柴田 知行

Cyclooxygenase-1 (COX-1) has been regarded as a constitutively expressed enzyme that generates prostaglandins for gastrointestinal integrity. We attempted to clarify the association between potentially functional polymorphisms (T-1676C and A-842G/C50T) in the COX-1 gene promoter and gastroduodenal disorders in a Japanese population. The study was performed with 480 stocked DNAs from subjects (gastric ulcers in 93 subjects and duodenal ulcers in 44) with no evidence of gastric malignancy. We employed the PCR-SSCP method to detect gene polymorphisms. The severity of histological chronic gastritis in antral biopsy specimens was classified according to the updated Sydney system. The T1676C polymorphism was not associated with either gastric mucosal atrophy or infiltration of inflammatory cells into gastric mucosa. In non-NSAID (non-steroidal antiinflammatory drug) users, male gender and Helicobacter pylori (HP) infection were significantly associated with both gastric and duodenal ulcers, whereas the -1676T allele carrier was significantly associated with only gastric ulcers (OR, 2.86; 95% Cl 1.29-6.34). In NSAID users, the number of -1676T alleles was significantly associated with developing gastroduodenal ulcers (OR, 5.80; 95% Cl, 1.59-21.1), whereas male gender and HP infection were not. The -842T/ C50T polymorphism was not detected in any of the 480 Japanese subjects. In conclusion, a carrier of the -1676T allele in the COX-1 gene promoter, as well as HP infection and male gender, seem to be significant risk factors for developing gastric ulcers, and the number of -1676T alleles was also a significant risk factor for the NSAID-induced ulcer, whereas the frequency of the A-842G polymorphism was thought to be very rare in the Japanese population.

Noncoding microRNAs regulate the expression of various mRNAs. We attempted to clarify the relationship between miR-27a genome polymorphism and chronic gastritis. The study was performed in 179 patients with no evidence of gastric malignancy. The severity of histologic chronic gastritis was classified according to the updated Sydney system. The frequency of miR-27a G allele was 34.6%. Although the frequencies of miR-27a G allele were increased in subjects with peptic ulcer or severe mucosal atrophy, no significant differences were seen. The miR-27a polymorphism showed an interaction with gender in relation to gastric mucosal atrophy (P =.090). In only male subjects, the miR-27a polymorphism was associated with the gastric mucosal atrophy (P =.039) and both atrophy and metaplasia scores in G/G group were significantly higher than those in the other groups. The miR-27a genome region polymorphism may be an important definitive factor to develop the gastric mucosal atrophy in Japanese male subjects.

Backgound: Trypsin acting at protease-activated receptor 2 (PAR2) contributes to a progression of malignant tumors. An abnormal DNA methylation has been recognized as an important molecular mechanism for the genesis of various types of cancers. We attempted to clarify the relationship between the promoter methylation of PAR2 and gastric cancer. Method: We estimated the methylation of the PAR2 promoter in both antral non-cancerous mucosa and cancer lesions in 94 patients with gastric cancer. We employed a methylation-specific PCR method. Results: Regarding the methylation ratio (MR) of antral-non-cancerous mucosa, no significant difference was despite among gender, age and Helicobacter pylori infection status, whereas MR increased rising inflammation scores. The MR of cancer lesions was significantly lower than that of antral non-cancerous mucosa. This finding was not dependent on tumor staging, but also histological classification. In venous invasion, lymph node metastasis, or peritoneal dissemination negative cases, this significant lower MR was also seen. Conclusion: The promoter methylation of PAR2 seems to be increased with a progression of chronic inflammation and has an inhibitory effect on carcinogenesis of the stomach.

Background and aim: Ulcerative colitis (UC) is a multifactorial disease resulting from a complex interaction of genetic and environmental factors. Identifying genetic variants that alter the innate immune response is highly relevant to understanding the pathogenesis of UC. The aim of this study was to investigate the association between CD14 and Toll-like receptor-2 (TLR2) genetic polymorphisms and chronic UC in Japanese patients. Methods: The study population consisted of 102 chronic UC patients and 146 healthy control subjects. Polymorphisms in the promoter at C-260T of CD14 gene were investigated by PCR restriction fragment length polymorphism, and -196 to -174 del of TLR2 was investigated by allele-specific PCR. Results: The frequencies of CD14 TT and T carrier were significantly higher in UC patients than in controls (TT: OR = 3.98, 95% CI 1.82-8.71, P = 0.0005; T carrier: OR = 2.98, 95% CI 1.47-6.01, P = 0.002). In addition, TT and T carrier were more closely associated with distal colitis phenotype (TT: OR = 7.78, 95% CI 2.14-28.28, P = 0.0007; T carrier: OR = 6.30, 95% CI 2.71-14.58, P = 0.005), onset after 20 years of age (TT: OR = 5.28, 95% CI 2.18-12.79; T carrier: OR = 3.79, 95% CI 1.67-8.59), chronic continuous type (TT: OR = 4.26, 95% CI 1.56-11.64; T carrier: OR = 3.09, 95% CI 1.33-7.82), and fewer than two hospitalizations (TT: OR = 4.44, 95% CI 1.81-10.89; T carrier: OR = 3.26, 95% CI 1.43-7.27). There was no significant difference in TLR2 -196 to -174 del/del and del/ins carrier frequencies between UC patients and healthy controls. However, these frequencies were significantly higher in steroid-dependant patients than in controls (del/del: OR = 6.08, 95% CI 1.41-26.21; del carrier: OR = 3.00, 95% CI 1.13-7.98). Conclusion: The results suggest that existence of a mutation in the CD14 gene is associated with an increased susceptibility to developing UC, especially chronic continuous distal colitis phenotypes that develop after 20 years of age. Furthermore, polymorphism of TLR2 may be related to an increased risk of intensive types such as steroid-dependent patients.

The mechanisms that cause chemoresistance of gastric cancer have yet to be elucidated. Taxanes and promising agents that were recently approved for treatment of advanced or recurrent gastric cancer. Mutations of beta-tubulin, which is a target of taxianes, have been shown to confer chemoresistance against these agents. The aim of the present study is to investigate the presence of mutations of the beta-tubulin in gastric cancer tissues. Sixty-six patients with advanced stage III or IV gastric cancer patients enrolled in this study. Paired samples of gastric cancer tissue and normal mucosa were obtained by endoscopy. The guanosine F-triphosphate (GTP)-binding site in exon 4 of the beta-tubulin gene was examined by polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) analysis, followed by sequencing of the products with abnormally shifted bands. SSCP analysis showed abnormal bands upstream of the GTP-binding site in 7 of the 66 patients, but sequence analysis found no nucleotide substitutions in these patients. Three variant bands were also detected down stream of the the GTP-binding site, but the sequences of the 3 products corresponded to those of two independent pseudogenes. Thus, none of the tumor samples showed mutation of the beta-tubulin exon 4 GTP-binding site. In conclusion, these findings suggest that mutations of the beta-tubulin gene are rare and are unlikely to be an important cause of taxane resistance to taxians.

The transcription factor Nrf2 regulates the Introduction expression of detoxifying and antioxidant genes. Three polymorphisms of the Nrf2 gene have been reported. We attempted to clarify the relationship between Nrf2 gene polymorphism and chronic gastritis in a Japanese population. The study was performed in 159 patients with no evidence of gastric malignancy on upper gastrointestinal endoscopy (mean age, 62.03 years; male:female ratio, 102:57; peptic ulcer diseases in 69 patients, and Helicobacter pylori (H. pylori) positivity in 73.0%). We employed the PCR-SSCP method to detect gene polymorphisms using DNA extracted from peripheral blood cells or from antral biopsy specimens obtained by endoscopy. The severity of the histological chronic gastritis in antral biopsy specimens was classified according to the updated Sydney system. Although the frequencies of the SNP(-686) and SNP(-650) A alleles were decreased in subjects with peptic ulcers or severe mucosal atrophy, no significant differences were seen. However, the number of -686 G alleles was correlated with both neutrophil activity and mononuclear cell infiltration (p=0.036 and p=0.010, respectively), while the -650 C/C genotype was an independent risk factor for mononuclear cell infiltration (p=0.021 by ANOVA). In addition, both the number of -686 G alleles and the -650 C/C genotype showed an interaction with H. pylori infection to promote the infiltration of mononuclear cells (p=0.037 by ANCOVA and p=0.041 by ANOVA, respectively). Nrf2 promoter polymorphisms are significantly associated with the development of gastric mucosal inflammation, either independently or by interacting with H. pylori infection.

Cationic metal and radionuclide contaminants can be extracted from soils to groundwater with sequestering agents such as EDTA. However, EDTA must then be removed from the groundwater, by advanced oxidation or specialized biological treatment. In this work, aqueous individual metal-EDTA solutions were aerated with steel wool for 25 h, at ambient pH, temperature, and pressure. Removal of approximately 99% of EDTA (0.09-1.78 mM); glyoxylic acid (0.153 mM); chelated Cd2+ (0.94 and 0.0952 mM), Pb2+ (0.0502 mM), and Hg2+ (0.0419 mM); and free chromate and vanadate was shown. EDTA was oxidized to glyoxylic acid and formaldehyde, and metals/metalloids were coprecipitated together with iron oxyhydroxide floc. Free arsenite and arsenate were each removed at 99.97%. Free Sr2+, and chelated Ni2+ were removed at 92% and 63%, respectively. Similar removals were obtained from mixtures, including 99.996 +/- 0.004% removal of total arsenic (95% confidence). Traces of iminodiacetic acid, nitrilotriacetic acid, and ethylenediaminetriacetic acid were detected after 25 h. Results are consistent with first-order, solution-phase oxidation of EDTA and glyoxylic acid by ferryl ion and H2O2, respectively, with inhibition due to sludge accumulation, and equilibrium metal coprecipitation. This ambient process, to our knowledge previously unknown, agrees with recently reported findings and shows promise for remediation of metals, metalloids, and radionuclides in wastewater, soil, and sediment.

Background: Aberrant DNA methylation is one of the major events in carcinogenesis. Promoter DNA methylation is also present in various non- neoplastic tissues including gastric epithelium as age- related phenomenon, suggesting that it occurs early in the process of tumorigenesis. Aim: We aimed to clarify the relationship of aberrant DNA methylation in nonneoplastic gastric epithelia with the risk of gastric cancer, Helicobactor pylori infection, and the degree of H. pylori - induced gastritis. Methods: 89 patients enrolled in this study. The status of aberrant DNA methylation was compared in two groups of patients: 43 cases with gastric cancer ( mean age 65.9 years [29-91], F: M = 0.30, intestinal type [ n = 25], diffuse type [ n =18]) and 46 age- and sex- matched patients without gastric cancer ( peptic ulcer diseases [ n = 11], gastritis [ n = 35]) as a control group. Genomic DNA was extracted directly from non- neoplastic epithelia of antral biopsies obtained by endoscopy. The promoter methylation status of the p14 and p21 genes was determined by methylation- specific- polymerase chain reaction ( MSP). The promoter methylation status of the p16 gene was quantified by digital densitographic analysis following MSP. The degree of gastritis in the antrum was assessed according to the updated Sydney system. The PG I/ II ratio was calculated based on the data of serum PG I and PG II levels measured by radioimmunoassay. Results: In all 89 subjects, CpG island methylation was found in 25.8% for p14, 52.8% for p16, 1.1% for p21. Among non- cancer patients, the methylation frequency of the p14 gene was significantly higher in H. pylori - positive than in H. pylori - negative patients ( 38.5 vs. 10.0%, p = 0.03). The mean (+/- SD) methylation levels of the p16 gene in non- neoplastic gastric epithelium was significantly higher in gastric cancer cases both in all patients and in H. pylori-positive patients ( 0.45 +/- 0.31 vs. 0.20 +/- 0.17; p = 0.019, 0.45 +/- 0.31 vs. 0.20 +/- 0.17; p = 0.016, respectively). The methylation level of the p16 gene was also associated with the presence of intestinal- type gastric cancer ( p = 0.017). The methylation level of the p16 gene was significantly higher in patients with intestinal metaplasia ( IM) than those without ( p = 0.04). Furthermore, the methylation level of the p16 gene was correlated with lower PG I/ II ratio ( p = 0.04). The methylation of the p21gene was found in only 1 patient with gastric cancer. Conclusions: Our data suggest that promoter of the p14 gene may be one of the specific regions whose methylation is closely associated with H. pylori infection. Methylation levels of the p16 gene seem to be accumulated in the progression of gastric mucosal atrophy and IM, and thus may be associated with the presence of gastric cancer especially for intestinaltype histopathology. Copyright (c) 2007 S. Karger AG, Basel.

Host genetic factors may play a key role in determining the long-term outcome of the Helicobacter pylori (H. pylori) infection. Toll-like receptor 4 (TLR4) mediated recognition of lipo-polysaccharide (LPS) is required for efficient recognition of gram-negative bacterial infections. The aim of this study is to investigate the effects of common polymorphisms of TLR4 Asp299Gly, Thr399Ile in patients with gastroduodenal diseases in Japanese population. The study was performed in 149 gastric cancer (GC) cases (mean age 64.0 +/- 12.4, M:F = 109:40) and 94 patients without evidence of GC (mean age 64.1 +/- 12.3, M:F = 65:25) as the control group. TLR4 Asp299Gly, Thr399Ile were determined by Polymerase chain reaction-length of polymorphisms (PCR-RFLP) in all the patients. Asp299Gly, Thr399Ile were not detected in all 243 patients enrolled in this study. In conclusion, our data suggest that TLR4 Asp299Gly, Thr399Ile are very rare in Japanese population and thus they may not be a important factor in determining the outcome of H. pylori infected individuals in Japan.

Trypsin acting at protease-activated receptor 2 (PAR2) has been reported to contribute to a progression of malignant tumors. In addition, a polymorphic form of PAR2 has been also reported to display reduced sensitivity to trypsin. However, a frequency of PAR2 polymorphism is unknown in Japan. The aim of the present study was to clarify a frequency of PAR2 polymorphism in Japan and to evaluate the relation between this polymorphism and gastric cancer. We estimated PAR2 T719C in 106 patients with gastric cancer (GC cases) and 96 patients without gastric cancer (non-GC cases). We employed a single-strand conformation polymorphism analysis after polymerase chain reaction (PCR-SSCP) for detecting of this polymorphism. There was no significant difference between GC and non-GC cases in the distribution of gender (M:F = 2.66 and 2.00, respectively) and age ( mean age = 66.12 and 63.50, respectively). Helicobacter pylori (H. pylori) positive ratio was 81.7% (GC cases: 92.0%, non-GC cases: 72.5%, p < 0.01). Single strand bands of 719C were not detected in all 202 patients by PCR-SSCP. In conclusion, a polymorphism F240S of PAR2 is very rare and does not contribute to the genesis and progression of gastric cancer in Japanese population.

1. No general consensus has been reached on the treatment of acute gastric lesions. The aims of the present study were to clarify the effects of sucralfate, cimetidine and rabeprazole monotherapies and combination therapies on acute gastric lesions from the viewpoint of connective tissue regeneration. 2. Gastric lesions were experimentally created by the oral administration of 50% ethanol-0.15 mol/L HCl to rats. After 30 min, the anti-ulcer agents sucralfate (100 mg/kg), cimetidine (20 mg/kg) and rabeprazole (2 mg/kg) were administered separately or in combination and the stomach was excised at different times to measure the level of hydroxyproline in the gastric mucosa and determine lesion index. Immunostaining against prolylhydroxylase was performed on some specimens. 3. In the control group, lesion index decreased linearly from 30 min after ethanol-HCl administration and the level of mucosal hydroxyproline peaked between 2 and 4 h later. Although sucralfate significantly promoted lesion healing, it had no effect on mucosal hydroxyproline level. Cimetidine suppressed increases in mucosal hydroxyproline and prolonged lesion healing, but these findings were reversed by combining cimetidine and sucralfate. Rabeprazole had no significant effect on lesion healing, but promoted lesion healing in combination with sucralfate. Immunohistochemical analysis showed that prolylhydroxylase was expressed in spindle cells that lined the glandular cells in a boundary area between normal and injured tissues. 4. Under conditions in which the effects of intragastric pH are minimal, sucralfate is superior to antisecretory agents in promoting the healing of acute gastric lesions.

Background and Aims: Proton pump inhibitors (PPI) and prostaglandin (PG) preparations are believed to both prevent NSAID-induced gastric ulcers and promote the delayed healing of gastric ulcers by NSAIDs, but it remains unclear which of these drugs is superior. The aim of this study was to clarify which achieved better healing of NSAID-induced gastric ulcers, not only with respect to epithelialization but also repair of the submucosal tissues. Methods: We used acetic acid to induce gastric ulcers in rats, and compared the changes between a control group, NSAID group, NSAID + PPI group and NSAID + PG group. After removing the stomach of each animal, an ulcer index was calculated and the Collagen content and type III Collagen content of granulation tissue were measured. We also studied fibroblast dynamics, including proliferation, Collagen synthesis, differentiation into myofibroblasts, and apoptosis. Results: Indomethacin prevented re-epithelialization of the ulcers, interfered with fibroblast function, and also delayed the replacement of type III Collagen. Omeprazole promoted epithelialization, but could not fully reverse the influence of indomethacin on granulation tissue maturation. A concomitant dose with misoprostol reversed it completely. Conclusions: From our point of view in this study in the use of experimental ulcers, it was thought that compensation of PG should have priority to gastric acid inhibition in terms of healing of NSAID-induced gastric ulcer. Copyright (c) 2006 S. Karger AG, Basel.

Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in Situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement.

The enteric pathogen Shigella utilizes host-encoded proteins to invade the gastrointestinal tract. Efficient invasion of host cells requires the stimulation of Rho-family GTPases and cytoskeletal alterations by Shigella-encoded IpaC [1]. Following invasion and lysis of the phagosome, Shigella exploits the host's actin-based polymerization machinery to assemble an actin tail that serves as the propulsive force required for spreading within and between cells (2,3]. The Shigella surface protein IcsA stimulates actin-tail formation by recruiting host-encoded N-WASP to drive Arp2/3-mediated actin assembly [4-7]. N-WASP is absolutely required for Shigella motility, but not for Shigella invasion [4, 6]. Although Rho-family GTPases have been implicated in both the invasion and motility of Shigella, the role of Cdc42, an N-WASP activator, in this process has been controversial [8, 9]. In these studies, we have examined the role of Cdc42 in Shigella invasion and actin-based motility using Cdc42-deficient cells. We demonstrate that Cdc42 is required for efficient Shigella invasion but reveal a minor Cdc42-independent pathway that can permit Shigella invasion. However, the actin-based motility of Shigella, as well as vaccinia, proceeds unperturbed in the absence of Cdc42. These data further support the involvement of distinct host-encoded proteins in the steps regulating invasion and intercellular spread of Shigella.