Ecdysteroid responses of estuarine crustaceans exposed through complete larval development to juvenile hormone agonist insecticides

Fenoxycarb and pyriproxyfen are insecticides that gain theirtoxicity by specifically acting as insect juvenile hormone agonists(JHA), and so are endocrine disruptors by design and effectivelyprevent larvae from maturing into adults. Efforts to assessthe environmental effects of JHAs on nontarget populations ofinvertebrates have resulted in the utilization of several establishedestuarine crustacean models. This work was conducted to testthe hypothesis that the mortality, inhibition of developmentand decreased fecundity reported previously in these animalsfrom JHA exposure coincides with abnormal circulating titersof ecdysteroids. Gravid female grass shrimp (Palaemonetes pugio)and mud crabs (Rhithropanopeus harrisii), species with differentdevelopmental plasticity and JHA tolerances, were collectedand held at wet lab conditions (20 ppt salinity, 25°C) untillarval release. Larvae were collected <12 hr after hatchand exposed to JHAs during a static renewal test through endof development with seawater or nominal concentrations of JHApreviously shown to induce significant developmental delaysand/or decreased body weights. Larvae were subsampled (10 larvae/sample,n = 2 to 8) at each developmental stage, lyophilized, and ecdysteroidsextracted by homogenization in 80% methanol and elution fromC18 Sep-Pak cartridges with 25%, 60% and 100% methanol to capturethe polar, free, and apolar conjugates, respectively, and thenquantified by ELISA. As was expected significant differencesin successful completion of development (larval survival), developmentalduration, and growth (dry weight) were observed. These physiologicalperturbations were linked with significantly altered ecdysteroidtiters, supporting a newly emerging theory that juvenoids possiblyact as anti-ecdysteroids through a novel molecular mechanisminvolving inhibition of ecdysteroid signaling.     

Gene delivery through cell culture substrate adsorbed DNA complexes

Efficient gene delivery is a fundamental goal of biotechnology and has numerous applications in both basic and applied science. Substrate-mediated delivery and reverse transfection enhance gene transfer by increasing the concentration of DNA in the cellular microenvironment through immobilizing a plasmid to a cell culture substrate prior to cell seeding. In this report, we examine gene delivery of plasmids that were complexed with cationic polymers (polyplexes) or lipids (lipoplexes) and subsequently immobilized to cell culture or biomaterial substrates by adsorption. Polyplexes and lipoplexes were adsorbed to either tissue culture polystyrene or serum-adsorbed tissue culture polystyrene. The quantity of DNA immobilized increased with time of exposure, and the deposition rate and final amount deposited depended upon the properties of the substrate and complex. For polyplexes, serum modification enhanced reporter gene expression up to 1500-fold relative to unmodified substrates and yielded equivalent or greater expression compared to bolus delivery. For lipoplexes, serum modification significantly increased the number of transfected cells relative to unmodified substrates yet provided similar levels of expression. Immobilized complexes transfect primary cells with improved cellular viability relative to bolus delivery. Finally, this substrate-mediated delivery approach was extended to a widely used biomaterial, poly(lactide-co-glycolide). Immobilization of DNA complexes to tissue culture polystyrene substrates can be a useful tool for enhancing gene delivery for in vitro studies. Additionally, adapting this system to biomaterials may facilitate application to fields such as tissue engineering.     

Transportable and Non-transportable Inhibitors of L-glutamate Uptake Produce Astrocytic Stellation and Increase EAAT2 Cell Surface Expression

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (d-Aspartate (d-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (l-CCG-III)) and non-transportable (dl-threo-β-benzyloxyaspartate (dl-TBOA)) inhibitors of Glu uptake in murine astrocytes. d-Asp (1 mM), l-CCG-III (0.5 mM) and dl-TBOA (0.5 mM) produced time-dependent (24–72 h) reductions in ³[H]d-Asp uptake (approximately 30–70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for l-CCG-III and dl-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.     

Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events

We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.     

Spatial Segregation of Congeneric Invaders in Central Pennsylvania, USA

Carduus acanthoides and Carduus nutans (plumeless and musk thistles) are among the most noxious weeds in the United States of America, presenting a serious challenge in cropping and pasture systems. Unfortunately, a lack of detailed spatial distribution information hampers both our ability to understand the factors affecting their invasive success, and the effectiveness of monitoring and management efforts. To examine patterns of distribution and co-occurrence at a local level, we sampled a 5000 km² area of central Pennsylvania that cut a transect across known areas of C. acanthoides and C. nutans infestation. A number of potential environmental explanatory variables were recorded and analyzed to examine whether they correlated with observed species distribution patterns. Patterns of forest density and spatial aggregation of the thistles were the primary covariates that significantly impacted both species’ distributions. The survey established that the frequency of sightings for each species diminished as the ranges converged, with only brief overlap: the two species are strongly negatively correlated in space. Understanding environmental correlates of infestation and the pattern of spatial dissociation of these two invasive species is an important step towards an improved understanding of the mechanisms underlying their invasive potential, and hence towards effective weed control.     

Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels

Calmodulin (CaM) is an essential, eukaryotic protein comprised of two highly homologous domains (N and C). CaM binds four calcium ions cooperatively, regulating a wide array of target proteins. A genetic screen of Paramecia by Kung [Kung, C. et al. (1992) Cell Calcium 13, 413-425] demonstrated that the domains of CaM have separable physiological roles: "under-reactive" mutations affecting calcium-dependent sodium currents mapped to the N-domain, while "over-reactive" mutations affecting calcium-dependent potassium currents localized to the C-domain of CaM. To determine whether and how these mutations affected intrinsic calcium-binding properties of CaM domains, phenylalanine fluorescence was used to monitor calcium binding to sites I and II (N-domain) and tyrosine fluorescence was used to monitor sites III and IV (C-domain). To explore interdomain interactions, binding properties of each full-length mutant were compared to those of its corresponding domain fragments. The calcium-binding properties of six under-reactive mutants (V35I/D50N, G40E, G40E/D50N, D50G, E54K, and G59S) and one over-reactive mutant (M145V) were indistinguishable from those of wild-type CaM, despite their deleterious physiological effects on ion-channel regulation. Four over-reactive mutants (D95G, S101F, E104K, and H135R) significantly decreased the calcium affinity of the C-domain. Of these, one (E104K) also increased the calcium affinity of the N-domain, demonstrating that the magnitude and direction of wild-type interdomain coupling had been perturbed. This suggests that, while some of these mutations alter calcium-binding directly, others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel.     

Fate of the initial follicle pool: empirical and mathematical evidence supporting its sufficiency for adult fertility

The importance of the initial follicle pool in fertility in female adult mammals has recently been debated. Utilizing a mathematical model of the dynamics of follicle progression (primordial to primary to secondary), we examined whether the initial follicle pool is sufficient for adult fertility through reproductive senescence in CD1 mice. Follicles in each stage were counted from postnatal day 6 through 12 months and data were fit to a series of first-order differential equations representing two mechanisms: an initial pool of primordial follicles as the only follicle source (fixed pool model), or an initial primordial follicle pool supplemented by germline stem cells (stem cell model). The fixed pool model fit the experimental data, accurately representing the maximum observed primary follicle number reached by 4-6 months of age. Although no germline stem cells could be identified by SSEA-1 immunostaining, the stem cell model was tested using a range of de novo primordial follicle production rates. The stem cell model failed to describe the observed decreases in follicles over time and did not parallel the accumulation and subsequent reduction in primary follicles during the early fertile lifespan of the mouse. Our results agree with established dogma that the initial endowment of ovarian follicles is not supplemented by an appreciable number of stem cells; rather, it is sufficient to ensure the fertility needs of the adult mouse.     

Integrating multiple disturbance aspects: management of an invasive thistle,Carduus nutans

Background and Aims

Disturbances occur in most ecological systems, and play an important role in biological invasions. We delimit five key disturbance aspects: intensity, frequency, timing, duration and extent. Few studies address more than one of these aspects, yet interactions and interdependence between aspects may lead to complex outcomes.

Methods

In a two-cohort experimental study, we examined how multiple aspects (intensity, frequency and timing) of a mowing disturbance regime affect the survival, phenology, growth and reproduction of an invasive thistle Carduus nutans (musk thistle).

Key Results

Our results show that high intensity and late timing strongly delay flowering phenology and reduce plant survival, capitulum production and plant height. A significant interaction between intensity and timing further magnifies the main effects. Unexpectedly, high frequency alone did not effectively reduce reproduction. However, a study examining only frequency and intensity, and not timing, would have erroneously attributed the importance of timing to frequency.

Conclusions

We used management of an invasive species as an example to demonstrate the importance of a multiple-aspect disturbance framework. Failure to consider possible interactions, and the inherent interdependence of certain aspects, could result in misinterpretation and inappropriate management efforts. This framework can be broadly applied to improve our understanding of disturbance effects on individual responses, population dynamics and community composition.     

Crosstalk between adenosine A1 and β1-adrenergic receptors regulates translocation of PKCε in isolated rat cardiomyocytes

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and β(1)-adrenergic agonists on function are complex in the heart.     

Hydrogel design for supporting neurite outgrowth and promoting gene delivery to maximize neurite extension

Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration.