Mutations at the Darkener of apricot (Doa) locus of Drosophila cause roughened eyes and increase transcript accumulation from the retrotransposon copia up to fourfold. Cloning of the gene and sequencing of cDNAs reveals that it encodes a putative serine/threonine protein kinase. Sequence data base searches identify it is a member of a novel highly conserved protein kinase family, with homologs in humans, mice, and Saccharomyces cerevisiae, not related to each other previously. Family members are characterized by a peptide motif reading EHLAMMERILG at kinase subdomain X, which is virtually 100% identical in all homologs. We therefore refer to this new family as the LAMMER protein kinases. As predicted from its primary sequence, Doa protein possesses intrinsic protein kinase activity when expressed in bacteria, as assayed via autophosphorylation. The gene is expressed throughout development, and both stage and tissue-specific RNAs are found. Its function is essential, because maternally deposited or zygotically transcribed mRNA is required for development to larval stages, and defects in segmentation and development of the nervous system are observed in embryos derived from heteroaUelic mothers. Doa function is also critical to Drosophila eye development, because the organization and development of pigment cells, bristles, and photoreceptors are affected in various mutant classes. In the most extreme cases that survive to adulthood, retinal photoreceptors degenerate prior to eclosion. These results demonstrate that the kinase encoded by Doa is required at multiple stages of development, for both differentiation and maintenance of specific cell types.

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Histolysis refers to a widespread disintegration of tissues that is morphologically distinct from apoptosis and often associated with the stimulation of autophagy. Here, we establish that a component of the apoptosome, and pivotal regulator of apoptosis, is also required for histolytic cell death. Using in vivo and ex vivo assays, we demonstrate a global apoptogenic requirement for dark, the fly ortholog of Apaf1, and show that a required focus of dark -organismal lethality maps to the central nervous system. We further demonstrate that the Dark protein itself is a caspase substrate and find that alterations of this cleavage site produced the first hypermorphic point mutation within the Apaf1/Ced-4 gene family. In a model of 'autophagic cell death', dark was essential for histolysis but dispensable for characteristic features of the autophagic program, indicating that the induction of autophagy occurs upstream or parallel to histolytic cell death. These results demonstrate that stimulation of autophagy per se is not a 'killing event' and, at the same time, establish that common effector pathways, regulated by the apoptosome, can underlie morphologically distinct forms of programmed cell death.KEY WORDS: Autophagy, Apoptosis, Drosophila, Histolysis, dark Development 133, 1457Development 133, -1465Development 133, (2006 DEVELOPMENT 1458 per se is not the mechanism of cell killing but lies upstream, or parallel to dark. These data establish that common effector pathways, regulated by the apoptosome, specify apoptotic and histolytic forms of PCD. MATERIALS AND METHODS MutagenesisTo isolate deletions that eliminate dark without compromising the function of adjacent neighboring genes, a P insertion associated with dark CD4 (Rodriguez et al., 1999) was remobilized and candidates were tested in trans to existing alleles and against lethal mutations in flanking genes. Promising 'hits' were screened by PCR. dark 82 failed to complement dark CD4 , but complements adjacent lethal alleles in the neighboring genes, RhoGEF and a new lethal P mutation in CG8963 that we fortuitously obtained in our first round of mutagenesis. Genomic PCR across the deletion junction and RT-PCR were used to validate the mutation and define the dark 82 lesion. yw was the parental wild-type strain for molecular analysis and for ex vivo hemocyte studies. RNA extraction and QRT-PCR were conducted as described by Gorski et al. (Gorski et al., 2003). Ages at 25°C were normalized from 18°C (Park et al., 1996). Genomic PCR and RT-PCR were performed as described by Chew et al. (Chew et al., 2004), with relevant gene-specific primers. Transgenic 'rescue' and genetic manipulationFull-length dark with 8ϫHis-tags at the N terminus and 3ϫMyc-tags at the C terminus was cloned into the BamHI/XhoI sites of the pFastBac1 vector (Invitrogen). The BamHI/XhoI insert was then subcloned into the pUAST vector to produce pUAST-dark WT . pUAST-dark V was generated by changing Aspartate 1292 to Alanine using a QuikChange Site-Directed Mutagenesis Kit (Stratagene). The ...

During Drosophila metamorphosis, larval tissues, such as the salivary glands, are histolysed whereas imaginal tissues differentiate into adult structures forming at eclosion a flyshaped adult.

The aim of our analysis was to compare the cost-effectiveness of high-dose intensity-modulated radiation therapy (IMRT) and hypofractionated intensity-modulated radiation therapy (HF-IMRT) versus conventional dose three-dimensional radiation therapy (3DCRT) for the treatment of localised prostate cancer. A Markov model was constructed to calculate the incremental quality-adjusted life years and costs. Transition probabilities, adverse events and utilities were derived from relevant systematic reviews. Microcosting in a large university hospital was applied to calculate cost vectors. The expected mean lifetime cost of patients undergoing 3DCRT, IMRT and HF-IMRT were 7,160 euros, 6,831 euros and 6,019 euros respectively. The expected quality-adjusted life years (QALYs) were 5.753 for 3DCRT, 5.956 for IMRT and 5.957 for HF-IMRT. Compared to 3DCRT, both IMRT and HF-IMRT resulted in more health gains at a lower cost. It can be concluded that high-dose IMRT is not only cost-effective compared to the conventional dose 3DCRT but, when used with a hypofractionation scheme, it has great cost-saving potential for the public payer and may improve access to radiation therapy for patients.

In contrast to the well defined mechanism of merocrine exocytosis, the mechanism of apocrine secretion, which was first described over 180 years ago, remains relatively uncharacterized. We identified apocrine secretory activity in the late prepupal salivary glands of Drosophila melanogaster just prior to the execution of programmed cell death (PCD). The excellent genetic tools available in Drosophila provide an opportunity to dissect for the first time the molecular and mechanistic aspects of this process. A prerequisite for such an analysis is to have pivotal immunohistochemical, ultrastructural, biochemical and proteomic data that fully characterize the process. Here we present data showing that the Drosophila salivary glands release all kinds of cellular proteins by an apocrine mechanism including cytoskeletal, cytosolic, mitochondrial, nuclear and nucleolar components. Surprisingly, the apocrine release of these proteins displays a temporal pattern with the sequential release of some proteins (e.g. transcription factor BR-C, tumor suppressor p127, cytoskeletal β-tubulin, non-muscle myosin) earlier than others (e.g. filamentous actin, nuclear lamin, mitochondrial pyruvate dehydrogenase). Although the apocrine release of proteins takes place just prior to the execution of an apoptotic program, the nuclear DNA is never released. Western blotting indicates that the secreted proteins remain undegraded in the lumen. Following apocrine secretion, the salivary gland cells remain quite vital, as they retain highly active transcriptional and protein synthetic activity.

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