Synthesis and functional analysis of novel bivalent estrogens.

The steroid hormone estrogen plays a critical role in female development and homeostasis. Estrogen mediates its effects through binding and activation of specific estrogen receptors alpha (ERalpha) and beta (ERbeta), members of the steroid/nuclear receptor family of ligand-induced transcription factors. Due to their intimate roles in genomic and nongenomic signaling pathways, these hormones and their receptors have been also implicated in the pathologies of a variety of cancers and metabolic disorders, and have been the target of large therapeutic development efforts. The binding of estrogen to its respective receptors initiates a cascade of events that include receptor dimerization, nuclear localization, DNA binding and recruitment of co-regulatory protein complexes. In this manuscript, we investigate the potential for manipulating steroid receptor gene expression activity through the development of bivalent steroid hormones that are predicted to facilitate hormone receptor dimerization events. Data are presented for the development and testing of novel estrogen dimers, linked through their C-17 moiety, that can activate estrogen receptor alpha (ERalpha)-mediated transcription events with efficacy and potency equal to or greater than that of ERalpha's cognate ligand, 17beta-estradiol. These bivalent estrogen structures open the door to the development of a variety of steroid therapeutics that could dramatically impact future drug development in this area.

Membrane and protein dynamics in virus-infected plant cells.

In terms of functional genomics research, Nicotiana benthamiana, more so than other model plants, is highly amenable to high-throughput methods, especially those employing virus-induced gene silencing and agroinfiltration. Furthermore, through recent and ongoing sequencing projects, there are now upward of 18,000 unique N. benthamiana ESTs to support functional genomics research. Despite these advances, the cell biology of N. benthamiana itself, and in the context of virus infection, lags behind that of other model systems. Therefore, to meet the challenges of diverse cell biology studies that will be derived from ongoing functional genomics projects, a series of methods relevant to the characterization of membrane and protein dynamics in virus-infected cells are provided here. The data presented here were derived from our studies with plant rhabdoviruses. However, the employed techniques should be broadly applicable within the field of plant virology. We report here on the use of a novel series of binary vectors for the transient or stable expression of autofluorescent protein fusions in plants. Use of these vectors in conjunction with advanced microscopy techniques such as fluorescent recovery after photobleaching and total internal fluorescence microscopy, has revealed novel insight into the membrane and protein dynamics of virus-infected cells.

Live-cell imaging of rhabdovirus-induced morphological changes in plant nuclear membranes.

Potato yellow dwarf virus (PYDV) and Sonchus yellow net virus (SYNV) belong to the genus Nucleorhabdovirus. These viruses replicate in nuclei of infected cells and mature virions accumulate in the perinuclear space after budding through the inner nuclear membrane. Infection of transgenic Nicotiana benthamiana 16c plants (which constitutively express green fluorescent protein (GFP) targeted to endomembranes) with PYDV or SYNV resulted in virus-specific patterns of accumulation of both GFP and membranes within nuclei. Using immunolocalization and a lipophilic fluorescent dye, we show that the sites of the relocalized membranes were coincident with foci of accumulation of the SYNV nucleocapsid protein. In contrast to the effects of PYDV and SYNV, inoculation of 16c plants with plus-strand RNA viruses did not result in accumulation of intranuclear GFP. Instead, such infections resulted in accumulation of GFP around nuclei, in a manner consistent with proliferation of the endoplasmic reticulum. We propose that the relocalization of GFP in 16c plants can be used to study sites of rhabdovirus accumulation in live cells. This study is the first to use live-cell imaging to characterize the effects of rhabdoviruses on plant nuclear membranes.