Our new PCR-based assay for 23S rRNA mutations of H. pylori is a useful method for detecting clarithromycin-resistant strains of H. pylori easily.

The ASP-PCR assay for 23S rRNA mutation of H. pylori is a useful method to detect clarithromycin-resistant strains of H. pylori easily.

Estrogenic activities of 20 selected pesticides-which are used for agricultural production as insecticides, fungicides and herbicides-were examined by estrogen receptor (ER)-dependent MCF-7 cell proliferation assay. Among them, chlordecone, dicofol, methoxychlor, gamma-HCH, fenarimol, EPN, triadimefon, and triadimenol had estrogenic activities, all of which were suppressed by the addition of pure antiestrogen ICI 182,780. The first 5 compounds exhibited binding capacities to ERalpha. The antiestrogenic activity of a compound was examined by estimating its suppressive effect on cell proliferation induced by 30 pM 17beta-estradiol. Strongly suspected antiestrogens were captan and myclobutanil, both of which were found to have the capacity to bind to ERalpha and which might exert their activities by competing at the level of ERalpha. Antiestrogenic activities of nitrofen, fenitrothion, fenarimol and triadimefon were also suggested. Affinities of the compounds for ERalpha and/or androgen receptor (AR) were lower than those of synthetic estrogen (diethylstilbestrol) and testosterone (mibolerone), respectively. Fenitrothion had the highest affinity to AR. Chlordecone, dicofol, methoxychlor, nitrofen, fenarimol, myclobutanil and pyridate had capacities to bind both ERalpha and AR. Chlordecone and pyridate were much more effective as competitors of estrogen binding to ERalpha than androgen binding to AR and, conversely, nitrofen was a more effective competitor of androgen binding to AR.

No abstract

Single-stranded DNA can change its structure dynamically in response to the presence of a complementary strand. The large structural change from the hairpin secondary structure to the double-stranded form leads to the chemistry of molecular beacons (MBs), or hairpin DNA oligomers having both a fluorescent and a quenching chromophore in the hairpin stem.[1] MBs can be used to report the presence of complementary strands by measuring the increasing fluorescence because of the decreasing energy transfer efficiency. Most DNA probes for fluorescent detection are covalently bound to fluorophores to make energy transfer effective. [1, 2] We have focused on an alternative way to link the fluorescence change with the structural changes of DNA that are induced during polymerase chain reaction (PCR) amplification.[3] We describe herein the chemical concept of noncovalent fluorescent DNA labeling by ligand binding to the secondary structure, which allows us to monitor PCR progress by measuring the change in fluorescence emitted from ligand-primer complexes in a homogeneous solution. This concept is not only complementary to that using fluorescent dye bound selectively to PCR product duplexes, [4] but it also expands the possibility of real-time PCR.The concept of DNA labeling by secondary-structureinducible ligand fluorescence is shown in Figure 1. PCR primers are labeled with a hairpin tag containing cytosine bulges (C-bulges). The molecule 2,7-diamino-1,8-naphthyridine (DANP) binds to a C-bulge in its protonated form (DANPH + ) and emits fluorescence at 430 nm with a 30 nm bathochromic shift from the fluorescence of free, unbound DANP.[5] We hypothesized that, as the PCR progresses, the hairpin tag will dissolve and be transformed into a duplex, thus resulting in the loss of the DANP binding site and a decrease in the fluorescence at 430 nm. Toward this end, we investigated the hairpin tags. These tags should identify the DANP binding site without disturbing the fluorescence efficiency, should not interfere with the PCR, should be transformed effectively into the duplex during PCR, and should be applicable to diverse primers. First, the sequence flanking the C-bulge producing the highest fluorescence intensity was investigated by measuring DANP fluorescence with duplexes having a C-bulge flanked by A-T and T-A base pairs. The G-C and C-G base pairs were omitted from the flanking base pairs because of their quenching of DANP fluorescence.[5b] The characteristic spectrum with a broad single peak at 430 nm was obtained for the A_A/TCT sequence. The relative fluorescence intensity at 450 nm for the four C-bulge duplexes and a fully matched duplex are shown in Figure S1 in the Supporting Information. The C-bulge in the A_A/TCT sequence enhanced DANP fluorescence by 7.6-fold compared with the fully matched duplex and by 82-fold compared with free, unbound DANP.We then designed hairpin tags comprising one to three A_A/TCT units separated by three to five base pairs and a T4 hairpin loop (Table 1). The intensity of DANP fluores...