Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.     

Changes in herbivore control in arable fields by detrital subsidies depend on predator species and vary in space

Prey from the decomposer subsystem may help sustain predator populations in arable fields. Adding organic residues to agricultural systems may therefore enhance pest control. We investigated whether resource addition (maize mulch) strengthens aboveground trophic cascades in winter wheat fields. Evaluating the flux of the maize-borne carbon into the food web after 9 months via stable isotope analysis allowed differentiating between prey in predator diets originating from the above- and belowground subsystems. Furthermore, we recorded aphid populations in predator-reduced and control plots of no-mulch and mulch addition treatments. All analyzed soil dwelling species incorporated maize-borne carbon. In contrast, only 2 out of 13 aboveground predator species incorporated maize carbon, suggesting that these 2 predators forage on prey from the above- and belowground systems. Supporting this conclusion, densities of these two predator species were increased in the mulch addition fields. Nitrogen isotope signatures suggested that these generalist predators in part fed on Collembola thereby benefiting indirectly from detrital resources. Increased density of these two predator species was associated by increased aphid control but the identity of predators responsible for aphid control varied in space. One of the three wheat fields studied even lacked aphid control despite of mulch-mediated increased density of generalist predators. The results suggest that detrital subsidies quickly enter belowground food webs but only a few aboveground predator species include prey out of the decomposer system into their diet. Variation in the identity of predator species benefiting from detrital resources between sites suggest that, depending on locality, different predator species are subsidised by prey out of the decomposer system and that these predators contribute to aphid control. Therefore, by engineering the decomposer subsystem via detrital subsidies, biological control by generalist predators may be strengthened.     

The ecological advantage of sexual reproduction in multicellular long-lived organisms

We present a model for the advantage of sexual reproduction in multicellular long-lived species in a world of structured resources in short supply. The model combines features of the Tangled Bank and the Red Queen hypothesis of sexual reproduction and is of broad applicability. The model is ecologically explicit with the dynamics of resources and consumers being modelled by differential equations. The life history of consumers is shaped by body mass-dependent rates as implemented in the metabolic theory of ecology. We find that over a broad range of parameters, sexual reproduction wins despite the two-fold cost of producing males, due to the advantage of producing offspring that can exploit underutilized resources. The advantage is largest when maturation and production of offspring set in before the resources of the parents become depleted, but not too early, due to the cost of producing males. The model thus leads to the dominance of sexual reproduction in multicellular animals living in complex environments, with resource availability being the most important factor affecting survival and reproduction.     

Skeletal Muscle Radio-Density Is an Independent Predictor of Response and Outcomes in Follicular Lymphoma Treated with Chemoimmunotherapy

Skeletal muscle radio-density (SMD) measures muscle radiation attenuation (in Hounsfield Units, HU) on computed tomography (CT) scans. Low SMD is prognostic of poor survival in melanoma, however its significance is unknown for hematologic malignancies. We performed a single institution, retrospective review of all follicular lymphoma (FL) patients who received chemoimmunotherapy from 2004–2009. Patient demographics, FL International Prognostic Index 1 (FLIPI-1), progression free (PFS) and overall survival (OS) were collected as primary endpoints. Objective response rates (ORR) were secondary. SMD was calculated using pre-treatment CT scans. In 145 patients reviewed, median values were age 59, FLIPI-1 of 2, stage III, and 8 chemoimmunotherapy cycles received. Median PFS for those with low SMD (<36.6 and <33.1 HU for patients with BMI ≤ 25 and > 25 kg/m², respectively) compared to those with high SMD was profoundly worse, 69.6 vs. 106.7 months (hazard ratio [HR] 1.85; p = 0.01), respectively. Median OS was not reached in patients with high SMD vs. 92.7 months in low SMD patients (HR 4.02; p = 0.0002). Multivariate analysis supported lower SMD’s OS detriment (HR = 3.40; p = 0.002) independent of FLIPI-1 (HR 1.46–2.76, p = 0.05) or gender. Low SMD predicted lower ORR, 83 vs. 96% (p = 0.01). SMD predicts survival independent of FLIPI-1 and potentially chemoimmunotherapy response. SMD is an inexpensive and powerful tool that can complement FLIPI-1.     

Evidence for DNA Damage Checkpoint Activation in Barrett Esophagus

Barrett esophagus is an epithelial metaplasia that predisposes to adenocarcinoma. Better markers of cancer risk are urgently needed to identify those patients who are likely to benefit most from emerging methods of endoscopic ablation. Disease progression is associated with genomic DNA changes (segmental gains, losses, or loss of heterozygosity). Although these changes are not easily assayed directly, we hypothesized that the underlying DNA damage should activate a DNA damage response (DDR), detectable by immunohistochemical (IHC) assays of checkpoint proteins and the resulting replicative phase cell cycle delays. Surgical specimens and endoscopic biopsies (N = 28) were subjected to IHC for the cell cycle markers cyclin A and phosphorylated histone H3 (P-H3), the DDR markers γH2AX and phosphorylated ATM/ATR substrates (P-ATM/ATRsub), and the DNA damage-responsive tumor suppressors p16 and p53. Correlations were made with histologic diagnoses. The fractions of cells that stained for cyclin A, P-H3, and γH2AX increased in parallel in dysplastic tissue, consistent with checkpoint-mediated cell cycle delays. Foci of nuclear γH2AX and P-ATM/ATRsub were demonstrated by standard and confocal immunofluorescence. Staining for p16 was more prevalent in early-stage disease with lower staining for γH2AX and P-H3. Staining for p53 was moderately increased in some early-stage disease and strongly increased in some advanced disease, consistent with checkpoint-mediated induction and mutational inactivation of p53, respectively. We suggest that IHC for DDR-associated markers may help stratify risk of disease progression in Barrett.     

Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.     

Utilization of prey from the decomposer system by generalist predators of grassland

We investigated the linkage between the detrital subsystem and generalist predators of meadow ecosystems by manipulating prey availability in two different ways: we increased resource availability for the decomposer subsystem and thereby decomposer prey by adding mulch materials (detritus enhancement), and we added fruitflies (Drosophila melanogaster, Diptera; prey enhancement) to fenced plots. Both supplemented materials significantly differed in their ¹³C/¹²C and ¹⁵N/¹⁴N ratios from those of the natural litter. We measured density responses of detritivorous, herbivorous and predaceous arthropods to the increased resource supply. We used ratios of natural stable isotopes of N and C in arthropod tissues to trace the flux from the added resources to consumers and to relate density responses of consumers to changes in resource supply. Effects of resource enhancement propagated through at least two trophic levels, resulting in higher densities of major decomposer and predator taxa. Effects of detritus enhancement were much stronger than those of prey enhancement. Signatures of δ¹³C proved density responses of Collembola taxa to be related to the added mulch materials. Among generalist predators, densities of juvenile wolf spiders (Lycosidae) responded more to detritus-enhancement than to prey-enhancement treatments. In contrast, the density of the web-building linyphiid and the non-web gnaphosid spiders remained unaffected. Each spider taxon, including those which did not respond numerically, was significantly enriched in ¹³C in detritus-enhancement treatments, suggesting that they gain energy from the decomposer system. Numbers of herbivores—cicadellids and aphids—were similar in each of the treatments, indicating that they were unaffected by changes in predator density. Our results indicate that the lack of a numerical response to resource supplementation is not necessarily due to the absence of a trophic linkage, but may be caused by compensatory changes in mortality factors such as cannibalism and intraguild predation.     

Fungal metabolic plasticity and sexual development mediate induced resistance to arthropod fungivory

Prey organisms do not tolerate predator attack passively but react with a multitude of inducible defensive strategies. Although inducible defence strategies are well known in plants attacked by herbivorous insects, induced resistance of fungi against fungivorous animals is largely unknown. Resistance to fungivory is thought to be mediated by chemical properties of fungal tissue, i.e. by production of toxic secondary metabolites. However, whether fungi change their secondary metabolite composition to increase resistance against arthropod fungivory is unknown. We demonstrate that grazing by a soil arthropod, Folsomia candida, on the filamentous fungus Aspergillus nidulans induces a phenotype that repels future fungivores and retards fungivore growth. Arthropod-exposed colonies produced significantly higher amounts of toxic secondary metabolites and invested more in sexual reproduction relative to unchallenged fungi. Compared with vegetative tissue and asexual conidiospores, sexual fruiting bodies turned out to be highly resistant against fungivory in facultative sexual A. nidulans. This indicates that fungivore grazing triggers co-regulated allocation of resources to sexual reproduction and chemical defence in A. nidulans. Plastic investment in facultative sex and chemical defence may have evolved as a fungal strategy to escape from predation.     

Tangled Bank dismissed too early

The Tangled Bank hypothesis has been one of the main theories to explain why most organisms reproduce sexually. It was most forcefully defended by Bell, who argued that genetically diverse offspring are able to extract more food from their environment than genetically identical clones. Due to the limited applicability of mathematical models and a lack of evidence that more sib‐competition leads to a larger advantage of sexual reproduction, the Tangled Bank hypothesis has since been abandoned by many authors in favor of the Red Queen hypothesis, which focuses on temporal environmental variation instead of spatial variation. Here, we argue that the rejection of the Tangled Bank hypothesis is based on a lack of appreciation of the importance of resources for determining the mode of reproduction, of the fundamental difference between the role of resources and the role of abiotic conditions, and of the negative feedback between resource consumption and resource availability. This negative feedback in fact leads to an ongoing temporal change in resource usage and thus connects the Tangled Bank concept to the Red Queen concept. We discuss recently introduced models that implement these ideas, and we suggest empirical studies on the relation between invasibility and genetic diversity of communities in order to test the Tangled Bank hypothesis more thoroughly.