The nucleotide sequence of the gyrA gene of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter skirrowii was determined. The deduced GyrA proteins are closely related to those of Wolinella succinogenes and Helicobacter pullorum, whereas those of Campylobacter species showed less sequence identity. The phylogenetic analysis of GyrA sequences provides a result similar to 16S rRNA gene sequence phylogenetic analysis and allows the discrimination among A. butzleri species. In addition, a Thr-->Ile mutation at amino acid 85 in the quinolone resistance-determining region was associated with ciprofloxacin resistance for two A. butzleri and one A. cryaerophilus ciprofloxacin-resistant strains.

A real-time PCR targeting the gyrase A subunit gene outside the quinolone resistance-determining region has been developed to detect Arcobacter species. The species identification was done by probe hybridization and melting curve analysis, using fluorescence resonance energy transfer technology. Discrimination between Arcobacter species was straightforward, as the corresponding melting points showed significant differences with the characteristic melting temperatures of 63.5°C, 58.4°C, 60.6°C, and 51.8°C for the Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter nitrofigilis type strains, respectively. The specificity of this assay was confirmed with pure cultures of 106 Arcobacter isolates from human clinical and veterinary specimens identified by phenotypic methods and 16S rRNA gene sequencing. The assay was then used to screen 345 clinical stool samples obtained from patients with diarrhea. The assay detected A. butzleri in four of these clinical samples (1.2%). These results were confirmed by a conventional PCR method targeting the 16S rRNA gene with subsequent sequencing of the PCR product. In conclusion, this real-time assay detects and differentiates Arcobacter species in pure culture as well as in the competing microbiota of the stool matrix. The assay is economical since only one biprobe is used and multiple Arcobacter species are identified in a single test.

The antiandrogenic effects of ring-DIMs suggest they may form the basis for the development of novel agents against hormone-sensitive prostate cancer, alone or in combination with other drugs.

SummaryWe recently reported that novel ring-substituted analogs of 3,3′-diindolylmethane (ring-DIMs) have anti-androgenic and growth inhibitory effects in androgen-dependent prostate cancer cells. The objectives of this study were to confirm the ability of 4,4′-and 7,7′-dibromo-and dichlorosubstituted ring-DIMs to inhibit androgen-stimulated proliferation of androgen-dependent LNCaP human prostate cancer cells using a non-invasive, real-time monitoring technique. In addition, their ability to induce apoptotic and necrotic cell death in androgen-dependent as well asindependent (PC-3) prostate cancer cells was studied. Prostate cancer cells were treated with increasing concentrations of DIM and ring-DIMs (0.3-30 μM) and effects on cell proliferation were measured in real-time using an xCELLigence cellular analysis system. Chromatin condensation and loss of membrane integrity were determined by Hoechst and propidium iodide staining, respectively. Apoptotic protein markers were measured by immuno-blotting and activation of caspases determined using selective fluorogenic substrates. Intra-and extracellular concentrations of DIM and ring-DIMs were assessed by electrospray ionization tandem mass CIHR Author ManuscriptCIHR Author Manuscript CIHR Author Manuscript spectrometry. Ring-DIMs inhibited androgen-stimulated LNCaP cell proliferation and induced apoptosis and necrosis in LNCaP and PC-3 cells with 2-4 fold greater potencies than DIM. DIM and the ring-DIMs increased caspases −3, −8 and −9 activity, elevated expression of Fas, FasL, DR4 and DR5 protein, and induced PARP cleavage in both cell lines. The cytotoxicity of the most potent ring-DIM, 4,4′-dibromoDIM, but not the other compounds was decreased by an inhibitor of caspase −3. The 4,4′-dibromoDIM was primarily found in the extracellular medium, whereas all other compounds were present to a much larger extent in the cell. In conclusion, ring-DIMs inhibited prostate cancer cell growth and induced cell death in LNCaP and PC-3 cells with greater potencies than DIM; they also structure-dependently activated different cell death pathways suggesting that these compounds have clinical potential as chemopreventive and chemotherapeutic agents in prostate cancer, regardless of hormone-dependency.

Human origins of DNA replication are specific sequences within the genome whereby DNA replication is initiated. A select group of proteins, known as the pre-replication (pre-RC) complex, in whose formation the Ku protein (Ku70/Ku86) was shown to play a role, bind to replication origins to initiate DNA replication. In this study, we have examined the involvement of Ku in breast tumorigenesis and tumor progression and found that the Ku protein expression levels in human breast metastatic (MCF10AC1a) cells were higher in the chromatin fraction compared to hyperplastic (MCF10AT) and normal (MCF10A) human breast cells, but remained constant in both the nuclear and cytoplasmic fractions. In contrast, in human intestinal cells, the Ku expression level was relatively constant for all cell fractions. Nascent DNA abundance and chromatin association of Ku70/86 revealed that the c-myc origin activity in MCF10AC1a is 2.5 to 5-fold higher than in MCF10AT and MCF10A, respectively, and Ku was bound to the c-myc origin more abundantly in MCF10AC1a, by approximately 1.5 to 4.2-fold higher than in MCF10AT and MCF10A, respectively. In contrast, similar nascent DNA abundance and chromatin association was found for all cell lines for the lamin B2 origin, associated with the constitutively active housekeeping lamin B2 gene. Electrophoretic mobility shift assays (EMSAs) performed on the nuclear extracts (NEs) of the three cell types revealed the presence of protein-DNA replication complexes on both the c-myc and lamin B2 origins, but an increase in binding activity was observed from normal, to transformed, to cancer cells for the c-myc origin, whereas no such difference was seen for the lamin B2 origin. Overall, the results suggest that increased Ku chromatin association, beyond wild type levels, alters cellular processes, which have been implicated in tumorigenesis.