Screening test for rapid food safety evaluation by menadione-catalysed chemiluminescent assay.

The chemiluminescent assay of menadione-catalysed H2O2 production by living mammalian cells was proposed to be useful for rapid food safety evaluation. The tested foods were extracted with water, ethanol and dimethylsulfoxide, and each extract was incubated with NIH3T3, Neuro-2a and HepG2 cells for 4h. Menadione-catalysed H2O2 production by living mammalian cells exposed to each extract was determined by the chemiluminescent assay requiring only 10 min, and the viability of the cells was estimated as percentage based on H2O2 production by intact cells. In this study the cytotoxicity of food was rated in order of inhibitory effect on H2O2 production by intact cells. The well known natural toxins such as Fusarium mycotoxin, tomato toxin tomatine, potato toxin solanine and marine toxins terodotoxin and brevetoxin could be detected by the above chemiluminescent assay.

Advantage of menadione-catalyzed chemiluminescent assay for the determination of viable mammalian cell number.

A chemiluminescent assay composed of TCPO [bis(2,4,6-trichlorophenyl)oxalate] and harmless rhodamine B is proposed to be superior in the determination of menadione-catalyzed hydrogen peroxide (H2O2) production by viable mammalian cells to that composed of TCPO and harmful pyrene [Anal. Biochem. 207 (1992) 255-260]. In tests, the proposed assay showed that the measurable concentration of H2O2 and the viable cell number ranged from 10⁻9 to 10⁻³ M and from 2 × 10² to 2 × 106 cells/100 µl/well in the presence of 10% bovine serum, respectively. The measuring time was approximately 10 min. On the other hand, the measurable cell numbers by the colorimetric WST-1 and MTT assays requiring several hours ranged only from 10³ to 104 cells/100 µl/well and from 104 to 105 cells/100 µl/well, respectively. The cytotoxicity of sodium dodecyl sulfate was also observed at intervals of 1 min by the proposed assay, but not by the above colorimetric assays.

Germline alterations in the CDH1 gene in familial gastric cancer in the Japanese population.

Germline point or small frameshift mutations of the CDH1 (E-cadherin) gene are known to cause familial gastric cancer (FGC), but the frequency of CDH1 mutations is low in Japanese patients with FGC. Because recent studies have reported germline large genomic deletions of CDH1 in European and Canadian patients with FGC, in the present study we examined DNA samples from 13 Japanese patients with FGC to determine whether similar germline changes were present in CDH1 in this population. Using a sequencing analysis, a 1-bp deletion (c.1212delC), leading to the production of a truncated protein (p.Asn405IlefsX12), was found in an FGC family; immunohistochemical analysis revealed the loss of CDH1 protein expression in the tumors in this family. Using a combination of multiplex ligation-dependent probe amplification (MLPA) and RT-PCR analyses, we also found a large genomic deletion (c.164-?_387+?del), leading to the loss of exon 3 and the production of a truncated protein (p.Val55GlyfsX38), in another FGC family. The functional effects of the detected mutations were examined using a slow aggregation assay. Significant impairment of cell-cell adhesion was detected in CHO-K1 cells expressing Ile405fsX12- and Gly55fsX38-type CDH1 compared with cells expressing wild-type CDH1. Our results suggest that the p.Asn405IlefsX12 and p.Val55GlyfsX38 mutations of the CDH1 gene contribute to carcinogenesis in patients with FGC. This is the first report of CDH1 germline truncating mutations in Japanese patients with FGC. Screening for large germline rearrangements should be included in CDH1 genetic testing for FGC.

Adenine DNA glycosylase activity of 14 human MutY homolog (MUTYH) variant proteins found in patients with colorectal polyposis and cancer.

Biallelic inactivating germline mutations in the base excision repair MUTYH (MYH) gene have been shown to predispose to MUTYH-associated polyposis (MAP), which is characterized by multiple colorectal adenomas and carcinomas. In this study, we successfully prepared highly homogeneous human MUTYH type 2 recombinant proteins and compared the DNA glycosylase activity of the wild-type protein and fourteen variant-type proteins on adenine mispaired with 8-hydroxyguanine, an oxidized form of guanine. The adenine DNA glycosylase activity of the p.I195V protein, p.G368D protein, p.M255V protein, and p.Y151C protein was 66.9%, 15.2%, 10.7%, and 4.5%, respectively, of that of the wild-type protein, and the glycosylase activity of the p.R154H, p.L360P, p.P377L, p.452delE, p.R69X, and p.Q310X proteins as well as of the p.D208N negative control form was extremely severely impaired. The glycosylase activity of the p.V47E, p.R281C, p.A345V, and p.S487F proteins, on the other hand, was almost the same as that of the wild-type protein. These results should be of great value in accurately diagnosing MAP and in fully understanding the mechanism by which MUTYH repairs DNA in which adenine is mispaired with 8-hydroxyguanine.

Absence of germline mono-allelic promoter hypermethylation of the CDH1 gene in gastric cancer patients.

BACKGROUND: Germline mono-allelic promoter hypermethylation of the MLH1 or MSH2 gene in families with hereditary nonpolyposis colorectal cancer has recently been reported. The purpose of this study was to evaluate if germline promoter hypermethylation of the tumor suppressor gene CDH1 (E-cadherin) might cause predisposition to gastric cancer. METHODS: We prepared two groups of samples, a group of blood samples from 22 patients with familial gastric cancer or early-onset gastric cancer selected from among 39 patients, and a group of non-cancerous gastric tissue samples from 18 patients with sporadic gastric cancer showing loss of CDH1 expression selected from among 159 patients. We then investigated the allele-specific methylation status of the CDH1 promoter by bisulfite sequencing of multiple clones. RESULTS: Although there was a difference between the methylation level of the two alleles in some samples, there was no mono-allelic promoter hypermethylation in any of the samples. CONCLUSION: These results suggest that germline mono-allelic hypermethylation of the CDH1 promoter is not a major predisposing factor for gastric cancer.

Altered expression of the human base excision repair gene NTH1 in gastric cancer.

A base excision repair enzyme, NTH1, has activity that is capable of removing oxidized pyrimidines, such as thymine glycol (Tg), from DNA. To clarify whether the NTH1 gene is involved in gastric carcinogenesis, we first examined the NTH1 expression level in eight gastric cancer cell lines, and the results showed that NTH1 expression was downregulated in all of them, including cell line AGS. Next, a comparison of excisional repair activity against Tg by empty vector-transfected AGS clones and FLAG-NTH1-expressing AGS clones showed that a low NTH1 expression level led to low capacity to repair the damaged base in the gastric epithelial cells. Reduced messenger RNA expression of NTH1 was also detected in 36% (18/50) of primary gastric cancers. Moreover, immunohistochemical analysis revealed that NTH1 was predominantly localized in the cytoplasm in 24% (12/50) of the primary gastric cancers in contrast to the nuclear localization in non-cancerous tissue, suggesting impaired excisional repair ability for nuclear DNA. No associations between clinicopathological factors and NTH1 expression level or localization pattern were detected in the gastric cancers. Next, we found two novel genetic polymorphisms, i.e. c.-163C>G and c.-241_-221del, in the NTH1 promoter region, and a luciferase assay showed that both were associated with reduced promoter activity. However, there were no associations between the polymorphisms and risk of gastric cancer in a gastric cancer case-control study. These findings suggested that downregulation of NTH1 expression and abnormal localization of NTH1 may be involved in the pathogenesis of a subset of gastric cancers.

Identification and characterization of a novel germline p53 mutation in a patient with glioblastoma and colon cancer.

Germline mutations in the p53 tumor suppressor gene have been identified in patients with Li-Fraumeni syndrome (LFS) and patients with Li-Fraumeni-like syndrome (LFL). However, to date, germline p53 mutations in patients not fulfilling the criteria of LFS or LFL have been reported only very rarely. In our study, a novel germline c.584T>C (p.Ile195Thr) mutation of the p53 gene was found in a 21-year-old male with a glioblastoma and colon cancer. He had no family history of cancer within second-degree relatives, and loss of the wild-type p53 allele and overexpression of p53 protein were observed in both tumors. Functional analyses revealed transactivation and growth suppressive function activities of the Thr195-type p53 to be impaired. These results suggest germline p53 mutations to possibly be responsible for a subset of young adult patient with multiple malignant tumors, even those not meeting the clinical criteria for LFS or LFL.

Inverse relationship between the length of the EGFR CA repeat polymorphism in lung carcinoma and protein expression of EGFR in the carcinoma.

BACKGROUND AND OBJECTIVES: There is a CA dinucleotide repeat polymorphism in the first intron of the epidermal growth factor receptor (EGFR) gene. Our aim was to examine the relationship between the CA repeat length in lung carcinoma and EGFR mutation, EGFR gene copy number, and EGFR protein expression level in the carcinoma. METHODS: We examined 168 lung carcinomas for the length of the CA repeat polymorphism by PCR-polyacrylamide gel electrophoresis analysis, for EGFR mutations by sequencing analysis, for EGFR gene copy number by fluorescence in situ hybridization analysis, and for EGFR protein expression by immunohistochemical analysis. RESULTS: When the carcinomas were divided into two groups according to the length of their CA repeat polymorphism, the EGFR protein expression level was found to be significantly higher in the shorter allele group than in the longer allele group (P = 0.0116), but its length was not associated with EGFR somatic mutations, high EGFR gene copy numbers, or clinicopathological factors, such as histological type or stage. CONCLUSIONS: The results suggested that the length of the EGFR CA repeat polymorphism in lung carcinoma is inversely related with level of EGFR protein expression in the carcinoma.

Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

PIK3CA mutation and amplification in human lung cancer.

To explore the significance of phosphatidylinositol-3-kinase, catalytic, alpha (PIK3CA) in the carcinogenesis in human lung, mutations and copy number changes were investigated in 148 Japanese patients with primary cancer of the lung. For biological validation, the effects of exogenously expressed wild-type and mutated PIK3CA were studied in an immortalized human airway epithelial cell line. Mutations in PIK3CA were found in five (3.6%) of the 139 available patients, and copy number gains were found in 21 (18.3%) of 115 patients, respectively. Overall, mutations or copy number gains were detected in 24 of the 106 patients (22.6%) for whom results in both analyses were available. The prevalence of copy number gains was higher in men, smokers, and in patients with squamous cell carcinoma than in the opposite categories. The copy number changes showed a trend toward higher prevalence in the earlier stages (P = 0.038). Interestingly, the presence of mutations and of copy number alterations were mutually exclusive in the present patients, implying that both entail equivalent oncogenic potential. Over-expressed wild-type PIK3CA and its two common mutants, K545E and H1047R, significantly enhanced the anchorage-independent growth activity and migration activity of immortalized airway epithelium 16HBE14o- cells, but the effects of the K545E and H1047R mutants were more remarkable than those of the wild-type. The present demonstrates an important role of PIK3CA in human lung carcinogenesis.

Association between CDH1 haplotypes and gastric cancer risk in a Japanese population.

OBJECTIVE: A c. -285C >A single nucleotide polymorphism (SNP) in the promoter region of the E-cadherin (CDH1) gene, which is a tumor suppressor in gastric cancer (GC), has been shown to decrease gene transcription, but GC case-control studies of this SNP have yielded controversial results. A haplotype study in an Italian population showed that haplotypes based on three SNPs, including the c. -285C >A, are associated with susceptibility to GC. Hence, the purpose of the present study was to carry out a more comprehensive genetic analysis of CDH1 using haplotype-tagging SNPs (htSNPs) in a Japanese case-control study to identify the CDH1 haplotype associated with susceptibility to GC in a Japanese population. MATERIAL AND METHODS: First, 11 SNPs in the CDH1 gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 30 healthy individuals. Haplotype frequencies were estimated with the expectation-maximization algorithm, and 7 common haplotypes of the CDH1 gene whose frequency was at least 3.3% were identified. Next, 5 htSNPs (c. -285C >A, c.48+6T >C, c.164 -3159T >C, c.2076C >T, and c.2296 -616G >C) were genotyped in a hospital-based case-control study of 148 GC patients and 292 age- and gender-matched healthy controls, and haplotype frequencies based on the 5 htSNPs were estimated. RESULTS: Although none of the 5 htSNPs was related to an overall risk of GC, frequencies of the ATCTG and CTTTG haplotypes were significantly higher and lower, respectively, in the GC cases than in the controls (p<0.05). CONCLUSIONS: These results suggest that the ATCTG and CTTTG CDH1 haplotypes may be associated with an increased risk and decreased risk, respectively, of GC in the Japanese population.

Identification and characterization of a novel germ line p53 mutation in familial gastric cancer in the Japanese population.

Germ line mutations of the p53 gene are known to cause Li-Fraumeni syndrome, and a germ line p53 mutation has recently been reported in a small subset of familial gastric cancer (FGC) in Europe and Korea. Although the incidence of gastric cancer is very high in Japan and familial clustering is not uncommon, there has been little information on the genetic factors of FGC. Therefore, to determine the role of germ line p53 mutations in FGC in the Japanese population in this study, we used sequencing analysis to examine 80 individuals from 35 Japanese FGC families without germ line CDH1 mutations for germ line p53 mutations. One missense (c.91G>A: p.Val31Ile) and two intronic germ line mutations were found, and transcriptional activity of the Ile31 mutant on p53-responsive genes was examined to determine the functional effect of the novel p.Val31Ile germ line mutation. A luciferase reporter assay showed that the transcriptional activity of p21 (CDKN1A) and MDM2 promoters but not of the BAX promoter was significantly lower in the Ile31-type p53 than in the wild-type (wt) p53. Next, doxycycline-regulated p53-inducible H1299 cell lines were established by applying a retrovirus-mediated gene transfer system to a p53-null human H1299 cell line. Under similar p53 expression conditions shown by western blot and immunofluorescence analyses, a cell proliferation assay showed that the Ile31-type p53 had significantly lower cell proliferation suppressing activity than wt p53. These results suggest that Ile31-type p53 may be partly involved in FGC because of its low transcriptional activity and low cell proliferation suppressing activity.

Effect of splice-site polymorphisms of the TMPRSS4, NPHP4 and ORCTL4 genes on their mRNA expression.

Genetic polymorphisms associated with structural changes of their gene product are important in terms of their potential relation with diseases. Therefore, in this study, splice-site variants of the transmembrane serine protease gene TMPRSS4, nephronophthisis gene NPHP4, and organic-cation transporter gene ORCTL4, were selected from the dbSNP single nucleotide polymorphism database as candidates to identify genetic polymorphisms associated with a structural change in their mRNA transcripts. The allele frequencies of the TMPRSS4 c.4-7A>G, NPHP4 c.2818-2A>T, and ORCTL4 c.517-2A>C polymorphisms in a Japanese population were determined to be 0.42, 0.10, and 0.27, respectively, by PCR-SSCP analysis. Next, the effect of these polymorphisms on the mode of pre-mRNA splicing was investigated by RT-PCR analysis followed by sequencing analysis. The TMPRSS4, NPHP4, and ORCTL4 polymorphisms were associated with the production of the r.4-6_4-1ins transcript, the r.2818_2823del and r.2818_2859del transcripts, and the r.517-94_517-1ins; r.517-2a>c and r.517_620del transcripts, respectively. Since the proteins encoded by all these transcripts are associated with relatively significant structural changes in the form amino acid insertion/deletion and premature termination, their functional ability may be greatly reduced. Our demonstration of structural changes in mRNA transcripts as a result of splice-site polymorphisms implies that they may be of biological significance in certain pathological conditions.

Analysis of oxidized epigallocatechin gallate by liquid chromatography/mass spectrometry.

(-)-Epigallocatechin gallate (EGCG) of catechins changes from non-colored at around neutral pH to yellow at higher pH region in aqueous solution. The pH-dependent oxidation of EGCG was analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). LC/MS/MS analysis of EGCG and its related compounds, (-)-epicatechin gallate (ECG) and (-)-epigallocatechin (EGC), successfully elucidated the structure relationship of EGCG solution involving the color change reaction at different pH conditions. The oxidation species produced at alkaline pH was detected at a different retention time from EGCG in the chromatograms of the EGCG sample. The oxidation species was found to correspond to M+14 (where M is the molecular weight of EGCG), which has two hydrogen atoms removed and addition of one oxygen atom to the gallyl moiety in the B-ring of EGCG.

The role of cyclooxygenase-2 (COX-2) in two different morphological stages of intestinal polyps in APC(Delta474) knockout mice.

The expression of COX-2 participates strongly in polyp formation of adenomatous polyposis coli (APC)-mutated mice. However, the mechanism of growth inhibition by COX-2 inhibition remains unclear. The aims of this study were to assess the role of COX-2 during the process of polyp formation in APC(Delta474) knockout mice. Starting at 4 weeks of age, the treated group (T group) were given a diet containing JTE-522, a selective COX-2 inhibitor, and the control group (C group) were given a control diet. At 12 weeks of age, mice were killed and polyps located in a proximal 10 cm of the small intestine were classified into two morphological stages: large adenomas (>300 microm in diameter) which lacked normal villous structure, and small adenomas (