Identification of a Lifespan Extending Mutation in the Schizosaccharomyces pombe Cyclin Gene clg1 + by Direct Selection of Long-Lived Mutants

Model organisms such as budding yeast, worms and flies have proven instrumental in the discovery of genetic determinants of aging, and the fission yeast Schizosaccharomyces pombe is a promising new system for these studies. We devised an approach to directly select for long-lived S. pombe mutants from a random DNA insertion library. Each insertion mutation bears a unique sequence tag called a bar code that allows one to determine the proportion of an individual mutant in a culture containing thousands of different mutants. Aging these mutants in culture allowed identification of a long-lived mutant bearing an insertion mutation in the cyclin gene clg1 ⁺. Clg1p, like Pas1p, physically associates with the cyclin-dependent kinase Pef1p. We identified a third Pef1p cyclin, Psl1p, and found that only loss of Clg1p or Pef1p extended lifespan. Genetic and co-immunoprecipitation results indicate that Pef1p controls lifespan through the downstream protein kinase Cek1p. While Pef1p is conserved as Pho85p in Saccharomyces cerevisiae, and as cdk5 in humans, genome-wide searches for lifespan regulators in S. cerevisiae have never identified Pho85p. Thus, the S. pombe system can be used to identify novel, evolutionarily conserved lifespan extending mutations, and our results suggest a potential role for mammalian cdk5 as a lifespan regulator.     

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution

Microvilli are found on the surface of many cell types, including the mammalian oocyte, where they are thought to act in initial contact of sperm and oocyte plasma membranes. CD9 is currently the only oocyte protein known to be required for sperm-oocyte fusion. We found CD9 is localized to the oocyte microvillar membrane using transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that CD9 null oocytes, which are unable to fuse with sperm, have an altered length, thickness and density of their microvilli. One aspect of this change in morphology was quantified using TEM by measuring the radius of curvature at the microvillar tips. A small radius of curvature is thought to promote fusibility and the radius of curvature of microvillar tips on CD9 wild-type oocytes was found to be half that of the CD9 null oocytes. We found that oocyte CD9 co-immunoprecipitates with two Ig superfamily cis partners, EWI-2 and EWI-F, which could have a role in linking CD9 to the oocyte microvillar actin core. We also examined latrunculin B-treated oocytes, which are known to have reduced fusion ability, and found altered microvillar morphology by SEM and TEM. Our data suggest that microvilli may participate in sperm-oocyte fusion. Microvilli could act as a platform to concentrate adhesion/fusion proteins and/or provide a membrane protrusion with a low radius of curvature. They may also have a dynamic interaction with the sperm that serves to capture the sperm cell and bring it into close contact with the oocyte plasma membrane.     

Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus

The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.     

Duck hunter activity,success, and satisfaction on public hunting areas

Because hunting disturbance can influence local distribution of ducks and their availability to hunters, managers often limit access to hunting areas to improve hunting success and satisfaction. Few studies have quantified the effectiveness of public area access restrictions on duck hunter activity, harvest, or hunters' satisfaction with their hunting experience. We used a cross-over design over 6 consecutive hunting seasons (2008–2009 through 2013–2014) on State Wildlife Areas (SWAs) in northeastern Colorado, USA, to compare the effects of restricted hunting access regulations and regulations without these restrictions on duck hunter activity, harvest success, harvest levels, and satisfaction. We also considered effects of SWA types, duck abundance, temperature, precipitation, use of equipment by duck hunting parties, and, for hunter satisfaction, hunting success, hunting parties' satisfaction with ducks seen, habitat conditions, crowding from other hunters, and SWA regulations. The number of days when duck hunters had access to restricted properties was about half that on unrestricted properties, and unrestricted properties were used by about twice as many duck hunting parties, but the mean number of hunting parties per available hunting day and mean party size were similar under the 2 types of regulations. Most (56%) duck hunting parties did not bag any ducks; hunting success (harvest of ≥1 duck by a hunting party) was best explained by a model that included a regulation type × hunting season interaction, a SWA type × month interaction, hunter density the previous day, an index of hunter investment (number of decoys used and whether dogs and calls were used), and temperature. Successful hunting parties harvested 1.92 ± 1.60 (SD) ducks/hunter/day (range = 0.1–7.0); the best model predicting the number of ducks harvested per hunter in successful parties included a regulation type × hunting season interaction, a SWA type × month interaction, hunter density the previous day, an index of hunter investment, temperature, and precipitation. Overall satisfaction of duck hunting parties with a day's hunt averaged 3.62 ± 1.20 based on a rank scale of 1 (very unsatisfied) to 5 (very satisfied), and was best explained by a model that included hunting party success; hunter investment; temperature; and satisfaction with duck numbers, habitat conditions, hunting regulations, and crowding. While greatly reducing days available for hunting, access restrictions were associated with improved chances of hunting parties successfully harvesting ≥1 duck in 5 of 6 hunting seasons, and substantially greater numbers of ducks harvested by hunters in successful parties in 1 of 6 hunting seasons. Restrictions did not have a strong direct effect on hunting parties' satisfaction with a day's hunt. Uncontrolled factors, including weather and use of equipment by hunters, had important influences on hunter success, harvest, and satisfaction. Managers should carefully assess hunting activity, hunter expectations and desires, and hunting area characteristics when considering access restrictions on public hunting areas.     

Production of Perennial Vegetation in an Oasis-desert Transition Zone in NW China - Allometric Estimation,and Assessment of Flooding and Use Effects

River oases at the southern fringe of the Taklamakan desert in NW China are surrounded by belts of spontaneous vegetation that protect the oases from sand drift. As an important source of forage, fuel and construction wood, this foreland vegetation is also a component part of the agricultural system of the oases but has been, and still is, destroyed through overuse. Within a broader study that aimed to provide a basis for a sustainable management of this foreland vegetation, biomass and production were studied in four vegetation types dominated either by Alhagi sparsifolia, Calligonum caput-medusae, Populus euphratica, or Tamarix ramosissima that were thought to occur under different regimes of natural flooding in the foreland of Qira (Cele) oasis, Xinjiang, NW China. Shoot biomass components were closely correlated to basal area (Calligonum, Populus, Tamarix) or shrub volume and projection area (Alhagi), enabling non-destructive estimation of stand biomass from shoot diameters or shrub dimensions with sufficient precision using allometric regression equations. Relationships between shoot basal area and biomass of the woody species (Calligonum, Populus and Tamarix) agreed with predictions by a theoretical model of plant vascular systems, suggesting that they are determined by hydraulic and mechanical requirements for shoot architecture. Average aboveground biomass densities of typical stands in late summer were 2.97 Mg/ha in Alhagi, 3.6 Mg/ha in a row plantation and 10.9 Mg/ha in homogenous stands of Calligonum, 22–29 Mg/ha in 22 year-old Populus forests and 1.9–3.1 Mg/ha in Tamarix-dominated vegetation. Annual aboveground production including wood and assimilation organs ranged from 2.11 to 11.3 Mg/ha in plantations of Calligonum, 3.17 to 6.12 Mg/ha in Populus, and 1.55 to 1.74 Mg/ha (based on total ground area) or 3.10 to 7.15 Mg/ha (in homogenous stands) in Tamarix. Production of Alhagi is equal to peak biomass. A thinning treatment simulating use by the local population enhanced productivity of Calligonum, Populus and Tamarix. A complete harvest of Alhagi in late August decreased production in the following year. An artificial flood irrigation treatment did not sufficiently increase soil water content except in the uppermost layer and had no clear beneficial effect on growth of the four species and even a negative effect on Alhagi, which was due to increased competition from annual species. As biomass and production with or without artificial irrigation were much higher than values expected for rain-fed desert vegetation at a mean annual precipitation of 35 mm, it is concluded that the existence of all vegetation types studied is probably based on permanent access to groundwater and that natural floods or precipitation do not contribute to their water supply. The effects of agricultural groundwater use in the oasis on groundwater in the foreland of the oasis need further study. Sustainable use of this productive vegetation is possible but requires proper management.