Daigo Tsubokawa

A monoclonal antibody, PGM34, against 6-sulfated blood-group H type 2 antigen, on the carbohydrate moiety of mucin

Mucin, a major component of mucus, is a highly O‐glycosylated, high‐molecular‐mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site‐specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide‐alditols obtained from pig gastric mucin were fractionated by successive gel‐filtration, ion‐exchange, and normal‐phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide‐alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucα1–2Galβ1–4GlcNAc(6SO3H)β1–6(Fucα1–2Galβ1–3)GalNAc‐ol and Fucα1–2Galβ1–4GlcNAc(6SO3H)β1–6(Galβ1–3)GalNAc‐ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucα1–2Galβ1–4GlcNAc(6SO3H)β‐ sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.

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.

Evaluation of bifidobacterial adhesion to acidic sugar chains of porcine colonic mucins

The aim of this study was to assess the adhesion of Bifidobacterium strains to acidic carbohydrate moieties of porcine colonic mucin. Mucins were extracted and purified via gel filtration chromatography followed by density-gradient ultracentrifugation. The presence of sulfated and sialylated carbohydrates in mucins was shown by enzyme-linked immunosorbent assays using PGM34 and HMC31 monoclonal antibodies (mAbs), respectively. Adhesion of Bifidobacterium strains to mucin preparations was markedly affected by the degree of purification. In eight of 22 strains, we observed increased adhesion to mucin preparations purified by ultracentrifugation. Moreover, in some of these eight strains, adhesion to mucin was reduced by pretreatment with sulfatase and/or sialidase, and competitively inhibited by pretreatment with PGM34 and/or HCM31 mAbs. Our results showed that some Bifidobacterium strains adhered to sulfo- and/or sialomucin and were able to recognize carbohydrate structures of the mAbs epitopes. Graphical Abstract The nitrogen source of the culture medium altered the alternative splicing pattern of serine-type carboxypeptidase ocpG mRNA in Aspergillus oryzae.

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.

Induction of Sda-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(GalNAcβ1-4)Galβ1-4GlcNAcβ-, and sulphated H type 2 antigen, Fucα1-2Galβ1-4GlcNAc(6SO₃H)β-, 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.

The monoclonal antibody HCM31 specifically recognises the Sda 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(GalNAcβ1‐4)Galβ1‐4GlcNAcβ‐ (Sda 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‐Sda antibodies. Taken together, these results strongly suggest that HCM31 specifically recognizes mucin‐type oligosaccharides with the Sda 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 Sda antigen.

Establishment of a novel tick- Babesia experimental infection model

Ticks are potent vectors of many deadly human and animal pathogens. Tick-borne babesiosis is a well-recognized malaria-like disease that occurs worldwide and recently has attracted increased attention as an emerging zoonosis. Although the proliferation of Babesia organisms is essential in the vectors, their detailed lifecycle with time information for migration in ticks remains unknown. A novel study model for the elucidation of the migration speed of Babesia parasites in their vector tick, Haemaphysalis longicornis, has been developed using an artificial feeding system with quantitative PCR method. The detectable DNA of Babesia parasites gradually disappeared in the tick midgut at 1 day post engorgement (DPE), and in contrary increased in other organs. The results indicated that the Babesia parasite passed the H. longicornis midgut within 24 hours post engorgement, migrated to the hemolymph, and then proliferated in the organs except the midgut. This time point may be an important curfew for Babesia parasites to migrate in the tick lumen. We also visualized the Babesia parasites in the experimentally infected ticks and in their eggs using IFAT for detecting their cytoskeletal structure, which suggested the successful tick infection and transovarial transmission of the parasite. This model will shed light on the further understanding of tick-Babesia interactions.

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.

Positive phototropism is accelerated in Biomphalaria glabrata snails by infection with Schistosoma mansoni

Parasite-induced behavioral changes in their hosts favor to complete the lifecycle of parasites. Schistosome infection is also known to cause physiological changes in infected freshwater snail intermediate hosts. Here, we report, a novel phenomenon in which Schistosoma mansoni, a highly debilitating worm affecting millions of people worldwide, alters the phototropic behavior of Biomphalaria glabrata, the vector snail. S. mansoni-infection enhanced positive phototropism of vector snails and infected snails spent significantly more time in light. Possibly, these behavioral changes help the parasite to be released efficiently from the infected intermediate hosts, and to infect mammalian hosts.

Initial development of Babesia ovata in the tick midgut.

The initial development of Babesia ovata in the midgut of the vector tick Haemaphysalis longicornis has been demonstrated through in vitro and in vivo studies. Although the research on the partial developmental cycles of B. ovata in the tick midgut was performed in our previous study by using ticks fed on experimentally B. ovata-infected cattle, detailed information on the developmental stages of B. ovata in H. longicornis was limited. This report describes the sequential development of stages of B. ovata in an in vitro study using B. ovata-infected erythrocytes and tick midgut contents. The in vivo study also confirmed the developmental stages in the midgut contents of artificially B. ovata-infected ticks. In this observation, we have recognized the distinct forms of B. ovata developmental stages in the tick midgut; the aggregation forms and ray bodies with shorter spikes and light-stained cytoplasm were shown by Giemsa staining. The similarities and differences of the stages as compared to previous reports have been discussed.