ATP-driven proton fluxes across membranes of secretory organelles

The ATP-dependent proton uptake by chromaffin granule membranes, lysosomes, and synaptosomes was examined. In synaptosomes the reaction was absolutely dependent on the presence of chloride, while in chromaffin granules chloride had a profound effect and in lysosomes only a minor effect. The presence of chloride markedly increases the rate of collapse of delta pH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone in all three organelles. Ascorbate with phenazine methosulfate uncoupled the ATP-dependent proton uptake by chromaffin granules, but had no effect on lysosomes and synaptosomes. Proton uptake by submitochondrial particles was about 50-fold more sensitive to dicyclohexylcarbodiimide than the proton uptake by chromaffin granule membranes. Chromaffin granule membranes were treated with 2 M sodium bromide to inactivate the mitochondrial ATPase. The treatment caused a complete inhibition of the ATP-dependent proton uptake. Solubilization of these membranes by sodium cholate, followed by reconstitution by cholate dilution revealed the ATP-dependent proton uptake of the system. It is concluded that the genuine ATPase enzyme of chromaffin granules is a proton translocator.     

Water-soluble contrast agents targeted at the estrogen receptor for molecular magnetic resonance imaging

Novel estrogen-conjugated pyridine-containing Gd(III) and Eu(III) contrast agents (EPTA-Gd/Eu) were designed and effectively synthesized. Convenient to administration and MRI experiments, both EPTA-Gd and EPTA-Eu are soluble in water. The EPTA-Gd selectively binds with a micromolar affinity to the estrogen receptor and induces proliferation of human breast cancer cells. The EPTA-Gd is not lethal and does not cause any adverse effects when administrated intravenously. It enhances T1 and T2 nuclear relaxation rates of water and serves as a selective contrast agent for localizing the estrogen receptor by MRI.     

Characteristics of sex-biased dispersal and gene flow in coastal river otters: implications for natural recolonization of extirpated populations

River otters (Lontra canadensis) were extirpated from much of their historic distribution because of exposure to pollution and urbanization, resulting in expansive reintroduction programmes that continue today for this and other species of otters worldwide. Bioaccumulation of toxins negatively affects fecundity among mustelids, but high vagility and different dispersal distances between genders may permit otter populations to recover from extirpation caused by localized environmental pollution. Without understanding the influence of factors such as social structure and sex-biased dispersal on genetic variation and gene flow among populations, effects of local extirpation and the potential for natural recolonization (i.e. the need for translocations) cannot be assessed. We studied gene flow among seven study areas for river otters (n = 110 otters) inhabiting marine environments in Prince William Sound, Alaska, USA. Using nine DNA microsatellite markers and assignment tests, we calculated immigration rates and dispersal distances and tested for isolation by distance. In addition, we radiotracked 55 individuals in three areas to determine characteristics of dispersal. Gender differences in sociality and spatial relationships resulted in different dispersal distances. Male river otters had greater gene flow among close populations (within 16-30 km) mostly via breeding dispersal, but both genders exhibited an equal, low probability of natal dispersal; and some females dispersed 60-90 km. These data, obtained in a coastal environment without anthropogenic barriers to dispersal (e.g. habitat fragmentation or urbanization), may serve as baseline data for predicting dispersal under optimal conditions. Our data may indicate that natural recolonization of coastal river otters following local extirpation could be a slow process because of low dispersal among females, and recolonization may be substantially delayed unless viable populations occurred nearby. Because of significant isolation by distance for male otters and low gene flow for females, translocations should be undertaken with caution to help preserve genetic diversity in this species.     

The Use of Phospholipid Fatty Acid Analysis to Measure Impact of Acid Rock Drainage on Microbial Communities in Sediments

The impact of acid rock drainage (ARD) and eutrophication on microbial communities in stream sediments above and below an abandoned mine site in the Adelaide Hills, South Australia, was quantified by PLFA analysis. Multivariate analysis of water quality parameters, including anions, soluble heavy metals, pH, and conductivity, as well as total extractable metal concentrations in sediments, produced clustering of sample sites into three distinct groups. These groups corresponded with levels of nutrient enrichment and/or concentration of pollutants associated with ARD. Total PLFA concentration, which is indicative of microbial biomass, was reduced by >70% at sites along the stream between the mine site and as far as 18 km downstream. Further downstream, however, recovery of the microbial abundance was apparent, possibly reflecting dilution effect by downstream tributaries. Total PLFA was >40% higher at, and immediately below, the mine site (0–0.1 km), compared with sites further downstream (2.5–18 km), even after accounting for differences in specific surface area of different sediment samples. The increased microbial population in the proximity of the mine source may be associated with the presence of a thriving iron-oxidizing bacteria community as a consequence of optimal conditions for these organisms while the lower microbial population further downstream corresponded with greater sediments metal concentrations. PCA of relative abundance revealed a number of PLFAs which were most influential in discriminating between ARD-polluted sites and the rest of the sites. These PLFA included the hydroxy fatty acids: 2OH12:0, 3OH12:0, 2OH16:0; the fungal marker: 18:26; the sulfate-reducing bacteria marker 10Me16:17; and the saturated fatty acids 12:0, 16:0, 18:0. Partial constrained ordination revealed that the environmental parameters with the greatest bearing on the PLFA profiles included pH, soluble aluminum, total extractable iron, and zinc. The study demonstrated the successful application of PLFA analysis to rapidly assess the toxicity of ARD-affected waters and sediments and to differentiate this response from the effects of other pollutants, such as increased nutrients and salinity.     

Modeling Behavior by Coastal River Otter (Lontra Canadensis) in Response to Prey Availability in Prince William Sound,Alaska: A Spatially-Explicit Individual-Based Approach

Effects of climate change on animal behavior and cascading ecosystem responses are rarely evaluated. In coastal Alaska, social river otters (Lontra Canadensis), largely males, cooperatively forage on schooling fish and use latrine sites to communicate group associations and dominance. Conversely, solitary otters, mainly females, feed on intertidal-demersal fish and display mutual avoidance via scent marking. This behavioral variability creates “hotspots” of nutrient deposition and affects plant productivity and diversity on the terrestrial landscape. Because the abundance of schooling pelagic fish is predicted to decline with climate change, we developed a spatially-explicit individual-based model (IBM) of otter behavior and tested six scenarios based on potential shifts to distribution patterns of schooling fish. Emergent patterns from the IBM closely mimicked observed otter behavior and landscape use in the absence of explicit rules of intraspecific attraction or repulsion. Model results were most sensitive to rules regarding spatial memory and activity state following an encounter with a fish school. With declining availability of schooling fish, the number of social groups and the time simulated otters spent in the company of conspecifics declined. Concurrently, model results suggested an elevation of defecation rate, a 25% increase in nitrogen transport to the terrestrial landscape, and significant changes to the spatial distribution of “hotspots” with declines in schooling fish availability. However, reductions in availability of schooling fish could lead to declines in otter density over time.     

Demographic and ecogeographic factors limit wild grapevine spread at the southern edge of its distribution range

The spatial distribution of plants is constrained by demographic and ecogeographic factors that determine the range and abundance of the species. Wild grapevine (Vitis vinifera ssp. sylvestris) is distributed from Switzerland in the north to Israel in the south. However, little is known about the ecogeographic constraints of this species and its genetic and phenotypic characteristics, especially at the southern edge of its distribution range in the Levant region. In this study, we explore the population structure of southern Levantine wild grapevines and the correlation between demographic and ecogeographic characteristics. Based on our genetic analysis, the wild grapevine populations in this region can be divided into two major subgroups in accordance with a multivariate spatial and ecogeographical clustering model. The identified subpopulations also differ in morphological traits, mainly leaf hairiness which may imply adaptation to environmental stress. The findings suggest that the Upper Jordan River population was spread to the Sea of Galilee area and that a third smaller subpopulation at the south of the Golan Heights may represent a distinguished gene pool or a recent establishment of a new population. A spatial distribution model indicated that distance to water sources, Normalized difference vegetation index, and precipitation are the main environmental factors constraining V. v. sylvestris distribution at its southern distribution range. These factors in addition to limited gene flow between populations prevent further spread of wild grapevines southwards to semi‐arid regions.     

Assessment of the spatial distribution of soil microbial communities in patchy arid and semi-arid landscapes of the Negev Desert using combined PLFA and DGGE analyses

Arid and semi-arid ecosystems are often characterized by vegetation patchiness and variable availability of resources. Phospholipid fatty acid (PLFA) and 16S rRNA gene fragment analyses were used to compare the bulk soil microbial community structure at patchy arid and semi-arid landscapes. Multivariate analyses of the PLFA data and the 16S rRNA gene fragments were in agreement with each other, suggesting that the differences between bulk soil microbial communities were primarily related to shrub vs intershrub patches, irrespective of climatic or site differences. This suggests that the mere presence of a living shrub is the dominant driving factor for the differential adaptation of the microbial communities. Lipid markers suggested as indicators of Gram-positive bacteria were higher in soils under the shrub canopies, while markers suggested as indicators of cyanobacteria and anaerobic bacteria were elevated in the intershrub soils. Secondary differences between soil microbial communities were associated with intershrub characteristics and to a lesser extent with the shrub species. This study provides an insight into the multifaceted nature of the factors that shape the microbial community structure in patchy desert landscapes. It further suggests that these drivers not only act in concert but also in a way that is dependent on the aridity level.     

Catalytic versatility and backups in enzyme active sites: the case of serum paraoxonase 1

The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential.     

Sociality in river otters: cooperative foraging or reproductive strategies?

We evaluated factors influencing social organization in coastalriver otters (Lontra canadensis) to test two hypotheses: groupformation is an antipredation strategy, or, alternatively,group information is related to cooperative foraging. Dataon group size, group composition, and sociality were obtainedthrough radiotracking 55 otters in Prince William Sound, Alaska, USA, from 1996 through 1998. For males, larger groups occurredafter the mating season and concurrent with availability ofschooling pelagic fishes. Stable isotope analysis revealedthat otters social in >10% of their locations had dietssignificantly higher in rapidly swimming pelagic fishes thandid less social otters, regardless of gender. In addition, ottersthat were more social had significantly smaller home rangesthan did less social otters, an observation consistent withincreased foraging efficiency through cooperative foraging.Discounting associations of females with young of the year,approximately 47% of females and only 24% of males were asocial.Among social otters, males were social in 46% of their locationsand 63% of that time occurred in all-male groups. Females wereonly social in 26% of locations and were in mixed-sex groups78% of that time. We hypothesize that the time-consuming taskof raising offspring prevents females from joining foraginggroups. When not raising young, females may join males to cooperativelyforage for better-quality prey (pelagic fishes), which wouldbe more difficult to acquire as a solitary forager.     

Variation of sesquiterpene lactones in Lactuca aculeata natural populations from Israel,Jordan and Turkey

A comparative phytochemical study of seven sesquiterpene lactones in natural populations of the wild lettuce Lactuca aculeata Boiss. (Asteraceae) was performed, based on 23 accessions derived from eight, two, and single localities from Israel, Jordan, and Turkey, respectively. The compounds were profiled and quantified in leaves and roots of the plants, grown from achenes (cypselas) in a greenhouse under controlled conditions, using reverse phase HPLC with on-line photodiode array detector. In the present study, L. aculeata was confirmed as a taxon strongly characterized by four dominant sesquiterpene lactones: 8-deoxylactucin, jacquinelin, crepidiaside B and lactuside A. An analysis of quantitative results of these four constituents led to the following conclusions: (i) the sesquiterpene lactone contents varied significantly, mainly within the populations; however, for two variables (crepidiaside B and lactuside A in root samples) the contents varied mostly between populations, (ii) these differences are likely to be genetically controlled since all accessions were grown under standardized glasshouse conditions. Accessions containing high levels of some sesquiterpene lactones were found in several populations. These traits should be conserved in situ and ex situ and utilized in further research and lettuce improvement.