New screening methods for probiotics with adhesion properties to sialic acid and sulphate residues in human colonic mucin using the Biacore assay.

AIMS: To determine the relationship between adhesive ability of probiotics and acidic residues in human colonic mucin, we developed a new screening method using Biacore to evaluate adherence of bacteria before and after sialic acid or sulphate residues were blocked or removed from mucin. METHODS AND RESULTS: Ten strains of lactobacilli and three strains of bifidobacteria isolated from human faeces were evaluated for their adhesive properties to soluble human colonic mucin (sHCM) using the Biacore binding assay. Three strains (Lactobacillus strain ME-522, Lact. gasseri ME-527 and Bifidobacterium bifidum MCC1092) showing significant adherence were selected. Decreased binding activities were observed after removing sialic acid of sHCM using sialidase. However, after removing the sulphate residue using sulphatase, the adhesion of ME-527 decreased; whereas the remaining two strains had increased adhesion. The adhesion of three probiotics significantly decreased after the sulphate residue was blocked by elution with barium chloride. CONCLUSIONS: A new evaluation method using the Biacore assay was developed to observe binding properties to the acidic residues of sHCM. Results indicated that there was a strong relationship between probiotic adhesion and acidic residues of sHCM. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing a screening method that quantitatively measures the binding between bacteria and acidic residues in sHCM using the Biacore binding assay; and provides a new method for the selection of probiotics in the future.

Identification of a new adhesin-like protein from Lactobacillus mucosae ME-340 with specific affinity to the human blood group A and B antigens.

AIMS: To identify and characterize a new adhesin-like protein of probiotics that show specific adhesion to human blood group A and B antigens. METHODS AND RESULTS: Using the BIACORE assay, the adhesion of cell surface components obtained from four lactobacilli strains that adhered to blood group A and B antigens was tested. Their components showed a significant adhesion to A and B antigens when compared to the bovine serum albumin (BSA) control. The 1 mol l(-1) GHCl fraction extracted from Lactobacillus mucosae ME-340 contained a 29-kDa band (Lam29) using SDS-PAGE. The N-terminal amino acid sequence and homology analysis showed that Lam29 was 90% similar to the substrate-binding protein of the ATP-binding cassette (ABC) transporter from Lactobacillus fermentum IFO 3956. The complete nucleotide sequence (858 bp) of Lam29 was determined and encoded a protein of 285 amino acid residues. Phylogenetic analysis and multiple sequence alignments indicated this protein may be related to the cysteine-binding transporter. CONCLUSIONS: The adhesion of ME-340 strain to blood group A and B antigens was mediated by Lam29 that is a putative component of ABC transporter as an adhesin-like protein. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus mucosae ME-340 expressing Lam29 may be useful for competitive exclusion of pathogens via blood group antigen receptors in the human gastrointestinal mucosa and in the development of new probiotic foods.

Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients.

Previous studies suggested that one or more genes on chromosome 5q21 are responsible for the inheritance of familial adenomatous polyposis (FAP) and Gardner's syndrome (GS), and contribute to tumor development in patients with noninherited forms of colorectal cancer. Two genes on 5q21 that are tightly linked to FAP (MCC and APC) were found to be somatically altered in tumors from sporadic colorectal cancer patients. One of the genes (APC) was also found to be altered by point mutation in the germ line of FAP and GS patients. These data suggest that more than one gene on chromosome 5q21 may contribute to colorectal neoplasia, and that mutations of the APC gene can cause both FAP and GS. The identification of these genes should aid in understanding the pathogenesis of colorectal neoplasia and in the diagnosis and counseling of patients with inherited predispositions to colorectal cancer.

Cell surface Lactobacillus plantarum LA 318 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) adheres to human colonic mucin.

AIMS: To characterize the adhesion molecule of Lactobacillus plantarum LA 318 that shows high adhesion to human colonic mucin (HCM). METHODS AND RESULTS: The adhesion test used the BIACORE assay where PBS-washed bacterial cells showed a significant decrease in adherence to HCM than distilled water-washed cells. A component in the PBS wash fraction adhered to the HCM and a main protein was detected as a c. 40-kDa band using SDS-PAGE. Using homology comparisons of the N-terminal amino acid sequences compared with sequence databases, this protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The DNA sequence of LA 318 GAPDH was 100% identical to the GAPDH (gapB) of L. plantarum WCFS1. The purified GAPDH adhered to HCM. CONCLUSIONS: We found the adhesin of L. plantarum LA 318 to HCM in its culture PBS wash fraction. The molecule was identified as GAPDH. Because LA 318 possesses the same adhesin as many pathogens, the lactobacilli GAPDH may compete with pathogens infecting the intestine. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing GAPDH expressed on the cell surface of lactobacilli adheres to mucin suggesting L. plantarum LA 318 adheres to HCM using GAPDH binding activity to colonize the human intestinal mucosa.

AURKA is one of the downstream targets of MAPK1/ERK2 in pancreatic cancer.

DUSP6/MKP-3, a specific inhibitor of MAPK1/ERK2, frequently loses its expression in primary pancreatic cancer tissues. This evidence suggests that constitutive activation of MAPK1 synergistically induced by frequent mutation of KRAS2 and the loss of function of DUSP6 plays key roles in pancreatic carcinogenesis and progression. By profiling of gene expressions associated with downregulation of MAPK1 induced by exogenous overexpression of DUSP6 in pancreatic cancer cells, we found that AURKA/STK15, the gene encoding Aurora-A kinase, which plays key roles in cellular mitosis, was among the downregulated genes along with its related genes, which included AURKB, TPX2 and CENPA. An association of expression and promoter activity of AURKA with MAPK activity was verified. Knockdown of ETS2 resulted in a reduction of AURKA expression. These results indicate that AURKA is a direct target of the MAPK pathway and that its overexpression in pancreatic cancer is induced by hyperactivation of the pathway, at least via ETS2.

Genome-wide profiling of promoter methylation in human.

DNA methylation in the promoter region of a gene is associated with a loss of that gene's expression and plays an important role in gene silencing. The inactivation of tumor-suppressor genes by aberrant methylation in the promoter region is well recognized in carcinogenesis. However, there has been little study in this area when it comes to genome-wide profiling of the promoter methylation. Here, we developed a genome-wide profiling method called Microarray-based Integrated Analysis of Methylation by Isoschizomers to analyse the DNA methylation of promoter regions of 8091 human genes. With this method, resistance to both the methylation-sensitive restriction enzyme HpaII and the methylation-insensitive isoschizomer MspI was compared between samples by using a microarray with promoter regions of the 8091 genes. The reliability of the difference in HpaII resistance was judged using the difference in MspI resistance. We demonstrated the utility of this method by finding epigenetic mutations in cancer. Aberrant hypermethylation is known to inactivate tumour suppressor genes. Using this method, we found that frequency of the aberrant promoter hypermethylation in cancer is higher than previously hypothesized. Aberrant hypomethylation is known to induce activation of oncogenes in cancer. Genome-wide analysis of hypomethylated promoter sequences in cancer demonstrated low CG/GC ratio of these sequences, suggesting that CpG-poor genes are sensitive to demethylation activity in cancer.

Enhancement of seed vigour following insecticide and phenolic elicitor treatment.

Thiamethoxam (CGA 293'343) is a novel broad-spectrum neonicotinoid insecticide. It is commercially used as a seed treatment under the trademark Cruiser (CRZ). Although many reports detail its insecticidal, plant-protecting properties, there are minimal reports concerning the effect on seed germination activities which can be key control points of seedling vigour. In this report, we investigated the effect of CRZ, fish protein hydrolysates (FPH; a known elicitor of pentose-phosphate pathway) and the combination of CRZ and FPH (CF) on seed vigour of pea, soybean and corn. Seed vigour was investigated by estimating germination percentage, shoot height, shoot weight, total soluble phenolic content, antioxidant content, G6PDH (glucose-6-phosphate dehydrogenase) activity, and GPX (guaiacol peroxidase) activity. Addition of FPH to CRZ (CF) seemed to have a slightly positive effect on seed vigour, especially, CF and FPH treatment for corn and FPH treatment for pea, suggesting that pre-sowing treatments may cause positive/negative effects on seed vigour, depending on the concentration of treatments. Further research will be needed to determine their effects and the optimal concentration for seed priming.

Clavicle fracture with osteomyelitis after neck dissection and post-operative radiotherapy: case report.

BACKGROUND: Non-traumatic bone fractures in cancer patients are usually pathological fractures due to bone metastases. In head and neck cancer patients, clavicle stress fractures may occur as a result of atrophy of the trapezius muscle after neck dissection in which the accessory nerve becomes structurally or functionally damaged. CASE REPORT: A 71-year-old man underwent modified radical neck dissection with accessory nerve preservation and post-operative radiotherapy for submandibular lymph node metastases of tongue cancer. Four weeks after the radiotherapy, a clavicle fracture, with osteomyelitis and abscess formation in the pectoralis major muscle, occurred. Unlike in simple stress fracture, long-term antibiotic administration and drainage surgery were required to suppress the inflammation. CONCLUSION: As seen in the present patient, clavicle stress fractures may occur even after neck dissection in which the accessory nerve is preserved, and may be complicated by osteomyelitis and abscess formation owing to risk factors such as radiotherapy, tracheostomy and contiguous infection.

The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2.

In human cells, hMLH1, hMLH3, hPMS1 and hPMS2 are four recognised and distinctive homologues of MutL, an essential component of the bacterial DNA mismatch repair (MMR) system. The hMLH1 protein forms three different heterodimers with one of the other MutL homologues. As a first step towards functional analysis of these molecules, we determined the interacting domains of each heterodimer and tried to understand their common features. Using a yeast two-hybrid assay, we show that these MutL homologues can form heterodimers by interacting with the same amino acid residues of hMLH1, residues 492-742. In contrast, three hMLH1 partners, hMLH3, hPMS1 and hPMS2 contain the 36 homologous amino acid residues that interact strongly with hMLH1. Contrary to the previous studies, these homologous residues reside at the N-terminal regions of three subdomains conserved in MutL homologues in many species. Interestingly, these residues in hPMS2 and hMLH3 may form coiled-coil structures as predicted by the MULTICOIL program. Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues. Therefore, the quantitative balance of these three MutL heterodimers may be important in their functions.

Identification of a 790-kilobase region of common allelic loss in chromosome 10q25-q26 in human endometrial cancer.

We previously identified two commonly deleted regions on chromosome bands 10q25-q26 in endometrial cancer: an 8-cM region and a 12-cM region. In the present study, we further studied the 8-cM region with 83 endometrial cancer specimens and 14 microsatellite markers and narrowed it down to a 1-cM region between D10S587 and D10S1723. This result was confirmed by two-color fluorescence in situ hybridization analysis. An association between histopathologically lower grade tumor and allelic loss (P = 0.03 by Fisher's exact test) was also observed. We also constructed a yeast artificial chromosome (YAC) contig and found that one YAC clone, which was 790 kb in size, harbored the whole 1-cM region of common allelic loss.

Disruption of the APC gene by a retrotransposal insertion of L1 sequence in a colon cancer.

The APC gene is responsible for familial adenomatous polyposis and is considered to be a tumor suppressor gene associated with development of sporadic colorectal tumors. Here we report the disruption of the APC gene caused by somatic insertion of a long interspersed repetitive element (LINE-1 sequence) into the last exon of the APC gene in a colon cancer. The inserted sequence was composed of a 3' portion of the LINE-1 consensus sequence and nearly 180 base pairs of polyadenylate tract. Furthermore, since an 8-base pair target site duplication was observed, retrotranscriptional insertion of an active LINE-1 sequence is suspected as the cause of this insertion event. This is the first report of the disruption of a tumor suppressor gene caused by somatic insertion of a mobile genetic element.

Loss of chromosome 18q is an early event in pancreatic ductal tumorigenesis.

Cytogenetic and molecular studies demonstrated that pancreatic cancer frequently shows specific chromosomal abnormalities, such as losses of 9p, 17p, and 18q, and gains of 8q and 20q. We have analyzed alterations in the copy number of specific chromosomal regions in cells from the pancreatic juices of 32 patients with various pancreatic disorders by fluorescence in situ hybridization (FISH) technique to pursue the possible clinical use of early diagnosis of pancreatic cancer. None of the chromosomal abnormalities were found in 13 specimens from individuals who had no neoplastic lesions. On the other hand, 12 specimens (63%) derived from the remaining 19 patients who had neoplastic lesions showed at least one chromosomal abnormality. Ten of these specimens were from pancreatic cancer patients; 7 cases (70%) showed chromosomal abnormalities. All but one of the 12 tumors with chromosomal abnormalities had loss of 18q. Furthermore, we detected a tumor in one patient in whom the routine cytological method and endoscopic retrograde chorangiopancreatography found nothing. Based on the results by FISH, we performed endoscopic ultrasonography and found a small serous cystadenoma in this patient. These results indicate that: (a) FISH analysis of cells from pancreatic juices obtained during endoscopic retrograde chorangiopancreatography is quite useful for detecting pancreatic ductal tumors; and (b) loss of chromosome 18q is one of the early genetic changes that provide very useful information in diagnosing pancreatic neoplasias.

The APC gene, responsible for familial adenomatous polyposis, is mutated in human gastric cancer.

Although gastric cancer is the most common cancer in the world, genetic changes during its carcinogenesis are not well understood. Since some gastric cancers are considered to originate from the intestinal metaplasia, it is likely that the adenomatous polyposis coli (APC) gene, the mutation of which causes adenomatous polyps in the colon, is associated with carcinogenesis of gastric cancer. Based on this idea, DNAs isolated from gastric cancers were examined by means of a RNase protection analysis coupled with polymerase chain reaction followed by sequencing of the polymerase chain reaction products. By screening nearly one-half of the coding region of the APC gene in 44 tumors, somatic mutations were detected in three tumors: a missense mutation, a nonsense mutation, and a 5-base pair deletion resulting in a frame shift which causes truncation of the gene product. These results suggest that the mutation of the APC gene also plays an important role during the carcinogenesis of at least some gastric cancers.

Frequent frameshift mutations of RIZ in sporadic gastrointestinal and endometrial carcinomas with microsatellite instability.

Many lines of evidence suggest that the retinoblastoma protein interacting zinc finger gene RIZ is a strong candidate for the tumor suppressor locus on 1p36, a region commonly deleted in many human cancers with chromosomal instability. In addition, a role for RIZ in tumors of the microsatellite instability pathway is suggested by frequent frameshift mutations in hereditary non-polyposis colorectal carcinomas. Here we studied RIZ mutations in sporadic cancers with microsatellite instability. Frameshift mutations in the two coding polyadenosine tracks of RIZ were found in 19 (48%) of 40 gastric carcinomas, 6 (33%) of 18 endometrial carcinomas, 14 (26%) of 51 of colorectal carcinomas, and 7 (54%) of 13 cell lines. Eleven tumor tissues showed biallelic inactivation of RIZ. In contrast, no frameshift mutations were found in 70 microsatellite stable tumors. These results suggest an important role for RIZ in sporadic cancers with microsatellite instability.

Expression of alpha-amylase isozymes in human thyroid tissues.

Expression of alpha-amylase genes in thyroid tissues was studied by assaying the total amylase activity as well as by using immunohistochemical and Northern blot analysis. The amylase genes expressed were determined by a combination of the polymerase chain reaction (PCR) and blot analysis using synthetic probes specific for the three known amylase isozyme complementary DNAs. The samples consisted of tissues from 18 human thyroid carcinomas (11 well-differentiated carcinomas, 2 poorly differentiated carcinomas, 1 anaplastic carcinoma, and 4 medullary carcinomas) and 9 specimens of nonmalignant thyroid tissue (2 were from nontumorous regions of resected glands and 7 were thyroid tissue from a patient with Graves' disease). Salivary-type amylase was expressed at a relatively high level in nonmalignant thyroid tissue and well-differentiated carcinoma and could be detected by Northern blot analysis. In poorly differentiated carcinoma, it was detected only by the PCR, while in anaplastic or medullary carcinoma, it was not detected even by the PCR. Thus, the expression of salivary-type amylase was characteristic of well-differentiated follicular cells. These observations suggest that salivary-type amylase expression may be a marker for identifying the histogenesis and stage of differentiation of thyroid cancer. In addition, the AMY2B gene product was detected in all different types of cells examined, although its expression was only detectable by the PCR. Pancreatic type amylase was not detected in any of the samples.

Frequent replication errors at microsatellite loci in tumors of patients with multiple primary cancers.

Nearly 10% of cancer patients develop a second primary cancer within 10 years after surgical removal of the first tumor. Hence, detection of a genetic risk for developing multiple primary tumors would be of clinical importance. To investigate whether a genetic defect(s) involving the mismatch repair system constitutes an important risk factor in patients with multiple primary cancers, we examined replication errors (RER) at microsatellite loci in 79 primary cancers which had developed among 38 patients with multiple primary cancers. The RER(+) phenotype was observed at five microsatellite loci on chromosomes 2, 3, 11, or 17 in tumors from 34 (89%) of 38 patients with multiple primary cancers but only in 19 tumors from 174 patients (11%) with a single primary cancer. Our results suggested that: (a) genetic instability may play an important role in development of multiple primary cancers, and (b) testing for RER in a primary cancer may be an appropriate approach to detection of patients at high risk for developing multiple primary cancers.

Correlation between the location of germ-line mutations in the APC gene and the number of colorectal polyps in familial adenomatous polyposis patients.

Recently we have isolated the adenomatous polyposis coli (APC) gene which causes familial adenomatous polyposis (FAP), and its germ-line mutations in a substantial number of FAP patients have been identified. On the basis of this information, we compared the location of germ-line mutations in the APC gene in 22 unrelated patients (12 of whom have been reported previously) with the number of colorectal polyps developed in FAP patients; 17 were sparse types and five were profuse types. All but one of the mutations were considered to cause truncation of the gene product by frame-shift due to deletion (14 cases) or nonsense mutation (seven cases). The location of the germ-line mutations seems to correlate with the two clinical types; germ-line mutations in five FAP patients with profuse polyps were observed between codon 1250 and codon 1464, whereas mutations in 17 FAP patients with fewer polyps were observed in the other regions of the APC gene. The result suggests that the number of colorectal polyps in FAP patients may be associated with a difference in the stability or biological function of the truncated APC protein.

Frequent somatic mutations of the APC gene in human pancreatic cancer.

The APC (adenomatous polyposis coli) gene is responsible for familial adenomatous polyposis and is also associated with the development of sporadic tumors of the colon and stomach. To investigate whether or not mutations of APC play any role in tumors arising in other organs, we examined somatic mutations of this gene in sporadic (nonfamilial) renal cell carcinomas, hepatocellular carcinomas, and cancers of the lung and pancreas. DNAs isolated from tumors were examined by means of a RNase protection analysis, coupled with the polymerase chain reaction followed by DNA sequencing of the polymerase chain reaction products. By screening a part of the APC coding region, we detected somatic mutations in four of ten pancreatic cancers; each of these mutations would yield a truncated APC product due to a 1- or 5-base pair deletion. These results imply that mutations in APC contribute to carcinogenesis in the pancreas.