SUMMARY Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled, due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.

Although it has been clear for >40 years that mesothelioma can be caused by asbestos, not all patients with this disease have a history of asbestos exposure. Other factors, including non-asbestos fibers and ionizing radiation, are known to cause malignant transformation of mesothelial cells. In addition, it is likely that genetics will play some role in susceptibility. Recently, it has been suggested that SV40 viral oncogenes could contribute to the carcinogenicity of asbestos. To better understand the role of SV40, we used the mesothelin promoter to construct MexTAg mice that express SV40 large T antigen (TAg) in the mesothelial compartment. We generated four MexTAg lines that carry high, intermediate, and low copy numbers of the transgene. All of these mice show a relatively low level of spontaneous tumor development. High-copy, 299h mice rapidly developed mesotheliomas when exposed to asbestos, and these tumors were faster growing and more invasive than those developing in wild-type and single-copy (266s) mice. In addition, we found a direct relationship between transgene copy number and survival after exposure to asbestos. A single copy of TAg was sufficient to immortalize mesothelial cells in vitro, but these cells did not show evidence of malignant transformation. In contrast, cell lines developed from mesothelial cells of animals carrying multiple copies of TAg were growth factor independent and could be cloned at limiting dilution in soft agar. These data provide the first in vivo demonstration of co-carcinogenicity between SV40 and asbestos. (Cancer Res 2006; 66(22): 10786-94)

Binary expression systems like the LexA-LexAop system provide a powerful experimental tool kit to study gene and tissue function in developmental biology, neurobiology, and physiology. However, the number of well-defined LexA enhancer trap insertions remains limited. In this study, we present the molecular characterization and initial tissue expression analysis of nearly 100 novel StanEx LexA enhancer traps, derived from the StanEx 1 index line. This includes 76 insertions into novel, distinct gene loci not previously associated with enhancer traps or targeted LexA constructs. Additionally, our studies revealed evidence for selective transposase-dependent replacement of a previously-undetected KP element on chromosome III within the StanEx 1 genetic background during hybrid dysgenesis, suggesting a molecular basis for the over-representation of LexA insertions at the NK7.1 locus in our screen. Production and characterization of novel fly lines were performed by students and teachers in experiment-based genetics classes within a geographically diverse network of public and independent high schools. Thus, unique partnerships between secondary schools and university-based programs have produced and characterized novel genetic and molecular resources in Drosophila for open-source distribution, and provide paradigms for development of science education through experience-based pedagogy.

With the recent discovery of a unique class of dual-specificity phosphatases that dephosphorylate glucans, we report an in vitro assay tailored for the detection of phosphatase activity against phosphorylated glucans. We demonstrate that in contrast to a general phosphatase assay utilizing a synthetic substrate, only phosphatases that possess glucan phosphatase activity liberate phosphate from the phosphorylated glucan amylopectin using the described assay. This assay is simple and cost-effective, providing reproducible results that clearly establish the presence or absence of glucan phosphatase activity. The assay described will be a useful tool in characterizing emerging members of the glucan phosphatase family.