Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor α (ERα) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERα binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5′ and 3′ ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERα binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERα-positive from ERα-negative breast tumors. The expression dynamics of the genes adjacent to ERα binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERα appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERα target genes. Unexpectedly, we found that only 22%–24% of the bona fide human ERα binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERα binding and gene regulation.

The organoplatinum complex containing ortho-metalated (S)-(1-(dimethylamino)ethyl)naphthalene as the chiral auxiliary has been used efficiently to promote the asymmetric [4 + 2] Diels-Alder reaction between diphenylvinylphosphine and 2-diphenylphosphinofuran to generate the chelating diphosphine exo-cycloadduct, 4(R),5(R)-bis(diphenylphosphino)-7oxabicyclo[2.2.1]hept-2-ene in 70% isolated yield. At room temperature, the reaction was complete in 5 d. However, the cycloaddition reaction proceeded at a significantly slower rate and exhibited a markedly lower stereoselectivity when the chiral platinum template was replaced by its organopalladium counterpart. The diphosphino-substituted oxanorbornene metal complexes are stable and the oxygen-carbon bonds are inert toward treatments with strong acids. Upon liberation from the metal ions, however, the chiral cycloadduct becomes unstable and undergoes the retro Diels-Alder reaction slowly to regenerate diphenylvinylphosphine and 2-diphenylphosphinofuran.

Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor a (ERa) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERa binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (.5 kb from 59 and 39 ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERa binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERa-positive from ERa-negative breast tumors. The expression dynamics of the genes adjacent to ERa binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERa appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERa target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERa binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERa binding and gene regulation.

The viability of three strains of probiotic Bifidobacterium lactis that were inoculated into UHT milk was examined with and without the presence of the yeast, Williopsis saturnus var. saturnus NCYC 22, in polypropylene tubes at 30 C. The B. lactis viable cell count for strains HN019 and BB-12 remained above 6.0 Log cfu/ml, while strain B94 had 5.7 Log cfu/ml after six weeks of incubation in the presence of the co-inoculated yeast. Incubating the bifidus milk without added yeast under anaerobic condition did not improve the survival of B. lactis HN019, indicating that oxygen removal may not be responsible for W. saturnus NCYC 22's viability enhancing property. The addition of yeast supernatant or non-viable yeast also did not show any stabilising effects, suggesting that physical contact and/or interaction between viable W. saturnus and B. lactis plays an important role in sustaining the viability of the probiotic. W. saturnus NCYC 22 could increase the survival of B. lactis in bifidus milk under ambient temperature regardless of the initial concentration of yeast cells inoculated due to yeast growth. This study demonstrated the viability enhancing effect of viable W. saturnus NCYC 22 on B. lactis HN019, which could help towards extending the shelf-life of dairy beverages containing probiotic bifidobacteria.