Spatial and Temporal Variability in Sediment Denitrification Within an Agriculturally Influenced Reservoir

Reservoirs are intrinsically linked to the rivers that feed them, creating a river–reservoir continuum in which water and sediment inputs are a function of the surrounding watershed land use. We examined the spatial and temporal variability of sediment denitrification rates by sampling longitudinally along an agriculturally influenced river–reservoir continuum monthly for 13 months. Sediment denitrification rates ranged from 0 to 63 μg N₂O g ash free dry mass of sediments (AFDM)⁻¹ h⁻¹ or 0–2.7 μg N₂O g dry mass of sediments (DM)⁻¹ h⁻¹ at reservoir sites, vs. 0–12 μg N₂O gAFDM⁻¹ h⁻¹ or 0–0.27 μg N₂O gDM⁻¹ h⁻¹ at riverine sites. Temporally, highest denitrification activity traveled through the reservoir from upper reservoir sites to the dam, following the load of high nitrate (NO₃⁻-N) water associated with spring runoff. Annual mean sediment denitrification rates at different reservoir sites were consistently higher than at riverine sites, yet significant relationships among theses sites differed when denitrification rates were expressed per gDM vs. per gAFDM. There was a significant positive relationship between sediment denitrification rates and NO₃⁻-N concentration up to a threshold of 0.88 mg NO₃⁻ -N l⁻¹, above which it appeared NO₃⁻-N was no longer limiting. Denitrification assays were amended seasonally with NO₃⁻-N and an organic carbon source (glucose) to determine nutrient limitation of sediment denitrification. While organic carbon never limited sediment denitrification, all sites were significantly limited by NO₃⁻-N during fall and winter when ambient NO ₃⁻-N was low.     

Cooperativity between the Ras-ERK and Rho-Rho kinase pathways in urokinase-type plasminogen activator-stimulated cell migration.

Binding of the urokinase-type plasminogen activator (uPA) to its receptor activates diverse cell signaling pathways. How these signals are integrated so that cell physiology is altered remains unclear. In this study, we demonstrated that migration of MCF-7 breast cancer cells and HT-1080 fibrosarcoma cells on serum-coated surfaces is stimulated by agents that activate ERK, including uPA, epidermal growth factor, and constitutively active MEK1. The promigratory activity of these agents was entirely blocked not only by the MEK-specific antagonist PD098059, but also by antagonists of the Rho-Rho kinase pathway, including Y-27632 and dominant-negative RhoA (RhoA-N19). uPA did not significantly increase the level of GTP-bound RhoA, suggesting that the constitutive activity of the Rho-Rho kinase pathway may be sufficient to support ERK-stimulated cell migration. Paradoxically, Y-27632 and RhoA-N19 increased ERK phosphorylation in MCF-7 cells, providing further evidence that ERK activation alone does not promote cell migration when Rho kinase is antagonized. When MCF-7 cell migration was stimulated by ERK-independent processes such as expression of the beta(3) integrin subunit or changing the substratum to type I collagen, Y-27632 and RhoA-N19 failed to inhibit the response. This study supports a model in which the Ras-ERK and Rho-Rho kinase pathways cooperate to promote cell migration. Neutralizing either pathway is sufficient to block the response to agents that stimulate cell migration by activating ERK.     

An Immortal Cell Culture Model of Hypothalamic Gonadotropin-Releasing Hormone Neurons

The neuroendocrine hypothalamus has been the object of intensive study in vivo and in tissue slices. However, using these models it is difficult to approach questions at the molecular and cellular level and to differentiate between direct effects and those mediated by other neurons. By using the regulatory domain of the rat gonadotropin-releasing hormone (GnRH) gene to target expression of the oncogene SV40 T antigen in transgenic mice, we have produced hypothalamic tumors which were cultured to produce clonal cell lines (GT1 cells). These cells express GnRH and many other neuronal markers, but do not express glial cell markers or other hypothalamic hormones. They have a distinctive neuronal phenotype, process the GnRH peptide accurately, and secrete GnRH in a pulsatile pattern. They respond to many neurotransmitters and neuromodulators including activin, norepinephrine, dopamine, nitric oxide, NMDA, and GABA, as well as GnRH itself. Thus, we have immortalized GnRH neurons by targeting oncogenesis to a defined population of neurons using the regulatory region of a gene which is expressed late in the differentiation of that cell lineage. The GT1 cell lines serve as an excellent model for molecular, pharmacological, electrophysiological, and biochemical investigations into the regulation of GnRH and the characteristics of a pure CNS neuronal population. Moreover, their derivation demonstrates the success of targeting tumorigenesis to specific differentiated neurons of the central nervous system in transgenic mice.     

An epidemiological study of oral contraceptives and breast cancer

During 1968-77, 707 women aged 16-50 years with newly diagnosed breast cancer and 707 matched controls were interviewed at eight teaching hospitals in London and Oxford about their use of oral contraceptives. Eighty-six of the patients with breast cancer were matched with controls with gall-bladder disease; these subjects were omitted from the main analyses, which thus related to 621 case-control pairs.The results were reassuring. A few statistically significant differences in oral contraceptive use were found between the breast cancer and control groups, but the data were subdivided in many ways, so that some “significant” differences would have been expected to occur by chance. The only subgroup in which the evidence for a positive association between pill use and breast cancer was at all convincing comprised women aged 46-50 years, but trends in those aged 41-45 were by and large in the opposite direction and results of combined analysis gave no cause for concern.Information on clinical stage was available for 487 patients with breast cancer treated before the end of 1975. Those who had never used oral contraceptives had appreciably more advanced tumours at presentation than those who had been using the pill during the year before detection of the lump, while past users of the pill occupied an intermediate position. This difference in staging was reflected in the pattern of survival. Oral contraceptives may have had a beneficial effect on tumour growth and spread, though diagnostic bias could not be definitely excluded.     

The enigma of number: why children find the meanings of even small number words hard to learn and how we can help them do better

Although number words are common in everyday speech, learning their meanings is an arduous, drawn-out process for most children, and the source of this delay has long been the subject of inquiry. Children begin by identifying the few small numerosities that can be named without counting, and this has prompted further debate over whether there is a specific, capacity-limited system for representing these small sets, or whether smaller and larger sets are both represented by the same system. Here we present a formal, computational analysis of number learning that offers a possible solution to both puzzles. This analysis indicates that once the environment and the representational demands of the task of learning to identify sets are taken into consideration, a continuous system for learning, representing and discriminating set-sizes can give rise to effective discontinuities in processing. At the same time, our simulations illustrate how typical prenominal linguistic constructions ("there are three balls") structure information in a way that is largely unhelpful for discrimination learning, while suggesting that postnominal constructions ("balls, there are three") will facilitate such learning. A training-experiment with three-year olds confirms these predictions, demonstrating that rapid, significant gains in numerical understanding and competence are possible given appropriately structured postnominal input. Our simulations and results reveal how discrimination learning tunes children's systems for representing small sets, and how its capacity-limits result naturally out of a mixture of the learning environment and the increasingly complex task of discriminating and representing ever-larger number sets. They also explain why children benefit so little from the training that parents and educators usually provide. Given the efficacy of our intervention, the ease with which it can be implemented, and the large body of research showing how early numerical ability predicts later educational outcomes, this simple discovery may have far-reaching consequences.     

Evolution of a major drug metabolizing enzyme defect in the domestic cat and other felidae: phylogenetic timing and the role of hypercarnivory

The domestic cat (Felis catus) shows remarkable sensitivity to the adverse effects of phenolic drugs, including acetaminophen and aspirin, as well as structurally-related toxicants found in the diet and environment. This idiosyncrasy results from pseudogenization of the gene encoding UDP-glucuronosyltransferase (UGT) 1A6, the major species-conserved phenol detoxification enzyme. Here, we established the phylogenetic timing of disruptive UGT1A6 mutations and explored the hypothesis that gene inactivation in cats was enabled by minimal exposure to plant-derived toxicants. Fixation of the UGT1A6 pseudogene was estimated to have occurred between 35 and 11 million years ago with all extant Felidae having dysfunctional UGT1A6. Out of 22 additional taxa sampled, representative of most Carnivora families, only brown hyena (Parahyaena brunnea) and northern elephant seal (Mirounga angustirostris) showed inactivating UGT1A6 mutations. A comprehensive literature review of the natural diet of the sampled taxa indicated that all species with defective UGT1A6 were hypercarnivores (>70% dietary animal matter). Furthermore those species with UGT1A6 defects showed evidence for reduced amino acid constraint (increased dN/dS ratios approaching the neutral selection value of 1.0) as compared with species with intact UGT1A6. In contrast, there was no evidence for reduced amino acid constraint for these same species within UGT1A1, the gene encoding the enzyme responsible for detoxification of endogenously generated bilirubin. Our results provide the first evidence suggesting that diet may have played a permissive role in the devolution of a mammalian drug metabolizing enzyme. Further work is needed to establish whether these preliminary findings can be generalized to all Carnivora.     

Transcribed microsatellite allele lengths are often correlated with gene expression in natural sunflower populations

Microsatellites are common in genomes of most eukaryotic species. Due to their high mutability, an adaptive role for microsatellites has been considered. However, little is known concerning the contribution of microsatellites towards phenotypic variation. We used populations of the common sunflower (Helianthus annuus) at two latitudes to quantify the effect of microsatellite allele length on phenotype at the level of gene expression. We conducted a common garden experiment with seed collected from sunflower populations in Kansas and Oklahoma followed by an RNA‐Seq experiment on 95 individuals. The effect of microsatellite allele length on gene expression was assessed across 3,325 microsatellites that could be consistently scored. Our study revealed 479 microsatellites at which allele length significantly correlates with gene expression (eSTRs). When irregular allele sizes not conforming to the motif length were removed, the number of eSTRs rose to 2,379. The percentage of variation in gene expression explained by eSTRs ranged from 1%–86% when controlling for population and allele‐by‐population interaction effects at the 479 eSTRs. Of these eSTRs, 70.4% are in untranslated regions (UTRs). A gene ontology (GO) analysis revealed that eSTRs are significantly enriched for GO terms associated with cis‐ and trans‐regulatory processes. Our findings suggest that a substantial number of transcribed microsatellites can influence gene expression.     

Functional Deficits in Pak5, Pak6 and Pak5/Pak6 Knockout Mice

The p21-activated kinases are effector proteins for Rho-family GTPases. PAK4, PAK5, and PAK6 are the group II PAKs associated with neurite outgrowth, filopodia formation, and cell survival. Pak4 knockout mice are embryonic lethal, while Pak5, Pak6, and Pak5/Pak6 double knockout mice are viable and fertile. Our previous work found that the double knockout mice exhibit locomotor changes and learning and memory deficits. We also found some differences with Pak5 and Pak6 single knockout mice and the present work further explores the potential differences of the Pak5 knockout and Pak6 knockout mice in comparison with wild type mice. The Pak6 knockout mice were found to weigh significantly more than the other genotypes. The double knockout mice were found to be less active than the other genotypes. The Pak5 knockout mice and the double knockout mice performed worse on the rotorod test. All the knockout genotypes were found to be less aggressive in the resident intruder paradigm. The double knockout mice were, once again, found to perform worse in the active avoidance assay. These results indicate, that although some behavioral differences are seen in the Pak5 and Pak6 single knockout mice, the double knockout mice exhibit the greatest changes in locomotion and learning and memory.     

Breast Cancer Antiestrogen Resistance 3 (BCAR3) Promotes Cell Motility by Regulating Actin Cytoskeletal and Adhesion Remodeling in Invasive Breast Cancer Cells

Metastatic breast cancer is incurable. In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility. Here, we focus on the role of the adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) in cellular processes that contribute to cell motility, including protrusion, adhesion remodeling, and contractility. Previous work from our group showed that elevated BCAR3 protein levels enhance cell migration, while depletion of BCAR3 reduces the migratory and invasive capacities of breast cancer cells. In the current study, we show that BCAR3 is necessary for membrane protrusiveness, Rac1 activity, and adhesion disassembly in invasive breast cancer cells. We further demonstrate that, in the absence of BCAR3, RhoA-dependent signaling pathways appear to predominate, as evidenced by an increase in RhoA activity, ROCK-mediated phosphorylation of myosin light chain II, and large ROCK/mDia1-dependent focal adhesions. Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling. Considering that BCAR3 protein levels are elevated in advanced breast cancer cell lines and enhance breast cancer cell motility, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process.     

Peripherally Administered Nanoparticles Target Monocytic Myeloid Cells,Secondary Lymphoid Organs and Tumors in Mice

Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide) nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d.) compared to intramuscular administration, leading to ∼50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma.