YND1 interacts with CDC55 and is a novel mediator of E4orf4-induced toxicity

Adenovirus E4orf4 (early region 4 open reading frame 4) protein induces protein phosphatase 2A-dependent non-classical apoptosis in mammalian cells and irreversible growth arrest in Saccharomyces cerevisiae. Oncogenic transformation sensitizes cells to E4orf4-induced cell death. To uncover additional components of the E4orf4 network required for induction of its unique mode of apoptosis, we used yeast genetics to select gene deletions conferring resistance to E4orf4. Deletion of YND1, encoding a yeast Golgi apyrase, conferred partial resistance to E4orf4. However, Ynd1p apyrase activity was not required for E4orf4-induced toxicity. Ynd1p and Cdc55p, the yeast protein phosphatase 2A-B subunit, contributed additively to E4orf4-induced toxicity. Furthermore, concomitant overexpression of one and deletion of the other was detrimental to yeast growth, demonstrating a functional interaction between the two proteins. YND1 and CDC55 also interacted genetically with CDC20 and CDH1/HCT1, encoding activating subunits of the anaphase-promoting complex/cyclosome. In addition to their functional interaction, Ynd1p and Cdc55p interacted physically, and this interaction was disrupted by E4orf4, which remained associated with both proteins. The results suggested that Ynd1p and Cdc55p share a common downstream target whose balanced modulation by the two E4orf4 partners is crucial to viability. Disruption of this balance by E4orf4 may lead to cell death. NTPDase-4/Lalp70/UDPase, the closest mammalian homologue of Ynd1p, associated with E4orf4 in mammalian cells, suggesting that the results in yeast are relevant to the mammalian system.     

A parturition-associated nonsynaptic coherent activity pattern in the developing hippocampus

Correlated neuronal activity is instrumental in the formation of networks, but its emergence during maturation is poorly understood. We have used multibeam two-photon calcium microscopy combined with targeted electrophysiological recordings in order to determine the development of population coherence from embryonic to postnatal stages in the hippocampus. At embryonic stages (E16-E19), synchronized activity is absent, and neurons are intrinsically active and generate L-type channel-mediated calcium spikes. At birth, small cell assemblies coupled by gap junctions spontaneously generate synchronous nonsynaptic calcium plateaus associated to recurrent burst discharges. The emergence of coherent calcium plateaus at birth is controlled by oxytocin, a maternal hormone initiating labour, and progressively shut down a few days later by the synapse-driven giant depolarizing potentials (GDPs) that synchronize the entire network. Therefore, in the developing hippocampus, delivery is an important signal that triggers the first coherent activity pattern, which is silenced by the emergence of synaptic transmission.     

High-yield isolation, expansion, and differentiation of murine bone marrow-derived mesenchymal stem cells using fibrin microbeads (FMB)

Transplantation of adult mesenchymal stem cells (MSCs) could provide a basis for tissue regeneration. MSCs are typically isolated from bone marrow (BM) based on their preferential adherence to plastic, although with low efficiency in terms of yield and purity. Extensive expansion is needed to reach a significant number of MSCs for any application. Fibrin microbeads (FMB) were designed to attach mesenchymal cells and to provide a matrix for their expansion. The current study was aimed at isolating a high yield of purified BM-derived mouse MSCs based on their preferential adherence and proliferation on FMB in suspension cultures. MSCs could be downloaded to plastics or further expanded on FMB. The yield of MSCs obtained by the FMB isolation technique was about one order of magnitude higher than that achieved by plastic adherence, suggesting that these cells are more abundant than previously reported. FMB-isolated cells were classified as MSCs by their fibroblastic morphology, self-renewal ability, and expression profile of their surface antigens, as examined by flow cytometry and immunostaining. In cell culture, the isolated MSCs could be induced to differentiate into three different mesodermal lineages, as demonstrated by histochemical stains and by RT-PCR analyses of tissue-specific genes. MSCs were also able to differentiate into osteocytes while still cultured on FMB. Our results suggest that FMB might serve as an efficient platform for the isolation, expansion, and differentiation of mouse BM-derived MSCs to be subsequently implanted for tissue regeneration.     

Effects of parathyroid hormone on exocrine pancreatic secretion in parathyroidectomized dogs

The effects of intravenous parathyroid hormone (PTH) on steady state Secretin-induced pancreatic secretion were studied in seven dogs before and after parathyroidectomy. Free flow of pancreatic juice was obtained by direct cannulation of the main pancreatic duct (the minor duct being ligated) : a gastric fistula prevented the entry of gastric acid into the duodenum. In the normal dog PTH caused a significant increase in volume and bicarbonate concentration, reciprocal change in chloride and no change in total protein concentration. The stimulatory effect of PTH was dose-dependent. In the parathyroidectomized dog, the basic Secretin-induced secretion was lower than the preoperative values, but PTH infusion caused a significant increase in volume of fluids and bicarbonate concentration, reciprocal change in chloride and no change in protein concentration. These results were not dependent on calcium blood level, and did not change after calcium injection to the hypocalcemic parathyroidectomized dog. It is suggested, that PTH may have a direct effect on pancreatic exocrine secretion.     

Epileptogenic actions of GABA and fast oscillations in the developing hippocampus

GABA excites immature neurons and inhibits adult ones, but whether this contributes to seizures in the developing brain is not known. We now report that in the developing, but not the adult, hippocampus, seizures beget seizures only if GABAergic synapses are functional. In the immature hippocampus, seizures generated with functional GABAergic synapses include fast oscillations that are required to transform a naive network to an epileptic one: blocking GABA receptors prevents the long-lasting sequels of seizures. In contrast, in adult neurons, full blockade of GABA(A) receptors generates epileptogenic high-frequency seizures. Therefore, purely glutamatergic seizures are not epileptogenic in the developing hippocampus. We suggest that the density of glutamatergic synapses is not sufficient for epileptogenesis in immature neurons; excitatory GABAergic synapses are required for that purpose. We suggest that the synergistic actions of GABA and NMDA receptors trigger the cascades involved in epileptogenesis in the developing hippocampus.