How Herbivore Browsing Strategy Affects Whole-Plant Photosynthetic Capacity

If a browse damage index indicates that a tree has been 50% browsed by herbivores, does this mean half the leaves are entirely eaten or are all the leaves half eaten? Were the affected leaves old or young? Large or small? In sunshine or shade? Understanding what effect browsing will have on the photosynthetic capacity and the plant’s survival ability clearly requires a greater understanding of browsing strategy across the canopy than can be given by a single index value. We developed stochastic models of leaf production, growth and consumption using data from kamahi (Weinmannia racemosa) trees in New Zealand which have been browsed by possums (Trichosurus vulpecula), to ascertain which of six feasible browsing strategies possums are most likely to be employing. We compared the area distribution of real fallen leaves to model output in order to select the best model, and used the model to predict the age distribution of leaves on the tree and thus infer its photosynthetic capability. The most likely browsing strategy that possums employ on kamahi trees is a preference for virgin (i.e. previously unbrowsed) leaves, consistent with the idea that browsing increases the production of chemical plant defences. More generally, our results show that herbivore browsing strategy can significantly change the whole-plant photosynthetic capability of any plant and hence its ability to survive, and therefore, herbivore damage indices should be used in conjunction with more detailed information about herbivore browsing strategy.     

Interspecific interactions and range limits: contrasts among interaction types

There is a great deal of interest in the effects of biotic interactions on geographic distributions. Nature contains many different types of biotic interactions (notably mutualism, commensalism, predation, amensalism, and competition), and it is difficult to compare the effects of multiple interaction types on species’ distributions. To resolve this problem, we analyze a general, flexible model of pairwise biotic interactions that can describe all interaction types. In the absence of strong positive feedback, a species’ ability to be present depends on its ability to increase in numbers when it is rare and the species it is interacting with is at equilibrium. This insight leads to counterintuitive conclusions. Notably, we often predict the same range limit when the focal species experiences competition, predation, or amensalism. Similarly, we often predict the same range margin or when the species experiences mutualism, commensalism, or benefits from prey. In the presence of strong positive density-dependent feedback, different species interactions produce different range limits in our model. In all cases, the abiotic environment can indirectly influence the impact of biotic interactions on range limits. We illustrate the implications of this observation by analyzing a stress gradient where biotic interactions are harmful in benign environments but beneficial in stressful environments. Our results emphasize the need to consider the effects of all biotic interactions on species’ range limits and provide a systematic comparison of when biotic interactions affect distributions.     

Plant structural complexity and mechanical defenses mediate predator–prey interactions in an odonate–bird system

Habitat‐forming species provide refuges for a variety of associating species; these refuges may mediate interactions between species differently depending on the functional traits of the habitat‐forming species. We investigated refuge provisioning by plants with different functional traits for dragonfly and damselfly (Odonata: Anisoptera and Zygoptera) nymphs emerging from water bodies to molt into their adult stage. During this period, nymphs experience high levels of predation by birds. On the shores of a small pond, plants with mechanical defenses (e.g., thorns and prickles) and high structural complexity had higher abundances of odonate exuviae than nearby plants which lacked mechanical defenses and exhibited low structural complexity. To disentangle the relative effects of these two potentially important functional traits on nymph emergence‐site preference and survival, we conducted two fully crossed factorial field experiments using artificial plants. Nymphs showed a strong preference for artificial plants with high structural complexity and to a lesser extent, mechanical defenses. Both functional traits increased nymph survival but through different mechanisms. We suggest that future investigations attempt to experimentally separate the elements contributing to structural complexity to elucidate the mechanistic underpinnings of refuge provisioning.     

Discovery of dual Axl/VEGF-R2 inhibitors as potential anti-angiogenic and anti-metastatic drugs for cancer chemotherapy

Axl tyrosine kinase has been shown to be involved in multiple pathways contributing to tumor development, angiogenesis, and metastasis. High Axl expression has been observed in many human tumors where it appears to confer aggressive tumor behavior. Here we present several series of dual Axl-VEGF-R2 kinase inhibitors based on extensive optimization of an acyl diaminotriazole. It was hypothesized that dual inhibition of these two receptor tyrosine kinases may have a synergistic affect in inhibiting tumor angiogenesis and metastasis. One of these molecules, R916562 showed comparable activity to Sunitinib in two mouse tumor xenograft models and a mouse corneal micropocket model.     

Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large‐scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal‐derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal‐derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design‐of‐experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two‐fold increase in OD₆₀₀ compared to YEB medium during a 4‐L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal‐derived components, thus facilitating the GMP‐compliant large‐scale transient expression of recombinant proteins in plants.     

Agrobacterium-mediated transformation of Pinus radiata organogenic tissue using vacuum-infiltration

An Agrobacterium tumefaciens-mediated transformation protocol was developed for detached cotyledons of Pinus radiata zygotic embryos resulting in up to 55% of cotyledons transiently expressing the reporter gene uidA. Transient expression of uidA was improved when detached cotyledons were pre-cultured on half strength medium containing cytokinin for 7 days, wounded by vortexing and then vacuum-infiltrated in a solution of A. tumefaciens. The transformation protocol was applied both to cotyledons and also to the apical meristematic dome which was the portion of the embryo remaining after cotyledons were detached, and from which the apical shoot and axillary shoots regenerate. Molecular analysis of putatively transformed shoots regenerated either adventitiously from cotyledons or via axillary shoots from apical domes, indicated the presence of uidA and nptII genes by PCR in some of these shoots. Biochemical analysis of putatively transformed shoots using nptII ELISA indicated that they contained the nptII enzyme. However, Southern hybridisation indicated stable integration of nptII only in one shoot which was regenerated from an apical dome. Shoots regenerated from cotyledons appeared to exhibit chimeric expression and were not stably transformed. Based on a comparison of time for regeneration, technical difficulty, molecular and biochemical analysis, apical domes may be more suitable as explants for transformation and subsequent regeneration of transclones than detached cotyledons.     

Association of chemokine-mediated block to HIV entry with coreceptor internalization.

Chemokines inhibit entry of HIV into CD4(+) T cells more effectively than into macrophages or transfected adherent cells. Here, we tested whether chemokine receptor internalization could account for cell type differences in the effectiveness of chemokines. Infection of CEM T cells expressing stably transduced wild-type CCR5 was much more readily inhibited by chemokine than were transduced HOS cells. This response correlated with the efficiency of CCR5 internalization. A mutated CCR5, termed M7-CCR5, in which the Ser/Thr phosphorylation sites in the cytoplasmic tail were changed to Ala, did not internalize in response to MIP-1alpha. M7-CCR5 was expressed at slightly higher levels than wild-type on stably transduced cell lines and was somewhat more potent as an HIV-1 coreceptor. The mutated receptor mobilized intracellular Ca(2+) in response to chemokine to a level 4-fold higher than did the wild type CCR5. Unexpectedly, the receptor was desensitized as efficiently as wild type, suggesting that desensitization does not require cytoplasmic tail phosphorylation. Entry of R5 HIV-1 reporter virus into cells stably expressing M7-CCR5 was largely resistant to blocking by MIP-1alpha. As much as 80% of entry inhibition was attributed to receptor internalization. Aminooxypentane (AOP)-MIP-1alpha was able to induce a low level of M7-CCR5 internalization in HOS and to weakly inhibit HIV-1 entry. Introduction of dominant negative dynamin into HOS cells reduced the ability of chemokine to inhibit infection. The inefficiency of internalization of chemokine receptors in some cell types could allow virus to replicate in vivo in the presence of endogenous chemokine. Last, M7-CCR5 is a useful tool for discriminating coreceptor internalization from binding site masking in the evaluation of small molecule inhibitors of HIV-1 entry.     

Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow.

We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin alpha(IIb)beta(3), involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin alpha(IIb)beta(3) engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin alpha(IIb)beta(3), in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin alpha(IIb)beta(3)-dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin alpha(IIb)beta(3) that serves to regulate the dynamics of platelet adhesion under flow.     

Linkages between tidal creek ecosystems and the landscape and demographic attributes of their watersheds

Twenty-three headwater tidal creeks draining watersheds representative of forested, suburban, urban, and industrial land cover were sampled along the South Carolina coast from 1994 to 2002 to: (1) evaluate the degree to which impervious land cover is an integrative watershed-scale indicator of stress; (2) synthesize and integrate the available data on linkages between land cover and tidal creek environmental quality into a conceptual model of the responses of tidal creeks to human development; and (3) use the model to develop recommendations for conserving and restoring tidal creek ecosystems. The following parameters were evaluated: human population density, land use, impervious cover, creek physical characteristics, water quality, sediment chemical contamination and grain size characteristics, benthic chlorophyll a levels, porewater ammonia concentration, fecal coliform concentration, and macrobenthic and nekton population and community characteristics.The conceptual model was developed and used to identify the linkages among watershed-scale stressors, physical and chemical exposures, and biological responses of tidal creeks to human development at the watershed scale. This model provides a visual representation of the manner in which human population growth is linked to changes in the physiochemical environment and ultimately the nursery habitat function of tidal creeks and the safety of seafood harvested from headwater tidal creeks. The ultimate stressor on the tidal creek ecosystem is the human population density in the watershed and associated increases in the amount of impervious land cover. Measurable adverse changes in the physical and chemical environment were observed when the impervious cover exceeded 10-20% including altered hydrography, changes in salinity variance, altered sediment characteristics, increased chemical contaminants, and increased fecal coliform loadings. Living resources responded when impervious cover exceeded 20-30%. The impacts on the living resources included reduced abundance of stress-sensitive macrobenthic taxa, reduced abundance of commercially and recreationally important shrimp, and altered food webs. Headwater tidal creeks appear to provide early warning of ensuing harm to larger tidal creeks, tidal rivers and estuaries, and the amount of impervious cover in a watershed appears to be an integrative measure of the adverse human alterations of the landscape. Through education and community involvement, a conservation ethic may be fostered that encourages the permanent protection of lands for the services they provide.     

Testing hypotheses for excess flower production and low fruit-to-flower ratios in a pollinating seed-consuming mutualism

Pollinator attraction, pollen limitation, resource limitation, pollen donation and selective fruit abortion have all been proposed as processes explaining why hermaphroditic plants commonly produce many more flowers than mature fruit. We conducted a series of experiments in Arizona to investigate low fruit-to-flower ratios in senita cacti, which rely exclusively on pollinating seed-consumers. Selective abortion of fruit based on seed predators is of particular interest in this case because plants relying on pollinating seed-consumers are predicted to have such a mechanism to minimize seed loss. Pollinator attraction and pollen dispersal increased with flower number, but fruit set did not, refuting the hypothesis that excess flowers increase fruit set by attracting more pollinators. Fruit set of natural- and hand-pollinated flowers were not different, supporting the resource, rather than pollen, limitation hypothesis. Senita did abort fruit, but not selectively based on pollen quantity, pollen donors, or seed predators. Collectively, these results are consistent with sex allocation theory in that resource allocation to excess flower production can increase pollen dispersal and the male fitness function of flowers, but consequently results in reduced resources available for fruit set. Inconsistent with sex allocation theory, however, fruit production and the female fitness function of flowers may actually increase with flower production. This is because excess flower production lowers pollinator-to-flower ratios and results in fruit abortion, both of which limit the abundance and hence oviposition rates, of pre-dispersal seed predators.