Over-expression of sirtuins (NAD+-dependent protein deacetylases) has been reported to increase lifespan in budding yeast, Caenorhabditis elegans and Drosophila melanogaster1-3. Studies of gene effects on ageing are vulnerable to confounding effects of genetic background4. We re-examined the reported effects of sirtuin over-expression on ageing and found that standardisation of genetic background and use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high level sir-2.1 over-expression1 abrogated the longevity increase, but not sir-2.1 over-expression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low copy number sir-2.1 over-expression2 also abrogated longevity. A Drosophila strain with ubiquitous over-expression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported3, but not relative to the appropriate transgenic controls, and nor was a new line with stronger over-expression of dSir2. These findings underscore the importance of controlling for genetic background and the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life extending effect of dietary restriction (DR) on ageing in Drosophila has also been reported to be dSir2 dependent3. We found that DR increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects on lifespan in C. elegans and Drosophila.

In C. elegans, increased lifespan in daf-2 insulin/IGF-1 receptor mutants is accompanied by up-regulation of the MDL-1 Mad basic helix-loop-helix leucine zipper transcription factor. Here we describe the role of mdl-1 in C. elegans germline proliferation and aging. The deletion allele mdl-1(tm311) shortened lifespan, and did so significantly more so in long-lived daf-2 mutants implying that mdl-1(+) contributes to effects of daf-2 on lifespan. mdl-1 mutant hermaphrodites also lay increased numbers of unfertilized oocytes. During aging, unfertilized oocytes in the uterus develop into tumors, whose development was accelerated by mdl-1(tm311). Opposite phenotypes were seen in daf-2 mutants, i.e. mdl-1 and daf-2 mutant germlines are hyperplastic and hypoplastic, respectively. Thus, MDL-1, like its mammalian orthologs, is an inhibitor of cell proliferation and growth that slows progression of an age-related pathology in C. elegans (uterine tumors). In addition, intestine-limited rescue of mdl-1 increased lifespan but not to wild type levels. Thus, mdl-1 likely acts both in the intestine and the germline to influence age-related mortality.

Neospora caninum is an apicomplexan parasite first mentioned in 1984 as a causative agent of neuromuscular disease in dogs. It is closely related to Toxoplasma gondii and Hammondia heydorni, and its subsequent description in 1988 has been, and still is, accompanied by discussions on the true phylogenetical status of the genus Neospora. N. caninum exhibits features that clearly distinguish this parasite from other members of the Apicomplexa, including distinct ultrastructural properties, genetic background, antigenic composition, host cell interactions, and the definition of the dog as a final host. Most importantly, N. caninum has a particular significance as a cause of abortion in cattle. In vitro culture has been indispensable for the isolation of this parasite and for investigations on the ultrastructural, cellular, and molecular characteristics of the different stages of N. caninum. Tissue culture systems include maintenance of N. caninum tachyzoites, which represent the rapidly proliferating stage in a large number of mammalian host cells, culture of parasites in organotypic brain slice cultures as a tool to investigate cerebral infection by N. caninum, and the use of techniques to induce the stage conversion from the tachyzoite stage to the slowly proliferating and tissue cyst-forming bradyzoite stage. This review will focus on the use of these tissue culture models as well as light- and electron-microscopical techniques for studies on N. caninum tachyzoites and bradyzoites, and on the physical interactions between parasites and host cells.

SNAP receptor (SNARE) and Sec1/Munc18 (SM) proteins are required for all intracellular membrane fusion events. SNAREs are widely believed to drive the fusion process, but the function of SM proteins remains unclear. To shed light on this, we screened for dominant-negative mutants of yeast Sec1 by random mutagenesis of a GAL1-regulated SEC1 plasmid. Mutants were identified on the basis of galactose-inducible growth arrest and inhibition of invertase secretion. This effect of dominant-negative sec1 was suppressed by overexpression of the vesicle (v)-SNAREs, Snc1 and Snc2, but not the target (t)-SNAREs, Sec9 and Sso2. The mutations isolated in Sec1 clustered in a hotspot within domain 3a, with F361 mutated in four different mutants. To test if this region was generally involved in SM protein function, the F361-equivalent residue in mammalian Munc18-1 (Y337) was mutated. Overexpression of the Munc18-1 Y337L mutant in bovine chromaffin cells inhibited the release kinetics of individual exocytosis events. The Y337L mutation impaired binding of Munc18-1 to the neuronal SNARE complex, but did not affect its binary interaction with syntaxin1a. Taken together, these data suggest that domain 3a of SM proteins has a functionally important role in membrane fusion. Furthermore, this approach of screening for dominant-negative mutants in yeast may be useful for other conserved proteins, to identify functionally important domains in their mammalian homologs.