The importance of food supply in high‐productivity ecosystems: Short‐term experimental tests with small rodents

Food availability is considered to be a primary factor affecting animal populations, yet few experimental tests have been performed to evaluate its actual importance in species‐rich ecosystems such as rainforests. It has been suggested that in such systems certain plant species may act as “keystone” resources for animals, but the importance of presumed keystone resources for populations has not been quantified experimentally. Using complementary seed removal and seed‐addition experiments, we determined how the supply of a presumed keystone resource, seeds of Araucaria angustifolia, affects short‐term demography of their main consumer group (small rodents) in a biodiversity hotspot, the Brazilian Atlantic Forest. We hypothesized that (i) the harvest of A. angustifolia seeds by human populations has negative impacts on rodents, and (ii) these seeds are a limiting resource for rodent populations. To test these hypotheses, we monitored populations of two species of numerically dominant rodents (Delomys dorsalis and Akodon montensis) within replicated control‐experimental plots. Manipulations of seed supply over 2 years had little effect on population size, body condition, survival, or reproduction of the two rodents, suggesting that, in the short‐term (within one generation), their populations are not food limited in Araucaria forests. Despite apparently having all the characteristics of a keystone resource, as currently defined in the literature, the seeds of A. angustifolia had limited influence on the short‐term demography of their main consumer group. In situations where purported keystone resources are seasonally abundant, their actual importance may be lower than generally assumed, and these resources then may have only localized and temporary effects on consumer populations.     

High community turnover and dispersal limitation relative to rapid climate change

Aim Many competing hypotheses seek to identify the mechanisms behind species richness gradients. Yet, the determinants of species turnover over broad scales are uncertain. We test whether environmental dissimilarity predicts biotic turnover spatially and temporally across an array of environmental variables and spatial scales using recently observed climate changes as a pseudo‐experimental opportunity. Location Canada. Methods We used an extensive database of observation records of 282 Canadian butterfly species collected between 1900 and 2010 to characterize spatial and temporal turnover based on Jaccard indices. We compare relationships between spatial turnover and differences in an array of relevant environmental conditions, including aspects of temperature, precipitation, elevation, primary productivity and land cover. Measurements were taken within 100‐, 200‐ and 400‐km grid cells, respectively. We tested the relative importance of each variable in predicting spatial turnover using bootstrap analysis. Finally, we tested for effects of temperature and precipitation change on temporal turnover, including distinctly accounting for turnover under individual species’ potential dispersal limitations. Results Temperature differences between areas correlate with spatial turnover in butterfly assemblages, independently of distance, sampling differences or the spatial resolution of the analysis. Increasing temperatures are positively related to biotic turnover within quadrats through time. Limitations on species dispersal may cause observed biotic turnover to be lower than expected given the magnitude of temperature changes through time. Main conclusions Temperature differences can account for spatial trends in biotic dissimilarity and turnover through time in areas where climate is changing. Butterfly communities are changing quickly in some areas, probably reflecting the dispersal capacities of individual species. However, turnover is lower through time than expected in many areas, suggesting that further work is needed to understand the factors that limit dispersal across broad regions. Our results illustrate the large‐scale effects of climate change on biodiversity in areas with strong environmental gradients.     

Changes in cell surface anionogenic groups induced by propranolol in Herpetomonas muscarum muscarum

The effects of propranolol (10(-3) mM) on the surface anionic groups of Herpetomonas muscarum muscarum were analysed by cell electrophoresis, by ultrastructural cytochemistry and by identification of sialic acids using paper chromatography. Differentiation of H. muscarum muscarum induced by propranolol treatment caused a significant increase in the net negative surface charge. Binding of cationized ferritin (CF) and colloidal iron hydroxide particles was observed at the cell surface of both untreated and propranolol-treated cells. In cells incubated in the presence of the drug the CF particles were distributed in all membrane regions. However, there were small areas where the particles were absent. In H. muscarum muscarum exposed to propranolol the density of residues of sialic acid per cell was higher, and the agglutinating activity with Sendai virus was more intense. However, the pattern of sialic acid, characterized by the presence of N-acetylneuraminic acid derivative, was not modified upon cell interaction with the drug. Treatment of both control and propranolol-treated protozoa with neuraminidase significantly reduced the surface charge. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface of H. muscarum muscarum.     

Herpetomonas samuelpessoai: Changes in cell shape and induction of differentiation by local anesthetic

Lidocaine, a local anesthetic, induces changes in morphology and motility of Herpetomonas samuelpessoai when incubated under nonproliferative conditions. The cells become rounded and immotile. These effects are dependent on time and temperature of incubation, concentration of lidocaine, and pH. Divalent cations (Ca²⁺, Mg²⁺, Ba²⁺, and Sr²⁺) reversed the effect of lidocaine. Lidocaine also induces the formation of membrane-bound cytoplasmic vacuoles as determined by morphometry applied to electron micrographs. Lidocaine had a dose-dependent effect on the growth of H. samuelpessoai in a chemically defined medium. At concentrations which did not interfere with cell growth, it induces the transformation of promastigote into opisthomastigote via paramastigote. It is suggested that lidocaine may be used as an inductor of differentiation in H. samuelpessoai opening the possibility of obtaining the three developmental stages of this trypanosomatid.     

Detection of cellular immunity with the soluble antigen of the fungus Sporothrix schenckii in the systemic form of the disease

Sporothrix schenckii is the etiologic agent of sporotrichosis, a mycosis of world-wide distribution more commonly occurring in tropical regions. The immunological mechanisms involved in the prevention and control of sporotrichosis are not fully understood but apparently include both the humoral and cellular responses. In the present investigation, cellular immunity was evaluated by in vivo and in vitro tests in mice infected with yeast-like forms of S. schenckii. The disease developed systemically and cellular immunity was evaluated for a period of 10 weeks. The soluble antigen utilized in the tests was prepared from yeast form of the fungus through the sonication (20 min: 10 sonications at 50 W at 2-min intervals). Delayed hypersensitivity and lymphocyte transformation tests showed that the cellular immune response was depressed between the 4th and 6th week of infection when the animals were challenged with the soluble fungal antigen. This depression frequently indicates worsening of the disease, with greater involvement of the host. This is a promising field of research for a better understanding of the pathogeny of this mycosis.     

Rethinking edge effects: the unaccounted role of geometric constraints

Edge effects strongly affect the abundance and distribution of organisms across landscapes, with wide‐ranging implications in ecology and conservation biology. The extensive literature on the subject has traditionally considered that edge effects result from the active avoidance or preference of organisms for certain portions of the habitat patch, assuming that abundance is uniform across a patch when environmental conditions are uniform. We demonstrate that this assumption is incorrect due to the so‐far ignored ‘geometric edge effect’ (GEE). In the absence of environmental gradients, abundance of any organism living in a bounded habitat patch will tend to be lower in areas located near the edges compared to areas in the centre of the patch, simply because the areas in the centre receive individuals from all directions, whereas areas near the edge do not receive individuals from outside the patch. This geometric effect was already known for species richness at large geographic scales, the mid‐domain effect, but its importance in the literature of edge effects remained neglected so far. Using simulations, we show that the GEE tends to reduce population abundance and community richness near the edges of bounded habitat patches, and that apparently neutral or negative responses to the edge may occur even when habitat quality is higher near the edges. A published study that detected significant edge effects is reanalyzed, demonstrating that interpreting observed abundance patterns without taking the GEE into account – as traditionally done in the vast literature on edge effects – could provide misleading conclusions. The incorporation of the GEE into sampling and analytical protocols of future studies could advance substantially our ability to understand and predict edge effects in heterogeneous landscapes.     

Microsatellite mutations in the offspring of irradiated parents 19 years after the Cesium-137 accident

In September of 1987, a radiotherapy unit containing 50.9 TBq of Cs¹³⁷Cl was removed from an abandoned radiotherapy clinic. This unit was subsequently disassembled leading to the most serious radiological accident yet to occur in the Western hemisphere. This event provides an opportunity to assess the genetic effects of ionizing radiation. We surveyed genetic variation of 12 microsatellite loci in 10 families of exposed individuals and their offspring and also in non-exposed families from the same area of Goias state. We found an increase in the number of new alleles in the offspring of the exposed individuals. The mutation rate was found to be higher in the exposed families compared to the control group. These results indicated that exposure to ionizing radiation can be detected in offspring of exposed individuals and also suggest that the elevated microsatellite mutation rate can be attributed to radioactive exposure.     

Simvastatin causes the formation of cholesterol-rich remnants in mice lacking apoE

Mice that lack apolipoprotein E (apoE) display a severe hypercholesterolemia, caused by the accumulation of apolipoprotein B-48 (apoB-48)-carrying remnants of chylomicrons and very-low-density lipoproteins in the plasma. Statins are potent inhibitors of cholesterol synthesis that, when administered to mice lacking apoE, cause paradoxical further increases in plasma cholesterol levels. In the present study, we examined the mechanisms responsible for this phenomenon. ApoE-deficient mice fed a chow diet containing simvastatin developed, as anticipated, an enhanced increase in plasma cholesterol and a decrease in plasma triglycerides. Fractionation of the plasma lipoproteins by FPLC revealed that the lipid changes were confined to the lipoprotein remnants. Western blot analysis of the remnants from the untreated and simvastatin-treated mice showed no differences in their apoB-48 content, indicating that both groups of animals accumulated similar numbers of remnant particles in the plasma. Following the injection of Triton WR-1339, the simvastatin-treated mice accumulated in the plasma significantly more cholesterol and significantly less triglycerides than the untreated animals. These results indicate that the enhanced hypercholesterolemia observed in apoE-deficient mice treated with simvastatin is not the result of an increased number of remnant particles in circulation but is caused by synthesis and secretion into the plasma of lipoproteins that are enriched in cholesterol and depleted of triglycerides.     

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.     

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as Candida albicans. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length Drosophila histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z'' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC₅₀ values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation in vivo, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.