RB and RB2/p130 genes demonstrate both specific and overlapping functions during the early steps of in vitro neural differentiation of marrow stromal stem cells

Marrow stromal stem cells (MSCs) are stem-like cells that are currently being tested for their potential use in cell therapy for a number of human diseases. MSCs can differentiate into both mesenchymal and nonmesenchymal lineages. In fact, in addition to bone, cartilage and fat, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes. RB and RB2/p130 genes are involved in the differentiation of several systems. For this reason, we evaluated the role of RB and RB2/p130 in the differentiation and apoptosis of MSCs under experimental conditions that allow for MSC differentiation toward the neuron-like phenotype. To this end, we ectopically expressed either RB or RB2/p130 and monitored proliferation, differentiation and apoptosis in rat primary MSC cultures induced to differentiate toward the neuron-like phenotype. Both RB and RB2/P130 decreased cell proliferation rate. In pRb-overexpressing cells, the arrest of cell growth was also observed in the presence of the HDAC-inhibitor TSA, suggesting that its antiproliferative activity does not rely upon the HDAC pathway, while the addition of TSA to pRb2/p130-overexpressing cells relieved growth inhibition. TUNEL reactions and studies on the expression of genes belonging to the Bcl-2 family showed that while RB protected differentiating MSCs from apoptosis, RB2/p130 induced an increase of apoptosis compared to controls. The effects of both RB and RB2/p130 on programmed cell death appeared to be HDAC- independent. Molecular analysis of neural differentiation markers and immunocytochemistry revealed that RB2/p130 contributes mainly to the induction of generic neural properties and RB triggers cholinergic differentiation. Moreover, the differentiation potentials of RB2/p130 and RB appear to rely, at least in part, on the activity of HDACs.     

Neuronal nitric oxide synthase: expression in rat parietal cells

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.     

Surviving starvation: Changes accompanying starvation tolerance in a bdelloid rotifer

Bdelloid rotifers survive desiccation and starvation by halting activity and entering a kind of dormancy. To understand the mechanisms of survival in the absence of food source, we studied the anatomical and ultrastructural changes occurring in a bdelloid species, Macrotrachela quadricornifera Milne 1886, after starvation for different periods. The starved rotifers present a progressive reduction of body size accompanied with a consistent reduction of the volume of the stomach syncytium, where lipid inclusions and digestive vacuoles tend to fade with prolonged starvation. Similar reduction occurs in the vitellarium gland, in which yolk granules progressively decrease in number and size. The changes observed in the syncytia of the stomach and the vitellarium suggest that during starvation M. quadricornifera uses resources diverted from the stomach syncytium first and from the vitellarium syncytium later, resources that are normally allocated to reproduction. The fine structure of starved bdelloids is compared with that of anhydrobiotic bdelloids, revealing that survival during either forms of dormancy is sustained by different physiological mechanisms. J. Morphol., 2011. © 2011 Wiley Periodicals, Inc.     

Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein

Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild‐type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.     

Downregulation of myosin II-B by siRNA alters the subcellular localization of the amyloid precursor protein and increases amyloid-beta deposition in N2a cells

The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-beta (Abeta) peptides and intraneuronal fibrillar structures. These pathological features may be functionally linked, but the mechanism by which Abeta accumulation relates to neuronal degeneration is still poorly understood. Abeta peptides are fragments cleaved from the amyloid precursor protein (APP), a transmembrane protein ubiquitously expressed in the nervous system. Although the proteolytic processing of APP has been implicated in AD, the physiological function of APP and the subcellular site of APP cleavages remain unknown. The overall structure of the protein and its fast anterograde transport along the axon support the idea that APP functions as a vesicular receptor for cytoskeletal motor proteins. In the current study, we test the hypothesis that myosin II, important contributor to the cytoskeleton of neuronal cells, may influence the trafficking and/or the processing of APP. Our results demonstrate that downregulation of myosin II-B, the major myosin isoform in neurons, is able to increase Abeta deposition, concomitantly altering the subcellular localization of APP. These new insights might be important for the understanding of the function of APP and provide a novel conceptual framework in which to analyze its pathological role.     

Disentangling the morphological stasis in two rotifer species of the <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> species complex

The taxonomic uncertainty surrounding cryptic species complexes has traditionally been resolved using lengthy experimental approaches, while, since the advent of PCR based techniques the number of cryptic species described in a variety of taxa is increasing steadily. Here we formally describe a new rotifer species of the Brachionus plicatilis complex: Brachionus manjavacas n.sp., disentangling what was known as a morphological stasis. Detailed morphological analyses demonstrated significant differences in body shape and size between B. manjavacas and B. plicatilis s.s., analysed by geometric morphometrics; unfortunately these statistical differences are not taxonomically reliable because of wide overlaps. Size and asymmetry of masticatory apparatus, named trophi, observed by SEM, gave similar results, with taxonomic ambiguity. Only the shape of small pieces of the trophi, named satellites, were consistently different between the species. On a strictly classical taxonomical basis it is absolutely useful to name new species on morphological bases, as we did, and to assess their status as distinct entities. Nevertheless, the two species are broadly similar; therefore, we do not suggest using the small differences in shape of satellites of trophi to identify the species for further ecological studies, but to continue discriminating them on genetic marker bases. Handling editor: K. Martens     

GLT-1 down-regulation induced by clozapine in rat frontal cortex is associated with synaptophysin up-regulation

In rat frontal cortex, extracellular levels of glutamate are raised by the anti-psychotic drug clozapine. We have recently shown that a significant reduction in the levels of the glutamate transporter GLT-1 may be one of the mechanisms responsible for this elevation. Here we studied whether GLT-1 down-regulation induced by chronic clozapine treatment is associated with changes in the expression of synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicular glutamate transporter 1 (VGLUT1), three major presynaptic proteins involved in neurotransmitter release. Quantitative high-resolution confocal microscopy studies in vivo showed that GLT-1 down-regulation is closely associated with a significant increase in synaptophysin, but not SNAP-25 and VGLUT1, expression. This was confirmed in vitro studies, and in western blotting studies of synaptophysin, SNAP-25 and VGLUT1. In addition, our results show that, following clozapine treatment, synaptophysin expression increases in the very cortical regions in which GLT-1 expression is down-regulated. These findings suggest that part of the effects of clozapine may be exerted via an action on the presynaptic machinery involved in neurotransmitter release.     

Morphology of Floscularia ringens (Rotifera, Monogononta) from egg to adult

Abstract. Floscularia ringens is a cosmopolitan, sessile rotifer (class Monogononta) that lives inside a tube it constructs from numerous small, rounded pellets. Adults of F. ringens produce parthenogenetic eggs that are retained within the tube. Upon hatching, juveniles remain within the maternal tube for a short time completing their development before swimming away. The free-swimming juvenile has a conical body, short foot, small corona, and mastax with trophi, but appears unable to feed. After a short time (<1 day), the young rotifer attaches permanently to a substrate and its morphology changes radically: the corona develops 4 wide lobes and the foot elongates, becoming slender and retractable. Once the corona has developed, the young animal begins to feed by producing filtering currents, and also starts to build its own tube. Here we report 4 new morphological details regarding this species. (1) A specialized epidermal groove is present on the trunk in front of the cloaca. (2) A small hole is located in the center of the inner surface of each pellet of the tube. (3) The muscles inside the foot are U-shaped in transverse section. (4) The size of the trophi remains unchanged during growth of the juvenile into an adult.