Selective detection of viable Helicobacter pylori using ethidium monoazide or propidium monoazide in combination with real-time polymerase chain reaction.

Because Helicobacter pylori has a role in the pathogenesis of gastric cancer, chronic gastritis and peptic ulcer disease, detection of its viable form is very important. The objective of this study was to optimize a PCR method using ethidium monoazide (EMA) or propidium monoazide (PMA) for selective detection of viable H. pylori cells in mixed samples of viable and dead bacteria. Before conducting the real-time PCR using SodB primers of H. pylori, EMA or PMA was added to suspensions of viable and/or dead H. pylori cells at concentrations between 1 and 100 µM. PMA at a concentration of 50 µM induced the highest DNA loss in dead cells with little loss of genomic DNA in viable cells. In addition, selective detection of viable cells in the mixtures of viable and dead cells at various ratios was possible with the combined use of PMA and real-time PCR. In contrast, EMA penetrated the membranes of both viable and dead cells and induced degradation of their genomic DNA. The findings of this study suggest that PMA, but not EMA, can be used effectively to differentiate viable H. pylori from its dead form.

MAM domain containing 2 is a potential breast cancer biomarker that exhibits tumour-suppressive activity.

OBJECTIVES: The aim of this study was to discover new potential biomarkers of breast cancer and investigate their cellular functions. MATERIALS AND METHODS: We analysed the gene expression profiles of matched pairs of breast tumour and normal tissues from 24 breast cancer patients. Tetracycline-inducible MAMDC2 expression system was established and used to evaluate cell proliferation in vitro and in vivo. MAMDC2-mediated signalling was determined using immunoblot analysis. RESULTS: We identified MAMDC2 as a down-regulated gene showing significant prognostic capability. Overexpression of MAMDC2 or treatment with MAMDC2-containing culture medium significantly inhibited the cell proliferation of T-47D cells. Furthermore, MAMDC2 expression reduced in vivo growth of T-47D xenograft tumours. MAMDC2 may exert its growth-inhibitory functions by attenuating the MAPK signalling pathway. CONCLUSION: We report that MAMDC2 has a tumour-suppressive role and, as a secretory protein, it might be useful as a biomarker for breast cancer treatment.

Velvet-mediated repression of ß-glucan synthesis in Aspergillus nidulans spores.

Beta-glucans are a heterologous group of fibrous glucose polymers that are a major constituent of cell walls in Ascomycetes and Basidiomycetes fungi. Synthesis of ß (1,3)- and (1,6)-glucans is coordinated with fungal cell growth and development, thus, is under tight genetic regulation. Here, we report that ß-glucan synthesis in both asexual and sexual spores is turned off by the NF-kB like fungal regulators VosA and VelB in Aspergillus nidulans. Our genetic and genomic analyses have revealed that both VosA and VelB are necessary for proper down-regulation of cell wall biosynthetic genes including those associated with ß-glucan synthesis in both types of spores. The deletion of vosA or velB results in elevated accumulation of ß-glucan in asexual spores. Double mutant analyses indicate that VosA and VelB play an inter-dependent role in repressing ß-glucan synthesis in asexual spores. In vivo chromatin immuno-precipitation analysis shows that both VelB and VosA bind to the promoter region of the ß-glucan synthase gene fksA in asexual spores. Similarly, VosA is required for proper repression of ß-glucan synthesis in sexual spores. In summary, the VosA-VelB hetero-complex is a key regulatory unit tightly controlling proper levels of ß-glucan synthesis in asexual and sexual spores.

Cloning, expression, and fibrin (ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4.

Previously, we purified a strong fibrinolytic enzyme (subtilisin DJ-4) from Bacillus sp. DJ-4 and characterized its enzymatic activity. Here, we cloned the gene subtilisin DJ-4, and determines its nucleotide sequence, which showed 97% identity with subtilisin BPN' from B. amyloliquefacens. Recombinant full-subtilisin DJ-4 (rf-subDJ-4) and mature-subtilisin DJ-4 (rm-subDJ-4) were expressed using a pET29 vector system, and their fibrin (ogen)olytic and plasminogen activator activities were studied. rf-subDJ-4 was found to have a higher stability to heat (60 degrees C) and to acidic conditions (pH 3.0-4.0) than the native subtilisin DJ-4 of Bacillus sp. DJ-4. The plasminogen activator activity of rf-subDJ-4 was 2.75 times greater than that of plasmin on a molar basis. And its specific activity (F/C, the ratio of fibrinolytic activity to caseinolytic activity) was 2.67 and 3.97 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively. rf-subDJ-4 rapidly hydrolyzed the Aalpha-, Bbeta-, and gamma-chains of fibrinogen within 5 min. But, unlike subtilisin BPN' at a very low concentration (50 ng), the gamma-chain was not cleaved. On the other hand, rm-subDJ-4 did not show enzyme activity.