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Aims/hypothesis The basic helix–loop–helix transcription factor neurogenin-3 (NGN3) commits the fates of pancreatic progenitors to endocrine cell types, but knowledge of the mechanisms regulating the choice between proliferation and differentiation of these progenitors is limited. Methods Using a chromatin immunoprecipitation cloning approach, we searched for direct targets of NGN3 and identified a zinc-finger transcription factor, OVO homologue-like 1 (OVOL1). Transactivation experiments were carried out to elucidate the functional role of NGN3 in Ovol1 gene expression. Embryonic and adult rodents pancreases were immunostained for OVOL1, Ki67 and NGN3. Results We showed that NGN3 negatively regulates transcription of Ovol1 in an E-box-dependent fashion. The presence of either NGN3 or NEUROD1, but not MYOD, reduced endogenous Ovol1 mRNA. OVOL1 was detected in pancreatic tissue around embryonic day 15.5, after which OVOL1 levels dramatically increased. In embryonic pancreas, OVOL1 protein levels were low in NGN3+ or Ki67+ cells, but high in quiescent differentiated cells. OVOL1 presence was maintained in adult pancreas, where it was detected in islets, pancreatic ducts and some acinar cells. Additionally OVOL1 presence was lacking in proliferating ductules in regenerating pancreas and induced in cells as they began to acquire their differentiated phenotype. Conclusions/interpretation The timing of OVOL1 appearance in pancreas and its increased levels in differentiated cells suggest that OVOL1 promotes the transition of cells from a proliferating, less-differentiated state to a quiescent more-differentiated state. We conclude that OVOL1, a downstream target of NGN3, may play an important role in regulating the balance between proliferation and differentiation of pancreatic cells.

PHGPx (phospholipid hydroperoxide glutathione peroxidase) is a selenoprotein present in at least three isoforms in testis: cytosolic, mitochondrial and nuclear. All of these derive from the same gene and are structurally related with the exception of the snPHGPx (sperm nucleus-specific form), which differs from the others due to the presence of an arginine-rich N-terminus. It has been demonstrated recently that this N-terminus is encoded by an alternative exon located in the first intron of the PHGPx gene. The expression of snPHGPx has been attributed either to an alternative pre-mRNA splicing or to the presence of a distinct promoter region. Nevertheless, the exact molecular mechanism by which the expression of snPHGPx occurs has not been demonstrated so far. Preliminary sequence analysis of the region located upstream of the alternative exon revealed some potential DNA-binding sites, one of which is specific to the binding of CREM (cAMP-response elementmodulator) transcription factors. By using electrophoretic mobility-shift assays, we demonstrated that both nuclear protein extract from highly purified rat spermatid cells and recombinant CREM-τ protein can specifically bind to this element. Furthermore, we cloned a 1059 bp comprising the intron and the alternative exon for snPHGPx in the pCAT®3 reporter vector. By transient transfection experiments, we demonstrated that the expression of the transcription factor CREM-τ can induce the activation of the reporter gene in NIH-3T3 cell line. These results were confirmed by chromatin immunoprecipitation experiments performed on highly purified rat spermatid cells.On the basis of these results, we demonstrate that snPHGPx expression is mediated by the transcription factor CREM-τ , which acts as a cis-acting element localized in the first intron of the PHGPx gene.

Helicobacter pylori is an important pathogen of the gastric system. The clinical outcome of infection is thought to be correlated with some genetic features of the bacterium. However, due to the extreme genetic variability of this organism, it is hard to draw definitive conclusions concerning its virulence factors. Here we describe a novel H. pylori gene which expresses an autolytic enzyme that is also capable of degrading the cell walls of both gram-positive and gram-negative bacteria. We designated this gene lys. We found this gene and observed its expression in a number of unrelated clinical strains, a fact that suggests that it is well conserved in the species. A comparison of the nucleotide sequences of lys and the hypothetical gene HP0339 from H. pylori strain ATCC 26695 revealed almost total identity, except for the presence of an insertion consisting of 24 nucleotides in the lys sequence. The coding sequences of lys and HP0339 show a high degree of homology with the coding sequence of bacteriophage T4 lysozyme. Because of this similarity, it was possible to model the three-dimensional structures of both the lys and HP0339 products.

Pancreatic beta-cell regeneration, for example, by inducing proliferation, remains an important goal in developing effective treatments for diabetes. However, beta cells have mainly been considered quiescent. This “static” view has recently been challenged by observations of relevant physiological conditions in which metabolic stress is compensated by an increase in beta-cell mass. Understanding the molecular mechanisms underlining these process could open the possibility of developing novel small molecules to increase beta-cell mass. Several cellular cell-cycle and signaling proteins provide attractive targets for high throughput screening, and recent advances in cell culture have enabled phenotypic screening for small molecule-induced beta-cell proliferation. We present here an overview of the current trends involving small-molecule approaches to induce beta-cell regeneration by proliferation.

1996/1997 IX Ciclo 1964 Versione digitalizzata della tesi di dottorato cartacea.