A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.     

A novel 96-well scintillation proximity assay for the measurement of apoptosis

The translocation of phospholipids across the plasma membrane has been widely documented as one of the earliest measurable biochemical events of apoptosis. Using fluorescently labelled annexin V, which preferentially binds phosphatidylserine (PS) in the presence of Ca²⁺, the externalization of PS can be measured and apoptosis quantified using flow cytometry. Conventional detection methods utilizing annexin V, while faster than in situ DNA end-labelling or DNA laddering, require extensive sample preparation which may compromise samples and makes rapid, high volume screening prohibitive. This paper describes a novel assay for the measurement of apoptosis based upon binding of radiolabelled annexin V to apoptotic cells attached to the growth surface of a 96-well scintillating microplate (Cytostar-T®). We compared measurements of apoptosis made by flow cytometry to those obtained with the scintillating microplate in three model systems, treatment of: mouse connective tissue (L-M) cells with lymphotoxin (LT), human lung carcinoma (H460) cells with Apo-2 ligand and human umbilical vein endothelial (HUVE) cells with staurosporine. In this assay, we compare both direct and indirect labelling methods by utilizing either iodinated annexin V or biotinylated annexin V/[³⁵S] streptavidin to radiolabel apoptotic cells. The signal detected is a direct consequence of the binding of annexin V to externalized PS on apoptotic cells and the proximity of the label to the base of the plate. Using this method, separation of bound and unbound radiolabel signal occurs directly within the well resulting in a sensitive assay that requires minimal manipulation and can accomodate a large number of samples.     

The structure of pyruvate kinase from Leishmania mexicana reveals details of the allosteric transition and unusual effector specificity.

Glycolysis occupies a central role in cellular metabolism, and is of particular importance for the catabolic production of ATP in protozoan parasites such as Leishmania and Trypanosoma. In these organisms pyruvate kinase plays a key regulatory role, and is unique in responding to fructose 2,6-bisphosphate as allosteric activator. The determination of the first eukaryotic pyruvate kinase crystal structure in the T-state is reported. A comparison of the leishmania and yeast R-state enzymes reveals fewer differences than the previous comparison of Escherichia coli T-state and rabbit muscle non-allosteric enzymes. Structural changes related to the allosteric transition can therefore be distinguished from those that are a consequence of the inherent wide structural divergence between bacterial and mammalian proteins. The allosteric transition involves significant changes in a tightly packed array of eight alpha helices at the interface near the catalytic site. At the other interface the allosteric transition appears to be accompanied by the bending of a ten-stranded intersubunit beta sheet adjacent to the effector site. Helix Calpha1 makes contacts to the N-terminal helical domain and bridges both interfaces. A comparison of the effector sites of the leishmania and yeast enzymes reveals the structural basis for the different effector specificity. Two loops comprising residues 443-453 and 480-489 adopt very different conformations in the two enzymes, and Lys453 and His480 that are a feature of trypanosomatid enzymes provide probable ligands for the 2-phospho group of the effector molecule. These differences offer an opportunity for the design of drugs that would bind to the trypanosomatid enzymes but not to those of the mammalian host.     

Molecular characterization of NOS in a mollusc: Expression in a giant modulatory neuron

Here we report on the molecular characterization of the first molluscan NOS in the CNS of the pond snail Lymnaea stagnalis. This Lymnaea NOS (Lym-nNOS) which is expressed preferentially in the CNS is most similar to mammalian neuronal NOS but contains tandem repeats of a seven amino acid motif not found in any other known NOS. We have localized Lym-nNOS to the serotonergic cerebral giant cells (CGCs) which modulate synaptic transmission within a neural network that generates feeding behavior. Our results suggest that the CGCs employ both NO and serotonin in the modulation of the central neural network underlying feeding. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 65–76, 1998     

Trace Metal Bioaccumulation in Epibenthic Macroinvertebrates (EMI) from the San Pedro Shelf,California

Trace metal/metalloid (As, Ag, Cd, Cr, Cu, Hg, Ni, Pb, Se, Zn) bioaccumulation was measured over a period of ten years (1985–1995) in five species of epibenthic macroinvertebrates (EMI) from the San Pedro Shelf, California. Four of the species are numerical dominants in the Southern California Bight (SCB) and two species are commercially harvested. Among three echinoderms, a predatory sea star yielded highest tissue concentrations compared to a sea cucumber and a sea urchin, and concentrations measured in a mantid shrimp exceeded those from a prawn. Comparison of trace metal bioaccumulation from the study area, including the ocean outfall, and elsewhere indicated that bioaccumulation in local EMI was generally low. Measurements from an outfall station and two reference stations were used to evaluate the spatial and temporal relationship between trace metal bioaccumulation and the wastewater discharge. It was concluded that there was no spatial or temporal relationship between EMI trace metal bioaccumulation and the discharge.     

Bayesian calibration of a stochastic,multiscale agent-based model for predicting in vitro tumor growth

Hybrid multiscale agent-based models (ABMs) are unique in their ability to simulate individual cell interactions and microenvironmental dynamics. Unfortunately, the high computational cost of modeling individual cells, the inherent stochasticity of cell dynamics, and numerous model parameters are fundamental limitations of applying such models to predict tumor dynamics. To overcome these challenges, we have developed a coarse-grained two-scale ABM (cgABM) with a reduced parameter space that allows for an accurate and efficient calibration using a set of time-resolved microscopy measurements of cancer cells grown with different initial conditions. The multiscale model consists of a reaction-diffusion type model capturing the spatio-temporal evolution of glucose and growth factors in the tumor microenvironment (at tissue scale), coupled with a lattice-free ABM to simulate individual cell dynamics (at cellular scale). The experimental data consists of BT474 human breast carcinoma cells initialized with different glucose concentrations and tumor cell confluences. The confluence of live and dead cells was measured every three hours over four days. Given this model, we perform a time-dependent global sensitivity analysis to identify the relative importance of the model parameters. The subsequent cgABM is calibrated within a Bayesian framework to the experimental data to estimate model parameters, which are then used to predict the temporal evolution of the living and dead cell populations. To this end, a moment-based Bayesian inference is proposed to account for the stochasticity of the cgABM while quantifying uncertainties due to limited temporal observational data. The cgABM reduces the computational time of ABM simulations by 93% to 97% while staying within a 3% difference in prediction compared to ABM. Additionally, the cgABM can reliably predict the temporal evolution of breast cancer cells observed by the microscopy data with an average error and standard deviation for live and dead cells being 7.61±2.01 and 5.78±1.13, respectively.     

Opening the black box: an open‐source release of Maxent

This software note announces a new open‐source release of the Maxent software for modeling species distributions from occurrence records and environmental data, and describes a new R package for fitting such models. The new release (ver. 3.4.0) will be hosted online by the American Museum of Natural History, along with future versions. It contains small functional changes, most notably use of a complementary log‐log (cloglog) transform to produce an estimate of occurrence probability. The cloglog transform derives from the recently‐published interpretation of Maxent as an inhomogeneous Poisson process (IPP), giving it a stronger theoretical justification than the logistic transform which it replaces by default. In addition, the new R package, maxnet, fits Maxent models using the glmnet package for regularized generalized linear models. We discuss the implications of the IPP formulation in terms of model inputs and outputs, treating occurrence records as points rather than grid cells and interpreting the exponential Maxent model (raw output) as as an estimate of relative abundance. With these two open‐source developments, we invite others to freely use and contribute to the software.     

Thermoregulatory behaviour explains countergradient variation in the upper thermal limit of a rainforest skink

The vulnerability of a terrestrial ectotherm to high environmental temperatures depends on the animal's thermal physiology and thermoregulatory behaviour. These variables – environment, physiology, and behaviour – interact with each other, complicating assessment of species vulnerability to global warming. We previously uncovered a counterintuitive pattern in rainforest sunskinks Lampropholis coggeri: a negative relationship between their critical thermal maximum (CT_max) and the temperature of their environment. Could this result be explained by a three‐way interaction between environment, physiology, and behaviour? Here we find that sunskink thermal preference is correlated positively with CT_max, but, importantly, skinks from hotter environments prefer lower temperatures than conspecifics from cooler environments. In an acclimation experiment, we find that CT_max is plastic and shifts in alignment with acclimation temperature. We also found heritable variation in this trait in a common garden study, but this variation was small relative to the plastic shifts observed in CT_max. Thus, our previous observation of a negative correlation between field CT_max and temperature is explained, at least in part, by the lizard's thermoregulatory behaviour: lizards from hot environments preferentially choose cool microenvironments, and their physiology acclimates to these cooler experienced temperatures. Our results suggest that behavioural adjustments to the environment can produce countergradient variation in physiological traits. More broadly, our work underscores the importance of interactions between environment, behaviour, and physiology in ectotherms. Understanding these interactions will be crucial in assessing vulnerability to climate change.