Regulation of Ca2+ signaling in mast cells by tyrosine-phosphorylated and unphosphorylated non-T cell activation linker

Engagement of the FcepsilonRI in mast cells and basophils leads to a rapid tyrosine phosphorylation of the transmembrane adaptors LAT (linker for activation of T cells) and NTAL (non-T cell activation linker, also called LAB or LAT2). NTAL regulates activation of mast cells by a mechanism, which is incompletely understood. Here we report properties of rat basophilic leukemia cells with enhanced or reduced NTAL expression. Overexpression of NTAL led to changes in cell morphology, enhanced formation of actin filaments and inhibition of the FcepsilonRI-induced tyrosine phosphorylation of the FcepsilonRI subunits, Syk kinase and LAT and all downstream activation events, including calcium and secretory responses. In contrast, reduced expression of NTAL had little effect on early FcepsilonRI-induced signaling events but inhibited calcium mobilization and secretory response. Calcium response was also repressed in Ag-activated cells defective in Grb2, a major target of phosphorylated NTAL. Unexpectedly, in cells stimulated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) ATPase, the amount of cellular NTAL directly correlated with the uptake of extracellular calcium even though no enhanced tyrosine phosphorylation of NTAL was observed. The combined data indicate that NTAL regulates FcepsilonRI-mediated signaling at multiple steps and by different mechanisms. At early stages NTAL interferes with tyrosine phosphorylation of several substrates and formation of signaling assemblies, whereas at later stages it regulates the activity of store-operated calcium channels through a distinct mechanism independent of enhanced NTAL tyrosine phosphorylation.     

Engagement of phospholipid scramblase 1 in activated cells: implication for phosphatidylserine externalization and exocytosis

Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca(2+)-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcepsilonRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcepsilonRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation.     

Selection on defensive traits in a sterile caste – caste evolution: a mechanism to overcome life‐history trade‐offs?

SUMMARY During development and evolution individuals generally face a trade-off between the development of weapons and gonads. In termites, characterized by reproductive division of labor, a caste evolved—the soldiers—which is completely sterile and which might be released from developmental trade-offs between weapons and testes. These soldiers are exclusively dedicated to defense. First, we investigated whether defensive traits are under selection in sterile termite soldiers using allometric analyses. In soldiers of the genus Cryptotermes phragmotic traits such as a sculptured and foreshortened head evolve rapidly but were also lost twice. Second, we compared the scaling relationships of these weapons with those in solitary insects facing a trade-off between weapons and gonads. Defensive traits consistently had lower slopes than nondefensive traits which supports the existence of stabilizing selection on soldier phragmotic traits in order to plug galleries. Moreover, soldier head widths were colony specific and correlated with the minimum gallery diameter of a colony. This can proximately be explained by soldiers developing from different instars. The scaling relationships of these termite soldiers contrast strikingly with those of weapons of solitary insects, which are generally exaggerated (i.e., overscaling) male traits. These differences may provide important insights into trait evolution. Trade-offs constraining the development of individuals may have been uncoupled in termites by evolving different castes, each specialized for one function. When individuals in social insect are "released" from developmental constraints through the evolution of castes, this certainly contributed to the ecological and evolutionary success of social insects.     

Slash Pile Burning Effects on Soil Biotic and Chemical Properties and Plant Establishment: Recommendations for Amelioration

Ponderosa pine forest restoration consists of thinning trees and reintroducing prescribed fire to reduce unnaturally high tree densities and fuel loads to restore ecosystem structure and function. A current issue in ponderosa pine restoration is what to do with the large quantity of slash that is created from thinning dense forest stands. Slash piling burning is currently the preferred method of slash removal because it allows land managers to burn large quantities of slash in a more controlled environment in comparison with broadcast burning slash. However burning slash piles is known to have adverse effects such as soil sterilization and exotic species establishment. This study investigated the effects of slash pile burning on soil biotic and chemical variables and early herbaceous succession on burned slash pile areas. Slash piles were created following tree thinning in two adjacent approximately 20‐ha ponderosa pine (Pinus ponderosa) restoration treatments in the Coconino National Forest near Flagstaff, Arizona. We selected 30 burned slash pile areas and sampled across a gradient of the burned piles for arbuscular mycorrhizal (AM) propagule densities, the soil seed bank, and soil chemical properties. In addition, we established five 1‐m² plots in each burned pile to quantify the effect of living soil (AM inoculum) and seeding amendments on early herbaceous succession in burned slash pile areas. The five treatments consisted of a control (no treatment), living soil (AM inoculum) amendment, sterilized soil (no AM inoculum) amendment, seed amendment, and a seed/soil (AM inoculum) amendment. Slash pile burning nearly eliminated populations of viable seeds and AM propagules and altered soil chemical properties. Amending scars with native seeds increased the cover of native forbs and grasses. Furthermore adding both seed and living soil more than doubled total native plant cover and decreased ruderal and exotic plant cover. These results indicate that seed/soil amendments that increase native forbs and grasses may enhance the rate of succession in burned slash pile areas by allowing these species to outcompete exotic and ruderal species also establishing at the site through natural regeneration.     

Sampling Techniques Influence Understory Plant Trajectories After Restoration: An Example from Ponderosa Pine Restoration

Abstract Although there is no one correct technique for sampling vegetation, the sampling design chosen may greatly influence the conclusions researchers can draw from restoration treatments. Considerations when designing vegetation sampling protocol include determining what sampling attributes to measure, the size and shape of the sampling plot, the number of replicates and their location within the study area, and the frequency of sampling. We installed 20 point‐intercept transects (50‐m long), 8 belt transects (10 × 50 m), 10 adapted Daubenmire transects (four 0.5 × 2‐m plots), and 4 modified‐Whittaker plots (20 × 50 m with smaller nested plots) in treatment and control units to measure understory herbaceous response in a forest restoration experiment that tested different treatments. Point‐intercept transects on average recorded at least twice as much plant cover as did adapted Daubenmire transects and modified‐Whittaker plots taken at the same location for all control and treatment units. Point‐intercept transects and adapted Daubenmire plots on average captured fewer rare and exotic species in the control and treatment units in comparison with the belt transects and modified‐Whittaker plots. Modified‐Whittaker plots captured the highest species richness in all units. Early successional understory response to restoration treatments was likely masked by the response of the herbaceous community to yearly climatic variation (dry vs. wet years). Species richness and abundance were higher in wet years than dry years for all control and treatment units. Our results illustrate that sampling techniques can greatly influence perceptions of understory plant trajectories and therefore the interpretation of whether restoration goals have been achieved. In addition, our results suggest that restoration monitoring needs to be conducted for a sufficient length of time so that restoration treatment responses can be detected.     

Isolation and characterization of a new dsRNA virus fromWickerhamia fluorescens

Virus-like particles (VLPs) were isolated from the yeastWickerhamia fluorescens strain CCY61-1-1. The VLPs are approximately 42 nm in diameter and contain only one species of dsRNA molecule. The apparent length of the dsRNA determined by native agarose gel electrophoresis was 4.6 kbp. Analysis of protein content of the VLPs showed them to contain one major capsid protein with an apparent molar mass of 74.5 kDa.     

Biogenesis of cytochrome c in Neurospora crassa. Synthesis of apocytochrome c, transfer to mitochondria and conversion to Holocytochrome c.

1. Precipitating antibodies specific for apocytochrome c and holocytochrome c, respectively, were employed to study synthesis and intracellular transport of cytochrome c in Neurospora in vitro. 2. Apocytochrome c as well as holocytochrome c were found to be synthesized in a cell-free homogenate. A precursor product relationship between the two components is suggested by kinetic experiments. 3. Apocytochrome c synthesized in vitro was found in the post-ribosomal fraction and not in the mitochondrial fraction, whereas holocytochrome c synthesized in vitro was mainly detected in the mitochondrial fraction. A precursor product relationship between postribosomal apocytochrome c and mitochondrial holocytochrome c is indicated by the labelling data. In the microsomal fraction both apocytochrome c and holocytochrome c were found in low amounts. Their labeling kinetics do not subbest a precursor role of microsomal apocytochrome c or holocytochrome c. 4. Formation of holocytochrome c from apocytochrome c was observed when postribosomal supernatant containing apocytochrome c synthesized in vitro was incubated with isolated mitochondria, but not when incubated in the absence of mitochondria. The cytochrome c formed under these conditions was detected in the mitochondria. 5. Conversion of labelled apocytochrome c synthesized in vitro to holocytochrome c during incubation of a postribosomal supernatant with isolated mitochondria was inhibited when excess isolated apocytochrome c, but not when holocytochrome c was added. 6. The data presented are interpreted to show that apocytochrome c is synthesized on cytoplasmic ribosomes and released into the supernatant. It is suggested that apocytochrome c migrates to the inner mitochondrial membrane, where the heme group is covalently linked to the apoprotein. The hypothesis is put forward that the concomitant change in conformation leads to trapping of holocytochrome c in the membrane. The problems of permeability of the outer mitochondrial membrane to apocytochrome c and the site and nature of the reaction by which the heme group is linked to the apoprotein are discussed.     

Thermal recovery after passage of the pulmonary circulation assessed by deconvolution

We determined the effect of H2O2 on both the physiological and biochemical lung changes seen in the adult sheep after endotoxin. Fourteen unanesthetized adult sheep with chronic lung lymph fistula were given Escherichia coli endotoxin (1 microgram/kg) over 30 min. Seven sheep were given catalase (32,500 U/kg body wt) as an intravenous bolus 30 min before endotoxin. Four sheep were given catalase alone. Oxidant lung changes were measured using arterial plasma conjugated dienes and lung tissue malondialdehyde (MDA) content, both reflecting the lipid peroxidation process. Animals were killed 5 h after endotoxin. We found that endotoxin alone caused an early increase in pulmonary arterial pressure lung lymph flow (QL), plasma thromboxane B2, 6-keto-prostaglandin F1 alpha, and plasma conjugated dienes. A decrease in cardiac output and arterial PO2 was also seen. A three- to four-fold increase in protein-rich QL was noted at 3-4 h as well as a continued increase in arterial conjugated dienes. Lung MDA and water content were also significantly increased from base line. Catalase pretreatment significantly attenuated both the physiological changes and the prostanoid and conjugated diene release. Lung MDA and water content also remained at base line. We conclude that H2O2 plays a major role in endotoxin-induced lung injury as well as the resulting lipid peroxidation process.