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The effects of the refractive-index mismatch in confocal laser scanning microscopy were extensively studied. The axial aberration induced in the case of fluorescent microspheres was measured. The data were used to take into account the mismatch-induced aberrations and to consider object-size influence. Then we focused on the effect of refractive-index mismatch on the effective system's point-spread function under different mismatch conditions and on depth of focusing. We experimentally verified that the peak of the point-spread function intensity profile decreases and the point-spread function itself progressively broadens as a function of the combined effect of the refractive-index mismatch and of the penetration depth, leading to a worsening of the system's overall performances. We also performed these same measurements by embedding subresolution beads in an oocyte's cytoplasm, which can be considered a turbid medium. We found evidence consistent with the previously developed theoretical model; in particular we found a strong dependence of the intensity peak on the focusing depth.

Neuroblastoma (NB) is a pediatric cancer characterized by remarkable cell heterogeneity within the tumor nodules. Here, we demonstrate that the synthesis of a pol III-transcribed noncoding (nc) RNA (NDM29) strongly restricts NB development by promoting cell differentiation, a drop of malignancy processes, and a dramatic reduction of the tumor initiating cell (TIC) fraction in the NB cell population. Notably, the overexpression of NDM29 also confers to malignant NB cells an unpredicted susceptibility to the effects of antiblastic drugs used in NB therapy. Altogether, these results suggest the induction of NDM29 expression as possible treatment to increase cancer cells vulnerability to therapeutics and the measure of its synthesis in NB explants as prognostic factor of this cancer type.-Castelnuovo, M., Massone, S., Tasso, R., Fiorino, G., Gatti, M., Robello, M., Gatta, E., Berger, A., Strub, K., Florio, T., Dieci, G., Cancedda, R., Pagano, A. An Alu-like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells. FASEB J. 24, 000 -000 (2010). www.fasebj.org

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The prion diseases are transmissible neurodegenerative pathologies characterized by the accumulation of altered forms of the prion protein (PrP), termed PrP(Sc), in the brain. Previous studies have shown that a synthetic peptide homologous to residues 106-126 of PrP (PrP 106-126) maintains many characteristics of PrP(Sc), i.e., the ability to form amyloid fibrils and to induce apoptosis in neurons. We have investigated the intracellular mechanisms involved in the cellular degeneration induced by PrP 106-126, using the GH3 cells as a model of excitable cells. When assayed in serum-deprived conditions (48 hr), PrP 106-126 (50 microM) induced cell death time-dependently, and this process showed the characteristics of the apoptosis. This effect was specific because a peptide with a scrambled sequence of PrP 106-126 was not effective. Then we performed microfluorimetric analysis of single cells to monitor intracellular calcium concentrations and showed that PrP 106-126 caused a complete blockade of the increase in the cytosolic calcium levels induced by K+ (40 mM) depolarization. Conversely, the scrambled peptide was ineffective. The L-type voltage-sensitive calcium channel blocker nicardipine (1 microM) also induced apoptosis in GH3 cells, suggesting that the blockade of Ca2+ entry through this class of calcium channels may cause GH3 apoptotic cell death. We thus analyzed, by means of electrophysiological studies, whether Prp 106-126 modulate L-type calcium channels activity and demonstrated that the apoptotic effect of PrP 106-126 was due to a dose-dependent inactivation of the L-type calcium channels. These data demonstrate that the prion protein fragment 106-126 induces a GH3 apoptotic cell death inducing a selective inhibition of the activity of the L-type voltage-sensitive calcium channels.

We used GH4C1 cells as a model to study the effects of the chemokine stromal cell-derived factor 1 (SDF1) in pituitary functions. In these cells, SDF1␣ induced proliferation and growth hormone secretion, suggesting a possible regulatory role for this chemokine at pituitary level. We evaluated the intracellular signaling involved in these effects: SDF1␣ increased cytosolic [Ca 2ϩ ] and activated Pyk2, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and large-conductance Ca 2ϩ -activated K ϩ channels (BK Ca ) channels. To correlate these intracellular effectors with the proliferative and secretory effects, we inhibited their activity using BAPTA-AM (Ca 2ϩ chelator), 2Ј-amino-3Ј-methoxyflavone (PD98059; a mitogen-activated protein kinase kinase inhibitor), salicylate (Pyk2 inhibitor), and tetraethyl ammonium (K ϩ channel blocker). All of these compounds reverted SDF1␣-induced proliferation, suggesting the involvement of multiple intracellular pathways. Conversely, only BAPTA-AM reverted growth hormone secretion. To identify a possible cross-talk and a molecular ordering among these pathways, we tested these antagonists on SDF1␣-dependent activation of ERK1/2, Pyk2, and BK Ca channels. From these experiments, we observed that the inhibition of [Ca 2ϩ ] i increase or BK Ca channel activity did not affect ERK1/2 activation by SDF1␣; Pyk2 activation was purely Ca 2ϩ -dependent, not involving ERK1/2 or BK Ca channels; and BK Ca channel activity was antagonized by Pyk2 but not by ERK1/2 inhibitors. These data suggest that an SDF1␣-dependent increase of [Ca 2ϩ ] i activates Pyk2, which in turn regulates BK Ca channel activity. Conversely, ERK1/2 activation is an independent phenomenon.In conclusion, we demonstrate that SDF1␣ causes both proliferation and growth hormone release from pituitary adenoma cells, suggesting that the activation of CXCR4 may represent a novel regulatory mechanism for growth hormone secretion and pituitary cell proliferation, which may contribute to pituitary adenoma development.