Adenosine A2A receptor activation reduces infarct size in the isolated, perfused mouse heart by inhibiting resident cardiac mast cell degranulation

Mast cells are found in the heart and contribute to reperfusion injury following myocardial ischemia. Since the activation of A2A adenosine receptors (A2AARs) inhibits reperfusion injury, we hypothesized that ATL146e (a selective A2AAR agonist) might protect hearts in part by reducing cardiac mast cell degranulation. Hearts were isolated from five groups of congenic mice: A2AAR+/+ mice, A2AAR(-/-) mice, mast cell-deficient (Kit(W-sh/W-sh)) mice, and chimeric mice prepared by transplanting bone marrow from A2AAR(-/-) or A2AAR+/+ mice to radiation-ablated A2AAR+/+ mice. Six weeks after bone marrow transplantation, cardiac mast cells were repopulated with >90% donor cells. In isolated, perfused hearts subjected to ischemia-reperfusion injury, ATL146e or CGS-21680 (100 nmol/l) decreased infarct size (IS; percent area at risk) from 38 +/- 2% to 24 +/- 2% and 22 +/- 2% in ATL146e- and CGS-21680-treated hearts, respectively (P < 0.05) and significantly reduced mast cell degranulation, measured as tryptase release into reperfusion buffer. These changes were absent in A2AAR(-/-) hearts and in hearts from chimeric mice with A2AAR(-/-) bone marrow. Vehicle-treated Kit(W-sh/W-sh) mice had lower IS (11 +/- 3%) than WT mice, and ATL146e had no significant protective effect (16 +/- 3%). These data suggest that in ex vivo, buffer-perfused hearts, mast cell degranulation contributes to ischemia-reperfusion injury. In addition, our data suggest that A2AAR activation is cardioprotective in the isolated heart, at least in part by attenuating resident mast cell degranulation.     

New techniques and findings in the study of a candidate allelochemical implicated in invasion success

Allelopathy has been hypothesized to promote the success of invasive plants. Support for the role of allelopathy in invasions has emerged from research on the candidate allelochemical (?)‐catechin, which is secreted by spotted knapweed. Here we describe new methods to quantify catechin in liquid and soil. With a new technique, we assayed catechin production by individual plants in liquid media and found levels up to two orders of magnitude less than previously reported. An acetone/water solution provided consistent recovery of catechin from soil, with percent recovery depending upon soil type. We evaluated soils from two spotted knapweed sites in Montana, USA, but found no measurable catechin. Idaho fescue, a native species reportedly sensitive to catechin, only exhibited slightly reduced growth at concentrations 10 times higher than previously reported to cause 100% mortality. Our results emphasize that more research is required to clarify the role of catechin in the invasion of spotted knapweed.     

Assessment of ecological risks in weed biocontrol: Input from retrospective ecological analyses

Prediction of the outcomes of natural enemy introductions remains the most fundamental challenge in biological control. Quantitative retrospective analyses of ongoing biocontrol projects provide a systematic strategy to evaluate and further develop ecological risk assessment. In this review, we highlight a crucial assumption underlying a continued reliance on the host specificity paradigm as a quantitative prediction of ecological risk, summarize the status of our retrospective analyses of nontarget effects of two weevils used against exotic thistles in North America, and discuss our prospective assessment of risk to a federally listed, threatened species (Cirsium pitcheri) based on those studies. Our analyses quantify the fact that host range and preference from host specificity tests are not sufficient to predict ecological impact if the introduced natural enemy is not strictly monophagous. The implicit assumption when such use is made of the host specificity data in risk assessment is that population impacts are proportional to relative preference and performance, the key components of host specificity. However, in concert with shifting awareness in the field, our studies demonstrate that the environment influences and can alter host use and population growth, leading to higher than expected direct impacts on the less preferred native host species at several spatial scales. Further, we have found that straightforward, easily anticipated indirect effects, on intraguild foragers as well as on the less preferred native host plant species, can be both widespread and significant. We conclude that intensive retrospective ecological studies provide some guidance for the quantitative prospective studies needed to assess candidate biological control agent dynamics and impacts and, so, contribute to improved rigor in the evaluation of total ecological risk to native species.     

Development of multiplexed protein profiling and detection using near infrared detection of reverse-phase protein microarrays

Protein microarrays have been recently employed for signal pathway profiling and high-throughput protein expression analysis. Reversephase arrays, where the array consists of immobilized analytes and lysates has especially shown promise in low abundance analyte detection and signal pathway profiling using phospho-specific antibodies. A limitation to current reverse phase array methodology is the inability to multiplex proteomic-based endpoints as each array can only report one analyte endpoint. In this study, we report on the use of a dual dye based approach that can effectively double the number of endpoints observed per array allowing, for example, both phosphospecific and total protein levels to be measured and analyzed at once. The method utilizes antibody bound dyes that emit in the infrared spectral region as a means of sensitive and specific detection.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

Association Between Body Fat Response to Exercise Training and Multilocus ADR Genotypes

Objectives : To examine the contribution of adrenergic receptor (ADR) gene polymorphisms and their gene‐gene interactions to the variability of exercise training‐induced body fat response. Research Methods and Procedures : This was an intervention study that used a volunteer sample of 70 healthy, sedentary men (n = 29) and postmenopausal women (n = 41) 50 to 75 years of age, with a BMI ≤37 kg/m², from the Washington, DC, metropolitan area. Participants completed 6 weeks of dietary stabilization (American Heart Association diet) before 24 weeks of supervised aerobic exercise training. Diet was maintained throughout the intervention. Change in percent total body fat, percent trunk fat, and fat mass by DXA in ADR genotype groups (Glu¹²/Glu⁹ α2b‐ADR, Trp64Arg β3‐ADR, and Gln27Glu β2‐ADR) at baseline and after 24 weeks of aerobic exercise training was measured. Results : In multivariate analysis (covariates: age, gender, and baseline value of phenotype), best fit models for percent total body and trunk fat response to exercise training retained main effects of all three ADR gene loci and the effects of each gene‐gene interaction (p = 0.009 and 0.003, respectively). Similarly, there was a trend for the fat mass response model (p = 0.03). The combined genetic factors explained 17.5% of the overall model variability for percent total body fat, 22% for percent trunk fat, and 10% for fat mass. Discussion : The body fat response to exercise training in older adults is associated with the combined effects of the Glu¹²/Glu⁹ α2b‐, Trp64Arg β3‐, and Gln27Glu β2‐ADR gene variants and their gene‐gene interactions.     

Assessment of Potential Predation Costs of Male Decoration and Courtship Display in Wolf Spiders Using Video Digitization and Playback

We used video digitization and playback techniques to examine the potential predation risk of leg decorations and visual displays of male wolf spiders, which vary across the genus Schizocosa (Araneae: Lycosidae). Video images of courting males of four Schizocosa species were modified by adding or removing tufts and presented on LCD minitelevisions to the wolf spider Hogna helluo, a common predator of Schizocosa. Predatory responses of H. helluo varied significantly among stimuli and were highest for S. ocreata (which has decorative tufts and leg-waving displays) and lower for S. rovneri, S. duplex, and S. uetzi (which lack decorations and visual displays). Removal of tufts from S. ocreata significantly reduced predatory responses of H. helluo, but addition of tufts to other species had no effect. Results suggest that leg decorations may increase detection of active leg-waving displays and thereby increase predation risk.     

Foliar physiology of yellow-poplar ( Liriodendron tulipifera L.) exposed to O3 and elevated CO2 over five seasons.

The chronic effects of ozone (O₃) alone or combined with elevated carbon dioxide (CO₂) on the foliar physiology of unfertilized field-grown yellow-poplar ( Liriodendron tulipifera L.) seedlings were studied from 1992 to 1996. Within open-top chambers, juvenile trees were exposed to the following: charcoal-filtered air (CF); 1× ambient ozone (1XO₃); 1.5× ambient ozone (1.5XO₃); 1.5× ambient ozone plus 700 ppm carbon dioxide (1.5XO₃+CO₂); or chamberless open-air (OA). Seasonal 24-h mean ambient O₃ concentrations ranged from 32 to 46 ppm over the five seasons. Averaged over 5 years, midseason net photosynthesis at saturating light ( A _sat) was reduced by 14% ( P =0.029) and stomatal conductance ( g _s) was reduced, albeit non-significant, by 13% ( P =0.096) in upper canopy foliage exposed to 1.5XO₃-air relative to CF controls. There were no significant differences over the 5 years in A _sat and g _s between trees grown in 1XO₃- and 1.5XO₃-air. Our results support the hypothesis that the magnitude of O₃ effects on A _sat and g _s decreases as saplings age. When averaged over the five seasons of exposure, total chlorophyll concentration ( chl) was not significantly affected by exposure to elevated O₃; however, in 1.5XO₃+CO₂-air, foliar chl was reduced by 33% relative to all others ( P <0.001). In 1.5XO₃+CO₂-air, A _sat was 1.4–1.9 times higher ( P <0.001) and g _s was 0.7 times lower ( P =0.022) than all others. O₃ uptake in juvenile trees exposed to elevated O₃ plus elevated CO₂ (1.5XO₃+CO₂-air) was most comparable to trees exposed to ambient air (1XO₃) throughout the study. These findings suggest that elevated CO₂ may minimize the negative effects of O₃ by reducing O₃ uptake through decreased stomatal conductance.     

Near-infrared fluorescence detection permits accurate imaging of loading controls for Western blot analysis

Housekeeping proteins are typically chosen as internal loading controls for Western blot analysis because of their high, relatively constant expression. It was previously reported that antibodies against beta-actin did not reliably identify differences in sample loading, and extended antibody incubations caused a failure to discriminate differences in target protein levels. Here, beta-actin and GAPDH were evaluated as loading controls using near-infrared fluorescence. A load-dependent response in signal intensity was observed over a 250-fold range of sample concentrations, with R(2) values as high as 0.9939. Longer antibody incubations continued to detect differences in protein level and load-dependent responses became more linear.