Maria Teresa Fernanda Locci

DjPum, a homologue of Drosophila Pumilio, is essential to planarian stem cell maintenance

As stem cells are rare and difficult to study in vivo in adults, the use of classical models of regeneration to address fundamental aspects of the stem cell biology is emerging. Planarian regeneration, which is based upon totipotent stem cells present in the adult – the so-called neoblasts– provides a unique opportunity to study in vivo the molecular program that defines a stem cell. The choice of a stem cell to self-renew or differentiate involves regulatory molecules that also operate as translational repressors, such as members of PUF proteins. In this study, we identified a homologue of the Drosophila PUF gene Pumilio (DjPum) in the planarian Dugesia japonica, with an expression pattern preferentially restricted to neoblasts. Through RNA interference (RNAi), we demonstrate that gene silencing of DjPum dramatically reduces the number of neoblasts, thus supporting the intriguing hypothesis that stem cell maintenance may be an ancestral function of PUF proteins.

Peroxisome proliferator-activated receptor (PPAR)γ is highly expressed in normal human pituitary gland

Objective: Expression of peroxisome proliferator-activated receptor (PPAR)γ in normal pituitary seems to be restricted to ACTH-secreting cells. The aim of the study was to evaluate the expression of PPARγ in normal human pituitary tissue and to study its localization in the pituitary secreting cells. Materials and methods: Normal pituitary tissue samples were obtained form 11 patients with non-secreting adenoma who underwent surgical excision of the tumor. Expression of PPAR? was evaluated by immunostaining and western blotting; localization of PPAR? in each pituitary secreting cell lineage was evaluated by double immunofluorescence using confocal microscopy. Pituitary non-functioning adenomas served as Controls. Results: PPARγ was highly expressed in all pituitary samples with a (mean±SD) 81±6.5% of stained cells; expression of PPARγ was confirmed by western blotting. Non-functioning pituitary adenomas had 74±11% PPAR? positive cells. Expression of PPARγ was either in cytoplasm or nuclei. In addition, treatment of GH3 cells, with a PPARγ ligand was associated with traslocation of the receptor from cytoplasm into the nucleus. Double immunostaining revealed that every pituitary secreting cell (GH, TSH, LH, FSH, PRL and ACTH) had PPARγ expressed. Discussion: The present study demonstrated that PPARγ is highly expressed in every normal pituitary secreting cell lineage. It can translocate into the nucleus by ligand binding; however, its role in pituitary hormone regulation remains to be elucidated.

Apoptosis is reduced in the colonic mucosa of patients with acromegaly.

Background  Patients with acromegaly have an increased risk of developing colonic tumours; reduced apoptosis is considered a leading mechanism in tumorigenesis. GH and IGF‐1 decrease apoptosis in several cell lines including human colonic adenocarcinoma, but it is unknown whether epithelial cells of colonic mucosa of patients with acromegaly have reduced apoptosis.

Autophagy-related protein LC3 and Beclin-1 in the first trimester of pregnancy

Autophagy is a degradation process that acts in response to environmental stressors. Recently, autophagy has been detected in normal term, preeclamptic and intrauterine growth-restricted placentas. The object of this work was to investigate the presence of autophagy in first trimester voluntary interruption of pregnancy placental villi by the expression of autophagy-related proteins, light chain 3 (LC3), and Beclin-1. In first trimester placental villi laser scanning confocal microscopy (LSCM) analysis revealed LC3 and Beclin-1 immunoreactivity prevalently located in villous cytotrophoblasts. Using LSCM, LC3, and Beclin-1 were localized to the cytoplasm of the trophoblast layer in human full-term placentas. Beclin-1 expression and LC3 activation were confirmed by western blotting. These data emphasize that autophagy activation is different among cytotrophoblasts and syncytiotrophoblasts depending on the gestational age and thus we speculate that autophagy might play a prosurvival role throughout human pregnancy.

Establishment and characterization of 4 new human pancreatic cancer cell lines: evidences of different tumor phenotypes.

Objectives: Pancreatic cancer still remains a challenge for its biological complexity and lack of effective therapeutic strategies. Establishing new pancreatic cancer cell lines is therefore of paramount importance to clarify its biology. Methods: We established and characterized 4 new pancreatic cancer cell lines (PP78, PP109, PP117, and PP161) according to their genetic (K-Ras, TP53, CDKN2A, and MADH4; DNA fingerprinting; karyotype), cytostructural (cytokeratins 7, 8, 18, and 19 vimentin, and ezrin), and functional profiles (doubling time; migration assay). Results: K-Ras, TP53, and CDKN2A gene alterations were detected in all 4 of them. Each cell line had a unique DNA profile revealed by DNA fingerprinting. A complex karyotype with numerous structural and numeric chromosomal abnormalities was present in each cell line. All 4 cell lines showed positivity for cytokeratins 7, 8, and 18. All but PP78 expressed cytokeratin 19, whereas vimentin was expressed only in PP117 and PP78 cells. A different ezrin cellular distribution was noticed in PP78 and PP117, being mostly located at membrane ruffles. This peculiar distribution was associated with the strongest migratory capability. Conclusions: Our results seem to confirm the pancreatic ductal adenocarcinoma heterogeneity; in fact, the same genetic abnormalities (K-Ras, TP53, and CDKN2A) may have different effects on tumor biology depending on cellular differentiation.

Yolk utilization in stick insects entails the release of vitellin polypeptides into the perivitelline fluid

This study investigates the developmental fate of vitellin (Vt) polypeptides generated by limited proteolysis in an insect embryo. To this end, a number of polyclonal (pAb) and monoclonal antibodies (mAb) were raised against the yolk sac and the perivitelline fluid of late embryos of the stick insect Carausius morosus. Two dimensional immuno gel electrophoresis and Western blotting demonstrate that polypeptides resulting from Vt processing are present both in the yolk sac and the perivitelline fluid. At the confocal microscope, different labelling patterns were detected in the ooplasm depending on the stage of development attained by the embryo. At early developmental stages, label is associated with large unsegmented portions of the fluid ooplasm. During embryonic development, the fluid ooplasm is gradually transformed into yolk granules by intervention of vitellophages. Prior to dorsal closure, the yolk sac is separated from the perivitelline fluid by interposition of serosa cells (the so called serosa membrane). Several mAbs raised against the perivitelline fluid react specifically with this membrane suggesting that the release of Vt polypeptides from the yolk sac occurs by intracellular transit through the serosa cells. By immunocytochemistry, gold label appears associated with the cell surface and a number of vacuoles of the serosa membrane. These data are interpreted as suggesting that Vt polypeptides resulting from limited proteolysis in stick insect embryos are not exhaustively degraded within the yolk sac, but are instead transferred transcytotically to the perivitelline fluid through the serosa membrane.

Serosa membrane plays a key role in transferring vitellin polypeptides to the perivitelline fluid in insect embryos

In mid‐embryogenesis, the stick insect Carausius morosus comes to be comprised of three distinct districts: the embryo proper, the yolk sac and the perivitelline fluid. A monolayered epithelium, the so‐called serosa membrane, encloses the yolk sac and its content of vitellophages and large yolk granules. During embryonic development, the yolk sac declines gradually in protein concentration due to Vt polypeptides undergoing limited proteolysis to yield a number of Vt cleavage products of lower molecular weights. mAbs 1D1 and 5H11 are monoclonal antibodies raised against some of the Vt cleavage products generated by this process in the yolk sac. At the confocal microscope, antibody fluorescence is initially associated with a few yolk granules, while it is gradually displaced in the cytosolic spaces of the vitellophages. With the proceeding of embryonic development, label appears also in the serosa membrane in the form of clustered dots. At the ultrastructural level, gold particles are initially associated with the vitellophages that are labeled on a few yolk granules and in the cytosolic space flanking the yolk granules. Subsequently, the serosa cells become labeled on vesicles close to the yolk granules or just underneath the plasma membrane. Inside the serosa cells, label is also associated with granules budding from the Golgi apparatus, but never with the intercellular channels percolating the serosa membrane. These observations are interpreted as indicating that Vt cleavage products leak out from the yolk granules into the cytosolic spaces of the vitellophages and are eventually transferred to the perivitelline fluid via transcytosis through the serosa cells.

Vitellin cleavage products are proteolytically degraded by ubiquitination in stick insect embryos

Vitellin polypeptides are proteolytically processed in ovarian follicles and embryos of the stick insect Carausius morosus. Data show that vitellin polypeptide A(3) of 54kDa is processed to yield polypeptide A(3)(*) of about 48kDa upon completion of ovarian development, whereas vitellin polypeptide A(2) of 90kDa yields polypeptide E(9) during embryonic development. As vitellin polypeptides are processed, polypeptides A(3)(*) and E(9) are transferred from the yolk granules to the cytosolic space of the vitellophages and start to express a ubiquitin reactivity. At the confocal microscope, anti-ubiquitin antibodies label specifically numerous small yolk granules and the cytosolic space of vitellophages. During embryonic development, ubiquitin carrying granules undergo acidification in much the same way as larger yolk granules. However, only these latter organelles are capable of converting a latent cysteine pro-protease into an active yolk protease upon acidification of their luminal space. These data are interpreted as indicating that ubiquitin-like polypeptides are restricted to small granules throughout ovarian and embryonic development, and that vitellin cleavage products are ubiquitinated following acidification of large yolk granules and transfer to the cytosolic space of the vitellophages.