Chloride ion transport and overexpression of TMEM16A in a guinea-pig asthma model.

BACKGROUND: TMEM16A, a Ca-activated Cl channel, regulates various physiological functions such as mucin secretion. However, the role of TMEM16A in hyper-secretion in asthma is not fully understood. OBJECTIVE: The aim of this study is to evaluate Cl ion transport via TMEM16A and determine the localization of TMEM16A in a guinea-pig asthma model. METHODS: Guinea-pigs were sensitized with ovalbumin (OVA) i.p. on Days 1 and 8. On Day 22, we assessed OVA challenge-induced Cl ion transport in the sensitized tracheas ex vivo in an Ussing chamber, compared with the non-sensitized tracheas. We then examined the effect of T16Ainh-A01, a TMEM16A inhibitor, on the increase in Cl ion transport. The tracheal epithelium was immunostained with an anti-TMEM16A antibody. Epithelial cells from guinea-pig tracheas were cultured at the air-liquid interface in the presence of IL-13 for in vitro study. We studied the effect of TMEM16A inhibitors on Ca-dependent agonist, uridine triphosphate (UTP)-induced increases in Cl ion transport in the cultured cells. The cells were immunostained with an anti-TMEM16A antibody, an anti-MUC5AC antibody and an anti-α-tubulin antibody. RESULTS: OVA challenge induced an increase in short circuit current within 1 min in the OVA-sensitized tracheas but not in the non-sensitized tracheas, which was inhibited by pretreatment of T16Ainh-A01. Sensitized tracheas showed goblet cell metaplasia with more positive TMEM16A immunostaining, particularly in the apical portion compared with the non-sensitized tracheas. The in vitro UTP-induced increase in Cl ion transport was strongly inhibited by pretreatment with T16Ainh-A01, benzbromarone, and niflumic acid. TMEM16A was positively immunostained at the apical portion and in the MUC5AC-positive area in IL-13-induced goblet cell metaplasia. CONCLUSIONS: Antigen challenge and Ca-dependent agonist treatment increased Cl ion transport via the overexpression of TMEM16A in goblet cell metaplasia in a guinea-pig asthma model. TMEM16A inhibitors may be useful for the treatment of hyper-secretion in asthma.

Predicting recurrence after curative resection for gastric cancer: External validation of the Italian Research Group for Gastric Cancer (GIRCG) prognostic scoring system.

BACKGROUND: Most nomograms for Gastric Cancer (GC) were developed to predict overall survival (OS) after curative resection. The Italian Research Group for Gastric Cancer (GIRCG) prognostic scoring system (PSS) was designed to predict the recurrence risk after curative treatment based on pathologic tumor stage and treatment performed (D1-D2/D3 lymphadenectomy). This study was carried out to externally validate the GIRCG's PSS. PATIENTS AND METHODS: Adopting the same criteria used by GIRCG to build the PSS, 185 patients with GC operated with curative intention were selected. The median follow-up period was 77.8 months (1.93-150.8) for all patients and 102.5 months (60.9-150.8) for patients free of disease. The NRI (net reclassification improvement) was calculated to estimate the overall improvement in the reclassification of patients using the PSS in place of the TNM stage system. RESULTS: GC recurrence occurred in 70 (37.8%) patients. The mean time to recurrence was 22.2 (range 1.9-98.1) months. For patients with recurrence, the gain in the proportion of reclassification was 0.257 (p < 0.001), indicating an improvement of 26%. For patients without recurrence, the gain in the proportion of reclassification was -0.122 (p < 0.001), indicating a worsening of 12%. The NRI calculated was 0.135 (p = 0.0527). CONCLUSION: The GIRCG's PSS, which predicts the likelihood of recurrence after radical surgical treatment for GC, is more accurate than TNM system to predict recurrence mainly for high-risk patients. Yet, the PSS does not have the same effectiveness for low-risk patients, overestimating the chance of recurrence occurs even for disease-free patients.

Identification of estrone-degrading Betaproteobacteria in activated sludge by microautoradiography fluorescent in situ hybridization.

UNLABELLED: Natural oestrogens, which are degraded but not completely removed in wastewater treatment plants, are suspected of causing the endocrine disruption of aquatic organisms in the receiving water body. While several bacterial isolates were reported to be oestrogen-degrading bacteria, our previous study implied that only the unidentified rod-shaped Betaproteobacteria in chains were responsible for estrone (E1) degradation by activated sludge especially at the sub-milligram per litre level. The Betaproteobacteria were suspected to be related to genera Sphaerotilus and Leptothrix according to morphological observations. Probe Spha823 was newly developed to target 16S rRNA gene clones obtained from activated sludge and closely related to the above genera. [(3) H]E1-incubated sludge samples showed that most of the (3) H-labelled cells hybridized with probe Spha823 by microautoradiography (MAR) fluorescent in situ hybridization. Spha823-defined cells were present in all three activated sludge samples tested, where they accounted for up to 3% of the total microbial biomass. Spha823-defined cells comprised 59·5-80·1% of the total MAR-positive cells, which suggested that the Sphaerotilus-Leptothrix-related bacteria were the most abundant micro-organisms involved in E1 degradation (at 200 µg l(-1) ) in the activated sludge samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Estrone (E1) is one of the natural estrogens, which can be degraded but is not always completely removed in wastewater treatment plants. E1 is suspected of causing the endocrine disruption of aquatic organisms in the receiving water body. We identified dominant E1-incorporating bacteria, which should include E1-degrading bacteria, in activated sludge treating domestic wastewater. Sphaerotilus-Leptothrix-related bacteria, which had never been reported in the previous attempts based on culture-dependent approach, occupied 60-80% of the E1-incorporating bacteria. This study demonstrates the identification of functionally active bacteria to degrade micro-pollutants at sub-milligram per litre level.

Characterization of monooxygenase gene diversity in benzene-amended soils.

AIM: To understand soil benzene monooxygenase gene diversity by clone library construction and microarray profiling. METHODS AND RESULTS: A primer set was designed, and benzene monooxygenase gene diversity was characterized in two benzene-amended soils. The dominant sequence types in the clone libraries were distinct between the two soils, and both sequences were assigned to novel clusters. Monooxygenase gene richness and diversity increased after benzene degradation. Oligonucleotide probes for microarray analysis were designed to detect a number of sequenced clones and reported monooxygenase genes. The microarray detected several genes that were not detected in the clone libraries of the same samples. Six probes were detected in more than one soil. CONCLUSIONS: The primer set designed in this study successfully detected diverse benzene monooxygenase genes. The level of diversity may have increased because the degradation of benzene differed from soil to soil. Microarrays have great potential in the comprehensive detection of gene richness as well as the elucidation of key genes for degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study introduces a new primer set that may be used to identify diverse benzene monooxygenase genes in the environment; moreover, it demonstrates the potential of microarray technology in the profiling of environmental samples.

Larger amounts of nitrite and nitrate-reducing bacteria in megaesophagus of Chagas' disease than in controls.

In the megaesophagus of Chagas' disease, chronic esophagitis is caused by stasis of swallowed food and saliva. In this environment, the overgrowth of aerobic and anaerobic bacteria, including nitrate-reducing bacteria, is observed. The reduction of nitrate into nitrite by the action of these bacteria has been associated with the formation of volatile nitrosamines in different situations of gastric bacterial overgrowth. We have hypothesized that this phenomenon could occur in the esophageal lumen of patients with megaesophagus. To evaluate the concentration of nitrite, the presence of volatile nitrosamines and the concentration of nitrate-reducing bacteria in the esophageal lumen of patients with non-advanced megaesophagus of Chagas' disease and in a group of patients without esophageal disease. Fifteen patients with non-advanced megaesophagus [megaesophagus group (MG)] and 15 patients without any esophageal disease [control group (CG)] were studied. Saliva samples were taken for nitrate and nitrite quantitative determination and esophageal stasis liquid samples were taken for nitrate and nitrite quantitative determination, volatile nitrosamines qualitative determination and reductive bacteria quantitative determination. MG and CG were equivalent in nitrate and nitrite saliva concentration and in nitrate esophageal concentration. Significant difference was found in nitrite (p = 0.003) and reductive bacteria concentration (p < 0.0001), both higher in MG. Volatile nitrosamines were identified in three MG patients and in none of the CG patients, but this was not significant (p = 0.113). There is a higher concentration of reductive bacteria in MG, responsible for the rise in nitrite concentration at the esophageal lumen and, eventually, for the formation of volatile nitrosamines.

Quantification of ammonia-oxidizing bacteria populations in full-scale sewage activated sludge systems and assessment of system variables affecting their performance.

This study carried out quantification of ammonia-oxidizing bacteria (AOB) populations in 12 full-scale sewage activated sludge systems that were different in ammonia removals and treatment processes during three different seasons. Experiment was divided into 3 parts: 1) analysis of AOB communities by PCR-DGGE-cloning-sequencing of 16S rRNA genes; 2) development of four real-time PCR primer sets for quantification of the particular AOB of interest; and 3) quantification of AOB populations by using the newly developed real-time PCR primer sets. The results suggested that all the primer sets gave good reproducibility and specificity for PCR amplification with the detection limits of 10(2) copies/PCR reaction. Although the 12 systems were different in several aspects, one of the identified sequence types of Nitrosomonas oligotropha cluster was the dominant AOB in every system and every season studied. However, the other sequence type of this cluster was not significantly involved in ammonia removals in the systems. The occurrence of N. communis cluster in the systems seemed to depend on the remaining oxygen concentrations in the sludge floc and thus the activity of aerobic heterotrophs in the aeration tanks. N. europaea-Nitrosococcus. mobilis solely existed in one A20 system of which the influent contained twice the chloride concentrations than those of other systems.

Distribution of ammonia-oxidizing bacteria in sewage activated sludge: analysis based on 16S rDNA sequence.

This study carried out analysis of ammonia-oxidizing bacteria (AOB) communities in 12 sewage activated sludge systems standing in eight sewage treatment plants located in Tokyo. The systems were different in the treatment process configuration: anaerobic/anoxic/aerobic (A20), anaerobic/aerobic (AO), and conventional activated sludge (AS) processes. AOB communities were analyzed by sequences of 16S rDNA amplicons, which were separated by denaturing gradient gel eletrophoresis (DGGE) after specific polymerase chain reaction (PCR) amplification. The results demonstrated that low ammonium concentrations in the influents of the 12 sewage activated sludge systems resulted in the dominance of Nitrosomonas oligotropha-like sequences. Further, Nitrosomonas europaea- and Nitrosomonas cryotolerans-like sequences were recovered from only one A20 system of which the influent contained higher ammonium and chloride concentrations than those of other systems. Nitrosomonas communis-like sequences were found in every A20 and AO system, but mostly not found in every AS system. In summary, influent characteristics and treatment process configuration affected the AOB communities in the 12 sewage activated sludge systems.

Degradation of polycyclic aromatic hydrocarbons by a newly isolated dibenzofuran-utilizing Janibacter sp. strain YY-1.

The dibenzofuran (DF)-utilizing bacterium strain YY-1 was newly isolated from soil. The isolate was identified as Janibacter sp. with respect to its 16S rDNA sequence and fatty acid profiles, as well as various physiological characteristics. In addition to DF, strain YY-1 could grow on fluorene and dibenzothiophene as sole sources of carbon and energy. It was also able to cometabolize a variety of polycyclic aromatic hydrocarbons including dibenzo- p-dioxin, phenanthrene, and anthracene. The major metabolites formed from DF, biphenyl, dibenzothiophene, and naphthalene were identified by using gas chromatography-mass spectrometry as 2,3,2'-trihydroxybiphenyl, biphenyl-dihydrodiol, dibenzothiophene 5-oxide, and coumarin, respectively. These results indicate that strain YY-1 can catalyze angular dioxygenation, lateral dioxygenation, and sulfoxidation.

Quantitative and rapid detection of the trichloroethylene-degrading bacterium Methylocystis sp. M in groundwater by real-time PCR.

We developed a method based on real-time PCR for the specific and rapid enumeration of a trichloroethylene-degrading methanotroph, Methylocystis sp. M, with the aim of monitoring the strain in groundwater. A primer set designed from the nucleotide sequence of the mmoC gene of a soluble methane monooxygenase (sMMO) gene cluster from Methylocystis sp. M was specific to amplify the DNA region from the strain and no PCR products were amplified with the sMMO gene clusters from six other methanotroph strains. The real-time PCR reliably quantified Methylocystis sp. M over at least five orders of magnitude (5x10(6) to 5x10(2 )cells/PCR tube, or 2x10(8) to 2x10(4 )cells/ml). Five cells of Methylocystis sp. M per PCR tube (2x10(2 )cells/ml) were detectable when the cells were suspended in distilled water. The concomitant presence of other methanotrophs in samples did not affect the reliability of enumeration; and recovery of the cells with a membrane filter enabled us to quantify cells of the strain in groundwater. This quantification procedure was completed within 3 h, including preparation time of environmental samples. We conclude that real-time PCR using the mmoC primer set can be used practically to analyze the behavior of Methylocystis sp. M at bioremediation sites.

Quantitative and specific detection of a trichloroethylene-degrading methanotroph, Methylocystis sp. strain M, by a most probable number-polymerase chain reaction method.

We developed a rapid and specific enumeration method for a trichloroethylene-degrading methanotroph, Methylocystis sp. strain M, based on a most probable number-polymerase chain reaction method for monitoring the bacterium at bioremediation sites. The primers designed for the mmoC gene of the soluble methane monooxygenase gene cluster were specific to strain M. Recovery of the cells with a membrane filter enabled us to detect strain M in trichloroethylene-contaminated groundwater. We used the enumeration method to monitor the number of strain M cells in effluent from soil columns supplied with trichloroethylene-contaminated groundwater. The number of strain M cells in the effluent depended on the amount of the strain M inoculated and the number of cells measured by the most probable number-polymerase chain reaction method was correlated with that measured by a culture method. The detection limit for strain M in effluent detected by MPN-PCR method was 4 to 8 x 10(2) cells/ml.

Distinct expression of CDX2 and GATA4/5, development-related genes, in human gastric cancer cell lines.

CDX2 is a tumor-suppressor homeobox gene involved in colon carcinogenesis, but its role in gastric cancer is unknown. Although GATA4, -5 and, -6 transcription factors have distinct functions in the regulation of gastrointestinal epithelial cell differentiation, there have been no reports regarding GATA4/5/6 alterations in gastrointestinal carcinomas. By using a semiquantitative reverse transcription-polymerase chain reaction assay, we studied the expression of gut development-related genes CDX2/1 and GATA4/5/6 in 11 human gastric cancer cell lines. The expression of CDX2 appeared to progressively decrease with the transition from well differentiated to poorly differentiated cancer cell lines. CDX1 was below detectable levels in all cell lines. The expression of GATA4 and GATA5 was undetectable in four and six cell lines, respectively, whereas the majority of the cell lines expressed GATA6 abundantly. These results suggest that CDX2 and GATA4/5 may be associated with the carcinogenesis of the stomach. Mol. Carcinog. 28:184-188, 2000.

Aerobic degradation of 1,1,1-trichloroethane by Mycobacterium spp. isolated from soil.

Two strains of 1,1,1-trichloroethane (TCA)-degrading bacteria, TA5 and TA27, were isolated from soil and identified as Mycobacterium spp. Strains TA5 and TA27 could degrade 25 and 75 mg. liter of TCA(-1) cometabolically in the presence of ethane as a carbon source, respectively. The compound 2,2,2-trichloroethanol was produced as a metabolite of the degradation process.

Genomic structure and alterations of homeobox gene CDX2 in colorectal carcinomas.

Expression of CDX2, a caudal-related homeobox gene, was found to be decreased in colorectal carcinomas. Heterozygous null mutant mice as to Cdx2 develop multiple intestinal adenomatous polyps. To clarify the role of CDX2 in colorectal carcinogenesis, we determined its genomic structure, and searched for mutations of CDX2 in 49 sporadic colorectal carcinomas and ten hereditary non-polyposis colorectal cancers (HNPCC) without microsatellite instability. None of them exhibited a mutation. We further examined 19 HNPCC carcinomas with microsatellite instability for mutations in a (G)7 repeat site within CDX2. One of them (5.3%) exhibited one G insertion. Loss of heterozygosity was observed in 2 of the 20 (10%) informative sporadic carcinomas, and in one of the three (33.3%) informative HNPCC cancers. These data indicate that CDX2 may play only a minor role in colorectal carcinogenesis.

Frequent allelic imbalance on chromosome 18q21 in early superficial colorectal cancers.

Genetic alterations in early superficial colorectal cancers have rarely been reported. In the present study, we searched for alterations in the APC and p53 genes in 27 superficial (20 depressed and 7 elevated) and 21 protruding colorectal cancers with submucosal invasion by means of PCR-single strand conformation polymorphism. Allelic imbalance (AI) on five loci, i.e., 1p34-36, 8p21-22, 14q32, 18q21 and 22q12-13, was also analyzed. Since a high incidence of 18q21 AI was detected in the superficial depressed cases, we further screened for alterations in Smad2, Smad4 and DCC. APC alterations were observed in three superficial depressed, one superficial elevated, and 11 protruding colorectal cancers, indicating that the frequency of APC alterations in superficial depressed cases was significantly lower than that in the protruding ones. There was no significant association between p53 alterations and macroscopic types. AI on 18q21 (13/20, 65%) was much higher than those on the other four loci in the superficial depressed cases. Moreover, the frequency of 18q21 AI in the superficial depressed cases was significantly higher than that in the protruding ones. Smad4 alterations were only detected in 1 of the 13 superficial depressed and 3 of the 17 protruding cases, while Smad2 and DCC alterations were not detected in any case examined. These data suggest that the carcinogenetic pathways of protruding and superficial depressed colorectal cancers are different, and that alterations of tumor suppressor gene(s) located on 18q21 other than Smad2, Smad4 and DCC might be associated with most superficial depressed colorectal cancers.

Genetic alterations are frequent in APC but rare in the TGF-beta type II receptor gene in cancer in adenomas of the colon.

Cancer in adenomas are thought to be an excellent model of colorectal carcinogenesis based on the adenoma-carcinoma sequence. We searched for alterations in the APC mutation cluster region, the whole coding regions of TGF-beta type II receptor (RII) and beta-catenin exon 3 in 16 cases of cancer in adenomas of the colon. Overexpression of the p53 protein was also analyzed. Nine of the 16 cases showed APC mutations in both the adenoma and cancer regions. Loss of heterozygosity in APC was found in one cancer in adenoma that had no mutation. p53 overexpression was detected in one adenoma and 10 cancerous regions, most of which also exhibited APC alterations. Two cases showed a missense mutation at codon 191 or loss of heterozygosity in TGF-beta RII in both the adenoma and cancer. Our data support the hypothesis that alterations of APC and p53 are responsible for most of the adenoma-carcinoma pathway, rather than TGF-beta RII alterations.

Proapoptotic gene BAX is frequently mutated in hereditary nonpolyposis colorectal cancers but not in adenomas.

BACKGROUND & AIMS: The p53 and BAX genes have been linked to apoptosis. p53 was not frequently found to be mutated in colorectal carcinomas with a microsatellite mutator phenotype, but frame-shift mutations in a tract of eight guanines within BAX were frequently found in these carcinomas. To understand the roles of these genes in hereditary nonpolyposis colorectal cancer (HNPCC) tumorigenesis, we examined whether BAX mutations occur in adenoma and carcinoma specimens from patients with HNPCC and also determined the frequencies of p53 mutations. METHODS: Thirteen colorectal adenomas and 24 adenocarcinomas from patients with HNPCC showing a microsatellite instability phenotype were screened by polymerase chain reaction followed by denaturing polyacrylamide gel electrophoresis and direct sequencing. RESULTS: Two of the 13 adenomas (15.4%) and 13 of the 24 adenocarcinomas (54.2%) showed mutation patterns and were confirmed to have frame-shift mutations at the BAX repeat site by direct sequencing. For p53, only 1 of the 24 adenocarcinomas (4.2%) showed a missense mutation. CONCLUSIONS: In HNPCC colorectal carcinomas, BAX was significantly (P = 0.024) more mutated than in adenomas. p53 was not frequently found to be mutated in these carcinomas. These data suggest that mutations in BAX, rather than mutations in p53, may contribute to the adenoma-carcinoma transition in HNPCC tumorigenesis.

Analyses of the APC and TGF-beta type II receptor genes, and microsatellite instability in mucosal colorectal carcinomas.

APC and transforming growth factor-beta type II receptor (TGF-beta RII) gene mutations, and microsatellite instability have been found in sporadic colorectal carcinomas. To clarify further the early alterations in colorectal carcinogenesis, we investigated these genetic changes in 23 protruding- and 24 superficial-type mucosal colorectal carcinomas. TGF-beta RII gene mutations and microsatellite instability were rarely found in these lesions. Nevertheless, APC was mutated in 16 of the 47 (34.0%) mucosal colorectal carcinomas and was significantly more frequently mutated in protruding- (I) and superficial elevated-type (IIa) (14/32, 43.8%) than in other superficial-type (IIa+IIc, IIb, IIc, and IIc+IIa) (2/ 15, 13.3%) mucosal colorectal carcinomas (P < 0.04). These results indicate that the APC gene may be involved from the beginning in the tumorigenesis of many early colorectal carcinomas, particularly of the protruding and superficial elevated types. However, there might be a distinct pathway for other superficial-type colorectal carcinomas, possibly not involving APC as an initial step of tumorigenesis.

Random amplified polymorphic DNA (RAPD) analyses for discriminating genotypes of Microcystis cyanobacteria.

Random amplified polymorphic DNA (RAPD) analysis was used to discriminate genotypes in five species of Microcystis cyanobacteria. Strains of each group with the identical allozyme genotype (T. Kato et al., Algol. Stud., 1991, 129-140; M. Watanabe, in "Toxic Microcystis," ed. by M.F. Watanabe et al., CRC Press, Tokyo, 1966, pp. 13-34) gave similar RAPD patterns characterizing the respective group. On the other hand, no similarities in RAPD patterns were observed among strains of which allozyme genotypes were different. A good accordance between the RAPD analysis and allozyme divergence indicated a high reliability of both methods for discrimination of the affiliated groups of Microcystis. Several amplified DNA fragments, which were expected to be markers for a particular taxon with identical allozyme genotype, were also observed on the RAPD patterns. Genetic homogeneities of M. novacekii, M. viridis, and M. wesenbergii were shown by RAPD analysis as well as the allozyme genotype. However, significant variations were observed in M. aeruginosa and M. ichthyoblabe in the levels of DNA and proteins (allozymes).

Characteristics of Escherichia coli HB101 and Pseudomonas putida PpY101 harboring a recombinant plasmid with tandem insertion of the mercury resistance operon.

We constructed the plasmid pSUPmer2 by inserting tandem copies of the mercury resistance (mer) operon into a broad host range-vector, and introduced it into Escherichia coli HB101 and Pseudomonas putida PpY101 to increase their mercury resistance. Strains harboring plasmid pSUPmer2 had higher mercury resistance and mercuric reductase activity than those strains harboring the plasmid pSUPmer which had one copy of the mer operon. Mercury resistance of P. putida PpY101 was significantly increased by tandem insertion of the mer operon.