Technical problems intrinsic to the purification of preribosome intermediates have limited our understanding of ribosome biosynthesis in humans. Addressing this issue is important given the implication of this biological process in human disease. Here we report a preribosome purification and tagging strategy that overcomes some of the existing technical difficulties. Using these tools, we find that the pre-40S precursors go through two distinct maturation phases inside the nucleolus and follow a regulatory step that precedes late maturation in the cytoplasm. This regulatory step entails the intertwined actions of both PARN (a metazoanspecific ribonuclease) and RRP12 (a phylogenetically conserved 40S biogenesis factor that has acquired additional functional features in higher eukaryotes). Together, these results demonstrate the usefulness of this purification method for the dissection of ribosome biogenesis in human cells. They also identify distinct maturation stages and metazoan-specific regulatory mechanisms involved in the generation of the human 40S ribosomal subunit.

Ex vivo islet cell culture in the presence of stimulating factors prior to transplantation is considered a good strategy in contrast to the short conclusion of islets transplantation. Previously, we demonstrated how T3 can increase b-cell function via specific activation of Akt; therefore we hypothesized that thyroid hormone T3 can be considered a promising candidate for the in vitro expansion of islet cell mass. Rat pancreatic islets have been isolated by the collagenase digestion and cultured in the presence or not of the thyroid hormone T3 10⁻⁷ M. Islets viability has been evaluated by the use of two different dyes, one cell-permeable green fluorescent dye and propidium iodide, and by the analysis of core cell damage upcoming. Moreover, islets function has been evaluated by insulin secretion. The ability of b-cells to counteract apoptosis induced by streptozotocin has been analyzed by TUNEL assay. We demonstrated that treatment of primary cultures of rat pancreatic islets with T3 results in augmented β-cell vitality with an increase of their functional properties. In addition, a sensible reduction of the core cell damage has been observed in the T3 treated islets, suggesting the preservation of the β-cells integrity during the culture period. Nonetheless, the insulin secretion is sensibly augmented after T3 stimulation. The strong increment shown in Akt activation suggests the involvement of this pathway in the observed phenomena. In conclusion we indicate T3 as a good factor to improve ex vivo islets cell culture.

Thyroid hormone action, widely recognized on cell proliferation and metabolism, has recently been related to the phosphoinositide 3 kinase (PI3K), an upstream regulator of the Akt kinase and the involvement of the thyroid hormone receptor beta1 has been hypothesized. The serine-threonine kinase Akt can regulate various substrates that drive cell mass proliferation and survival. Its action has also been characterized in pancreatic beta-cells. We previously demonstrated that Akt activity and its activation in the insulinoma cell line hCM could be considered a specific target of the non-genomic action of T3. In this study we analyzed the molecular pathways involved in the regulation of cell proliferation, survival, size, and protein synthesis by T3 in a stable TRbeta1 interfered insulinoma cell line, derived from the hCM, and evidenced a strong regulation of both physiological and molecular events by T3 mediated by the thyroid hormone receptor beta1. We showed that the thyroid receptor beta1 mediates the T3 regulation of the cdk4.cyc D1.p21(CIP1).p27(KIP1) complex formation and activity. In addition TRbeta1 is essential for the T3 upregulation of the Akt targets beta-catenin, p70S6K, and for the phosphorylation of Bad and mTOR. We demonstrated that the beta1 receptor mediates the T3 upregulation of protein synthesis and cell size, together with the cell proliferation and survival, playing a crucial role in the T3 regulation of the PI3K/Akt pathway.