Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications

Post-translational modifications (PTMs) of histones play a role in modifying chromatin structure for DNA-templated processes in the eukaryotic nucleus, such as transcription, replication, recombination and repair; thus, histone PTMs are considered major players in the epigenetic control of these processes. Linking specific histone PTMs to gene expression is an arduous task requiring large amounts of highly purified and natively modified histones to be analyzed by various techniques. We have developed robust and complementary procedures, which use strong protein denaturing conditions and yield highly purified core and linker histones from unsynchronized proliferating, M-phase arrested and butyrate-treated cells, fully preserving their native PTMs without using enzyme inhibitors. Cell hypotonic swelling and lysis, nuclei isolation/washing and chromatin solubilization under mild conditions are bypassed to avoid compromising the integrity of histone native PTMs. As controls for our procedures, we tested the most widely used conventional methodologies and demonstrated that they indeed lead to drastic histone dephosphorylation. Additionally, we have developed methods for preserving acid-labile histone modifications by performing non-acid extractions to obtain highly purified H3 and H4. Importantly, isolation of histones H3, H4 and H2A/H2B is achieved without the use of HPLC. Functional supercoiling assays reveal that both hyper- and hypo-phosphorylated histones can be efficiently assembled into polynucleosomes. Notably, the preservation of fully phosphorylated mitotic histones and their assembly into polynucleosomes should open new avenues to investigate an important but overlooked question: the impact of mitotic phosphorylation in chromatin structure and function

Noonops.

48 pages : illustrations (some color) ; 26 cm. Part of the oonopid PBI project. Cf. acknowledgments.A new genus, Noonops, is established to contain 23 species of soft-bodied, New World oonopine spiders that differ from those of Oonops Templeton and similar genera in having the male palpal bulb fused to the cymbium, and from those of Wanops Chamberlin and Ivie and Oonopoides Bryant in having shorter legs. Six specific names are transferred from Oonops to Noonops: O. floridanus (Chamberlin and Ivie) from Florida and Georgia (chosen as the type species), O. gertschi Chickering from the Bahama Islands (which is placed as a junior synonym of N. floridanus), O. furtivus Gertsch from Texas and Tamaulipas, O. sonora Gertsch and Davis from Arizona, California, Sonora, Baja California, and Baja California Sur, O. puebla Gertsch and Davis from Puebla, and O. chilapensis Chamberlin and Ivie from Guerrero. Males of N. sonora and females of N. furtivus are described for the first time; 18 new species are described: N. ocotillo, N. mortero, N. joshua, N. skinner, N. coachella, and N. californicus from Arizona and southern California and N. willisi, N. mesa, N. naci, N. tarantula, N. miraflores, N. culiacan, N. taxquillo, N. chapul, N. beattyi, N. iviei, N. tonila, and N. minutus from Mexico

Soy isoflavones, estrogen therapy, and breast cancer risk: analysis and commentary

There has been considerable investigation of the potential for soyfoods to reduce risk of cancer, and in particular cancer of the breast. Most interest in this relationship is because soyfoods are essentially a unique dietary source of isoflavones, compounds which bind to estrogen receptors and exhibit weak estrogen-like effects under certain experimental conditions. In recent years the relationship between soyfoods and breast cancer has become controversial because of concerns – based mostly on in vitro and rodent data – that isoflavones may stimulate the growth of existing estrogen-sensitive breast tumors. This controversy carries considerable public health significance because of the increasing popularity of soyfoods and the commercial availability of isoflavone supplements. In this analysis and commentary we attempt to outline current concerns regarding the estrogen-like effects of isoflavones in the breast focusing primarily on the clinical trial data and place these concerns in the context of recent evidence regarding estrogen therapy use in postmenopausal women. Overall, there is little clinical evidence to suggest that isoflavones will increase breast cancer risk in healthy women or worsen the prognosis of breast cancer patients. Although relatively limited research has been conducted, and the clinical trials often involved small numbers of subjects, there is no evidence that isoflavone intake increases breast tissue density in pre- or postmenopausal women or increases breast cell proliferation in postmenopausal women with or without a history of breast cancer. The epidemiologic data are generally consistent with the clinical data, showing no indication of increased risk. Furthermore, these clinical and epidemiologic data are consistent with what appears to be a low overall breast cancer risk associated with pharmacologic unopposed estrogen exposure in postmenopausal women. While more research is required to definitively allay concerns, the existing data should provide some degree of assurance that isoflavone exposure at levels consistent with historical Asian soyfood intake does not result in adverse stimulatory effects on breast tissue