Complete functional rescue of the ABCA1-/- mouse by human BAC transgenesis

Humanized mouse models are useful tools to explore the functional and regulatory differences between human and murine orthologous genes. We have combined a bioinformatics approach and an in vivo approach to assess the functional and regulatory differences between the human and mouse ABCA1 genes. Computational analysis identified significant differences in potential regulatory sites between the human and mouse genes. The effect of these differences was assessed in vivo, using a bacterial artificial chromosome transgenic humanized ABCA1 mouse model that expresses the human gene in the absence of mouse ABCA1. Humanized mice expressed human ABCA1 protein at levels similar to wild-type mice and fully compensated for cholesterol efflux activity and lipid levels seen in ABCA1-deficient mice. Liver X receptor agonist administration resulted in significant increases in HDL values associated with parallel increases in the hepatic ABCA1 protein and mRNA levels in the humanized ABCA1 mice, as seen in the wild-type animals. Our studies indicate that despite differences in potential regulatory regions, the human ABCA1 gene is able to functionally fully compensate for the mouse gene. Our humanized ABCA1 mice can serve as a useful model system for functional analysis of the human ABCA1 gene in vivo and can be used for the generation of potential new therapeutics that target HDL metabolism.     

Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response

In this study, genome-wide expression analyses were used to study the response of Saccharomyces cerevisiae to stress throughout a 15-day wine fermentation. Forty per cent of the yeast genome significantly changed expression levels to mediate long-term adaptation to fermenting grape must. Among the genes that changed expression levels, a group of 223 genes was identified, which was designated as fermentation stress response (FSR) genes that were dramatically induced at various points during fermentation. FSR genes sustain high levels of induction up to the final time point and exhibited changes in expression levels ranging from four- to 80-fold. The FSR is novel; 62% of the genes involved have not been implicated in global stress responses and 28% of the FSR genes have no functional annotation. Genes involved in respiratory metabolism and gluconeogenesis were expressed during fermentation despite the presence of high concentrations of glucose. Ethanol, rather than nutrient depletion, seems to be responsible for entry of yeast cells into the stationary phase.     

Spiking neural network simulation: numerical integration with the Parker-Sochacki method

Mathematical neuronal models are normally expressed using differential equations. The Parker-Sochacki method is a new technique for the numerical integration of differential equations applicable to many neuronal models. Using this method, the solution order can be adapted according to the local conditions at each time step, enabling adaptive error control without changing the integration timestep. The method has been limited to polynomial equations, but we present division and power operations that expand its scope. We apply the Parker-Sochacki method to the Izhikevich ‘simple’ model and a Hodgkin-Huxley type neuron, comparing the results with those obtained using the Runge-Kutta and Bulirsch-Stoer methods. Benchmark simulations demonstrate an improved speed/accuracy trade-off for the method relative to these established techniques.

Chemosensory afference in the tentacle nerve of Lymnaea stagnalis

Although the neural control of behavior has been extensively studied in gastropods, basic gaps remain in our understanding of how sensory stimuli are processed. In particular, there is only patchy evidence regarding the functional roles of sense organs and the extensive peripheral nervous system they contain. Our goal was to use extracellular electrophysiological recordings to confirm the chemosensory role of the tentacles in the great pond snail, Lymnaea stagnalis. Employing a special twin-channel suction electrode to improve signal-to-noise ratio, we applied three food odors (derived from earthworm-based food pellets, algae-based pellets, and fresh lettuce) to a reduced preparation of the tentacle while recording neuronal activity in the tentacle nerve. Responses were assessed by comparing average spike frequencies produced in response to saline flow with and without odors. We report stronger neuronal responses to earthworm-based food odors and weaker responses to algae-based food odors. There were no clear neuronal responses produced when lettuce food odor or control saline was applied to the tentacle. Overall, our results provide strong evidence for the chemosensory role of the tentacles in navigation behavior by L. stagnalis. Although it is unclear whether the differences in neuronal responses to different odors are a technical consequence of our recording system or a genuine feature of the snail sensory system, these results are a useful foundation for further study of peripheral nervous system function in gastropods.     

Cloning and functional analysis of the Sry-related HMG box gene, Sox18

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii) an intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor -2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue.     

Use of Sandwich Cultures for the Study of Feeding in the Hexactinellid Sponge Rhabdocalyptus dawsoni (Lambe, 1892)

Abstract Fragments of sponge tissue were cultured between glass slides and coverslips, permitting direct observation of cytoplasmic movements and tissue organization in vitro. The cut surfaces healed and the cultures lived for periods of several weeks. Cytoplasmic organization appeared similar to that described from study of sectioned material. Uptake of food particles (Escherichia coli, Isochrysis galbana) and latex beads took place primarily in the region of the flagellated chambers. Cytoplasmic streams were seen throughout the preparation and may serve for distribution of nutrients in these syncytial animals. It is proposed that the sandwich cultures are valid models of the intact sponge. Copyright © 1996 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.