Kentaro Sugano

Identification of glycolipid receptors for Helicobacter pylori by TLC-immunostaining

Helicobacter pylori has been identified as a causative agent in active chronic gastritis. The receptor for this bacteria, however, is not known. It is likely that the receptor molecules may be glycosphingolipids* as shown in the cases of other bacteria. We explored this possibility by a thin‐layer chromatography (TLC)‐immunostaining method. Among glycosphingolipids extracted from human gastric mucosa, intact Helicobacter pylori specifically bound to PSO3‐GalCer and II3NeuAc‐LacCer, whereas no specific binding to neutral glycosphingolipids, which share the same ceramide moiety with PSO3‐GalCer or II3NeuAc‐LacCer, was demonstrated. Sonicated bacteria could still bind to II3NeuAc‐LacCer with comparable affinity. In contrast, the binding of bacteria of PSO3‐GalCer was greatly diminished upon sonication. These results suggest that each of the oligosaccharide moieties of II3NeuAc‐LarCer and PSO3‐GalCer may be specifically recognized by different ligand molecules of Helicobacter pylori.

Distinct Roles of Smad2-, Smad3-, and ERK-dependent Pathways in Transforming Growth Factor-β1 Regulation of Pancreatic Stellate Cellular Functions

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-β1 (TGF-β1) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-β1 mRNA expression level or the amount of autocrine TGF-β1 peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant-negative Smad2/3 attenuated PSC proliferation enhanced by dominant-negative Smad2/3 expression without altering their activation. Exogenous TGF-β1 increased TGFβ1 mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-β1, and consequently attenuated TGF-β1 enhancement of its own mRNA expression in PSCs. We propose that TGF-β1 differentially regulates PSC activation, proliferation, and TGF-β1 mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.

OLIGOMER FORMATION OF HISTAMINE H2 RECEPTORS EXPRESSED IN SF9 AND COS7 CELLS

A histamine H2 receptor, which had been mutated at its glycosylation site and tagged at its N‐terminus with an HA tag (HA‐H2 receptor), was expressed in Sf9 cells and COS7 cells. Immunoprecipitation and immunoblotting of HA–H2 receptors with αHA antibody revealed four bands of 31.5±2.5 kDa, 59.0±6.0 kDa, 80.5±4.5 kDa and 120 kDa. These bands were also detected by immunoblot using anti‐H2 receptor serum (C‐terminus). In addition, H2 receptors without the HA‐tag co‐immunoprecipitated with HA‐tagged H2 receptors devoid of the 51 C‐terminal amino acids, via immunoprecipitation with αHA antibody, when the two receptors were coexpressed. These results suggest that H2 receptors are present as receptor oligomers and that the C‐terminal portion is not involved in the formation of these oligomers.

Systematic analysis of glycosphingolipids in the human gastrointestinal tract: Enrichment of sulfatides with hydroxylated longer-chain fatty acids in the gastric and duodenal mucosa

The composition of the glycosphingolipids of the human gastrointestinal tract was studied. The major neutral glycosphingolipids were ceramide monohexosides (e.g., GalCer, GlcCer), LacCer, Gb3Cer, Gb4Cer and more polar ones with more than four sugars, whereas neither Gg3Cer nor Gg4Cer were present. The acidic glycosphingolipids consisted of sulfatides and gangliosides such as GM3, GM1, GD3 and GD1a. Also a large amount of sulfatides was found in the gastric mucosa and duodenum. The concentrations of sulfatides in the fundic mucosa, antral mucosa and duodenum amounted to 416.0, 933.8 and 682.9 nmol/g of dry weight, respectively, exceeding those in the gastric mucosa and kidney of other mammals. The major molecular species of the sulfatides were identified as I3SO3-GalCer with hydroxylated longer-chain fatty acids based on the analyses by gas-liquid chromatography and negative ion fast-atom bombardment mass spectrometry. In contrast, gangliosides in these regions showed a tendency to be lower than sulfatides, and the molar ratios of sulfatides to gangliosides were about 2.0, whereas those in other parts were less than 0.5. A high content of sulfatides in the gastric and duodenal mucosa, where mucosa is easily insulted by acid, pepsin and bile salts, may be closely related to their roles in mucosal protection.

Role of the C Terminus in Histamine H2 Receptor Signaling, Desensitization, and Agonist-induced Internalization

To evaluate the role of the histamine H2 receptor C terminus in signaling, desensitization, and agonist-induced internalization, canine H2 receptors with truncated C termini were generated. Wild-type (WT) and truncated receptors were tagged at their N termini with a hemagglutinin (HA) epitope and expressed in COS7 cells. Most of the C-terminal intracellular tail could be truncated (51 of 70 residues, termed T308mutant) without loss of functions: cAMP production, tiotidine binding, and plasma membrane targeting. In fact, the T308 mutant produced more cAMP than the WT when cell-surface expression per cell was equivalent. Pretreatment of cells with 10−5 m histamine desensitized cAMP productions via WT and T308 receptors to similar extents. Incubation of cells expressing WT receptors with 10−5 m histamine reduced cell-surface anti-HA antibody binding by approximately 30% (by 30 min, t 1/2 ∼ 15 min), but did not affect the B max of tiotidine in membrane fractions, which represents total receptor amounts, suggesting that WT receptors were internalized from the cell surface. In contrast, no internalization was observed with T308 receptors following histamine treatment. A mutant with a deletion of the 30 C-terminal amino acids, termed T329, was functional but was as potent as the WT in terms of cAMP production. Apart from being desensitized by histamine, the internalization of the receptor was indistinguishable from that of the WT. Internalization was observed in the T320 but not in T313 mutant, narrowing the region involved in internalization to that between Glu314 and Asn320 (ETSLRSN). Of these seven residues, either Thr315, Ser316, or both, were replaced with Ala. Thr315 and Ser316 are conserved among species. The mutation at Thr315 (but not that at Ser316) abolished internalization. Taken together, these results demonstrate that Thr315 is involved in agonist-induced internalization. Furthermore, the finding that T308 receptors were desensitized in the absence of internalization suggests that internalization and desensitization are meditated by independent mechanisms.

Gastrin stimulates the growth of gastric pit with less-differentiated features.

Gastrin stimulates the growth of gastric mucosa by increasing mostly its glandular region but is not known to induce the growth of a pit region where its major constituent cells, gastric surface mucous (GSM) cells, turn over rapidly. To investigate the effect of gastrin on GSM cells, we generated hypergastrinemic mice by expressing a human gastrin transgene. We obtained a hypergastrinemic mouse line whose average serum gastrin level is 671 ± 252 pg/ml (normal level <150 pg/ml). Gastrin-positive cells were found in the fundic mucosa. The gastric mucosa exhibited hypertrophic growth, which was characterized by an elongated pit with an active proliferative zone, but the glandular region containing parietal cells was normal or reduced in size. The GSM cells contained fewer mucous granules than those of control littermates and lost reactivity to the GSM cell-specific cholera toxin β-subunit lectin. GSM cells along the foveolar region and many mucous neck cells became Alcian blue positive, suggesting the appearance of sialomucin in these cells. We suggest that gastrin stimulates the growth of the proliferative zone of gastric glands, which results in the elongation of the pit region whose GSM cells exhibit less-differentiated features.Gastrin stimulates the growth of gastric mucosa by increasing mostly its glandular region but is not known to induce the growth of a pit region where its major constituent cells, gastric surface mucous (GSM) cells, turn over rapidly. To investigate the effect of gastrin on GSM cells, we generated hypergastrinemic mice by expressing a human gastrin transgene. We obtained a hypergastrinemic mouse line whose average serum gastrin level is 671 +/- 252 pg/ml (normal level <150 pg/ml). Gastrin-positive cells were found in the fundic mucosa. The gastric mucosa exhibited hypertrophic growth, which was characterized by an elongated pit with an active proliferative zone, but the glandular region containing parietal cells was normal or reduced in size. The GSM cells contained fewer mucous granules than those of control littermates and lost reactivity to the GSM cell-specific cholera toxin beta-subunit lectin. GSM cells along the foveolar region and many mucous neck cells became Alcian blue positive, suggesting the appearance of sialomucin in these cells. We suggest that gastrin stimulates the growth of the proliferative zone of gastric glands, which results in the elongation of the pit region whose GSM cells exhibit less-differentiated features.

Susceptibility of ganglioside GM1 to a new bacterial neuraminidase

Recently, a new bacterial neuraminidase (N-acetylneuraminate glycohydrolase (EC 3.2.1.1 8)) was isolated and purified from the culture filtrate of a non-pathogenic bacterium, Arthrobacter ureafaciens [1 ]. This preparation was reported to liberate Nacetylneuraminic acid (NeuNAc) from substrates containing the terminal a,2~3, a,2~6 and ct,2~8 linkages of the NeuNAc residue, and to show no activities of contaminating enzymes, such as protease, NeuNAcaldolase and other glycosidases [ 1 ]. However, the action of this neuraminidase on gangliosides has not yet been studied in detail. This report described the specific action of this new neuraminidase on ganglioside GM~, which was believed to be resistant to various neuraminidases of viral, bacterial and mammalian origin [2-5] . The new bacterial neuraminidase hydrolyzed not only ganglioside GM1 but an oligosaccharide having the same saccharide structure as GMI. Addition of . detergents, especially bile salts, resulted in a marked

Pancreatic carcinoma accompanied by pseudocyst: Report of two cases

Two cases of pancreatic cancer accompanied by pseudocyst are reported. Case 1 was a 60-year-old man who was admitted to our hospital complaining of left lower abdominal discomfort. A cystic lesion, about 3 cm in diameter, was found in the pancreatic tail by ultrasonography (US) and computed tomography (CT). No signs of chronic pancreatitis were found. At operation, an elastic, hard, white tumor, about 1 cm in diameter, was felt adjacent to the cystic lesion on the duodenal side. Histologically, this tumor was a duct cell carcinoma with an adjacent pseudocyst upstream of the pancreas. Case 2 was a 57-year-old man who complained of back pain and loss of body weight. US and CT examination revealed a cystic lesion, 11×7 cm in size, in the tail of the pancreas. Histological examination of the resected speciment revealed both a duct cell carcinoma, 3 cm in size, in the body of the pancreas and a pseudocyst, 9 cm in size. Pseudocysts accompanying carcinoma are thought to develop from obstruction of the pancreatic duct by the carcinoma, followed by intraductal high pressure and disruption of ductules upstream of the pancreas. Thus, we should pay careful attention to pseudocyst of the pancreas, especially when signs of diffuse chronic inflammation cannot be found, to help identify duct cell carcinoma in the early stage. Further detailed examinations of the cyst fluid or pancreatic juice, such as cytology, tumor marker determinations, or establishment of K-ras codon 12 mutation, are needed.

Production of bioactive gastrin from the non-endocrine cell lines CHO and COS-7

We made a mutated progastrin cDNA construct that contains a cleavage site (‐Arg−4‐Arg−3‐Lys−2 ‐Arg−1) specific for the Kex2‐like endoprotease furin, located ahead of the bioactive gastrin. For expressing the mutated progastrin cDNA, we used two non‐endocrine cell lines, CHO and COS‐7. CHO cells exhibit amidating enzyme activity and levels of amidation enzyme mRNA as high as those in the pituitary‐derived endocrine cell line GH3, whereas COS‐7 cells have far less amidating activity and lower amounts of mRNA. Mutant progastrin‐expressing CHO cells produced mostly amidated gastrin. Gel filtration showed the size of this gastrin corresponded to that of the synthetic human gastrin‐17. In contrast, COS‐7 cells produced glycine‐extended gastrin and only a small amount of amidated gastrin. The difference in the amount of amidated gastrin products produced by the two non‐endocrine cell lines is due to differing amounts of the amidation enzyme contained in each cell line.

Localization of the histamine H2 receptor, a target for antiulcer drugs, in gastric parietal cells

Background/Aims: Histamine H₂ receptor antagonists are widely used for the treatment of peptic ulcer disorders. However, whether the H₂ receptor is present in parietal or immune cells in the lamina propria remains controversial. This study is designed to determine the H₂ receptor localization immunohistochemically using an antibody against the newly cloned mouse histamine H₂ receptor. Methods: We cloned the mouse histamine H₂ receptor gene and generated a specific antipeptide antibody against the C terminus. Immunohistochemical studies were performed with this antibody and with a monoclonal antibody against H⁺/K⁺ adenosine triphosphatase (ATPase). Results: Histamine H₂ receptors were localized on the plasma membrane and on the cytoplasm just beneath the plasma membrane on the basolateral sides of gastric cells. Confocal microscopy of double-stained sections using the monoclonal antibody against H⁺/K⁺ ATPase, a specific parietal cell marker, showed that histamine H₂ receptors colocalized with H⁺/K⁺ ATPase. No specific histamine H₂ receptor immunoreactivities were observed in the submucosal regions. Conclusion: The H₂ receptor is localized in the gastric parietal cell.