A continuous fluorescence assay for protein kinase C

A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation at pCa = 6.2), phosphatidylserine, diacylglycerol, or phorbol-12-myristate-13-acetate (half-maximal stimulation at 2 nM) and ATP, and correlates well (r = 0.997) with [32P]phosphate incorporation into the peptide. This fluorescence assay allows detection of 0.02 nM PKC, while similar concentrations of cyclic AMP-dependent or type II calmodulin-dependent protein kinases produced no change in peptide fluorescence. The method can be used to assay purified PKC as well as activity in crude brain homogenates. Incubation of PKC with staurosporine inhibits the fluorescence decrease with an IC50 of 2 nM. Thus the fluorescence decrease that occurs in the acrylodan-peptide provides a continuous fluorescence assay for PKC activity.     

Determining the Effects of Cattle Grazing Treatments on Yosemite Toads (Anaxyrus [=Bufo] canorus) in Montane Meadows

Amphibians are experiencing a precipitous global decline, and population stability on public lands with multiple uses is a key concern for managers. In the Sierra Nevada Mountains (California, USA), managers have specifically identified livestock grazing as an activity that may negatively affect Yosemite toads due to the potential overlap of grazing with toad habitat. Grazing exclusion from Yosemite toad breeding and rearing areas and/or entire meadows have been proposed as possible management actions to alleviate the possible impact of cattle on this species. The primary objective of this study was to determine if different fencing treatments affect Yosemite toad populations. We specifically examined the effect of three fencing treatments on Yosemite toad breeding pool occupancy, tadpoles, and young of the year (YOY). Our hypothesis was that over the course of treatment implementation (2006 through 2010), Yosemite toad breeding pool occupancy and early life stage densities would increase within two fencing treatments relative to actively grazed meadows due to beneficial changes to habitat quality in the absence of grazing. Our results did not support our hypothesis, and showed no benefit to Yosemite toad presence or early life stages in fenced or partially fenced meadows compared to standard USDA Forest Service grazing levels. We found substantial Yosemite toad variation by both meadow and year. This variation was influenced by meadow wetness, with water table depth significant in both the tadpole and YOY models.     

Competition and succession among coral endosymbionts

Host species often support a genetically diverse guild of symbionts, the identity and performance of which can determine holobiont fitness under particular environmental conditions. These symbiont communities are structured by a complex set of potential interactions, both positive and negative, between the host and symbionts and among symbionts. In reef‐building corals, stable associations with specific symbiont species are common, and we hypothesize that this is partly due to ecological mechanisms, such as succession and competition, which drive patterns of symbiont winnowing in the initial colonization of new generations of coral recruits. We tested this hypothesis using the experimental framework of the de Wit replacement series and found that competitive interactions occurred among symbionts which were characterized by unique ecological strategies. Aposymbiotic octocoral recruits within high‐ and low‐light environments were inoculated with one of three Symbiodiniaceae species as monocultures or with cross‐paired mixtures, and we tracked symbiont uptake using quantitative genetic assays. Priority effects, in which early colonizers excluded competitive dominants, were evidenced under low light, but these early opportunistic species were later succeeded by competitive dominants. Under high light, a more consistent competitive hierarchy was established in which competitive dominants outgrew and limited the abundance of others. These findings provide insight into mechanisms of microbial community organization and symbiosis breakdown and recovery. Furthermore, transitions in competitive outcomes across spatial and temporal environmental variation may improve lifetime host fitness.     

Multiple defender effects: synergistic coral defense by mutualist crustaceans

The majority of our understanding of mutualisms comes from studies of pairwise interactions. However, many hosts support mutualist guilds, and interactions among mutualists make the prediction of aggregate effects difficult. Here, we apply a factorial experiment to interactions of 'guard' crustaceans that defend their coral host from seastar predators. Predation was reduced by the presence of mutualists (15% reduction in predation frequency and 45% in volume of coral consumed). The frequency of attacks with both mutualists was lower than with a single species, but it did not differ significantly from the expected frequency of independent effects. In contrast, the combined defensive efficacy of both mutualist species reduced the volume of coral tissue lost by 73%, significantly more than the 38% reduction expected from independent defensive efforts, suggesting the existence of a cooperative synergy in defensive behaviors of 'guard' crustaceans. These emergent 'multiple defender effects' are statistically and ecologically analogous to the emergent concept of 'multiple predator effects' known from the predation literature.     

Transient exposure of dendritic cells to maturation stimuli is sufficient to induce complete phenotypic maturation while preserving their capacity to respond to subsequent restimulation

Dendritic cells (DC) are activated by pathogens, cytokines and activated T cells. We investigated the impact of a transient initial DC stimulation on the kinetics of maturation using a combination of double-stranded RNA and TNFalpha and subsequent restimulation by T cell-derived stimuli. Transient stimulation of DC was sufficient to start an irreversible program of phenotypic maturation which proceeded in the absence of the initial stimulus. Transiently stimulated DC secreted lower amounts of IL-12 during the 48-h period of the first stimulation than cells activated for 48 h. Although both DC preparations expressed the same level of maturation-associated markers at 48 h, DC stimulated for shorter periods preserved higher sensitivity to boosting upon subsequent stimulation by T cell-derived signals. We showed that DC initially stimulated for shorter periods were more potent stimulators of T lymphocytes and they induced a more polarized Th1 response. These results indicate that short exposure of DC to maturation stimuli enables an efficient defensive immune response induction by differentially regulating phenotypic maturation and cytokine production of DC.