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  • Author or Editor: M. Sass x
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The process of autophagy, or bulk degradation of cellular proteins and organelles through an autophagosomic-lysosomal pathway constantly functions in all eukary- otic cells. Also a type of physiological cell death exists, which is best characterized with the strenghtening of the autophagic process, but no DNA degradation or caspase activation can be detected, in contrast to apoptosis [2]. Autophagy can be promoted in various ways: addiction of certain drugs (like vin- blastine [6]), hormones (like 20-hidroxy-ecdysone [3]) or simply nutrition d&epriva- tion [5] leads to the increased amount of proteins degraded by lysosomal enzymes. The isolation and cloning of yeast autophagy mutants gives an excellent opportu- nity to examine their putative homologs in Drosophila melanogaster. Fourteen genes have been identified in Saccharomyces cerevisiae required for autophagy [5], based on several mutant phenotypes, &like the sorting problems of vacuolar enzymes such as carboxypeptidase Y or aminopeptidase I, or the less of viability and the inability of degrading cytosolic proteins like fatty acid synthase during starvation. Nine of them (apg5, apg6, apg7, apg12, aut1, aut2, aut7, aut9, vps4) appear to have clear homologs in the fly and human genome, using the BLAST tools at http://work- bench.sdsc.edu, http://www.ncbi.nlm.nih.gov and http://www.fruitfly.org (BLASTN, TBLASTN services) for sequence similarity searches. The sequence alignment of the yeast, fly and human proteins can be seen in Figure 1. The high degree of similarity suggests existing homology among these genes, although new and lost functions were identified in some cases [7]. Remarkably, vps4 exists in two slightly different copies in human, and aut7 exists in multiple different copies as well (two in Drosophila and three in Homo), suggesting different roles, or at least different regulation. As expected, fly and human genes are much more simi- lar to each other than to the yeast homolog, promising that Drosophila experiments will better contribute to the understanding of the roles of these genes in detail in high- er eukaryotes. The precise function of these genes is still unclear, however, molecu-

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The intersegmental muscles (ISMs) of tobacco hornworm, Manduca sexta are a well-characterised model system for examining the biochemical changes that accompany programmed cell death during develop- ment. When the ISMs become committed to die, there are dramatic increases in both the ubiquitin- expression, and ubiquitin-dependent proteolysis. Since the 26S proteasome is responsible for ATP/ubiq- uitin-dependent proteolysis in cells, we examined its enzymatic properties. Specific chymotrypsin-like proteolytic activity of 26S proteasomes isolated from ISM is four times higher than that of surviving flight muscle (FM). However, specific activity does not change between developmental stages within ISM or FM. The difference between proteolytic capacity of the two kinds of muscles is even higher when the ISM become committed to die because 26S proteasome content of ISM increases just before cell death. These observations underline the role of 26S proteasome in programmed cell death.

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Two novel proteins with apparent molecular weight of 38 (Manduca sexta midgut MsM38) and 46kDa (MsM46) were isolated from midgut homogenates in wandering stage Manduca sexta larvae and both of them were found to be present exclusively in this tissue on Western blots. Immunocytochemical studies revealed that both proteins are expressed in the regenerative cells however, their distribution pattern is clearly different. MsM38 is localized in the cytoplasm of resting regenerative cells during the feeding period, and is accumulated in the calcospherits at the beginning of the wandering period. Along with the delamination of the larval epithelium, this protein is released apically from these vesicles. The antiserum labels an additional 76 kDa protein in the wandering larval midgut homogenates. The appearance of this 76 kDa protein coincides with the accumulation of the immunopositive material in the calcospherits. MsM46 is similarly distributed during the feeding period in the cytoplasm of regenerative cells. At the beginning of the wandering period it accumulates around the newly forming large apical vacuoles, that are released at the time of complete delamination of the larval epithelium. In parallel with this process MsM46, and another 40 kDa protein, that becomes labeled from this period on Western blots appeares on the apical microvillar projections. Thus both isolated proteins are directed apically from different compartments, that raises the possibility of a dual apical routing pathway in regenerative cells.

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The pattern of cuticle protein synthesis by the epidermis of insects changes during the last larval, pupal and adult development, leading to an alteration in cuticular stucture and feature. We have isolated a pro- tein that had an apparent molecular mass of 33.1 kD from larval cuticle of Manduca sexta. Synthesis, transport and accumulation of MsCP33.1 were followed during metamorphosis by immunoblots and immunocytochemical methods using the antibody developed against this protein. Our data prove that the presence of MsCP33.1 in the larval cuticle is general while its appearance in the pupal or adult integu- ment is restricted only in the cuticle of wings and apodemes. We established that the synthesis of 33.1 kD protein is negatively regulated by moulting hormone (20-hydroxyecdysone). Possible roles for this cuticular protein are discussed.

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Scolexin is one of the bacterial induced hemolymph proteins of tobacco hornworm (Manduca sexta) lar- vae, that has hemocyte coagulation-provoking activity. The 72 kDa scolexin complex is composed of two 36 kDa subunits. To examine the protein secretory pathways in insect epithelia a polyclonal antibody was raised against the 36 kDa hemolymph protein. This MsH36 antibody recognised a 36 and a 72 kDa pro- tein in tissue homogenates. On the basis of the characteristic labelling pattern observed on immunoblots and immunocytochemical sections we concluded that the 36 kDa protein in the hemolymph, in the midgut and in the epidermis was identical with the scolexin subunit. In present paper we report a labelling shift in the midgut epithelium between goblet and columnar cells that may be controlled by the hormonal system. A 72 kDa protein showed similar epitops and molecular weight to the scolexin com- plex and was detected in epidermis and in cuticle under both reducing and non-reducing conditions. Tissue localization of 36 kDa and 72 kDa MsH36 antibody labelling proteins indicated the possibility that the epidermal cells produce two kinds of scolexin-like proteins. The complex composed of 36 kDa subunits are transported basolaterally into the circulation and display hemocyte coagulation inducing activity while the 72 kDa form contains two subunits linked covalently secreted apically into the cuticle.

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