Expression Profile Analysis of Neurodegenerative Disease: Advances in Specificity and Resolution

Microarray technology has become a common tool for developing expression profiles. Initially used in the analysis of cells lines and homogeneous tissues, this platform has been applied to more diverse tissues, such as the brain. Several neural disorders have already been profiled by microarrays using relatively large amounts of tissue. This data has unveiled many genes with differential expression between normal and diseased tissue that could potentially be used as gene markers for these afflictions. Because of the heterogeneity of the CNS, it is likely that small differences between gene expression in these studies would be enhanced by the sampling of a subset of cells based on these newly characterized gene markers. Subtraction of normal, unaffected cells from the sample may also result in a more accurate profile of a diseased cell. Expression profile studies from several neuropathological states are presented, with emphasis placed on those studies using small samples of cellular material and those using specialized methods of cell isolation and RNA amplification.     

Trophic interactions and population growth rates: describing patterns and identifying mechanisms

While the concept of population growth rate has been of central importance in the development of the theory of population dynamics, few empirical studies consider the intrinsic growth rate in detail, let alone how it may vary within and between populations of the same species. In an attempt to link theory with data we take two approaches. First, we address the question ''what growth rate patterns does theory predict we should see in time-series?'' The models make a number of predictions, which in general are supported by a comparative study between time-series of harvesting data from 352 red grouse populations. Variations in growth rate between grouse populations were associated with factors that reflected the quality and availability of the main food plant of the grouse. However, while these results support predictions from theory, they provide no clear insight into the mechanisms influencing reductions in population growth rate and regulation. In the second part of the paper, we consider the results of experiments, first at the individual level and then at the population level, to identify the important mechanisms influencing changes in individual productivity and population growth rate. The parasitic nematode Trichostrongylus tenuis is found to have an important influence on productivity, and when incorporated into models with their patterns of distribution between individuals has a destabilizing effect and generates negative growth rates. The hypothesis that negative growth rates at the population level were caused by parasites was demonstrated by a replicated population level experiment. With a sound and tested model framework we then explore the interaction with other natural enemies and show that in general they tend to stabilize variations in growth rate. Interestingly, the models show selective predators that remove heavily infected individuals can release the grouse from parasite-induced regulation and allow equilibrium populations to rise. By contrast, a tick-borne virus that killed chicks simply leads to a reduction in the equilibrium. When humans take grouse they do not appear to stabilize populations and this may be because many of the infective stages are available for infection before harvesting commences. In our opinion, an understanding of growth rates and population dynamics is best achieved through a mechanistic approach that includes a sound experimental approach with the development of models. Models can be tested further to explore how the community of predators and others interact with their prey.     

Pattern does not equal process: what does patch occupancy really tell us about metapopulation dynamics?

Patch occupancy surveys are commonly used to parameterize metapopulation models. If isolation predicts patch occupancy, this is generally attributed to a balance between distance-dependent recolonization and spatially independent extinctions. We investigated whether similar patterns could also be generated by a process of spatially correlated extinctions following a unique colonization event (analogous to nonequilibrium processes in island biogeography). We simulated effects of spatially correlated extinctions on patterns of patch occupancy among pikas (Ochotona princeps) at Bodie, California, using randomly located extinction disks to represent the likely effects of predation. Our simulations produced similar patterns to those cited as evidence of balanced metapopulation dynamics. Simulations using a variety of disk sizes and patch configurations confirmed that our results are potentially applicable to a broad range of species and sites. Analyses of the observed patterns of patch occupancy at Bodie revealed little evidence of rescue effects and strong evidence that most recolonizations are ephemeral in nature. Persistence will be overestimated if static or declining patterns of patch occupancy are mistakenly attributed to dynamically stable metapopulation processes. Consequently, simple patch occupancy surveys should not be considered as substitutes for detailed experimental tests of hypothesized population processes, particularly when conservation concerns are involved.     

The influence of n-alkanols and cholesterol on the duration and conductance of gramicidin single channels in monoolein bilayers

The mean lifetime of gramicidin A channels in bilayers formed from monoolein and squalane was sharply reduced by the absorption of a range of n-alkanols and cholesterol. Results are shown for n-hexanol, n-octanol, n-decanol, n-dodecanol, n-tetradecanol, n-hexadecanol, n-octadecanol and cholesterol. The longer chain n-alkanols were apparently more effective than the shorter members and cholesterol was the most effective of the substances examined. The single channel conductance was also affected, though to a much lesser extent than the mean channel lifetime, the n-alkanols producing increases and cholesterol a decrease. It is suggested that membrane fluidity changes are not likely to be primarily responsible for the reductions in channel lifetimes but that the bilayer tension, which is known to be increased by n-octanol, could be significant.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.     

The blockage of the electrical conductance in a pore-containing membrane by the n-alkanes.

1. In monooelein bilayers made highly conducting by the addition of a fixed amount of o-pyromellitylgramicidin, the membrane conductance has been shown to be strongly dependent on the chain length of the n-alkane with which the membrane is in equilibrium. Thus for n-hexadecane, the conductance is larger by approx. 10(4) times than it is for n-octane. This result is independent of whether the polypeptide is introduced via the aqueous or lipid phases. 2. The observed conductance variations have been accounted for in terms of a mechanism (outlined in earlier publications) which is based on the thickness and tension changes produced in bilayers by the adsorption of n-alkanes. Essentially quantitative agreement between theory and experiment is found.     

Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.