. In addition, the plerocercoid body was lysed by Se23kCP, which facilitated larval invasion of host tissue. Our findings suggest that Se23kCP induces host tissue invasion and migration, and might be an essential molecule for sparganosis onset and progression.

Discrimination of rat Brunner's gland carbohydrate antigens by site-specific monoclonal antibodies.

Mucus produced and secreted by gastrointestinal mucosa contains various types of mucins equipped with unique sugar chains considered to play critical roles in protecting mucous membranes; therefore, the identification and verification of mucin sugar chains is important for understanding the underlying mechanisms. In our previous work, we generated three monoclonal antibodies (mAbs), RGM22, RGM26, and RGM42, which recognize sugar chains in rat gastric mucin. Here, we immunohistochemically analyzed the rat gastrointestinal mucosa and found that the antigens recognized by RGM22 and RGM42 were expressed in the rat antrum and Brunner's glands, whereas that recognized by RGM26 was detected in the antrum, but rarely in Brunner's glands. We found that these antibodies reacted with porcine gastric mucin-derived oligosaccharides bearing a common structure: GalNAcα1-3(Fucα1-2)Galß1-4GlcNAcß1-6GalNAc-ol. Moreover, epitope analysis revealed that RGM42 and RGM22 recognized α-linked GalNAc and GalNAcα1-3Gal, respectively, on the GalNAcα1-3(Fucα1-2)Gal structure, whereas RGM26 was specific for GalNAcα1-3(Fucα1-2)Gal. These results indicate that rat Brunner's glands express specific antigens bearing GalNAcα1-3Gal that are recognized by RGM22 and RGM42. Thus, RGM22, RGM26, and RGM42 with their unique antigen specificities could be useful tools for investigation of oligosaccharide diversity among mucins.

Malonic acid suppresses mucin-type O-glycan degradation during hydrazine treatment of glycoproteins.

Hydrazine treatment is frequently used for releasing mucin-type O-glycans (O-glycans) from glycoproteins because the method provides O-glycans that retain a reducible GalNAc at their reducing end, which is available for fluorescent labeling. However, many O-glycans are degraded by "peeling" during this treatment. In the current study, it was found that malonic acid suppressed O-glycan degradation during hydrazine treatment of bovine fetuin or porcine gastric mucin in both the gas and liquid phases. This is paradoxical because the release of O-glycans from glycoproteins occurs under alkaline conditions. However, malonic acid seems to prevent the degradation through its acidic property given that other weak acids also prevented the degradation. Accordingly, disodium malonate did not suppress O-glycan degradation. Application of this method to rat gastric mucin demonstrated that the majority of the major O-glycans obtained in the presence of malonic acid were intact, whereas those obtained in the absence of malonic acid were degraded. These results suggest that hydrazine treatment in the presence of malonic acid would allow glycomic analysis of native mucin glycoproteins.

Induction of Sd(a)-sialomucin and sulfated H-sulfomucin in mouse small intestinal mucosa by infection with parasitic helminth.

Mucin is a major component of mucus on gastrointestinal mucosa. Mucin alteration in the host is considered to be the principal event for expulsion of intestinal helminths. However, it is unclear what mucin alterations are induced by various helminth infections. In this study, the alterations of mouse small intestinal mucin after infection with two nematodes, Nippostrongylus brasiliensis and Heligmosomoides polygyrus, which parasitize the jejunal epithelium, and a cestode, Vampirolepis nana, which parasitizes the ileal epithelium, were examined biochemically and histologically using two anti-mucin monoclonal antibodies (mAbs), HCM31 and PGM34, which recognize Sd(a) antigen, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß-, and sulphated H type 2 antigen, Fucα1-2Galß1-4GlcNAc(6SO3H)ß-, respectively. The goblet cell mucins that reacted with HCM31 increased conspicuously on the jejunal mucosa concurrently with expulsion of N. brasiliensis. Increased levels of HCM31-reactive mucins were observed in the jejunal mucosa after H. polygyrus infection, despite the ongoing parasitism. Goblet cell mucins that reacted with PGM34 increased on the ileal mucosa during V. nana parasitism. Small intestinal goblet cells reacting with the two mAbs were not observed in non-infected mice, although sialomucins and sulfomucins were abundantly present. Additionally, the number of ileal goblet cells that reacted with the two mAbs was increased at the time of expulsion of heterophyid trematode. These results indicate that the type of specific acidic mucins expressed after infection varies among species of intestinal helminth, and, furthermore, that the relationship with worm expulsion is also different.

Essential role of gastric gland mucin in preventing gastric cancer in mice.

Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycans) having terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc). Previously, we identified human α1,4-N-acetylglucosaminyltransferase (α4GnT), which is responsible for the O-glycan biosynthesis and characterized αGlcNAc function in suppressing Helicobacter pylori in vitro. In the present study, we engineered A4gnt(-/-) mice to better understand its role in vivo. A4gnt(-/-) mice showed complete lack of αGlcNAc expression in gastric gland mucin. Surprisingly, all the mutant mice developed gastric adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absence of H. pylori infection. Microarray and quantitative RT-PCR analysis revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Il-11, and Hgf in the gastric mucosa of A4gnt(-/-) mice. Further supporting an important role for this O-glycan in cancer progression, we also observed significantly reduced αGlcNAc in human gastric adenocarcinoma and adenoma. Our results demonstrate that the absence of αGlcNAc triggers gastric tumorigenesis through inflammation-associated pathways in vivo. Thus, αGlcNAc-terminated gastric mucin plays dual roles in preventing gastric cancer by inhibiting H. pylori infection and also suppressing tumor-promoting inflammation.

Rapid and specific alterations of goblet cell mucin in rat airway and small intestine associated with resistance against Nippostrongylus brasiliensis reinfection.

The intestinal parasitic nematode Nippostrongylus brasiliensis is expelled rapidly from the rat in reinfection challenge compared with that of the primary infection owing to the host defense mechanisms raised against the pre-intestinal- and intestinal-stage larvae. We examined the relationship between the mucin alterations in airway and jejunal mucosae and the worm expulsion after third-stage larva reinfection. When rats had been inoculated with fourth-stage larvae and immunized with only the intestinal-stage worms for more than 8 days, the challenge larvae were expelled during the intestinal stage along with a rapid increase of the specific sialomucin in jejunal mucosa, without any effect on the bronchial mucus. When rats had been infected with third-stage larvae and immunized with only the pre-intestinal stage larvae by killing with antihelminthic, the challenge larvae were rejected during the pre-intestinal stage along with marked goblet cell hyperplasia and Muc5AC mucin hyperproduction on the bronchial mucosa, but not as a result of jejunal mucin alteration. Taking these finding together, immunization with pre-intestinal- and intestinal-stage worms independently increases the airway and intestinal goblet cell mucins, respectively, and in both cases, the mucin alterations may contribute to rapid worm expulsion upon reinfection.

The monoclonal antibody HCM31 specifically recognises the Sd(a) tetrasaccharide in goblet cell mucin.

Rat small intestinal goblet cell mucins reacting with monoclonal antibody HCM31 increase significantly during regeneration from experimental mucosal damage and at the period of expulsion of parasitic nematode, Nippostrongylus brasiliensis (N.b). The reduction in reactivity of HCM31 with mucin upon neuraminidase treatment, suggested that HCM31 recognizes sialylated oligosaccharide on mucin. HCM31-reactive sialomucins are therefore considered to play an important role in the physiological and pathological changes in the gastrointestinal mucosa. To determine the epitope for HCM31, oligosaccharide-alditols reacted with HCM31 were obtained from the small intestinal mucins of N.b-infected rats and purified by ion-exchange chromatography followed by normal-phase HPLC. Two HCM31-reactive oligosaccharide-alditols were obtained. Analyses using tandem mass spectrometry and NMR spectroscopy showed that these oligosaccharides were core 4 mucin-type oligosaccharides having a common tetrasaccharide sequence, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß- (Sd(a) blood group antigen). These structures were not found in the small intestinal mucin oligosaccharides from uninfected rats. This epitope specificity of HCM31 was also confirmed using previously established anti-GM2 and anti-Sd(a) antibodies. Taken together, these results strongly suggest that HCM31 specifically recognizes mucin-type oligosaccharides with the Sd(a) tetrasaccharide sequence. Immunohistochemical examination of human gastrointestinal tracts showed that HCM31 site-specifically stained the goblet cells in normal sigmoid colon and normal rectum, but the goblet cells stained with HCM31 were reduced in the corresponding cancer tissues. HCM31 seems to be useful for diagnosis of colonic cancer and for examining the function of secretory-type mucin with Sd(a) antigen.

Protective effect of geranylgeranylacetone against loxoprofen sodium-induced small intestinal lesions in rats.

Nonsteroidal anti-inflammatory drugs induce small intestinal ulcers but the preventive measures against it remain unknown. So we evaluated the effect of geranylgeranylacetone (GGA), a mucosal protectant, on both the mucus content and loxoprofen sodium-induced lesions in the rat small intestine. Normal male Wistar rats were given GGA (200 or 400mg/kg p.o.) and euthanized 3h later for measurement of mucin content and immunoreactivity. Other Wistar rats were given loxoprofen sodium (30mg/kg s.c.) and euthanized 24h later. GGA (30-400mg/kg p.o.) was administered twice: 30min before and 6h after loxoprofen sodium. The total mucin content of the small intestinal mucosa increased, especially the ratio of sialomucin, which increased approximately 20% more than the control level after a single dose of GGA. Loxoprofen sodium provoked linear ulcers along the mesenteric margin of the distal jejunum, accompanied by an increase in enterobacterial translocation. Treatment of the animals with GGA dose-dependently prevented the development of intestinal lesions, and bacterial translocation following loxoprofen sodium was also significantly decreased. GGA protects the small intestine against loxoprofen sodium-induced lesions, probably by inhibiting enterobacterial invasion of the mucosa as a result of the increase in the mucosal barrier.

Vulnerable sites and changes in mucin in the rat small intestine after non-steroidal anti-inflammatory drugs administration.

BACKGROUND AND AIMS: The location of mucosal damage and changes in mucin content in the rat small intestine following administration of non-steroidal anti-inflammatory drugs (NSAIDs) have not been well elucidated. METHODS: After subcutaneous administration of loxoprofen sodium (10-40 mg/kg), the small intestinal mucosa of male Wistar rats was evaluated macroscopically, histologically, and immunohistochemically by measuring the total mucin content and immunoreactivity for anti-mucin monoclonal antibody, HCM31, 1, 3, 7, and 14 days later. Changes in the number of enterobacteria invading the mucosa around the lesions were also determined. RESULTS: Loxoprofen sodium induced erosions and ulcers along the mesenteric margin of the distal jejunum. Early (≤6 h) mucosal lesions were small and round, located between the branches of the mesenteric arteries. In the jejunum, there was a transient increase in the total mucin content, and HCM31-positive mucin in the mucosa around the ulcers increased significantly on days 3 and 7, but in the ileum there were no marked changes and few ulcers. Bacterial translocation following loxoprofen sodium administration significantly increased, according to the site of the intestinal lesions. CONCLUSIONS: Vascularly compromised sites along the jejunal mesenteric margin are vulnerable to NSAIDs-induced damage and show increased numbers of enterobacteria in the NSAIDs-treated mucosa. Increased sialomucin content in the mucus around the lesions may play an important role in the healing of NSAIDs-induced intestinal lesions.

Nippostrongylus brasiliensis: increase of sialomucins reacting with anti-mucin monoclonal antibody HCM31 in rat small intestinal mucosa with primary infection and reinfection.

Infections with the parasitic helminth, Nippostrongylus brasiliensis, cause changes in rat small intestinal goblet cell mucin, particularly in the peripheral sugar residues of oligosaccharide. These changes may correlate with expulsion. In this study, we examined changes in mucin oligosaccharides caused by primary infection and reinfection with N. brasiliensis, using two monoclonal antibodies, HCM31 and PGM34, that react with sialomucin and sulfomucin, respectively. Enzyme-linked immunosorbent assay of jejunal mucins showed that the relative reactivity of mucins with HCM31, but not PGM34, increased up to 16 days after primary infection and 6 days after reinfection, the times when the worms were expelled from the rats. Immunohistochemical studies confirmed that goblet cells stained with HCM31 greatly increased at the time of worm expulsion. These results indicate that the marked increase observed in HCM31-reactive sialomucins may be related to expulsion of the worms.

Effects of indomethacin on the rat small intestinal mucosa: immunohistochemical and biochemical studies using anti-mucin monoclonal antibodies.

BACKGROUND: The luminal surface of the gastrointestinal tract is covered by a viscoelastic gel layer that acts as a protective barrier against the intraluminal environment. Because the situation of the small intestine has not been elucidated to the same degree as other sections, in this study, we investigated the effects of indomethacin on the rat small intestinal mucosa. METHODS: Male Wistar rats were given indomethacin 10 mg/kg s-c and sacrificed 1, 3, 7, or 14 days later. The small intestine was opened along the anti-mesenteric side, and examined macroscopically. Total mucin content in the small intestinal epithelium was measured and immunoreactivity was examined using anti-mucin monoclonal antibodies HCM31 and PGM34. RESULTS: Indomethacin caused punched out and linear ulcers located mostly along the mesenteric margin of the distal jejunum with sparing of the ileum. Histological examination showed sialomucin recognized by HCM31 increased on day 3 especially in the regenerating epithelium around the ulcer edge. Furthermore, the surface mucous gel layer displayed a multilaminated pattern, consisting of non-sulfated sialomucin-rich layers and sulfated mucin-rich layers, where both mucins had the common core protein, MUC2. Biochemical measurements also showed the total mucin content of the jejunum increased transiently and HCM31-positive mucin increased approximately 4 times greater than baseline on day 3, but no marked changes were observed in the ileum, with few ulcers observed. CONCLUSIONS: Indomethacin administration causes quantitative and qualitative change in jejunal mucin. In particular, sialomucin plays an important role in regenerating epithelium during the healing process following indomethacin-induced mucosal damage.

Evaluation of conditions for release of mucin-type oligosaccharides from glycoproteins by hydrazine gas treatment.

By using commercially available anhydrous hydrazine in the gas-phase, mucin-type oligosaccharides were released from porcine gastric mucin (PGM) and bovine fetuin. The data indicated that a certain amount of the oligosaccharides from PGM were further degraded. Despite this, the HPLC elution profile of the anthranilic acid (AA)-derivatized oligosaccharides obtained by the treatment with hydrazine at 65 degrees C for 6 h resembled those obtained from the alkaline-borohydride treatment, except for the additional disaccharide fractions derived from the core 1 side of the oligosaccharides by further degradation. The other degraded products derived from the core 2 side could not be derivatized by AA, therefore, not visible by fluorescence detection. Liberation of the oligosaccharides was incomplete by the hydrazine treatment for 6 h. Although almost complete liberation was achieved by extending the treatment to 18 h, the degraded products also increased. In this case, the addition of barium oxide to the reaction vessel decreased the degree of further degradation. Results similar to PGM were obtained from bovine fetuin, but with less degradation. Application of this method for the analysis of rat gastric mucin (RGM) obtained from the corpus and antral region revealed that RGM has a large oligosaccharide portion on the core 1 side.

Effects of combination treatment with famotidine and methylmethionine sulfonium chloride on the mucus barrier of rat gastric mucosa.

BACKGROUND AND AIM: In Japan, peptic ulcer disease (PUD) is treated clinically with a combination of a mucosal protectant and acid suppressants, but there is scant information regarding the effects of these drugs on normal gastric mucus cells. In the present study, the effects of co-administration of methylmethionine sulfonium chloride (MMSC) and famotidine on rat gastric mucus cells were investigated using both biochemical and histological methods. METHODS: Rats were divided into four groups: controls were given carboxymethylcellulose orally once daily for 7 days and the second, third and fourth groups were treated similarly with famotidine (famotidine group), MMSC (MMSC group) or famotidine plus MMSC (combination group). After killing the rats on the 8th day, the stomachs were removed and the biosynthesis and amount of mucin in different areas of the gastric mucosa were compared among groups. Using anti-mucin monoclonal antibodies, the mucin content and immunoreactivity were also compared. RESULTS: Both the biosynthesis and accumulation of mucin were significantly decreased in the famotidine group, but increased in the MMSC and combination groups. The amount and immunoreactivity of surface mucus cell-derived mucin were both reduced in the famotidine group, and increased in the MMSC and combination groups. There was no difference among the groups in the content and immunoreactivity of gland mucus cell-derived mucin. CONCLUSION: Famotidine-induced suppression of gastric surface mucus cell function is prevented by combined treatment with MMSC, raising the possibility of a more effective cure of PUD.

Effects of tea catechins on the gastrointestinal mucosa in rats.

Although tea catechins are known to exert a potent antiulcer effect on the alimentary tract, there is scant information concerning their effects on normal mucus cell functions. Using original anti-mucin monoclonal antibodies, we studied the influences of long-term administration of catechins on the quantity and quality of mucin in rat gastrointestinal mucosa. Administration of 0.5% tea catechins significantly increased the mucin content of the ileum, but not the stomach. An enzyme-linked immunosorbent assay (ELISA) showed no remarkable qualitative changes in gastric mucin, but a selective increase and decrease in sulfo- and sialomucins, respectively, in the ileum of rats administered catechins. The ELISA results were consistent with both the immunohistochemical findings and the high-iron diamine-alcian blue staining pattern. These findings indicate that tea catechins modulate ileal mucin metabolism in the ileal mucosa, suggesting that further studies focusing on the ileal epithelium will assist in further elucidation of the mechanism of catechin effects.

Changes in the mucus barrier of the rat during 5-fluorouracil-induced gastrointestinal mucositis.

OBJECTIVE: A frequent complication of antineoplastic chemotherapy (CT) is gastrointestinal (GI) mucositis. Although clinically this mucositis can be treated, data on the effect of CTon the mucosal defense mechanisms are scant, so the effects of 5-fluorouracil (5-FU) on mucin, one of the principal defense factors of the GI mucosa, were investigated. MATERIAL AND METHODS: 5-FU was administered orally to rats at a dose of 50 mg/kg once daily for 5 days. Using anti-mucin monoclonal antibodies, the immunoreactivity in different areas of the rats' GI tracts was compared, as well as the mucin content. Changes in the GI mucin during the process of recovery from the injury were also investigated. Immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) was used to determine whether or not the effects of 5-FU on cell proliferation contributed to the changes in mucin. RESULTS: 5-FU caused significant alterations of the immunoreactivity and content of mucin in the rat GI mucosa, especially in the jejunum. The jejunal mucin content was most markedly reduced on day 1 after drug withdrawal, and increased thereafter. By day 7, the content had transiently but significantly increased approximately 1.5-fold, and returned to the basal level by day 13. The number of PCNA-positive cells strikingly decreased at day 1, but by day 7 had increased approximately 2-fold, compared with the control. CONCLUSION: The activation of mucus cells in the jejunum, if appropriately manipulated, could lead to more effective prevention of CT-induced GI mucositis.