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RNA/DNA hybrids form when RNA hybridizes with its template DNA generating a three-stranded structure known as the R-loop. Knowledge of how they form and resolve, as well as their functional roles, is limited. Here, by pull-down assays followed by mass spectrometry, we identified 803 proteins that bind to RNA/DNA hybrids. Because these proteins were identified using in vitro assays, we confirmed that they bind to R-loops in vivo. They include proteins that are involved in a variety of functions, including most steps of RNA processing. The proteins are enriched for K homology (KH) and helicase domains. Among them, more than 300 proteins preferred binding to hybrids than double-stranded DNA. These proteins serve as starting points for mechanistic studies to elucidate what RNA/DNA hybrids regulate and how they are regulated.

Water-level management is widespread and illustrates how contemporary climate can interact directly and indirectly with numerous biological and abiotic factors to influence reproductive success of wildlife species. We studied common loons, an iconic waterbird sensitive to timing and magnitude of water-level changes during the breeding season, using a before-after-control-impact design on large lakes in Voyageurs National Park (Minnesota, USA), to assess the effect of anthropogenic changes in hydroregime on their nesting success and productivity. We examined multiple competing a priori hypotheses in an information-theoretic framework, and predicted that magnitude of changes in loon productivity would be greater in the Namakan Reservoir, where water-level management was altered to mimic a more natural hydroregime, than in Rainy Lake, where management remained relatively unchanged. We determined outcomes from 278 nests during 2004–2006 by performing boat-based visits every 3–5 days, and measuring hydrologic, vegetative, and microtopographic covariates. Relative to comparably collected data for 260 total loon pairs during 1983–1986, productivity (chicks hatched/territorial pair) increased 95% in the Namakan Reservoir between the 2 time periods. Nest success declined in both lakes over the 2 study periods but less so in the Namakan Reservoir than in Rainy Lake. Flooding was a primary cause of nest failures (though second nests were less likely to flood). Nest predation appears to have increased considerably between the 2 study periods. Top-ranked models suggested that timing of nest initiation, probability of nest flooding, probability of nest stranding, and probability of nest success were each related to 2–4 factors, including date of initiation, timing of initiation relative to peak water levels, changes in the elevation of the nest edge, maximum water-level change between initiation and peak water levels, and maximum water-level change between initiation and nest outcome. The top model for all variables except stranding each garnered ≥82% of total model weight. Results demonstrate that water-level management can be altered to benefit productivity of common loons. However, nuanced interactions between land-use change, invasive species, human development, recreation, climate change, and recovery of top predators may often complicate both management decisions and interpretation of water-level impacts on wildlife. © 2013 The Wildlife Society.

FOXC1 and FOXC2 are forkhead transcription factors that play essential roles during development and physiology. Despite their critical role, the mechanisms that regulate the function of these factors remain poorly understood. We have identified conserved motifs within a previously defined N-terminal negative regulatory region of FOXC1/C2 that conforms to the definition of synergy control or SC motifs. Because such motifs inhibit the activity of transcription factors by serving as sites of post-translational modification by small ubiquitin-like modifier (SUMO), we have examined whether FOXC1/C2 are targets of SUMOylation and probed the functional significance of this modification. We find that endogenous FOXC1 forms modified by SUMO2/3 can be detected. Moreover, in cell culture, all three SUMO isoforms are readily conjugated to FOXC1 and FOXC2. The modification can be reconstituted in vitro with purified components and can be reversed in vitro by treatment with the SUMO protease SENP2. SUMOylation of FOXC1 and FOXC2 occurs primarily on one consensus synergy control motif with minor contributions of a second, more degenerate site. Notably, although FOXC1 is also phosphorylated at multiple sites, disruption of sites immediately downstream of the SC motifs does not influence SUMOylation. Consistent with a negative functional role, SUMOylation-deficient mutants displayed higher transcriptional activity when compared with wild type forms despite comparable protein levels and subcellular localization. Thus, the findings demonstrate that SC motifs mediate the inhibitory function of this region by serving as sites for SUMOylation and reveal a novel mechanism for acute and reversible regulation of FOXC1/C2 function.

Communication on a cellular level--defined as chemical signaling, sensing, and response--is an essential and universal component of all living organisms and the framework that unites all ecosystems. Evolutionarily conserved signaling "webs," existing both within an organism and between organisms, rely on efficient and accurate interpretation of chemical signals by receptors. Therefore, endocrine-disrupting chemicals (EDCs), which have been shown to disrupt hormone signaling in laboratory animals and exposed wildlife, may have broader implications for disrupting signaling webs that have yet to be identified as possible targets. In this article, I explore common evolutionary themes of chemical signaling (e.g., estrogen signaling in vertebrates and phytoestrogen signaling from plants to symbiotic soil bacteria) and show that such signaling systems are targets of disruption by EDCs. Recent evolutionary phylogenetic data have shown that the estrogen receptor (ER) is the ancestral receptor from which all other steroid receptors have evolved. In addition to binding endogenous estrogens, ERs also bind phytoestrogens, an ability shared in common with nodulation D protein (NodD) receptors found in Rhizobium soil bacteria. Recent data have shown that many of the same synthetic and natural environmental chemicals that disrupt endocrine signaling in vertebrates also disrupt phytoestrogen-NodD receptor signaling in soil bacteria, which is necessary for nitrogen-fixing symbiosis. Bacteria-plant symbiosis is an unexpected target of EDCs, and other unexpected nontarget species may also be vulnerable to EDCs found in the environment.

Some organochlorine pesticides and other synthetic chemicals mimic hormones in representatives of each vertebrate class, including mammals, reptiles, amphibians, birds, and fish. These compounds are called endocrine-disrupting chemicals (EDCs). Similarly, hormonelike signaling has also been observed when vertebrates are exposed to plant chemicals called phytoestrogens. Previous research has shown the mechanism of action for EDCs and phytoestrogens is as unintended ligands for the estrogen receptor (ER). Although pesticides have been synthesized to deter insects and weeds, plants produce phytoestrogens to deter herbivores, as attractant cues for insects, and as recruitment signals for symbiotic soil bacteria. Our data present the first evidence that some of the same organochlorine pesticides and EDCs known to disrupt endocrine signaling through ERs in exposed wildlife and humans also disrupt the phytoestrogen signaling that leguminous plants use to recruit Sinorhizobium meliloti soil bacteria for symbiotic nitrogen fixation. Here we report that a variety of EDCs and pesticides commonly found in agricultural soils interfere with the symbiotic signaling necessary for nitrogen fixation, suggesting that the principles underlying endocrine disruption may have more widespread biological and ecological importance than had once been thought.

The oestrogen receptor (ER) has proven to be an extraordinarily successful target for breast cancer treatment and prevention. The clinical use of tamoxifen, a nonsteroidal antioestrogen, demonstrated (1) that the strategic use of adjuvant tamoxifen in ER-positive patients could save lives and (2) that a selective ER modulator (SERM) could reduce the incidence of breast cancer in high-risk women. The ER is now the target for new and safer therapies such as the aromatase inhibitors and the pure antioestrogens that either block oestrogen synthesis or destroy the ER. However, the use of raloxifene, a SERM to prevent osteoporosis with the potential to prevent breast cancer has introduced a new dimension into preventive oncology. The widespread use of endocrine modulators (SERMs, aromatase inhibitors, and pure antioestrogens) raised the question of drug resistance. It is now clear that endocrine resistance can evolve through stages. Once a breast tumour becomes resistant to SERMs, the growth is stimulated by either the SERM or oestrogen. This is why an aromatase inhibitor is effective following SERM resistance and withdrawal. However, the extended use of repeated endocrine therapies now supersensitized the cells to oestrogen that causes apoptosis through the ER. We suggest that future clinical treatment strategies incorporate an 'oestrogen purge' to both enhance the actions of chemotherapy or completely reverse endocrine resistance and restore endocrine sensitivity. These new data build on the idea that breast cancer can be controlled as a chronic disease and will permit patients to live long and productive lives during targeted maintenance treatment.

The effect of human activities on animal populations is widely debated, particularly since a recent decision by the U.S. Department of the Interior to ban snowmobiles from national parks. Immunoassays of fecal glucocorticoid levels provide a sensitive and noninvasive method of measuring the physiological stress responses of wildlife to disturbances. We tested for associations between snowmobile activity and glucocorticoid levels in an elk (Cervus elaphus) population in Yellowstone National Park and wolf (Canis lupus) populations in Yellowstone, Voyageurs, and Isle Royale national parks. For wolves, comparisons among populations and years showed that fecal glucocorticoid levels were higher in areas and times of heavy snowmobile use. For elk, day-to-day variation in fecal glucocorticoid levels paralleled variation in the number of snowmobiles after we controlled for the effects of weather and age. Also for elk, glucocorticoid concentrations were higher in response to snowmobiles than to wheeled vehicles after we controlled for the effects of age, weather, and number of vehicles. Despite these stress responses, there was no evidence that current levels of snowmobile activity are affecting the population dynamics of either species in these locations.

Mice lacking natriuretic peptide receptor A (NPRA) have marked cardiac hypertrophy and chamber dilatation disproportionate to their increased blood pressure (BP), suggesting, in support of previous in vitro data, that the NPRA system moderates the cardiac response to hypertrophic stimuli. Here, we have followed the changes in cardiac function in response to altered mechanical load on the heart of NPRA-null mice (Npr1‐/‐). Chronic treatment with either enalapril, furosemide, hydralazine, or losartan were all effective in reducing and maintaining BP at normal levels without affecting heart weight/body weight. In the reverse direction, we used transverse aortic constriction (TAC) to induce pressure overload. In the Npr1 ‐/‐ mice, TAC resulted in a 15-fold increase in atrial natriuretic peptide (ANP) expression, a 55% increase in left ventricular weight/body weight (LV/BW), dilatation of the LV, and significant decline in cardiac function. In contrast, banded Npr1 +/+ mice showed only a threefold increase in ANP expression, an 11% increase in LV/BW, a 0.2 mm decrease in LV end diastolic dimension, and no change in fractional shortening. The activation of mitogen-activated protein kinases that occurs in response to TAC did not differ in the Npr1+/+ and Npr1‐/‐ mice. Taken together, these results suggest that the NPRA system has direct antihypertrophic actions in the heart, independent of its role in BP control.

Natriuretic peptides, produced in the heart, bind to the natriuretic peptide receptor A (NPRA) and cause vasodilation and natriuresis important in the regulation of blood pressure. We here report that mice lacking a functional Npr1 gene coding for NPRA have elevated blood pressures and hearts exhibiting marked hypertrophy with interstitial fibrosis resembling that seen in human hypertensive heart disease. Echocardiographic evaluation of the mice demonstrated a compensated state of systemic hypertension in which cardiac hypertrophy and dilatation are evident but with no reduction in ventricular performance. Nevertheless, sudden death, with morphologic evidence indicative in some animals of congestive heart failure and in others of aortic dissection, occurred in all 15 male mice lacking Npr1 before 6 months of age, and in one of 16 females in our study. Thus complete absence of NPRA causes hypertension in mice and leads to cardiac hypertrophy and, particularly in males, lethal vascular events similar to those seen in untreated human hypertensive patients.

SYNOPSIS. The topic of endocrine disruption and the broad range of physiological effects caused by endocrine disrupting chemicals (EDCs) can only be meaningfully framed within an ecological and evolutionary context. Environmental pollutants and EDCs operate by disrupting the ‘‘chemical communication’’ that coordinates signaling within an organism. Here we discuss how EDCs are also able to disrupt the chemical communication between plants and soil bacteria necessary for initiating nitrogen-fixing symbiosis. We also examine, through examples of pollutant-related impacts on a wide range of invertebrates, the need for identifying emerging targets of EDCs. We suggest broadening the defined field of endocrine disruption to encompass the effects of synthetic chemicals that interfere with signaling and communication, not only within an organism, but also between organisms and linking ecosystems. The ecological consequences of failing to recognize novel targets of chemical pollutants and EDCs may be a net loss of biological diversity and a further imbalance of the global nitrogen cycle.

Endocrine disrupting chemicals (EDCs) include organochlorine pesticides, plastics manufacturing by-products, and certain herbicides[1]. These chemicals have been shown to disrupt hormonal signaling in exposed wildlife, lab animals, and mammalian cell culture by binding to estrogen receptors (ER-α and ER-β) and affecting the expression of estrogen responsive genes[2,3]. Additionally, certain plant chemicals, termed phytoestrogens, are also able to bind to estrogen receptors and modulate gene expression, and as such also may be considered EDCs[4]. One example of phytoestrogen action is genistein, a phytochemical produced by soybeans, binding estrogen receptors, and changing expression of estrogen responsive genes which certain studies have linked to a lower incidence of hormonally related cancers in Japanese populations[5]. Why would plants make compounds that are able to act as estrogens in the human body? Obviously, soybeans do not intentionally produce phytoestrogens to prevent breast cancer in Japanese women.

An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expression of yeast enhanced green fluorescent protein (yEGFP), for the determination of estrogenic activity in extracts of calf urine samples. Urine samples were spiked with 1 and 5 ngmL(-1) 17beta-estradiol and 17alpha-ethynylestradiol, 10 and 50 ngmL(-1) mestranol, and 100 ngmL(-1) testosterone and progesterone. Sample extracts of blank and spiked urine samples were prepared at our laboratory and sent to seven laboratories together with a reagent blank, a DMSO blank, and eight 17beta-estradiol stock solutions in DMSO ranging in concentration from 0 to 545 ngmL(-1). Sample extracts and standards were coded and tested blindly. A decision limit (CCalpha) was determined based on the response of seven blank urine samples. Signals of the negative controls, e.g. urine samples spiked with 100 ngmL(-1) testosterone or progesterone, were all below the determined CCalpha and were thus screened as compliant. Positive controls, i.e. the urine samples spiked at two levels with 17beta-estradiol, 17alpha-ethynylestradiol and mestranol, were almost all screened as suspect, i.e. gave signals above the determined CCalpha. Determined EC(50) values calculated from the 17beta-estradiol dose-response curves obtained by the seven laboratories ranged from 0.59 to 0.95 nM.

Unprecedented agricultural intensification and increased crop yield will be necessary to feed the burgeoning world population, whose global food demand is projected to double in the next 50 years. Although grain production has doubled in the past four decades, largely because of the widespread use of synthetic nitrogenous fertilizers, pesticides, and irrigation promoted by the “Green Revolution,” this rate of increased agricultural output is unsustainable because of declining crop yields and environmental impacts of modern agricultural practices. The last 20 years have seen diminishing returns in crop yield in response to increased application of fertilizers, which cannot be completely explained by current ecological models. A common strategy to reduce dependence on nitrogenous fertilizers is the production of leguminous crops, which fix atmospheric nitrogen via symbiosis with nitrogen-fixing rhizobia bacteria, in rotation with nonleguminous crops. Here we show previously undescribed in vivo evidence that a subset of organochlorine pesticides, agrichemicals, and environmental contaminants induces a symbiotic phenotype of inhibited or delayed recruitment of rhizobia bacteria to host plant roots, fewer root nodules produced, lower rates of nitrogenase activity, and a reduction in overall plant yield at time of harvest. The environmental consequences of synthetic chemicals compromising symbiotic nitrogen fixation are increased dependence on synthetic nitrogenous fertilizer, reduced soil fertility, and unsustainable long-term crop yields.

To study a gene interaction network, we developed a gene-targeting strategy that allows efficient and stable genomic integration of multiple genetic constructs at distinct target loci in the yeast genome. This gene-targeting strategy uses a modular plasmid with a recyclable selectable marker and a multiple cloning site into which the gene of interest is cloned, flanked by two long regions of homology to the target genomic locus that are generated using adaptamer primers. We used this strategy to integrate into a single yeast strain components of the oestrogen receptor (ER) signalling network, comprising the human ERα and three reporter genes driven by oestrogen response elements (EREs). The engineered strain contains multiple reporters of ligand-dependent receptor signalling, providing sensitive, reproducible, rapid, low-cost quantitative assays of ERα activity in order to screen potential receptor agonists. Further, because two of the ERE-driven reporter genes are required for growth in deficient media, the strain's growth rate—and therefore its fitness—depends on ligand-induced ERα activity. This evolvable oestrogen receptor activity sensor (EERAS) can therefore provide the foundation of a long-term experimental evolution strategy to elucidate ER structure–function relations and ligand-receptor evolution. Copyright © 2007 John Wiley & Sons, Ltd.

Breast cancer is the most common cancer affecting women worldwide (1). In the United States alone, an estimated 211,000 new cases of breast cancer will be diagnosed in 2003 (2). It is estimated that 1 in every 8 American women will be diagnosed with breast cancer within the course of her lifetime (2). With more sensitive and accurate means of early detection and an ever-increasing number of drugs available to treat breast cancer, it is likely that women diagnosed today will live longer and may need more than one type of cancer therapy. Many cellular factors mediate breast transformation and tumor growth including growth factors, members of phosphorylation signaling cascades, oncogenes, and nuclear hormone receptors. While each of these factors has a role in the development of breast cancer, the steroid hormone estrogen is the primary promotional factor. Epidemiologic evidence has shown that a woman’s overall lifetime exposure to endogenous estrogen, increased by early menarche, late menopause, and nulliparity, is the primary risk factor for developing breast cancer (3).

We have advanced the view that estrogens activate the estrogen receptor (ER) alpha complex differently. A group of planar (estradiol, genistein, and coumestrol) and nonplanar (methoxychlor and its mono- and didemethylated phenolic metabolites) environmental estrogens, which are all full estrogens in MCF-7 breast cancer cell proliferation assays, was shown to segregate discretely into planar and nonplanar groups. These groups were delineated using a novel assay of mutant ER cDNAs stably transfected into MDA-MB-231 cells and the activation of the transforming growth factor alpha target gene in situ that putatively describes the external shape of the ER complex. Planar compounds activate estrogen action through the two traditional activation functions (AFs), AF1 and AF2, in the ER. In contrast, nonplanar compounds can activate estrogen action through AF1 and the amino acids Asp-351 and Asp-538, which are exposed when helix 12 silences AF2. The observation that class I (planar) and class II (nonplanar) compounds have different mechanisms of estrogen action may have important implications for tissue selective modulation of the ER.

Targeted disruption of the gene for natriuretic peptide receptor-A (NPR-A) worsens pulmonary hypertension and right ventricular hypertrophy during hypoxia, but its effect on left ventricular mass and systemic pressures is not known. We examined the effect of 3 wk of hypobaric hypoxia (0.5 atm) on right and left ventricular pressure and mass in mice with 2 (wild type), 1, or 0 copies of Npr1, the gene that encodes for NPR-A in mice. Under normoxic conditions, right ventricular peak pressure (RVPP) was greater in 0 than in 2 copy mice, but there were no genotype-related differences in carotid artery PP (CAPP). The left ventricular free wall weight-to-body weight (LV/body wt) ratio was greater in 0 than in 2 copy mice and there was a trend toward a greater right ventricular weight-to-body weight (RV/body wt) ratio. Three weeks of hypoxia increased RVPP and RV/body wt in all genotypes. The increase in RVPP was similar in all genotypes (11–14 mmHg), but the hypoxia-induced increase in RV/body wt was more than twice as great in 0 copy mice than in 2 copy mice (1.11 ± 0.06 to 2.65 ± 0.46 vs. 0.96 ± 0.04 to 1.4 ± 0.09, P < 0.05). Chronic hypoxia had no effect on CAPP in any genotype and did not effect LV/body wt in 1 or 2 copy mice, but increased LV/body wt 41% in 0 copy mice. We conclude that absent expression of NPR-A worsens right ventricular hypertrophy and causes left ventricular hypertrophy during exposure to chronic hypoxia without increasing pulmonary or systemic arterial pressure responses.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) exert their physiological actions by binding to natriuretic peptide receptor A (NPRA), a receptor guanylate cyclase (rGC) that synthesizes cGMP in response to both ligands. The family of rGCs is rapidly expanding, and it is plausible that there might be additional, as yet undiscovered, rGCs whose function is to provide alternative signalling pathways for one or both of these peptides, particularly given the low affinity of NPRA for BNP. We have investigated this hypothesis, using a genetically modified (knockout) mouse in which the gene encoding NPRA has been disrupted. Enzyme assays and NPRA-specific Western blots performed on tissues from wild-type mice demonstrate that ANP-activated cGMP synthesis provides a good index of NPRA protein expression, which ranges from maximal in adrenal gland, lung, kidney, and testis to minimal in heart and colon. In contrast, immunoreactive NPRA is not detectable in tissues isolated from NPRA knockout animals and ANP- and BNP-stimulatable GC activities are markedly reduced in all mutant tissues. However, testis and adrenal gland retain statistically significant, high-affinity responses to BNP. This residual response to BNP cannot be accounted for by natriuretic peptide receptor B, or any other known mammalian rGC, suggesting the presence of a novel receptor in these tissues that prefers BNP over ANP.

Lipopolysaccharides (LPS) and capsular polysaccharides (K antigens) may influence the interaction of rhizobia with their specific hosts; therefore, we conducted a comparative analysis of Sinorhizobium fredii and Sinorhizobium meliloti , which are genetically related, yet symbiotically distinct, nitrogen-fixing microsymbionts of legumes. We found that both species typically produce strain-specific K antigens that consist of 3-deoxy- d - manno -2-octulosonic acid (Kdo), or other 1-carboxy-2-keto-3-deoxy sugars (such as sialic acid), and hexoses. The K antigens of each strain are distinguished by glycosyl composition, anomeric configuration, acetylation, and molecular weight distribution. One consistent difference between the K antigens of S. fredii and those of S. meliloti is the presence of N -acetyl groups in the polysaccharides of the latter. In contrast to the K antigens, the LPS of Sinorhizobium spp. are major common antigens. Rough (R) LPS is the predominant form of LPS produced by cultured cells, and some strains release almost no detectable smooth (S) LPS upon extraction. Sinorhizobium spp. are delineated into two major RLPS core serogroups, which do not correspond to species (i.e., host range). The O antigens of the SLPS, when present, have similar degrees of polymerization and appear to be structurally conserved throughout the genus. Interestingly, one strain was found to be distinct from all others: S. fredii HH303 produces a unique K antigen, which contains galacturonic acid and rhamnose, and the RLPS did not fall into either of the RLPS core serogroups. The results of this study indicate that the conserved S- and RLPS of Sinorhizobium spp. lack the structural information necessary to influence host specificity, whereas the variable K antigens may affect strain-cultivar interactions.

Nitrogen fixation is a symbiotic process initiated by chemical signals from legumes that are recognized by soil bacteria. Here we show that some endocrine-disrupting chemicals (EDCs)1,2,3, so called because of their effect on hormone-signalling pathways in animal cells, also interfere with the symbiotic signalling that leads to nitrogen fixation. Our results raise the possibility that these phytochemically activated pathways may have features in common with hormonal signalling in vertebrates, thereby extending the biological and ecological impact of EDCs.

Are last names harder to pronounce than words in that they do not always follow standard English pronunciation rules? If they are, then names would require special treatment in text‐to‐speech translation systems. A database was developed of last names and associated pronunciations of Los Alamos National Laboratory employees. An algorithm was developed for scoring the accuracy of a hypothesized pronunciation when compared against ground truth (the employees' own pronunciations of their names). Using the rules in Dectalk, the following sets of data were compared with respect to the accuracy of the rules: names versus words, English names versus non‐English names, and non‐English names versus words. Significant differences were found among the various conditions. [Work supported by DOE Contract W‐7405‐ENG‐36.]