Cortical bone dynamics, strength, and densitometry after induction of emphysema in hamsters.

Recent evidence suggests that patients suffering from chronic obstructive pulmonary disease are also at an increased risk of developing osteoporosis. The pathophysiological mechanism(s) linking these progressive diseases is unknown. The goal of this investigation was to determine whether there were alterations in bone mineral density and content, cortical bone structure and strength, and indexes of bone formation and resorption in the elastase-induced emphysematous hamster. At 3 wk after induction of emphysema, the femoral bone mineral content was 8% less (P = 0.026) and the femoral fracture strength was 6% less (P = 0.032) in the emphysematous hamster than in controls. The cortical area was 8.4% less (P = 0.013) and the periosteal mineral appositional rate was 27% less (P = 0.05) than in controls. Additionally, the endocortical eroded surface in the emphysematous group was about twice that in the control group (P = 0.003). Differences in some indexes of bone formation and resorption, paralleled by differences in bone structure and strength, were observed 3 wk after induction of emphysema. These differences in skeletal metabolism and strength may help explain some of the skeletal changes associated with chronic obstructive pulmonary disease in humans.     

Scapula form and locomotion in chimpanzee evolution

A number of primatologists have followed Coolidge (Am. J. Phys. Anthropol. 18:1–57, 1933) in suggesting that 1) there are significant shape differences in scapula form between pygmy and common chimpanzees, 2) scapulae of P. paniscus resemble those of hylobatids more than do those of P. troglodytes, and 3) therefore pygmy chimpanzees may exhibit a greater component of arm-swinging and other arboreal behaviors than common chimpanzees. In this paper I utilize a comparative analysis of ontogenetic allometries of linear dimensions to determine shape differences in the scapulae of adult pygmy and common chimpanzees and to clarify size-related changes in shape resulting from ontogenetic scaling, i.e., the differential extension of common patterns of growth allometry. Results demonstrate that the scapulae of adult P. paniscus are relatively narrower (in a direction approximately perpendicular to the scapula spine) than those of P. troglodytes, supporting Coolidge's original claim. The allometric analysis further demonstrates, however, that the two chimpanzee species exhibit ontogenetic scaling for all proportions of the scapula examined. Thus, adult pygmy chimpanzees have the scapula proportions observed in small adult and subadult P. troglodytes of comparable scapula size. The implications of this finding for past claims concerning differences in locomotor behavior between the species are discussed. This work lends additional support to previous studies that have demonstrated a high frequency of ontogenetic scaling within the genus Pan and a pedomorphic or juvenilized morphology in the pygmy chimpanzee.     

Modeling of benzocaine analog interactions with the D4S6 segment of NaV4.1 voltage-gated sodium channels

Local anesthetics (LAs) are compounds that inhibit the propagation of action potentials in excitable tissues by blocking voltage-gated Na+ channels. Mutagenesis studies have demonstrated that several amino acid residues are important sites of LA interaction with the channel, but these studies provide little information regarding the molecular forces that govern drug-binding interactions, including the binding orientation of drugs. We used computational methods to construct a simple model of benzocaine analog binding with the D4S6 segment of rat skeletal muscle (NaV4.1) sodium channels. The model revealed that four hydrophobic residues form a binding cavity for neutral LAs, and docking studies indicated that increasing hydrophobicity among the benzocaine analogs allowed a better fit within the binding cavity. The similarities between our simple model and published experimental data suggested that modeling of LA interactions with sodium channels, along with experimental approaches, could further enhance our understanding of LA interactions with sodium channels.     

Thermal perception thresholds recorded using method of limits change over brief time intervals

Quantitative Sensory Testing (QST) of thermal perception thresholds assesses small afferent nerve function. QST has also been widely used to investigate the effects of interventions on the perception of activity within these nerve fibres, often over brief time periods. The natural variation in perception thresholds over brief time periods has not been determined, however, complicating accurate identification of induced changes. The present study therefore investigated changes in thermal perception threshold values within a 1-h period. Twenty-four healthy women volunteers aged 18-28 years (mean 20.6, SD 2.8) undertook cold sensation (CS), warm sensation (WS), cold pain (CP), and hot pain (HP) perception threshold measurements on the thenar eminence of the dominant hand during six 8-min experimental cycles. The order of stimulus presentation was randomized within pre-selected criteria. An adaptation temperature of 32 degrees C, a rate of temperature change of 0.5 degrees C/s, a 3 cm x 3 cm thermode, and a method of limits algorithm were used. Separate two-way ANOVAs with repeated measures showed statistically significant changes over time for WS, CS, and HP (p < 0.05), but not for CP (p = 0.232). The results indicate that WS, CS, and HP perception thresholds change significantly with repeated testing over a 1-h period. These results should be carefully considered when assessing the importance of observed changes in thermal perception thresholds. In research trials exclusion of a control group would be a fundamental flaw.     

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.