From aging to virulence: forging connections through the study of copper homeostasis in eukaryotic microorganisms

Recent years have witnessed an explosion in the breadth of investigations on transition metal homeostasis and the subsequent depth of our understanding of metals in biology. Many genes and proteins that serve in the uptake, distribution, sensing and detoxification of one such transition metal, copper, have been identified. Through genetic and biochemical studies, the molecular details of copper uptake are being elucidated, and evidence suggests a largely conserved mechanism for copper acquisition and distribution from yeast to humans. Investigations of the mitochondrial copper pathway reveal the complexity surrounding copper delivery to cytochrome oxidase and highlight additional roles for some of the participants in copper homeostasis, such as a copper chaperone that influences the subcellular distribution of its target for copper incorporation. Furthermore, our understanding of the structure and function of copper transporters, chaperones and cupro-proteins, coupled with the emergence of additional model systems, is providing surprising examples of the integration of copper homeostasis with other physiological and pathophysiological processes and states, such as cancer, aging and virulence.     

Circadian control of neurogenesis

The life-long addition of new neurons has been documented in many regions of the vertebrate and invertebrate brain, including the hippocampus of mammals (Altman and Das, 1965; Eriksson et al., 1998; Jacobs et al., 2000), song control nuclei of birds (Alvarez-Buylla et al., 1990), and olfactory pathway of rodents (Lois and Alvarez-Buylla, 1994), insects (Cayre et al., 1996) and crustaceans (Harzsch and Dawirs, 1996; Sandeman et al., 1998; Harzsch et al., 1999; Schmidt, 2001). The possibility of persistent neurogenesis in the neocortex of primates is also being widely discussed (Gould et al., 1999; Kornack and Rakic, 2001). In these systems, an effort is underway to understand the regulatory mechanisms that control the timing and rate of neurogenesis. Hormonal cycles (Rasika et al., 1994; Harrison et al., 2001), serotonin (Gould, 1999; Brezun and Daszuta, 2000; Beltz et al., 2001), physical activity (Van Praag et al., 1999) and living conditions (Kemperman and Gage, 1999; Sandeman and Sandeman, 2000) influence the rate of neuronal proliferation and survival in a variety of organisms, suggesting that mechanisms controlling life-long neurogenesis are conserved across a range of vertebrate and invertebrate species. The present article extends these findings by demonstrating circadian control of neurogenesis. Data show a diurnal rhythm of neurogenesis among the olfactory projection neurons in the crustacean brain, with peak proliferation during the hours surrounding dusk, the most active period for lobsters. These data raise the possibility that light-controlled rhythms are a primary regulator of neuronal proliferation, and that previously-demonstrated hormonal and activity-driven influences over neurogenesis may be secondary events in a complex circadian control pathway.     

Molecular determinants of sugar transport regulation by ATP

Intracellular ATP inhibits human erythrocyte net sugar transport by binding cooperatively to the glucose transport protein (GluT1). ATP binding produces altered transporter affinity for substrate and promotes substrate occlusion within a post-translocation vestibule formed by GluT1 cytosolic domains. The accompanying paper (Cloherty, E. K., Levine, K. B., Graybill, C., and Carruthers, A. (2002) Biochemistry 41, 12639-12651) demonstrates that reduced intracellular pH promotes high-affinity ATP binding to GluT1 but inhibits ATP-modulation of GluT1-mediated sugar transport. The present study explores the role of GluT1 residues 326-343 (a proposed GluT1 ATP-binding site subdomain) in GluT1 ATP binding by using alanine scanning mutagenesis. Cos-7 and HEK cells were transfected with a cDNA encoding full-length human GluT1 terminating in a carboxyl-terminal hemagglutinin (HA)-His6 epitope. The transporter (GluT1.HA.H6) is expressed at the surface of both cell-types and is catalytically active. In HEK cells, both parental GluT1- and GluT1.HA.H6-mediated sugar transport are acutely sensitive to cellular metabolic inhibition. Isolated, detergent-solubilized GluT1.HA.H6 is photolabeled by [gamma-32P]-azidoATP in an ATP-protectable manner. Alanine substitution of E329 or G332/R333/R334 enhances GluT1.HA.H6 [gamma-32P]azidoATP photoincorporation but blocks acute modulation of net sugar transport by cellular metabolic inhibition. These actions resemble those of reduced pH on ATP binding to and modulation of red cell GluT1. It is proposed that cooperative nucleotide binding to GluT1 and nucleotide modulation of GluT1-mediated sugar transport are regulated by a proton-sensitive saltbridge (Glu329-Arg333/334).     

Patterns of activity and fatality of migratory bats at a wind energy facility in Alberta,Canada

Studying migratory behavior of bats is challenging. Thus, most information regarding their migratory behavior is anecdotal. Recently, however, fatalities of migratory bats at some wind energy facilities across North America have provided the opportunity and impetus to study bat migration at fine spatial and temporal scales. Using acoustic monitoring and carcass searches, we examined temporal and spatial variation in activity levels and fatality rates of bats at a wind energy facility in southern Alberta, Canada. Our goals were to better understand the influence of weather variables and turbine location on the activity and fatality of hoary bats (Lasiurus cinereus) and silver-haired bats (Lasionycteris noctivagans), and to use that understanding to predict variation in fatality rates at wind energy facilities and recommend measures to reduce fatalities. Overall activity of migratory bats and of silver-haired bats increased in low wind speeds and warm ambient temperatures, and was reduced when the wind was from the North or Northeast, whereas hoary bat activity increased with falling barometric pressure. Fatalities of migratory bats in general increased with increased activity of migratory bats, increased moon illumination, and falling barometric pressure and were influenced by the interaction between barometric pressure change and activity. Fatalities of silver-haired bats increased with increased activity, moon illumination, and winds from the south-east. Hoary bat fatalities increased with falling barometric pressure. Our results indicate that both the activity and fatality of migratory bats are affected by weather variables, but that species differ in their responses to environmental conditions. Spatially, fatalities were not influenced by the position of turbines within a turbine row, but were influenced by the location of turbines within the facility. Our findings have implications for our understanding of bat migration and efforts to reduce fatalities at wind energy facilities. To maximize the reduction of bat fatalities, operators of wind energy facilities could incorporate migratory bats' response to environmental variables, such as barometric pressure and fraction of moon illuminated, into their existing mitigation strategies. © 2011 The Wildlife Society.     

Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ubiquity of microbes creates a copious supply of potential contaminants in a laboratory. To ensure experimental success, the number of contaminants on equipment and work surfaces must be minimized. Common among many experiments in microbiology are techniques involving the measurement and transfer of cultures containing bacterial cells or viral particles. To do so without contacting non-sterile surfaces or contaminating sterile media requires (1) preparing a sterile workspace, (2) precisely setting and accurately reading instruments for aseptic transfer of liquids, and (3) properly manipulating instruments, cultures flasks, bottles and tubes within a sterile field. Learning these procedures calls for training and practice. At first, actions should be slow, deliberate, and controlled with the goal being for aseptic technique to become second nature when working at the bench. Here we present the steps for measuring volumes using serological pipettes and micropipettors within a sterile field created by a Bunsen burner. Volumes range from microliters (μl) to milliliters (ml) depending on the instrument used. Liquids commonly transferred include sterile broth or chemical solutions as well as bacterial cultures and phage stocks. By following these procedures, students should be able to: •Work within the sterile field created by the Bunsen burner flame. •Use serological pipettes without compromising instrument sterility.• Aspirate liquids with serological pipettes, precisely reading calibrated volumes by aligning the meniscus formed by the liquid to the graduation marks on the pipette. •Keep culture bottles, flasks, tubes and their respective caps sterile during liquid transfers. •Identify different applications for plastic versus glass serological pipettes. •State accuracy limitations for micropipettors. •Precisely and accurately set volumes on micropipettors. •Know how to properly use the first and second stop on a micropipettor to aspirate and transfer correct volumes.     

Discovery of a novel class of isoxazoline voltage gated sodium channel blockers

Analogs of the previously reported voltage gated sodium channel blocker CDA54 were prepared in which one of the amide functions was replaced with aromatic and non-aromatic heterocycles. Replacement of the amide with an aromatic heterocycle resulted in significant loss of sodium channel blocking activity, while non-aromatic heterocycle replacements were well tolerated.     

Demand for sucrose in the genetically obese Zucker (fa/fa) rat

Obese Zucker rats (fa/fa) eat more food than lean controls in free-feeding conditions, which strongly influences their phenotypic expression. Few studies, however, characterize their food consumption in environments that are more representative of foraging conditions, e.g., how effort plays a role in food procurement. This study examined the reinforcing efficacy of sucrose in obese Zucker rats by varying the responses required to obtain single sucrose pellets. Male Zucker rats (15 lean, 14 obese) lever-pressed under eight fixed ratio (FR) schedules of sucrose reinforcement, in which the number of lever-presses required to gain access to a single sucrose pellet varied from 1 to 300. Linear and exponential demand equations, which characterize the value of a reinforcer by its sensitivity to price (FR), were fit to the number of food reinforcers and responses made. Free food consumption was also examined. Obese Zuckers, compared to leans, consumed more food under free-feeding conditions. Moreover, they had higher levels of consumption and response output, but only at low FR values. Both groups were equally sensitive to price increases at higher FR values. This suggests that environmental conditions may interact with genes in the expression of food reinforcer efficacy.