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Sample records for ectopic granule cells

  1. Morphological aspects of the ectopic granule-like cellular populations in the albino rat hippocampal formation: a Golgi study.

    PubMed

    Martí-Subirana, A; Soriano, E; García-Verdugo, J M

    1986-02-01

    Rat hippocampal formation was examined by the Golgi impregnation method. Three different ectopic granule-like populations of cells were differentiated: (1) Ectopic granule-like cells of the regio inferior, located in the stratum radiatum; one or two dendrites arose from the cell body and ran towards either the molecular layer of the fascia dentata or the stratum lacunosum-moleculare of the hippocampus, where they branched into secondary and tertiary dendrites. (2) Ectopic granule-like cells of the hilar region; this cell population showed bipolar and monopolar types of dendritic tree. Unipolar cells had dendrites oriented towards the granular layer where they branched profusely. Dendritic arborisation of bipolar cells was confined to the hilus. (3) Ectopic granule-like cells of the molecular layer; they showed several structural appearances depending on their location within the layer. Axonal tracts of ectopic granule-like cells gave rise to numerous collaterals; the main branch ran to the CA4 and CA3 hippocampal subregions. Several 'en passant' and mossy-like boutons were shown along this path.

  2. Morphological aspects of the ectopic granule-like cellular populations in the albino rat hippocampal formation: a Golgi study.

    PubMed Central

    Martí-Subirana, A; Soriano, E; García-Verdugo, J M

    1986-01-01

    Rat hippocampal formation was examined by the Golgi impregnation method. Three different ectopic granule-like populations of cells were differentiated: (1) Ectopic granule-like cells of the regio inferior, located in the stratum radiatum; one or two dendrites arose from the cell body and ran towards either the molecular layer of the fascia dentata or the stratum lacunosum-moleculare of the hippocampus, where they branched into secondary and tertiary dendrites. (2) Ectopic granule-like cells of the hilar region; this cell population showed bipolar and monopolar types of dendritic tree. Unipolar cells had dendrites oriented towards the granular layer where they branched profusely. Dendritic arborisation of bipolar cells was confined to the hilus. (3) Ectopic granule-like cells of the molecular layer; they showed several structural appearances depending on their location within the layer. Axonal tracts of ectopic granule-like cells gave rise to numerous collaterals; the main branch ran to the CA4 and CA3 hippocampal subregions. Several 'en passant' and mossy-like boutons were shown along this path. Images Figs. 2-7 Figs. 9-12 Figs. 14-16 PMID:2447048

  3. RNA Granules in Germ Cells

    PubMed Central

    Voronina, Ekaterina; Seydoux, Geraldine; Sassone-Corsi, Paolo; Nagamori, Ippei

    2011-01-01

    “Germ granules” are cytoplasmic, nonmembrane-bound organelles unique to germline. Germ granules share components with the P bodies and stress granules of somatic cells, but also contain proteins and RNAs uniquely required for germ cell development. In this review, we focus on recent advances in our understanding of germ granule assembly, dynamics, and function. One hypothesis is that germ granules operate as hubs for the posttranscriptional control of gene expression, a function at the core of the germ cell differentiation program. PMID:21768607

  4. Clonal Analysis of Newborn Hippocampal Dentate Granule Cell Proliferation and Development in Temporal Lobe Epilepsy123

    PubMed Central

    LaSarge, Candi L.; McAuliffe, John J.

    2015-01-01

    Abstract Hippocampal dentate granule cells are among the few neuronal cell types generated throughout adult life in mammals. In the normal brain, new granule cells are generated from progenitors in the subgranular zone and integrate in a typical fashion. During the development of epilepsy, granule cell integration is profoundly altered. The new cells migrate to ectopic locations and develop misoriented “basal” dendrites. Although it has been established that these abnormal cells are newly generated, it is not known whether they arise ubiquitously throughout the progenitor cell pool or are derived from a smaller number of “bad actor” progenitors. To explore this question, we conducted a clonal analysis study in mice expressing the Brainbow fluorescent protein reporter construct in dentate granule cell progenitors. Mice were examined 2 months after pilocarpine-induced status epilepticus, a treatment that leads to the development of epilepsy. Brain sections were rendered translucent so that entire hippocampi could be reconstructed and all fluorescently labeled cells identified. Our findings reveal that a small number of progenitors produce the majority of ectopic cells following status epilepticus, indicating that either the affected progenitors or their local microenvironments have become pathological. By contrast, granule cells with “basal” dendrites were equally distributed among clonal groups. This indicates that these progenitors can produce normal cells and suggests that global factors sporadically disrupt the dendritic development of some new cells. Together, these findings strongly predict that distinct mechanisms regulate different aspects of granule cell pathology in epilepsy. PMID:26756038

  5. Vaccine adjuvants: Tailor-made mast-cell granules

    NASA Astrophysics Data System (ADS)

    Gunzer, Matthias

    2012-03-01

    Mast cells induce protective immune responses through secretion of stimulatory granules. Microparticles modelled after mast-cell granules are now shown to replicate and enhance the functions of their natural counterparts and to direct the character of the resulting immunity.

  6. PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure.

    PubMed

    LaSarge, Candi L; Santos, Victor R; Danzer, Steve C

    2015-03-01

    Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offsets the reduction in boutons per axon length. These morphological changes predict a net increase in granule cell innervation of CA3. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell communication with CA3. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy.

  7. PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure

    PubMed Central

    LaSarge, Candi L.; Santos, Victor R; Danzer, Steve C.

    2015-01-01

    Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offset the reduction in boutons per axon length. These morphological changes predicts a net increase in granule cell >> CA3 innervation. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell >> CA3 communication. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy. PMID:25600212

  8. Insulin granules. Insulin secretory granules control autophagy in pancreatic β cells.

    PubMed

    Goginashvili, Alexander; Zhang, Zhirong; Erbs, Eric; Spiegelhalter, Coralie; Kessler, Pascal; Mihlan, Michael; Pasquier, Adrien; Krupina, Ksenia; Schieber, Nicole; Cinque, Laura; Morvan, Joëlle; Sumara, Izabela; Schwab, Yannick; Settembre, Carmine; Ricci, Romeo

    2015-02-20

    Pancreatic β cells lower insulin release in response to nutrient depletion. The question of whether starved β cells induce macroautophagy, a predominant mechanism maintaining energy homeostasis, remains poorly explored. We found that, in contrast to many mammalian cells, macroautophagy in pancreatic β cells was suppressed upon starvation. Instead, starved β cells induced lysosomal degradation of nascent secretory insulin granules, which was controlled by protein kinase D (PKD), a key player in secretory granule biogenesis. Starvation-induced nascent granule degradation triggered lysosomal recruitment and activation of mechanistic target of rapamycin that suppressed macroautophagy. Switching from macroautophagy to insulin granule degradation was important to keep insulin secretion low upon fasting. Thus, β cells use a PKD-dependent mechanism to adapt to nutrient availability and couple autophagy flux to secretory function.

  9. Proteoglycans support proper granule formation in pancreatic acinar cells.

    PubMed

    Aroso, Miguel; Agricola, Brigitte; Hacker, Christian; Schrader, Michael

    2015-10-01

    Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas.

  10. Synaptic representation of locomotion in single cerebellar granule cells

    PubMed Central

    Powell, Kate; Mathy, Alexandre; Duguid, Ian; Häusser, Michael

    2015-01-01

    The cerebellum plays a crucial role in the regulation of locomotion, but how movement is represented at the synaptic level is not known. Here, we use in vivo patch-clamp recordings to show that locomotion can be directly read out from mossy fiber synaptic input and spike output in single granule cells. The increase in granule cell spiking during locomotion is enhanced by glutamate spillover currents recruited during movement. Surprisingly, the entire step sequence can be predicted from input EPSCs and output spikes of a single granule cell, suggesting that a robust gait code is present already at the cerebellar input layer and transmitted via the granule cell pathway to downstream Purkinje cells. Thus, synaptic input delivers remarkably rich information to single neurons during locomotion. DOI: http://dx.doi.org/10.7554/eLife.07290.001 PMID:26083712

  11. Monoamine-containing granulated cells in the frog lung.

    PubMed

    Wasano, K; Yamamoto, T

    1978-10-17

    The epithelium of the primary bronchus of the frog lung has been studied by fluorescence and electron microscopy. Clusters of five to ten, ovoid, brilliantly yellow fluorescent cells were observed in the basal portion of the epithelium. These cells contained numerous electron-dense granules of variable shape and size. The granules gave a positive argentaffin reaction at the ultrastructural level, suggesting a possible existence of monoamines in the granules. In addition, synaptic contact between the intraepithelial nerves and the cells, which was characterized by the aggregation of the granules toward the presynaptic membrane thickening of the cell, was also noted. These data are discussed in relation to similar studies in birds and mammals, and a possible function of these cells suggested.

  12. Ectopic Atoh1 expression drives Merkel cell production in embryonic, postnatal and adult mouse epidermis.

    PubMed

    Ostrowski, Stephen M; Wright, Margaret C; Bolock, Alexa M; Geng, Xuehui; Maricich, Stephen M

    2015-07-15

    Merkel cells are mechanosensitive skin cells whose production requires the basic helix-loop-helix transcription factor Atoh1. We induced ectopic Atoh1 expression in the skin of transgenic mice to determine whether Atoh1 was sufficient to create additional Merkel cells. In embryos, ectopic Atoh1 expression drove ectopic expression of the Merkel cell marker keratin 8 (K8) throughout the epidermis. Epidermal Atoh1 induction in adolescent mice similarly drove widespread K8 expression in glabrous skin of the paws, but in the whisker pads and body skin ectopic K8+ cells were confined to hair follicles and absent from interfollicular regions. Ectopic K8+ cells acquired several characteristics of mature Merkel cells in a time frame similar to that seen during postnatal development of normal Merkel cells. Although ectopic K8+ cell numbers decreased over time, small numbers of these cells remained in deep regions of body skin hair follicles at 3 months post-induction. In adult mice, greater numbers of ectopic K8+ cells were created by Atoh1 induction during anagen versus telogen and following disruption of Notch signaling by conditional deletion of Rbpj in the epidermis. Our data demonstrate that Atoh1 expression is sufficient to produce new Merkel cells in the epidermis, that epidermal cell competency to respond to Atoh1 varies by skin location, developmental age and hair cycle stage, and that the Notch pathway plays a key role in limiting epidermal cell competency to respond to Atoh1 expression.

  13. Somatically expressed germ-granule components, PGL-1 and PGL-3, repress programmed cell death in C. elegans.

    PubMed

    Al-Amin, Mohammad; Min, Hyemin; Shim, Yhong-Hee; Kawasaki, Ichiro

    2016-01-01

    We previously reported that germline apoptosis in C. elegans increased by loss of PGL-1 and PGL-3, members of a family of constitutive germ-granule components, from germ cells in adult hermaphrodite gonads. In this study, we found that somatic apoptosis was reduced in synthetic multivulva class B (synMuv B) mutants due to ectopic expression of PGL-1 and PGL-3 in the soma. In synMuv B-mutant somatic cells, CED-4 expression level was reduced due to ectopic expression of PGL-1. Furthermore, in contrast to wild type, somatic apoptosis in synMuv B mutants increased following DNA damage in a SIR-2.1-dependent manner. Intriguingly, somatic apoptosis was repressed not only in synMuv B mutants but also by ectopically expressing pgl-1 and/or pgl-3 transgenes in wild-type somatic cells. Our study demonstrates that germ-granule components, PGL-1 and PGL-3, can serve as negative regulators of apoptosis not only in the germline but also in the soma in C. elegans. PMID:27650246

  14. Somatically expressed germ-granule components, PGL-1 and PGL-3, repress programmed cell death in C. elegans

    PubMed Central

    Al-Amin, Mohammad; Min, Hyemin; Shim, Yhong-Hee; Kawasaki, Ichiro

    2016-01-01

    We previously reported that germline apoptosis in C. elegans increased by loss of PGL-1 and PGL-3, members of a family of constitutive germ-granule components, from germ cells in adult hermaphrodite gonads. In this study, we found that somatic apoptosis was reduced in synthetic multivulva class B (synMuv B) mutants due to ectopic expression of PGL-1 and PGL-3 in the soma. In synMuv B-mutant somatic cells, CED-4 expression level was reduced due to ectopic expression of PGL-1. Furthermore, in contrast to wild type, somatic apoptosis in synMuv B mutants increased following DNA damage in a SIR-2.1-dependent manner. Intriguingly, somatic apoptosis was repressed not only in synMuv B mutants but also by ectopically expressing pgl-1 and/or pgl-3 transgenes in wild-type somatic cells. Our study demonstrates that germ-granule components, PGL-1 and PGL-3, can serve as negative regulators of apoptosis not only in the germline but also in the soma in C. elegans. PMID:27650246

  15. Virtual cell and tissue dynamics of ectopic activation of the ventricles

    NASA Astrophysics Data System (ADS)

    Benson, Alan P.; Halley, Graeme; Li, Pan; Tong, Wing C.; Holden, Arun V.

    2007-03-01

    Cardiac ventricular cells and tissues are normally excitable, and are activated by propagating waves of excitation that are initiated in the specialized pacemaking region of the heart. However, isolated or repetitive activity can be initiated at abnormal (ectopic) sites in the ventricles. To trigger an endogenous ectopic beat, there must be a compact focus of cells with changed membrane excitation parameters and kinetics, which initiate activity by after-depolarizations triggered by propagating activity, or that have bifurcated into autorhythmicity. This ectopic focus needs to be large enough, and adequately coupled, to drive the surrounding tissue. We investigate the initiation of ectopic excitation in computational models of human ventricular cells triggered by after-depolarizations and by up/down regulation of specific membrane conductance systems, and the propagation and evolution of ectopic activity in homogeneous or heterogeneous and isotropic, anisotropic, or orthotropic tissues.

  16. Characterization of Mast Cell Secretory Granules and Their Cell Biology

    PubMed Central

    Azouz, Nurit Pereg; Hammel, Ilan

    2014-01-01

    Exocytosis and secretion of secretory granule (SG) contained inflammatory mediators is the primary mechanism by which mast cells exert their protective immune responses in host defense, as well as their pathological functions in allergic reactions and anaphylaxis. Despite their central role in mast cell function, the molecular mechanisms underlying the biogenesis and secretion of mast cell SGs remain largely unresolved. Early studies have established the lysosomal nature of the mast cell SGs and implicated SG homotypic fusion as an important step occurring during both their biogenesis and compound secretion. However, the molecular mechanisms that account for key features of this process largely remain to be defined. A novel high-resolution imaging based methodology allowed us to screen Rab GTPases for their phenotypic and functional impact and identify Rab networks that regulate mast cell secretion. This screen has identified Rab5 as a novel regulator of homotypic fusion of the mast cell SGs that thereby regulates their size and cargo composition. PMID:24988214

  17. Cerebellar granule cell migration and the effects of alcohol.

    PubMed

    Jiang, Yulan; Kumada, Tatsuro; Cameron, D Bryant; Komuro, Hitoshi

    2008-01-01

    In the developing brain the majority of neurons migrate from their birthplace to their final destination. This active movement is essential for the formation of cortical layers and nuclei. The impairment of migration does not affect the viability of neurons but often results in abnormal differentiation. The proper migration of neurons requires the orchestrated activities of multiple cellular and molecular events, such as pathway selection, the activation of specific receptors and channels, and the assembly and disassembly of cytoskeletal components. The migration of neurons is very vulnerable to exposure to environmental toxins, such as alcohol. In this article, we will focus on recent developments in the migration of cerebellar granule cells. First, we will describe when, where and how granule cells migrate through different cortical layers to reach their final destination. Second, we will present how internal programs control the sequential changes in granule cell migration. Third, we will review the roles of external guidance cues and transmembrane signals in granule cell migration. Finally, we will reveal mechanisms by which alcohol exposure impairs granule cell migration. PMID:18075250

  18. Abscisic acid induces ectopic outgrowth in epidermal cells through cortical microtubule reorganization in Arabidopsis thaliana

    PubMed Central

    Takatani, Shogo; Hirayama, Takashi; Hashimoto, Takashi; Takahashi, Taku; Motose, Hiroyasu

    2015-01-01

    Abscisic acid (ABA) regulates seed maturation, germination and various stress responses in plants. The roles of ABA in cellular growth and morphogenesis, however, remain to be explored. Here, we report that ABA induces the ectopic outgrowth of epidermal cells in Arabidopsis thaliana. Seedlings of A. thaliana germinated and grown in the presence of ABA developed ectopic protrusions in the epidermal cells of hypocotyls, petioles and cotyledons. One protrusion was formed in the middle of each epidermal cell. In the hypocotyl epidermis, two types of cell files are arranged alternately into non-stoma cell files and stoma cell files, ectopic protrusions being restricted to the non-stoma cell files. This suggests the presence of a difference in the degree of sensitivity to ABA or in the capacity of cells to form protrusions between the two cell files. The ectopic outgrowth was suppressed in ABA insensitive mutants, whereas it was enhanced in ABA hypersensitive mutants. Interestingly, ABA-induced ectopic outgrowth was also suppressed in mutants in which microtubule organization was compromised. Furthermore, cortical microtubules were disorganized and depolymerized by the ABA treatment. These results suggest that ABA signaling induces ectopic outgrowth in epidermal cells through microtubule reorganization. PMID:26068445

  19. Ectopic Cerebellar Cell Migration Causes Maldevelopment of Purkinje Cells and Abnormal Motor Behaviour in Cxcr4 Null Mice

    PubMed Central

    Huang, Guo-Jen; Edwards, Andrew; Tsai, Cheng-Yu; Lee, Yi-Shin; Peng, Lei; Era, Takumi; Hirabayashi, Yoshio; Tsai, Ching-Yen; Nishikawa, Shin-Ichi; Iwakura, Yoichiro; Chen, Shu-Jen; Flint, Jonathan

    2014-01-01

    SDF-1/CXCR4 signalling plays an important role in neuronal cell migration and brain development. However, the impact of CXCR4 deficiency in the postnatal mouse brain is still poorly understood. Here, we demonstrate the importance of CXCR4 on cerebellar development and motor behaviour by conditional inactivation of Cxcr4 in the central nervous system. We found CXCR4 plays a key role in cerebellar development. Its loss leads to defects in Purkinje cell dentritogenesis and axonal projection in vivo but not in cell culture. Transcriptome analysis revealed the most significantly affected pathways in the Cxcr4 deficient developing cerebellum are involved in extra cellular matrix receptor interactions and focal adhesion. Consistent with functional impairment of the cerebellum, Cxcr4 knockout mice have poor coordination and balance performance in skilled motor tests. Together, these results suggest ectopic the migration of granule cells impairs development of Purkinje cells, causes gross cerebellar anatomical disruption and leads to behavioural motor defects in Cxcr4 null mice. PMID:24516532

  20. Ectopic KNOX Expression Affects Plant Development by Altering Tissue Cell Polarity and Identity[OPEN

    PubMed Central

    Rebocho, Alexandra B.

    2016-01-01

    Plant development involves two polarity types: tissue cell (asymmetries within cells are coordinated across tissues) and regional (identities vary spatially across tissues) polarity. Both appear altered in the barley (Hordeum vulgare) Hooded mutant, in which ectopic expression of the KNOTTED1-like Homeobox (KNOX) gene, BKn3, causes inverted polarity of differentiated hairs and ectopic flowers, in addition to wing-shaped outgrowths. These lemma-specific effects allow the spatiotemporal analysis of events following ectopic BKn3 expression, determining the relationship between KNOXs, polarity, and shape. We show that tissue cell polarity, based on localization of the auxin transporter SISTER OF PINFORMED1 (SoPIN1), dynamically reorients as ectopic BKn3 expression increases. Concurrently, ectopic expression of the auxin importer LIKE AUX1 and boundary gene NO APICAL MERISTEM is activated. The polarity of hairs reflects SoPIN1 patterns, suggesting that tissue cell polarity underpins oriented cell differentiation. Wing cell files reveal an anisotropic growth pattern, and computational modeling shows how polarity guiding growth can account for this pattern and wing emergence. The inverted ectopic flower orientation does not correlate with SoPIN1, suggesting that this form of regional polarity is not controlled by tissue cell polarity. Overall, the results suggest that KNOXs trigger different morphogenetic effects through interplay between tissue cell polarity, identity, and growth. PMID:27553356

  1. Different subsets of newborn granule cells: a possible role in epileptogenesis?

    PubMed

    Bielefeld, Pascal; van Vliet, Erwin A; Gorter, Jan A; Lucassen, Paul J; Fitzsimons, Carlos P

    2014-01-01

    Several factors, including epileptic seizures, can strongly stimulate ongoing neurogenesis in the adult hippocampus. Although adult-born granule cells generated after seizure activity have different physiological properties from their normal counterparts, they integrate into the existing, mature network of the adult hippocampal dentate gyrus. However, the exact role of the neurogenic response during epilepsy and its possible involvement in epileptogenesis have remained elusive. Here, we discuss recent studies shedding new light on the interplay between epilepsy and neurogenesis, and try to explain discrepancies in this literature by proposing seizure severity-dependent induction of two subsets of newborn cells with different properties. We hypothesise that a low seizure intensity would stimulate neurogenesis to a 'physiological plasticity' level and have few pathological consequences. In contrast, a high initial seizure intensity may induce a specific subset of altered and/or ectopically located new granule cells with different electrophysiological properties that could initiate hyperexcitatory recurrent networks that could, in turn, contribute to chronic epilepsy. This hypothesis may clarify previously contradictory data in the literature, and could thereby aid in our understanding of the role of neurogenesis in epileptogenesis, and open up promising avenues for therapeutic intervention.

  2. Ectopic Pregnancy

    MedlinePlus

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  3. Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

    SciTech Connect

    Baconnais, S.; Delavoie, F. |; Zahm, J.M.; Milliot, M.; Castillon, N.; Terryn, C.; Banchet, V.; Michel, J.; Danos, O.; Merten, M.; Chinet, T.; Zierold, K.; Bonnet, N.; Puchelle, E. , E-Mail: edith.puchelle@univ-reims.fr; Balossier, G.

    2005-10-01

    The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na{sup +} absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na{sup +}, Mg{sup 2+}, P, S and Cl{sup -}) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR{sub inh}-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

  4. Signal transduction pathways in mast cell granule-mediated endothelial cell activation.

    PubMed Central

    Chi, Luqi; Stehno-Bittel, Lisa; Smirnova, Irina; Stechschulte, Daniel J; Dileepan, Kottarappat N

    2003-01-01

    BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen

  5. Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum

    PubMed Central

    Haldipur, Parthiv; Sivaprakasam, Iswariya; Periasamy, Vinod; Govindan, Subashika; Mani, Shyamala

    2015-01-01

    ABSTRACT The plane of division of granule neuron progenitors (GNPs) was analysed with respect to the pial surface in P0 to P14 cerebellum and the results showed that there was a significant bias towards the plane of cell division being parallel to pial surface across this developmental window. In addition, the distribution of β-Catenin in anaphase cells was analysed, which showed that there was a significant asymmetry in the distribution of β-Catenin in dividing GNPs. Further, inhibition of Sonic Hedgehog (Shh) signalling had an effect on plane of cell division. Asymmetric distribution of β-Catenin was shown to occur towards the source of a localized extracellular cue. PMID:25979710

  6. Rapid Feedforward Inhibition and Asynchronous Excitation Regulate Granule Cell Activity in the Mammalian Main Olfactory Bulb

    PubMed Central

    Burton, Shawn D.

    2015-01-01

    Granule cell-mediated inhibition is critical to patterning principal neuron activity in the olfactory bulb, and perturbation of synaptic input to granule cells significantly alters olfactory-guided behavior. Despite the critical role of granule cells in olfaction, little is known about how sensory input recruits granule cells. Here, we combined whole-cell patch-clamp electrophysiology in acute mouse olfactory bulb slices with biophysical multicompartmental modeling to investigate the synaptic basis of granule cell recruitment. Physiological activation of sensory afferents within single glomeruli evoked diverse modes of granule cell activity, including subthreshold depolarization, spikelets, and suprathreshold responses with widely distributed spike latencies. The generation of these diverse activity modes depended, in part, on the asynchronous time course of synaptic excitation onto granule cells, which lasted several hundred milliseconds. In addition to asynchronous excitation, each granule cell also received synchronous feedforward inhibition. This inhibition targeted both proximal somatodendritic and distal apical dendritic domains of granule cells, was reliably recruited across sniff rhythms, and scaled in strength with excitation as more glomeruli were activated. Feedforward inhibition onto granule cells originated from deep short-axon cells, which responded to glomerular activation with highly reliable, short-latency firing consistent with tufted cell-mediated excitation. Simulations showed that feedforward inhibition interacts with asynchronous excitation to broaden granule cell spike latency distributions and significantly attenuates granule cell depolarization within local subcellular compartments. Collectively, our results thus identify feedforward inhibition onto granule cells as a core feature of olfactory bulb circuitry and establish asynchronous excitation and feedforward inhibition as critical regulators of granule cell activity. SIGNIFICANCE

  7. Newborn granule cells in the ageing dentate gyrus

    PubMed Central

    Morgenstern, Nicolás A; Lombardi, Gabriela; Schinder, Alejandro F

    2008-01-01

    The dentate gyrus of the hippocampus generates neurons throughout life, but adult neurogenesis exhibits a marked age-dependent decline. Although the decrease in the rate of neurogenesis has been extensively documented in the ageing hippocampus, the specific characteristics of dentate granule cells born in such a continuously changing environment have received little attention. We have used retroviral labelling of neural progenitor cells of the adult mouse dentate gyrus to study morphological properties of neurons born at different ages. Dendritic spine density was measured to estimate glutamatergic afferent connectivity. Fully mature neurons born at the age of 2 months display ∼2.3 spines μm−1 and maintain their overall morphology and spine density in 1-year-old mice. Surprisingly, granule cells born in 10-month-old mice, at which time the rate of neurogenesis has decreased by ∼40-fold, reach a density of dendritic spines similar to that of neurons born in young adulthood. Therefore, in spite of the sharp decline in cell proliferation, differentiation and overall neuronal number, the ageing hippocampus presents a suitable environment for new surviving neurons to reach a high level of complexity, comparable to that of all other dentate granule cells. PMID:18565998

  8. Ectopic Pregnancy

    MedlinePlus

    ... 5 Things to Know About Zika & Pregnancy Ectopic Pregnancy KidsHealth > For Parents > Ectopic Pregnancy Print A A ... lower back pain continue What Causes an Ectopic Pregnancy? An ectopic pregnancy usually happens because a fertilized ...

  9. FIB/SEM cell sectioning for intracellular metal granules characterization

    NASA Astrophysics Data System (ADS)

    Milani, Marziale; Brundu, Claudia; Santisi, Grazia; Savoia, Claudio; Tatti, Francesco

    2009-05-01

    Focused Ion Beams (FIBs) provide a cross-sectioning tool for submicron dissection of cells and subcellular structures. In combination with Scanning Electron Microscope (SEM), FIB provides complementary morphological information, that can be further completed by EDX (Energy Dispersive X-ray Spectroscopy). This study focus onto intracellular microstructures, particularly onto metal granules (typically Zn, Cu and Fe) and on the possibility of sectioning digestive gland cells of the terrestrial isopod P. scaber making the granules available for a compositional analysis with EDX. Qualitative and quantitative analysis of metal granules size, amount and distribution are performed. Information is made available of the cellular storing pattern and, indirectly, metal metabolism. The extension to human level is of utmost interest since some pathologies of relevance are metal related. Apart from the common metal-overload-diseases (hereditary hemochromatosis, Wilson's and Menkes disease) it has been demonstrated that metal in excess can influence carcinogenesis in liver, kidney and breast. Therefore protocols will be established for the observation of mammal cells to improve our knowledge about the intracellular metal amount and distribution both in healthy cells and in those affected by primary or secondary metal overload or depletion.

  10. Calcium dynamics in the secretory granules of neuroendocrine cells.

    PubMed

    Alvarez, Javier

    2012-01-01

    Cellular Ca(2+)signaling results from a complex interplay among a variety of Ca(2+) fluxes going across the plasma membrane and across the membranes of several organelles, together with the buffering effect of large numbers of Ca(2+)-binding sites distributed along the cell architecture. Endoplasmic and sarcoplasmic reticulum, mitochondria and even nucleus have all been involved in cellular Ca(2+) signaling, and the mechanisms for Ca(2+) uptake and release from these organelles are well known. In neuroendocrine cells, the secretory granules also constitute a very important Ca(2+)-storing organelle, and the possible role of the stored Ca(2+) as a trigger for secretion has attracted considerable attention. However, this possibility is frequently overlooked, and the main reason for that is that there is still considerable uncertainty on the main questions related with granular Ca(2+) dynamics, e.g., the free granular [Ca(2+)], the physical state of the stored Ca(2+) or the mechanisms for Ca(2+) accumulation and release from the granules. This review will give a critical overview of the present state of knowledge and the main conflicting points on secretory granule Ca(2+) homeostasis in neuroendocrine cells.

  11. Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers.

    PubMed

    Chai, Yoke Chin; Roberts, Scott J; Desmet, Eline; Kerckhofs, Greet; van Gastel, Nick; Geris, Liesbet; Carmeliet, Geert; Schrooten, Jan; Luyten, Frank P

    2012-04-01

    Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell-host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca(2+)) and phosphate (P(i)) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca(2+) and P(i) supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (μCT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell-host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials. PMID:22269651

  12. Disinhibition of olfactory bulb granule cells accelerates odour discrimination in mice

    PubMed Central

    Nunes, Daniel; Kuner, Thomas

    2015-01-01

    Granule cells are the dominant cell type of the olfactory bulb inhibiting mitral and tufted cells via dendrodendritic synapses; yet the factors regulating the strength of their inhibitory output, and, therefore, their impact on odour discrimination, remain unknown. Here we show that GABAAR β3-subunits are distributed in a somatodendritic pattern, mostly sparing the large granule cell spines also known as gemmules. Granule cell-selective deletion of β3-subunits nearly abolishes spontaneous and muscimol-induced currents mediated by GABAA receptors in granule cells, yet recurrent inhibition of mitral cells is strongly enhanced. Mice with disinhibited granule cells require less time to discriminate both dissimilar as well as highly similar odourants, while discrimination learning remains unaffected. Hence, granule cells are controlled by an inhibitory drive that in turn tunes mitral cell inhibition. As a consequence, the olfactory bulb inhibitory network adjusts the speed of early sensory processing. PMID:26592770

  13. Ectopic Paratubal Adrenal Cell Rest Associated with Mucinous Cystadenoma of Ovary

    PubMed Central

    Dey, Soumit; Ray, Prasenjit Sen; Sarkar, Ranu; Bhattacharyya, Palas

    2015-01-01

    Ectopic adrenal cortex is a rare entity. Usually found in male children; commonly located around kidney, retroperitoneum, spermatic cord and para-testicular region. Rarely, adults with heterotopic adrenal glands are described. Incidence in females is very less; though sometimes detected accidentally in hysterectomy specimens. We describe a case of ectopic adrenal cortical cell in paratubal region in a patient with mucinous cyst adenoma of ovary. A 26-year-old female presented with complains of menstrual irregularities and abdominal discomfort for 6 months. Investigations suggested a right ovarian cyst. Right ovarian cystectomy with partial salpingectomy was performed; histopathology revealed mucinous cyst adenoma. Sections from tube showed presence of ectopic adrenal cortical rest in the paratubal region, incidentally discovered on microscopy. We present this case because of its rarity in females, interesting presentation with another unrelated gynaecological pathology, its potentiality for malignant transformation and possible complications. PMID:26557532

  14. Control over the morphology and segregation of Zebrafish germ cell granules during embryonic development

    PubMed Central

    Strasser, Markus J; Mackenzie, Natalia C; Dumstrei, Karin; Nakkrasae, La-Iad; Stebler, Jürg; Raz, Erez

    2008-01-01

    Background Zebrafish germ cells contain granular-like structures, organized around the cell nucleus. These structures share common features with polar granules in Drosophila, germinal granules in Xenopus and chromatoid bodies in mice germ cells, such as the localization of the zebrafish Vasa, Piwi and Nanos proteins, among others. Little is known about the structure of these granules as well as their segregation in mitosis during early germ-cell development. Results Using transgenic fish expressing a fluorescently labeled novel component of Zebrafish germ cell granules termed Granulito, we followed the morphology and distribution of the granules. We show that whereas these granules initially exhibit a wide size variation, by the end of the first day of development they become a homogeneous population of medium size granules. We investigated this resizing event and demonstrated the role of microtubules and the minus-end microtubule dependent motor protein Dynein in the process. Last, we show that the function of the germ cell granule resident protein the Tudor domain containing protein-7 (Tdrd7) is required for determination of granule morphology and number. Conclusion Our results suggest that Zebrafish germ cell granules undergo a transformation process, which involves germ cell specific proteins as well as the microtubular network. PMID:18507824

  15. AMPA receptors in cerebellar granule cells during development in culture.

    PubMed

    Hack, N J; Sluiter, A A; Balázs, R

    1995-06-27

    The survival and maturation of differentiating cerebellar granule cells in culture are known to be promoted by excitatory amino acids (EAAs) which, however, compromise the survival of mature cells. In contrast to the trophic effect, the toxic effect of alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropiate (AMPA) could only be elicited when the desensitisation of AMPA receptors was blocked, cyclothiazide being used in this study. Nevertheless, even under these conditions, toxicity induced by AMPA in contrast to kainate was, at 9 DIV, only half of the maximal toxicity attained by 13-16 DIV. Since cellular responses to AMPA depend so dramatically on the maturational stage of granule cells, we examined here whether this characteristic is related to developmental changes in AMPA receptor properties, which may result from changes in the subunit composition of the receptor. In contrast to toxicity, AMPA-induced 45Ca2+ influx (determined in the presence of cyclothiazide and the NMDA receptor blocker MK-801) reached a maximum already at 9 DIV. This also applied to a fraction of the 45Ca2+ uptake which persisted either after Cd2+ application or under Na(+)-free conditions and therefore presumably was mediated directly through AMPA receptor channels. Quantitative analysis of Western blots showed that the amounts of GluR4 and to a lesser extent GluR2/3/4c are substantial already at 2 DIV, remaining fairly constant until 9 DIV, followed by an increase by 16 DIV. However GluR1, which is hardly detectable in granule cells in vivo and is also low early in vitro, increased almost linearly with cultivation time.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. In vivo imaging of dendritic pruning in dentate granule cells.

    PubMed

    Gonçalves, J Tiago; Bloyd, Cooper W; Shtrahman, Matthew; Johnston, Stephen T; Schafer, Simon T; Parylak, Sarah L; Tran, Thanh; Chang, Tina; Gage, Fred H

    2016-06-01

    We longitudinally imaged the developing dendrites of adult-born mouse dentate granule cells (DGCs) in vivo and found that they underwent over-branching and pruning. Exposure to an enriched environment and constraint of dendritic growth by disrupting Wnt signaling led to increased branch addition and accelerated growth, which were, however, counteracted by earlier and more extensive pruning. Our results indicate that pruning is regulated in a homeostatic fashion to oppose excessive branching and promote a similar dendrite structure in DGCs. PMID:27135217

  17. LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration

    PubMed Central

    Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

    2015-01-01

    The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1Atoh1 CKO) to investigate the function of LKB1 in cerebellar development. The LKB1Atoh1 CKO mice displayed motor dysfunction. In the LKB1Atoh1 CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1Atoh1 CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development. PMID:26549569

  18. Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

    PubMed

    Yokawa, Satoru; Furuno, Tadahide; Suzuki, Takahiro; Inoh, Yoshikazu; Suzuki, Ryo; Hirashima, Naohide

    2016-09-01

    Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 μm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 μm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity. PMID:27262873

  19. Accumulation of abnormal adult-generated hippocampal granule cells predicts seizure frequency and severity

    PubMed Central

    Hester, Michael S.; Danzer, Steve C.

    2013-01-01

    Accumulation of abnormally integrated, adult-born, hippocampal dentate granule cells (DGC) is hypothesized to contribute to the development of temporal lobe epilepsy (TLE). DGCs have long been implicated in TLE, as they regulate excitatory signaling through the hippocampus and exhibit neuroplastic changes during epileptogenesis. Furthermore, DGCs are unusual in that they are continually generated throughout life, with aberrant integration of new cells underlying the majority of restructuring in the dentate during epileptogenesis. While it is known that these abnormal networks promote abnormal neuronal firing and hyperexcitability, it has yet to be established whether they directly contribute to seizure generation. If abnormal DGCs do contribute, a reasonable prediction would be that the severity of epilepsy will be correlated with the number or load of abnormal DGCs. To test this prediction, we utilized a conditional, inducible transgenic mouse model to fate-map adult-generated DGCs. Mossy cell loss, also implicated in epileptogenesis, was assessed as well. Transgenic mice rendered epileptic using the pilocarpine-status epilepticus model of epilepsy were monitored 24/7 by video/EEG for four weeks to determine seizure frequency and severity. Positive correlations were found between seizure frequency and: 1) the percentage of hilar ectopic DGCs, 2) the amount of mossy fiber sprouting and 3) the extent of mossy cell death. In addition, mossy fiber sprouting and mossy cell death were correlated with seizure severity. These studies provide correlative evidence in support of the hypothesis that abnormal DGCs contribute to the development of TLE, and also support a role for mossy cell loss. PMID:23699504

  20. The input-output transformation of the hippocampal granule cells: from grid cells to place fields.

    PubMed

    de Almeida, Licurgo; Idiart, Marco; Lisman, John E

    2009-06-10

    Grid cells in the rat medial entorhinal cortex fire (periodically) over the entire environment. These cells provide input to hippocampal granule cells whose output is characterized by one or more small place fields. We sought to understand how this input-output transformation occurs. Available information allows simulation of this process with no freely adjustable parameters. We first examined the spatial distribution of excitation in granule cells produced by the convergence of excitatory inputs from randomly chosen grid cells. Because the resulting summation depends on the number of inputs, it is necessary to use a realistic number (approximately 1200) and to take into consideration their 20-fold variation in strength. The resulting excitation maps have only modest peaks and valleys. To analyze how this excitation interacts with inhibition, we used an E%-max (percentage of maximal suprathreshold excitation) winner-take-all rule that describes how gamma-frequency inhibition affects firing. We found that simulated granule cells have firing maps that have one or more place fields whose size and number approximates those observed experimentally. A substantial fraction of granule cells have no place fields, as observed experimentally. Because the input firing rates and synaptic properties are known, the excitatory charge into granule cells could be calculated (2-3 pC) and was found to be only somewhat larger than required to fire granule cells (1 pC). We conclude that the input-output transformation of dentate granule does not depend strongly on synaptic modification; place field formation can be understood in terms of simple summation of randomly chosen excitatory inputs, in conjunction with a winner-take-all network mechanism.

  1. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures.

    PubMed

    Mélida, Hugo; Largo-Gosens, Asier; Novo-Uzal, Esther; Santiago, Rogelio; Pomar, Federico; García, Pedro; García-Angulo, Penélope; Acebes, José Luis; Álvarez, Jesús; Encina, Antonio

    2015-04-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment. PMID:25735403

  2. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures.

    PubMed

    Mélida, Hugo; Largo-Gosens, Asier; Novo-Uzal, Esther; Santiago, Rogelio; Pomar, Federico; García, Pedro; García-Angulo, Penélope; Acebes, José Luis; Álvarez, Jesús; Encina, Antonio

    2015-04-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

  3. Conversion of quiescent niche cells to somatic stem cells causes ectopic niche formation in the Drosophila testis

    PubMed Central

    Hétié, Phylis; de Cuevas, Margaret; Matunis, Erika

    2014-01-01

    Summary Adult stem cells reside in specialized regulatory microenvironments, or niches, where local signals ensure stem cell maintenance. The Drosophila testis contains a well-characterized niche wherein signals from post-mitotic hub cells promote maintenance of adjacent germline stem cells and somatic cyst stem cells (CySCs). Hub cells were considered to be terminally differentiated; here we show that they can give rise to CySCs. Genetic ablation of CySCs triggers hub cells to transiently exit quiescence, delaminate from the hub, and convert into functional CySCs. Ectopic Cyclin D-Cdk4 expression in hub cells is also sufficient to trigger their conversion into CySCs. In both cases, this conversion causes the formation of multiple ectopic niches over time. Therefore, our work provides a model for understanding how oncogenic mutations in quiescent niche cells could promote loss of quiescence, changes in cell fate, and aberrant niche expansion more generally. PMID:24746819

  4. Nanoimaging granule dynamics and subcellular structures in activated mast cells using soft X-ray tomography

    PubMed Central

    Chen, Huan-Yuan; Chiang, Dapi Meng-Lin; Lin, Zi-Jing; Hsieh, Chia-Chun; Yin, Gung-Chian; Weng, I.-Chun; Guttermann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Lai, Lee-Jene; Liu, Fu-Tong

    2016-01-01

    Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level. PMID:27748356

  5. Sparse activity of identified dentate granule cells during spatial exploration

    PubMed Central

    Diamantaki, Maria; Frey, Markus; Berens, Philipp; Preston-Ferrer, Patricia; Burgalossi, Andrea

    2016-01-01

    In the dentate gyrus – a key component of spatial memory circuits – granule cells (GCs) are known to be morphologically diverse and to display heterogeneous activity profiles during behavior. To resolve structure–function relationships, we juxtacellularly recorded and labeled single GCs in freely moving rats. We found that the vast majority of neurons were silent during exploration. Most active GCs displayed a characteristic spike waveform, fired at low rates and showed spatial activity. Primary dendritic parameters were sufficient for classifying neurons as active or silent with high accuracy. Our data thus support a sparse coding scheme in the dentate gyrus and provide a possible link between structural and functional heterogeneity among the GC population. DOI: http://dx.doi.org/10.7554/eLife.20252.001 PMID:27692065

  6. Ectopic expression of single transcription factors directs differentiation of a medaka spermatogonial cell line.

    PubMed

    Thoma, Eva C; Wagner, Toni U; Weber, Isabell P; Herpin, Amaury; Fischer, Andreas; Schartl, Manfred

    2011-08-01

    The capability to form all cell types of the body is a unique feature of stem cells. However, many questions remain concerning the mechanisms regulating differentiation potential. The derivation of spermatogonial cell lines (SGs) from mouse and human, which can differentiate across germ-layer borders, suggested male germ cells as a potential stem cell source in addition to embryonic stem cells. Here, we present a differentiation system using an SG of the vertebrate model organism Oryzias latipes (medaka). We report differentiation of this cell line into 4 different ectodermal and mesodermal somatic cell types. In addition to differentiation into adipocytes by retinoic acid treatment, we demonstrate for the first time that directed differentiation of an SG can be induced by ectopic expression of single transcription factors, completely independent of culture conditions. Transient transfection with mitf-m, a transcription factor that has been shown to induce differentiation into melanocytes in medaka embryonic stem cells, resulted in the formation of the same cell type in spermatogonia. Similarly, the formation of neuron-like cells and matrix-depositing osteoblasts was induced by ectopic expression of mash1 and cbfa1, respectively. Interestingly, we found that the expression of all mentioned fate-inducing transcription factors leads to recapitulation of the temporal pattern of marker gene expression known from in vivo studies. PMID:21090990

  7. Transient expression of Ngn3 in Xenopus endoderm promotes early and ectopic development of pancreatic beta and delta cells

    PubMed Central

    Oropeza, Daniel; Horb, Marko

    2012-01-01

    Promoting ectopic development of pancreatic beta cells from other cell types is one of the strategies being pursued for the treatment of diabetes. To achieve this, a detailed outline of the molecular lineage that operates in pancreatic progenitor cells to generate beta cells over other endocrine cell types is necessary. Here, we demonstrate that early transient expression of the endocrine progenitor bHLH protein Neurogenin 3 (Ngn3) favors the promotion of pancreatic beta and delta cell fates over an alpha cell fate, while later transient expression promotes ectopic development of all three endocrine cell fates. We found that short-term activation of Ngn3 in Xenopus laevis endoderm just after gastrulation was sufficient to promote both early and ectopic development of beta and delta cells. By examining gene expression changes four hours after Ngn3 activation we identified several new downstream targets of Ngn3. We show that several of these are required for the promotion of ectopic beta cells by Ngn3 as well as for normal beta cell development. These results provide new detail regarding the Ngn3 transcriptional network operating in endocrine progenitor cells to specify a beta cell phenotype and should help define new approaches to promote ectopic development of beta cells for diabetes therapy. PMID:22121111

  8. Characterization of the T-cell subpopulations in the granulation tissues of chronic suppurative otitis media

    PubMed Central

    WANG, BING; CHENG, YING; XU, MIN

    2016-01-01

    The present study aimed to investigate the potential involvement of specific T-cell subpopulations in granulation tissue formation in chronic suppurative otitis media (CSOM). Fifteen patients with CSOM were enrolled in this study. Granulation tissues were obtained from the middle ear cavity. Hematoxylin and eosin staining was performed for histopathological observation, and different T-cell subpopulations were characterized by immunohistochemistry. No evident association was identified between granulation tissue formation and disease course. The number of cluster of differentiation 8+ (CD8+) T cells, forkhead box P3+ (FOXP3+) regulatory T (Treg) cells and OX40+ T cells were significantly higher in granulation tissues from patients with ear discharge within the last 6 months compared to those without (P<0.05). Fresh granulation tissues had more CD8+ T cells and FOXP3+ Treg cells compared to the mature granulation tissues (P<0.05). There was a differential abundance of specific T-cell subpopulations in the granulation tissues in CSOM with different disease courses or with ear discharge, suggesting that T cell-mediated cellular immunity is involved in lesion formation of CSOM. PMID:27313854

  9. Lkb1 deletion promotes ectopic lipid accumulation in muscle progenitor cells and mature muscles.

    PubMed

    Shan, Tizhong; Zhang, Pengpeng; Bi, Pengpeng; Kuang, Shihuan

    2015-05-01

    Excessive intramyocellular triglycerides (muscle lipids) are associated with reduced contractile function, insulin resistance, and Type 2 diabetes, but what governs lipid accumulation in muscle is unclear. Here we report a role of Lkb1 in regulating lipid metabolism in muscle stem cells and their descendent mature muscles. We used Myod(Cre) and Lkb1(flox/flox) mice to specifically delete Lkb1 in myogenic cells including stem and differentiated cells, and examined the lipid accumulation and gene expression of myoblasts cultured from muscle stem cells (satellite cells). Genetic deletion of Lkb1 in myogenic progenitors led to elevated expression of lipogenic genes and ectopic lipid accumulation in proliferating myoblasts. Interestingly, the Lkb1-deficient myoblasts differentiated into adipocyte-like cells upon adipogenic induction. However, these adipocyte-like cells maintained myogenic gene expression with reduced ability to form myotubes efficiently. Activation of AMPK by AICAR prevented ectopic lipid formation in the Lkb1-null myoblasts. Notably, Lkb1-deficient muscles accumulated excessive lipids in vivo in response to high-fat diet feeding. These results demonstrate that Lkb1 acts through AMPK to limit lipid deposition in muscle stem cells and their derivative mature muscles, and point to the possibility of controlling muscle lipid content using AMPK activating drugs.

  10. Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

    SciTech Connect

    Sontag, Ryan L.; Weber, Thomas J.

    2012-05-04

    In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.

  11. Ectopic differentiation of melanocyte stem cells is influenced by genetic background.

    PubMed

    Harris, Melissa L; Levy, Denise J; Watkins-Chow, Dawn E; Pavan, William J

    2015-03-01

    Hair graying in mouse is attributed to the loss of melanocyte stem cell function and the progressive depletion of the follicular melanocyte population. Single-gene, hair graying mouse models have pointed to a number of critical pathways involved in melanocyte stem cell biology; however, the broad range of phenotypic variation observed in human hair graying suggests that additional genetic variants involved in this process may yet be discovered. Using a sensitized approach, we ask here whether natural genetic variation influences a predominant cellular mechanism of hair graying in mouse, melanocyte stem cell differentiation. We developed an innovative method to quantify melanocyte stem cell differentiation by measuring ectopically pigmented melanocyte stem cells in response to the melanocyte-specific transgene Tg(Dct-Sox10). We make the novel observation that the production of ectopically pigmented melanocyte stem cells varies considerably across strains. The success of sensitizing for melanocyte stem cell differentiation by Tg(Dct-Sox10) sets the stage for future investigations into the genetic basis of strain-specific contributions to melanocyte stem cell biology.

  12. Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus.

    PubMed

    John, Danielle; Shelukhina, Irina; Yanagawa, Yuchio; Deuchars, Jim; Henderson, Zaineb

    2015-03-19

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7*nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7*nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2-3 week-old Wistar rats, and 2-9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7*nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7*nicotinic receptor modulator, which were blocked by a specific α7*nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7*nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7*nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain.

  13. Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus

    PubMed Central

    John, Danielle; Shelukhina, Irina; Yanagawa, Yuchio; Deuchars, Jim; Henderson, Zaineb

    2015-01-01

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7⁎nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7⁎nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2–3 week-old Wistar rats, and 2–9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7⁎nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7⁎nicotinic receptor modulator, which were blocked by a specific α7⁎nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7⁎nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7⁎nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain. PMID:25553616

  14. Granule changes of human skin mast cells characteristic of piecemeal degranulation and associated with recovery during wound healing in situ.

    PubMed

    Dvorak, A M; Kissell, S

    1991-02-01

    Human skin mast cells (HSMC) in situ were examined by electron microscopy of surgical biopsy specimens obtained from a wide variety of circumstances. From these studies, it is apparent that the general ultrastructural morphology of normal HSMC is similar to that of human mast cells from other sites, except that the most prevalent granule pattern is that of crystal granules. Scroll granules, particle granules, and mixed granules can also occur in HSMC. Cytoplasmic lipid bodies occur in HSMC extremely rarely, unlike mast cells from lung and gut. Circumstances in which piecemeal degranulation (PMD) of HSMC occurs, such as bullous pemphigoid, examined at high magnifications, revealed typical, focal geographic losses from cytoplasmic granules, often leaving completely empty granule containers in the cytoplasm. Crystal portions of mixed granules were uniquely susceptible to granule losses typified by PMD. Cytoplasmic smooth vesicles were prominent in PMD. These structures were either empty or contained granule-like dense materials and were free in the cytoplasm or attached to granules. HSMC present in wound healing revealed recovery from PMD losses. Typically these granules contained numerous irregular foci of markedly dense new granule products within empty or partially empty granule containers. The morphology of PMD and recovery of HSMC in vivo is contrasted with the morphology of anaphylactic degranulation (AND) and recovery of human mast cells ex vivo.

  15. Ectopic TBX1 suppresses thymic epithelial cell differentiation and proliferation during thymus organogenesis.

    PubMed

    Reeh, Kaitlin A G; Cardenas, Kim T; Bain, Virginia E; Liu, Zhijie; Laurent, Micheline; Manley, Nancy R; Richie, Ellen R

    2014-08-01

    The thymus and parathyroid glands arise from a shared endodermal primordium in the third pharyngeal pouch (3rd pp). Thymus fate is specified in the ventral 3rd pp between E9.5 and E11, whereas parathyroid fate is specified in the dorsal domain. The molecular mechanisms that specify fate and regulate thymus and parathyroid development are not fully delineated. Previous reports suggested that Tbx1 is required for thymus organogenesis because loss of Tbx1 in individuals with DiGeorge syndrome and in experimental Tbx1 deletion mutants is associated with thymus aplasia or hypoplasia. However, the thymus phenotype is likely to be secondary to defects in pharyngeal pouch formation. Furthermore, the absence of Tbx1 expression in the thymus-fated domain of the wild-type 3rd pp suggested that Tbx1 is instead a negative regulator of thymus organogenesis. To test this hypothesis, we generated a novel mouse strain in which expression of a conditional Tbx1 allele was ectopically activated in the thymus-fated domain of the 3rd pp. Ectopic Tbx1 expression severely repressed expression of Foxn1, a transcription factor that marks the thymus-fated domain and is required for differentiation and proliferation of thymic epithelial cell (TEC) progenitors. By contrast, ectopic Tbx1 did not alter the expression pattern of Gcm2, a transcription factor restricted to the parathyroid-fated domain and required for parathyroid development. Ectopic Tbx1 expression impaired TEC proliferation and arrested TEC differentiation at an early progenitor stage. The results support the hypothesis that Tbx1 negatively regulates TEC growth and differentiation, and that extinction of Tbx1 expression in 3rd pp endoderm is a prerequisite for thymus organogenesis.

  16. Ectopic TBX1 suppresses thymic epithelial cell differentiation and proliferation during thymus organogenesis

    PubMed Central

    Reeh, Kaitlin A. G.; Cardenas, Kim T.; Bain, Virginia E.; Liu, Zhijie; Laurent, Micheline; Manley, Nancy R.; Richie, Ellen R.

    2014-01-01

    The thymus and parathyroid glands arise from a shared endodermal primordium in the third pharyngeal pouch (3rd pp). Thymus fate is specified in the ventral 3rd pp between E9.5 and E11, whereas parathyroid fate is specified in the dorsal domain. The molecular mechanisms that specify fate and regulate thymus and parathyroid development are not fully delineated. Previous reports suggested that Tbx1 is required for thymus organogenesis because loss of Tbx1 in individuals with DiGeorge syndrome and in experimental Tbx1 deletion mutants is associated with thymus aplasia or hypoplasia. However, the thymus phenotype is likely to be secondary to defects in pharyngeal pouch formation. Furthermore, the absence of Tbx1 expression in the thymus-fated domain of the wild-type 3rd pp suggested that Tbx1 is instead a negative regulator of thymus organogenesis. To test this hypothesis, we generated a novel mouse strain in which expression of a conditional Tbx1 allele was ectopically activated in the thymus-fated domain of the 3rd pp. Ectopic Tbx1 expression severely repressed expression of Foxn1, a transcription factor that marks the thymus-fated domain and is required for differentiation and proliferation of thymic epithelial cell (TEC) progenitors. By contrast, ectopic Tbx1 did not alter the expression pattern of Gcm2, a transcription factor restricted to the parathyroid-fated domain and required for parathyroid development. Ectopic Tbx1 expression impaired TEC proliferation and arrested TEC differentiation at an early progenitor stage. The results support the hypothesis that Tbx1 negatively regulates TEC growth and differentiation, and that extinction of Tbx1 expression in 3rd pp endoderm is a prerequisite for thymus organogenesis. PMID:25053428

  17. Ectopic Pregnancy

    MedlinePlus

    ... woman is pregnant. If you have an ectopic pregnancy, the fertilized egg grows in the wrong place, ... tubes. The result is usually a miscarriage. Ectopic pregnancy can be a medical emergency if it ruptures. ...

  18. Internalized Tau sensitizes cells to stress by promoting formation and stability of stress granules

    PubMed Central

    Brunello, Cecilia A.; Yan, Xu; Huttunen, Henri J.

    2016-01-01

    Stress granules are membrane-less RNA- and RNA-binding protein-containing complexes that are transiently assembled in stressful conditions to promote cell survival. Several stress granule-associated RNA-binding proteins have been associated with neurodegenerative diseases. In addition, a close link was recently identified between the stress granule core-nucleating protein TIA-1 and Tau. Tau is a central pathological protein in Alzheimer’s disease and other tauopathies, and misfolded, aggregated Tau is capable of propagating pathology via cell-to-cell transmission. Here we show that following internalization hyperphosphorylated extracellular Tau associates with stress granules in a TIA-1 dependent manner. Cytosolic Tau normally only weakly interacts with TIA-1 but mutations mimicking abnormal phosphorylation promote this interaction. We show that internalized Tau significantly delays normal clearance of stress granules in the recipient cells sensitizing them to secondary stress. These results suggest that secreted Tau species may have properties, likely related to its hyperphosphorylation and oligomerization, which promote pathological association of internalized Tau with stress granules altering their dynamics and reducing cell viability. We suggest that stress granules and TIA-1 play a central role in the cell-to-cell transmission of Tau pathology. PMID:27460788

  19. Ectopic Expression of Testis Germ Cell Proteins in Cancer and Its Potential Role in Genomic Instability.

    PubMed

    Nielsen, Aaraby Yoheswaran; Gjerstorff, Morten Frier

    2016-01-01

    Genomic instability is a hallmark of human cancer and an enabling factor for the genetic alterations that drive cancer development. The processes involved in genomic instability resemble those of meiosis, where genetic material is interchanged between homologous chromosomes. In most types of human cancer, epigenetic changes, including hypomethylation of gene promoters, lead to the ectopic expression of a large number of proteins normally restricted to the germ cells of the testis. Due to the similarities between meiosis and genomic instability, it has been proposed that activation of meiotic programs may drive genomic instability in cancer cells. Some germ cell proteins with ectopic expression in cancer cells indeed seem to promote genomic instability, while others reduce polyploidy and maintain mitotic fidelity. Furthermore, oncogenic germ cell proteins may indirectly contribute to genomic instability through induction of replication stress, similar to classic oncogenes. Thus, current evidence suggests that testis germ cell proteins are implicated in cancer development by regulating genomic instability during tumorigenesis, and these proteins therefore represent promising targets for novel therapeutic strategies.

  20. Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults

    SciTech Connect

    Gianotti, A.J.; Clark, D.T.; Dash, J. )

    1991-04-01

    A source of confusion has existed since the turn of the century about the reddish brown, weakly birefringent 'sphaerocrystals' located in the intestines of strongyle nematodes, Strongylus and Ancylostoma. X-ray diffraction and energy dispersive spectrometric analyses were used for accurate determination of the crystalline order and elemental composition of the granules in the canine hookworm Ancylostoma caninum. The composition of the intestinal pigmented granules was identified unequivocally as zinc sulfide. It seems most probable that the granules serve to detoxify high levels of metallic ions (specifically zinc) present due to the large intake of host blood.

  1. Formation of tRNA granules in the nucleus of heat-induced human cells.

    PubMed

    Miyagawa, Ryu; Mizuno, Rie; Watanabe, Kazunori; Ijiri, Kenichi

    2012-02-01

    The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA(Met) (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA(Met) was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules. PMID:22244871

  2. Formation of tRNA granules in the nucleus of heat-induced human cells

    SciTech Connect

    Miyagawa, Ryu; Mizuno, Rie; Watanabe, Kazunori; Ijiri, Kenichi

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. Black-Right-Pointing-Pointer tRNAs form the unique granules in the nucleus. Black-Right-Pointing-Pointer tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA{sup Met} (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA{sup Met} was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

  3. Engineering secondary metabolism in maize cells by ectopic expression of transcription factors

    PubMed Central

    Grotewold, E; Chamberlin, M; Snook, M; Siame, B; Butler, L; Swenson, J; Maddock, S; Clair, GS; Bowen, B

    1998-01-01

    Manipulation of plant natural product biosynthesis through genetic engineering is an attractive but technically challenging goal. Here, we demonstrate that different secondary metabolites can be produced in cultured maize cells by ectopic expression of the appropriate regulatory genes. Cell lines engineered to express the maize transcriptional activators C1 and R accumulate two cyanidin derivatives, which are similar to the predominant anthocyanin found in differentiated plant tissues. In contrast, cell lines that express P accumulate various 3-deoxy flavonoids. Unexpectedly, P-expressing cells in culture also accumulate phenylpropanoids and green fluorescent compounds that are targeted to different subcellular compartments. Two endogenous biosynthetic genes (c2 and a1, encoding chalcone synthase and flavanone/dihydroflavonol reductase, respectively) are independently activated by ectopic expression of either P or C1/R, and there is a dose-response relationship between the transcript level of P and the degree to which c2 or a1 is expressed. Our results support a simple model showing how the gene encoding P may act as a quantitative trait locus controlling insecticidal C-glycosyl flavone level in maize silks, and they suggest how p1 might confer a selective advantage against insect predation in maize. PMID:9596632

  4. Prox1 postmitotically defines dentate gyrus cells by specifying granule cell identity over CA3 pyramidal cell fate in the hippocampus.

    PubMed

    Iwano, Tomohiko; Masuda, Aki; Kiyonari, Hiroshi; Enomoto, Hideki; Matsuzaki, Fumio

    2012-08-01

    The brain is composed of diverse types of neurons that fulfill distinct roles in neuronal circuits, as manifested by the hippocampus, where pyramidal neurons and granule cells constitute functionally distinct domains: cornu ammonis (CA) and dentate gyrus (DG), respectively. Little is known about how these two types of neuron differentiate during hippocampal development, although a set of transcription factors that is expressed in progenitor cells is known to be required for the survival of granule cells. Here, we demonstrate in mice that Prox1, a transcription factor constitutively expressed in the granule cell lineage, postmitotically functions to specify DG granule cell identity. Postmitotic elimination of Prox1 caused immature DG neurons to lose the granule cell identity and in turn terminally differentiate into the pyramidal cell type manifesting CA3 neuronal identity. By contrast, Prox1 overexpression caused opposing effects on presumptive hippocampal pyramidal cells. These results indicate that the immature DG cell has the potential to become a granule cell or a pyramidal cell, and Prox1 defines the granule cell identity. This bi-potency is lost in mature DG cells, although Prox1 is still required for correct gene expression in DG granule cells. Thus, our data indicate that Prox1 acts as a postmitotic cell fate determinant for DG granule cells over the CA3 pyramidal cell fate and is crucial for maintenance of the granule cell identity throughout the life.

  5. Persistent Mullerian Duct Syndrome with Embryonal Cell Carcinoma along with Ectopic Cross Fused Kidney

    PubMed Central

    Bharath, NR Manju; Narayana, V; Raja, V Om Pramod Kumar; Jambula, Pranav Reddy

    2016-01-01

    Persistent Mullerian Duct Syndrome (PMDS) is a form of internal male pseudohermaphroditism, where there is normal development of male secondary sexual characters, along with the presence of bilateral fallopian tubes and uterus. Majority of these cases go undetected and some cases are accidentally diagnosed while investigating for other problems. Cross fused renal ectopia is a condition where one kidney lies in the opposite side, fused to the other kidney. We present an extremely rare case of a phenotypical male presenting with mass per abdomen and bilateral cryptorchidism, turned out to have uterus with bilateral fallopian tubes, ectopic cross fused right kidney and Embryonal cell carcinoma of left undescended testis. PMID:26894123

  6. Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells.

    PubMed

    Fu, Zhanyan; Vicini, Stefano

    2009-09-01

    Neuroligins (NLGs) are postsynaptic cell adhesion molecules that are thought to function in synaptogenesis. To investigate the role of NLGs on synaptic transmission once the synapse is formed, we transfected neuroligin-2 (NLG-2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABA(A) (gamma-aminobutyric acid) receptor mediated miniature postsynaptic currents (mIPSCs). NLG-2 transfected cells had mIPSCs with faster decay than matching GFP expressing controls at young culture ages (days in vitro, DIV7-8). Down-regulation of NLG-2 by the isoform specific shRNA-NLG-2 resulted in an opposite effect. We and others have shown that the switch of alpha subunits of GABA(A)Rs from alpha2/3 to alpha1 underlies developmental speeding of the IPSC decay in various CNS regions, including the cerebellum. To assess whether the reduced decay time of mIPSCs by NLG-2 is due to the recruitment of more alpha1 containing GABA(A)Rs at the synapses, we examined the prolongation of current decay by the Zolpidem, which has been shown to preferentially enhance the activity of alpha1 subunit-containing GABA channel. The application of Zolpidem resulted in a significantly greater prolongation kinetics of synaptic currents in NLG-2 over-expressing cells than control cells, suggesting that NLG-2 over-expression accelerates synapse maturation by promoting incorporation of the alpha1 subunit-containing GABA(A)Rs at postsynaptic sites in immature cells. In addition, the effect of NLG-2 on the speeding of decay time course of synaptic currents was abolished when we used CGC cultures from alpha1-/- mice. Lastly, to exclude the possibility that the fast decay of mIPSCs induced by NLG-2 could be also due to the impacts of NLG-2 on the GABA transient in synaptic cleft, we measured the sensitivity of mIPSCs to the fast-off competitive antagonists TPMPA. We found that TPMPA similarly inhibits mIPSCs in control and NLG-2 over-expressing CGCs both at young age (DIV8) and old age (DIV14) of

  7. Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells

    PubMed Central

    Fu, Zhanyan; Vicini, Stefano

    2009-01-01

    Neuroligins (NLGs) are postsynaptic cell adhesion molecules that are thought to function in synaptogenesis. To investigate the role of NLGs on synaptic transmission once the synapse is formed, we transfected neuroligin-2(NLG2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABAA (γ-aminobutyric acid) receptor mediated miniature postsynaptic currents (mISPCs). NLG2 transfected cells had mIPSCs with faster decay than matching GFP expressing controls at young culture ages (days in vitro, DIV 7-8). Down-regulation of NLG2 by the isoform specific shRNA-NLG2 resulted in an opposite effect. We and others have shown that the switch of α subunits of GABAA Rs from α2/3 to α1 underlies developmental speeding of the IPSC decay in various CNS regions, including the cerebellum. To assess whether the reduced decay time of mIPSCs by NLG2 is due to the recruitment of more α1 containing GABAARs at the synapses, we examined the prolongation of current decay by the zolpidem, which has been shown to preferentially enhance the activity of α1 subunit containing GABA channel. The application of zolpidem resulted in a significantly greater prolongation kinetics of synaptic currents in NLG2 over-expressing cells than control cells, suggesting that NLG2 over-expression accelerates synapse maturation by promoting incorporation of the α1 subunit-containing GABAARs at postsynaptic sites in immature cells. In addition, the effect of NLG2 on the speeding of decay time course of synaptic currents was abolished when we used CGC cultures from α1-/- mice. Lastly, to exclude the possibility that the fast decay of mIPSCs induced by NLG2 could be also due to the impacts of NLG2 on the GABA transient in synaptic cleft, we measured the sensitivity of mIPSCs to the fast-off competitive antagonists TPMPA. We found that TPMPA similarly inhibits mIPSCs in control and NLG2 over-expressing CGCs both at young age (DIV8) and old age (DIV14) of cultures. However, we confirm our previous

  8. Bone homing of mesenchymal stem cells by ectopic alpha 4 integrin expression.

    PubMed

    Kumar, Sanjay; Ponnazhagan, Selvarangan

    2007-12-01

    The pluripotent nature of mesenchymal stem cells (MSC) widens their potential for tissue regeneration and as vehicles for cell therapy in molecular medicine. Although the MSC are relatively easier to obtain and propagate in culture, a major impediment remains in their engraftment to target tissues on autologous transfer. We report here that transient, ectopic expression of alpha4 integrin (CD49d) on MSC greatly increases bone homing in an immunocompetent mouse model. Heterodimerization of the alpha4 integrin with endogenous beta1 integrin (CD29) was confirmed to influence this targeting. In addition to retaining their stem cell property, the engrafted MSC were also found to form osteoblasts and osteocytes in the growth plate of recipient mouse limb bones (femur/tibia) in vivo. These findings provide evidence for a novel strategy to achieve bone homing of genetically engineered MSC, which may broadly benefit in targeted therapies for osteopenic bone defects and cancer bone metastasis.

  9. Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma.

    PubMed

    Finkin, Shlomi; Yuan, Detian; Stein, Ilan; Taniguchi, Koji; Weber, Achim; Unger, Kristian; Browning, Jeffrey L; Goossens, Nicolas; Nakagawa, Shigeki; Gunasekaran, Ganesh; Schwartz, Myron E; Kobayashi, Masahiro; Kumada, Hiromitsu; Berger, Michael; Pappo, Orit; Rajewsky, Klaus; Hoshida, Yujin; Karin, Michael; Heikenwalder, Mathias; Ben-Neriah, Yinon; Pikarsky, Eli

    2015-12-01

    Ectopic lymphoid-like structures (ELSs) are often observed in cancer, yet their function is obscure. Although ELSs signify good prognosis in certain malignancies, we found that hepatic ELSs indicated poor prognosis for hepatocellular carcinoma (HCC). We studied an HCC mouse model that displayed abundant ELSs and found that they constituted immunopathological microniches wherein malignant hepatocyte progenitor cells appeared and thrived in a complex cellular and cytokine milieu until gaining self-sufficiency. The egress of progenitor cells and tumor formation were associated with the autocrine production of cytokines previously provided by the niche. ELSs developed via cooperation between the innate immune system and adaptive immune system, an event facilitated by activation of the transcription factor NF-κB and abolished by depletion of T cells. Such aberrant immunological foci might represent new targets for cancer therapy. PMID:26502405

  10. Competition from newborn granule cells does not drive axonal retraction of silenced old granule cells in the adult hippocampus

    PubMed Central

    Lopez, Carla M.; Pelkey, Kenneth A.; Chittajallu, Ramesh; Nakashiba, Toshiaki; Tóth, Katalin; Tonegawa, Susumu; McBain, Chris J.

    2012-01-01

    In the developing nervous system synaptic refinement, typified by the neuromuscular junction where supernumerary connections are eliminated by axon retraction leaving the postsynaptic target innervated by a single dominant input, critically regulates neuronal circuit formation. Whether such competition-based pruning continues in established circuits of mature animals remains unknown. This question is particularly relevant in the context of adult neurogenesis where newborn cells must integrate into preexisting circuits, and thus, potentially compete with functionally mature synapses to gain access to their postsynaptic targets. The hippocampus plays an important role in memory formation/retrieval and the dentate gyrus (DG) subfield exhibits continued neurogenesis into adulthood. Therefore, this region contains both mature granule cells (old GCs) and immature recently born GCs that are generated throughout adult life (young GCs), providing a neurogenic niche model to examine the role of competition in synaptic refinement. Recent work from an independent group in developing animals indicated that embryonically/early postnatal generated GCs placed at a competitive disadvantage by selective expression of tetanus toxin (TeTX) to prevent synaptic release rapidly retracted their axons, and that this retraction was driven by competition from newborn GCs lacking TeTX. In contrast, following 3–6 months of selective TeTX expression in old GCs of adult mice we did not observe any evidence of axon retraction. Indeed ultrastructural analyses indicated that the terminals of silenced GCs even maintained synaptic contact with their postsynaptic targets. Furthermore, we did not detect any significant differences in the electrophysiological properties between old GCs in control and TeTX conditions. Thus, our data demonstrate a remarkable stability in the face of a relatively prolonged period of altered synaptic competition between two populations of neurons within the adult brain

  11. A hypothesis for temporal coding of young and mature granule cells

    PubMed Central

    Rangel, Lara M.; Quinn, Laleh K.; Chiba, Andrea A.; Gage, Fred H.; Aimone, James B.

    2013-01-01

    While it has been hypothesized that adult neurogenesis (NG) plays a role in the encoding of temporal information at long time-scales, the temporal relationship of immature cells to the highly rhythmic network activity of the hippocampus has been largely unexplored. Here, we present a theory for how the activity of immature adult-born granule cells relates to hippocampal oscillations. Our hypothesis is that theta rhythmic (5–10 Hz) excitatory and inhibitory inputs into the hippocampus could differentially affect young and mature granule cells due to differences in intrinsic physiology and synaptic inhibition between the two cell populations. Consequently, immature cell activity may occur at broader ranges of theta phase than the activity of their mature counterparts. We describe how this differential influence on young and mature granule cells could separate the activity of differently aged neurons in a temporal coding regime. Notably, this process could have considerable implications on how the downstream CA3 region interprets the information conveyed by young and mature granule cells. To begin to investigate the phasic behavior of granule cells, we analyzed in vivo recordings of the rat dentate gyrus (DG), observing that the temporal behavior of granule cells with respect to the theta rhythm is different between rats with normal and impaired levels of NG. Specifically, in control animals, granule cells exhibit both strong and weak coupling to the phase of the theta rhythm. In contrast, the distribution of phase relationships in NG-impaired rats is shifted such that they are significantly stronger. These preliminary data support our hypothesis that immature neurons could distinctly affect the temporal dynamics of hippocampal encoding. PMID:23717259

  12. Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice

    SciTech Connect

    Otsuru, Satoru; Tamai, Katsuto . E-mail: tamai@gts.med.osaka-u.ac.jp; Yamazaki, Takehiko; Yoshikawa, Hideki; Kaneda, Yasufumi

    2007-03-09

    Recent studies have suggested the existence of osteoblastic cells in the circulation, but the origin and role of these cells in vivo are not clear. Here, we examined how these cells contribute to osteogenesis in a bone morphogenetic protein (BMP)-induced model of ectopic bone formation. Following lethal dose-irradiation and subsequent green fluorescent protein-transgenic bone marrow cell-transplantation (GFP-BMT) in mice, a BMP-2-containing collagen pellet was implanted into muscle. Three weeks later, a significant number of GFP-positive osteoblastic cells were present in the newly generated ectopic bone. Moreover, peripheral blood mononuclear cells (PBMNCs) from the BMP-2-implanted mouse were then shown to include osteoblast progenitor cells (OPCs) in culture. Passive transfer of the PBMNCs isolated from the BMP-2-implanted GFP-mouse to the BMP-2-implanted nude mouse led to GFP-positive osteoblast accumulation in the ectopic bone. These data provide new insight into the mechanism of ectopic bone formation involving bone marrow-derived OPCs in circulating blood.

  13. Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.

    PubMed

    Sánchez, J; Buendía, A J; Salinas, J; Bernabé, A; Rodolakis, A; Stewart, I J

    1996-09-01

    Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells.

  14. Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.

    PubMed Central

    Sánchez, J; Buendía, A J; Salinas, J; Bernabé, A; Rodolakis, A; Stewart, I J

    1996-01-01

    Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells. PMID:8751945

  15. DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells

    PubMed Central

    Spike, Caroline A.; Bader, Jason; Reinke, Valerie; Strome, Susan

    2008-01-01

    P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs). PMID:18234720

  16. Depletion of primary cilia from mature dentate granule cells impairs hippocampus-dependent contextual memory

    PubMed Central

    Rhee, Soyoung; Kirschen, Gregory W.; Gu, Yan; Ge, Shaoyu

    2016-01-01

    The primary cilium, a sensory organelle, regulates cell proliferation and neuronal development of dentate granule cells in the hippocampus. However, its role in the function of mature dentate granule cells remains unknown. Here we specifically depleted and disrupted ciliary proteins IFT20 and Kif3A (respectively) in mature dentate granule cells and investigated hippocampus-dependent contextual memory and long-term plasticity at mossy fiber synapses. We found that depletion of IFT20 in these cells significantly impaired context-dependent fear-related memory. Furthermore, we tested synaptic plasticity of mossy fiber synapses in area CA3 and found increased long-term potentiation upon depletion of IFT20 or disruption of Kif3A. Our findings suggest a role of primary cilia in the memory function of mature dentate granule cells, which may result from abnormal mossy fiber synaptic plasticity. A direct link between the primary cilia of mature dentate granule cells and behavior will require further investigation using independent approaches to manipulate primary cilia. PMID:27678193

  17. Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity.

    PubMed

    Sutton, Vivien R; Brennan, Amelia J; Ellis, Sarah; Danne, Jill; Thia, Kevin; Jenkins, Misty R; Voskoboinik, Ilia; Pejler, Gunnar; Johnstone, Ricky W; Andrews, Daniel M; Trapani, Joseph A

    2016-03-01

    The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.

  18. The ectopic expression of Snail in MDBK cells does not induce epithelial-mesenchymal transition

    PubMed Central

    IZAWA, GENYA; KOBAYASHI, WAKAKO; HARAGUCHI, MISAKO; SUDO, AKIHARU; OZAWA, MASAYUKI

    2015-01-01

    Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as by the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail expression induces EMT in Madin-Darby canine kidney (MDCK) cells and the human epidermoid carcinoma cell line, A431. Snail is a zinc finger transcription factor and triggers EMT by suppressing E-cadherin expression. In the present study, to broaden our knowledge of Snail-induced EMT, we generated stable Snail transfectants using Madin-Darby bovine kidney (MDBK) cells. Contrary to the MDCK or A431 cells examined in our previous studies, the MDBK cells transfected with the Snail construct maintained an epithelial morphology and showed no sign of reduced cell-cell adhesiveness compared to the control cells. Consistent with these observations, the down-regulation of epithelial marker proteins, e.g. E-cadherin and desmoglein, and the upregulation of mesenchymal marker proteins, e.g., N-cadherin and fibronectin, were not detected. Furthermore, the E-cadherin promoter was not methylated. Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT. As previously demonstrated, in MDCK cells, Snail expression is accompanied by the increased expression of other EMT-inducing transcription factors, e.g., Slug and zinc finger E-box-binding homeobox 1 (ZEB1). However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors. Thus, it is possible that the failure to upregulate other EMT-related transcription factors may explain the lack of Snail-mediated induction of EMT in MDBK cells. PMID:25998899

  19. The ectopic expression of Snail in MDBK cells does not induce epithelial-mesenchymal transition.

    PubMed

    Izawa, Genya; Kobayashi, Wakako; Haraguchi, Misako; Sudo, Akiharu; Ozawa, Masayuki

    2015-07-01

    Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as by the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail expression induces EMT in Madin-Darby canine kidney (MDCK) cells and the human epidermoid carcinoma cell line, A431. Snail is a zinc finger transcription factor and triggers EMT by suppressing E-cadherin expression. In the present study, to broaden our knowledge of Snail‑induced EMT, we generated stable Snail transfectants using Madin-Darby bovine kidney (MDBK) cells. Contrary to the MDCK or A431 cells examined in our previous studies, the MDBK cells transfected with the Snail construct maintained an epithelial morphology and showed no sign of reduced cell-cell adhesiveness compared to the control cells. Consistent with these observations, the downregulation of epithelial marker proteins, e.g. E-cadherin and desmoglein, and the upregulation of mesenchymal marker proteins, e.g., N-cadherin and fibronectin, were not detected. Furthermore, the E-cadherin promoter was not methylated. Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT. As previously demonstrated, in MDCK cells, Snail expression is accompanied by the increased expression of other EMT-inducing transcription factors, e.g., Slug and zinc finger E-box-binding homeobox 1 (ZEB1). However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors. Thus, it is possible that the failure to upregulate other EMT-related transcription factors may explain the lack of Snail-mediated induction of EMT in MDBK cells.

  20. Olfactory experiences dynamically regulate plasticity of dendritic spines in granule cells of Xenopus tadpoles in vivo

    PubMed Central

    Zhang, Li; Huang, Yubin; Hu, Bing

    2016-01-01

    Granule cells, rich in dendrites with densely punctated dendritic spines, are the most abundant inhibitory interneurons in the olfactory bulb. The dendritic spines of granule cells undergo remodeling during the development of the nervous system. The morphological plasticity of the spines’ response to different olfactory experiences in vivo is not fully known. In initial studies, a single granule cell in Xenopus tadpoles was labeled with GFP plasmids via cell electroporation; then, morphologic changes of the granule cell spines were visualized by in vivo confocal time-lapse imaging. With the help of long-term imaging, the total spine density, dynamics, and stability of four types of dendritic spines (mushroom, stubby, thin and filopodia) were obtained. Morphological analysis demonstrated that odor enrichment produced a remarkable increase in the spine density and stability of large mushroom spine. Then, with the help of short-term imaging, we analyzed the morphological transitions among different spines. We found that transitions between small spines (thin and filopodia) were more easily influenced by odor stimulation or olfactory deprivation. These results indicate that different olfactory experiences can regulate the morphological plasticity of different dendritic spines in the granule cell. PMID:27713557

  1. Ectopic porcine spermatogenesis in murine subcutis: tissue grafting versus cell-injection methods

    PubMed Central

    Watanabe, Takeshi; Hayashi, Hirofumi; Kita, Kaoru; Kubota, Yoshinobu; Ogawa, Takehiko

    2009-01-01

    Fragments of testis tissue from immature animals grow and develop spermatogenesis when grafted onto subcutaneous areas of immunodeficient mice. The same results are obtained when dissociated cells from immature testes of rodents are injected into the subcutis of nude mice. Those cells reconstitute seminiferous tubules and facilitate spermatogenesis. We compared these two methods, tissue grafting and cell-injection methods, in terms of the efficiency of spermatogenesis in the backs of three strains of immunodeficient mice, using neonatal porcine testicular tissues and cells as donor material. Nude, severe combined immunodeficient (SCID) and NOD/Shi-SCID, IL-2Rγcnull (NOG) mice were used as recipients. At 10 months after surgery, the transplants were examined histologically. Both grafting and cell-injection methods resulted in porcine spermatogenesis on the backs of recipient mice; the percentage of spermatids present in the transplants was 67% and 22%, respectively. Using the grafting method, all three strains of mice supported the same extent of spermatogenesis. As for the cell-injection method, although SCID mice were the best host for supporting reconstitution and spermatogenesis, any difference from the other strains was not significant. As NOG mice did not show any better results, the severity of immunodeficiency seemed to be irrelevant for supporting xeno-ectopic spermatogenesis. Our results confirmed that tubular reconstitution is applicable to porcine testicular cells. This method as well as the grafting method would be useful for studying spermatogenesis in different kinds of animals. PMID:19137001

  2. Ectopic micronodular thymoma with lymphoid stroma in the cervical region: a rare case associated with Langerhans cells proliferation.

    PubMed

    Yu, Min; Meng, Yuan; Xu, Bin; Zhao, Lin; Zhang, Qingfu

    2016-01-01

    Micronodular thymoma (MNT) with lymphoid stroma is a rare thymic epithelial neoplasm with the characteristics of multiple nodules separated by abundant lymphoid stroma. MNTs mainly arise in the anterior mediastinum and thymus, while ectopic MNTs are extremely rarely seen. Here, we report an ectopic MNT that occurred in the neck of a 62-year-old woman. There were also scattered eosinophilic granulocytes and S100(+)/CD1a(+) Langerhans cells within the tumor. This case provides a better understanding of such rare, poorly understood cases. PMID:27486334

  3. Ectopic micronodular thymoma with lymphoid stroma in the cervical region: a rare case associated with Langerhans cells proliferation

    PubMed Central

    Yu, Min; Meng, Yuan; Xu, Bin; Zhao, Lin; Zhang, Qingfu

    2016-01-01

    Micronodular thymoma (MNT) with lymphoid stroma is a rare thymic epithelial neoplasm with the characteristics of multiple nodules separated by abundant lymphoid stroma. MNTs mainly arise in the anterior mediastinum and thymus, while ectopic MNTs are extremely rarely seen. Here, we report an ectopic MNT that occurred in the neck of a 62-year-old woman. There were also scattered eosinophilic granulocytes and S100+/CD1a+ Langerhans cells within the tumor. This case provides a better understanding of such rare, poorly understood cases. PMID:27486334

  4. Small-cell Lung Cancer in a Young Adult Nonsmoking Patient with Ectopic Adrenocorticotropin (ACTH) Production.

    PubMed

    Aoki, Masahiko; Fujisaka, Yasuhito; Tokioka, Satoshi; Hirai, Ai; Henmi, Yujiro; Inoue, Yosuke; Narabayashi, Ken; Yamano, Takeshi; Tamura, Yosuke; Egashira, Yutaro; Higuchi, Kazuhide

    2016-01-01

    Cushing's syndrome due to young small-cell lung cancer (SCLC) is recognized as being extremely rare. We herein present the case of a 35-year-old nonsmoking man who presented with thirst and polyuria. Laboratory examinations showed hyperglycemia, hypokalemia and liver enzyme elevation. Imaging examinations revealed the presence of multiple liver tumors and lymph node swelling. The levels of serum neuroendocrine tumor markers were elevated. The patient was diagnosed with SCLC based on the pathological examination of a biopsy specimen from the right supraclavicular lymph node. The physical findings, including proximal myopathy, truncal obesity and pigmentation suggested high levels of glucocorticoids. An immunohistochemical examination of the tumor showed that it was positive for adrenocorticotropin (ACTH). An endocrinological investigation allowed for the definitive diagnosis of SCLC with ectopic ACTH production. PMID:27181543

  5. Ectopic recombination within homologous immunoglobulin mu gene constant regions in a mouse hybridoma cell line.

    PubMed Central

    Baker, M D; Read, L R

    1992-01-01

    We have transferred a pSV2neo vector containing the wild-type constant region of the immunoglobulin mu gene (C mu) into the mutant hybridoma igm482, which bears a 2-bp deletion in the third constant-region exon of its haploid chromosomal mu gene (C mu 3). Independent igm482 transformants contain the wild-type immunoglobulin C mu region stably integrated in ectopic chromosomal positions. We report here that the wild-type immunoglobulin C mu region can function as the donor sequence in a gene conversion event which corrects the 2-bp deletion in the mutant igm482 chromosomal C mu 3 exon. The homologous recombination event restores normal immunoglobulin M production in the mutant cell. Images PMID:1406631

  6. Protein kinases are associated with multiple, distinct cytoplasmic granules in quiescent yeast cells.

    PubMed

    Shah, Khyati H; Nostramo, Regina; Zhang, Bo; Varia, Sapna N; Klett, Bethany M; Herman, Paul K

    2014-12-01

    The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival.

  7. Protein Kinases Are Associated with Multiple, Distinct Cytoplasmic Granules in Quiescent Yeast Cells

    PubMed Central

    Shah, Khyati H.; Nostramo, Regina; Zhang, Bo; Varia, Sapna N.; Klett, Bethany M.; Herman, Paul K.

    2014-01-01

    The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival. PMID:25342717

  8. Dentate Gyrus Granule Cell Firing Patterns Can Induce Mossy Fiber Long-Term Potentiation In Vitro

    PubMed Central

    Mistry, Rajen; Dennis, Siobhan; Frerking, Matthew; Mellor, Jack R.

    2010-01-01

    Hippocampal granule cells transmit information about behaviourally-relevant stimuli to CA3 pyramidal cells via mossy fiber synapses. These synapses express a form of long-term potentiation (mfLTP) that is non-Hebbian and does not require NMDA receptors. mfLTP is thought to be induced and expressed presynaptically, hence, the major determinant of whether mfLTP occurs is activity in the granule cells. However, it remains unclear whether mfLTP can be induced by activity patterns that granule cells exhibit in vivo, and — if so — what context generates these patterns. To address these issues, we examined granule cell activity from in vivo recordings from rats during performance of a delayed nonmatch-to-sample (DNMS) task and found that granule cells exhibit a wide range of spike patterns. In vitro slice experiments in mice demonstrated that some, but not all, of these patterns of activity could induce mfLTP. By further defining the activity thresholds for mfLTP in hippocampal slices, we found that mfLTP can only be induced by spike patterns that fire in high frequency bursts with a low average firing frequency. Using this information, we then screened for supra-threshold bursts of activity during the DNMS task. In a subset of cells, supra-threshold bursts occurred preferentially during the sampling phase of the task. If supra-threshold bursting took place later, during the delay phase, task performance was disrupted. We conclude that mfLTP can be induced by granule cell spike patterns during a memory task, and that the timing of mfLTP induction can predict task performance. PMID:20635414

  9. Identification of miRNAs differentially expressed in human epilepsy with or without granule cell pathology.

    PubMed

    Zucchini, Silvia; Marucci, Gianluca; Paradiso, Beatrice; Lanza, Giovanni; Roncon, Paolo; Cifelli, Pierangelo; Ferracin, Manuela; Giulioni, Marco; Michelucci, Roberto; Rubboli, Guido; Simonato, Michele

    2014-01-01

    The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets.

  10. Identification of miRNAs Differentially Expressed in Human Epilepsy with or without Granule Cell Pathology

    PubMed Central

    Paradiso, Beatrice; Lanza, Giovanni; Roncon, Paolo; Cifelli, Pierangelo; Ferracin, Manuela; Giulioni, Marco; Michelucci, Roberto; Simonato, Michele

    2014-01-01

    The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets. PMID:25148080

  11. Seizure-Induced Motility of Differentiated Dentate Granule Cells Is Prevented by the Central Reelin Fragment.

    PubMed

    Orcinha, Catarina; Münzner, Gert; Gerlach, Johannes; Kilias, Antje; Follo, Marie; Egert, Ulrich; Haas, Carola A

    2016-01-01

    Granule cell dispersion (GCD) represents a pathological widening of the granule cell layer in the dentate gyrus and it is frequently observed in patients with mesial temporal lobe epilepsy (MTLE). Recent studies in human MTLE specimens and in animal epilepsy models have shown that a decreased expression and functional inactivation of the extracellular matrix protein Reelin correlates with GCD formation, but causal evidence is still lacking. Here, we used unilateral kainate (KA) injection into the mouse hippocampus, an established MTLE animal model, to precisely map the loss of reelin mRNA-synthesizing neurons in relation to GCD along the septotemporal axis of the epileptic hippocampus. We show that reelin mRNA-producing neurons are mainly lost in the hilus and that this loss precisely correlates with the occurrence of GCD. To monitor GCD formation in real time, we used organotypic hippocampal slice cultures (OHSCs) prepared from mice which express enhanced green fluorescent protein (eGFP) primarily in differentiated dentate granule cells. Using life cell microscopy we observed that increasing doses of KA resulted in an enhanced motility of eGFP-positive granule cells. Moreover, KA treatment of OHSC resulted in a rapid loss of Reelin-producing interneurons mainly in the hilus, as observed in vivo. A detailed analysis of the migration behavior of individual eGFP-positive granule cells revealed that the majority of these neurons actively migrate toward the hilar region, where Reelin-producing neurons are lost. Treatment with KA and subsequent addition of the recombinant R3-6 Reelin fragment significantly prevented the movement of eGFP-positive granule cells. Together, these findings suggest that GCD formation is indeed triggered by a loss of Reelin in hilar interneurons. PMID:27516734

  12. Seizure-Induced Motility of Differentiated Dentate Granule Cells Is Prevented by the Central Reelin Fragment

    PubMed Central

    Orcinha, Catarina; Münzner, Gert; Gerlach, Johannes; Kilias, Antje; Follo, Marie; Egert, Ulrich; Haas, Carola A.

    2016-01-01

    Granule cell dispersion (GCD) represents a pathological widening of the granule cell layer in the dentate gyrus and it is frequently observed in patients with mesial temporal lobe epilepsy (MTLE). Recent studies in human MTLE specimens and in animal epilepsy models have shown that a decreased expression and functional inactivation of the extracellular matrix protein Reelin correlates with GCD formation, but causal evidence is still lacking. Here, we used unilateral kainate (KA) injection into the mouse hippocampus, an established MTLE animal model, to precisely map the loss of reelin mRNA-synthesizing neurons in relation to GCD along the septotemporal axis of the epileptic hippocampus. We show that reelin mRNA-producing neurons are mainly lost in the hilus and that this loss precisely correlates with the occurrence of GCD. To monitor GCD formation in real time, we used organotypic hippocampal slice cultures (OHSCs) prepared from mice which express enhanced green fluorescent protein (eGFP) primarily in differentiated dentate granule cells. Using life cell microscopy we observed that increasing doses of KA resulted in an enhanced motility of eGFP-positive granule cells. Moreover, KA treatment of OHSC resulted in a rapid loss of Reelin-producing interneurons mainly in the hilus, as observed in vivo. A detailed analysis of the migration behavior of individual eGFP-positive granule cells revealed that the majority of these neurons actively migrate toward the hilar region, where Reelin-producing neurons are lost. Treatment with KA and subsequent addition of the recombinant R3–6 Reelin fragment significantly prevented the movement of eGFP-positive granule cells. Together, these findings suggest that GCD formation is indeed triggered by a loss of Reelin in hilar interneurons. PMID:27516734

  13. Seizure-Induced Motility of Differentiated Dentate Granule Cells Is Prevented by the Central Reelin Fragment.

    PubMed

    Orcinha, Catarina; Münzner, Gert; Gerlach, Johannes; Kilias, Antje; Follo, Marie; Egert, Ulrich; Haas, Carola A

    2016-01-01

    Granule cell dispersion (GCD) represents a pathological widening of the granule cell layer in the dentate gyrus and it is frequently observed in patients with mesial temporal lobe epilepsy (MTLE). Recent studies in human MTLE specimens and in animal epilepsy models have shown that a decreased expression and functional inactivation of the extracellular matrix protein Reelin correlates with GCD formation, but causal evidence is still lacking. Here, we used unilateral kainate (KA) injection into the mouse hippocampus, an established MTLE animal model, to precisely map the loss of reelin mRNA-synthesizing neurons in relation to GCD along the septotemporal axis of the epileptic hippocampus. We show that reelin mRNA-producing neurons are mainly lost in the hilus and that this loss precisely correlates with the occurrence of GCD. To monitor GCD formation in real time, we used organotypic hippocampal slice cultures (OHSCs) prepared from mice which express enhanced green fluorescent protein (eGFP) primarily in differentiated dentate granule cells. Using life cell microscopy we observed that increasing doses of KA resulted in an enhanced motility of eGFP-positive granule cells. Moreover, KA treatment of OHSC resulted in a rapid loss of Reelin-producing interneurons mainly in the hilus, as observed in vivo. A detailed analysis of the migration behavior of individual eGFP-positive granule cells revealed that the majority of these neurons actively migrate toward the hilar region, where Reelin-producing neurons are lost. Treatment with KA and subsequent addition of the recombinant R3-6 Reelin fragment significantly prevented the movement of eGFP-positive granule cells. Together, these findings suggest that GCD formation is indeed triggered by a loss of Reelin in hilar interneurons.

  14. Luciferase labeling for multipotent stromal cell tracking in spinal fusion versus ectopic bone tissue engineering in mice and rats.

    PubMed

    Geuze, Ruth E; Prins, Henk-Jan; Öner, F Cumhur; van der Helm, Yvonne J M; Schuijff, Leontine S; Martens, Anton C; Kruyt, Moyo C; Alblas, Jacqueline; Dhert, Wouter J A

    2010-11-01

    Tissue engineering of bone, by combining multipotent stromal cells (MSCs) with osteoconductive scaffolds, has not yet yielded any clinically useful applications so far. The fate and contribution of the seeded cells are not sufficiently clarified, especially at clinically relevant locations. Therefore, we investigated cell proliferation around the spine and at ectopic sites using noninvasive in vivo bioluminescence imaging (BLI) in relation to new bone formation. Goat MSCs were lentivirally transduced to express luciferase. After showing both correlation between MSC viability and BLI signal as well as survival and osteogenic capacity of these cells ectopically in mice, they were seeded on ceramic scaffolds and implanted in immunodeficient rats at two levels in the spine for spinal fusion as well as subcutaneously. Nontransduced MSCs were used as a control group. All rats were monitored at day 1 and after that weekly until termination at week 7. In mice a BLI signal was observed during the whole observation period, indicating survival of the seeded MSCs, which was accompanied by osteogenic differentiation in vivo. However, these same MSCs showed a different response in the rat model, where the BLI signal was present until day 14, both in the spine and ectopically, indicating that MSCs were able to survive at least 2 weeks of implantation. Only when the signal was still present after the total implantation period ectopically, which only occurred in one rat, new bone was formed extensively and the implanted MSCs were responsible for this bone formation. Ectopically, neither a reduced proliferative group (irradiated) nor a group in which the cells were devitalized by liquid nitrogen and the produced extracellular matrix remained (matrix group) resulted in bone formation. This suggests that the release of soluble factors or the presence of an extracellular matrix is not enough to induce bone formation. For the spinal location, the question remains whether the implanted

  15. Ectopic pregnancy

    MedlinePlus

    Tubal pregnancy; Cervical pregnancy; Tubal ligation - ectopic pregnancy ... In most pregnancies, the fertilized egg travels through the fallopian tube to the womb (uterus). If the movement of the egg ...

  16. Dependence of structure stability and integrity of aerobic granules on ATP and cell communication.

    PubMed

    Jiang, Bo; Liu, Yu

    2013-06-01

    Aerobic granules are dense and compact microbial aggregates with various bacterial species. Recently, aerobic granulation technology has been extensively explored for treatment of municipal and industrial wastewaters. However, little information is currently available with regard to their structure stability and integrity at levels of energy metabolism and cell communication. In the present study, a typical chemical uncoupler, 3,3',4',5-tetrachlorosalicylanilide with the power to dissipate proton motive force and subsequently inhibit adenosine triphosphate (ATP) generation, was used to investigate possible roles of ATP and cell communication in maintaining the structure stability and integrity of aerobic granules. It was found that inhibited ATP synthesis resulted in the reduced production of autoinducer-2 and N-acylhomoserine lactones essential for cell communication, while lowered extracellular polymeric substance (EPS) production was also observed. As a consequence, aerobic granules appeared to break up. This study showed that ATP-dependent quorum sensing and EPS were essential for sustaining the structure stability and integrity of aerobic granules. PMID:23011346

  17. Stimulation of mast cells leads to cholesterol accumulation in macrophages in vitro by a mast cell granule-mediated uptake of low density lipoprotein

    SciTech Connect

    Kokkonen, J.O.; Kovanen, P.T.

    1987-04-01

    The uptake of low density lipoprotein (LDL) by cultured mouse macrophages was markedly promoted by isolated rat mast cell granules present in the culture medium. The granule-mediated uptake of /sup 125/I-LDL enhanced the rate of cholesteryl ester synthesis in the macrophages, the result being accumulation of cholesteryl esters in these cells. Binding of LDL to the granules was essential for the granule-mediated uptake of LDL by macrophages, for the uptake process was prevented by treating the granules with avidin or protamine chloride or by treating LDL with 1,2-cyclohexanedione, all of which inhibit the binding of LDL to the granules. Inhibition of granule phagocytosis by the macrophages with cytochalasin B also abolished the granule-mediated uptake of LDL. Finally, mouse macrophage monolayers and LDL were incubated in the presence of isolated rat serosal mast cells. Stimulation of the mast cells with compound 48/80, a degranulating agent, resulted in dose-dependent release of secretory granules from the mast cells and a parallel increase in /sup 14/C cholesteryl ester synthesis in the macrophages. The results show that, in this in vitro model, the sequence of events leading to accumulation of cholesteryl esters in macrophages involves initial stimulation of mast cells, subsequent release of their secretory granules, binding of LDL to the exocytosed granules, and, finally, phagocytosis of the LDL-containing granules by macrophages.

  18. Hematopoietic Stem Cell Regeneration Enhanced by Ectopic Expression of ROS-detoxifying Enzymes in Transplant Mice

    PubMed Central

    Miao, Weimin; XuFeng, Richard; Park, Moo-Rim; Gu, Haihui; Hu, Linping; Kang, Jin Wook; Ma, Shihui; Liang, Paulina H; Li, Yanxin; Cheng, Haizi; Yu, Hui; Epperly, Michael; Greenberger, Joel; Cheng, Tao

    2013-01-01

    High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo. PMID:23295952

  19. Role of the planar cell polarity pathway in regulating ectopic hair cell-like cells induced by Math1 and testosterone treatment.

    PubMed

    Yang, Xiao-Yu; Jin, Kai; Ma, Rui; Yang, Juan-Mei; Luo, Wen-Wei; Han, Zhao; Cong, Ning; Ren, Dong-Dong; Chi, Fang-Lu

    2015-07-30

    Planar cell polarity (PCP) signaling regulates cochlear extension and coordinates orientation of sensory hair cells in the inner ear. Retroviral-mediated introduction of the Math1 transcription factor leads to the transdifferentiation of some mature supporting cells into hair cells. Testosterone, a gonadal sex steroid hormone, is associated with neuroprotection and regeneration in Central Nervous System (CNS) development. Experiments were performed in vitro using Ad5-EGFP-Math1/Ad5-Math1 in neonatal mouse cochleas. Establishment of ectopic hair-cell like cell(HCLC) polarity in the lesser epithelial ridge (LER) with or without testosterone-3-(O-carboxymethyl) oxime bovine serum albumin (testosterone-BSA) treatment was investigated to determine the role of the PCP pathway in regulating ectopic regenerated (HCLCs) through induction by Math1 and testosterone treatment. After Math1 infection, new ectopic regenerated HCLCs were detected in the LER. After the HCLCs developed actin-rich stereocilia, the basal bodies moved from the center to the distal side. Moreover, the narrower, non-sensory LER region meant that the convergent extension (CE) was also established after transfection with Math1. After 9 days of in vitro testosterone-BSA treatment, more Edu(+), Sox2(+), and HCLC cells were observed in the LER with an accompanying downregulation of E-cadherin. Interestingly, the CE of the Ad5-EGFP-math1 treated LER is altered, but the intrinsic cellular polarity of the HCLCs is not obviously changed. In summary, our results indicate that PCP signaling is involved in the development of ectopic HCLCs and the CE of the ectopic sensory region is altered by testosterone-BSA through downregulation of cell-cell adhesion. Testosterone-BSA and Math1 treatment could promote an increase in HCLCs in the LER through proliferation and transdifferentiation.

  20. Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells.

    PubMed

    Giraud, Matthieu; Yoshida, Hideyuki; Abramson, Jakub; Rahl, Peter B; Young, Richard A; Mathis, Diane; Benoist, Christophe

    2012-01-10

    Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.

  1. Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells*

    PubMed Central

    Cabeza, Jose M.; Acosta, Jorge; Alés, Eva

    2013-01-01

    In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified “large-capacitance flickers” that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent “compound cavicapture,” most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event. PMID:23709219

  2. Degranulation of eosinophilic granule cells in neurofibromas and gastrointestinal tract in the bicolor damselfish.

    PubMed

    Schmale, Michael C; Vicha, Dale; Cacal, Saul M

    2004-07-01

    Damselfish neurofibromatosis (DNF) is a neoplastic disease affecting bicolor damselfish (Stegastes partitus Poey) on Florida reefs. Previous studies have demonstrated high densities of eosinophilic granule containing cells (EGC), the proposed equivalent of mast cells in fishes, in neurofibromas and malignant peripheral nerve sheath tumors (mpnst) in DNF. These lesions are similar to those in the disease neurofibromatosis type 1 (NF1) in humans, which contain large numbers of mast cells. In the present study, experiments were conducted to measure the response of EGC in these tumors as well as in the submucosa of the digestive tract to the mast cell degranulating agent compound 48/80. Degranulation of these cells was visible by light microscopy and characterized by conspicuous swelling of granules and often by the presence of free granules adjacent to the EGC. Degranulation occurred by release of intact granules (diacytosis), as reported in other fishes, rather than by fusion of granules with the cell membrane (exocytosis) as reported in mast cells in mammals. Baseline levels of EGC exhibiting degranulation ranged from 20-26% in the submucosa to 30% in tumors. Within 1-2h of exposure to compound 48/80, significant increases in average levels of degranulation were observed, to 67% in the gut and 72% in tumors. Degranulation was significantly more extensive in the tumors than in the gut. The outermost edges of the tumors contained significantly higher densities of EGC but these cells exhibited lower rates of degranulation than those in the inner regions of tumors. These observations support the hypothesis that the EGC present in neurofibromas and mpnst in DNF are equivalent to the mast cell component in neurofibromas in NF1.

  3. TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells.

    PubMed

    Ohara-Imaizumi, Mica; Nishiwaki, Chiyono; Kikuta, Toshiteru; Nagai, Shintaro; Nakamichi, Yoko; Nagamatsu, Shinya

    2004-07-01

    We imaged and analysed the motion of single insulin secretory granules near the plasma membrane in live pancreatic beta-cells, from normal and diabetic Goto-Kakizaki (GK) rats, using total internal reflection fluorescence microscopy (TIRFM). In normal rat primary beta-cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from 'newcomers'. In diabetic GK rat beta-cells, the number of fusion events from previously docked granules were markedly reduced, and, in contrast, the fusion from newcomers was still preserved. The dynamic change in the number of docked insulin granules showed that, in GK rat beta-cells, the total number of docked insulin granules was markedly decreased to 35% of the initial number after glucose stimulation. Immunohistochemistry with anti-insulin antibody observed by TIRFM showed that GK rat beta-cells had a marked decline of endogenous insulin granules docked to the plasma membrane. Thus our results indicate that the decreased number of docked insulin granules accounts for the impaired insulin release during the first phase of insulin release in diabetic GK rat beta-cells.

  4. Addition of Adipose-Derived Stem Cells to Mesenchymal Stem Cell Sheets Improves Bone Formation at an Ectopic Site

    PubMed Central

    Wang, Zhifa; Li, Zhijin; Dai, Taiqiang; Zong, Chunlin; Liu, Yanpu; Liu, Bin

    2016-01-01

    To determine the effect of adipose-derived stem cells (ADSCs) added to bone marrow-derived mesenchymal stem cell (MSC) sheets on bone formation at an ectopic site. We isolated MSCs and ADSCs from the same rabbits. We then prepared MSC sheets for implantation with or without ADSCs subcutaneously in the backs of severe combined immunodeficiency (SCID) mice. We assessed bone formation at eight weeks after implantation by micro-computed tomography and histological analysis. In osteogenic medium, MSCs grew to form multilayer sheets containing many calcium nodules. MSC sheets without ADSCs formed bone-like tissue; although neo-bone and cartilage-like tissues were sparse and unevenly distributed by eight weeks after implantation. In comparison, MSC sheets with ADSCs promoted better bone regeneration as evidenced by the greater density of bone, increased mineral deposition, obvious formation of blood vessels, large number of interconnected ossified trabeculae and woven bone structures, and greater bone volume/total volume within the composite constructs. Our results indicate that although sheets of only MSCs have the potential to form tissue engineered bone at an ectopic site, the addition of ADSCs can significantly increase the osteogenic potential of MSC sheets. Thus, the combination of MSC sheets with ADSCs may be regarded as a promising therapeutic strategy to stimulate bone regeneration. PMID:26848656

  5. Addition of Adipose-Derived Stem Cells to Mesenchymal Stem Cell Sheets Improves Bone Formation at an Ectopic Site.

    PubMed

    Wang, Zhifa; Li, Zhijin; Dai, Taiqiang; Zong, Chunlin; Liu, Yanpu; Liu, Bin

    2016-01-01

    To determine the effect of adipose-derived stem cells (ADSCs) added to bone marrow-derived mesenchymal stem cell (MSC) sheets on bone formation at an ectopic site. We isolated MSCs and ADSCs from the same rabbits. We then prepared MSC sheets for implantation with or without ADSCs subcutaneously in the backs of severe combined immunodeficiency (SCID) mice. We assessed bone formation at eight weeks after implantation by micro-computed tomography and histological analysis. In osteogenic medium, MSCs grew to form multilayer sheets containing many calcium nodules. MSC sheets without ADSCs formed bone-like tissue; although neo-bone and cartilage-like tissues were sparse and unevenly distributed by eight weeks after implantation. In comparison, MSC sheets with ADSCs promoted better bone regeneration as evidenced by the greater density of bone, increased mineral deposition, obvious formation of blood vessels, large number of interconnected ossified trabeculae and woven bone structures, and greater bone volume/total volume within the composite constructs. Our results indicate that although sheets of only MSCs have the potential to form tissue engineered bone at an ectopic site, the addition of ADSCs can significantly increase the osteogenic potential of MSC sheets. Thus, the combination of MSC sheets with ADSCs may be regarded as a promising therapeutic strategy to stimulate bone regeneration.

  6. Birbeck Granules Are Subdomains of Endosomal Recycling Compartment in Human Epidermal Langerhans Cells, Which Form Where Langerin Accumulates

    PubMed Central

    Mc Dermott, Ray; Ziylan, Umit; Spehner, Danièle; Bausinger, Huguette; Lipsker, Dan; Mommaas, Mieke; Cazenave, Jean-Pierre; Raposo, Graça; Goud, Bruno; de la Salle, Henri; Salamero, Jean; Hanau, Daniel

    2002-01-01

    Birbeck granules are unusual rod-shaped structures specific to epidermal Langerhans cells, whose origin and function remain undetermined. We investigated the intracellular location and fate of Langerin, a protein implicated in Birbeck granule biogenesis, in human epidermal Langerhans cells. In the steady state, Langerin is predominantly found in the endosomal recycling compartment and in Birbeck granules. Langerin internalizes by classical receptor-mediated endocytosis and the first Birbeck granules accessible to endocytosed Langerin are those connected to recycling endosomes in the pericentriolar area, where Langerin accumulates. Drug-induced inhibition of endocytosis results in the appearance of abundant open-ended Birbeck granule-like structures appended to the plasma membrane, whereas inhibition of recycling induces Birbeck granules to merge with a tubular endosomal network. In mature Langerhans cells, Langerin traffic is abolished and the loss of internal Langerin is associated with a concomitant depletion of Birbeck granules. Our results demonstrate an exchange of Langerin between early endosomal compartments and the plasma membrane, with dynamic retention in the endosomal recycling compartment. They show that Birbeck granules are not endocytotic structures, rather they are subdomains of the endosomal recycling compartment that form where Langerin accumulates. Finally, our results implicate ADP-ribosylation factor proteins in Langerin trafficking and the exchange between Birbeck granules and other endosomal membranes. PMID:11809842

  7. Synaptic action of ethanol on cerebellar auditory granule cells reveals acute tolerance

    SciTech Connect

    Huang, C.M.; Liu, G.; Huang, R.H. )

    1991-03-11

    The cerebellum is very sensitive to acute intoxication by ethanol. The authors have recorded electrophysiological responses of granule cells to auditory stimulation from the posterior cerebellar vermis of cats before and after a relatively low dose of ethanol. Auditory responses of granule cells were severely inhibited by ethanol at a transient, peak ethanol concentration of 15-18 mM in the cerebrospinal fluid (CSF). Thereafter, the clearance of ethanol from CSF followed an exponential time course, with 50% of the CSF ethanol being cleared with every passing hour. Auditory responses of granule cells returned to control levels within 60-90 minutes, despite the presence of a DSF ethanol concentration at 8-10mM, indicating acute tolerance. Moreover, a second, identical dose of ethanol, delivered two hours after the first dose produced an attenuated inhibition in the auditory response of cerebellar granule cells. The inhibition took a longer time to be evident but a shorter time to recover than that followed by the first dose of ethanol.

  8. Structural, mass and elemental analyses of storage granules in methanogenic archaeal cells

    PubMed Central

    Toso, Daniel B.; Henstra, Anne M.; Gunsalus, Robert P.; Zhou, Z. Hong

    2013-01-01

    Summary Storage granules are an important component of metabolism in many organisms spanning the bacterial, eukaryal and archaeal domains, but systematic analysis of their organization inside cells is lacking. In this study, we identify and characterize granulelike inclusion bodies in a methanogenic archaeon, Methanospirillum hungatei, an anaerobic microorganism that plays an important role in nutrient recycling in the ecosystem. Using cryo electron microscopy, we show that granules in mature M. hungatei are amorphous in structure with a uniform size. Energy dispersive X-ray spectroscopy analysis establishes that each granule is a polyphosphate body (PPB) that consists of high concentrations of phosphorous and oxygen, and increased levels of iron and magnesium. By scanning transmission electron tomography, we further estimate that the mass density within a PPB is a little less than metal titanium at room temperature and is about four times higher than that of the surrounding cytoplasm. Finally, three-dimensional cryo electron tomography reveals that PPBs are positioned off-centre in their radial locations relative to the cylindrical axis of the cell, and almost uniformly placed near cell ends. This positioning ability points to a genetic program that spatially and temporally directs the accumulation of polyphosphate into a storage granule, perhaps for energy-consuming activities, such as cell maintenance, division or motility. PMID:21854518

  9. Rat basophilic leukemia cells express syntaxin-3 and VAMP-7 in granule membranes.

    PubMed

    Hibi, T; Hirashima, N; Nakanishi, M

    2000-04-29

    In neuronal cells, it is generally agreed that SNARE proteins underlie the release of neurotransmitter. It is controversial, however, whether they also work functionally in the degranulation of RBL-2H3 cells because the expression of SNARE proteins has not been confirmed and the degranulation is not inhibited by tetanus toxin which cleaves one of SNARE proteins, VAMP-2. We investigated the expression and the localization of SNARE proteins including VAMP-7 which is insensitive to tetanus toxin. RT-PCR analysis showed the existence of SNARE proteins, including syntaxin-2, -3, -4, SNAP-23, VAMP-2, and VAMP-7. Experiments using GFP-conjugated proteins revealed that VAMP-7 was localized only in granule membranes, whereas syntaxin-3 was in both the plasma and granule membranes. Upon antigen stimulation, these proteins in granule membranes moved to the cell surface due to the fusion of granules with the plasma membrane. The results suggest the involvement of SNARE proteins in the degranulation of RBL-2H3 cells.

  10. Morphological changes among hippocampal dentate granule cells exposed to early kindling-epileptogenesis.

    PubMed

    Singh, Shatrunjai P; He, Xiaoping; McNamara, James O; Danzer, Steve C

    2013-12-01

    Temporal lobe epilepsy is associated with changes in the morphology of hippocampal dentate granule cells. These changes are evident in numerous models that are associated with substantial neuron loss and spontaneous recurrent seizures. By contrast, previous studies have shown that in the kindling model, it is possible to administer a limited number of stimulations sufficient to produce a lifelong enhanced sensitivity to stimulus evoked seizures without associated spontaneous seizures and minimal neuronal loss. Here we examined whether stimulation of the amygdala sufficient to evoke five convulsive seizures (class IV or greater on Racine's scale) produce morphological changes similar to those observed in models of epilepsy associated with substantial cell loss. The morphology of GFP-expressing granule cells from Thy-1 GFP mice was examined either 1 day or 1 month after the last evoked seizure. Interestingly, significant reductions in dendritic spine density were evident 1 day after the last seizure, the magnitude of which had diminished by 1 month. Further, there was an increase in the thickness of the granule cell layer 1 day after the last evoked seizure, which was absent a month later. We also observed an increase in the area of the proximal axon, which again returned to control levels a month later. No differences in the number of basal dendrites were detected at either time point. These findings demonstrate that the early stages of kindling epileptogenesis produce transient changes in the granule cell body layer thickness, molecular layer spine density, and axon proximal area, but do not produce striking rearrangements of granule cell structure.

  11. Model cerebellar granule cells can faithfully transmit modulated firing rate signals

    PubMed Central

    Rössert, Christian; Solinas, Sergio; D'Angelo, Egidio; Dean, Paul; Porrill, John

    2014-01-01

    A crucial assumption of many high-level system models of the cerebellum is that information in the granular layer is encoded in a linear manner. However, granule cells are known for their non-linear and resonant synaptic and intrinsic properties that could potentially impede linear signal transmission. In this modeling study we analyse how electrophysiological granule cell properties and spike sampling influence information coded by firing rate modulation, assuming no signal-related, i.e., uncorrelated inhibitory feedback (open-loop mode). A detailed one-compartment granule cell model was excited in simulation by either direct current or mossy-fiber synaptic inputs. Vestibular signals were represented as tonic inputs to the flocculus modulated at frequencies up to 20 Hz (approximate upper frequency limit of vestibular-ocular reflex, VOR). Model outputs were assessed using estimates of both the transfer function, and the fidelity of input-signal reconstruction measured as variance-accounted-for. The detailed granule cell model with realistic mossy-fiber synaptic inputs could transmit information faithfully and linearly in the frequency range of the vestibular-ocular reflex. This was achieved most simply if the model neurons had a firing rate at least twice the highest required frequency of modulation, but lower rates were also adequate provided a population of neurons was utilized, especially in combination with push-pull coding. The exact number of neurons required for faithful transmission depended on the precise values of firing rate and noise. The model neurons were also able to combine excitatory and inhibitory signals linearly, and could be replaced by a simpler (modified) integrate-and-fire neuron in the case of high tonic firing rates. These findings suggest that granule cells can in principle code modulated firing-rate inputs in a linear manner, and are thus consistent with the high-level adaptive-filter model of the cerebellar microcircuit. PMID:25352777

  12. Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

    NASA Technical Reports Server (NTRS)

    Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.; Rhodes, Christopher J.

    2002-01-01

    The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.

  13. Ectopic Kidney

    MedlinePlus

    ... Human Development March of Dimes National Office MedlinePlus Kidney and Urologic Disease Organizations Many organizations provide support ... Organizations​​ . (PDF, 345 KB)​​​​​ Alternate Language URL Ectopic Kidney Page Content On this page: What is an ...

  14. Ectopic hypophyseal hormonal cells in benign cystic teratoma of the ovary. Light microscopic histochemical dye staining and immunoperoxidase cytochemistry.

    PubMed

    McKeel, D W; Askin, F B

    1978-03-01

    Histologic study of the hypophyseal component of a benign cystic ovarian teratoma disclosed elements that resembled sellar adenohypophysis, pars intermedia, and neurohypophysis. Histochemical dye methods revealed secretory cells with cytologic and granule-staining characteristics of somatotrophs, mammotrophs, melanocorticotrophs, and thyrotrophs. Nongranulated follicular cells and salivary gland rest cells also were present. Indirect immunoperoxidase staining with monospecific antisera to anterior pituitary hormones revealed abundant prolactin-containing cells, which comprised more than 50% of all chromophilic cells, as well as numerous cells that contained growth hormone and thyroid-stimulating hormone. Gonadotrophic cells could not be demonstrated by either tinctorial stains or immunostaining.

  15. Upregulation of Nanog and Sox-2 genes following ectopic expression of Oct-4 in amniotic fluid mesenchymal stem cells.

    PubMed

    Wang, Kai-Hung; Kao, An-Pei; Chang, Chia-Cheng; Lin, Ta-Chin; Kuo, Tsung-Cheng

    2015-01-01

    Octamer-binding transcription factor 4 (Oct-4), an important gene regulating stem cell pluripotency, is well-known for its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. The aim of this study was to assess the effect of ectopic expression of Oct human amniotic fluid stem cells. We developed a novel method for isolation of putative human amniotic fluid-derived multipotent stem cells. These cells showing mesenchymal stem cell phenotypes (human amniotic fluid-derived mesenchymal stem cells, hAFMSCs) were transfected with a plasmid carrying genes for Oct-4 and the green fluorescent protein (GFP). The stably transfected cells, hAFMSCs-Oct4/GFP, were selected by using G418 and found to express the GFP reporter gene under the control of Oct-4 promoter. We found that hAFMSCs developed by our method possess very high self-renewal ability (about 78 cumulative population doublings) and multilineage differentiation potency. Significantly, the hAFMSCs-Oct4/GFP cells showed enhanced expression of the three major pluripotency genes Oct-4, Nanog, and Sox-2, and increased colony-forming ability and growth rate compared with the parental hAFMSCs. We demonstrated that the ectopic expression of Oct-4 gene in hAFMSCs with high self-renewal ability could upregulate Nanog and Sox-2 gene expression and enhance cell growth rate and colony-forming efficiency. Therefore, the ectopic expression of Oct-4 could be a strategy to develop pluripotency in hAFMSCs for clinical applications.

  16. Local postsynaptic voltage-gated sodium channel activation in dendritic spines of olfactory bulb granule cells.

    PubMed

    Bywalez, Wolfgang G; Patirniche, Dinu; Rupprecht, Vanessa; Stemmler, Martin; Herz, Andreas V M; Pálfi, Dénes; Rózsa, Balázs; Egger, Veronica

    2015-02-01

    Neuronal dendritic spines have been speculated to function as independent computational units, yet evidence for active electrical computation in spines is scarce. Here we show that strictly local voltage-gated sodium channel (Nav) activation can occur during excitatory postsynaptic potentials in the spines of olfactory bulb granule cells, which we mimic and detect via combined two-photon uncaging of glutamate and calcium imaging in conjunction with whole-cell recordings. We find that local Nav activation boosts calcium entry into spines through high-voltage-activated calcium channels and accelerates postsynaptic somatic depolarization, without affecting NMDA receptor-mediated signaling. Hence, Nav-mediated boosting promotes rapid output from the reciprocal granule cell spine onto the lateral mitral cell dendrite and thus can speed up recurrent inhibition. This striking example of electrical compartmentalization both adds to the understanding of olfactory network processing and broadens the general view of spine function.

  17. Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis.

    PubMed

    Serafini, Barbara; Rosicarelli, Barbara; Magliozzi, Roberta; Stigliano, Egidio; Aloisi, Francesca

    2004-04-01

    Multiple sclerosis (MS) is characterized by synthesis of oligoclonal immunoglobulins and the presence of B-cell clonal expansions in the central nervous system (CNS). Because ectopic lymphoid tissue generated at sites of chronic inflammation is thought to be important in sustaining immunopathological processes, we have investigated whether structures resembling lymphoid follicles could be identified in the CNS of MS patients. Sections from post-mortem MS brains and spinal cords were screened using immunohistochemistry for the presence of CD20+ B-cells, CD3+ T-cells, CD138+ plasma cells and CD21+, CD35+ follicular dendritic cells, and for the expression of lymphoid chemokines (CXCL 13, CCL21) and peripheral node addressin (PNAd). Lymphoid follicle-like structures containing B-cells, T-cells and plasma cells, and a network of follicular dendritic cells producing CXCL13 were observed in the cerebral meninges of 2 out of 3 patients with secondary progressive MS, but not in relapsing remitting and primary progressive MS. We also show that proliferating B-cells are present in intrameningeal follicles, a finding which is suggestive of germinal center formation. No follicle-like structures were detected in parenchymal lesions. The formation of ectopic lymphoid follicies in the meninges of patients with MS could represent a critical step in maintaining humoral autoimmunity and in disease exacerbation.

  18. On the origins of the universal dynamics of endogenous granules in mammalian cells.

    PubMed

    Vanapalli, Siva A; Li, Yixuan; Mugele, Frieder; Duits, Michel H G

    2009-12-01

    Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

  19. Dendritic morphology, synaptic transmission, and activity of mature granule cells born following pilocarpine-induced status epilepticus in the rat

    PubMed Central

    Gao, Fei; Song, Xueying; Zhu, Dexiao; Wang, Xiaochen; Hao, Aijun; Nadler, J. Victor; Zhan, Ren-Zhi

    2015-01-01

    To understand the potential role of enhanced hippocampal neurogenesis after pilocarpine-induced status epilepticus (SE) in the development of epilepsy, we quantitatively analyzed the geometry of apical dendrites, synaptic transmission, and activation levels of normotopically distributed mature newborn granule cells in the rat. SE in male Sprague-Dawley rats (between 6 and 7 weeks old) lasting for more than 2 h was induced by an intraperitoneal injection of pilocarpine. The complexity, spine density, miniature post-synaptic currents, and activity-regulated cytoskeleton-associated protein (Arc) expression of granule cells born 5 days after SE were studied between 10 and 17 weeks after CAG-GFP retroviral vector-mediated labeling. Mature granule cells born after SE had dendritic complexity similar to that of granule cells born naturally, but with denser mushroom-like spines in dendritic segments located in the outer molecular layer. Miniature inhibitory post-synaptic currents (mIPSCs) were similar between the controls and rats subjected to SE; however, smaller miniature excitatory post-synaptic current (mEPSC) amplitude with a trend toward less frequent was found in mature granule cells born after SE. After maturation, granule cells born after SE did not show denser Arc expression in the resting condition or 2 h after being activated by pentylenetetrazol-induced transient seizure activity than vicinal GFP-unlabeled granule cells. Thus our results suggest that normotopic granule cells born after pilocarpine-induced SE are no more active when mature than age-matched, naturally born granule cells. PMID:26500490

  20. Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons.

    PubMed

    Srivastava, Rupali; Kumar, Manoj; Peineau, Stéphane; Csaba, Zsolt; Mani, Shyamala; Gressens, Pierre; El Ghouzzi, Vincent

    2013-04-01

    Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

  1. Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells

    PubMed Central

    Madroñal, Noelia; Delgado-García, José M.; Fernández-Guizán, Azahara; Chatterjee, Jayanta; Köhn, Maja; Mattucci, Camilla; Jain, Apar; Tsetsenis, Theodoros; Illarionova, Anna; Grinevich, Valery; Gross, Cornelius T.; Gruart, Agnès

    2016-01-01

    The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories. PMID:26988806

  2. Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells.

    PubMed

    Madroñal, Noelia; Delgado-García, José M; Fernández-Guizán, Azahara; Chatterjee, Jayanta; Köhn, Maja; Mattucci, Camilla; Jain, Apar; Tsetsenis, Theodoros; Illarionova, Anna; Grinevich, Valery; Gross, Cornelius T; Gruart, Agnès

    2016-01-01

    The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories. PMID:26988806

  3. Sphingomyelin derivatives increase the frequency of microvesicle and granule fusion in chromaffin cells.

    PubMed

    García-Martínez, V; Montes, M A; Villanueva, J; Gimenez-Molina, Y; de Toledo, G A; Gutiérrez, L M

    2015-06-01

    Sphingomyelin derivatives like sphingosine have been shown to enhance secretion in a variety of systems, including neuroendocrine and neuronal cells. By studying the mechanisms underlying this effect, we demonstrate here that sphingomyelin rafts co-localize strongly with synaptosomal-associated protein of 25Kda (SNAP-25) clusters in cultured bovine chromaffin cells and that they appear to be linked in a dynamic manner. In functional terms, when cultured rat chromaffin cells are treated with sphingomyelinase (SMase), producing sphingomyelin derivatives, the secretion elicited by repetitive depolarizations is enhanced. This increase was independent of cell size and it was significant 15min after initiating stimulation. Interestingly, by evaluating the membrane capacitance we found that the events in control untreated cells corresponded to two populations of microvesicles and granules, and the fusion of both these populations is clearly enhanced after treatment with SMase. Furthermore, SMase does not increase the size of chromaffin granules. Together, these results strongly suggest that SNARE-mediated exocytosis is enhanced by the generation of SMase derivatives, reflecting an increase in the frequency of fusion of both microvesicles and chromaffin granules rather than an increase in the size of these vesicles.

  4. With regards to the presence of iron granules in plasma cells.

    PubMed

    D'Angelo, G; Giardini, C; Zanco, M D

    1991-12-01

    We report on the occasional presence of iron granules in plasma cells in two male patients respectively 64 and 71 years old, both with excessive drinking habits. One patient also had liver cirrhosis. In both patients the bone-marrow biopsy showed a macrocytic anemia without megaloblasts. We refer the morphologic data because the cases reported are not many and the presence of iron granules in plasma cells was a curious and rare aspect. The most important feature appearing from the data issued is the gap concerning both the source and mechanism that cause this phenomenon. Some investigations have suggested that the plasma cell iron is located in mitochondria, others have noted that iron granules were located between the Golgi region and the rough endoplasmic reticulum. Moreover, the morphologic data are not related to the number of plasma cells in the bone-marrow and there is no causal relation between alcoholic abuse and plasma cell iron. The first problem is common, the second is rare.

  5. Enterovirus 71 induces anti-viral stress granule-like structures in RD cells.

    PubMed

    Zhu, Yuanmei; Wang, Bei; Huang, He; Zhao, Zhendong

    2016-08-01

    Stress granules (SGs) are dynamic cytoplasmic granules formed in response to a variety of stresses, including viral infection. Several viruses can modulate the formation of SG with different effects, but the relationship between SG formation and EV71 infection is poorly understood. In this study, we report that EV71 inhibits canonical SGs formation in infected cells and induces the formation of novel RNA granules that were distinguished from canonical SGs in composition and morphology, which we termed 'SG like structures'. Our results also demonstrated that EV71 triggered formation of SG-like structures is dependent on PKR and eIF2α phosphorylation and requires ongoing cellular mRNA synthesis. Finally, we found that SG-like structures are antiviral RNA granules that promote cellular apoptosis and suppress EV71 propagation. Taken together, our findings explain the formation mechanism of SG-like structures induced by EV71 and shed light on virus-host interaction and molecular mechanism underlying EV71 pathogenesis.

  6. Enterovirus 71 induces anti-viral stress granule-like structures in RD cells.

    PubMed

    Zhu, Yuanmei; Wang, Bei; Huang, He; Zhao, Zhendong

    2016-08-01

    Stress granules (SGs) are dynamic cytoplasmic granules formed in response to a variety of stresses, including viral infection. Several viruses can modulate the formation of SG with different effects, but the relationship between SG formation and EV71 infection is poorly understood. In this study, we report that EV71 inhibits canonical SGs formation in infected cells and induces the formation of novel RNA granules that were distinguished from canonical SGs in composition and morphology, which we termed 'SG like structures'. Our results also demonstrated that EV71 triggered formation of SG-like structures is dependent on PKR and eIF2α phosphorylation and requires ongoing cellular mRNA synthesis. Finally, we found that SG-like structures are antiviral RNA granules that promote cellular apoptosis and suppress EV71 propagation. Taken together, our findings explain the formation mechanism of SG-like structures induced by EV71 and shed light on virus-host interaction and molecular mechanism underlying EV71 pathogenesis. PMID:27216457

  7. [Ectopic calcification].

    PubMed

    Fukumoto, Seiji

    2014-02-01

    Calcium deposition can be observed in many tissues in addition to bones and teeth which physiologically calcify. This unphysiological calcification can damage several organs. It has been shown that vascular calcification which is a risk factor for cardiovascular events develops through similar mechanisms to physiological calcification. Further studies to clarify detailed mechanisms of calcification are necessary to develop measures that inhibit unphysiological ectopic calcification without affecting physiological calcification in bones and teeth.

  8. Properties of AMPA receptors expressed in rat cerebellar granule cell cultures: Ca2+ influx studies.

    PubMed

    Hack, N; Balázs, R

    1995-09-01

    Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), in terms of AMPA-induced toxicity or 45Ca2+ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of 45Ca2+ accumulation. 45Ca2+ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall 45Ca2+ influx. These included Ca2+ uptake through NMDA-receptor channels, voltage-sensitive Ca2+ channels, and via Na+/Ca2+ exchange. However, nearly one-fifth of the total 45Ca2+ influx remained unaccounted for and this estimate was similar to 45Ca2+ influx observed under Na(+)-free conditions. This observation suggested that a significant proportion of the Ca2+ flux passes through the AMPA-receptor channel proper, a view supported by Co2+ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro.

  9. Elemental levels in mast cell granules differ in sections from normal and diabetic rats: an X-ray microanalysis study

    SciTech Connect

    Kendall, M.D.

    1988-03-01

    Mast cells around the thymus of rats stain red with alcian blue and safranin indicating that the mast cells are probably of the peritoneal (connective tissue) type. After the onset of streptozotocin induced diabetes some cells contain both red and blue granules and blue staining cells may appear. X-ray microanalysis of frozen freeze-dried sections from diabetic male CSE Wistar rats showed electron dense granules to have similar amounts of S to normal rat mast cell granules but reduced levels of Na, Mg, P, Cl and K. Two cells also had electron lucent granules with very high levels of Na, Cl, K and Ca and reduced concentrations of S. The differences in elemental composition suggest that the mast cells from diabetic rats are not immature, but are related to the condition of induced diabetes, and that granules of very different composition can occur within a single cell. X-ray microanalysis has given an insight into mast cell granule elemental content which was not possible by conventional biochemical methods.

  10. Structural Plasticity of Dentate Granule Cell Mossy Fibers During the Development of Limbic Epilepsy

    PubMed Central

    Danzer, Steve C.; He, Xiaoping; Loepke, Andreas W.; McNamara, James O.

    2009-01-01

    Altered granule cell≫CA3 pyramidal cell synaptic connectivity may contribute to the development of limbic epilepsy. To explore this possibility, granule cell giant mossy fiber bouton plasticity was examined in the kindling and pilocarpine models of epilepsy using green fluorescent protein-expressing transgenic mice. These studies revealed significant increases in the frequency of giant boutons with satellite boutons 2 days and 1 month after pilocarpine status epilepticus, and increases in giant bouton area at 1 month. Similar increases in giant bouton area were observed shortly after kindling. Finally, both models exhibited plasticity of mossy fiber giant bouton filopodia, which contact GABAergic interneurons mediating feedforward inhibition of CA3 pyramids. In the kindling model, however, all changes were fleeting, having resolved by 1 month after the last evoked seizure. Together, these findings demonstrate striking structural plasticity of granule cell mossy fiber synaptic terminal structure in two distinct models of adult limbic epileptogenesis. We suggest that these plasticities modify local connectivities between individual mossy fiber terminals and their targets, inhibitory interneurons, and CA3 pyramidal cells potentially altering the balance of excitation and inhibition during the development of epilepsy. PMID:19294647

  11. Serotonin modulates the excitatory synaptic transmission in the dentate granule cells.

    PubMed

    Nozaki, Kanako; Kubo, Reika; Furukawa, Yasuo

    2016-06-01

    Serotonergic fibers from the raphe nuclei project to the hippocampal formation, the activity of which is known to modulate the inhibitory interneurons in the dentate gyrus. On the other hand, serotonergic modulation of the excitatory synapses in the dentate gyrus is not well examined. In the present study, we examined the effects of 5-HT on the excitatory postsynaptic potentials (EPSPs) in the dentate granule cells evoked by the selective stimulation of the lateral perforant path (LPP), the medial perforant path (MPP), or the mossy cell fibers (MCF). 5-HT depressed the amplitude of unitary EPSPs (uEPSPs) evoked by the stimulation of LPP or MPP, whereas uEPSPs evoked by MCF stimulation were little affected. The effect was partly explained by the decrease of the resting membrane resistance following the activation of 5-HT1A receptors, which was confirmed by computer simulations. We also found that the probability of evoking uEPSP by LPP stimulation but not MPP or MCF stimulation was reduced by 5-HT and that the paired-pulse ratio of LPP-evoked EPSP but not that of MPP- or MCF-evoked ones was increased by 5-HT. These effects were blocked by 5-HT2 antagonist, suggesting that the transmitter release in the LPP-granule cell synapse is inhibited by the activation of 5-HT2 receptors. The present results suggest that 5-HT can modulate the EPSPs in the dentate granule cells by at least two distinct mechanisms. PMID:26961099

  12. Double-strand break-induced recombination between ectopic homologous sequences in somatic plant cells.

    PubMed Central

    Puchta, H

    1999-01-01

    Homologous recombination between ectopic sites is rare in higher eukaryotes. To test whether double-strand breaks (DSBs) can induce ectopic recombination, transgenic tobacco plants harboring two unlinked, nonfunctional homologous parts of a kanamycin resistance gene were produced. To induce homologous recombination between the recipient locus (containing an I-SceI site within homologous sequences) and the donor locus, the rare cutting restriction enzyme I-SceI was transiently expressed via Agrobacterium in these plants. Whereas without I-SceI expression no recombination events were detectable, four independent recombinants could be isolated after transient I-SceI expression, corresponding to approximately one event in 10(5) transformations. After regeneration, the F1 generation of all recombinants showed Mendelian segregation of kanamycin resistance. Molecular analysis of the recombinants revealed that the resistance gene was indeed restored via homologous recombination. Three different kinds of reaction products could be identified. In one recombinant a classical gene conversion without exchange of flanking markers occurred. In the three other cases homologous sequences were transferred only to one end of the break. Whereas in three cases the ectopic donor sequence remained unchanged, in one case rearrangements were found in recipient and donor loci. Thus, ectopic homologous recombination, which seems to be a minor repair pathway for DSBs in plants, is described best by recombination models that postulate independent roles for the break ends during the repair process. PMID:10388832

  13. Myeloperoxidase and crystalline bodies in the granules of DMBA-induced rat chloroma cells.

    PubMed

    Ioachim, H L; Keller, S; Sabbath, M; Andersson, B; Dorsett, B; Essner, E

    1972-01-01

    Chloroma (chloroleukemia) was induced in a splenectomized rat by repeatedly administering dimethylbenz(a)anthracene (DMBA) and was serially transplanted thereafter. Composed of immature myeloid cells, the tumor imparted a green discoloration to the tissues that it infiltrated extensively. Chloroma cells fluoresced red in ultraviolet light, produced a characteristic curve in spectrophotometry, and contained large amounts of myeloperoxidase. They included numerous intracytoplasmic granules of both types A and B, which contained occasional crystalline bars. Permanent lines of chloroma cells were established in tissue culture. These cells, while maintaining their initial morphology, ceased producing myeloperoxidase and subsequently induced white tumors when they were isotransplanted.

  14. Tethering of ICAM on target cells is required for LFA-1-dependent NK cell adhesion and granule polarization

    PubMed Central

    Gross, Catharina C.; Brzostowski, Joseph A.; Liu, Dongfang; Long, Eric O.

    2013-01-01

    αLβ2 integrin (LFA-1) has an important role in the formation of T cell and NK cell cytotoxic immunological synapses and in target cell killing. Binding of LFA-1 to ICAM on target cells promotes not only adhesion, but also polarization of cytolytic granules in NK cells. Here we tested whether LFA-1-dependent NK cell responses are regulated by the distribution and mobility of ICAM at the surface of target cells. We show that depolymerization of F-actin in NK-sensitive target cells abrogated LFA-1-dependent conjugate formation and granule polarization in primary NK cells. Degranulation, which is not controlled by LFA-1, was not impaired. Fluorescence recovery after photobleaching experiments and particle tracking by total internal reflection fluorescence microscopy revealed that ICAM-1 and ICAM-2 were distributed in largely immobile clusters. ICAM clusters were maintained and became highly mobile after actin depolymerization. Moreover, reducing ICAM-2 mobility on an NK-resistant target cell through expression of ezrin, an adapter molecule that tethers proteins to the actin cytoskeleton, enhanced LFA-1-dependent adhesion and granule polarization. Finally, while NK cells kept moving over freely diffusible ICAM-1 on a lipid bilayer, they bound and spread over solid-phase ICAM-1. We conclude that tethering, rather than clustering of ICAM promotes proper signaling by LFA-1 in NK cells. Our findings suggest that the lateral diffusion of integrin ligands on cells may be an important determinant of susceptibility to lysis by cytotoxic lymphocytes. PMID:20675589

  15. Effects of zinc chloride on the RNP structures in HEp-2 cells: accumulation of perichromatin granules

    SciTech Connect

    Cervera, J.; Baguena-Cervellera, R.; Martinez, A.

    1985-12-01

    The effects of zinc on the ribonucleoprotein (RNP) constituents of HEp-2 cells have been analyzed. Pulse-chase autoradiographic experiments show a preferential inhibition of nucleolar RNA synthesis and a block in the transport of nucleolar and extranucleolar RNA in zinc-treated cells. Concomitantly with the disturbance in RNA metabolism and in protein synthesis, nucleolar condensation, accumulation of perichromatin granules and fibrils, condensation of interchromatin fibrils, and appearance of dense granular bodies occur. Accumulation of perichromatin fibrils and condensation of interchromatin fibrils appear to be related to the block in the transport of heterogeneous nuclear RNA. Depletion of certain proteins required for the assembly of RNP particles could share in the abnormal behavior of RNA and lead to the accumulation of perichromatin granules and the appearance of dense granular bodies.

  16. Insulin granule trafficking in beta-cells: mathematical model of glucose-induced insulin secretion.

    PubMed

    Bertuzzi, Alessandro; Salinari, Serenella; Mingrone, Geltrude

    2007-07-01

    A mathematical model that represents the dynamics of intracellular insulin granules in beta-cells is proposed. Granule translocation and exocytosis are controlled by signals assumed to be essentially related to ATP-to-ADP ratio and cytosolic Ca(2+) concentration. The model provides an interpretation of the roles of the triggering and amplifying pathways of glucose-stimulated insulin secretion. Values of most of the model parameters were inferred from available experimental data. The numerical simulations represent a variety of experimental conditions, such as the stimulation by high K(+) and by different time courses of extracellular glucose, and the predicted responses agree with published experimental data. Model capacity to represent data measured in a hyperglycemic clamp was also tested. Model parameter changes that may reflect alterations of beta-cell function present in type 2 diabetes are investigated, and the action of pharmacological agents that bind to sulfonylurea receptors is simulated.

  17. Effects of eugenol on granule cell dispersion in a mouse model of temporal lobe epilepsy.

    PubMed

    Jeong, Kyoung Hoon; Lee, Dong-Seok; Kim, Sang Ryong

    2015-09-01

    Granule cell dispersion (GCD), a structural abnormality, is characteristic of temporal lobe epilepsy (TLE). Eugenol (EUG) is an essential component of medicinal herbs and is suggested to exert anticonvulsant activity. However, it is unclear whether EUG ameliorates the abnormal morphological changes in granule cells induced by epileptic insults. In the present study, we examined whether intraperitoneal injection of EUG attenuated increased seizure activity and GCD following intrahippocampal injection of kainic acid (KA). Our results showed that EUG significantly increased the seizure threshold, resulting in delayed seizure onset, and reduced GCD in KA-induced epilepsy. Moreover, EUG treatment significantly attenuated KA-induced activation of mammalian target of rapamycin complex 1 (mTORC1), which is involved in GCD development, in the dentate gyrus (DG). These results suggest that EUG may have beneficial effects in the treatment of epilepsy through its ability to inhibit GCD via suppression of KA-induced mTORC1 activation in the hippocampal DG in vivo.

  18. In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells.

    PubMed

    Lee, Kyung Hoon; Lee, Won Young; Do, Jung Tae; Park, Chan Kyu; Kim, Nam Hyung; Kim, Jin Hoi; Chung, Hak Jae; Kim, Dong Woon; Song, Hyuk

    2016-08-01

    Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells.

  19. In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells.

    PubMed

    Lee, Kyung Hoon; Lee, Won Young; Do, Jung Tae; Park, Chan Kyu; Kim, Nam Hyung; Kim, Jin Hoi; Chung, Hak Jae; Kim, Dong Woon; Song, Hyuk

    2016-08-01

    Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells. PMID:27328332

  20. Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells.

    PubMed

    Rudolf, Rüdiger; Kögel, Tanja; Kuznetsov, Sergei A; Salm, Thorsten; Schlicker, Oliver; Hellwig, Andrea; Hammer, John A; Gerdes, Hans-Hermann

    2003-04-01

    Neuroendocrine secretory granules, the storage organelles for neuropeptides and hormones, are formed at the trans-Golgi network, stored inside the cell and exocytosed upon stimulation. Previously, we have reported that newly formed secretory granules of PC12 cells are transported in a microtubule-dependent manner from the trans-Golgi network to the F-actin-rich cell cortex, where they undergo short directed movements and exhibit a homogeneous distribution. Here we provide morphological and biochemical evidence that myosin Va is associated with secretory granules. Expression of a dominant-negative tail domain of myosin Va in PC12 cells led to an extensive clustering of secretory granules close to the cell periphery, a loss of their cortical restriction and a strong reduction in their motility in the actin cortex. Based on this data we propose a model that implies a dual transport system for secretory granules: after microtubule-dependent delivery to the cell periphery, secretory granules exhibit a myosin Va-dependent transport leading to their restriction and even dispersal in the F-actin-rich cortex of PC12 cells. PMID:12615975

  1. Specific localization of membrane dipeptidase and dipeptidyl peptidase IV in secretion granules of two different pancreatic islet cells.

    PubMed

    Grondin, G; Hooper, N M; LeBel, D

    1999-04-01

    Endocrine cells require several protein convertases to process the precursors of hormonal peptides that they secrete. In addition to the convertases, which have a crucial role in the maturation of prohormones, many other proteases are present in endocrine cells, the roles of which are less well established. Two of these proteases, dipeptidyl peptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19), have been immunocytochemically localized in the endocrine pancreas of the pig. Membrane dipeptidase was present exclusively in cells of the islet of Langerhans that were positive for the pancreatic polypeptide, whereas dipeptidyl peptidase IV was restricted to cells positive for glucagon. Both enzymes were observed in the content of secretory granules and therefore would be released into the interstitial space as the granules undergo exocytosis. At this location they could act on secretions of other islet cells. The relative concentration of dipeptidyl peptidase IV was lower in dense glucagon granules, where the immunoreactivity to glucagon was higher, and vice versa for light granules. This suggests that, in A-cells, dipeptidyl peptidase IV could be sent for degradation in the endosomal/lysosomal compartment during the process of granule maturation or could be removed from granules for continuous release into the interstitial space. The intense proteolytic activity that takes place in the endocrine pancreas could produce many potential dipeptide substrates for membrane dipeptidase. (J Histochem Cytochem 47:489-497, 1999)

  2. The histone deacetylase Hos2 forms an Hsp42-dependent cytoplasmic granule in quiescent yeast cells.

    PubMed

    Liu, I-Chun; Chiu, Sheng-Wen; Lee, Hsin-Yi; Leu, Jun-Yi

    2012-04-01

    One of many physiological adjustments in quiescent cells is spatial regulation of specific proteins and RNA important for the entry to or exit from the stationary phase. By examining the localization of epigenetic-related proteins in Saccharomyces cerevisiae, we observed the formation of a reversible cytosolic "stationary-phase granule" (SPG) by Hos2, a nuclear histone deacetylase. In the stationary phase, hos2 mutants display reduced viability. Additionally, they exhibit a significant delay when recovering from stationary phase. Hos2 SPGs also contained Hst2, a Sir2 homologue, and several stress-related proteins, including Set3, Yca1, Hsp26, Hsp42, and some known components of stress granules. However, Hos2 SPG formation does not depend on the formation of stress granules or processing bodies. The absence or presence of glucose is sufficient to trigger assembly or disassembly of Hos2 SPGs. Among the identified components of Hos2 SPGs, Hsp42 is the first and last member observed in the Hos2 SPG assembly and disassembly processes. Hsp42 is also vital for the relocalization of the other components to Hos2 SPGs, suggesting that Hsp42 plays a central role in spatial regulation of proteins in quiescent cells.

  3. Study of Mast Cells and Granules from Primo Nodes Using Scanning Ionic Conductance Microscopy.

    PubMed

    Yoo, Yeong-Yung; Jung, Goo-Eun; Kwon, Hee-Min; Bae, Kyoung-Hee; Cho, Sang-Joon; Soh, Kwang-Sup

    2015-12-01

    Acupuncture points have a notable characteristic in that they have a higher density of mast cells (MCs) compared with nonacupoints in the skin, which is consistent with the augmentation of the immune function by acupuncture treatment. The primo vascular system, which was proposed as the anatomical structure of the acupuncture points and meridians, also has a high density of MCs. We isolated the primo nodes from the surfaces of internal abdominal organs, and the harvested primo nodes were stained with toluidine blue. The MCs were easily recognized by their stained color and their characteristic granules. The MCs were classified into four stages according to the degranulation of histamine granules in the MCs. Using conventional optical microscopes details of the degranulation state of MCs in each stage were not observable. However, we were able to investigate the distribution of the granules on the surfaces of the MCs in each stage, and to demonstrate the height profiles and three-dimensional structures of the MCs without disturbance of the cell membrane by using the scanning ion conductance microscopy.

  4. Synthetic mast-cell granules as adjuvants to promote and polarize immunity in lymph nodes

    NASA Astrophysics Data System (ADS)

    St. John, Ashley L.; Chan, Cheryl Y.; Staats, Herman F.; Leong, Kam W.; Abraham, Soman N.

    2012-03-01

    Granules of mast cells (MCs) enhance adaptive immunity when, on activation, they are released as stable particles. Here we show that submicrometre particles modelled after MC granules augment immunity when used as adjuvants in vaccines. The synthetic particles, which consist of a carbohydrate backbone with encapsulated inflammatory mediators such as tumour necrosis factor, replicate attributes of MCs in vivo including the targeting of draining lymph nodes and the timed release of the encapsulated mediators. When used as an adjuvant during vaccination of mice with haemagglutinin from the influenza virus, the particles enhanced adaptive immune responses and increased survival of mice on lethal challenge. Furthermore, differential loading of the particles with the cytokine IL-12 directed the character of the response towards Th1 lymphocytes. The synthetic MC adjuvants replicate and enhance the functions of MCs during vaccination, and can be extended to polarize the resulting immunity.

  5. Bax inactivation in lurcher mutants rescues cerebellar granule cells but not purkinje cells or inferior olivary neurons.

    PubMed

    Selimi, F; Vogel, M W; Mariani, J

    2000-07-15

    Lurcher is a gain-of-function mutation in the delta2 glutamate receptor gene (Grid2) that turns the receptor into a leaky ion channel. The expression of the Lurcher gene in heterozygous (Grid2(Lc/+)) mutants induces the death of almost all Purkinje cells starting from the second postnatal week. Ninety percent of the granule cells and 60-75% of the inferior olivary neurons die because of the loss of their target neurons, the Purkinje cells. The apoptotic nature of the neurodegeneration has been demonstrated previously by the presence of activated caspase-3 and DNA fragmentation. Bax, a pro-apoptotic gene of the Bcl-2 family, has been shown to be involved in developmental neuronal death. To study the role of Bax in Grid2(Lc/+) neurodegeneration, double mutants with Grid2(Lc/)+ mice and Bax knock-out mice (Bax-/-) were generated. Bax deletion had no effect on the death of Purkinje cells and inferior olivary neurons, although a temporary rescue of some Purkinje cells could be detected in P15 Grid2(Lc/)+;Bax-/- animals. From postnatal day 15 (P15) to P60, the number of granule cells in Grid2(Lc/)+;Bax-/-mice did not significantly change and was significantly increased compared with the number found in Grid2(Lc/)+;Bax+/+ mice. Granule cell number in P60 Grid2(Lc/)+;Bax-/- mice corresponded to 70% of the number found in wild-type mice. Our results show that Bax inactivation in Grid2(Lc/+) mice does not rescue intrinsic Purkinje cell death or the target-related cell death of olivary neurons, but Bax inactivation does inhibit persistently target-related cell death in cerebellar granule cells.

  6. Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor.

    PubMed

    Zanin, Juan Pablo; Abercrombie, Elizabeth; Friedman, Wilma J

    2016-07-19

    Cerebellar granule cell progenitors (GCP) proliferate extensively in the external granule layer (EGL) of the developing cerebellum prior to differentiating and migrating. Mechanisms that regulate the appropriate timing of cell cycle withdrawal of these neuronal progenitors during brain development are not well defined. The p75 neurotrophin receptor (p75(NTR)) is highly expressed in the proliferating GCPs, but is downregulated once the cells leave the cell cycle. This receptor has primarily been characterized as a death receptor for its ability to induce neuronal apoptosis following injury. Here we demonstrate a novel function for p75(NTR) in regulating proper cell cycle exit of neuronal progenitors in the developing rat and mouse EGL, which is stimulated by proNT3. In the absence of p75(NTR), GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits.

  7. Cell secretion mediated by granule-associated vesicle transport: a glimpse at evolution.

    PubMed

    Crivellato, Enrico; Nico, Beatrice; Gallo, Valentina Patrizia; Ribatti, Domenico

    2010-07-01

    Regulated secretion allows extrusion of cell products stored in specialized membrane-bound organelles called secretory granules or secretory vesicles. Regulated secretion provides basic functions in living organisms, and in a phylogenetic perspective, it is recognizable in the most primitive eukaryotic forms. This article is an attempt to trace the evolutionary history of a special type of secretory pattern, which has been referred to as vesicle-mediated degranulation or piecemeal degranulation (PMD). First described in the early 70s of the last century in inflammatory cells, such as the basophils, mast cells, and eosinophils, this regulated secretory route has subsequently been recognized in endocrine cells, in particular in the chromaffin cells of the adrenal medulla. This vesicle-mediated degranulation is held to mobilize small and specific aliquots of granule-associated material for selective paracrine or endocrine transport to the cell exterior. PMD has been identified in many vertebrate classes. By contrast, no data are available for invertebrates. We speculate that this pattern of cell secretion emerged early in phylogenesis, when the first metazoans appeared. In this review article, we will first revise the concept of vesicle-mediated degranulation in the light of the most recent experimental discoveries and theoretical implications. Then, the distribution of this secretory mode among vertebrates and its molecular basis will be highlighted. Finally, the potential occurrence of PMD in invertebrates, its biological significance from an evolutionary perspective and the future direction of investigations will be briefly sketched.

  8. The survival of cultured mouse cerebellar granule cells is not dependent on elevated potassium-ion concentration.

    PubMed

    Mogensen, H S; Hack, N; Balázs, R; Jørgensen, O S

    1994-08-01

    The effects of K(+)-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from degenerated cells was removed by prior DNAase treatment. These DNA estimates of cell numbers were comparable with values obtained by direct counting of fluorescein diacetate-stained viable cells. In agreement with previous studies, the survival of rat granule cells was promoted by increasing the concentration of K+ in the medium from 5 to 25 mM throughout a 7-day culture period. In contrast, mouse granule cells survived in culture containing 'low' K+ (5 or 10 mM), as well as in the presence of 'high' K+ (25 mM). On the other hand, several biochemical parameters in mouse granule cells were markedly increased by cultivation in 'high' as compared with 'low' K(+)-containing media, demonstrated by increased fluorescein diacetate esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented 2-deoxy-D-glucose accumulation and increased N-methyl-D-aspartate-evoked 45Ca2+ influx. It was concluded that although cultivation in 'high' K+ promotes biochemical differentiation in mouse cerebellar granule cells, these cells differ from their rat counterparts in that they do not develop a survival requirement for K(+)-induced membrane depolarization.

  9. Differential dendritic Ca2+ signalling in young and mature hippocampal granule cells

    PubMed Central

    Stocca, Gabriella; Schmidt-Hieber, Christoph; Bischofberger, Josef

    2008-01-01

    Neuronal activity is critically important for development and plasticity of dendrites, axons and synaptic connections. Although Ca2+ is an important signal molecule for these processes, not much is known about the regulation of the dendritic Ca2+ concentration in developing neurons. Here we used confocal Ca2+ imaging to investigate dendritic Ca2+ signalling in young and mature hippocampal granule cells, identified by the expression of the immature neuronal marker polysialated neural cell adhesion molecule (PSA-NCAM). Using the Ca2+-sensitive fluorescent dye OGB-5N, we found that both young and mature granule cells showed large action-potential evoked dendritic Ca2+ transients with similar amplitude of ∼200 nm, indicating active backpropagation of action potentials. However, the decay of the dendritic Ca2+ concentration back to baseline values was substantially different with a decay time constant of 550 ms in young versus 130 ms in mature cells, leading to a more efficient temporal summation of Ca2+ signals during theta-frequency stimulation in the young neurons. Comparison of the peak Ca2+ concentration and the decay measured with different Ca2+ indicators (OGB-5N, OGB-1) in the two populations of neurons revealed that the young cells had an ∼3 times smaller endogenous Ca2+-binding ratio (∼75 versus∼220) and an ∼10 times slower Ca2+ extrusion rate (∼170 s−1versus∼1800 s−1). These data suggest that the large dendritic Ca2+ signals due to low buffer capacity and slow extrusion rates in young granule cells may contribute to the activity-dependent growth and plasticity of dendrites and new synaptic connections. This will finally support differentiation and integration of young neurons into the hippocampal network. PMID:18591186

  10. Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro; Nakamichi, Yoko; Nishiwaki, Chiyono; Kawakami, Hayato; Nagamatsu, Shinya

    2009-12-04

    To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic {beta} cells, the regulated biphasic exocytosis from two types of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.

  11. Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy.

    PubMed

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro; Nakamichi, Yoko; Nishiwaki, Chiyono; Kawakami, Hayato; Nagamatsu, Shinya

    2009-12-01

    To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic beta cells, the regulated biphasic exocytosis from two types of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.

  12. Radiosensitizing Effects of Ectopic miR-101 on Non-Small-Cell Lung Cancer Cells Depend on the Endogenous miR-101 Level

    SciTech Connect

    Chen, Susie; Wang Hongyan; Ng, Wooi Loon; Curran, Walter J.; Wang Ya

    2011-12-01

    Purpose: Previously, we showed that ectopic miR-101 could sensitize human tumor cells to radiation by targeting ATM and DNA-PK catalytic subunit (DNA-PKcs) to inhibit DNA repair, as the endogenous miR-101 levels are low in tumors in general. However, the heterogeneity of human cancers may result in an exception. The purpose of this study was to test the hypothesis that a few tumor cell lines with a high level of endogenous miR-101 would prove less response to ectopic miR-101. Methods and Materials: Fourteeen non-small-cell lung cancer (NSCLC) cell lines and one immortalized non-malignant lung epithelial cell line (NL20) were used for comparing endogenous miR-101 levels by real-time reverse transcription-polymerase chain reaction. Based on the different miR-101 levels, four cell lines with different miR-101 levels were chosen for transfection with a green fluorescent protein-lentiviral plasmid encoding miR-101. The target protein levels were measured by using Western blotting. The radiosensitizing effects of ectopic miR-101 on these NSCLC cell lines were determined by a clonogenic assay and xenograft mouse model. Results: The endogenous miR-101 level was similar or lower in 13 NSCLC cell lines but was 11-fold higher in one cell line (H157) than in NL20 cells. Although ectopic miR-101 efficiently decreased the ATM and DNA-PKcs levels and increased the radiosensitization level in H1299, H1975, and A549 cells, it did not change the levels of the miR-101 targets or radiosensitivity in H157 cells. Similar results were observed in xenograft mice. Conclusions: A small number of NSCLC cell lines could have a high level of endogenous miR-101. The ectopic miR-101 was able to radiosensitize most NSCLC cells, except for the NSCLC cell lines that had a much higher endogenous miR-101 level. These results suggest that when we choose one miRNA as a therapeutic tool, the endogenous level of the miRNA in each tumor should be considered.

  13. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans

    PubMed Central

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S.; Greenstein, David; Navarro, Rosa E.

    2016-01-01

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline—the immortal cell lineage required for sexual reproduction—protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. PMID:26865701

  14. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

    PubMed

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S; Greenstein, David; Navarro, Rosa E

    2016-01-01

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline-the immortal cell lineage required for sexual reproduction-protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. PMID:26865701

  15. Distinct Integrin-Dependent Signals Define Requirements for Lytic Granule Convergence and Polarization in Natural Killer Cells

    PubMed Central

    Hsu, Hsiang-Ting; Orange, Jordan S.

    2015-01-01

    Lytic granules in natural killer (NK) cells represent a dangerous cargo that is targeted for secretion to destroy diseased cells. The appropriate management of these organelles enables the mounting of a precise and valuable host defense. The process of NK cell adhesion to a target cell through engagement of the integrin LFA-1 (lymphocyte function–associated antigen 1) promotes lytic granule organization through complex cellular mechanics and a signaling pathway characterized by Zhang et al. in this issue of Science Signaling. A limited, partially overlapping set of signaling molecules can be distinguished for their ability to promote the convergence of NK cell lytic granules on the microtubule organizing center and their polarization as they progress en masse toward the interface with a target cell. PMID:25292212

  16. Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells

    PubMed Central

    Brennecke, Philip; Reyes, Alejandro; Pinto, Sheena; Rattay, Kristin; Nguyen, Michelle; Küchler, Rita; Huber, Wolfgang; Kyewski, Bruno; Steinmetz, Lars M.

    2015-01-01

    Expression of tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for self-tolerance induction and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA-sequencing and provide evidence for numerous recurring TRA co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process, which might involve local re-modeling of chromatin and thus ensures a comprehensive representation of the immunological self. PMID:26237553

  17. N-methyl-D-aspartate promotes the survival of cerebellar granule cells in culture.

    PubMed

    Balázs, R; Jørgensen, O S; Hack, N

    1988-11-01

    Our previous studies on the survival-promoting influence of elevated concentrations of extracellular K+ ([K+]e) on cultured cerebellar granule cells led to the proposal that depolarization in vitro mimics the effect of the earliest afferent inputs received by the granule cells in vivo. This, in turn, might be mediated through the stimulation of excitatory amino acid receptors, in particular the N-methyl-D-aspartate-preferring subtype gating ion channels which are also permeable to Ca2+. Here we report that N-methyl-D-aspartate indeed has a dramatic effect on the survival in culture of cells derived from dissociated cerebella of 7-8-day-old rats and cultured in media containing 'low' [K+]e (5-15 mM). In addition to the visual inspection of the cultures, the effect of N-methyl-D-aspartate was quantitatively evaluated, using estimates related to the number of viable cells (determination of DNA and of reduction rate of a tetrazolium salt). Furthermore, proteins which are relatively enriched in either nerve cells (neuronal cell adhesion molecule, D3-protein and synaptin) or in glia (glutamine synthetase) were also measured. The findings showed that the rescue of cells by N-methyl-D-aspartate involved primarily nerve cells and that the survival requirement for N-methyl-D-aspartate, as for high K+, developed between 2 and 4 days in vitro. The effect depended on both the concentration of N-methyl-D-aspartate and the degree of depolarization of the cells: both the potency and the efficacy of N-methyl-D-aspartate were increased as [K+]e was raised from 5 to 15 mM, at which range K+ on its own has little if any influence on granule cell survival. These characteristics are consistent with the voltage-dependence of ion conductance through the N-methyl-D-aspartate receptor-linked channel. The most pronounced effect of N-methyl-D-aspartate was obtained in the presence of 15 mM K+, when cell survival approached that obtained in 'control' cultures (grown in 25 mM K

  18. Generation and Characterization of an Nse-CreERT2 Transgenic Line Suitable for Inducible Gene Manipulation in Cerebellar Granule Cells

    PubMed Central

    Pohlkamp, Theresa; Steller, Laura; May, Petra; Günther, Thomas; Schüle, Roland; Frotscher, Michael

    2014-01-01

    We created an Nse-CreERT2 mouse line expressing the tamoxifen-inducible CreERT2 recombinase under the control of the neuron-specific enolase (Nse) promoter. By using Cre reporter lines we could show that this Nse-CreERT2 line has recombination activity in the granule cells of all cerebellar lobules as well as in postmitotic granule cell precursors in the external granular layer of the developing cerebellum. A few hippocampal dentate gyrus granule cells showed Cre-mediated recombination as well. Cre activity could be induced in both the developing and adult mouse brain. The established mouse line constitutes a valuable tool to study the function of genes expressed by cerebellar granule cells in the developing and adult brain. In combination with reporter lines it is a useful model to analyze the development and maintenance of the cerebellar architecture including granule cell distribution, migration, and the extension of granule cell fibers in vivo. PMID:24950299

  19. Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

    SciTech Connect

    Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.; Paulsen, Ragnhild E.

    2010-09-15

    Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

  20. Glucose Toxic Effects on Granulation Tissue Productive Cells: The Diabetics' Impaired Healing

    PubMed Central

    Berlanga-Acosta, Jorge; Schultz, Gregory S.; López-Mola, Ernesto; Guillen-Nieto, Gerardo; García-Siverio, Marianela; Herrera-Martínez, Luis

    2013-01-01

    Type 2 diabetes mellitus is a metabolic noncommunicable disease with an expanding pandemic magnitude. Diabetes predisposes to lower extremities ulceration and impairs the healing process leading to wound chronification. Diabetes also dismantles innate immunity favoring wound infection. Amputation is therefore acknowledged as one of the disease's complications. Hyperglycemia is the proximal detonator of systemic and local toxic effectors including proinflammation, acute-phase proteins elevation, and spillover of reactive oxygen and nitrogen species. Insulin axis deficiency weakens wounds' anabolism and predisposes to inflammation. The systemic accumulation of advanced glycation end-products irreversibly impairs the entire physiology from cells-to-organs. These factors in concert hamper fibroblasts and endothelial cells proliferation, migration, homing, secretion, and organization of a productive granulation tissue. Diabetic wound bed may turn chronically inflammed, procatabolic, and an additional source of circulating pro-inflammatory cytokines, establishing a self-perpetuating loop. Diabetic fibroblasts and endothelial cells may bear mitochondrial damages becoming prone to apoptosis, which impairs granulation tissue cellularity and perfusion. Endothelial progenitor cells recruitment and tubulogenesis are also impaired. Failure of wound reepithelialization remains a clinical challenge while it appears to be biologically multifactorial. Ulcer prevention by primary care surveillance, education, and attention programs is of outmost importance to reduce worldwide amputation figures. PMID:23484099

  1. Glucose toxic effects on granulation tissue productive cells: the diabetics' impaired healing.

    PubMed

    Berlanga-Acosta, Jorge; Schultz, Gregory S; López-Mola, Ernesto; Guillen-Nieto, Gerardo; García-Siverio, Marianela; Herrera-Martínez, Luis

    2013-01-01

    Type 2 diabetes mellitus is a metabolic noncommunicable disease with an expanding pandemic magnitude. Diabetes predisposes to lower extremities ulceration and impairs the healing process leading to wound chronification. Diabetes also dismantles innate immunity favoring wound infection. Amputation is therefore acknowledged as one of the disease's complications. Hyperglycemia is the proximal detonator of systemic and local toxic effectors including proinflammation, acute-phase proteins elevation, and spillover of reactive oxygen and nitrogen species. Insulin axis deficiency weakens wounds' anabolism and predisposes to inflammation. The systemic accumulation of advanced glycation end-products irreversibly impairs the entire physiology from cells-to-organs. These factors in concert hamper fibroblasts and endothelial cells proliferation, migration, homing, secretion, and organization of a productive granulation tissue. Diabetic wound bed may turn chronically inflammed, procatabolic, and an additional source of circulating pro-inflammatory cytokines, establishing a self-perpetuating loop. Diabetic fibroblasts and endothelial cells may bear mitochondrial damages becoming prone to apoptosis, which impairs granulation tissue cellularity and perfusion. Endothelial progenitor cells recruitment and tubulogenesis are also impaired. Failure of wound reepithelialization remains a clinical challenge while it appears to be biologically multifactorial. Ulcer prevention by primary care surveillance, education, and attention programs is of outmost importance to reduce worldwide amputation figures.

  2. Altered patterning of dentate granule cell mossy fiber inputs onto CA3 pyramidal cells in limbic epilepsy

    PubMed Central

    McAuliffe, John J.; Bronson, Stefanie L.; Hester, Michael S.; Murphy, Brian L.; Dahlquist-Topalá, Renée; Richards, David A.; Danzer, Steve C.

    2009-01-01

    Impaired gating by hippocampal dentate granule cells may promote the development of limbic epilepsy by facilitating seizure spread through the hippocampal trisynaptic circuit. The second synapse in this circuit, the dentate granule cell≫CA3 pyramidal cell connection, may be of particular importance because pathological changes occurring within the dentate likely exert their principal effect on downstream CA3 pyramids. Here, we utilized GFP-expressing mice and immunolabeling for the zinc transporter ZnT-3 to reveal the pre- and postsynaptic components of granule cell≫CA3 pyramidal cell synapses following pilocarpine-epileptogenesis. Confocal analyses of these terminals revealed that while granule cell presynaptic giant boutons increased in size and complexity one month after status epilepticus, individual thorns making up the postsynaptic thorny excrescences of the CA3 pyramidal cells were reduced in number. This reduction, however, was transient, and three months after status, thorn density recovered. This recovery was accompanied by a significant change in the distribution of thorns along pyramidal cells dendrites. While thorns in control animals tended to be tightly clustered, thorns in epileptic animals were more evenly distributed. Computational modeling of thorn distributions predicted an increase in the number of boutons required to cover equivalent numbers of thorns in epileptic vs. control mice. Confirming this prediction, ZnT-3 labeling of presynaptic giant boutons apposed to GFP-expressing thorns revealed a near doubling in bouton density, while the number of individual thorns per bouton was reduced by half. Together, these data provide clear evidence of novel plastic changes occurring within the epileptic hippocampus. PMID:20014385

  3. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    PubMed

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.

  4. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    PubMed

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D. PMID:26845357

  5. Pattern of rise in subplasma membrane Ca2+ concentration determines type of fusing insulin granules in pancreatic beta cells.

    PubMed

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Nakamichi, Yoko; Nishiwaki, Chiyono; Sakurai, Takashi; Nagamatsu, Shinya

    2009-07-31

    We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca2+ concentration ([Ca2+](PM)) with the Ca2+ indicator Fura Red in rat beta cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca2+](PM) caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca2+](PM) induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca2+](PM) rise directly determines the types of fusing granules.

  6. [Microspectrofluorometric analysis of the effect of centrophenoxine on lipofuscin granules of hybridoma (Retrovirus-transformed) cells].

    PubMed

    Tatariunas, A B

    1990-04-01

    The increase of the own luminescence lipofuscin granules (LG) in the course of single and repeated ultraviolet (UV) excitations (365 nm) in hybridoma (retrovirus transformed) cells cultured with or without 5 x 10(-4) M centrophenoxine (CP) was studied by microspectrofluorometric method. It was shown that CP influences only the rate of photochemical changes of chromophores in LG. Kinetic patterns of the own luminescence intensity of LG at the wavelength of 540 nm during excitation by UV permit one to suggest the occurrence of the cyclic chromophore changes.

  7. Differentiation and developmental origin of cerebellar granule neuron ectopia in protein O-mannose UDP-N-acetylglucosaminyl transferase 1 knockout mice.

    PubMed

    Li, X; Zhang, P; Yang, Y; Xiong, Y; Qi, Y; Hu, H

    2008-03-18

    The cerebellar cortex of protein O-mannose UDP-N-acetylglucosaminyl transferase 1 (POMGnT1) knockout mice contains discrete clusters of granule neurons that fail to migrate from the external germinal layer (EGL) to the internal granule cell layer (IGL). To test the hypothesis that the breaches in the pial basement membrane and glia limitans contribute to the formation of such heterotopias, POMGnT1 deficient mice were used to examine the mechanisms underlying these migration defects. The basement membrane, glia limitans, and granule neuron development were assessed with protein markers and immunofluorescent microscopy. Further, the integrity of the pial basement membrane, and granule neuron differentiation state were assessed by electron microscopy. Localized breaches in pial basement membrane and disruptions in the glia limitans were strongly associated with ectopia of EGL cells. In such ectopias, Bergmann glia fibers were retracted and disorganized with very few protruded into the ectopic area. Thus, migration failure was correlated with a compromised Bergmann glia scaffold. Nevertheless, the ectopic EGL cells showed characteristics of differentiated granule neurons and formed synapses with mossy fibers. Altogether, these results suggest that pial basement membrane breaches and glia limitans disruptions are the underlying causes of cerebellar granule neuron ectopia in POMGnT1 knockout mice. Moreover, migration into the IGL is not required for granule cell acquisition of certain differentiated characteristics.

  8. TIA1 oxidation inhibits stress granule assembly and sensitizes cells to stress-induced apoptosis

    PubMed Central

    Arimoto-Matsuzaki, Kyoko; Saito, Haruo; Takekawa, Mutsuhiro

    2016-01-01

    Cytoplasmic stress granules (SGs) are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of misfolded proteins, and that are formed in response to certain types of stress including ER stress. SG formation contributes to cell survival not only by suppressing translation but also by sequestering some apoptosis regulatory factors. Because cells can be exposed to various stresses simultaneously in vivo, the regulation of SG assembly under multiple stress conditions is important but unknown. Here we report that reactive oxygen species (ROS) such as H2O2 oxidize the SG-nucleating protein TIA1, thereby inhibiting SG assembly. Thus, when cells are confronted with a SG-inducing stress such as ER stress caused by protein misfolding, together with ROS-induced oxidative stress, they cannot form SGs, resulting in the promotion of apoptosis. We demonstrate that the suppression of SG formation by oxidative stress may underlie the neuronal cell death seen in neurodegenerative diseases. PMID:26738979

  9. Glutamate-induced protein phosphorylation in cerebellar granule cells: role of protein kinase C.

    PubMed

    Eboli, M L; Mercanti, D; Ciotti, M T; Aquino, A; Castellani, L

    1994-10-01

    Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells. 32P-labelled cells have been stimulated with 100 microM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells. PMID:7891841

  10. Spatiotemporal profile of dendritic outgrowth from newly born granule cells in the adult rat dentate gyrus.

    PubMed

    Shapiro, Lee A; Upadhyaya, Pooja; Ribak, Charles E

    2007-05-29

    Neurogenesis in the adult dentate gyrus occurs in the subgranular zone where newborn neurons (NNs) migrate a short distance into the granule cell layer and extend their rudimentary apical dendritic processes upon a radial glial scaffold. Using doublecortin (DCX) immunocytochemistry, these growing dendrites can be visualized because dendritic growth cones, including filipodia and lamellipodia, are labeled in both light and electron microscopic preparations. To study the rate of dendritic outgrowth of newborn dentate granule cells, single injections of 5-bromo-2-deoxyuridine (BrdU) with different survival times were combined with double immunolabeling for BrdU and DCX. At the earliest time points (4 and 12 h after BrdU injections), a rudimentary process can be observed to emanate from BrdU/DCX double-labeled cells. By 48 h the dendrites first appeared in the molecular layer. By 96 h after BrdU injection, these apical dendrites extended into the middle of the molecular layer where they ramified. The calculated rate of dendritic growth for NNs was about 15 microm per day for the first 3 days, and then a doubling in length occurred at 4 and 5 days that coincided with a retraction of the basal dendrite. In addition, electron microscopy of DCX-labeled apical dendrites showed that they were much thinner (1/4 to 1/3 the size) in diameter than unlabeled, mature apical dendrites and that they had developing synapses on them in the molecular layer.

  11. Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Hedgehog-induced medulloblastoma

    PubMed Central

    Schüller, Ulrich; Heine, Vivi M.; Mao, Junhao; Kho, Alvin T.; Dillon, Allison K.; Han, Young-Goo; Huillard, Emmanuelle; Sun, Tao; Ligon, Azra H.; Qian, Ying; Ma, Qiufu; Alvarez-Buylla, Arturo; McMahon, Andrew P.; Rowitch, David H.; Ligon, Keith L.

    2008-01-01

    Origins of the brain tumor, medulloblastoma, from stem cells or restricted progenitor cells are unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted CNS progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNP) derive from hGFAP+ and Olig2+ RL progenitors. Hh activation in a spectrum of early and late stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and that they retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors. PMID:18691547

  12. Ectopic lymphoid tissues and local immunity

    PubMed Central

    Carragher, Damian M.; Rangel-Moreno, Javier; Randall, Troy D.

    2008-01-01

    Ectopic or tertiary lymphoid tissues develop at sites of inflammation or infection in peripheral, non-lymphoid organs. These tissues are architecturally similar to conventional secondary lymphoid organs, with separated B and T cell areas, specialized populations of dendritic cells, well-differentiated stromal cells and high endothelial venules. Ectopic lymphoid tissues are often associated with the local pathology that results from chronic infection or chronic inflammation. However, there are also examples in which ectopic lymphoid tissues appear to contribute to local protective immune responses. Here we review how ectopic lymphoid structures develop and function in the context of local immunity and pathology. PMID:18243731

  13. Distribution and phenotypes of unipolar brush cells in relation to the granule cell system of the rat cochlear nuclear nucleus

    PubMed Central

    Diño, Maria. R.; Mugnaini, Enrico

    2009-01-01

    In most mammals the cochlear nuclear complex (CN) contains a distributed system of granule cells (GCS), whose parallel fiber axons innervate the dorsal cochlear nucleus (DCN). Like their counterpart in cerebellum, CN granules are innervated by mossy fibers of various origins. The GCS is complemented by unipolar brush (UBCs) and Golgi cells, and by stellate and cartwheel cells of the DCN. This cerebellum-like microcircuit modulates the activity of the DCN’s main projection neurons, the pyramidal, giant and tuberculoventral neurons, and is thought to improve auditory performance by integrating acoustic and proprioceptive information. In this paper, we focus on the UBCs, a chemically heterogeneous neuronal population, using antibodies to calretinin, mGluR1α epidermal growth factor substrate 8 (Eps8) and the transcription factor Tbr2. Eps8 and Tbr2 labeled most of the CN’s UBCs, if not the entire population, while calretinin and mGluR1α distinguished two largely separate subsets with overlapping distributions. By double labeling with antibodies to Tbr2 and the α6 GABAA-receptor subunit, we found that UBCs populate all regions of the GCS and occur at remarkably high densities in the DCN and subpeduncular corner, but rarely in the lamina. Although GCS subregions likely share the same microcircuitry, their dissimilar UBC densities suggest they may be functionally distinct. UBCs and granules are also present in regions previously not included in the GCS, namely the rostrodorsal magnocellular portions of VCN, vestibular nerve root, trapezoid body, spinal tract and sensory and principal nuclei of the trigeminal nerve, and cerebellar peduncles. The UBC’s dendritic brush receives AMPA- and NMDA-mediated input from an individual mossy fiber, favoring singularity of input, and its axon most likely forms several mossy fiber-like endings that target numerous granule cells and other UBCs, as in the cerebellum. The UBCs therefore, may amplify afferent signals temporally and

  14. Inferior ectopic bone formation of mesenchymal stromal cells from adipose tissue compared to bone marrow: rescue by chondrogenic pre-induction.

    PubMed

    Brocher, J; Janicki, P; Voltz, P; Seebach, E; Neumann, E; Mueller-Ladner, U; Richter, W

    2013-11-01

    Human mesenchymal stromal cells derived from bone marrow (BMSC) and adipose tissue (ATSC) represent a valuable source of progenitor cells for cell therapy and tissue engineering. While ectopic bone formation is a standard activity of human BMSC on calcium phosphate ceramics, the bone formation capacity of human ATSC has so far been unclear. The objectives of this study were to assess the therapeutic potency of ATSC for bone formation in an ectopic mouse model and determine molecular differences by standardized comparison with BMSC. Although ATSC contained less CD146(+) cells, exhibited better proliferation and displayed similar alkaline phosphatase activity upon osteogenic in vitro differentiation, cells did not develop into bone-depositing osteoblasts on β-TCP after 8weeks in vivo. Additionally, ATSC expressed less BMP-2, BMP-4, VEGF, angiopoietin and IL-6 and more adiponectin mRNA, altogether suggesting insufficient osteochondral commitment and reduced proangiogenic activity. Chondrogenic pre-induction of ATSC/β-TCP constructs with TGF-β and BMP-6 initiated ectopic bone formation in >75% of samples. Both chondrogenic pre-induction and the osteoconductive microenvironment of β-TCP were necessary for ectopic bone formation by ATSC pointing towards a need for inductive conditions/biomaterials to make this more easily accessible cell source attractive for future applications in bone regeneration.

  15. The perinuclear factor, a rheumatoid arthritis-specific autoantigen, is not present in keratohyalin granules of cultured buccal mucosa cells.

    PubMed Central

    Hoet, R M; Voorsmit, R A; Van Venrooij, W J

    1991-01-01

    Rheumatoid arthritis patients have antibodies in their serum directed against the perinuclear factor, a protein component present in keratohyalin granules in the cytoplasm of human buccal mucosa cells. The anti-perinuclear factor (APF) can only be detected by an indirect immunofluorescence test performed on fresh buccal mucosa cells from 'selected donors'. To obtain a more reliable antigen source and to gain more insight into the origin and nature of the perinuclear factor we attempted to culture perinuclear factor-containing buccal mucosa cells. Here we describe the successful culturing of such cells, which, however, did not contain keratohyalin granules nor the perinuclear factor. By adding the phorbol ester 12-o-tetradecanoylphorbol-13-acetate (TPA) we were able to induce keratohyalin granules in both cultured primary buccal mucosa cells and a squamous carcinoma cell line of the cheek (SqCC/Y1). These induced keratohyalin granules do contain the protein profilaggrin, which in vivo, in fresh buccal mucosa cells, co-localizes with the perinuclear factor. However, we were not able to demonstrate the presence of the perinuclear factor, not even after induction of terminal differentiation of the cultured cells nor after Epstein-Barr virus infection. Our results suggest that the perinuclear factor, in contrast to profilaggrin, is not an integral component of buccal mucosa cells. Images Fig. 1 PMID:1849807

  16. Cholinergic afferent stimulation induces axonal function plasticity in adult hippocampal granule cells.

    PubMed

    Martinello, Katiuscia; Huang, Zhuo; Lujan, Rafael; Tran, Baouyen; Watanabe, Masahiko; Cooper, Edward C; Brown, David A; Shah, Mala M

    2015-01-21

    Acetylcholine critically influences hippocampal-dependent learning. Cholinergic fibers innervate hippocampal neuron axons, dendrites, and somata. The effects of acetylcholine on axonal information processing, though, remain unknown. By stimulating cholinergic fibers and making electrophysiological recordings from hippocampal dentate gyrus granule cells, we show that synaptically released acetylcholine preferentially lowered the action potential threshold, enhancing intrinsic excitability and synaptic potential-spike coupling. These effects persisted for at least 30 min after the stimulation paradigm and were due to muscarinic receptor activation. This caused sustained elevation of axonal intracellular Ca(2+) via T-type Ca(2+) channels, as indicated by two-photon imaging. The enhanced Ca(2+) levels inhibited an axonal KV7/M current, decreasing the spike threshold. In support, immunohistochemistry revealed muscarinic M1 receptor, CaV3.2, and KV7.2/7.3 subunit localization in granule cell axons. Since alterations in axonal signaling affect neuronal firing patterns and neurotransmitter release, this is an unreported cellular mechanism by which acetylcholine might, at least partly, enhance cognitive processing. PMID:25578363

  17. In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

    SciTech Connect

    Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B. . E-mail: jbarnett@hsc.wvu.edu

    2007-06-01

    The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

  18. Initial loss but later excess of GABAergic synapses with dentate granule cells in a rat model of temporal lobe epilepsy.

    PubMed

    Thind, Khushdev K; Yamawaki, Ruth; Phanwar, Ibanri; Zhang, Guofeng; Wen, Xiling; Buckmaster, Paul S

    2010-03-01

    Many patients with temporal lobe epilepsy display neuron loss in the dentate gyrus. One potential epileptogenic mechanism is loss of GABAergic interneurons and inhibitory synapses with granule cells. Stereological techniques were used to estimate numbers of gephyrin-positive punctae in the dentate gyrus, which were reduced short-term (5 days after pilocarpine-induced status epilepticus) but later rebounded beyond controls in epileptic rats. Stereological techniques were used to estimate numbers of synapses in electron micrographs of serial sections processed for postembedding GABA-immunoreactivity. Adjacent sections were used to estimate numbers of granule cells and glutamic acid decarboxylase-positive neurons per dentate gyrus. GABAergic neurons were reduced to 70% of control levels short-term, where they remained in epileptic rats. Integrating synapse and cell counts yielded average numbers of GABAergic synapses per granule cell, which decreased short-term and rebounded in epileptic animals beyond control levels. Axo-shaft and axo-spinous GABAergic synapse numbers in the outer molecular layer changed most. These findings suggest interneuron loss initially reduces numbers of GABAergic synapses with granule cells, but later, synaptogenesis by surviving interneurons overshoots control levels. In contrast, the average number of excitatory synapses per granule cell decreased short-term but recovered only toward control levels, although in epileptic rats excitatory synapses in the inner molecular layer were larger than in controls. These findings reveal a relative excess of GABAergic synapses and suggest that reports of reduced functional inhibitory synaptic input to granule cells in epilepsy might be attributable not to fewer but instead to abundant but dysfunctional GABAergic synapses.

  19. NMDA receptor-dependent CREB activation in survival of cerebellar granule cells during in vivo and in vitro development.

    PubMed

    Monti, Barbara; Marri, Lucia; Contestabile, Antonio

    2002-10-01

    During both in vivo and in vitro development, cerebellar granule cells depend on the activity of the NMDA glutamate receptor subtype for survival and full differentiation. With the present results, we demonstrate that CREB activation, downstream of the NMDA receptor, is a necessary step to ensure survival of these neurons. The levels of CREB expression and activity increase progressively during the second week of postnatal cerebellar development and the phosphorylated form of CREB is localized selectively to cerebellar granule cells during the critical developmental stages examined. Chronically blocking the NMDA receptor through systemic administration of the competitive antagonist, CGP 39551, during the in vivo critical developmental period, between 7-11 postnatal days, results in increased apoptotic elimination of differentiating granule neurons in the cerebellum [Monti & Contestabile, Eur. J. Neurosci., 12, 3117-3123 (2000)]. We report here that this event is accompanied by a significant decrease of CREB phosphorylation in the cerebellum of treated rat pups. When cerebellar granule neurons are explanted and maintained in dissociated cultures, the levels of CREB phosphorylation increase with differentiation, similar to that which happens during in vivo development. When granule cells are kept in non-trophic conditions, their viability is affected and both CREB phosphorylation and transcriptional activity are decreased significantly. The neuronal viability and the deficiency of CREB activity, are both rescued by the pharmacological activation of the NMDA receptor. These results provide good circumstantial evidence for a functional link between the NMDA receptor and CREB activity in promoting neuronal survival during development.

  20. Telocytes as a Source of Progenitor Cells in Regeneration and Repair Through Granulation Tissue.

    PubMed

    Díaz-Flores, Lucio; Gutiérrez, Ricardo; Pino García, Maria; González, Miriam; Díaz-Flores, Lucio; Francisco Madrid, Juan

    2016-01-01

    This review outlines the role of CD34+ stromal cells/telocytes (CD34+ SC/TCs) in repair and considers the following issues. Firstly, the conceptual aspects of repair, including regeneration and repair through granulation tissue (RTGT) as two types of repair, RTGT stages (inflammatory, proliferative, and remodeling), and tissue in repair as a substrate to assess the in vivo behavior of activated CD34+ SC/TCs. Subsequently, current knowledge of CD34+ SC/TCs, such as identification, characteristics, and functions, as well as possible stages (quiescent and activated) are taken into account. We then consider the role in regeneration of quiescent CD34+ SC/TCs (in unperturbed physiological conditions) as a nurse of stem cells (e.g., in the heart, skin, respiratory tree, gastrointestinal tract, liver, eye, and choroid plexus). Special attention is paid to the characteristics of activated CD34+ SC/TCs and the overlapping steps of activation with and without loss of CD34 expression and with and without gain of αSMA expression. With this contribution, we establish the role of CD34+ SC/TCs as progenitor cells and as a source of fibroblasts and myofibroblasts in repair through granulation tissue, fibrosis, and tumor stroma. Activated CD34+ SC/TCs in encapsulation and other processes (e.g., Reinke's edema, cutaneous myxoid cyst, mixomatous mitral valve degeneration, and fibrous papula of the face) are also outlined. Finally, similarities between modifications of CD34+ SC/TCs during in vivo activation and of multipotent mesenchymal stromal/stem cells in culture are examined in order to correlate the growing literature on CD34+ SC/TCs and the exponential research in cultured mesenchymal stromal/stem cells.

  1. Transgenic Expression of Ad4BP/SF-1 in Fetal Adrenal Progenitor Cells Leads to Ectopic Adrenal Formation

    PubMed Central

    Zubair, Mohamad; Oka, Sanae; Parker, Keith L.; Morohashi, Ken-ichirou

    2009-01-01

    Deficiency of adrenal 4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1; NR5A1) impairs adrenal development in a dose-dependent manner, whereas overexpression of Ad4BP/SF-1 is associated with adrenocortical tumorigenesis. Despite its essential roles in adrenal development, the mechanism(s) by which Ad4BP/SF-1 regulates this process remain incompletely understood. We previously identified a fetal adrenal enhancer (FAdE) that stimulates Ad4BP/SF-1 expression in the fetal adrenal gland by a two-step mechanism in which homeobox proteins initiate Ad4BP/SF-1 expression, which then maintains FAdE activity in an autoregulatory loop. In the present study, we examined the effect of transgenic expression of Ad4BP/SF-1 controlled by FAdE on adrenal development. When Ad4BP/SF-1 was overexpressed using a FAdE-Ad4BP/SF-1 transgene, FAdE activity expanded outside of its normal field, resulting in increased adrenal size and the formation of ectopic adrenal tissue in the thorax. The increased size of the adrenal gland did not result from a corresponding increase in cell proliferation, suggesting rather that the increased levels of Ad4BP/SF-1 may divert uncommitted precursors to the steroidogenic lineage. The effects of FAdE-controlled Ad4BP/SF-1 overexpression in mice provide a novel model of ectopic adrenal formation that further supports the critical role of Ad4BP/SF-1 in the determination of steroidogenic cell fate in vivo. PMID:19628584

  2. The role of calcium and cyclic nucleotide signaling in cerebellar granule cell migration under normal and pathological conditions.

    PubMed

    Komuro, Yutaro; Galas, Ludovic; Lebon, Alexis; Raoult, Emilie; Fahrion, Jennifer K; Tilot, Amanda; Kumada, Tasturo; Ohno, Nobuhiko; Vaudry, David; Komuro, Hitoshi

    2015-04-01

    In the developing brain, immature neurons migrate from their sites of origin to their final destination, where they reside for the rest of their lives. This active movement of immature neurons is essential for the formation of normal neuronal cytoarchitecture and proper differentiation. Deficits in migration result in the abnormal development of the brain, leading to a variety of neurological disorders. A myriad of extracellular guidance molecules and intracellular effector molecules is involved in controlling the migration of immature neurons in a cell type, cortical layer and birth-date-specific manner. To date, little is known about how extracellular guidance molecules transfer their information to the intracellular effector molecules, which regulate the migration of immature neurons. In this article, to fill the gap between extracellular guidance molecules and intracellular effector molecules, using the migration of cerebellar granule cells as a model system of neuronal cell migration, we explore the role of second messenger signaling (specifically Ca(2+) and cyclic nucleotide signaling) in the regulation of neuronal cell migration. We will, first, describe the cortical layer-specific changes in granule cell migration. Second, we will discuss the roles of Ca(2+) and cyclic nucleotide signaling in controlling granule cell migration. Third, we will present recent studies showing the roles of Ca(2+) and cyclic nucleotide signaling in the deficits in granule cell migration in mouse models of fetal alcohol spectrum disorders and fetal Minamata disease.

  3. N-methyl-D-aspartate promotes the survival of cerebellar granule cells: pharmacological characterization.

    PubMed

    Balázs, R; Hack, N; Jørgensen, O S; Cotman, C W

    1989-07-01

    The survival of cerebellar granule cells in culture is promoted by chronic exposure to N-methyl-D-aspartate (NMDA). The effect is due to the stimulation of 'conventional' NMDA receptor-ionophore complex: it is concentration dependent, voltage dependent and blocked by the selective antagonists D-2-amino-5-phosphonovalerate, D-2-amino-7-phosphonoheptanoate, dextromethorphan and (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imin emaleate (MK 801). The most potent antagonist tested was MK-801. In contrast, non-selective antagonists, including kynurenate, were much less effective. Further, the trophic effect of NMDA is not reproduced by ibotenate or quinolinate at the concentration range tested. It could also be shown that glutamate released into the culture medium is responsible for limited cell survival in the absence of NMDA.

  4. Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

    PubMed

    Chang, Hsin-Fang; Bzeih, Hawraa; Schirra, Claudia; Chitirala, Praneeth; Halimani, Mahantappa; Cordat, Emmanuelle; Krause, Elmar; Rettig, Jens; Pattu, Varsha

    2016-09-15

    CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

  5. Disruption of centrifugal inhibition to olfactory bulb granule cells impairs olfactory discrimination

    PubMed Central

    Nunez-Parra, Alexia; Maurer, Robert K.; Krahe, Krista; Smith, Richard S.; Araneda, Ricardo C.

    2013-01-01

    Granule cells (GCs) are the most abundant inhibitory neuronal type in the olfactory bulb and play a critical role in olfactory processing. GCs regulate the activity of principal neurons, the mitral cells, through dendrodendritic synapses, shaping the olfactory bulb output to other brain regions. GC excitability is regulated precisely by intrinsic and extrinsic inputs, and this regulation is fundamental for odor discrimination. Here, we used channelrhodopsin to stimulate GABAergic axons from the basal forebrain selectively and show that this stimulation generates reliable inhibitory responses in GCs. Furthermore, selective in vivo inhibition of GABAergic neurons in the basal forebrain by targeted expression of designer receptors exclusively activated by designer drugs produced a reversible impairment in the discrimination of structurally similar odors, indicating an important role of these inhibitory afferents in olfactory processing. PMID:23959889

  6. Maturation and Functional Integration of New Granule Cells into the Adult Hippocampus.

    PubMed

    Toni, Nicolas; Schinder, Alejandro F

    2015-12-04

    The adult hippocampus generates functional dentate granule cells (GCs) that release glutamate onto target cells in the hilus and cornus ammonis (CA)3 region, and receive glutamatergic and γ-aminobutyric acid (GABA)ergic inputs that tightly control their spiking activity. The slow and sequential development of their excitatory and inhibitory inputs makes them particularly relevant for information processing. Although they are still immature, new neurons are recruited by afferent activity and display increased excitability, enhanced activity-dependent plasticity of their input and output connections, and a high rate of synaptogenesis. Once fully mature, new GCs show all the hallmarks of neurons generated during development. In this review, we focus on how developing neurons remodel the adult dentate gyrus and discuss key aspects that illustrate the potential of neurogenesis as a mechanism for circuit plasticity and function.

  7. Glycosylphosphatidylinositol is involved in the membrane attachment of proteins in granules of chromaffin cells.

    PubMed Central

    Fouchier, F; Bastiani, P; Baltz, T; Aunis, D; Rougon, G

    1988-01-01

    Incubation at 37 degrees C or treatment of granule membranes of chromaffin cells with Staphylococcus aureus phosphatidylinositol-specific phospholipase C converted from an amphiphilic to a hydrophilic form two proteins with molecular masses of 82 and 68 kDa respectively. Their release is time- and enzyme-concentration-dependent. We showed that they were immunoreactive with an anti-(cross-reacting determinant) antibody known to be revealed only after removal of a diacylglycerol anchor. Furthermore, the action of HNO2 suggests the presence of a non-acetylated glucosamine residue in the determinant. This is one of the first reports suggesting that a glycosylphosphatidylinositol anchor might exist in membranes other than the plasma membrane. We showed that the 68 kDa protein is probably not the subunit of dopamine (3,4-dihydroxyphenethylamine) beta-hydroxylase, an enzyme present in granules in both soluble and membrane-associated forms. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:2851974

  8. Revisiting the single cell protein application of Cupriavidus necator H16 and recovering bioplastic granules simultaneously.

    PubMed

    Kunasundari, Balakrishnan; Murugaiyah, Vikneswaran; Kaur, Gurjeet; Maurer, Frans H J; Sudesh, Kumar

    2013-01-01

    Cupriavidus necator H16 (formerly known as Hydrogenomonas eutropha) was famous as a potential single cell protein (SCP) in the 1970s. The drawback however was the undesirably efficient accumulation of non-nutritive polyhydroxybutyrate (PHB) storage compound in the cytoplasm of this bacterium. Eventually, competition from soy-based protein resulted in SCP not receiving much attention. Nevertheless, C. necator H16 remained in the limelight as a producer of PHB, which is a material that resembles commodity plastics such as polypropylene. PHB is a 100% biobased and biodegradable polyester. Although tremendous achievements have been attained in the past 3 decades in the efficient production of PHB, this bioplastic is still costly. One of the main problems has been the recovery of PHB from the cell cytoplasm. In this study, we showed for the first time that kilogram quantities of PHB can be easily recovered in the laboratory without the use of any solvents and chemicals, just by using the cells as SCP. In addition, the present study also demonstrated the safety and tolerability of animal model used, Sprague Dawley given lyophilized cells of C. necator H16. The test animals readily produced fecal pellets that were whitish in color, as would be expected of PHB granules. The pellets were determined to contain about 82-97 wt% PHB and possessed molecular mass of around 930 kg/mol. The PHB granules recovered biologically possessed similar molecular mass compared to chloroform extracted PHB [950 kg/mol]. This method now allows the production and purification of substantial quantities of PHB for various experimental trials. The method reported here is easy, does not require expensive instrumentation, scalable and does not involve extensive use of solvents and strong chemicals.

  9. Revisiting the single cell protein application of Cupriavidus necator H16 and recovering bioplastic granules simultaneously.

    PubMed

    Kunasundari, Balakrishnan; Murugaiyah, Vikneswaran; Kaur, Gurjeet; Maurer, Frans H J; Sudesh, Kumar

    2013-01-01

    Cupriavidus necator H16 (formerly known as Hydrogenomonas eutropha) was famous as a potential single cell protein (SCP) in the 1970s. The drawback however was the undesirably efficient accumulation of non-nutritive polyhydroxybutyrate (PHB) storage compound in the cytoplasm of this bacterium. Eventually, competition from soy-based protein resulted in SCP not receiving much attention. Nevertheless, C. necator H16 remained in the limelight as a producer of PHB, which is a material that resembles commodity plastics such as polypropylene. PHB is a 100% biobased and biodegradable polyester. Although tremendous achievements have been attained in the past 3 decades in the efficient production of PHB, this bioplastic is still costly. One of the main problems has been the recovery of PHB from the cell cytoplasm. In this study, we showed for the first time that kilogram quantities of PHB can be easily recovered in the laboratory without the use of any solvents and chemicals, just by using the cells as SCP. In addition, the present study also demonstrated the safety and tolerability of animal model used, Sprague Dawley given lyophilized cells of C. necator H16. The test animals readily produced fecal pellets that were whitish in color, as would be expected of PHB granules. The pellets were determined to contain about 82-97 wt% PHB and possessed molecular mass of around 930 kg/mol. The PHB granules recovered biologically possessed similar molecular mass compared to chloroform extracted PHB [950 kg/mol]. This method now allows the production and purification of substantial quantities of PHB for various experimental trials. The method reported here is easy, does not require expensive instrumentation, scalable and does not involve extensive use of solvents and strong chemicals. PMID:24205250

  10. Revisiting the Single Cell Protein Application of Cupriavidus necator H16 and Recovering Bioplastic Granules Simultaneously

    PubMed Central

    Kunasundari, Balakrishnan; Murugaiyah, Vikneswaran; Kaur, Gurjeet; Maurer, Frans H. J.; Sudesh, Kumar

    2013-01-01

    Cupriavidus necator H16 (formerly known as Hydrogenomonas eutropha) was famous as a potential single cell protein (SCP) in the 1970s. The drawback however was the undesirably efficient accumulation of non-nutritive polyhydroxybutyrate (PHB) storage compound in the cytoplasm of this bacterium. Eventually, competition from soy-based protein resulted in SCP not receiving much attention. Nevertheless, C. necator H16 remained in the limelight as a producer of PHB, which is a material that resembles commodity plastics such as polypropylene. PHB is a 100% biobased and biodegradable polyester. Although tremendous achievements have been attained in the past 3 decades in the efficient production of PHB, this bioplastic is still costly. One of the main problems has been the recovery of PHB from the cell cytoplasm. In this study, we showed for the first time that kilogram quantities of PHB can be easily recovered in the laboratory without the use of any solvents and chemicals, just by using the cells as SCP. In addition, the present study also demonstrated the safety and tolerability of animal model used, Sprague Dawley given lyophilized cells of C. necator H16. The test animals readily produced fecal pellets that were whitish in color, as would be expected of PHB granules. The pellets were determined to contain about 82-97 wt% PHB and possessed molecular mass of around 930 kg/mol. The PHB granules recovered biologically possessed similar molecular mass compared to chloroform extracted PHB [950 kg/mol]. This method now allows the production and purification of substantial quantities of PHB for various experimental trials. The method reported here is easy, does not require expensive instrumentation, scalable and does not involve extensive use of solvents and strong chemicals. PMID:24205250

  11. Stress Granules Modulate SYK to Cause Microglial Cell Dysfunction in Alzheimer's Disease.

    PubMed

    Ghosh, Soumitra; Geahlen, Robert L

    2015-11-01

    Microglial cells in the brains of Alzheimer's patients are known to be recruited to amyloid-beta (Aβ) plaques where they exhibit an activated phenotype, but are defective for plaque removal by phagocytosis. In this study, we show that microglia stressed by exposure to sodium arsenite or Aβ(1-42) peptides or fibrils form extensive stress granules (SGs) to which the tyrosine kinase, SYK, is recruited. SYK enhances the formation of SGs, is active within the resulting SGs and stimulates the production of reactive oxygen and nitrogen species that are toxic to neuronal cells. This sequestration of SYK inhibits the ability of microglial cells to phagocytose Escherichia coli or Aβ fibrils. We find that aged microglial cells are more susceptible to the formation of SGs; and SGs containing SYK and phosphotyrosine are prevalent in the brains of patients with severe Alzheimer's disease. Phagocytic activity can be restored to stressed microglial cells by treatment with IgG, suggesting a mechanism to explain the therapeutic efficacy of intravenous IgG. These studies describe a mechanism by which stress, including exposure to Aβ, compromises the function of microglial cells in Alzheimer's disease and suggest approaches to restore activity to dysfunctional microglial cells.

  12. Generation of human-induced pluripotent stem cells from gut mesentery-derived cells by ectopic expression of OCT4/SOX2/NANOG.

    PubMed

    Li, Yang; Zhao, Hongxi; Lan, Feng; Lee, Andrew; Chen, Liu; Lin, Changsheng; Yao, Yuanqing; Li, Lingsong

    2010-06-01

    Induced pluripotent stem (iPS) cells have been generated from human somatic cells by ectopic expression of defined transcription factors. Application of this approach in human cells may have enormous potential to generate patient-specific pluripotent stem cells. However, traditional methods of reprogramming in human somatic cells involve the use of oncogenes c-MYC and KLF4, which are not applicable to clinical translation. In the present study, we investigated whether human fetal gut mesentery-derived cells (hGMDCs) could be successfully reprogrammed into induced pluripotent stem (iPS) cells by OCT4, SOX2, and NANOG alone. We used lentiviruses to express OCT4, SOX2, NANOG, in hGMDCs, then generated iPS cells that were identified by morphology, presence of pluripotency markers, global gene expression profile, DNA methylation status, capacity to form embryoid bodies (EBs), and terotoma formation. iPS cells resulting from hGMDCs were similar to human embryonic stem (ES) cells in morphology, proliferation, surface markers, gene expression, and epigenetic status of pluripotent cell-specific genes. Furthermore, these cells were able to differentiate into cell types of all three germ layers both in vitro and in vivo, as shown by EB and teratoma formation assays. DNA fingerprinting showed that the human iPS cells were derived from the donor cells, and are not a result of contamination. Our results provide proof that hGMDCs can be reprogrammed into pluripotent cells by ectopic expression of three factors (OCT4, SOX2, and NANOG) without the use of oncogenes c-MYC and KLF4. PMID:20698766

  13. Shaping the landscape of the Escherichia coli chromosome: replication-transcription encounters in cells with an ectopic replication origin

    PubMed Central

    Ivanova, Darja; Taylor, Toni; Smith, Sarah L.; Dimude, Juachi U.; Upton, Amy L.; Mehrjouy, Mana M.; Skovgaard, Ole; Sherratt, David J.; Retkute, Renata; Rudolph, Christian J.

    2015-01-01

    Each cell division requires the unwinding of millions of DNA base pairs to allow chromosome duplication and gene transcription. As DNA replication and transcription share the same template, conflicts between both processes are unavoidable and head-on collisions are thought to be particularly problematic. Surprisingly, a recent study reported unperturbed cell cycle progression in Escherichia coli cells with an ectopic replication origin in which highly transcribed rrn operons were forced to be replicated opposite to normal. In this study we have re-generated a similar strain and found the doubling time to be twice that of normal cells. Replication profiles of this background revealed significant deviations in comparison to wild-type profiles, particularly in highly transcribed regions and the termination area. These deviations were alleviated by mutations that either inactivate the termination area or destabilise RNA polymerase complexes and allow their easier displacement by replication forks. Our data demonstrate that head-on replication-transcription conflicts are highly problematic. Indeed, analysis of the replication profile of the previously published E. coli construct revealed a chromosomal rearrangement that alleviates replication-transcription conflicts in an intriguingly simple way. Our data support the idea that avoiding head-on collisions has significantly contributed to shaping the distinct architecture of bacterial chromosomes. PMID:26160884

  14. Sorafenib, a multikinase inhibitor, induces formation of stress granules in hepatocarcinoma cells

    PubMed Central

    Adjibade, Pauline; St-Sauveur, Valérie Grenier; Huberdeau, Miguel Quevillon; Fournier, Marie-Josée; Savard, Andreanne; Coudert, Laetitia; Khandjian, Edouard W.; Mazroui, Rachid

    2015-01-01

    Stress granules (SGs) are cytoplasmic RNA multimeric bodies that form under stress conditions known to inhibit translation initiation. In most reported stress cases, the formation of SGs was associated with the cell recovery from stress and survival. In cells derived from cancer, SGs formation was shown to promote resistance to either proteasome inhibitors or 5-Fluorouracil used as chemotherapeutic agents. Despite these studies, the induction of SGs by chemotherapeutic drugs contributing to cancer cells resistance is still understudied. Here we identified sorafenib, a tyrosine kinase inhibitor used to treat hepatocarcinoma, as a potent chemotherapeutic inducer of SGs. The formation of SGs in sorafenib-treated hepatocarcionoma cells correlates with inhibition of translation initiation; both events requiring the phosphorylation of the translation initiation factor eIF2α. Further characterisation of the mechanism of sorafenib-induced SGs revealed PERK as the main eIF2α kinase responsible for SGs formation. Depletion experiments support the implication of PERK-eIF2α-SGs pathway in hepatocarcinoma cells resistance to sorafenib. This study also suggests the existence of an unexpected complex regulatory balance between SGs and phospho-eIF2α where SGs dampen the activation of the phospho-eIF2α-downstream ATF4 cell death pathway. PMID:26556863

  15. Stress Granules contribute to α-globin homeostasis in differentiating erythroid cells

    PubMed Central

    Ghisolfi, Laura; Dutt, Shilpee; McConkey, Marie E.; Ebert, Benjamin L.; Anderson, Paul

    2012-01-01

    Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin protein and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2α and inhibiting the translation of globin protein when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2α-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells. PMID:22452989

  16. Sorafenib, a multikinase inhibitor, induces formation of stress granules in hepatocarcinoma cells.

    PubMed

    Adjibade, Pauline; St-Sauveur, Valérie Grenier; Quevillon Huberdeau, Miguel; Fournier, Marie-Josée; Savard, Andreanne; Coudert, Laetitia; Khandjian, Edouard W; Mazroui, Rachid

    2015-12-22

    Stress granules (SGs) are cytoplasmic RNA multimeric bodies that form under stress conditions known to inhibit translation initiation. In most reported stress cases, the formation of SGs was associated with the cell recovery from stress and survival. In cells derived from cancer, SGs formation was shown to promote resistance to either proteasome inhibitors or 5-Fluorouracil used as chemotherapeutic agents. Despite these studies, the induction of SGs by chemotherapeutic drugs contributing to cancer cells resistance is still understudied. Here we identified sorafenib, a tyrosine kinase inhibitor used to treat hepatocarcinoma, as a potent chemotherapeutic inducer of SGs. The formation of SGs in sorafenib-treated hepatocarcionoma cells correlates with inhibition of translation initiation; both events requiring the phosphorylation of the translation initiation factor eIF2α. Further characterisation of the mechanism of sorafenib-induced SGs revealed PERK as the main eIF2α kinase responsible for SGs formation. Depletion experiments support the implication of PERK-eIF2α-SGs pathway in hepatocarcinoma cells resistance to sorafenib. This study also suggests the existence of an unexpected complex regulatory balance between SGs and phospho-eIF2α where SGs dampen the activation of the phospho-eIF2α-downstream ATF4 cell death pathway.

  17. Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor

    PubMed Central

    Zanin, Juan Pablo; Abercrombie, Elizabeth; Friedman, Wilma J

    2016-01-01

    Cerebellar granule cell progenitors (GCP) proliferate extensively in the external granule layer (EGL) of the developing cerebellum prior to differentiating and migrating. Mechanisms that regulate the appropriate timing of cell cycle withdrawal of these neuronal progenitors during brain development are not well defined. The p75 neurotrophin receptor (p75NTR) is highly expressed in the proliferating GCPs, but is downregulated once the cells leave the cell cycle. This receptor has primarily been characterized as a death receptor for its ability to induce neuronal apoptosis following injury. Here we demonstrate a novel function for p75NTR in regulating proper cell cycle exit of neuronal progenitors in the developing rat and mouse EGL, which is stimulated by proNT3. In the absence of p75NTR, GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits. DOI: http://dx.doi.org/10.7554/eLife.16654.001 PMID:27434667

  18. FHF-independent conduction of action potentials along the leak-resistant cerebellar granule cell axon

    PubMed Central

    Dover, Katarzyna; Marra, Christopher; Solinas, Sergio; Popovic, Marko; Subramaniyam, Sathyaa; Zecevic, Dejan; D'Angelo, Egidio; Goldfarb, Mitchell

    2016-01-01

    Neurons in vertebrate central nervous systems initiate and conduct sodium action potentials in distinct subcellular compartments that differ architecturally and electrically. Here, we report several unanticipated passive and active properties of the cerebellar granule cell's unmyelinated axon. Whereas spike initiation at the axon initial segment relies on sodium channel (Nav)-associated fibroblast growth factor homologous factor (FHF) proteins to delay Nav inactivation, distal axonal Navs show little FHF association or FHF requirement for high-frequency transmission, velocity and waveforms of conducting action potentials. In addition, leak conductance density along the distal axon is estimated as <1% that of somatodendritic membrane. The faster inactivation rate of FHF-free Navs together with very low axonal leak conductance serves to minimize ionic fluxes and energetic demand during repetitive spike conduction and at rest. The absence of FHFs from Navs at nodes of Ranvier in the central nervous system suggests a similar mechanism of current flux minimization along myelinated axons. PMID:27666389

  19. Ectopic Lignification in the Flax lignified bast fiber1 Mutant Stem Is Associated with Tissue-Specific Modifications in Gene Expression and Cell Wall Composition[C][W

    PubMed Central

    Chantreau, Maxime; Portelette, Antoine; Dauwe, Rebecca; Kiyoto, Shingo; Crônier, David; Morreel, Kris; Arribat, Sandrine; Neutelings, Godfrey; Chabi, Malika; Boerjan, Wout; Yoshinaga, Arata; Mesnard, François; Grec, Sebastien; Chabbert, Brigitte; Hawkins, Simon

    2014-01-01

    Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply. PMID:25381351

  20. Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

    PubMed

    Chantreau, Maxime; Portelette, Antoine; Dauwe, Rebecca; Kiyoto, Shingo; Crônier, David; Morreel, Kris; Arribat, Sandrine; Neutelings, Godfrey; Chabi, Malika; Boerjan, Wout; Yoshinaga, Arata; Mesnard, François; Grec, Sebastien; Chabbert, Brigitte; Hawkins, Simon

    2014-11-01

    Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

  1. Deorphanization and characterization of the ectopically expressed olfactory receptor OR51B5 in myelogenous leukemia cells

    PubMed Central

    Manteniotis, S; Wojcik, S; Göthert, J R; Dürig, J; Dührsen, U; Gisselmann, G; Hatt, H

    2016-01-01

    The ectopic expression of olfactory receptors (ORs) in the human body has been of major interest in the past decade. Several studies have reported the expression of ORs not only in healthy tissues such as heart, sperm or skin cells, but also in cancerous tissues of the liver, prostate or intestine. In the present study, we detected the expression of OR51B5 in the chronic myelogenous leukemia (CML) cell line K562 and in white blood cell samples of clinically diagnosed acute myelogenous leukemia (AML) patients by reverse transcription-PCR and immunocytochemical staining. The known OR51B5 ligand isononyl alcohol increased the levels of intracellular Ca2+ in both AML patient blood cells and K562 cells. With calcium imaging experiments, we characterized in greater detail the OR51B5-mediated signaling pathway. Here, we observed an involvement of adenylate cyclase and the downstream L-type and T-type calcium channels. In addition, the activation of OR51B5 leads to an inhibition of cell proliferation in K562 cells. In western blot experiments, we found that incubation with isononyl alcohol led to a reduction in p38-MAPK (mitogen-activated protein kinase) phosphorylation that might be responsible for the decreased cell proliferation. In the present study, we characterized the OR51B5-mediated signaling pathway downstream of the activation with isononyl alcohol, which leads to reduced proliferation and therefore provide a novel pharmacological target for CML and AML, the latter of which remains difficult to treat. PMID:27551504

  2. Object/Context-Specific Memory Deficits Associated with Loss of Hippocampal Granule Cells after Adrenalectomy in Rats

    ERIC Educational Resources Information Center

    Spanswick, Simon C.; Sutherland, Robert J.

    2010-01-01

    Chronic adrenalectomy (ADX) causes a gradual and selective loss of granule cells in the dentate gyrus (DG) of the rat. Here, we administered replacement corticosterone to rats beginning 10 wk after ADX. We then tested them in three discrimination tasks based on object novelty, location, or object/context association. Only during testing of the…

  3. PERK Activation Promotes Medulloblastoma Tumorigenesis by Attenuating Premalignant Granule Cell Precursor Apoptosis.

    PubMed

    Ho, Yeung; Li, Xiting; Jamison, Stephanie; Harding, Heather P; McKinnon, Peter J; Ron, David; Lin, Wensheng

    2016-07-01

    Evidence suggests that activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress negatively or positively influences cell transformation by regulating apoptosis. Patched1 heterozygous deficient (Ptch1(+/-)) mice reproduce human Gorlin's syndrome and are regarded as the best animal model to study tumorigenesis of the sonic hedgehog subgroup of medulloblastomas. It is believed that medulloblastomas in Ptch1(+/-) mice results from the transformation of granule cell precursors (GCPs) in the developing cerebellum. Here, we determined the role of PERK signaling on medulloblastoma tumorigenesis by assessing its effects on premalignant GCPs and tumor cells. We found that PERK signaling was activated in both premalignant GCPs in young Ptch1(+/-) mice and medulloblastoma cells in adult mice. We demonstrated that PERK haploinsufficiency reduced the incidence of medulloblastomas in Ptch1(+/-) mice. Interestingly, PERK haploinsufficiency enhanced apoptosis of premalignant GCPs in young Ptch1(+/-) mice but had no significant effect on medulloblastoma cells in adult mice. Moreover, we showed that the PERK pathway was activated in medulloblastomas in humans. These results suggest that PERK signaling promotes medulloblastoma tumorigenesis by attenuating apoptosis of premalignant GCPs during the course of malignant transformation. PMID:27181404

  4. Transient degradation of NF-kappaB proteins in macrophages after interaction with mast cell granules.

    PubMed Central

    Ito, N; Li, Y; Suzuki, T; Stechschulte, D J; Dileepan, K N

    1998-01-01

    The exposure of the macrophage cell line, J774 to mast cell granules (MCG) led to the formation of altered nuclear transcription factor proteins (NF-kappaBx), which had faster electrophoretic mobility than the p50 homodimer of NF-KB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-kappaBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBalpha remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of mouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-KB proteins. The alterations in NF-KB proteins did not affect the expression of inducible nitric oxide synthase (iNOS) or TNF-alpha mRNA inJ774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-KB proteins with associated inhibition of iNOS or TNF-alpha mRNA expression. PMID:9927232

  5. The Phospholipase D2 Knock Out Mouse Has Ectopic Purkinje Cells and Suffers from Early Adult-Onset Anosmia

    PubMed Central

    Zhang, Qifeng; Smethurst, Elizabeth; Segonds-Pichon, Anne; Schrewe, Heinrich; Wakelam, Michael J. O.

    2016-01-01

    Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA), a lipid messenger molecule involved in a number of cellular events including, through its membrane curvature properties, endocytosis. The PLD2 knock out (PLD2KO) mouse has been previously reported to be protected from insult in a model of Alzheimer's disease. We have further analysed a PLD2KO mouse using mass spectrophotometry of its lipids and found significant differences in PA species throughout its brain. We have examined the expression pattern of PLD2 which allowed us to define which region of the brain to analyse for defect, notably PLD2 was not detected in glial-rich regions. The expression pattern lead us to specifically examine the mitral cells of olfactory bulbs, the Cornus Amonis (CA) regions of the hippocampus and the Purkinje cells of the cerebellum. We find that the change to longer PA species correlates with subtle architectural defect in the cerebellum, exemplified by ectopic Purkinje cells and an adult-onset deficit of olfaction. These observations draw parallels to defects in the reelin heterozygote as well as the effect of high fat diet on olfaction. PMID:27658289

  6. MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development.

    PubMed

    Constantin, Lena; Constantin, Myrna; Wainwright, Brandon J

    2016-03-01

    The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies. PMID:26773048

  7. Morphometric studies of secretory granule formation in mouse pancreatic acinar cells. Dissecting the early structural changes following pilocarpine injection

    PubMed Central

    HAMMEL, ILAN; SHOR-HAZAN, OSNAT; ELDAR, TORA; AMIHAI, DINA; LEW, SYLVIA

    1999-01-01

    Secretory granule formation in pancreatic acinar cells is known to involve massive membrane flow. In previous studies we have undertaken morphometry of the regranulation mechanism in these cells and in mast cells as a model for cellular membrane movement. In our current work, electron micrographs of pancreatic acinar cells from ICR mice were taken at several time points after extensive degranulation induced by pilocarpine injection in order to investigate the volume changes of rough endoplasmic reticulum (RER), nucleus, mitochondria and autophagosomes. At 2–4 h after stimulation, when the pancreatic cells demonstrated a complete loss of granules, this was accompanied by an increased proportion of autophagosomal activity. This change primarily reflected a greatly increased proportion of profiles retaining autophagic vacuoles containing recognisable cytoplasmic structures such as mitochondria, granule profiles and fragments of RER. The mitochondrial structures reached a significant maximal size 4 h following injection (before degranulation 0.178±0.028 μm3; at 4 h peak value, 0.535±0.109 μm3). Nucleus size showed an early volume increase approaching a maximum value 2 h following degranulation. The regranulation span was thus divided into 3 stages. The first was the membrane remodelling stage (0–2 h). During this period the volume of the RER and secretory granules was greatly decreased. At the intermediate stage (2–4 h) a significant increase of the synthesis zone was observed within the nucleus. The volume of the mitochondria was increasing. At the last step, the major finding was a significant granule accumulation in parallel with an active Golgi zone. PMID:10227666

  8. Application of acid-treated yeast cell wall (AYC) as a pharmaceutical additive. III. AYC aqueous coating onto granules and film formation mechanism of AYC.

    PubMed

    Yuasa, Hiroshi; Kaneshige, Junichi; Ozeki, Tetsuya; Kasai, Takahide; Eguchi, Takahiro; Ishiwaki, Naomu

    2002-04-26

    From the viewpoint of effective utilization of natural resources and development of new pharmaceutical materials, acid-treated yeast cell wall (AYC) was prepared via a novel approach involving acidification of brewers' yeast cell wall. AYC aqueous dispersion containing 5% (w/v) AYC and 0.5% (w/v) glycerol was prepared. Subsequently, AYC was coated onto core granules containing acetaminophen (AAP). Spray mist size under various spray conditions and viscosity of the AYC aqueous dispersion at various AYC concentrations were measured. AYC spray mists were optically observed. The surface of AYC cast film and AYC-coated granules were observed with a confocal scanning laser microscope. We attempted to show the utility of AYC as a novel material for granule coating, following the tablet coating in our previous report. In addition, the film formation mechanism of AYC was investigated. A smooth surface of the AYC-coated granules was obtained at a coating ratio of only 5%, which generally requires approximately 15-30% coating against the core granule weight, with no aggregation. These results are attributable to the fact that the granules were coated with a large number of small mists of AYC and the coating progressed efficiently, and the thin film layer of AYC was formed on the granules by mutual tangling of the hydrogel layers of AYC polysaccharides. AAP release from AYC-coated granules was obviously rapid, suggesting the high utility of AYC as a coating material for the rapidly releasing granules.

  9. Growth conditions differentially modulate the vulnerability of developing cerebellar granule cells to excitatory amino acids.

    PubMed

    Resink, A; Hack, N; Boer, G J; Balázs, R

    1994-08-29

    The survival of immature nerve cells in a cerebellar culture, predominantly excitatory granule cells, is known to be promoted by chronic exposure to high K+ (> 20 mM) or glutamate (Glu) receptor agonists. These treatments are believed to mimic the in vivo effect of the incoming glutamatergic afferents, the mossy fibres. Here we report that with maturation the cells become vulnerable to excitatory amino acids (EAAs) and that the characteristics of EAA sensitivity are dependent on the environmental influences being either "trophic" (25 mM K+ or 140 microM NMDA, K25 or K10 + NMDA) or "non-trophic" (10 mM K+, K10). Toxicity was assayed routinely at 9 days in vitro (DIV) after 24 h exposure to EAAs. Under all the tested conditions, the effect of Glu was mediated exclusively through NMDA receptors. However, the efficacy and potency of Glu were high in K25- and K10 + NMDA-grown cells compared with K10-grown cells. Growth conditions had the same influence on NMDA as on Glu-induced toxicity, but with the following special features: (1) in comparison with K25 cells, the potency of NMDA was significantly lower in K10 + NMDA cells. The K10 + NMDA cultures behaved as if they were completely insensitive to the NMDA which is present in their growth medium. (2) The K10-grown cells were not vulnerable to NMDA, unless the cell membrane was depolarised by shifting the cells into K25 medium. The efficacy of NMDA became then similar to that in K25 cultures, although the potency was about 7-fold less. Thus NMDA receptors can be activated by the depolarisation of K10 cells, implying the operation of Mg2+ blockade of the channel at normal resting membrane potential. Although non-NMDA receptors did not seem to be involved in Glu toxicity, cells were vulnerable to kainate, which killed significantly more cells than Glu (about 80% vs 70%). This was partly due to the resistance of GABAergic interneurons present in the cultures to Glu- or NMDA-induced toxicity. In contrast to the effects of

  10. MAPK Phosphatase AP2C3 Induces Ectopic Proliferation of Epidermal Cells Leading to Stomata Development in Arabidopsis

    PubMed Central

    Kazanaviciute, Vaiva; Magyar, Zoltan; Ayatollahi, Zahra; Unterwurzacher, Verena; Choopayak, Chonnanit; Boniecka, Justyna; Murray, James A. H.; Bogre, Laszlo; Meskiene, Irute

    2010-01-01

    In plant post-embryonic epidermis mitogen-activated protein kinase (MAPK) signaling promotes differentiation of pavement cells and inhibits initiation of stomata. Stomata are cells specialized to modulate gas exchange and water loss. Arabidopsis MAPKs MPK3 and MPK6 are at the core of the signaling cascade; however, it is not well understood how the activity of these pleiotropic MAPKs is constrained spatially so that pavement cell differentiation is promoted only outside the stomata lineage. Here we identified a PP2C-type phosphatase termed AP2C3 (Arabidopsis protein phosphatase 2C) that is expressed distinctively during stomata development as well as interacts and inactivates MPK3, MPK4 and MPK6. AP2C3 co-localizes with MAPKs within the nucleus and this localization depends on its N-terminal extension. We show that other closely related phosphatases AP2C2 and AP2C4 are also MAPK phosphatases acting on MPK6, but have a distinct expression pattern from AP2C3. In accordance with this, only AP2C3 ectopic expression is able to stimulate cell proliferation leading to excess stomata development. This function of AP2C3 relies on the domains required for MAPK docking and intracellular localization. Concomitantly, the constitutive and inducible AP2C3 expression deregulates E2F-RB pathway, promotes the abundance and activity of CDKA, as well as changes of CDKB1;1 forms. We suggest that AP2C3 downregulates the MAPK signaling activity to help maintain the balance between differentiation of stomata and pavement cells. PMID:21203456

  11. Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

    PubMed Central

    Yuasa, Masato; Yamada, Tsuyoshi; Taniyama, Takashi; Masaoka, Tomokazu; Xuetao, Wei; Yoshii, Toshitaka; Horie, Masaki; Yasuda, Hiroaki; Uemura, Toshimasa; Okawa, Atsushi; Sotome, Shinichi

    2015-01-01

    We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2. PMID:25659106

  12. Protective effect of fangchinoline on cyanide-induced neurotoxicity in cultured rat cerebellar granule cells.

    PubMed

    Cho, Soon Ok; Seong, Yeon Hee

    2002-06-01

    The present study was performed to examine the effect of fangchinoline, a bis- benzylisoquinoline alkaloid, which exhibits the characteristics of a Ca2+ channel blocker, on cyanide-induced neurotoxicity using cultured rat cerebellar granule neurons. NaCN produced a concentration-dependent reduction of cell viability, which was blocked by MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, verapamil, L-type Ca2+ channel blocker, and L-NAME, a nitric oxide synthase inhibitor. Pretreatment with fangchinoline over a concentration range of 0.1 to 10 microM significantly decreased the NaCN-induced neuronal cell death, glutamate release into medium, and elevation of [Ca2+]i and oxidants generation. These results suggest that fangchinoline may mitigate the harmful effects of cyanide-induced neuronal cell death by interfering with [Ca2+]i influx, due to its function as a Ca2+ channel blocker, and then by inhibiting glutamate release and oxidants generation. PMID:12135109

  13. Thioredoxin/thioredoxin reductase system involvement in cerebellar granule cell apoptosis.

    PubMed

    Bobba, A; Casalino, E; Petragallo, V A; Atlante, A

    2014-10-01

    The involvement of thioredoxin/thioredoxin reductase system has been investigated in cerebellar granule cells (CGCs), a cellular system in which neurons are induced in apoptosis by the physiological stimulus of lowering extracellular potassium. Clarifying the sequence of events that occur during apoptosis is a critical issue as it can lead to the identification of those key events that, if blocked, can slow down or reverse the death process. The results reported in this work show that TrxR is involved in the early phase of CGC apoptosis with an increase in activity that coincides with the increased expression of the TrxR1 isoform and guarantees the maintenance of adequate level of Trx in its reduced, active form. However, in late apoptosis, when about 50 % of cells are dead, partial proteolysis of TrxR1 by calpain occurs and the reduction of TrxR1 mRNA, together with the overall decrease in TrxR activity, contribute to increase the levels of the oxidized form of Trx. When the reduced form of Trx is externally added to apoptotic cultures, a significant reduction in cell death is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survival of CGCs. PMID:25055978

  14. Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester.

    PubMed

    Jebelli, Joseph; Piers, Thomas; Pocock, Jennifer

    2015-01-01

    Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique - described here - is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis(13,14). Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer's or Parkinson's disease.

  15. Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells

    PubMed Central

    Redhai, Siamak; Hellberg, Josephine E. E. U.; Wainwright, Mark; Perera, Sumeth W.; Castellanos, Felix; Kroeger, Benjamin; Gandy, Carina; Leiblich, Aaron; Corrigan, Laura; Hilton, Thomas; Patel, Benjamin; Fan, Shih-Jung; Hamdy, Freddie; Goberdhan, Deborah C. I.

    2016-01-01

    Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion. PMID:27727275

  16. Differential inhibition onto developing and mature granule cells generates high-frequency filters with variable gain

    PubMed Central

    Pardi, María Belén; Ogando, Mora Belén; Schinder, Alejandro F; Marin-Burgin, Antonia

    2015-01-01

    Adult hippocampal neurogenesis provides the dentate gyrus with heterogeneous populations of granule cells (GC) originated at different times. The contribution of these cells to information encoding is under current investigation. Here, we show that incoming spike trains activate different populations of GC determined by the stimulation frequency and GC age. Immature GC respond to a wider range of stimulus frequencies, whereas mature GC are less responsive at high frequencies. This difference is dictated by feedforward inhibition, which restricts mature GC activation. Yet, the stronger inhibition of mature GC results in a higher temporal fidelity compared to that of immature GC. Thus, hippocampal inputs activate two populations of neurons with variable frequency filters: immature cells, with wide‐range responses, that are reliable transmitters of the incoming frequency, and mature neurons, with narrow frequency response, that are precise at informing the beginning of the stimulus, but with a sparse activity. DOI: http://dx.doi.org/10.7554/eLife.08764.001 PMID:26163657

  17. Kruppel-Like Factor 4 Regulates Granule Cell Pax6 Expression and Cell Proliferation in Early Cerebellar Development

    PubMed Central

    Zhang, Peter; Ha, Thomas; Larouche, Matt; Swanson, Douglas; Goldowitz, Dan

    2015-01-01

    Kruppel-like factor 4 (Klf4) is a transcription factor that regulates many important cellular processes in stem cell biology, cancer, and development. We used histological and molecular methods to study the expression of Klf4 in embryonic development of the normal and Klf4 knockout cerebellum. We find that Klf4 is expressed strongly in early granule cell progenitor development but tails-off considerably by the end of embryonic development. Klf4 is also co-expressed with Pax6 in these cells. In the Klf4-null mouse, which is perinatal lethal, Klf4 positively regulates Pax6 expression and regulates the proliferation of neuronal progenitors in the rhombic lip, external granular layer and the neuroepithelium. This paper is the first to describe a role for Klf4 in the cerebellum and provides insight into this gene’s function in neuronal development. PMID:26226504

  18. Development of a Cell-Based Fluorescence Polarization Biosensor Using Preproinsulin to Identify Compounds That Alter Insulin Granule Dynamics.

    PubMed

    Yi, Na Young; He, Qingping; Caligan, Thomas B; Smith, Ginger R; Forsberg, Lawrence J; Brenman, Jay E; Sexton, Jonathan Z

    2015-11-01

    Diabetes currently affects 9.3% of the U.S. population totaling $245 billion annually in U.S. direct and indirect healthcare costs. Current therapies for diabetes are limited in their ability to control blood glucose and/or enhance insulin sensitivity. Therefore, innovative and efficacious therapies for diabetes are urgently needed. Herein we describe a fluorescent insulin reporter system (preproinsulin-mCherry, PPI-mCherry) that tracks live-cell insulin dynamics and secretion in pancreatic β-cells with utility for high-content assessment of real-time insulin dynamics. Additionally, we report a new modality for sensing insulin granule packaging in conventional high-throughput screening (HTS), using a hybrid cell-based fluorescence polarization (FP)/internal FRET biosensor using the PPI-mCherry reporter system. We observed that bafilomycin, a vacuolar H(+) ATPase inhibitor and inhibitor of insulin granule formation, significantly increased mCherry FP in INS-1 cells with PPI-mCherry. Partial least squares regression analysis demonstrated that an increase of FP by bafilomycin is significantly correlated with a decrease in granularity of PPI-mCherry signal in the cells. The increased FP by bafilomycin is due to inhibition of self-Förster resonant energy transfer (homo-FRET) caused by the increased mCherry intermolecular distance. FP substantially decreases when insulin is tightly packaged in the granules, and the homo-FRET decreases when insulin granule packaging is inhibited, resulting in increased FP. We performed pilot HTS of 1782 FDA-approved small molecules and natural products from Prestwick and Enzo chemical libraries resulting in an overall Z'-factor of 0.52 ± 0.03, indicating the suitability of this biosensor for HTS. This novel biosensor enables live-cell assessment of protein-protein interaction/protein aggregation in live cells and is compatible with conventional FP plate readers. PMID:26505612

  19. SORCS1 is necessary for normal insulin secretory granule biogenesis in metabolically stressed β cells.

    PubMed

    Kebede, Melkam A; Oler, Angie T; Gregg, Trillian; Balloon, Allison J; Johnson, Adam; Mitok, Kelly; Rabaglia, Mary; Schueler, Kathryn; Stapleton, Donald; Thorstenson, Candice; Wrighton, Lindsay; Floyd, Brendan J; Richards, Oliver; Raines, Summer; Eliceiri, Kevin; Seidah, Nabil G; Rhodes, Christopher; Keller, Mark P; Coon, Joshua L; Audhya, Anjon; Attie, Alan D

    2014-10-01

    We previously positionally cloned Sorcs1 as a diabetes quantitative trait locus. Sorcs1 belongs to the Vacuolar protein sorting-10 (Vps10) gene family. In yeast, Vps10 transports enzymes from the trans-Golgi network (TGN) to the vacuole. Whole-body Sorcs1 KO mice, when made obese with the leptin(ob) mutation (ob/ob), developed diabetes. β Cells from these mice had a severe deficiency of secretory granules (SGs) and insulin. Interestingly, a single secretagogue challenge failed to consistently elicit an insulin secretory dysfunction. However, multiple challenges of the Sorcs1 KO ob/ob islets consistently revealed an insulin secretion defect. The luminal domain of SORCS1 (Lum-Sorcs1), when expressed in a β cell line, acted as a dominant-negative, leading to SG and insulin deficiency. Using syncollin-dsRed5TIMER adenovirus, we found that the loss of Sorcs1 function greatly impairs the rapid replenishment of SGs following secretagogue challenge. Chronic exposure of islets from lean Sorcs1 KO mice to high glucose and palmitate depleted insulin content and evoked an insulin secretion defect. Thus, in metabolically stressed mice, Sorcs1 is important for SG replenishment, and under chronic challenge by insulin secretagogues, loss of Sorcs1 leads to diabetes. Overexpression of full-length SORCS1 led to a 2-fold increase in SG content, suggesting that SORCS1 is sufficient to promote SG biogenesis. PMID:25157818

  20. Abnormal differentiation of newborn granule cells in age-related working memory impairments.

    PubMed

    Nyffeler, Myriel; Yee, Benjamin K; Feldon, Joram; Knuesel, Irene

    2010-11-01

    Age-related declines in spatial memory have been linked to abnormal functional properties and connectivity of newborn granule cells. However, the relationship between adult neurogenesis, aging, and cognitive performance seems more complex than previously anticipated, likely due to the difficulty of disentangling alterations related to training as such and those associated with cognitive performance. Here, we investigated how different aspects of adult neurogenesis might be related to training, age and cognitive performance amongst aged subjects by comparing behaviourally naïve and tested rats of 3, 6, 24mo of age. We separated aged rats into learning-impaired and -unimpaired groups based on their performance in the Morris water maze to investigate neurogenesis-related morphological and neurochemical changes. We report an age-related decline in cell proliferation and maturation independent of cognitive performance and testing. We confirm an age-related altered differentiation of newborn neurons which was particularly prominent in learning-impaired rats. This was associated with an abnormally prolonged expression of the early progenitor marker Nestin, potentially also affecting maturation, survival/integration of newborn neurons into existing neuronal networks, which might underlie the individual differences in cognitive performance during aging.

  1. Distinct roles of NMDA receptors at different stages of granule cell development in the adult brain

    PubMed Central

    Mu, Yangling; Zhao, Chunmei; Toni, Nicolas; Yao, Jun; Gage, Fred H

    2015-01-01

    NMDA receptor (NMDAR)-dependent forms of synaptic plasticity are thought to underlie the assembly of developing neuronal circuits and to play a crucial role in learning and memory. It remains unclear how NMDAR might contribute to the wiring of adult-born granule cells (GCs). Here we demonstrate that nascent GCs lacking NMDARs but rescued from apoptosis by overexpressing the pro-survival protein Bcl2 were deficient in spine formation. Insufficient spinogenesis might be a general cause of cell death restricted within the NMDAR-dependent critical time window for GC survival. NMDAR loss also led to enhanced mushroom spine formation and synaptic AMPAR activity throughout the development of newborn GCs. Moreover, similar elevated synapse maturation in the absence of NMDARs was observed in neonate-generated GCs and CA1 pyramidal neurons. Together, these data suggest that NMDAR operates as a molecular monitor for controlling the activity-dependent establishment and maturation rate of synaptic connections between newborn neurons and others. DOI: http://dx.doi.org/10.7554/eLife.07871.001 PMID:26473971

  2. SORCS1 is necessary for normal insulin secretory granule biogenesis in metabolically stressed β cells

    PubMed Central

    Kebede, Melkam A.; Oler, Angie T.; Gregg, Trillian; Balloon, Allison J.; Johnson, Adam; Mitok, Kelly; Rabaglia, Mary; Schueler, Kathryn; Stapleton, Donald; Thorstenson, Candice; Wrighton, Lindsay; Floyd, Brendan J.; Richards, Oliver; Raines, Summer; Eliceiri, Kevin; Seidah, Nabil G.; Rhodes, Christopher; Keller, Mark P.; Coon, Joshua L.; Audhya, Anjon; Attie, Alan D.

    2014-01-01

    We previously positionally cloned Sorcs1 as a diabetes quantitative trait locus. Sorcs1 belongs to the Vacuolar protein sorting-10 (Vps10) gene family. In yeast, Vps10 transports enzymes from the trans-Golgi network (TGN) to the vacuole. Whole-body Sorcs1 KO mice, when made obese with the leptinob mutation (ob/ob), developed diabetes. β Cells from these mice had a severe deficiency of secretory granules (SGs) and insulin. Interestingly, a single secretagogue challenge failed to consistently elicit an insulin secretory dysfunction. However, multiple challenges of the Sorcs1 KO ob/ob islets consistently revealed an insulin secretion defect. The luminal domain of SORCS1 (Lum-Sorcs1), when expressed in a β cell line, acted as a dominant-negative, leading to SG and insulin deficiency. Using syncollin-dsRed5TIMER adenovirus, we found that the loss of Sorcs1 function greatly impairs the rapid replenishment of SGs following secretagogue challenge. Chronic exposure of islets from lean Sorcs1 KO mice to high glucose and palmitate depleted insulin content and evoked an insulin secretion defect. Thus, in metabolically stressed mice, Sorcs1 is important for SG replenishment, and under chronic challenge by insulin secretagogues, loss of Sorcs1 leads to diabetes. Overexpression of full-length SORCS1 led to a 2-fold increase in SG content, suggesting that SORCS1 is sufficient to promote SG biogenesis. PMID:25157818

  3. The effects of neurotrophin-3 and brain-derived neurotrophic factor on cerebellar granule cell movement and neurite extension in vitro.

    PubMed

    Tanaka, S; Sekino, Y; Shirao, T

    2000-01-01

    Migration of the granule cells is a major stage of cerebellar maturation. Granule cells express neurotrophins and their receptors; however, their role in cell migration has not been defined. In this study we investigated the effects of exogenous neurotrophins on the movement and neurite extension of granule cells from glial-free cerebellar cell reaggregates in vitro. Our results provide direct evidence that neurotrophin-3 and brain-derived neurotrophic factor differentially affect the granule cells. Neurotrophin-3 significantly affected granule cell movements by decreasing the migration index (the ratio of the number of cells that moved further than half the neurite length) and the speed of cell soma movement, but did not affect neurite length or growth cone migration. In contrast, brain-derived neurotrophic factor and neurotrophin-4 acted on growing neurites and growth cones by significantly increasing neurite length and the speed of growth cone migration, but had no effect either on the migration index or on the speed of the cell soma movement. The results suggest that neurotrophins differentially affect neurite extension and the movements of cerebellar granule cells. PMID:10842017

  4. Quantification of endocrine cells and ultrastructural study of insulin granules in the large intestine of opossum Didelphis aurita (Wied-Neuwied, 1826).

    PubMed

    dos Santos, Daiane Cristina Marques; Cupertino, Marli do Carmo; Fialho, Maria do Carmo Queiroz; Barbosa, Alfredo Jose Afonso; Fonseca, Cláudio Cesar; Sartori, Sirlene Souza Rodrigues; da Matta, Sérgio Luis Pinto

    2014-02-01

    This study aimed to investigate the distribution of argyrophil, argentaffin, and insulin-immunoreactive endocrine cells in the large intestine of opossums (Didelphis aurita) and to describe the ultrastructure of the secretory granules of insulin-immunoreactive endocrine cells. Fragments of the large intestine of 10 male specimens of D. aurita were collected, processed, and subjected to staining, immunohistochemistry, and transmission electron microscopy. The argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells were sparsely distributed in the intestinal glands of the mucous layer, among other cell types of the epithelium in all regions studied. Proportionally, the argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells represented 62.75%, 36.26%, and 0.99% of the total determined endocrine cells of the large intestine, respectively. Quantitatively, there was no difference between the argyrophil and the argentaffin endocrine cells, whereas insulin-immunoreactive endocrine cells were less numerous. The insulin-immunoreactive endocrine cells were elongated or pyramidal, with rounded nuclei of irregularly contoured, and large amounts of secretory granules distributed throughout the cytoplasm. The granules have different sizes and electron densities and are classified as immature and mature, with the mature granules in predominant form in the overall granular population. In general, the granule is shown with an external electron-lucent halo and electron-dense core. The ultrastructure pattern in the granules of the insulin-immunoreactive endocrine cells was similar to that of the B cells of pancreatic islets in rats.

  5. Overexpressed TPX2 causes ectopic formation of microtubular arrays in the nuclei of acentrosomal plant cells.

    PubMed

    Petrovská, Beáta; Jerábková, Hana; Kohoutová, Lucie; Cenklová, Vera; Pochylová, Žaneta; Gelová, Zuzana; Kocárová, Gabriela; Váchová, Lenka; Kurejová, Michaela; Tomastíková, Eva; Binarová, Pavla

    2013-11-01

    TPX2 performs multiple roles in microtubule organization. Previously, it was shown that plant AtTPX2 binds AtAurora1 kinase and colocalizes with microtubules in a cell cycle-specific manner. To elucidate the function of TPX2 further, this work analysed Arabidopsis cells overexpressing AtTPX2-GFP. Distinct arrays of bundled microtubules, decorated with AtTPX2-GFP, were formed in the vicinity of the nuclear envelope and in the nuclei of overexpressing cells. The microtubular arrays showed reduced sensitivity to anti-microtubular drugs. TPX2-mediated formation of nuclear/perinuclear microtubular arrays was not specific for the transition to mitosis and occurred independently of Aurora kinase. The fibres were not observed in cells with detectable programmed cell death and, in this respect, they differed from TPX2-dependent microtubular assemblies functioning in mammalian apoptosis. Colocalization and co-purification data confirmed the interaction of importin with AtTPX2-GFP. In cells with nuclear foci of overexpressed AtTPX2-GFP, strong nuclear signals for Ran and importin diminished when microtubular arrays were assembled. This observation suggests that TPX2-mediated microtubule formation might be triggered by a Ran cycle. Collectively, the data suggest that in the acentrosomal plant cell, in conjunction with importin, overexpressed AtTPX2 reinforces microtubule formation in the vicinity of chromatin and the nuclear envelope.

  6. Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography.

    PubMed

    Xue, Yanhong; Zhao, Wei; Du, Wen; Zhang, Xiang; Ji, Gang; Ying, Wang; Xu, Tao

    2012-07-01

    Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.

  7. Reelin Regulates the Maturation of Dendritic Spines, Synaptogenesis and Glial Ensheathment of Newborn Granule Cells

    PubMed Central

    Bosch, Carles; Masachs, Nuria; Exposito-Alonso, David; Martínez, Albert; Teixeira, Cátia M.; Fernaud, Isabel; Pujadas, Lluís; Ulloa, Fausto; Comella, Joan X.; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2016-01-01

    The Reelin pathway is essential for both neural migration and for the development and maturation of synaptic connections. However, its role in adult synaptic formation and remodeling is still being investigated. Here, we investigated the impact of the Reelin/Dab1 pathway on the synaptogenesis of newborn granule cells (GCs) in the young-adult mouse hippocampus. We show that neither Reelin overexpression nor the inactivation of its intracellular adapter, Dab1, substantially alters dendritic spine numbers in these neurons. In contrast, 3D-electron microscopy (focused ion beam milling/scanning electron microscope) revealed that dysregulation of the Reelin/Dab1 pathway leads to both transient and permanent changes in the types and morphology of dendritic spines, mainly altering mushroom, filopodial, and branched GC spines. We also found that the Reelin/Dab1 pathway controls synaptic configuration of presynaptic boutons in the dentate gyrus, with its dysregulation leading to a substantial decrease in multi-synaptic bouton innervation. Lastly, we show that the Reelin/Dab1 pathway controls astroglial ensheathment of synapses. Thus, the Reelin pathway is a key regulator of adult-generated GC integration, by controlling dendritic spine types and shapes, their synaptic innervation patterns, and glial ensheathment. These findings may help to better understanding of hippocampal circuit alterations in neurological disorders in which the Reelin pathway is implicated. Significance Statement The extracellular protein Reelin has an important role in neurological diseases, including epilepsy, Alzheimer's disease and psychiatric diseases, targeting hippocampal circuits. Here we address the role of Reelin in the development of synaptic contacts in adult-generated granule cells (GCs), a neuronal population that is crucial for learning and memory and implicated in neurological and psychiatric diseases. We found that the Reelin pathway controls the shapes, sizes, and types of dendritic

  8. Tracing CD34+ Stromal Fibroblasts in Palatal Mucosa and Periodontal Granulation Tissue as a Possible Cell Reservoir for Periodontal Regeneration.

    PubMed

    Roman, Alexandra; Páll, Emőke; Mihu, Carmen M; Petruţiu, Adrian S; Barbu-Tudoran, Lucian; Câmpian, Radu S; Florea, Adrian; Georgiu, Carmen

    2015-08-01

    The aim of the present research was to trace CD34+ stromal fibroblastic cells (CD34+ SFCs) in the palatal connective tissue harvested for muco-gingival surgical procedures and in granulation tissues from periodontal pockets using immunohistochemical and transmission electron microscopy. Immunohistochemical analysis targeted the presence of three antigens: CD31, α-smooth muscle actin (α-SMA), and CD34. In the palate, CD31 staining revealed a colored inner ring of the vessels representing the endothelium, α-SMA+ was located in the medial layer of the vasculature, and CD34 was intensely expressed by endothelial cells and artery adventitial cells (considered to be CD34+ SFCs). Granulation tissue showed the same pattern for CD31+ and α-SMA, but a different staining pattern for CD34. Ultrastructural examination of the palatal tissue highlighted perivascular cells with fibroblast-like characteristics and pericytes in close spatial relationship to endothelial cells. The ultrastructural evaluation of granulation tissue sections confirmed the presence of neovasculature and the inflammatory nature of this tissue. The present study traced the presence of CD34+ SFCs and of pericytes in the palatal connective tissue thus highlighting once more its intrinsic regenerative capabilities. The clinical and systemic factors triggering mobilization and influencing the fate of local CD34+SCFs and other progenitors are issues to be further investigated.

  9. AMP Kinase Activation Alters Oxidant-Induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization.

    PubMed

    Mahboubi, Hicham; Koromilas, Antonis E; Stochaj, Ursula

    2016-10-01

    Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK), which functions as a stress sensor and is transiently activated by adverse physiologic conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation but limits their fusion into larger granules. PMID:27430620

  10. Cognitive enhancing treatment with a PPARγ agonist normalizes dentate granule cell presynaptic function in Tg2576 APP mice.

    PubMed

    Nenov, Miroslav N; Laezza, Fernanda; Haidacher, Sigmund J; Zhao, Yingxin; Sadygov, Rovshan G; Starkey, Jonathan M; Spratt, Heidi; Luxon, Bruce A; Dineley, Kelly T; Denner, Larry

    2014-01-15

    Hippocampal network hyperexcitability is considered an early indicator of Alzheimer's disease (AD) memory impairment. Some AD mouse models exhibit similar network phenotypes. In this study we focused on dentate gyrus (DG) granule cell spontaneous and evoked properties in 9-month-old Tg2576 mice that model AD amyloidosis and cognitive deficits. Using whole-cell patch-clamp recordings, we found that Tg2576 DG granule cells exhibited spontaneous EPSCs that were higher in frequency but not amplitude compared with wild-type mice, suggesting hyperactivity of DG granule cells via a presynaptic mechanism. Further support of a presynaptic mechanism was revealed by increased I-O relationships and probability of release in Tg2576 DG granule cells. Since we and others have shown that activation of the peroxisome proliferator-activated receptor gamma (PPARγ) axis improves hippocampal cognition in mouse models for AD as well as benefitting memory performance in some humans with early AD, we investigated how PPARγ agonism affected synaptic activity in Tg2576 DG. We found that PPARγ agonism normalized the I-O relationship of evoked EPSCs, frequency of spontaneous EPSCs, and probability of release that, in turn, correlated with selective expression of DG proteins essential for presynaptic SNARE function that are altered in patients with AD. These findings provide evidence that DG principal cells may contribute to early AD hippocampal network hyperexcitability via a presynaptic mechanism, and that hippocampal cognitive enhancement via PPARγ activation occurs through regulation of presynaptic vesicular proteins critical for proper glutamatergic neurotransmitter release, synaptic transmission, and short-term plasticity.

  11. Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution

    PubMed Central

    Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-ichiro

    2016-01-01

    Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules. PMID:27796315

  12. Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata).

    PubMed

    Cavalcante, Moisés C M; de Andrade, Leonardo R; Du Bocage Santos-Pinto, Claudia; Straus, Anita H; Takahashi, Hélio K; Allodi, Silvana; Pavão, Mauro S G

    2002-03-01

    In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.

  13. Weaver mutant mouse cerebellar granule cells respond normally to chronic depolarization.

    PubMed

    Bjerregaard, A; Mogensen, H S; Hack, N; Balázs, R; Jørgensen, O S

    1997-04-01

    We studied the effects of chronic K(+)-induced membrane depolarization and treatment with N-methyl-D-aspartate (NMDA) on cerebellar granule cells (CGCs) from weaver mutant mice and non-weaver litter-mates. The weaver mutation is a Gly-to-Ser substitution in a conserved region of the Girk2 G protein-coupled inward rectifying potassium channel [Patil N., Cox D. R., Bhat D., Faham M., Myers R. M. and Peterson A. S. (1995) Nature Genet. 11, 126-129] which induces early death of CGCs. The biochemical differentiation of CGCs was estimated as the rate of 2-deoxy-D-glucose accumulation and the expression of neural cell adhesion molecule (NCAM). High (25 mM) K+ ion concentration or treatment with NMDA greatly promoted the biochemical differentiation of both weaver mutant and non-weaver litter-mate mouse CGCs. In contrast to the marked effect on biochemical differentiation in both weaver and non-weaver mice CGSs, chronic high K+ treatment only had limited effect on survival. The survival of weaver mutant mouse CGCs in medium containing 5 mM K+ ions was very low, only 20% of the plated cells surviving at 7 days after plating, as opposed to the 50% for non-weaver CGCs. Chronic high K+ treatment improved the relative survival of weaver mutant mouse CGCs 1.6 2.2-fold and that of non-weaver CGCs 1.2-1.4-fold; the same number of CGCs (about 20% of the plated cells) were rescued by high K+ in both types of culture. The findings indicate that, in culture weaver mutant mouse, CGCs have a normal response to membrane depolarization and that the normal function of the Girk2 potassium channel is not critical for the survival of differentiated CGCs.

  14. Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines.

    PubMed

    Firulli, Beth A.; Virshup, David M.; Firulli, Anthony B.

    2004-01-01

    Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, it was suspected that HAND1 was being phosphorylated during trophoblast giant cell differentiation and that coexpression of a constitutively active kinase with HAND1 resulted in changes in the proteins dimerization profile. In order to accurately document HAND1 phosphorylation and identify the resides being modified, we employed metabolic cell labeling with (32)P of tissue culture cells coexpressing a Flag-epitope tagged HAND1 along with a number of active kinases and phosphatase subunits. We generated phosphopeptide maps of the phosphorylated HAND1 using the methods described below and linked these modifications to changes in HAND1 biological function.

  15. Inhibition of Protease-Activated Receptor 1 Does not Affect Dendritic Homeostasis of Cultured Mouse Dentate Granule Cells.

    PubMed

    Schuldt, Gerlind; Galanis, Christos; Strehl, Andreas; Hick, Meike; Schiener, Sabine; Lenz, Maximilian; Deller, Thomas; Maggio, Nicola; Vlachos, Andreas

    2016-01-01

    Protease-activated receptors (PARs) are widely expressed in the central nervous system (CNS). While a firm link between PAR1-activation and functional synaptic and intrinsic neuronal properties exists, studies on the role of PAR1 in neural structural plasticity are scarce. The physiological function of PAR1 in the brain remains not well understood. We here sought to determine whether prolonged pharmacologic PAR1-inhibition affects dendritic morphologies of hippocampal neurons. To address this question we employed live-cell microscopy of mouse dentate granule cell dendrites in 3-week old entorhino-hippocampal slice cultures prepared from Thy1-GFP mice. A subset of cultures were treated with the PAR1-inhibitor SCH79797 (1 μM; up to 3 weeks). No major effects of PAR1-inhibition on static and dynamic parameters of dentate granule cell dendrites were detected under control conditions. Granule cells of PAR1-deficient slice cultures showed unaltered dendritic morphologies, dendritic spine densities and excitatory synaptic strength. Furthermore, we report that PAR1-inhibition does not prevent dendritic retraction following partial deafferentation in vitro. Consistent with this finding, no major changes in PAR1-mRNA levels were detected in the denervated dentate gyrus (DG). We conclude that neural PAR1 is not involved in regulating the steady-state dynamics or deafferentation-induced adaptive changes of cultured dentate granule cell dendrites. These results indicate that drugs targeting neural PAR1-signals may not affect the stability and structural integrity of neuronal networks in healthy brain regions. PMID:27378862

  16. Inhibition of Protease-Activated Receptor 1 Does not Affect Dendritic Homeostasis of Cultured Mouse Dentate Granule Cells

    PubMed Central

    Schuldt, Gerlind; Galanis, Christos; Strehl, Andreas; Hick, Meike; Schiener, Sabine; Lenz, Maximilian; Deller, Thomas; Maggio, Nicola; Vlachos, Andreas

    2016-01-01

    Protease-activated receptors (PARs) are widely expressed in the central nervous system (CNS). While a firm link between PAR1-activation and functional synaptic and intrinsic neuronal properties exists, studies on the role of PAR1 in neural structural plasticity are scarce. The physiological function of PAR1 in the brain remains not well understood. We here sought to determine whether prolonged pharmacologic PAR1-inhibition affects dendritic morphologies of hippocampal neurons. To address this question we employed live-cell microscopy of mouse dentate granule cell dendrites in 3-week old entorhino-hippocampal slice cultures prepared from Thy1-GFP mice. A subset of cultures were treated with the PAR1-inhibitor SCH79797 (1 μM; up to 3 weeks). No major effects of PAR1-inhibition on static and dynamic parameters of dentate granule cell dendrites were detected under control conditions. Granule cells of PAR1-deficient slice cultures showed unaltered dendritic morphologies, dendritic spine densities and excitatory synaptic strength. Furthermore, we report that PAR1-inhibition does not prevent dendritic retraction following partial deafferentation in vitro. Consistent with this finding, no major changes in PAR1-mRNA levels were detected in the denervated dentate gyrus (DG). We conclude that neural PAR1 is not involved in regulating the steady-state dynamics or deafferentation-induced adaptive changes of cultured dentate granule cell dendrites. These results indicate that drugs targeting neural PAR1-signals may not affect the stability and structural integrity of neuronal networks in healthy brain regions. PMID:27378862

  17. Early B-cell-specific inactivation of ATM synergizes with ectopic CyclinD1 expression to promote pre-germinal center B-cell lymphomas in mice.

    PubMed

    Yamamoto, K; Lee, B J; Li, C; Dubois, R L; Hobeika, E; Bhagat, G; Zha, S

    2015-06-01

    Ataxia telangiectasia-mutated (ATM) kinase is a master regulator of the DNA damage response. ATM is frequently inactivated in human B-cell non-Hodgkin lymphomas, including ~50% of mantle cell lymphomas (MCLs) characterized by ectopic expression of CyclinD1. Here we report that early and robust deletion of ATM in precursor/progenitor B cells causes cell autonomous, clonal mature B-cell lymphomas of both pre- and post-germinal center (GC) origins. Unexpectedly, naive B-cell-specific deletion of ATM is not sufficient to induce lymphomas in mice, highlighting the important tumor suppressor function of ATM in immature B cells. Although EμCyclinD1 is not sufficient to induce lymphomas, EμCyclinD1 accelerates the kinetics and increases the incidence of clonal lymphomas in ATM-deficient B-cells and skews the lymphomas toward pre-GC-derived small lymphocytic neoplasms, sharing morphological features of human MCL. This is in part due to CyclinD1-driven expansion of ATM-deficient naive B cells with genomic instability, which promotes the deletions of additional tumor suppressor genes (i.e. Trp53, Mll2, Rb1 and Cdkn2a). Together these findings define a synergistic function of ATM and CyclinD1 in pre-GC B-cell proliferation and lymphomagenesis and provide a prototypic animal model to study the pathogenesis of human MCL. PMID:25676421

  18. Ectopic mesodermal expression promoted by the eight-cell stage Bufo arenarum subequatorial cytoplasm.

    PubMed

    Casal, F C; Manes, M E

    1999-07-01

    Mesodermal determinants were investigated by cytoplasmic transfer and blastomere isolation in the eight-cell stage of Bufo arenarum. Their existence was confirmed by assaying the subequatorial cytoplasm's ability to respecify the developmental potency of animal quartets. The gray subequatorial cytoplasm, but not animal cytoplasm, is able to divert the ectodermal fate of animal quartets to several mesodermal components. The source of the transplanted cytoplasm was important in determining the category of the resulting structures. Ventral subequatorial cytoplasm from ventrovegetal blastomeres generated ventral derivatives, namely erythrocytes and mesenchyma. Dorsal subequatorial cytoplasm from dorsovegetal blastomeres produced dorsolateral derivatives, such as notochord, muscle, nephric tubules, and coelomic epithelium, including mesenchyma. On the other hand, transfer of vegetal pole cytoplasm to animal quartets resulted in the formation of groups of endoderm-like cells dispersed among epidermal cells. However, the presence of such cells did not cause any mesodermal induction. The present findings suggest the existence of cytoplasmic information responsible for mesodermal specification. The alternative hypothesis that animal blastomeres become mesoderm due to vegetal induction is questioned.

  19. Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis.

    PubMed

    Chen, Su-Fen; Liang, Ke; Yin, Dong-Mei; Ni, Di-An; Zhang, Zhi-Guo; Ruan, Yong-Ling

    2016-12-01

    There are several hypotheses that explain stomatal behavior. These include the concept of osmoregulation mediated by potassium and its counterions malate and chlorine and the more recent starch-sugar hypothesis. We have previously reported that the activity of the sucrose cleavage enzyme, vacuolar invertase (VIN), is significantly higher in guard cells than in other leaf epidermal cells and its activity is correlated with stomatal aperture. Here, we examined whether VIN indeed controls stomatal movement under normal and drought conditions by transforming Arabidopsis with a tobacco vacuolar invertase inhibitor homolog (Nt-inhh) under the control of an abscisic acid-sensitive and guard cell-specific promoter (AtRab18). The data obtained showed that guard cells of transgenic Arabidopsis plants had lower VIN activity, stomatal aperture and conductance than that of wild-type plants. Moreover, the transgenic plants also displayed higher drought tolerance than wild-type plants. The data indicate that VIN is a promising target for manipulating stomatal function to increase drought tolerance.

  20. Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis.

    PubMed

    Chen, Su-Fen; Liang, Ke; Yin, Dong-Mei; Ni, Di-An; Zhang, Zhi-Guo; Ruan, Yong-Ling

    2016-12-01

    There are several hypotheses that explain stomatal behavior. These include the concept of osmoregulation mediated by potassium and its counterions malate and chlorine and the more recent starch-sugar hypothesis. We have previously reported that the activity of the sucrose cleavage enzyme, vacuolar invertase (VIN), is significantly higher in guard cells than in other leaf epidermal cells and its activity is correlated with stomatal aperture. Here, we examined whether VIN indeed controls stomatal movement under normal and drought conditions by transforming Arabidopsis with a tobacco vacuolar invertase inhibitor homolog (Nt-inhh) under the control of an abscisic acid-sensitive and guard cell-specific promoter (AtRab18). The data obtained showed that guard cells of transgenic Arabidopsis plants had lower VIN activity, stomatal aperture and conductance than that of wild-type plants. Moreover, the transgenic plants also displayed higher drought tolerance than wild-type plants. The data indicate that VIN is a promising target for manipulating stomatal function to increase drought tolerance. PMID:26899912

  1. Gray platelet syndrome. Demonstration of alpha granule membranes that can fuse with the cell surface.

    PubMed Central

    Rosa, J P; George, J N; Bainton, D F; Nurden, A T; Caen, J P; McEver, R P

    1987-01-01

    Platelets from patients with the gray platelet syndrome have decreased recognizable alpha granules and are markedly deficient in some alpha-granule secretory proteins. Using immunocytochemical techniques with antibodies to an alpha-granule membrane protein, GMP-140, we identified the membranes of intracellular vesicles in gray platelets as alpha-granule membranes. Gray platelets contained normal amounts of GMP-140 as measured by electroimmunoassay. The activation of gray platelets with thrombin caused GMP-140 to be redistributed to the plasma membrane surface, as in normal platelets. In agreement with previous studies, an endogenously synthesized secretory protein, platelet factor 4, was undetectable in gray platelets. However, the alpha-granule proteins albumin and IgG, which are thought to be derived from endocytosis of plasma proteins into megakaryocytes, were present in substantial quantities and were secreted efficiently from gray platelets. Therefore, the fundamental defect in the gray platelet syndrome may be in the targeting of endogenously synthesized secretory proteins to developing alpha granules in megakaryocytes. Images PMID:2443536

  2. Tactile responses in the granule cell layer of cerebellar folium crus IIa of freely behaving rats

    NASA Technical Reports Server (NTRS)

    Hartmann, M. J.; Bower, J. M.

    2001-01-01

    We recorded activity from the granule cell layer (GCL) of cerebellar folium Crus IIa as freely moving rats engaged in a variety of natural behaviors, including grooming, eating, and free tactile exploration. Multiunit responses in the 1000-4500 Hz range were found to be strongly correlated with tactile stimulation of lip and whisker (perioral) regions. These responses occurred regardless of whether the stimulus was externally or self-generated and during both active and passive touch. In contrast, perioral movements that did not tactually stimulate this region of the face (e.g., chewing) produced no detectable increases in GCL activity. In addition, GCL responses were not correlated with movement extremes. When rats used their lips actively for palpation and exploration, the tactile responses in the GCL were not detectably modulated by ongoing jaw movements. However, active palpation and exploratory behaviors did result in the largest and most continuous bursts of GCL activity: responses were on average 10% larger and 50% longer during palpation and exploration than during grooming or passive stimulation. Although activity levels differed between behaviors, the position and spatial extent of the peripheral receptive field was similar over all behaviors that resulted in tactile input. Overall, our data suggest that the 1000-4500 Hz multiunit responses in the Crus IIa GCL of awake rats are correlated with tactile input rather than with movement or any movement parameter and that these responses are likely to be of particular importance during the acquisition of sensory information by perioral structures.

  3. Spatial information enhanced by non-spatial information in hippocampal granule cells.

    PubMed

    Hayakawa, Hirofumi; Samura, Toshikazu; Kamijo, Tadanobu Chuyo; Sakai, Yutaka; Aihara, Takeshi

    2015-02-01

    The hippocampus organizes sequential memory composed of non-spatial information (such as objects and odors) and spatial information (places). The dentate gyrus (DG) in the hippocampus receives two types of information from the lateral and medial entorhinal cortices. Non-spatial and spatial information is delivered respectively to distal and medial dendrites (MDs) of granule cells (GCs) within the molecular layer in the DG. To investigate the role of the association of those two inputs, we measured the response characteristics of distal and MDs of a GC in a rat hippocampal slice and developed a multi-compartment GC model with dynamic synapses; this model reproduces the response characteristics of the dendrites. Upon applying random inputs or input sequences generated by a Markov process to the computational model, it was found that a high-frequency random pulse input to distal dendrites (DDs) and, separately, regular burst inputs to MDs were effective for inducing GC activation. Furthermore, when the random and theta burst inputs were simultaneously applied to the respective dendrites, the pattern discrimination for theta burst input to MDs that caused slight GC activation was enhanced in the presence of random input to DDs. These results suggest that the temporal pattern discrimination of spatial information is originally involved in a synaptic characteristic in GCs and is enhanced by non-spatial information input to DDs. Consequently, the co-activation of two separate inputs may play a crucial role in the information processing on dendrites of GCs by usefully combing each temporal sequence.

  4. Improved performance of microbial fuel cell using combination biocathode of graphite fiber brush and graphite granules

    NASA Astrophysics Data System (ADS)

    Zhang, Guo-dong; Zhao, Qing-liang; Jiao, Yan; Zhang, Jin-na; Jiang, Jun-qiu; Ren, Nanqi; Kim, Byung Hong

    2011-08-01

    The efficiency and sustainability of microbial fuel cell (MFC) are heavily dependent on the cathode performance. We show here that the use of graphite fiber brush (GBF) together with graphite granules (GGs) as a basal material for biocathode (MFC reactor type R1) significantly improve the performance of a MFC compared with MFCs using GGs (MFC reactor type R2) or GFB (MFC reactor type R3) individually. Compared with R3, the use of the combination biocathode (R1) can shorten the start-up time by 53.75%, improve coulombic efficiencies (CEs) by 21.0 ± 2.7% at external resistance (REX) of 500 Ω, and increase maximum power densities by 38.2 ± 12.6%. Though the start-up time and open circuit voltage (OCV) of the reactor R2 are similar to R1, the CE (REX = 500 Ω) and maximum power density of R2 are 21.4 ± 1.7% and 38.2 ± 15.6% lower than that of R1. Fluorescence in situ hybridization (FISH) analyses indicate the bacteria on cathodes of R1 and R2 are richer than that of R3. Molecular taxonomic analyses reveal that the biofilm formed on the biocathode surface is dominated by strains belonging to Nitrobacter, Achromobacter, Acinetobacter, and Bacteroidetes. Combination of GFB and GGs as biocathode material in MFC is more efficient and can achieve sustainable electricity recovery from organic substances, which substantially increases the viability and sustainability of MFCs.

  5. Porous hydroxyapatite and biphasic calcium phosphate ceramics promote ectopic osteoblast differentiation from mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Zhang, Lingli; Hanagata, Nobutaka; Maeda, Megumi; Minowa, Takashi; Ikoma, Toshiyuki; Fan, Hongsong; Zhang, Xingdong

    2009-04-01

    Because calcium phosphate (Ca-P) ceramics have been used as bone substitutes, it is necessary to investigate what effects the ceramics have on osteoblast maturation. We prepared three types of Ca-P ceramics with different Ca-P ratios, i.e. hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP), and biphasic calcium phosphate (BCP) ceramics with dense-smooth and porous structures. Comprehensive gene expression microarray analysis of mouse osteoblast-like cells cultured on these ceramics revealed that porous Ca-P ceramics considerably affected the gene expression profiles, having a higher potential for osteoblast maturation. In the in vivo study that followed, porous Ca-P ceramics were implanted into rat skeletal muscle. Sixteen weeks after the implantation, more alkaline-phosphatase-positive cells were observed in the pores of hydroxyapatite and BCP, and the expression of the osteocalcin gene (an osteoblast-specific marker) in tissue grown in pores was also higher in hydroxyapatite and BCP than in β-TCP. In the pores of any Ca-P ceramics, 16 weeks after the implantation, we detected the expressions of marker genes of the early differentiation stage of chondrocytes and the complete differentiation stage of adipocytes, which originate from mesenchymal stem cells, as well as osteoblasts. These marker gene expressions were not observed in the muscle tissue surrounding the implanted Ca-P ceramics. These observations indicate that porous hydroxyapatite and BCP had a greater potential for promoting the differentiation of mesenchymal stem cells into osteoblasts than β-TCP.

  6. In Vivo Ectopic Bone Formation by Devitalized Mineralized Stem Cell Carriers Produced Under Mineralizing Culture Condition

    PubMed Central

    Chai, Yoke Chin; Geris, Liesbet; Bolander, Johanna; Pyka, Grzegorz; Van Bael, Simon; Luyten, Frank P.

    2014-01-01

    Abstract Functionalization of tissue engineering scaffolds with in vitro–generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca2+) and phosphate (PO43−) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano

  7. In vivo ectopic bone formation by devitalized mineralized stem cell carriers produced under mineralizing culture condition.

    PubMed

    Chai, Yoke Chin; Geris, Liesbet; Bolander, Johanna; Pyka, Grzegorz; Van Bael, Simon; Luyten, Frank P; Schrooten, Jan

    2014-12-01

    Functionalization of tissue engineering scaffolds with in vitro-generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca(2+)) and phosphate (PO4 (3-)) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography

  8. In vivo ectopic bone formation by devitalized mineralized stem cell carriers produced under mineralizing culture condition.

    PubMed

    Chai, Yoke Chin; Geris, Liesbet; Bolander, Johanna; Pyka, Grzegorz; Van Bael, Simon; Luyten, Frank P; Schrooten, Jan

    2014-12-01

    Functionalization of tissue engineering scaffolds with in vitro-generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca(2+)) and phosphate (PO4 (3-)) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography

  9. The homing of bone marrow MSCs to non-osseous sites for ectopic bone formation induced by osteoinductive calcium phosphate

    PubMed Central

    Song, Guodong; Habibovic, Pamela; Bao, Chongyun; Hu, Jing; van Blitterswijk, Clemens A.; Yuan, Huipin; Chen, Wenchuan; Xu, Hockin H.K.

    2013-01-01

    Osteoinductive biomaterials are promising for bone repair. There is no direct proof that bone marrow mesenchymal stem cells (BMSCs) home to non-osseous sites and participate in ectopic bone formation induced by osteoinductive bioceramics. The objective of this study was to use a sex-mismatched beagle dog model to investigate BMSC homing via blood circulation to participate in ectopic bone formation via osteoinductive biomaterial. BMSCs of male dogs were injected into female femoral marrow cavity. The survival and stable chimerism of donor BMSCs in recipients were confirmed with polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). Biphasic calcium phosphate (BCP) granules were implanted in dorsal muscles of female dogs. Y chromosomes were detected in samples harvested from female dogs which had received male BMSCs. At 4 weeks, cells with Y-chromosomes were distributed in the new bone matrix throughout the BCP granule implant. At 6 weeks, cells with Y chromosomes were present in newly mineralized woven bone. TRAP positive osteoclast-like cells were observed in 4-week implants, and the number of such cells decreased from 4 to 6 weeks. These results show that osteoprogenitors were recruited from bone marrow and homed to ectopic site to serve as a cell source for calcium phosphate-induced bone formation. In conclusion, BMSCs were demonstrated to migrate from bone marrow through blood circulation to non-osseous bioceramic implant site to contribute to ectopic bone formation in a canine model. BCP induced new bone in muscles without growth factor delivery, showing excellent osteoinductivity that could be useful for bone tissue engineering. PMID:23298780

  10. Ectopic Expression of BnaC.CP20.1 Results in Premature Tapetal Programmed Cell Death in Arabidopsis.

    PubMed

    Song, Liping; Zhou, Zhengfu; Tang, Shan; Zhang, Zhiqiang; Xia, Shengqian; Qin, Maomao; Li, Bao; Wen, Jing; Yi, Bin; Shen, Jinxiong; Ma, Chaozhi; Fu, Tingdong; Tu, Jinxing

    2016-09-01

    Tapetal programmed cell death (PCD) is essential in pollen grain development, and cysteine proteases are ubiquitous enzymes participating in plant PCD. Although the major papain-like cysteine proteases (PLCPs) have been investigated, the exact functions of many PLCPs are still poorly understood in PCD. Here, we identified a PLCP gene, BnaC.CP20.1, which was closely related to XP_013596648.1 from Brassica oleracea. Quantitative real-time PCR analysis revealed that BnaC.CP20.1 expression was down-regulated in male-sterile lines in oilseed rape, suggesting a connection between this gene and male sterility. BnaC.CP20.1 is especially active in the tapetum and microspores in Brassica napus from the uninucleate stage until formation of mature pollen grains during anther development. On expression of BnaC.CP20.1 prior to the tetrad stage, BnA9::BnaC.CP20.1 transgenic lines in Arabidopsis thaliana showed a male-sterile phenotype with shortened siliques containing fewer or no seeds by self-crossing. Scanning electron microscopy indicated that the reticulate exine was defective in aborted microspores. Callose degradation was delayed and microspores were not released from the tetrad in a timely fashion. Additionally, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay indicated that BnaC.CP20.1 ectopic expression led to premature tapetal PCD. Transmission electron microscopy analyses further demonstrated that the pollen abortion was due to the absence of tectum connections to the bacula in the transgenic anthers. These findings suggest that timely expression of BnaC.CP20.1 is necessary for tapetal degeneration and pollen wall formation. PMID:27388342

  11. Ectopic Expression of BnaC.CP20.1 Results in Premature Tapetal Programmed Cell Death in Arabidopsis.

    PubMed

    Song, Liping; Zhou, Zhengfu; Tang, Shan; Zhang, Zhiqiang; Xia, Shengqian; Qin, Maomao; Li, Bao; Wen, Jing; Yi, Bin; Shen, Jinxiong; Ma, Chaozhi; Fu, Tingdong; Tu, Jinxing

    2016-09-01

    Tapetal programmed cell death (PCD) is essential in pollen grain development, and cysteine proteases are ubiquitous enzymes participating in plant PCD. Although the major papain-like cysteine proteases (PLCPs) have been investigated, the exact functions of many PLCPs are still poorly understood in PCD. Here, we identified a PLCP gene, BnaC.CP20.1, which was closely related to XP_013596648.1 from Brassica oleracea. Quantitative real-time PCR analysis revealed that BnaC.CP20.1 expression was down-regulated in male-sterile lines in oilseed rape, suggesting a connection between this gene and male sterility. BnaC.CP20.1 is especially active in the tapetum and microspores in Brassica napus from the uninucleate stage until formation of mature pollen grains during anther development. On expression of BnaC.CP20.1 prior to the tetrad stage, BnA9::BnaC.CP20.1 transgenic lines in Arabidopsis thaliana showed a male-sterile phenotype with shortened siliques containing fewer or no seeds by self-crossing. Scanning electron microscopy indicated that the reticulate exine was defective in aborted microspores. Callose degradation was delayed and microspores were not released from the tetrad in a timely fashion. Additionally, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay indicated that BnaC.CP20.1 ectopic expression led to premature tapetal PCD. Transmission electron microscopy analyses further demonstrated that the pollen abortion was due to the absence of tectum connections to the bacula in the transgenic anthers. These findings suggest that timely expression of BnaC.CP20.1 is necessary for tapetal degeneration and pollen wall formation.

  12. Effects of formaldehyde exposure on granule cell number and volume of dentate gyrus: a histopathological and stereological study.

    PubMed

    Aslan, Huseyin; Songur, Ahmet; Tunc, Ayten Turkkani; Ozen, Oguz Aslan; Bas, Orhan; Yagmurca, Murat; Turgut, Mehmet; Sarsilmaz, Mustafa; Kaplan, Suleyman

    2006-11-29

    The hippocampal formation is a complex region of the brain related to memory and learning. The purpose of the present study was to determine whether exposure of neonatal rats to formaldehyde (FA) had either early or delayed effects on the numbers of granule cells in the dentate gyrus (DG). After birth, the neonatal male Wistar rats were exposed throughout a 30-day period to various concentrations of FA: 0 (control group), 6 ppm (low concentration group) and 12 ppm (high concentration group). This was done by placing them for 6 h/day and 5 days per week in a glass chamber containing FA vapor. Then, five animals from each group were anesthetized and decapitated on postnatal day (PND) 30, and the remaining five animals were sacrificed on PND 90 by intracardiac perfusion using 10% neutral buffered FA solution. The Cavalieri principle of stereological approaches was used to determine the volume of the DG in these sections. The optical fractionator counting method was used to estimate the total number of granule cells in the DG. The appearance of granule cells was normal under light microscopy in all PND 30 and PND 90 groups. There were significant age-related reductions in the volume of the DG at PND 90 irrespective of which group was examined. Significant age-related neuron loss was also determined at PND 90 compared to that at PND 30. Rats treated with a high concentration FA were found to have fewer granule cells than either the animals treated with a low concentration FA or the control group (p<0.01 and p<0.01, respectively) at PND 90 but not at PND 30. These findings clearly indicate that granule cells in the DG may be vulnerable to stress and the concentration of FA to which they are exposed during early postnatal life, and also that a neurotoxic effect of high dose FA on cell number is only seen after a long time period. These results may explain why some disorders do not appear until later life. PMID:17011527

  13. Evidence that zymogen granules are not a physiologically relevant calcium pool. Defining the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells.

    PubMed

    Yule, D I; Ernst, S A; Ohnishi, H; Wojcikiewicz, R J

    1997-04-01

    A key event leading to exocytosis of pancreatic acinar cell zymogen granules is the inositol 1,4,5-trisphosphate (InsP3)-mediated release of Ca2+ from intracellular stores. Studies using digital imaging microscopy and laser-scanning confocal microscopy have indicated that the initial release of Ca2+ is localized to the apical region of the acinar cell, an area of the cell dominated by secretory granules. Moreover, a recent study has shown that InsP3 is capable of releasing Ca2+ from a preparation enriched in secretory granules (Gerasimenko, O., Gerasimenko, J., Belan, P., and Petersen, O. H., (1996) Cell 84, 473-480). In the present study, we have investigated the possibility that zymogen granules express InsP3 receptors and are thus Ca2+ release sites. Immunofluorescence staining, obtained with antisera specific to types I, II, or III InsP3 receptors and analyzed by confocal fluorescence microscopy revealed that all InsP3 receptor types were present in acinar cells. The type II receptor localized exclusively to an area close to or at the luminal plasma membrane. While types I and III InsP3 receptors displayed a similar luminal distribution, these receptors were also present at low levels in nuclei. The localization of InsP3 receptor was in marked contrast to the distribution of amylase, a zymogen granule content protein. In a zymogen granule fraction prepared in an identical manner to the aforementioned report demonstrating InsP3-induced Ca2+ release, immunoblotting demonstrated the presence of types I, II, and III InsP3 receptors. Ca2+ release from this preparation in response to InsP3, but not thapsigargin, could also be demonstrated. In contrast, when the zymogen granules were further purified on a Percoll gradient, InsP3 receptors were undetectable, and InsP3 failed to release Ca2+. Transmission electron microscopy performed on both preparations showed that the Percoll-purified granule preparation consisted of essentially pure zymogen granules, whereas the

  14. The Flexibility of Ectopic Lipids

    PubMed Central

    Loher, Hannah; Kreis, Roland; Boesch, Chris; Christ, Emanuel

    2016-01-01

    In addition to the subcutaneous and the visceral fat tissue, lipids can also be stored in non-adipose tissue such as in hepatocytes (intrahepatocellular lipids; IHCL), skeletal (intramyocellular lipids; IMCL) or cardiac muscle cells (intracardiomyocellular lipids; ICCL). Ectopic lipids are flexible fuel stores that can be depleted by physical exercise and repleted by diet. They are related to obesity and insulin resistance. Quantification of IMCL was initially performed invasively, using muscle biopsies with biochemical and/or histological analysis. 1H-magnetic resonance spectroscopy (1H-MRS) is now a validated method that allows for not only quantifying IMCL non-invasively and repeatedly, but also assessing IHCL and ICCL. This review summarizes the current available knowledge on the flexibility of ectopic lipids. The available evidence suggests a complex interplay between quantitative and qualitative diet, fat availability (fat mass), insulin action, and physical exercise, all important factors that influence the flexibility of ectopic lipids. Furthermore, the time frame of the intervention on these parameters (short-term vs. long-term) appears to be critical. Consequently, standardization of physical activity and diet are critical when assessing ectopic lipids in predefined clinical situations. PMID:27649157

  15. The Flexibility of Ectopic Lipids.

    PubMed

    Loher, Hannah; Kreis, Roland; Boesch, Chris; Christ, Emanuel

    2016-01-01

    In addition to the subcutaneous and the visceral fat tissue, lipids can also be stored in non-adipose tissue such as in hepatocytes (intrahepatocellular lipids; IHCL), skeletal (intramyocellular lipids; IMCL) or cardiac muscle cells (intracardiomyocellular lipids; ICCL). Ectopic lipids are flexible fuel stores that can be depleted by physical exercise and repleted by diet. They are related to obesity and insulin resistance. Quantification of IMCL was initially performed invasively, using muscle biopsies with biochemical and/or histological analysis. ¹H-magnetic resonance spectroscopy (¹H-MRS) is now a validated method that allows for not only quantifying IMCL non-invasively and repeatedly, but also assessing IHCL and ICCL. This review summarizes the current available knowledge on the flexibility of ectopic lipids. The available evidence suggests a complex interplay between quantitative and qualitative diet, fat availability (fat mass), insulin action, and physical exercise, all important factors that influence the flexibility of ectopic lipids. Furthermore, the time frame of the intervention on these parameters (short-term vs. long-term) appears to be critical. Consequently, standardization of physical activity and diet are critical when assessing ectopic lipids in predefined clinical situations. PMID:27649157

  16. Misfolded N-CoR is Linked to the Ectopic Reactivation of CD34/Flt3-Based Stem-Cell Phenotype in Promyelocytic and Monocytic Acute Myeloid Leukemia

    PubMed Central

    Nin, Dawn Sijin; Li, Feng; Visvanathan, Sridevi; Khan, Matiullah

    2015-01-01

    Nuclear receptor co-repressor (N-CoR) is the key component of generic co-repressor complex essential for the transcriptional control of genes involved in cellular hemostasis. We have recently reported that N-CoR actively represses Flt3, a key factor of hematopoietic stem cells (HSC) self-renewal and growth, and that de-repression of Flt3 by the misfolded N-CoR plays an important role in the pathogenesis of promyelocytic and monocytic acute myeloid leukemia (AML). The leukemic cells derived from the promyelocytic and monocytic AML are distinctly characterized by the ectopic reactivation of stem cell phenotypes in relatively committed myeloid compartment. However, the molecular mechanism underlying this phenomenon is not known. Here, we report that N-CoR function is essential for the commitment of primitive hematopoietic cells to the cells of myeloid lineage and that loss of N-CoR function due to misfolding is linked to the ectopic reactivation of generic stem cell phenotypes in promyelocytic and monocytic AML. Analysis of N-CoR and Flt3 transcripts in mouse hematopoietic cells revealed a positive correlation between N-CoR level and the commitment of myeloid cells and an inverse correlation between N-CoR and Flt3 levels in primitive as well as committed myeloid cells. Enforced N-CoR expression in mouse HSCs inhibited their growth and self-renewal potentials and promoted maturation toward cells of myeloid lineage, suggesting a role of N-CoR in the commitment of cells of myeloid lineage. In contrast to AML cells with natively folded N-CoR, primary and secondary promyelocytic and monocytic AML cells harboring the misfolded N-CoR were highly positive for Flt3 and myeloid antigen-based HSC marker CD34. Genetic and therapeutic restoration of N-CoR conformation significantly down-regulated the CD34 levels in monocytic AML cells, suggesting an important role of N-CoR in the suppression of CD34-based HSC phenotypes. These findings collectively suggest that N-CoR is crucial

  17. Blockade of excitatory synaptogenesis with proximal dendrites of dentate granule cells following rapamycin treatment in a mouse model of temporal lobe epilepsy

    PubMed Central

    Yamawaki, Ruth; Thind, Khushdev; Buckmaster, Paul S.

    2014-01-01

    Inhibiting the mTOR signaling pathway with rapamycin blocks granule cell axon (mossy fiber) sprouting after epileptogenic injuries, including pilocarpine-induced status epilepticus. However, it remains unclear whether axons from other types of neurons sprout into the inner molecular layer and synapse with granule cell dendrites despite rapamycin treatment. If so, other aberrant positive-feedback networks might develop. To test this possibility stereological electron microscopy was used to estimate numbers of excitatory synapses in the inner molecular layer per hippocampus in pilocarpine-treated control mice, in mice 5 d after pilocarpine-induced status epilepticus, and after status epilepticus and daily treatment beginning 24 h later with rapamycin or vehicle for 2 months. The optical fractionator method was used to estimate numbers of granule cells in Nissl-stained sections so that numbers of excitatory synapses in the inner molecular layer per granule cell could be calculated. Control mice had an average of 2280 asymmetric synapses in the inner molecular layer per granule cell, which was reduced to 63% of controls 5 d after status epilepticus, recovered to 93% of controls in vehicle-treated mice 2 months after status epilepticus, but remained at only 63% of controls in rapamycin-treated mice. These findings reveal that rapamycin prevented excitatory axons from synapsing with proximal dendrites of granule cells and raise questions about the recurrent excitation hypothesis of temporal lobe epilepsy. PMID:25234294

  18. Cell adhesion, ammonia removal and granulation of autotrophic nitrifying sludge facilitated by N-acyl-homoserine lactones.

    PubMed

    Li, An-Jie; Hou, Bao-Lian; Li, Mei-Xi

    2015-11-01

    In this study, six N-acyl-homoserine lactone (AHL) molecules (C6-HSL, C8-HSL, C10-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL and 3-oxo-C10-HSL) were each dosed into a bioreactor and seeded using autotrophic nitrifying sludge (ANS). The effects of the AHLs on cell adhesion, nitrification and sludge granulation were investigated. The results indicated that the efficiencies of cell adhesion and ammonia removal both had a close correlation with the side chain length and β position substituent group of the AHLs. The best-performing AHL in terms of accelerating bacterial attached-growth was 3-oxo-C6-HSL, whereas C6-HSL outperformed the others in terms of the ammonia degradation rate. The addition of 3-oxo-C6-HSL or C6-HSL increased the biomass growth rate, microbial activity, extracellular proteins and nitrifying bacteria, which can accelerate the formation of nitrifying granules. Consequently, selecting AHL molecules that could improve bacteria in attached-growth mode and nitrification efficiency simultaneously will most likely facilitate the rapid granulation of nitrifying sludge.

  19. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

    PubMed

    Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

    2013-04-17

    After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.

  20. Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato.

    PubMed

    Albacete, Alfonso; Cantero-Navarro, Elena; Großkinsky, Dominik K; Arias, Cintia L; Balibrea, María Encarnación; Bru, Roque; Fragner, Lena; Ghanem, Michel E; González, María de la Cruz; Hernández, Jose A; Martínez-Andújar, Cristina; van der Graaff, Eric; Weckwerth, Wolfram; Zellnig, Günther; Pérez-Alfocea, Francisco; Roitsch, Thomas

    2015-02-01

    Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the

  1. Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato

    PubMed Central

    Albacete, Alfonso; Cantero-Navarro, Elena; Großkinsky, Dominik K.; Arias, Cintia L.; Balibrea, María Encarnación; Bru, Roque; Fragner, Lena; Ghanem, Michel E.; González, María de la Cruz; Hernández, Jose A.; Martínez-Andújar, Cristina; van der Graaff, Eric; Weckwerth, Wolfram; Zellnig, Günther; Pérez-Alfocea, Francisco; Roitsch, Thomas

    2015-01-01

    Drought stress conditions modify source–sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of

  2. Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato.

    PubMed

    Albacete, Alfonso; Cantero-Navarro, Elena; Großkinsky, Dominik K; Arias, Cintia L; Balibrea, María Encarnación; Bru, Roque; Fragner, Lena; Ghanem, Michel E; González, María de la Cruz; Hernández, Jose A; Martínez-Andújar, Cristina; van der Graaff, Eric; Weckwerth, Wolfram; Zellnig, Günther; Pérez-Alfocea, Francisco; Roitsch, Thomas

    2015-02-01

    Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the

  3. Synaptic fatigue at the naive perforant path-dentate granule cell synapse in the rat.

    PubMed

    Abrahamsson, Therése; Gustafsson, Bengt; Hanse, Eric

    2005-12-15

    Synaptic activation at low frequency is often used to probe synaptic function and synaptic plasticity, but little is known about how such low-frequency activation itself affects synaptic transmission. In the present study, we have examined how the perforant path-dentate granule cell (PP-GC) synapse adapts to low-frequency activation from a previously non-activated (naive) state. Stimulation at 0.2 Hz in acute slices from developing rats (7-12 days old) caused a gradual depression of the AMPA EPSC (at -80 mV) to about half within 50 stimuli. This synaptic fatigue was unaffected by the NMDA and metabotropic glutamate (mGlu) receptor antagonists d-AP5 and LY-341495. A smaller component of this synaptic fatigue was readily reversible when switching to very low-frequency stimulation (0.033-0.017 Hz) and is attributed to a reversible decrease in release probability, which is probably due to depletion of readily releasable vesicles. Thus, it was expressed to the same extent by AMPA and NMDA EPSCs, and was associated with a decrease in quantal content (measured as 1/CV(2)) with no change in the paired-pulse ratio. The larger component of the synaptic fatigue was not readily reversible, was selective for AMPA EPSCs and was associated with a decrease in 1/CV(2), thus probably representing silencing of AMPA signalling in a subset of synapses. In adult rats (> 30 days old), the AMPA silencing had disappeared while the low-frequency depression remained unaltered. The present study has thus identified two forms of synaptic plasticity that contribute to fatigue of synaptic transmission at low frequencies at the developing PP-GC synapse; AMPA silencing and a low-frequency depression of release probability.

  4. Formation of ectopic osteogenesis in weightlessness

    NASA Technical Reports Server (NTRS)

    1977-01-01

    An ectopic osteogenesis experiment aboard the Cosmos-936 biosatellite is described. Decalcified, lyophilized femur and tibia were implanted under the fascia or in the anterior wall of the abdomen in rats. Bone formation before and after the tests is described and illustrated. The extent of formation of ectopic bone in weightlessness did not differ significantly from that in the ground controls, but the bone marrow of the ectopic bone of the flight rats consisted exclusively of fat cells. The deficit of support-muscle loading was considered to cause the disturbance in skeletal bone tissue development.

  5. Nonspecific cytotoxic cells of teleosts are armed with multiple granzymes and other components of the granule exocytosis pathway.

    PubMed

    Praveen, Kesavannair; Leary, John H; Evans, Donald L; Jaso-Friedmann, Liliana

    2006-03-01

    Granzymes are members of the serine protease family and major components of cytotoxic granules of professional killer cells. Multiple granzymes have been identified from human and rodents with different substrate specificities. Although the significance of granzymes A and B in cell-mediated cytotoxicity has been extensively investigated, recent reports suggest that other granzymes may have either equal or greater importance in mediating cell death. Studies on the evolution of these closely related proteases were hindered by the lack of sequence and biochemical information of granzymes from "lower vertebrates." Here we report the generation of a catalytically active recombinant granzyme identified in the cytotoxic cells of an ectothermic vertebrate. Fully active, soluble recombinant catfish granzyme-1 (CFGR-1) was generated using a yeast-based expression system. In vitro enzyme kinetic assays using various thiobenzyl ester substrates verified its tryptase activity in full agreement with previous observations by sequence comparison and molecular modeling. The tryptase activity that was secreted from catfish NCC during an in vitro cytotoxicity assay strongly correlated with the cytotoxicity induced by these cells. Evidence for additional granzymes with different substrate specificities in NCC was obtained by analysis of the protease activity of supernatants collected from in vitro cytotoxicity assays. Searches of the catfish EST database further confirmed the presence of teleost granzymes with different substrate specificities. Granzyme activity measurements suggested a predominance of chymase and tryptase activities in NCC. Further proof that the granule exocytosis pathway is one of the cytotoxic mechanisms in NCC was provided by the expression of granule components perforin, granulysin and serglycin detected by RT-PCR analysis. These results demonstrate the evidence for a parallel evolution of effector molecules of cell-mediated cytotoxicity in teleosts. PMID

  6. Experience-Dependent Regulation of Dentate Gyrus Excitability by Adult-Born Granule Cells

    PubMed Central

    Park, Eun Hye; Burghardt, Nesha S.; Dvorak, Dino; Hen, René

    2015-01-01

    Behavioral studies have established a role for adult-born dentate granule cells in discriminating between similar memories. However, it is unclear how these cells mediate memory discrimination. Excitability is enhanced in maturing adult-born neurons, spurring the hypothesis that the activity of these cells “directly” encodes and stores memories. An alternative hypothesis posits that maturing neurons “indirectly” contribute to memory encoding by regulating excitation–inhibition balance. We evaluated these alternatives by using dentate-sensitive active place avoidance tasks to assess experience-dependent changes in dentate field potentials in the presence and absence of neurogenesis. Before training, X-ray ablation of adult neurogenesis-reduced dentate responses to perforant-path stimulation and shifted EPSP-spike coupling leftward. These differences were unchanged after place avoidance training with the shock zone in the initial location, which both groups learned to avoid equally well. In contrast, sham-treated mice decreased dentate responses and shifted EPSP-spike coupling leftward after the shock zone was relocated, whereas X-irradiated mice failed to show these changes in dentate function and were impaired on this test of memory discrimination. During place avoidance, excitation–inhibition coupled neural synchrony in dentate local field potentials was reduced in X-irradiated mice, especially in the θ band. The difference was most prominent during conflict learning, which is impaired in the X-irradiated mice. These findings indicate that maturing adult-born neurons regulate both functional network plasticity in response to memory discrimination and dentate excitation–inhibition coordination. The most parsimonious interpretation of these results is that adult neurogenesis indirectly regulates hippocampal information processing. SIGNIFICANCE STATEMENT Adult-born neurons in the hippocampal dentate gyrus are important for flexibly using memories, but

  7. Intraventricular administration of endoneuraminidase-N facilitates ectopic migration of subventricular zone-derived neural progenitor cells into 6-OHDA lesioned striatum of mice.

    PubMed

    Li, Chen; Zhang, Yong-Xin; Yang, Chun; Hao, Fei; Chen, Sha-Sha; Hao, Qiang; Lu, Tao; Qu, Ting-Yu; Zhao, Li-Ru; Duan, Wei-Ming

    2016-03-01

    Polysialic acid (PSA), a carbohydrate polymer associated with the neural cell adhesion molecule (NCAM), plays an important role in the migration, differentiation and maturation of neuroblasts. Endoneuraminidase-N (Endo-N) can specifically cleave PSA from NCAM. The objective of the present study was to examine: the effect of Endo-N on characteristics of subventricular zone (SVZ)-derived neural progenitor cells (NPCs) in vitro; whether intraventricular administration of Endo-N could increase ectopic migration of SVZ-derived NPCs into 6-hydroxydopamine (6-OHDA)-lesioned striatum, and whether migrated NPCs could differentiate into neuronal and glial cells. In in vitro study, Endo-N was found to inhibit the migration of NPCs, and to enhance the differentiation of NPCs. In in vivo study, mice sequentially received injections of 6-OHDA into the right striatum, Endo-N into the right lateral ventricle, and bromodeoxyuridine (BrdU) intraperitoneally. The data showed that intraventricular injections of Endo-N disorganized the normal structure of the rostral migratory stream (RMS), and drastically increased the number of BrdU-immunoreactive (IR) cells in 6-OHDA-lesioned striatum. In addition, a number of BrdU-IR cells were double labeled for doublecortin (DCX), NeuN or glial fibrillary acidic protein (GFAP). The results suggest that interruption of neuroblast chain pathway with Endo-N facilitates ectopic migration of SVZ-derived NPCs into the lesioned striatum, and migrated NPCs can differentiate into neurons and astrocytes. PMID:26724216

  8. Cell Division Mode Change Mediates the Regulation of Cerebellar Granule Neurogenesis Controlled by the Sonic Hedgehog Signaling

    PubMed Central

    Yang, Rong; Wang, Minglei; Wang, Jia; Huang, Xingxu; Yang, Ru; Gao, Wei-Qiang

    2015-01-01

    Summary Symmetric and asymmetric divisions are important for self-renewal and differentiation of stem cells during neurogenesis. Although cerebellar granule neurogenesis is controlled by sonic hedgehog (SHH) signaling, whether and how this process is mediated by regulation of cell division modes have not been determined. Here, using time-lapse imaging and cell culture from neuronal progenitor-specific and differentiated neuron-specific reporter mouse lines (Math1-GFP and Dcx-DsRed) and Patched+/− mice in which SHH signaling is activated, we find evidence for the existence of symmetric and asymmetric divisions that are closely associated with progenitor proliferation and differentiation. While activation of the SHH pathway enhances symmetric progenitor cell divisions, blockade of the SHH pathway reverses the cell division mode change in Math1-GFP;Dcx-DsRed;Patched+/− mice by promoting asymmetric divisions or terminal neuronal symmetric divisions. Thus, cell division mode change mediates the regulation of cerebellar granule neurogenesis controlled by SHH signaling. PMID:26527387

  9. Anion exchange polymer coated graphite granule electrodes for improving the performance of anodes in unbuffered microbial fuel cells

    NASA Astrophysics Data System (ADS)

    Wang, Xu; Li, Dengfeng; Mao, Xuhui; Yu, Eileen Hao; Scott, Keith; Zhang, Enren; Wang, Dihua

    2016-10-01

    In this paper, graphite granule composite electrodes are prepared for microbial fuel cells (MFCs) by coating commercial graphite granules with the mixture of quaternary DABCO polysulfone or Nafion ion exchange polymer and carbon black. The results of electrochemical impedance spectroscopy (EIS) suggest that the addition of carbon black could significantly improve the electrical conductivity of graphite granule anodes. When phosphate buffer solution (PBS) is replaced by NaCl solution, the current densities of the pristine anode, 0.08 g Nafion coated anode and 0.16 g QDPSU coated anode decrease by 52.6%, 20.6% and 10.3% at -0.2 V (vs. Ag/AgCl), respectively. The solution resistance of ion exchange polymer coated anodes is more stable in comparison with that of pristine anode. After 40 operational days, the performance drop of 0.16 g QDPSU coated anode when switching the solution from PBS to NaCl is still smaller than that of pristine anode. However, 0.08 g Nafion coated anode shows the similar performance in NaCl solution to the pristine anode after long term operation. This study reveals that QDPSU anion exchange polymer is more suitable for the anode modification. The QDPSU coated anode promises a great potential for three-dimensional anode based MFCs to treat domestic wastewater.

  10. Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats

    PubMed Central

    1992-01-01

    The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats. PMID:1371773

  11. Effects of spaced learning in the water maze on development of dentate granule cells generated in adult mice.

    PubMed

    Trinchero, Mariela F; Koehl, Muriel; Bechakra, Malik; Delage, Pauline; Charrier, Vanessa; Grosjean, Noelle; Ladeveze, Elodie; Schinder, Alejandro F; Abrous, D Nora

    2015-11-01

    New dentate granule cells (GCs) are generated in the hippocampus throughout life. These adult-born neurons are required for spatial learning in the Morris water maze (MWM). In rats, spatial learning shapes the network by regulating their number and dendritic development. Here, we explored whether such modulatory effects exist in mice. New GCs were tagged using thymidine analogs or a GFP-expressing retrovirus. Animals were exposed to a reference memory protocol for 10-14 days (spaced training) at different times after newborn cells labeling. Cell proliferation, cell survival, cell death, neuronal phenotype, and dendritic and spine development were examined using immunohistochemistry. Surprisingly, spatial learning did not modify any of the parameters under scrutiny including cell number and dendritic morphology. These results suggest that although new GCs are required in mice for spatial learning in the MWM, they are, at least for the developmental intervals analyzed here, refractory to behavioral stimuli generated in the course of learning in the MWM.

  12. Inactivating and non-inactivating dihydropyridine-sensitive Ca2+ channels in mouse cerebellar granule cells.

    PubMed Central

    Slesinger, P A; Lansman, J B

    1991-01-01

    1. Granule cells were dissociated from mouse cerebellum and grown in vitro. Currents through single Ca2+ channels were recorded from the cell body with the patch clamp technique. 2. Voltage steps to 0 mV produced brief channel openings with a mean open time of approximately 0.5 ms. The single-channel conductance measured from the amplitude of the single-channel current with 90 mM-Ba2+ in the patch electrode was 22 pS. 3. The probability of Ca2+ channel opening increased with test potentials more positive than -30 mV, with half-activation near 0 mV, and followed the Boltzmann relation for the activation of whole-cell Ca2+ current. 4. Voltage steps to potentials more positive than 0 mV produced more channel activity at the beginning than at the end of the voltage step. The average of the single-channel currents decayed to a non-zero level with a time course similar to that of the whole-cell Ca2+ current. 5. The amplitude as well as the decay of the mean current measured during a test pulse to 0 mV was reduced as the holding potential was made more positive than approximately -90 mV. The change in the open channel probability with holding potential followed the Boltzmann relation which described the inactivation of the whole-cell Ca2+ current. 6. Ca2+ channel activity persisted for over several minutes after excising the patch from the cell body when intracellular cyclic AMP was increased. After patch excision, the number of functional channels decreased to a level where only one channel at a time was active. Ca2+ channel openings appeared as either short bursts at the beginning of the voltage step or long bursts that lasted throughout the pulse. 7. Exposing the cell to the dihydropyridine agonist +(S)-202-791 markedly increased the fraction of sweeps with long openings and produced a non-decaying mean current that was approximately 5 times larger than control. In a fraction of the sweeps, however, long openings occurred more frequently at the beginning than at the

  13. Transcriptional Profiling of Newly Generated Dentate Granule Cells Using TU Tagging Reveals Pattern Shifts in Gene Expression during Circuit Integration1,2

    PubMed Central

    Chatzi, Christina; Shen, Rongkun; Goodman, Richard H.

    2016-01-01

    Abstract Despite representing only a small fraction of hippocampal granule cells, adult-generated newborn granule cells have been implicated in learning and memory (Aimone et al., 2011). Newborn granule cells undergo functional maturation and circuit integration over a period of weeks. However, it is difficult to assess the accompanying gene expression profiles in vivo with high spatial and temporal resolution using traditional methods. Here we used a novel method [“thiouracil (TU) tagging”] to map the profiles of nascent mRNAs in mouse immature newborn granule cells compared with mature granule cells. We targeted a nonmammalian uracil salvage enzyme, uracil phosphoribosyltransferase, to newborn neurons and mature granule cells using retroviral and lentiviral constructs, respectively. Subsequent injection of 4-TU tagged nascent RNAs for analysis by RNA sequencing. Several hundred genes were significantly enhanced in the retroviral dataset compared with the lentiviral dataset. We compared a selection of the enriched genes with steady-state levels of mRNAs using quantitative PCR. Ontology analysis revealed distinct patterns of nascent mRNA expression, with newly generated immature neurons showing enhanced expression for genes involved in synaptic function, and neural differentiation and development, as well as genes not previously associated with granule cell maturation. Surprisingly, the nascent mRNAs enriched in mature cells were related to energy homeostasis and metabolism, presumably indicative of the increased energy demands of synaptic transmission and their complex dendritic architecture. The high spatial and temporal resolution of our modified TU-tagging method provides a foundation for comparison with steady-state RNA analyses by traditional transcriptomic approaches in defining the functional roles of newborn neurons. PMID:27011954

  14. Inactivation of calcium currents in granule cells cultured from mouse cerebellum.

    PubMed Central

    Slesinger, P A; Lansman, J B

    1991-01-01

    1. Cells dissociated from mouse cerebellum were grown in vitro. Ca2+ channel currents were recorded from granule cells with the patch-clamp technique under conditions which suppressed currents through Na+ and K+ channels and minimized run-down of current through Ca2+ channels. 2. A strong depolarizing voltage step from a hyperpolarized holding potential produced inward Ca2+ channel current that decayed exponentially to a non-zero level. Inward current decayed to approximately 40% of its peak amplitude (range 20-90%). 3. The inward current increased in amplitude when Ca2+ was replaced with Ba2+ or after raising the concentration of extracellular Ba2+, but the rate of decay of current was unaffected. 4. The current-voltage (I-V) relation showed that peak or sustained current increased with voltage pulses more positive than approximately -30 mV, reached a maximum amplitude near +20 mV and became progressively smaller with larger depolarizations. 5. The tail currents produced after rapidly repolarizing the membrane potential to -70 mV from a positive test pulse decayed along a single exponential time course with a time constant of approximately 0.5 ms. The amplitude of tail current measured at a fixed repolarization potential increased as the pre-pulse was made more positive and reached a maximum with pre-pulses more positive than +40 mV. A plot of normalized amplitude of the tail current as a function of the pre-pulse potential was fitted with a Boltzmann relation with V1/2 = approximately + 8 mV and steepness k = 14 mV. 6. Shifting the holding potential to more positive potentials reduced the amplitude of the Ca2+ channel current elicited by the fixed voltage step and abolished the decay of the inward current. The peak current was normalized to the maximum peak current elicited from a very negative holding potential and plotted as a function of holding potential. The points were fitted with a Boltzmann relation for inactivation with V1/2 = approximately -57 mV and

  15. Heterologous processing of rat prosomatostatin to somatostatin-14 by PC2: requirement for secretory cell but not the secretion granule.

    PubMed Central

    Galanopoulou, A S; Seidah, N G; Patel, Y C

    1995-01-01

    The role of PC2 in prosomatostatin (PSS) processing was investigated in GH3/GH4C1 pituitary cells. These cells are sparsely granulated, express different amounts of PC2 and no PC1. We described heterologous processing of rat PSS (rPSS) co-expressed with PC2 in stably transfected cells, correlate PC2 protein levels under different conditions of transfection with efficiency of PSS processing to somatostatin-14 (SS-14), determine the effect of modulating cell granularity on enzyme expression and PSS processing, and compare the relative potency of PC2 with that of PC1, PSS and cleavage products were monitored by HPLC and radioimmunoassay of SS-like immunoreactivity (SSLI). Radioimmunoassay analysis of N-terminal PC2-like immunoreactivity (PC2 LI) in GH4C1:rPSS, GH4C1:rPSS + PC2 and GH3:rPSS transfectants showed a gradient of PC2 protein of 1:2.6:3.4 in cell extracts and 1:4.7:9 in secretion media from these cells respectively. The concentration of PC2 protein correlated with SS-14 conversion efficiency was 36 +/- 3% in GH4C1:rPSS cells, 56 +/- 7% in GH4C1:rPSS-PC2 cells and 100% in GH3:rPSS cells. Treatment of GH4C1:rPSS + PC2 cells with epidermal growth factor, insulin, and beta-estradiol to induce granules, significantly increased basal and forskolin-stimulated co-release of SS LI and PC2 LI, but had no influence on SS-14 processing efficiency. Hormone treatment led to a small increase in the ratio of mature PC2 (68 kDa) to proPC2 (75 kDa) forms. PC1 stably transfected in GH4C1 cells produced significantly greater SS-14 conversion (62% in cells, 66% in media) compared with PC2 transfectants (53% in cells, 47% in media) These results provide the first proof that PC2 can effect dibasic processing of mammalian PSS, and, along with PC1, qualifies as an authentic SS-14 convertase. The activity of PC2 requires the milieu of the secretory cell but not the secretory granule. Images Figure 1 Figure 2 Figure 3 Figure 6 PMID:7575441

  16. Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

    PubMed

    Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

    2014-03-01

    Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression.

  17. The granule cell density of the dentate gyrus following administration of Urtica dioica extract to young diabetic rats.

    PubMed

    Fazeli, S A; Gharravi, A M; Ghafari, S; Jahanshahi, M; Golalipour, M J

    2008-08-01

    Urtica dioica L. Stinging nettle has long been known worldwide as a medicinal plant. To study the benefits of the nettle in diabetic encephalopathy, the granule cell density of the dentate gyrus of diabetic rats was studied following administration of Urtica dioica extract. A total of 24 male albino Wistar rats were allocated equally to normal, diabetic, preventive and treatment groups. Hyperglycaemia was induced by streptozotocin (80 mg/kg) in the animals of the diabetic and treatment groups. One week after injection of the streptozotocin the animals in the treatment group received a hydroalcoholic extract of Urtica dioica (100 mg/kg/day) for 4 weeks intraperitoneally. The rats of the preventive group received hydroalcoholic extract of U. dioica (100 mg/kg/day) IP for the first 5 days and an injection of streptozotocin (80 mg/kg) on the 6th day. After 5 weeks of study all the rats were sacrificed and coronal sections were taken from the dorsal hippocampal formation of the right cerebral hemispheres and stained with cresyl violet. The area densities of the granule cells were measured and compared in the four groups. The density was lower in the diabetic rats compared with the controls (p > 0.05). The preventive group showed lower cell density than the controls (p > 0.05). The densities in the treated rats were higher than in the diabetic rats (p > 0.05). Furthermore, the control and treated rats showed similar densities (p > 0.05). It seems that U. dioica extract can help compensate for granule cell loss in the diabetic rat dentate gyrus, which can ameliorate cognitive impairment in diabetes. However, preventive use of the extract showed no significant benefit. PMID:18828102

  18. The neurotrophic activity of PACAP on rat cerebellar granule cells is associated with activation of the protein kinase A pathway and c-fos gene expression.

    PubMed

    Vaudry, D; Basille, M; Anouar, Y; Fournier, A; Vaudry, H; Gonzalez, B J

    1998-12-11

    In vitro studies have shown that PACAP promotes cell survival and neurite outgrowth in immature cerebellar granule cells. In the present study, we have examined the transduction pathways involved in the neurotrophic activity of PACAP. Incubation of cultured granule cells with graded concentrations of PACAP produced a dose-dependent increase in c-fos mRNA level. The effects of PACAP on c-fos gene expression and granule cell survival were both mimicked by dbcAMP but not by PMA. The maximum effect of PACAP on c-fos gene expression was observed after 1 h of treatment. Similar effects of the peptide on granule cell survival were observed whether the cells were continuously incubated with PACAP for 48 h or only exposed to PACAP during 1 h. The PKA inhibitor H89 significantly reduced the effect of PACAP on c-fos mRNA level, whereas the specific PKC inhibitor chelerytrine had no effect. These data indicate that the action of PACAP on cerebellar granule cell survival and c-fos gene expression are both mediated through the adenylyl cyclase/PKA pathway.

  19. Stimulation of the N-methyl-D-aspartate receptor has a trophic effect on differentiating cerebellar granule cells.

    PubMed

    Balázs, R; Hack, N; Jørgensen, O S

    1988-04-22

    N-methyl-D-aspartate (NMDA) supplementation of cerebellar cultures enriched in granule neurones (about 90%) prevented the extensive cell loss which occurs when cultivation takes place, in serum containing media, in the presence of 'low' K+ (5-15 mM). Estimation of tetanus toxin receptors and N-CAM contents indicated that NMDA rescued primarily nerve cells. The influence of NMDA in promoting cell survival was blocked by the receptor antagonist, 2-amino-5-phosphonovalerate. The effect depended both on the concentration of NMDA and on the degree of depolarization of cells, the affinity in the presence of 15 mM K+ being similar to that of NMDA receptor binding. The results attest a new role for excitatory amino acid transmitters by showing that they can exert a stage-dependent trophic action on developing nerve cells.

  20. Hydrogen sulfide induces hyperpolarization and decreases the exocytosis of secretory granules of rat GH3 pituitary tumor cells.

    PubMed

    Mustafina, Alsu N; Yakovlev, Aleksey V; Gaifullina, Aisylu Sh; Weiger, Thomas M; Hermann, Anton; Sitdikova, Guzel F

    2015-10-01

    The aim of the present study was to evaluate the effects of hydrogen sulfide (H2S) on the membrane potential, action potential discharge and exocytosis of secretory granules in neurosecretory pituitary tumor cells (GH3). The H2S donor - sodium hydrosulfide (NaHS) induced membrane hyperpolarization, followed by truncation of spontaneous electrical activity and decrease of the membrane resistance. The NaHS effect was dose-dependent with an EC50 of 152 μM (equals effective H2S of 16-19 μM). NaHS effects were not altered after inhibition of maxi conductance calcium-activated potassium (BK) channels by tetraethylammonium or paxilline, but were significantly reduced after inhibition or activation of ATP-dependent potassium channels (KATP) by glibenclamide or by diazoxide, respectively. In whole-cell recordings NaHS increased the amplitude of KATP currents, induced by hyperpolarizing pulses and subsequent application of glibenclamide decreased currents to control levels. Using the fluorescent dye FM 1-43 exocytosis of secretory granules was analyzed in basal and stimulated conditions (high K(+) external solution). Prior application of NaHS decreased the fluorescence of the cell membrane in both conditions which links with activation of KATP currents (basal secretion) and activation of KATP currents and BK-currents (stimulated exocytosis). We suggest that H2S induces hyperpolarization of GH3 cells by activation of KATP channels which results in a truncation of spontaneous action potentials and a decrease of hormone release.

  1. Effervescent Granules Prepared Using Eucommia ulmoides Oliv. and Moso Bamboo Leaves: Hypoglycemic Activity in HepG2 Cells

    PubMed Central

    Li, Xiang-Zhou

    2016-01-01

    Eucommia ulmoides Oliv. (E. ulmoides Oliv.) and moso bamboo (Phyllostachys pubescens) leaves are used as folk medicines in central-western China to treat diabetes. To investigate the hypoglycemic activity of the effervescent granules prepared using E. ulmoides Oliv. and moso bamboo leaves (EBEG) in HepG2 cells, EBEG were prepared with 5% of each of polysaccharides and chlorogenic acids from moso bamboo and E. ulmoides Oliv. leaves, respectively. HepG2 cells cultured in a high-glucose medium were classified into different groups. The results displayed EBEG-treated cells showed better glucose utilization than the negative controls; thus, the hypoglycemic effect of EBEG was much greater than that of granules prepared using either component alone, thereby indicating that this effect was due to a synergistic action of the components. Further, glucose consumption levels in the cells treated with EBEG (156.35% at 200 μg/mL) and the positive controls (metformin, 162.29%; insulin, 161.52%) were similar. Thus, EBEG exhibited good potential for use as a natural antidiabetic agent. The hypoglycemic effect of EBEG could be due to the synergistic action of polysaccharides from the moso bamboo leaves and chlorogenic acids from E. ulmoides Oliv. leaves via the inhibition of alpha-glucosidase and glucose-6-phosphate displacement enzyme. PMID:27656239

  2. Effervescent Granules Prepared Using Eucommia ulmoides Oliv. and Moso Bamboo Leaves: Hypoglycemic Activity in HepG2 Cells.

    PubMed

    Li, Xiang-Zhou; Zhang, Sheng

    2016-01-01

    Eucommia ulmoides Oliv. (E. ulmoides Oliv.) and moso bamboo (Phyllostachys pubescens) leaves are used as folk medicines in central-western China to treat diabetes. To investigate the hypoglycemic activity of the effervescent granules prepared using E. ulmoides Oliv. and moso bamboo leaves (EBEG) in HepG2 cells, EBEG were prepared with 5% of each of polysaccharides and chlorogenic acids from moso bamboo and E. ulmoides Oliv. leaves, respectively. HepG2 cells cultured in a high-glucose medium were classified into different groups. The results displayed EBEG-treated cells showed better glucose utilization than the negative controls; thus, the hypoglycemic effect of EBEG was much greater than that of granules prepared using either component alone, thereby indicating that this effect was due to a synergistic action of the components. Further, glucose consumption levels in the cells treated with EBEG (156.35% at 200 μg/mL) and the positive controls (metformin, 162.29%; insulin, 161.52%) were similar. Thus, EBEG exhibited good potential for use as a natural antidiabetic agent. The hypoglycemic effect of EBEG could be due to the synergistic action of polysaccharides from the moso bamboo leaves and chlorogenic acids from E. ulmoides Oliv. leaves via the inhibition of alpha-glucosidase and glucose-6-phosphate displacement enzyme. PMID:27656239

  3. Effervescent Granules Prepared Using Eucommia ulmoides Oliv. and Moso Bamboo Leaves: Hypoglycemic Activity in HepG2 Cells

    PubMed Central

    Li, Xiang-Zhou

    2016-01-01

    Eucommia ulmoides Oliv. (E. ulmoides Oliv.) and moso bamboo (Phyllostachys pubescens) leaves are used as folk medicines in central-western China to treat diabetes. To investigate the hypoglycemic activity of the effervescent granules prepared using E. ulmoides Oliv. and moso bamboo leaves (EBEG) in HepG2 cells, EBEG were prepared with 5% of each of polysaccharides and chlorogenic acids from moso bamboo and E. ulmoides Oliv. leaves, respectively. HepG2 cells cultured in a high-glucose medium were classified into different groups. The results displayed EBEG-treated cells showed better glucose utilization than the negative controls; thus, the hypoglycemic effect of EBEG was much greater than that of granules prepared using either component alone, thereby indicating that this effect was due to a synergistic action of the components. Further, glucose consumption levels in the cells treated with EBEG (156.35% at 200 μg/mL) and the positive controls (metformin, 162.29%; insulin, 161.52%) were similar. Thus, EBEG exhibited good potential for use as a natural antidiabetic agent. The hypoglycemic effect of EBEG could be due to the synergistic action of polysaccharides from the moso bamboo leaves and chlorogenic acids from E. ulmoides Oliv. leaves via the inhibition of alpha-glucosidase and glucose-6-phosphate displacement enzyme.

  4. Interactions between Inhibitory Interneurons and Excitatory Associational Circuitry in Determining Spatio-Temporal Dynamics of Hippocampal Dentate Granule Cells: A Large-Scale Computational Study

    PubMed Central

    Hendrickson, Phillip J.; Yu, Gene J.; Song, Dong; Berger, Theodore W.

    2015-01-01

    This paper reports on findings from a million-cell granule cell model of the rat dentate gyrus that was used to explore the contributions of local interneuronal and associational circuits to network-level activity. The model contains experimentally derived morphological parameters for granule cells, which each contain approximately 200 compartments, and biophysical parameters for granule cells, basket cells, and mossy cells that were based both on electrophysiological data and previously published models. Synaptic input to cells in the model consisted of glutamatergic AMPA-like EPSPs and GABAergic-like IPSPs from excitatory and inhibitory neurons, respectively. The main source of input to the model was from layer II entorhinal cortical neurons. Network connectivity was constrained by the topography of the system, and was derived from axonal transport studies, which provided details about the spatial spread of axonal terminal fields, as well as how subregions of the medial and lateral entorhinal cortices project to subregions of the dentate gyrus. Results of this study show that strong feedback inhibition from the basket cell population can cause high-frequency rhythmicity in granule cells, while the strength of feedforward inhibition serves to scale the total amount of granule cell activity. Results furthermore show that the topography of local interneuronal circuits can have just as strong an impact on the development of spatio-temporal clusters in the granule cell population as the perforant path topography does, both sharpening existing clusters and introducing new ones with a greater spatial extent. Finally, results show that the interactions between the inhibitory and associational loops can cause high frequency oscillations that are modulated by a low-frequency oscillatory signal. These results serve to further illustrate the importance of topographical constraints on a global signal processing feature of a neural network, while also illustrating how rich

  5. Caprin-1 is a target of the deafness gene Pou4f3 and is recruited to stress granules in cochlear hair cells in response to ototoxic damage.

    PubMed

    Towers, Emily R; Kelly, John J; Sud, Richa; Gale, Jonathan E; Dawson, Sally J

    2011-04-01

    The POU4 family of transcription factors are required for survival of specific cell types in different sensory systems. Pou4f3 is essential for the survival of auditory sensory hair cells and several mutations in human POU4F3 cause hearing loss. Thus, genes regulated by Pou4f3 are likely to be essential for hair cell survival. We performed a subtractive hybridisation screen in an inner-ear-derived cell line to find genes with differential expression in response to changes in Pou4f3 levels. The screen identified the stress-granule-associated protein Caprin-1 as being downregulated by Pou4f3. We demonstrated that this regulation occurs through the direct interaction of Pou4f3 with binding sites in the Caprin-1 5' flanking sequence, and describe the expression pattern of Caprin-1 mRNA and protein in the cochlea. Moreover, we found Caprin-1-containing stress granules are induced in cochlear hair cells following aminoglycoside-induced damage. This is the first report of stress granule formation in mammalian hair cells and suggests that the formation of Caprin-1-containing stress granules is a key damage response to a clinically relevant ototoxic agent. Our results have implications for the understanding of aminoglycoside-induced hearing loss and provide further evidence that stress granule formation is a fundamental cellular stress response.

  6. Pattern of rise in subplasma membrane Ca{sup 2+} concentration determines type of fusing insulin granules in pancreatic {beta} cells

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Nakamichi, Yoko; Nishiwaki, Chiyono; Sakurai, Takashi; Nagamatsu, Shinya

    2009-07-31

    We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca{sup 2+} concentration ([Ca{sup 2+}]{sub PM}) with the Ca{sup 2+} indicator Fura Red in rat {beta} cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca{sup 2+}]{sub PM} caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca{sup 2+}]{sub PM} induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca{sup 2+}]{sub PM} rise directly determines the types of fusing granules.

  7. Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients

    PubMed Central

    Bagdonaite, Ieva; Wandall, Hans H.; Litvinov, Ivan V.; Nastasi, Claudia; Becker, Jürgen C.; Dabelsteen, Sally; Geisler, Carsten; Bonefeld, Charlotte M.; Zhang, Qian; Wasik, Mariusz A.; Zhou, Youwen; Sasseville, Denis; Ødum, Niels; Woetmann, Anders

    2015-01-01

    CD22 is a member of the Sialic acid-binding Ig-like lectin (Siglec) family of lectins described to be exclusively present in B lymphocytes and B cell-derived neoplasms. Here, we describe a novel splice form of CD22 (designated CD22ΔN), which lacks the N-terminal domain as demonstrated by exon-specific RT-PCR and differential recognition by anti-CD22 antibodies. Importantly, CD22ΔN mRNA is expressed in skin lesions from 39 out of 60 patients with cutaneous T cell lymphoma (CTCL), whereas few patients (6 out of 60) expresses full-length, wild type CD22 (CD22wt). In addition, IHC staining of tumor biopsies confirmed the expression of CD22 in CD4+ T cells. Moreover, four out of four malignant T cell lines express CD22: Two cell lines express CD22ΔN (MyLa2059 and PB2B) and two express CD22wt (MAC-1 and MAC-2A). siRNA-mediated silencing of CD22 impairs proliferation and survival of malignant T cells, demonstrating a functional role for both CD22ΔN and CD22wt in these cells. In conclusion, we provide the first evidence for an ectopic expression of CD22 and a novel splice variant regulating malignant proliferation and survival in CTCL. Analysis of expression and function of CD22 in cutaneous lymphomas may form the basis for development of novel targeted therapies for our patients. PMID:25957418

  8. Hydroxylated polychlorinated biphenyls increase reactive oxygen species formation and induce cell death in cultured cerebellar granule cells

    SciTech Connect

    Dreiem, Anne Rykken, Sidsel; Lehmler, Hans-Joachim; Robertson, Larry W.; Fonnum, Frode

    2009-10-15

    Polychlorinated biphenyls (PCBs) are persistent organic pollutants that bioaccumulate in the body, however, they can be metabolized to more water-soluble products. Although they are more readily excreted than the parent compounds, some of the metabolites are still hydrophobic and may be more available to target tissues, such as the brain. They can also cross the placenta and reach a developing foetus. Much less is known about the toxicity of PCB metabolites than about the parent compounds. In the present study, we have investigated the effects of eight hydroxylated (OH) PCB congeners (2'-OH PCB 3, 4-OH PCB 14, 4-OH PCB 34, 4'-OH PCB 35, 4-OH PCB 36, 4'-OH PCB 36, 4-OH PCB 39, and 4'-OH PCB 68) on reactive oxygen species (ROS) formation and cell viability in rat cerebellar granule cells. We found that, similar to their parent compounds, OH-PCBs are potent ROS inducers with potency 4-OH PCB 14 < 4-OH PCB 36 < 4-OH PCB 34 < 4'-OH PCB 36 < 4'-OH PCB 68 < 4-OH PCB 39 < 4'-OH PCB 35. 4-OH PCB 36 was the most potent cell death inducer, and caused apoptotic or necrotic morphology depending on concentration. Inhibition of ERK1/2 kinase with U0126 reduced both cell death and ROS formation, suggesting that ERK1/2 activation is involved in OH-PCB toxicity. The results indicate that the hydroxylation of PCBs may not constitute a detoxification reaction. Since OH-PCBs like their parent compounds are retained in the body and may be more widely distributed to sensitive tissues, it is important that not only the levels of the parent compounds but also the levels of their metabolites are taken into account during risk assessment of PCBs and related compounds.

  9. BOULE, a Deleted in Azoospermia Homolog, Is Recruited to Stress Granules in the Mouse Male Germ Cells

    PubMed Central

    Kim, Byunghyuk; Rhee, Kunsoo

    2016-01-01

    High temperature adversely affects normal development of male germ cells in mammals. Acute heat stress induces the formation of stress granules (SGs) in a set of male germ cells, and the SGs have been proposed to protect those cells from heat-induced apoptosis. DAZL, one of DAZ (Deleted in Azoospermia) family proteins, was shown to be an essential component of SGs, which is required for SG formation in the mouse testis. In the present study, we asked whether BOULE, the founding member of DAZ family proteins, is a component of the SGs. We show that BOULE is recruited to the SGs upon heat stress, and that these SGs are developmental stage-specific. These results suggest that DAZ family proteins may have conserved roles in the SGs of male germ cells. PMID:27632217

  10. Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate.

    PubMed

    Lavial, Fabrice; Acloque, Hervé; Bachelard, Elodie; Nieto, M Angela; Samarut, Jacques; Pain, Bertrand

    2009-06-01

    When they are derived from blastodermal cells of the pre-primitive streak in vitro, the pluripotency of Chicken Embryonic Stem Cells (cESC) can be controlled by the cPouV and Nanog genes. These cESC can differentiate into derivatives of the three germ layers both in vitro and in vivo, but they only weakly colonize the gonads of host embryos. By contrast, non-cultured blastodermal cells and long-term cultured chicken primordial germ cells maintain full germline competence. This restriction in the germline potential of the cESC may result from either early germline determination in the donor embryos or it may occur as a result of in vitro culture. We are interested in understanding the genetic determinants of germline programming. The RNA binding protein Cvh (Chicken Vasa Homologue) is considered as one such determinant, although its role in germ cell physiology is still unclear. Here we show that the exogenous expression of Cvh, combined with appropriate culture conditions, induces cESC reprogramming towards a germ cell fate. Indeed, these cells express the Dazl, Tudor and Sycp3 germline markers, and they display improved germline colonization and adopt a germ cell fate when injected into recipient embryos. Thus, our results demonstrate that Vasa can drive ES cell differentiation towards the germ cell lineage, both in vitro and in vivo.

  11. Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

    SciTech Connect

    Fujita-Yoshigaki, Junko . E-mail: yoshigaki.junko@nihon-u.ac.jp; Katsumata, Osamu; Matsuki, Miwako; Yoshigaki, Tomoyoshi; Furuyama, Shunsuke; Sugiya, Hiroshi

    2006-05-26

    Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.

  12. Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis.

    PubMed

    Scharfman, H E; Goodman, J H; Sollas, A L

    2000-08-15

    A group of neurons with the characteristics of dentate gyrus granule cells was found at the hilar/CA3 border several weeks after pilocarpine- or kainic acid-induced status epilepticus. Intracellular recordings from pilocarpine-treated rats showed that these "granule-like" neurons were similar to normal granule cells (i. e., those in the granule cell layer) in membrane properties, firing behavior, morphology, and their mossy fiber axon. However, in contrast to normal granule cells, they were synchronized with spontaneous, rhythmic bursts of area CA3 pyramidal cells that survived status epilepticus. Saline-treated controls lacked the population of granule-like cells at the hilar/CA3 border and CA3 bursts. In rats that were injected after status epilepticus with bromodeoxyuridine (BrdU) to label newly born cells, and also labeled for calbindin D(28K) (because it normally stains granule cells), many double-labeled neurons were located at the hilar/CA3 border. Many BrdU-labeled cells at the hilar/CA3 border also were double-labeled with a neuronal marker (NeuN). Taken together with the recent evidence that granule cells that are born after seizures can migrate into the hilus, the results suggest that some newly born granule cells migrate as far as the CA3 cell layer, where they become integrated abnormally into the CA3 network, yet they retain granule cell intrinsic properties. The results provide insight into the physiological properties of newly born granule cells in the adult brain and suggest that relatively rigid developmental programs set the membrane properties of newly born cells, but substantial plasticity is present to influence their place in pre-existing circuitry.

  13. Bestrophin1 Channels are Insensitive to Ethanol and Do not Mediate Tonic GABAergic Currents in Cerebellar Granule Cells

    PubMed Central

    Diaz, Marvin R.; Wadleigh, Aya; Hughes, Benjamin A.; Woodward, John J.; Valenzuela, C. Fernando

    2012-01-01

    The granule cell layer of the cerebellum functions in spatio-temporal encoding of information. Granule cells (GCs) are tonically inhibited by spillover of GABA released from Golgi cells and this tonic inhibition is facilitated by acute ethanol. Recently, it was demonstrated that a specialized Ca2+-activated anion-channel, bestrophin1 (Best1), found on glial cells, can release GABA that contributes up to 50–75% of the tonic GABAergic current. However, it is unknown if ethanol has any actions on Best1 function. Using whole-cell electrophysiology, we found that recombinant Best1 channels expressed in HEK-293 cells were insensitive to 40 and 80 mM ethanol. We attempted to measure the Best1-mediated component of the tonic current in slices using 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We confirmed that this agent blocks recombinant Best1 channels. Unexpectedly, we found that NPPB significantly potentiated the tonic current and the area and decay of GABAA-mediated spontaneous inhibitory post-synaptic currents (IPSCs) in GCs in rodent slices under two different recording conditions. To better isolate the Best1-dependent tonic current component, we blocked the Golgi cell component of the tonic current with tetrodotoxin and found that NPPB similarly and significantly potentiated the tonic current amplitude and decay time of miniature IPSCs. Two other Cl−-channel blockers were also tested: 4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) showed no effect on GABAergic transmission, while niflumic acid (NFA) significantly suppressed the tonic current noise, as well as the mIPSC frequency, amplitude, and area. These data suggest that acute ethanol exposure does not modulate Best1 channels and these findings serve to challenge recent data indicating that these channels participate in the generation of tonic GABAergic currents in cerebellar GCs. PMID:22275879

  14. Characterization of Ectopic Colonies That Form in Widespread Areas of the Nervous System with Neural Stem Cell Transplants into the Site of a Severe Spinal Cord Injury

    PubMed Central

    Sharp, Kelli G.; Yee, Kelly Matsudaira; Hatch, Maya N.; Bonner, Joseph F.

    2014-01-01

    We reported previously the formation of ectopic colonies in widespread areas of the nervous system after transplantation of fetal neural stem cells (NSCs) into spinal cord transection sites. Here, we characterize the incidence, distribution, and cellular composition of the colonies. NSCs harvested from E14 spinal cords from rats that express GFP were treated with a growth factor cocktail and grafted into the site of a complete spinal cord transection. Two months after transplant, spinal cord and brain tissue were analyzed histologically. Ectopic colonies were found at long distances from the transplant in the central canal of the spinal cord, the surface of the brainstem and spinal cord, and in the fourth ventricle. Colonies were present in 50% of the rats, and most rats had multiple colonies. Axons extended from the colonies into the host CNS. Colonies were strongly positive for nestin, a marker for neural precursors, and contained NeuN-positive cells with processes resembling dendrites, GFAP-positive astrocytes, APC/CC1-positive oligodendrocytes, and Ki-67-positive cells, indicating ongoing proliferation. Stereological analyses revealed an estimated 21,818 cells in a colony in the fourth ventricle, of which 1005 (5%) were Ki-67 positive. Immunostaining for synaptic markers (synaptophysin and VGluT-1) revealed large numbers of synaptophysin-positive puncta within the colonies but fewer VGluT-1 puncta. Continuing expansion of NSC-derived cell masses in confined spaces in the spinal cord and brain could produce symptoms attributable to compression of nearby tissue. It remains to be determined whether other cell types with self-renewing potential can also form colonies. PMID:25319698

  15. 5-HT1A Receptors on Mature Dentate Gyrus Granule Cells are Critical for the Antidepressant Response

    PubMed Central

    Samuels, Benjamin Adam; Anacker, Christoph; Hu, Alice; Levinstein, Marjorie R.; Pickenhagen, Anouchka; Tsetsenis, Theodore; Madroñal, Noelia; Donaldson, Zoe R.; Drew, Liam John; Dranovsky, Alex; Gross, Cornelius T.; Tanaka, Kenji F.; Hen, René

    2015-01-01

    Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants, but the mechanisms by which they influence behavior are only partially resolved. Adult hippocampal neurogenesis is necessary for some of the responses to SSRIs, but it is unknown whether the mature dentate gyrus granule cells (mature DG GCs) also contribute. We deleted Serotonin 1A receptor (5HT1AR; a receptor required for the SSRI response) specifically from DG GCs and found that the effects of the SSRI fluoxetine on behavior and the Hypothalamic-Pituitary-Adrenal (HPA) axis were abolished. By contrast, mice lacking 5HT1ARs only in young adult born granule cells (abGCs) showed normal fluoxetine responses. Importantly, 5HT1AR deficient mice engineered to express functional 5HT1ARs only in DG GCs responded to fluoxetine, indicating that 5HT1ARs in DG GCs are sufficient to mediate an antidepressant response. Taken together, these data indicate that both mature DG GCs and young abGCs must be engaged for an antidepressant response. PMID:26389840

  16. Glomerular and Mitral-Granule Cell Microcircuits Coordinate Temporal and Spatial Information Processing in the Olfactory Bulb

    PubMed Central

    Cavarretta, Francesco; Marasco, Addolorata; Hines, Michael L.; Shepherd, Gordon M.; Migliore, Michele

    2016-01-01

    The olfactory bulb processes inputs from olfactory receptor neurons (ORNs) through two levels: the glomerular layer at the site of input, and the granule cell level at the site of output to the olfactory cortex. The sequence of action of these two levels has not yet been examined. We analyze this issue using a novel computational framework that is scaled up, in three-dimensions (3D), with realistic representations of the interactions between layers, activated by simulated natural odors, and constrained by experimental and theoretical analyses. We suggest that the postulated functions of glomerular circuits have as their primary role transforming a complex and disorganized input into a contrast-enhanced and normalized representation, but cannot provide for synchronization of the distributed glomerular outputs. By contrast, at the granule cell layer, the dendrodendritic interactions mediate temporal decorrelation, which we show is dependent on the preceding contrast enhancement by the glomerular layer. The results provide the first insights into the successive operations in the olfactory bulb, and demonstrate the significance of the modular organization around glomeruli. This layered organization is especially important for natural odor inputs, because they activate many overlapping glomeruli. PMID:27471461

  17. Semi-quantitative monitoring of confluence of adherent mesenchymal stromal cells on calcium-phosphate granules by using widefield microscopy images.

    PubMed

    Piccinini, Filippo; Pierini, Michela; Lucarelli, Enrico; Bevilacqua, Alessandro

    2014-10-01

    The analysis of cell confluence and proliferation is essential to design biomaterials and scaffolds to use as bone substitutes in clinical applications. Accordingly, several approaches have been proposed in the literature to estimate the area of the scaffold covered by cells. Nevertheless, most of the approaches rely on sophisticated equipment not employed for routine analyses, while the rest of them usually do not provide significant statistics about the cell distribution. This research aims at studying confluence and proliferation of mesenchymal stromal cells (MSC) adherent on OSPROLIFE(®), a commercial biomaterial in the form of granules. In particular, we propose a Computer Vision approach that can routinely be employed to monitor the surface of the single granules covered by cells because only a standard widefield fluorescent microscope is required. In order to acquire significant statistics data, we analyse wide-area images built by using MicroMos v2.0, an updated version of a previously published software specific for stitching brightfield and phase-contrast images manually acquired via a widefield microscope. In particular, MicroMos v2.0 permits to build accurate "mosaics" of fluorescent images, after correcting vignetting and photo-bleaching effects, providing a consistent representation of a sample region containing numerous granules. Then, our method allows to make automatically a statistically significant estimate of the percentage of the area of the single granules covered by cells. Finally, by analysing hundreds of granules at different time intervals we also obtained reliable data regarding cell proliferation, confirming that not only MSC adhere onto the OSPROLIFE(®) granules, but even proliferate over time.

  18. Granule cell ascending axon excitatory synapses onto Golgi cells implement a potent feedback circuit in the cerebellar granular layer.

    PubMed

    Cesana, Elisabetta; Pietrajtis, Katarzyna; Bidoret, Céline; Isope, Philippe; D'Angelo, Egidio; Dieudonné, Stéphane; Forti, Lia

    2013-07-24

    The function of inhibitory interneurons within brain microcircuits depends critically on the nature and properties of their excitatory synaptic drive. Golgi cells (GoCs) of the cerebellum inhibit cerebellar granule cells (GrCs) and are driven both by feedforward mossy fiber (mf) and feedback GrC excitation. Here, we have characterized GrC inputs to GoCs in rats and mice. We show that, during sustained mf discharge, synapses from local GrCs contribute equivalent charge to GoCs as mf synapses, arguing for the importance of the feedback inhibition. Previous studies predicted that GrC-GoC synapses occur predominantly between parallel fibers (pfs) and apical GoC dendrites in the molecular layer (ML). By combining EM and Ca(2+) imaging, we now demonstrate the presence of functional synaptic contacts between ascending axons (aa) of GrCs and basolateral dendrites of GoCs in the granular layer (GL). Immunohistochemical quantification estimates these contacts to be ∼400 per GoC. Using Ca(2+) imaging to identify synaptic inputs, we show that EPSCs from aa and mf contacts in basolateral dendrites display similarly fast kinetics, whereas pf inputs in the ML exhibit markedly slower kinetics as they undergo strong filtering by apical dendrites. We estimate that approximately half of the local GrC contacts generate fast EPSCs, indicating their basolateral location in the GL. We conclude that GrCs, through their aa contacts onto proximal GoC dendrites, define a powerful feedback inhibitory circuit in the GL.

  19. Impact of Chromogranin A deficiency on catecholamine storage, catecholamine granule morphology and chromaffin cell energy metabolism in vivo.

    PubMed

    Pasqua, Teresa; Mahata, Sumana; Bandyopadhyay, Gautam K; Biswas, Angshuman; Perkins, Guy A; Sinha-Hikim, Amiya P; Goldstein, David S; Eiden, Lee E; Mahata, Sushil K

    2016-03-01

    Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40 % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200 nm) than in WT mice (200-350 nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47 % increase in DCV/DC ratio, implying vesicle swelling due to increased osmotically active free catecholamines. Upon challenge with 2 U/kg insulin, there was a diminution in adrenomedullary EPI, no change in NE and a very large increase in the EPI and NE precursor dopamine (DA), consistent with increased catecholamine biosynthesis during prolonged secretion. We found dilated mitochondrial cristae, endoplasmic reticulum and Golgi complex, as well as increased synaptic mitochondria, synaptic vesicles and glycogen granules in Chga-KO mice compared to WT mice, suggesting that decreased granulogenesis and catecholamine storage in CgA-deficient mouse adrenal medulla is compensated by increased VMAT-dependent catecholamine update into storage vesicles, at the expense of enhanced energy expenditure by the chromaffin cell. PMID:26572539

  20. Granule Associated Serine Proteases of Hematopoietic Cells – An Analysis of Their Appearance and Diversification during Vertebrate Evolution

    PubMed Central

    Akula, Srinivas; Thorpe, Michael; Boinapally, Vamsi; Hellman, Lars

    2015-01-01

    Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish. PMID:26569620

  1. Apoptosis of cerebellar granule cells induced by organotin compounds found in drinking water: involvement of MAP kinases.

    PubMed

    Mundy, William R; Freudenrich, Theresa M

    2006-01-01

    Mono- and dialkyl organotin compounds are used primarily as heat stabilizers in polyvinyl chloride (PVC) plastics. Recently, monomethyltin (MMT), dimethyltin (DMT), monobutyltin (MBT), and dibutyltin (DBT) have been detected in water from homes and businesses served by PVC pipes. While trialkyl organotins such as trimethyltin (TMT) and triethyltin (TET) are well known neurotoxicants, the toxicity of the mono- and dialkyl organotins is not well described. The present study compared the cytotoxicity of organotins found in drinking water with the known neurotoxicant TMT in primary cultures of cerebellar granule cells, and examined the role of MAP kinase signaling in organotin-induced cell death. Twenty-four hour exposure to TMT resulted in a concentration-dependent decrease in cell viability with an EC(50) of 3 microM. Exposure to MMT, DMT, and MBT at concentrations up to 10 microM had no effect. DBT, however, was very potent, and decreased cell viability with an EC(50) of 0.3 microM. Staining of organotin-treated cerebellar granule cells with the nuclear dye Syto-13 revealed that TMT and DBT, but not MMT, DMT, or MBT, produced condensation and fragmentation of chromatin characteristic of apoptosis. TMT- and DBT-induced apoptosis was confirmed using TUNEL staining and measurement of PARP cleavage. Activation of MAP kinase pathways was examined after 6 h of exposure to the organotins which induced apoptosis. Both TMT and DBT activated ERK1/2, but only TMT activated the JNK/c-Jun and p38 pathways. Pharmacologic blockade of JNK/c-Jun and p38 activation significantly decreased apoptosis produced by TMT, but not by DBT. These results show that DBT is a potent neurotoxicant in vitro, but unlike TMT, does not induce cell death via activation of MAP kinase signaling.

  2. Regulation of secretory granule size by the precise generation and fusion of unit granules

    PubMed Central

    Hammel, Ilan; Lagunoff, David; Galli, Stephen J

    2010-01-01

    Abstract Morphometric evidence derived from studies of mast cells, pancreatic acinar cells and other cell types supports a model in which the post-Golgi processes that generate mature secretory granules can be resolved into three steps: (1) fusion of small, Golgi-derived progranules to produce immature secretory granules which have a highly constrained volume; (2) transformation of such immature granules into mature secretory granules, a process often associated with a reduction in the maturing granule’s volume, as well as changes in the appearance of its content and (3) fusion of secretory granules of the smallest size, termed ‘unit granules’, forming granules whose volumes are multiples of the unit granule’s volume. Mutations which perturb this process can cause significant pathology. For example, Chediak–Higashi syndrome / lysosomal trafficking regulator (CHS)/(Lyst) mutations result in giant secretory granules in a number of cell types in human beings with the Chediak–Higashi syndrome and in ‘beige’ (Lystbg/Lystbg) mice. Analysis of the secretory granules of mast cells and pancreatic acinar cells in Lyst-deficient beige mice suggests that beige mouse secretory granules retain the ability to fuse randomly with other secretory granules no matter what the size of the fusion partners. By contrast, in normal mice, the pattern of granule–granule fusion occurs exclusively by the addition of unit granules, either to each other or to larger granules. The normal pattern of fusion is termed unit addition and the fusion evident in cells with CHS/Lyst mutations is called random addition. The proposed model of secretory granule formation has several implications. For example, in neurosecretory cells, the secretion of small amounts of cargo in granules constrained to a very narrow size increases the precision of the information conveyed by secretion. By contrast, in pancreatic acinar cells and mast cells, large granules composed of multiple unit granules

  3. Particle size of hydroxyapatite granules calcified from red algae affects the osteogenic potential of human mesenchymal stem cells in vitro.

    PubMed

    Weissenboeck, Martina; Stein, Elisabeth; Undt, Gerhard; Ewers, Rolf; Lauer, Gunter; Turhani, Dritan

    2006-01-01

    Hydroxyapatite (HA) microparticles as a carrier in an injectable tissue-engineered bone filler are considered promising candidates for the treatment of small bone defects in the craniomaxillofacial region. HA granules calcified from red algae, varying in size, were evaluated in vitro for their suitability to be used as a carrier for human mesenchymal stem cells (hMSCs). Three groups of granules were produced in grain sizes of 10-100, 200-500 and 600-1,000 mum. After seeding and culturing hMSCs under osteogenic differentiation conditions onto HA particles for 3, 6 and 9 days, cellular proliferation (tetrazolium salt, XTT), alkaline phosphatase (ALP)-specific activity and total protein synthesis were investigated. The osteoblastic phenotype of the cells was evaluated by assaying the bone-specific genes osteocalcin, osteopontin and collagen type I. XTT assay revealed significantly higher (p < 0.01) proliferation of cells grown on the smallest grain size after 9 days of culture. Regarding ALP-specific activity, significantly higher levels of activity were detected in cells grown on the smallest grain size. Different grain sizes had no significant effects on the secretion of osteocalcin and osteopontin. Collagen type I production was significantly higher (p < 0.05) in cells grown on the biggest grain size in comparison with the two other grain sizes. These results show that the particle size of HA microparticles affects the osteogenic potential of cultured hMSCs and lead to the conclusion that particle size has differential effects on ALP-specific activity and collagen type I production.

  4. Conditional deletion of α-CaMKII impairs integration of adult-generated granule cells into dentate gyrus circuits and hippocampus-dependent learning.

    PubMed

    Arruda-Carvalho, Maithe; Restivo, Leonardo; Guskjolen, Axel; Epp, Jonathan R; Elgersma, Ype; Josselyn, Sheena A; Frankland, Paul W

    2014-09-01

    New granule cells are continuously integrated into hippocampal circuits throughout adulthood, and the fine-tuning of this process is likely important for efficient hippocampal function. During development, this integration process is critically regulated by the α-calcium/calmodulin-dependent protein kinase II (α-CaMKII), and here we ask whether this role is conserved in the adult brain. To do this, we developed a transgenic strategy to conditionally delete α-CaMKII from neural progenitor cells and their progeny in adult mice. First, we found that the selective deletion of α-CaMKII from newly generated dentate granule cells led to an increase in dendritic complexity. Second, α-CaMKII deletion led to a reduction in number of mature synapses and cell survival. Third, consistent with altered morphological and synaptic development, acquisition of one-trial contextual fear conditioning was impaired after deletion of α-CaMKII from newly generated dentate granule cells. Previous work in Xenopus identified α-CaMKII as playing a key role in the stabilization of dendritic and synaptic structure during development. The current study indicates that α-CaMKII plays a plays a similar, cell-autonomous role in the adult hippocampus and, in addition, reveals that the loss of α-CaMKII from adult-generated granule cells is associated with impaired hippocampus-dependent learning.

  5. LXR agonist rescued the deficit in the proliferation of the cerebellar granule cells induced by dexamethasone.

    PubMed

    Bian, Xuting; Zhong, Hongyu; Li, Fen; Cai, Yulong; Li, Xin; Wang, Lian; Fan, Xiaotang

    2016-09-01

    Dexamethasone (DEX) exposure during early postnatal life produces permanent neuromotor and intellectual deficits and stunts cerebellar growth. The liver X receptor (LXR) plays important roles in CNS development. However, the effects of LXR on the DEX-mediated impairment of cerebellar development remain undetermined. Thus, mice were pretreated with LXR agonist TO901317 (TO) and were later exposed to DEX to evaluate its protective effects on DEX-mediated deficit during cerebellar development. The results showed that an acute exposure of DEX on postnatal day 7 resulted in a significant impairment in cerebellar development and decreased the proliferation of granule neuron precursors in the external granule layer of cerebellum. This effect was attenuated by pretreatment with TO. We further found that the decrease in the proliferation caused by DEX occurred via up-regulation of glucocorticoid receptor and p27kip1, which could be partially prevented by LXR agonist pretreatment. Overall, our results suggest that LXR agonist pretreatment could protect against DEX-induced deficits in cerebellar development in postnatal mice and may thus be perspective recruited to counteract such GC side effects. PMID:27369072

  6. Population imaging at subcellular resolution supports specific and local inhibition by granule cells in the olfactory bulb

    PubMed Central

    Wienisch, Martin; Murthy, Venkatesh N.

    2016-01-01

    Information processing in early sensory regions is modulated by a diverse range of inhibitory interneurons. We sought to elucidate the role of olfactory bulb interneurons called granule cells (GCs) in odor processing by imaging the activity of hundreds of these cells simultaneously in mice. Odor responses in GCs were temporally diverse and spatially disperse, with some degree of non-random, modular organization. The overall sparseness of activation of GCs was highly correlated with the extent of glomerular activation by odor stimuli. Increasing concentrations of single odorants led to proportionately larger population activity, but some individual GCs had non-monotonic relations to concentration due to local inhibitory interactions. Individual dendritic segments could sometimes respond independently to odors, revealing their capacity for compartmentalized signaling in vivo. Collectively, the response properties of GCs point to their role in specific and local processing, rather than global operations such as response normalization proposed for other interneurons. PMID:27388949

  7. Granule cell dispersion is not a predictor of surgical outcome in temporal lobe epilepsy with mesial temporal sclerosis.

    PubMed

    da Costa Neves, Rafael Scarpa; Jardim, Anaclara Prada; Caboclo, Luís Otávio; Lancellotti, Carmen; Marinho, Taissa Ferrari; Hamad, Ana Paula; Marinho, Murilo; Centeno, Ricardo; Cavalheiro, Esper Abrão; Scorza, Carla Alessandra; Targas Yacubian, Elza Márcia

    2013-01-01

    The aim of this retrospective study of a series of patients with mesial temporal lobe epilepsy (MTLE) and mesial temporal sclerosis (MTS) was to analyze the association of granule cell dispersion (GCD) with surgical prognosis, patterns of MTS and clinical data. Hippocampal specimens from 66 patients with MTLE and unilateral MTS and from 13 controls were studied. Quantitative neuropathological evaluation was performed on NeuN-stained hippocampal sections. Patients' clinical data, types of MTS and surgical outcome were reviewed. GCD occurred in 45.5% of cases and was not correlated with clinical variable. More severe neuronal loss was observed in patients with GCD. Except for MTS Type 2 - observed only in four no- GCD patients - groups did not differ with respect to the types of MTS. Surgical outcome was similar in both groups. In conclusion, GCD was associated with the degree of hippocampal cell loss, but was not a predictor of surgical outcome.

  8. Fine structure of the cell clusters in the cochlear nerve root: stellate, granule, and mitt cells offer insights into the synaptic organization of local circuit neurons.

    PubMed

    Hutson, K A; Morest, D K

    1996-07-29

    The small cell shell of the cochlear nucleus contains a complex integrative machinery which can be used to study the roles of interneurons in sensory processing. The cell clusters in the cochlear nerve root of the chinchilla provide the simplest example of this structure. Reported here are the neuronal architecture and synaptic organization of the three principal cell types and the three distinctive neuropil structures that could be characterized with the Nissl and Golgi methods and electron microscopy. Granule cells were characterized by several dendrites with claw-like terminals that received synaptic contacts from multiple excitatory mossy fiber rosettes. Given their relatively large number and their prolific parallel fiber synapses, the granule cells provide a suitable substrate for a tangential spread of excitatory activity, which could build to considerable proportions. The mitt cells had a thickened, single dendrite, its terminal branches arranged in a shape reminiscent of a baseball catcher's mitt. The dendritic mitt enclosed an enormous, convoluted mossy fiber rosette forming many excitatory synapses on just one cell. This could provide for a discrete, comparatively fast input-output relay of signals. Small stellate cells had longer, radiating dendrites that engaged the synaptic nests. These nests were strung in long strands, containing heterogeneous synapses from putative excitatory and inhibitory inputs. Given the prevalence of the synaptic nests, the small stellate cells appear to have the greatest integrative capacity. They provide the main output of the synaptic nests.

  9. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells

    SciTech Connect

    Stevens, R.L.; Austen, K.F. ); Fox, C.C.; Lichtenstein, L.M. )

    1988-04-01

    The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of {sup 35}S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although ({sup 35}S)heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate ({sup 35}S)sulfate into separate heparin and chondroitin sulfate proteoglycans in an {approx}2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin ({sup 35}S)sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin ({sup 35}S)sulfate E proteoglycans and the ({sup 35}S)heparin proteoglycans were exocytosed from the ({sup 35}S)sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of {sup 35}S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.

  10. The effect of gallium nitride on long-term culture induced aging of neuritic function in cerebellar granule cells.

    PubMed

    Chen, Chi-Ruei; Young, Tai-Horng

    2008-04-01

    Gallium nitride (GaN) has been developed for a variety of microelectronic and optical applications due to its unique electric property and chemical stability. In the present study, n-type and p-type GaN were used as substrates to culture cerebellar granule neurons to examine the effect of GaN on cell response for a long-term culture period. It was found that GaN could rapidly induce cultured neurons to exhibit a high phosphorylated Akt level after 20h of incubation. It was assumed that the anti-apoptotic effect of Akt phosphorylation could be correlated with cell survival, neurite growth and neuronal function for up to 35 days of incubation. Morphological studies showed GaN induced larger neuronal aggregates and neurite fasciculation to exhibit a dense fiber network after 8 days of incubation. Western blot analysis and immunocytochemical characterization showed that GaN still exhibited the expression of neurite growth and function, such as high levels of GAP-43, synapsin I and synaptophysin even after 35 days of incubation. In addition, survival of cerebellar granule neurons on GaN was improved by the analysis of lactate dehydrogenase (LDH) release from damaged cells. These results indicated that neuronal connections were formed on GaN by a gradual process from Akt activation and cell aggregation to develop neurite growth, fasciculation and function. Therefore, GaN offers a good model system to identify a well-characterized pattern of neuronal behavior for a long-term culture period, consistent with the development of a neurochip requiring the integration of biological system and semiconductor material.

  11. Ectopic bone regeneration by human bone marrow mononucleated cells, undifferentiated and osteogenically differentiated bone marrow mesenchymal stem cells in beta-tricalcium phosphate scaffolds.

    PubMed

    Ye, Xinhai; Yin, Xiaofan; Yang, Dawei; Tan, Jian; Liu, Guangpeng

    2012-07-01

    case at 8 weeks. Overall, this study suggests that ectopic osteogenesis of cell/scaffold composites is more dependent on the in vitro expansion condition, and osteo-differentiated BMSCs hold the highest potential concerning in vivo bone regeneration.

  12. Ectopic Hidradenoma Papilliferum

    PubMed Central

    Rosmaninho, Aristóteles David Neiva; de Almeida, Maria Teresa Duarte Pinto; Costa, Vírgilio; Sanches, Maria Madalena Vasconcelos; Lopes, Carlos; Selores Gomes Meirinhos, Maria Manuela

    2010-01-01

    Hidradenoma papilliferum is a rare tumor that occurs almost exclusively in females on the anogenital area. Rare cases of ectopic (nongenital) hidradenoma papilliferum have been described. The lesions usually present as an asymptomatic slow-growing, red, firm, mobile, well-delimitated nodule that grows for a long time before resection. We describe a case of an 26-year-old man that presented with an enlarging nodule on his right eyelid. The histological findings revealed a hidradenoma papilliferum. So far, among the very few reports of ectopic hidradenoma papilliferum, only a very small number were localized to the eyelid. PMID:21197082

  13. Scar Ectopic Pregnancy.

    PubMed

    Patel, Madhuri Arvind

    2015-12-01

    Scar ectopic pregnancy is the rarest form of ectopic pregnancy and has been increasingly diagnosed all over the world. This is a life-threatening form of abnormal implantation of embryo within the myometrium and fibrous tissues in a previous scar on the uterus, especially following caesarean section. With the increasing rate of caesarean section, there is a substantial increase in this condition with better understanding of this disease. The early and accurate diagnosis with timely management can prevent pregnancy complications such as haemorrhage, uterine rupture and can preserve fertility.

  14. PTBP1 is required for glucose-stimulated cap-independent translation of insulin granule proteins and Coxsackieviruses in beta cells

    PubMed Central

    Knoch, Klaus-Peter; Nath-Sain, Suchita; Petzold, Antje; Schneider, Hendryk; Beck, Mike; Wegbrod, Carolin; Sönmez, Anke; Münster, Carla; Friedrich, Anne; Roivainen, Merja; Solimena, Michele

    2014-01-01

    Glucose and GLP-1 stimulate not only insulin secretion, but also the post-transcriptional induction of insulin granule biogenesis. This process involves the nucleocytoplasmic translocation of the RNA binding protein PTBP1. Binding of PTBP1 to the 3′-UTRs of mRNAs for insulin and other cargoes of beta cell granules increases their stability. Here we show that glucose enhances also the binding of PTBP1 to the 5′-UTRs of these transcripts, which display IRES activity, and their translation exclusively in a cap-independent fashion. Accordingly, glucose-induced biosynthesis of granule cargoes was unaffected by pharmacological, genetic or Coxsackievirus-mediated inhibition of cap-dependent translation. Infection with Coxsackieviruses, which also depend on PTBP1 for their own cap-independent translation, reduced instead granule stores and insulin release. These findings provide insight into the mechanism for glucose-induction of insulin granule production and on how Coxsackieviruses, which have been implicated in the pathogenesis of type 1 diabetes, can foster beta cell failure. PMID:25061557

  15. Mutant human FUS Is ubiquitously mislocalized and generates persistent stress granules in primary cultured transgenic zebrafish cells.

    PubMed

    Acosta, Jamie Rae; Goldsbury, Claire; Winnick, Claire; Badrock, Andrew P; Fraser, Stuart T; Laird, Angela S; Hall, Thomas E; Don, Emily K; Fifita, Jennifer A; Blair, Ian P; Nicholson, Garth A; Cole, Nicholas J

    2014-01-01

    FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS), a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C) human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG) inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons.

  16. Prenatal alcohol exposure alters synaptic activity of adult hippocampal dentate granule cells under conditions of enriched environment.

    PubMed

    Kajimoto, Kenta; Valenzuela, C Fernando; Allan, Andrea M; Ge, Shaoyu; Gu, Yan; Cunningham, Lee Anna

    2016-08-01

    Prenatal alcohol exposure (PAE) results in fetal alcohol spectrum disorder (FASD), which is characterized by a wide range of cognitive and behavioral deficits that may be linked to impaired hippocampal function and adult neurogenesis. Preclinical studies in mouse models of FASD indicate that PAE markedly attenuates enrichment-mediated increases in the number of adult-generated hippocampal dentate granule cells (aDGCs), but whether synaptic activity is also affected has not been studied. Here, we utilized retroviral birth-dating coupled with whole cell patch electrophysiological recordings to assess the effects of PAE on enrichment-mediated changes in excitatory and inhibitory synaptic activity as a function of DGC age. We found that exposure to an enriched environment (EE) had no effect on baseline synaptic activity of 4- or 8-week-old aDGCs from control mice, but significantly enhanced the excitatory/inhibitory ratio of synaptic activity in 8-week-old aDGCs from PAE mice. In contrast, exposure to EE significantly enhanced the excitatory/inhibitory ratio of synaptic activity in older pre-existing DGCs situated in the outer dentate granule cell layer (i.e., those generated during embryonic development; dDGCs) in control mice, an effect that was blunted in PAE mice. These findings indicate distinct electrophysiological responses of hippocampal DGCs to behavioral challenge based on cellular ontogenetic age, and suggest that PAE disrupts EE-mediated changes in overall hippocampal network activity. These findings may have implications for future therapeutic targeting of hippocampal dentate circuitry in clinical FASD. © 2016 Wiley Periodicals, Inc. PMID:27009742

  17. Radiation-induced alterations in synaptic neurotransmission of dentate granule cells depend on the dose and species of charged particles.

    PubMed

    Marty, V N; Vlkolinsky, R; Minassian, N; Cohen, T; Nelson, G A; Spigelman, I

    2014-12-01

    The evaluation of potential health risks associated with neuronal exposure to space radiation is critical for future long duration space travel. The purpose of this study was to evaluate and compare the effects of low-dose proton and high-energy charged particle (HZE) radiation on electrophysiological parameters of the granule cells in the dentate gyrus (DG) of the hippocampus and its associated functional consequences. We examined excitatory and inhibitory neurotransmission in DG granule cells (DGCs) in dorsal hippocampal slices from male C57BL/6 mice at 3 months after whole body irradiation with accelerated proton, silicon or iron particles. Multielectrode arrays were used to investigate evoked field synaptic potentials, an extracellular measurement of synaptic excitability in the perforant path to DG synaptic pathway. Whole-cell patch clamp recordings were used to measure miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) in DGCs. Exposure to proton radiation increased synaptic excitability and produced dose-dependent decreases in amplitude and charge transfer of mIPSCs, without affecting the expression of γ-aminobutyric acid type A receptor α2, β3 and γ2 subunits determined by Western blotting. Exposure to silicon radiation had no significant effects on synaptic excitability, mEPSCs or mIPSCs of DGCs. Exposure to iron radiation had no effect on synaptic excitability and mIPSCs, but significantly increased mEPSC frequency at 1 Gy, without changes in mEPSC kinetics, suggesting a presynaptic mechanism. Overall, the data suggest that proton and HZE exposure results in radiation dose- and species-dependent long-lasting alterations in synaptic neurotransmission, which could cause radiation-induced impairment of hippocampal-dependent cognitive functions.

  18. Recombinant Escherichia coli produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein

    PubMed Central

    Bäckström, B Thomas; Brockelbank, Jane A; Rehm, Bernd HA

    2007-01-01

    Background Fluorescence activated cell sorting (FACS) is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. Beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. As the individual suspension bead passes through the sensing region of the FACS machine, fluorescent signals are acquired and analysed. Currently, antigens are tediously purified and chemically cross-linked to preformed beads. Purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. As an alternative, we genetically engineered Escherichia coli to produce biopolyester (polyhdroxyalkanoate=PHA) granules displaying diagnostically relevant antigens in their native conformation and suitable for FACS analysis. Results Hybrid genes were constructed, which encode either the mouse interleukin-2 (IL2) or the myelin oligodendrocyte glycoprotein (MOG) fused via an enterokinase site providing linker region to the C terminus of the PHA granule associated protein PhaP, respectively. The hybrid genes were expressed in PHA-accumulating recombinant E. coli. MOG and IL2 fusion proteins were abundantly attached to PHA granules and were identified by MALDI-TOF/MS analysis and N terminal sequencing. A more abundant second fusion protein of either MOG or IL2 resulted from an additional N terminal fusion, which did surprisingly not interfere with attachment to PHA granule. PHA granules displaying either IL2 or MOG were used for FACS using monoclonal anti-IL2 or anti-MOG antibodies conjugated to a fluorescent dye. FACS analysis showed significant and specific binding of respective antibodies. Enterokinase treatment of IL2 displaying PHA granules enabled removal of IL2 as monitored by FACS analysis. Mice were immunized with either MOG or OVA (ovalbumin) and the respective sera were

  19. Effects of spaced learning in the water maze on development of dentate granule cells generated in adult mice.

    PubMed

    Trinchero, Mariela F; Koehl, Muriel; Bechakra, Malik; Delage, Pauline; Charrier, Vanessa; Grosjean, Noelle; Ladeveze, Elodie; Schinder, Alejandro F; Abrous, D Nora

    2015-11-01

    New dentate granule cells (GCs) are generated in the hippocampus throughout life. These adult-born neurons are required for spatial learning in the Morris water maze (MWM). In rats, spatial learning shapes the network by regulating their number and dendritic development. Here, we explored whether such modulatory effects exist in mice. New GCs were tagged using thymidine analogs or a GFP-expressing retrovirus. Animals were exposed to a reference memory protocol for 10-14 days (spaced training) at different times after newborn cells labeling. Cell proliferation, cell survival, cell death, neuronal phenotype, and dendritic and spine development were examined using immunohistochemistry. Surprisingly, spatial learning did not modify any of the parameters under scrutiny including cell number and dendritic morphology. These results suggest that although new GCs are required in mice for spatial learning in the MWM, they are, at least for the developmental intervals analyzed here, refractory to behavioral stimuli generated in the course of learning in the MWM. PMID:25740272

  20. Delayed coupling to feedback inhibition during a critical period for the integration of adult-born granule cells.

    PubMed

    Temprana, Silvio G; Mongiat, Lucas A; Yang, Sung M; Trinchero, Mariela F; Alvarez, Diego D; Kropff, Emilio; Giacomini, Damiana; Beltramone, Natalia; Lanuza, Guillermo M; Schinder, Alejandro F

    2015-01-01

    Developing granule cells (GCs) of the adult dentate gyrus undergo a critical period of enhanced activity and synaptic plasticity before becoming mature. The impact of developing GCs on the activity of preexisting dentate circuits remains unknown. Here we combine optogenetics, acute slice electrophysiology, and in vivo chemogenetics to activate GCs at different stages of maturation to study the recruitment of local target networks. We show that immature (4-week-old) GCs can efficiently drive distal CA3 targets but poorly activate proximal interneurons responsible for feedback inhibition (FBI). As new GCs transition toward maturity, they reliably recruit GABAergic feedback loops that restrict spiking of neighbor GCs, a mechanism that would promote sparse coding. Such inhibitory loop impinges only weakly in new cohorts of young GCs. A computational model reveals that the delayed coupling of new GCs to FBI could be crucial to achieve a fine-grain representation of novel inputs in the dentate gyrus. PMID:25533485

  1. Restricted diffusion of calretinin in cerebellar granule cell dendrites implies Ca2+-dependent interactions via its EF-hand 5 domain

    PubMed Central

    Arendt, Oliver; Schwaller, Beat; Brown, Edward B; Eilers, Jens; Schmidt, Hartmut

    2013-01-01

    Ca2+-binding proteins (CaBPs) are important regulators of neuronal Ca2+ signalling, acting either as buffers that shape Ca2+ transients and Ca2+ diffusion and/or as Ca2+ sensors. The diffusional mobility represents a crucial functional parameter of CaBPs, describing their range-of-action and possible interactions with binding partners. Calretinin (CR) is a CaBP widely expressed in the nervous system with strong expression in cerebellar granule cells. It is involved in regulating excitability and synaptic transmission of granule cells, and its absence leads to impaired motor control. We quantified the diffusional mobility of dye-labelled CR in mouse granule cells using two-photon fluorescence recovery after photobleaching. We found that movement of macromolecules in granule cell dendrites was not well described by free Brownian diffusion and that CR diffused unexpectedly slow compared to fluorescein dextrans of comparable size. During bursts of action potentials, which were associated with dendritic Ca2+ transients, the mobility of CR was further reduced. Diffusion was significantly accelerated by a peptide embracing EF-hand 5 of CR. Our results suggest long-lasting, Ca2+-dependent interactions of CR with large and/or immobile binding partners. These interactions render CR a poorly mobile Ca2+ buffer and point towards a Ca2+ sensor function of CR. PMID:23732647

  2. Control of Granule Mobility and Exocytosis by Ca2+-Dependent Formation of F-Actin in Pancreatic Duct Epithelial Cells

    PubMed Central

    Jung, Seung-Ryoung; Kim, Mean-Hwan; Hille, Bertil; Koh, Duk-Su

    2009-01-01

    Elevation of intracellular Ca2+ concentration ([Ca2+]i) triggers exocytosis of secretory granules in pancreatic duct epithelia. In this study, we find that the signal also controls granule movement. Motions of fluorescently labeled granules stopped abruptly after a [Ca2+]i increase, kinetically coincident with formation of filamentous actin (F-actin) in the whole cytoplasm. At high resolution, the new F-actin meshwork was so dense that cellular structures of granule size appeared physically trapped in it. Depolymerization of F-actin with latrunculin B blocked both the F-actin formation and the arrest of granules. Interestingly, when monitored with total internal reflection fluorescence microscopy, the immobilized granules still moved slowly and concertedly toward the plasma membrane. This group translocation was abolished by blockers of myosin. Exocytosis measured by microamperometry suggested that formation of a dense F-actin meshwork inhibited exocytosis at small Ca2+ rises <1 μm. Larger [Ca2+]i rises increased exocytosis because of the co-ordinate translocation of granules and fusion to the membrane. We propose that the Ca2+-dependent freezing of granules filters out weak inputs but allows exocytosis under stronger inputs by controlling granule movements. PMID:19192247

  3. Maturation time of new granule cells in the dentate gyrus of adult macaque monkeys exceeds six months.

    PubMed

    Kohler, Shawn J; Williams, Nancy I; Stanton, Gregory B; Cameron, Judy L; Greenough, William T

    2011-06-21

    We studied two groups of adult macaque monkeys to determine the time course of adult neurogenesis in the dentate gyrus of the hippocampus. In the first group, six adult monkeys (Macaca mulatta) received a single injection of the thymidine analog BrdU (75 mg/kg), which is incorporated into replicating DNA and serves as a marker for new cell birth. Brain tissue was collected 48 h, 2 wk, and 6 wk after BrdU injection to examine the initial stages of neurogenesis. Because mature neurons were not evident at 6 wk, we examined tissue collected over a longer time course in a second study. In this study, eight monkeys (Macaca fascicularis) who were subjects in a separate exercise study received 10 weekly injections of BrdU (75 mg/kg), and brain tissue was collected at 16 and 28 wk from the first injection. Based on the timing of expression of neuronal cell markers (βIII-tubulin, doublecortin, NeuN), the extent of dendritic arborization, and acquisition of mature cell body morphology, we show that granule cell maturation in the dentate gyrus of a nonhuman primate is protracted over a minimum of a 6-mo time period, more than 6 times longer than in rodents. The lengthened time course for new cell maturation in nonhuman primates may be appropriate for preservation of neural plasticity over their longer life span and is relevant to our understanding of antidepressant and other therapies that have been linked to neurogenesis in humans.

  4. Casein Kinase 1δ Is an APC/CCdh1 Substrate that Regulates Cerebellar Granule Cell Neurogenesis

    PubMed Central

    Penas, Clara; Govek, Eve-Ellen; Fang, Yin; Ramachandran, Vimal; Daniel, Mark; Wang, Weiping; Maloof, Marie E.; Rahaim, Ronald J.; Bibian, Mathieu; Kawauchi, Daisuke; Finkelstein, David; Han, Jeng-Liang; Long, Jun; Li, Bin; Robbins, David J.; Malumbres, Marcos; Roussel, Martine F.; Roush, William R.; Hatten, Mary E.; Ayad, Nagi G.

    2015-01-01

    SUMMARY Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of central nervous system progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/CCdh1) ubiquitin ligase, and conditional deletion of the APC/CCdh1 activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/CCdh1 also downregulates CK1δ during cell cycle exit. Therefore, we conclude that APC/CCdh1 controls CK1δ levels to balance proliferation and cell cycle exit in the developing central nervous system. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a new therapeutic target. PMID:25843713

  5. The cytokine interleukin-1beta reduces the docking and fusion of insulin granules in pancreatic beta-cells, preferentially decreasing the first phase of exocytosis.

    PubMed

    Ohara-Imaizumi, Mica; Cardozo, Alessandra K; Kikuta, Toshiteru; Eizirik, Decio L; Nagamatsu, Shinya

    2004-10-01

    The prediabetic period in type I diabetes mellitus is characterized by the loss of first phase insulin release. This might be due to islet infiltration mediated by mononuclear cells and local release of cytokines, but the mechanisms involved are unknown. To determine the role of cytokines in insulin exocytosis, we have presently utilized total internal reflection fluorescence microscopy (TIRFM) to image and analyze the dynamic motion of single insulin secretory granules near the plasma membrane in live beta-cells exposed for 24 h to interleukin (IL)-1beta or interferon (IFN)-gamma. Immunohistochemistry observed via TIRFM showed that the number of docked insulin granules was decreased by 60% in beta-cells treated with IL-1beta, while it was not affected by exposure to IFN-gamma. This effect of IL-1beta was paralleled by a 50% reduction in the mRNA and the number of clusters of SNAP-25 in the plasma membrane. TIRF images of single insulin granule motion during a 15-min stimulation by 22 mm glucose in IL-1beta-treated beta-cells showed a marked reduction in the fusion events from previously docked granules during the first phase insulin release. Fusion from newcomers, however, was well preserved during the second phase of insulin release of IL-1beta-treated beta-cells. The present observations indicate that IL-1beta, but not IFN-gamma, has a preferential inhibitory effect on the first phase of glucose-induced insulin release, mostly via an action on previously docked granules. This suggests that beta-cell exposure to immune mediators during the course of insulitis might be responsible for the loss of first phase insulin release.

  6. Periodontal ligament versus bone marrow mesenchymal stem cells in combination with Bio-Oss scaffolds for ectopic and in situ bone formation: A comparative study in the rat.

    PubMed

    Yu, Bo-Han; Zhou, Qian; Wang, Zuo-Lin

    2014-01-30

    The aim of this study was to compare the osteogenic effects of periodontal ligament stem cells (PDLSCs) versus bone marrow mesenchymal stem cells (BMMSCs) in combination with Bio-Oss scaffolds on subcutaneous and critical-size defects in the immunodeficient rat calvarium. PDLSCs and BMMSCs were obtained from the same canine donor. Twenty-four rats were randomly assigned to one of four experimental groups (n = 6 each): group A (no-graft negative control), group B (Bio-Oss positive control), group C (BMMSC/Bio-Oss test group), and group D (PDLSC/Bio-Oss test group). Eight weeks post-transplantation, ectopic and in situ bone regeneration was evaluated by micro-computed tomography (µ-CT), histology, histomorphometry, and immunohistochemistry. The stem cell/Bio-Oss constructs were significantly superior to the controls in terms of their ability to promote osteogenesis (p < 0.01), while the PDLSC/Bio-Oss construct tended to be superior to the BMMSC/Bio-Oss construct. Thus, engineered stem cell/Bio-Oss complexes can successfully reconstruct critical-size defects in rats, and PDLSCs and BMMSCs are both suitable as seed cells.

  7. Expression Analysis of SOX14 during Retinoic Acid Induced Neural Differentiation of Embryonal Carcinoma Cells and Assessment of the Effect of Its Ectopic Expression on SOXB Members in HeLa Cells

    PubMed Central

    Popovic, Jelena; Stanisavljevic, Danijela; Schwirtlich, Marija; Klajn, Andrijana; Marjanovic, Jelena; Stevanovic, Milena

    2014-01-01

    SOX14 is a member of the SOXB2 subgroup of transcription factors implicated in neural development. Although the first SOX14 gene in vertebrates was cloned and characterized more than a decade ago and its expression profile during development was revealed in various animal model systems, the role of this gene during neural development is largely unknown. In the present study we analyzed the expression of SOX14 in human NT2/D1 and mouse P19 pluripotent embryonal carcinoma cells. We demonstrated that it is expressed in both cell lines and upregulated during retinoic acid induced neural differentiation. We showed that SOX14 was expressed in both neuronal and non-neuronal differentiated derivatives, as revealed by immunocytochemistry. Since it was previously proposed that increased SOXB2 proteins level interfere with the activity of SOXB1 counteracting partners, we compared expression patterns of SOXB members during retinoic acid induction of embryonal carcinoma cells. We revealed that upregulation of SOX14 expression is accompanied by alterations in the expression patterns of SOXB1 members. In order to analyze the potential cross-talk between them, we generated SOX14 expression construct. The ectopic expression of SOX14 was demonstrated at the mRNA level in NT2/D1, P19 and HeLa cells, while an increased level of SOX14 protein was detected in HeLa cells only. By transient transfection experiments in HeLa cells we showed for the first time that ectopic expression of SOX14 repressed SOX1 expression, whereas no significant effect on SOX2, SOX3 and SOX21 was observed. Data presented here provide an insight into SOX14 expression during in vitro neural differentiation of embryonal carcinoma cells and demonstrate the effect of its ectopic expression on protein levels of SOXB members in HeLa cells. Obtained results contribute to better understanding the role of one of the most conserved SOX proteins. PMID:24637840

  8. Pregnancy-associated ectopic decidua.

    PubMed

    Zaytsev, P; Taxy, J B

    1987-07-01

    Extrauterine formation of decidua of stromal cells has been well described, particularly in the cervix and ovary. Sporadic reports have documented decidua formation of peritoneal surfaces and lymph nodes as well as ovarian ectopic decidua in pregnant patients. The apparent hormonal mechanism of this phenomenon suggests a relationship to endometriosis. Between 1983 and 1986, tissue from 10 pregnant patients (acquired from nine at cesarean section and from one at appendectomy) demonstrated submesothelial decidua formation in the form of microscopic nodules and diffuse cell arrangements in an edematous stroma. Some cases showed mild chronic inflammation. An intracytoplasmic lipofusin-type pigment was observed in one case. Four cases had diffuse omental involvement: two with small amounts of free peritoneal blood and two with peritoneal adhesions. Three cases were associated with paratubal cysts: two of these occurred in the uterine and appendiceal serosa, respectively, and one case showed involvement of a retroperitoneal lymph node associated with a pheochromocytoma. No specific gross observations were noted at surgery. There was no prior or subsequent evidence of endometriosis. A review of 958 elective tubal ligations performed between 1983 and 1985 demonstrated 52 examples (5.5%) of serosal decidua formation. Ectopic stromal decidua formation in pregnancy is: (a) a physiologic phenomenon related to the possible specialized sensitivity of the superficial coelomic stroma to progesterone; and (b) diffusely distributed in the peritoneum although it is a clinicopathologic process distinct from endometriosis.

  9. Influence of platelet-rich plasma on osteogenic differentiation of mesenchymal stem cells and ectopic bone formation in calcium phosphate ceramics.

    PubMed

    Kasten, Philip; Vogel, Julia; Luginbühl, Reto; Niemeyer, Philipp; Weiss, Stefan; Schneider, Sven; Kramer, Martin; Leo, Albrecht; Richter, Wiltrud

    2006-01-01

    Platelet-rich plasma (PRP) contains a mixture of growth factors that play an important role in wound and fracture healing. While PRP enhanced bone formation by autogenous cancellous bone grafts, its influence in combination with different bone substitutes remained unknown. This study evaluated the effect of PRP on osteogenic differentiation and ectopic bone formation of human mesenchymal stem cells (MSC) in distinct resorbable calcium phosphate ceramics. Calcium-deficient hydroxyapatite (CDHA) blocks with a large specific surface area (48 m2/g) and beta-tricalcium phosphate (beta-TCP) with a low specific surface area (<0.5 m2/g) were loaded with 2 x 10(5) bone marrow-derived MSC. Half of the specimens were treated with 5-fold concentrated PRP. Biocomposites were implanted subcutaneously into SCID mice or kept under osteogenic culture conditions for 2 weeks before implantation. The addition of PRP increased the specific alkaline phosphatase (ALP) activity (p = 0.012) in undifferentiated MSC/CDHA composites but not in MSC/beta-TCP composites. Osteogenic preinduction was ineffective for CDHA and reduced ALP activity of beta-TCP composites significantly at explantation. Ectopic bone formation was stronger in MSC/CDHA (7/32) compared to MSC/beta-TCP (2/30) composites, but no influence of PRP was evident. In conclusion, the effect of PRP depended on the type of ceramic and the differentiation status of the MSC, and enhanced ALP activity of MSC on the high surface scaffold CDHA only, but PRP did not improve osteogenesis in our setting.

  10. HAP1 helps to regulate actin-based transport of insulin-containing granules in pancreatic β cells.

    PubMed

    Wang, Zhiyong; Peng, Ting; Wu, Hongnian; He, Jun; Li, He

    2015-07-01

    Huntingtin-associated protein 1 (HAP1) is enriched in neurons and binds to polyglutamine-expanded huntingtin. It consists of two alternatively spliced isoforms, HAP1A and HAP1B, which differ only in their short C-terminal sequences. Both HAP1A and HAP1B have been also detected in pancreatic β cells, where the loss of HAP1 impairs glucose-stimulated insulin secretion. Here, we use time-lapse laser scanning confocal microscopy to provide direct evidence that HAP1A, but not HAP1B, co-localizes and co-migrates with insulin-containing vesicles and actin-based myosin Va motor protein in the INS-1 pancreatic β cell line. Knocking down HAP1 expression using small interfering RNA significantly inhibited actin-based transport of insulin vesicles following glucose stimulation. Co-immunoprecipitation experiments demonstrated interaction between HAP1A, myosin Va, and phogrin, a transmembrane protein in insulin-containing vesicles. Stimulating INS-1 cells with glucose increased the association of HAP1A with myosin Va, while silencing HAP1 expression reduced the association of myosin Va with phogrin after glucose stimulation, without affecting levels of myosin Va or actin. Our results provide real-time evidence in living cells that HAP1 may help regulate transport of insulin-containing secretory granules along cortical actin filaments. This also raises the possibility that HAP1 may play an important role in actin-based secretory vesicle trafficking in neurons. PMID:25744490

  11. Cooperative antiproliferative effect of coordinated ectopic expression of DLC1 tumor suppressor protein and silencing of MYC oncogene expression in liver cancer cells: Therapeutic implications

    PubMed Central

    Yang, Xuyu; Zhou, Xiaoling; Tone, Paul; Durkin, Marian E.; Popescu, Nicholas C.

    2016-01-01

    Human hepatocellular carcinoma (HCC) is one of the most common types of cancer and has a very poor prognosis; thus, the development of effective therapies for the treatment of advanced HCC is of high clinical priority. In the present study, the anti-oncogenic effect of combined knockdown of c-Myc expression and ectopic restoration of deleted in liver cancer 1 (DLC1) expression was investigated in human liver cancer cells. Expression of c-Myc in human HCC cells was knocked down by stable transfection with a Myc-specific short hairpin (sh) RNA vector. DLC1 expression in Huh7 cells was restored by adenovirus transduction, and the effects of DLC1 expression and c-Myc knockdown on Ras homolog gene family, member A (RhoA) levels, cell proliferation, soft agar colony formation and cell invasion were measured. Downregulation of c-Myc or re-expression of DLC1 led to a marked reduction in RhoA levels, which was associated with decreases in cell proliferation, soft agar colony formation and invasiveness; this inhibitory effect was augmented with a combination of DLC1 transduction and c-Myc suppression. To determine whether liver cell-specific delivery of DLC1 was able to enhance the inhibitory effect of c-Myc knockdown on tumor growth in vivo, DLC1 vector DNA complexed with galactosylated polyethylene glycol-linear polyethyleneimine was administered by tail vein injection to mice bearing subcutaneous xenografts of Huh7 cells transfected with shMyc or control shRNA. A cooperative inhibitory effect of DLC1 expression and c-Myc knockdown on the growth of Huh7-derived tumors was observed, suggesting that targeted liver cell delivery of DLC1 and c-Myc shRNA may serve as a possible gene therapy modality for the treatment of human HCC. PMID:27446476

  12. Isolation of neuromelanin granules.

    PubMed

    Tribl, Florian

    2008-12-01

    Neuromelanin granules are pigmented organelles in the human midbrain that give name to a brain area, substantia nigra pars compacta, which macroscopically appears as a dark brown region in the midbrain due to the insoluble pigment neuromelanin. The substantia nigra pars compacta massively degenerates in Parkinson's disease and gives rise to severely disabling movement symptoms. It has been suggested that neuromelanin granules play an important role in the neurodegenerative events in Parkinson's disease: redox-active iron is bound to neuromelanin and thereby retained within this compartment, but in Parkinson's disease it is thought to be increasingly released into the cytosol, promoting oxidative stress. This unit includes a methodological workflow for the isolation of neuromelanin granules from the human midbrain. This top-down approach (describes an approach that reduces the complexity of the sample stepwise from the level of tissue to cell, and from cell to organelle) encompasses the organelle isolation by sequential density gradient centrifugation and the assessment of the isolation efficacy by western blotting. PMID:19085988

  13. Cholesteatoma in ectopic kidney.

    PubMed

    Karabulut, Yasemin Yuyucu; Tek, Mesut; Eti, Neslihan; Akbay, Erdem

    2016-09-01

    Cholesteatoma in the urinary system is a rarely seen benign condition. Rosina firstly defined this condition in the year 1953. Histopathologically it is characterized with keratinization, and squamous metaplasia of urothelial epithelium associated with desquamation of keratinized layers. Flank pain is the most common symptom that is caused by elimination of keratinous material. In our case we will discuss cholesteatoma developed in an ectopic kidney which has not been described in the literature before. PMID:27635299

  14. Cholesteatoma in ectopic kidney

    PubMed Central

    Karabulut, Yasemin Yuyucu; Tek, Mesut; Eti, Neslihan; Akbay, Erdem

    2016-01-01

    Cholesteatoma in the urinary system is a rarely seen benign condition. Rosina firstly defined this condition in the year 1953. Histopathologically it is characterized with keratinization, and squamous metaplasia of urothelial epithelium associated with desquamation of keratinized layers. Flank pain is the most common symptom that is caused by elimination of keratinous material. In our case we will discuss cholesteatoma developed in an ectopic kidney which has not been described in the literature before. PMID:27635299

  15. Blue- and green-absorbing visual pigments of Drosophila: ectopic expression and physiological characterization of the R8 photoreceptor cell-specific Rh5 and Rh6 rhodopsins.

    PubMed

    Salcedo, E; Huber, A; Henrich, S; Chadwell, L V; Chou, W H; Paulsen, R; Britt, S G

    1999-12-15

    Color discrimination requires the input of different photoreceptor cells that are sensitive to different wavelengths of light. The Drosophila visual system contains multiple classes of photoreceptor cells that differ in anatomical location, synaptic connections, and spectral sensitivity. The Rh5 and Rh6 opsins are expressed in nonoverlapping sets of R8 cells and are the only Drosophila visual pigments that remain uncharacterized. In this study, we ectopically expressed Rh5 and Rh6 in the major class of photoreceptor cells (R1-R6) and show them to be biologically active in their new environment. The expression of either Rh5 or Rh6 in "blind" ninaE(17) mutant flies, which lack the gene encoding the visual pigment of the R1-R6 cells, fully rescues the light response. Electrophysiological analysis showed that the maximal spectral sensitivity of the R1-R6 cells is shifted to 437 or 508 nm when Rh5 or Rh6, respectively, is expressed in these cells. These spectral sensitivities are in excellent agreement with intracellular recordings of the R8p and R8y cells measured in Calliphora and Musca. Spectrophotometric analyses of Rh5 and Rh6 in vivo by microspectrophotometry, and of detergent-extracted pigments in vitro, showed that Rh5 is reversibly photoconverted to a stable metarhodopsin (lambda(max) = 494 nm), whereas Rh6 appears to be photoconverted to a metarhodopsin (lambda(max) = 468 nm) that is less thermally stable. Phylogenetically, Rh5 belongs to a group of short-wavelength-absorbing invertebrate visual pigments, whereas Rh6 is related to a group of long-wavelength-absorbing pigments and is the first member of this class to be functionally characterized. PMID:10594055

  16. ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons.

    PubMed

    Lenzi, Jessica; De Santis, Riccardo; de Turris, Valeria; Morlando, Mariangela; Laneve, Pietro; Calvo, Andrea; Caliendo, Virginia; Chiò, Adriano; Rosa, Alessandro; Bozzoni, Irene

    2015-07-01

    Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUS(R514S) and FUS(R521C) patient fibroblasts, whereas in the case of the severe FUS(P525L) mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis.

  17. Molecular characterization of mouse lens epithelial cell lines and their suitability to study RNA granules and cataract associated genes.

    PubMed

    Terrell, Anne M; Anand, Deepti; Smith, Sylvie F; Dang, Christine A; Waters, Stephanie M; Pathania, Mallika; Beebe, David C; Lachke, Salil A

    2015-02-01

    The discovery of cytosolic RNA granule (RG) component proteins associated with human cataract has initiated investigations on post-transcriptional mechanisms of gene expression control in the lens. Application of established mouse lens epithelial cell lines (LECs) can provide rapid insights on RG function in lens cells, especially because mouse mutants in several RG components are not available. However, although these LECs represent potential reagents for such analyses, they are uncharacterized for lens gene expression or RG formation. Therefore, a detailed molecular and cellular characterization of three permanent mouse LECs 17EM15, 21EM15 and αTN4 is performed in this study. Comparative analysis between microarray gene expression datasets on LEC 21EM15 and iSyTE lens tissue demonstrates that 30% of top 200 iSyTE identified lens-enriched genes are expressed in these cells. Majority of these candidates are independently validated to either have lens expression, function or linkage to cataract. Moreover, analysis of microarray data with genes described in Cat-Map, an online database of cataract associated genes and loci, demonstrates that 131 genes linked to cataract loci are expressed in 21EM15 cells. Furthermore, gene expression in LECs is compared to isolated lens epithelium or fiber cells by qRT-PCR and by comparative analyses with publically available epithelium or fiber-specific microarray and RNA-seq (sequencing) datasets. Expression of select candidate genes was validated by regular and real-time quantitative RT-PCR. Expression of lens epithelium-enriched genes Foxe3, Pax6, Anxa4 and Mcm4 is up-regulated in LEC lines, compared to isolated lens fiber cells. Moreover, similar to isolated lens epithelium, all three LECs exhibit down-regulation of fiber cell-expressed genes Crybb1, Mip and Prox1 when compared to fiber cells. These data indicate that the LEC lines exhibit greater similarity to lens epithelium than to fiber cells. Compared to non-lens cell line

  18. Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis.

    PubMed

    Wang, Qishan; Bag, Jnanankur

    2006-02-17

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including alpha-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of the MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis.

  19. Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis

    SciTech Connect

    Wang Qishan; Bag, Jnanankur . E-mail: jbag@uoguelph.ca

    2006-02-17

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including {alpha}-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of the MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis.

  20. Interactive effects involving different classes of excitatory amino acid receptors and the survival of cerebellar granule cells in culture.

    PubMed

    Balázs, R; Hack, N; Jørgensen, O S

    1990-01-01

    Differentiating granule cells develop survival requirements in culture which can be met by treatment with high K+ or N-methyl-D-aspartate (NMDA) and, according to our recent findings, also with low concentrations of kainic acid (KA, 50 microM). We have now attempted to elucidate the mechanism(s) underlying the trophic effect of KA. KA rescue of cells was completely suppressed by blockers of voltage-sensitive calcium channels, such as nifedipine in low concentrations (5 x 10(-7) M), indicating that the promotion of cell survival is mediated through the activation of these channels by membrane depolarization. Thus the trophic influences of KA and NMDA share a common mechanism, increased Ca2+ influx (albeit through different routes), a conclusion that is supported by the observation that the effects of these agonists at concentrations causing maximal promotion of cell survival were not additive. Interactive effects involving different classes of excitatory amino acid receptors were revealed by the potentiation of the KA rescue of cells by the NMDA receptor antagonists, 2-amino 5-phosphonovalerate (APV) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate (MK-801), which on their own failed to promote, but rather reduced cell survival. The potentiation of the KA effect by the competitive NMDA antagonist APV was counteracted by the weak NMDA agonist, quinolinic acid. These observations suggest that KA alone has both trophic and toxic effects, the latter being mediated secondarily through an NMDA-like glutamate receptor, which is distinct from the conventional NMDA, KA and quisqualate preferring subtypes.

  1. Adverse effects of 2,4-dichlorophenoxyacetic acid on rat cerebellar granule cell cultures were attenuated by amphetamine.

    PubMed

    Bongiovanni, B; Ferri, A; Brusco, A; Rassetto, M; Lopez, L M; Evangelista de Duffard, A M; Duffard, R

    2011-05-01

    2,4-Dichlorophenoxyacetic acid (2,4-D), a worldwide-used herbicide, has been shown to produce a wide range of adverse effects in the health--from embryotoxicity and teratogenicity to neurotoxicity--of animals and humans. In this study, neuronal morphology and biochemical events in rat cerebellar granule cell (CGC) cultures have been analyzed to define some of the possible mechanisms involved in 2,4-D-induced cell death. For that purpose, amphetamine (AMPH) that has been shown to accelerate the recovery of several functions in animals with brain injury has been used as a pharmacologycal tool and was also investigated as a possible protecting agent. Addition of 2,4-D to CGC cultures produced a drastic decrease in cell viability, in association with an increased incidence of necrosis and apoptosis, and an increased level of reactive oxygen species, a decrease in glutathione content, and an abnormal activity of some enzymes with respect to the control group. The adverse effects of 2,4-D were partly attenuated in presence of AMPH. Some deleterious effects on several ultrastructural features of the cells, as well as the enhanced incidence of apoptosis, were partially preserved in AMPH-protected cultures as compared with those which were exposed to 2,4-D alone. The collected evidences (1) confirms the previously observed, deleterious effects of 2.4D on the same or a similar model; (2) suggests that the 2,4-D-induced apoptosis could have been mediated by or associated to an oxidative imbalance in the affected cells, and (3) shows some evidence of a protective effect of AMPH on 2,4-D-induced cell death, which could have been exerted through a reduction in the oxidative stress.

  2. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

    PubMed

    Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-03-10

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

  3. Corruption of the dentate gyrus by "dominant" granule cells: Implications for dentate gyrus function in health and disease.

    PubMed

    Scharfman, Helen E; Myers, Catherine E

    2016-03-01

    The dentate gyrus (DG) and area CA3 of the hippocampus are highly organized lamellar structures which have been implicated in specific cognitive functions such as pattern separation and pattern completion. Here we describe how the anatomical organization and physiology of the DG and CA3 are consistent with structures that perform pattern separation and completion. We then raise a new idea related to the complex circuitry of the DG and CA3 where CA3 pyramidal cell 'backprojections' play a potentially important role in the sparse firing of granule cells (GCs), considered important in pattern separation. We also propose that GC axons, the mossy fibers, already known for their highly specialized structure, have a dynamic function that imparts variance--'mossy fiber variance'--which is important to pattern separation and completion. Computational modeling is used to show that when a subset of GCs become 'dominant,' one consequence is loss of variance in the activity of mossy fiber axons and a reduction in pattern separation and completion in the model. Empirical data are then provided using an example of 'dominant' GCs--subsets of GCs that develop abnormally and have increased excitability. Notably, these abnormal GCs have been identified in animal models of disease where DG-dependent behaviors are impaired. Together these data provide insight into pattern separation and completion, and suggest that behavioral impairment could arise from dominance of a subset of GCs in the DG-CA3 network. PMID:26391451

  4. Corruption of the dentate gyrus by "dominant" granule cells: Implications for dentate gyrus function in health and disease.

    PubMed

    Scharfman, Helen E; Myers, Catherine E

    2016-03-01

    The dentate gyrus (DG) and area CA3 of the hippocampus are highly organized lamellar structures which have been implicated in specific cognitive functions such as pattern separation and pattern completion. Here we describe how the anatomical organization and physiology of the DG and CA3 are consistent with structures that perform pattern separation and completion. We then raise a new idea related to the complex circuitry of the DG and CA3 where CA3 pyramidal cell 'backprojections' play a potentially important role in the sparse firing of granule cells (GCs), considered important in pattern separation. We also propose that GC axons, the mossy fibers, already known for their highly specialized structure, have a dynamic function that imparts variance--'mossy fiber variance'--which is important to pattern separation and completion. Computational modeling is used to show that when a subset of GCs become 'dominant,' one consequence is loss of variance in the activity of mossy fiber axons and a reduction in pattern separation and completion in the model. Empirical data are then provided using an example of 'dominant' GCs--subsets of GCs that develop abnormally and have increased excitability. Notably, these abnormal GCs have been identified in animal models of disease where DG-dependent behaviors are impaired. Together these data provide insight into pattern separation and completion, and suggest that behavioral impairment could arise from dominance of a subset of GCs in the DG-CA3 network.

  5. Granule cell excitability regulates gamma and beta oscillations in a model of the olfactory bulb dendrodendritic microcircuit.

    PubMed

    Osinski, Bolesław L; Kay, Leslie M

    2016-08-01

    Odors evoke gamma (40-100 Hz) and beta (20-30 Hz) oscillations in the local field potential (LFP) of the mammalian olfactory bulb (OB). Gamma (and possibly beta) oscillations arise from interactions in the dendrodendritic microcircuit between excitatory mitral cells (MCs) and inhibitory granule cells (GCs). When cortical descending inputs to the OB are blocked, beta oscillations are extinguished whereas gamma oscillations become larger. Much of this centrifugal input targets inhibitory interneurons in the GC layer and regulates the excitability of GCs, which suggests a causal link between the emergence of beta oscillations and GC excitability. We investigate the effect that GC excitability has on network oscillations in a computational model of the MC-GC dendrodendritic network with Ca(2+)-dependent graded inhibition. Results from our model suggest that when GC excitability is low, the graded inhibitory current mediated by NMDA channels and voltage-dependent Ca(2+) channels (VDCCs) is also low, allowing MC populations to fire in the gamma frequency range. When GC excitability is increased, the activation of NMDA receptors and other VDCCs is also increased, allowing the slow decay time constants of these channels to sustain beta-frequency oscillations. Our model argues that Ca(2+) flow through VDCCs alone could sustain beta oscillations and that the switch between gamma and beta oscillations can be triggered by an increase in the excitability state of a subpopulation of GCs. PMID:27121582

  6. Different ataxin-3 amyloid aggregates induce intracellular Ca(2+) deregulation by different mechanisms in cerebellar granule cells.

    PubMed

    Pellistri, Francesca; Bucciantini, Monica; Invernizzi, Gaetano; Gatta, Elena; Penco, Amanda; Frana, Anna Maria; Nosi, Daniele; Relini, Annalisa; Regonesi, Maria Elena; Gliozzi, Alessandra; Tortora, Paolo; Robello, Mauro; Stefani, Massimo

    2013-12-01

    This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca(2+) levels and the abnormal Ca(2+) signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca(2+) responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca(2+) response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.

  7. Studies on cell migration, adenylate cyclase and membrane-coating granules in the buccal epithelium of the zinc-deficient rabbit, including the influence of isoproterenol.

    PubMed

    Chen, S Y

    1988-01-01

    Cell migration was slightly increased; cytochemical reaction deposits of adenylate cyclase and the area density of membrane-coating granules (MCG) were significantly increased. Upon isoproterenol stimulation, the MCG area density was significantly increased, whereas the cell migration rate was unchanged. Thus in zinc deficiency, there may be a simultaneous increase in the production and secretion of MCGs, in adenylate cyclase activity, and in cell migration. The non-significantly increased cell migration rate may not keep pace with the significantly increased cell-production rate, resulting in thickening of the epithelium.

  8. Ectopic miR-125a Expression Induces Long-Term Repopulating Stem Cell Capacity in Mouse and Human Hematopoietic Progenitors.

    PubMed

    Wojtowicz, Edyta E; Lechman, Eric R; Hermans, Karin G; Schoof, Erwin M; Wienholds, Erno; Isserlin, Ruth; van Veelen, Peter A; Broekhuis, Mathilde J C; Janssen, George M C; Trotman-Grant, Aaron; Dobson, Stephanie M; Krivdova, Gabriela; Elzinga, Jantje; Kennedy, James; Gan, Olga I; Sinha, Ankit; Ignatchenko, Vladimir; Kislinger, Thomas; Dethmers-Ausema, Bertien; Weersing, Ellen; Alemdehy, Mir Farshid; de Looper, Hans W J; Bader, Gary D; Ritsema, Martha; Erkeland, Stefan J; Bystrykh, Leonid V; Dick, John E; de Haan, Gerald

    2016-09-01

    Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols. PMID:27424784

  9. GGPPS-mediated Rab27A geranylgeranylation regulates β cell dysfunction during type 2 diabetes development by affecting insulin granule docked pool formation.

    PubMed

    Jiang, Shan; Shen, Di; Jia, Wen-Jun; Han, Xiao; Shen, Ning; Tao, Weiwei; Gao, Xiang; Xue, Bin; Li, Chao-Jun

    2016-01-01

    Loss of first-phase insulin secretion associated with β cell dysfunction is an independent predictor of type 2 diabetes mellitus (T2DM) onset. Here we found that a critical enzyme involved in protein prenylation, geranylgeranyl pyrophosphate synthase (GGPPS), is required to maintain first-phase insulin secretion. GGPPS shows a biphasic expression pattern in islets of db/db mice during the progression of T2DM: GGPPS is increased during the insulin compensatory period, followed by a decrease during β cell dysfunction. Ggpps deletion in β cells results in typical T2DM β cell dysfunction, with blunted glucose-stimulated insulin secretion and consequent insulin secretion insufficiency. However, the number and size of islets and insulin biosynthesis are unaltered. Transmission electron microscopy shows a reduced number of insulin granules adjacent to the cellular membrane, suggesting a defect in docked granule pool formation, while the reserve pool is unaffected. Ggpps ablation depletes GGPP and impairs Rab27A geranylgeranylation, which is responsible for the docked pool deficiency in Ggpps-null mice. Moreover, GGPPS re-expression or GGPP administration restore glucose-stimulated insulin secretion in Ggpps-null islets. These results suggest that GGPPS-controlled protein geranylgeranylation, which regulates formation of the insulin granule docked pool, is critical for β cell function and insulin release during the development of T2DM.

  10. Modulation of Stromal Cell-Derived Factor-1/CXC Chemokine Receptor 4 Axis Enhances rhBMP-2-Induced Ectopic Bone Formation

    PubMed Central

    Wise, Joel K.; Sumner, Dale Rick

    2012-01-01

    Enhancement of in vivo mobilization and homing of endogenous mesenchymal stem cells (MSCs) to an injury site is an innovative strategy for improvement of bone tissue engineering and repair. The present study was designed to determine whether mobilization by AMD3100 and/or local homing by delivery of stromal cell-derived factor-1 (SDF-1) enhances recombinant human bone morphogenetic protein-2 (rhBMP-2) induced ectopic bone formation in an established rat model. Rats received an injection of either saline or AMD3100 treatment 1 h before harvesting of bone marrow for in vitro colony-forming unit-fibroblasts (CFU-F) culture or the in vivo subcutaneous implantation of absorbable collagen sponges (ACSs) loaded with saline, recombinant human bone morphogenetic protein-2 (rhBMP-2), SDF-1, or the combination of SDF-1 and rhBMP-2. AMD3100 treatment resulted in a significant decrease in CFU-F number, compared with saline, which confirmed that a single systemic AMD3100 treatment rapidly mobilized MSCs from the bone marrow. At 28 and 56 days, bone formation in the explanted ACS was assessed by microcomputed tomography (μCT) and histology. At 28 days, AMD3100 and/or SDF-1 had no statistically significant effect on bone volume (BV) or bone mineral content (BMC), but histology revealed more active bone formation with treatment of AMD3100, loading of SDF-1, or the combination of both AMD3100 and SDF-1, compared with saline-treated rhBMP-2 loaded ACS. At 56 days, the addition of AMD3100 treatment, loading of SDF-1, or the combination of both resulted in a statistically significant stimulatory effect on BV and BMC, compared with the saline-treated rhBMP-2 loaded ACS. Histology of the 56-day ACS were consistent with the μCT analysis, exhibiting more mature and mineralized bone formation with AMD3100 treatment, SDF-1 loading, or the combination of both, compared with the saline-treated rhBMP-2 loaded ACS. The present study is the first that provides evidence of the efficacy of AMD

  11. More Docked Vesicles and Larger Active Zones at Basket Cell-to-Granule Cell Synapses in a Rat Model of Temporal Lobe Epilepsy

    PubMed Central

    Yamawaki, Ruth; Thind, Khushdev

    2016-01-01

    Temporal lobe epilepsy is a common and challenging clinical problem, and its pathophysiological mechanisms remain unclear. One possibility is insufficient inhibition in the hippocampal formation where seizures tend to initiate. Normally, hippocampal basket cells provide strong and reliable synaptic inhibition at principal cell somata. In a rat model of temporal lobe epilepsy, basket cell-to-granule cell (BC→GC) synaptic transmission is more likely to fail, but the underlying cause is unknown. At some synapses, probability of release correlates with bouton size, active zone area, and number of docked vesicles. The present study tested the hypothesis that impaired GABAergic transmission at BC→GC synapses is attributable to ultrastructural changes. Boutons making axosomatic symmetric synapses in the granule cell layer were reconstructed from serial electron micrographs. BC→GC boutons were predicted to be smaller in volume, have fewer and smaller active zones, and contain fewer vesicles, including fewer docked vesicles. Results revealed the opposite. Compared with controls, epileptic pilocarpine-treated rats displayed boutons with over twice the average volume, active zone area, total vesicles, and docked vesicles and with more vesicles closer to active zones. Larger active zones in epileptic rats are consistent with previous reports of larger amplitude miniature IPSCs and larger BC→GC quantal size. Results of this study indicate that transmission failures at BC→GC synapses in epileptic pilocarpine-treated rats are not attributable to smaller boutons or fewer docked vesicles. Instead, processes following vesicle docking, including priming, Ca2+ entry, or Ca2+ coupling with exocytosis, might be responsible. SIGNIFICANCE STATEMENT One in 26 people develops epilepsy, and temporal lobe epilepsy is a common form. Up to one-third of patients are resistant to currently available treatments. This study tested a potential underlying mechanism for previously reported

  12. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope

    PubMed Central

    Lamm, Christian E.; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-01-01

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell’s nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein. PMID:26978388

  13. An Ectopic Pelvic Kidney

    PubMed Central

    Bhoil, Rohit; Sood, Dinesh; Singh, Yash Paul; Nimkar, Kshama; Shukla, Anurag

    2015-01-01

    Summary Background If a kidney does not ascend as it should in normal fetal development, it remains in the pelvic area and is called a pelvic kidney. Often a person with a pelvic kidney will go through his/her whole life unaware of this condition, unless it is discovered during neonatal kidney ultrasound screening or if complications arise later in life due to this or a completely different reason and the condition is noted during investigations. Generally, this is not a harmful condition but it can lead to complications like in our case. With appropriate testing and treatment, if needed, an ectopic kidney should cause no serious long-term health complications and all that may be required for the patient is reassurance with advice to follow up at regular intervals. Case Report A 28-year-old male presented with recurrent pain in his lower left abdomen for one month and an episode of hematuria 3 days earlier accompanied by an attack of acute pain lasting for 3–4 hours. He gave a history of passing 2 small (about 5 mm each) calculi in his urine after the occurrence of hematuria, following which pain decreased in intensity. No history of fever was present. Conclusions Although a simple ectopic kidney seldom causes symptoms, the association of malrotation of the renal pelvis with calculus increases the risk of hematuria and/or hydronephrosis, presenting with colicky pain as in the present case. The clinician should be aware of these in such a case. If asymptomatic, no treatment is required. However, the patient should be advised to have follow-up ultrasounds at regular intervals to detect complications like calculus, hydronephrosis, etc. With appropriate testing and treatment, if required, an ectopic kidney should not cause serious long-term health complications. PMID:26413178

  14. Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish

    PubMed Central

    Takeuchi, Miki; Yamaguchi, Shingo; Yonemura, Shigenobu; Kakiguchi, Kisa; Sato, Yoshikatsu; Higashiyama, Tetsuya; Shimizu, Takashi; Hibi, Masahiko

    2015-01-01

    Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets. PMID:26451951

  15. Simulation of Ectopic Pacemakers in the Heart: Multiple Ectopic Beats Generated by Reentry inside Fibrotic Regions

    PubMed Central

    Gouvêa de Barros, Bruno; Weber dos Santos, Rodrigo; Lobosco, Marcelo; Alonso, Sergio

    2015-01-01

    The inclusion of nonconducting media, mimicking cardiac fibrosis, in two models of cardiac tissue produces the formation of ectopic beats. The fraction of nonconducting media in comparison with the fraction of healthy myocytes and the topological distribution of cells determines the probability of ectopic beat generation. First, a detailed subcellular microscopic model that accounts for the microstructure of the cardiac tissue is constructed and employed for the numerical simulation of action potential propagation. Next, an equivalent discrete model is implemented, which permits a faster integration of the equations. This discrete model is a simplified version of the microscopic model that maintains the distribution of connections between cells. Both models produce similar results when describing action potential propagation in homogeneous tissue; however, they slightly differ in the generation of ectopic beats in heterogeneous tissue. Nevertheless, both models present the generation of reentry inside fibrotic tissues. This kind of reentry restricted to microfibrosis regions can result in the formation of ectopic pacemakers, that is, regions that will generate a series of ectopic stimulus at a fast pacing rate. In turn, such activity has been related to trigger fibrillation in the atria and in the ventricles in clinical and animal studies. PMID:26583127

  16. Ectopic Lymphoid Structures: Powerhouse of Autoimmunity

    PubMed Central

    Corsiero, Elisa; Nerviani, Alessandra; Bombardieri, Michele; Pitzalis, Costantino

    2016-01-01

    Ectopic lymphoid structures (ELS) often develop at sites of inflammation in target tissues of autoimmune diseases, such as rheumatoid arthritis, Sjögren’s syndrome, multiple sclerosis, myasthenia gravis, and systemic lupus erythematosus. ELS are characterized by the formation of organized T/B cells aggregates, which can acquire follicular dendritic cells network supporting an ectopic germinal center response. In this review, we shall summarize the mechanisms that regulate the formation of ELS in tertiary lymphoid organs, with particular emphasis on the role of lymphoid chemokines in both formation and maintenance of ELS, the role of emerging positive and negative regulators of ELS development and function, including T follicular helper cells and IL-27, respectively. Finally, we shall discuss the main functions of ELS in supporting the affinity maturation, clonal selection, and differentiation of autoreactive B cells contributing to the maintenance and perpetuation of humoral autoimmunity. PMID:27799933

  17. Evidence of the survival of ectopically transplanted oral mucosal epithelial stem cells after repeated wounding of cornea.

    PubMed

    Sugiyama, Hiroaki; Yamato, Masayuki; Nishida, Kohji; Okano, Teruo

    2014-08-01

    Tissue engineering has become an essential tool in the development of regenerative medicine. We have developed cell sheet-based techniques for use in regenerative medicine that have already been successfully used in clinical applications. Native corneal epithelium is produced from limbal stem cells located in the transition zone between the cornea and the bulbar conjunctiva. Limbal stem cell deficiency (LSCD) is a severe defect of the limbal stem cells leading to vision loss due to conjunctival epithelial invasion and neovascularization. Rabbit LSCD models were treated with transplantable autologous oral mucosal epithelial cell (OEC) sheets fabricated on temperature-responsive cell culture surfaces, after which, the ocular surfaces were clear and smooth with no observable defects. The central part of the reconstructed ocular surface was scraped and wounded, after which proliferating epithelial cells covered the scraped area within a few days. The ocular surfaces were clear and smooth even after repeated scrapings and consisted of only OECs or heterogeneously mixed with corneal epithelial cells. This study demonstrates that transplanted cell sheets containing oral mucosal epithelial stem cells could reconstruct the ocular surface to maintain cornea homeostasis; moreover, they provide an ideal microenvironment to support the proliferation of remaining native limbal stem cells.

  18. Aggregation of maternal pigment granules is induced by the cytosolic discoidin domain of the Xenopus Del1 protein.

    PubMed

    Tsabar, Nir; Gefen, Aharon; Elias, Sarah; Frank, Dale

    2005-05-01

    Xenopus oocytes generate pigment granules (melanosomes) that predominantly localize to the animal hemisphere cortex. During embryonic development, these granules are located near the membranes of outer layer ectoderm cells. We report a novel phenotype found during an expression cloning screen in Xenopus laevis embryos. The phenotype is characterized by dissociation of pigment granules from the cell membrane to form large central aggregates. This phenomenon was induced by a truncated form of the Xenopus Del1 (XDel1) protein that contains only the C-terminal discoidin (D2) domain. This truncated form of XDel1 localized to membranes as shown by a chimeric enhanced green fluorescent protein construct. Although a similar localization occurred in immature oocytes, dissociation of pigment granules was limited to the oocyte vegetal hemisphere. The full-length XDel1 cDNA was cloned, and XDel1 mRNA expression was found to be ubiquitous and continuous from early oocyte to tail bud stages, with a transient enrichment in the cement gland. Ectopic expression of various deletion or full-length constructs or antisense morpholino oligonucleotides did not induce any significant developmental phenotypes.

  19. Serpinin: A Novel Chromogranin A-Derived, Secreted Peptide Up-Regulates Protease Nexin-1 Expression and Granule Biogenesis in Endocrine Cells

    PubMed Central

    Koshimizu, Hisatsugu; Cawley, Niamh X.; Kim, Taeyoon; Yergey, Alfred L.

    2011-01-01

    Previously we demonstrated that chromogranin A (CgA) promoted secretory granule biogenesis in endocrine cells by stabilizing and preventing granule protein degradation in the Golgi, through up-regulation of expression of the protease inhibitor, protease nexin-1 (PN-1). However, the mechanism by which CgA signals the increase of PN-1 expression is unknown. Here we identified a 2.9-kDa CgA-C-terminus peptide, which we named serpinin, in conditioned media from AtT-20 cells, a corticotroph cell line, which up-regulated PN-1 mRNA expression. Serpinin was secreted from AtT-20 cells upon high potassium stimulation and increased PN-1 mRNA transcription in these cells, in an actinomycin D-inhibitable manner. CgA itself and other CgA-derived peptides, when added to AtT-20 cell media, had no effect on PN-1 expression. Treatment of AtT-20 cells with 10 nm serpinin elevated cAMP levels and PN-1 mRNA expression, and this effect was inhibited by a protein kinase A inhibitor, 6–22 amide. Serpinin and a cAMP analog, 8-bromo-cAMP, promoted the translocation of the transcription factor Sp1 into the nucleus, which is known to drive PN-1 expression. Additionally, an Sp1 inhibitor, mithramycin A inhibited the serpinin-induced PN-1 mRNA up-regulation. Furthermore, a luciferase reporter assay demonstrated serpinin-induced up-regulation of PN-1 promoter activity in an Sp1-dependent manner. When added to CgB-transfected 6T3 cells, a mutant AtT20 cell line, serpinin induced granule biogenesis as evidenced by the presence of CgB puncta accumulation in the processes and tips. Our findings taken together show that serpinin, a novel CgA-derived peptide, is secreted upon stimulation of corticotrophs and plays an important autocrine role in up-regulating PN-1-dependent granule biogenesis via a cAMP-protein kinase A-Sp1 pathway to replenish released granules. PMID:21436258

  20. Polyphenols differentially inhibit degranulation of distinct subsets of vesicles in mast cells by specific interaction with granule-type-dependent SNARE complexes

    PubMed Central

    Yang, Yoosoo; Oh, Jung-Mi; Heo, Paul; Shin, Jae Yoon; Kong, Byoungjae; Shin, Jonghyeok; Lee, Ji-Chun; Oh, Jeong Su; Park, Kye Won; Lee, Choong Hwan; Shin, Yeon-Kyun; Kweon, Dae-Hyuk

    2016-01-01

    Anti-allergic effects of dietary polyphenols were extensively studied in numerous allergic disease models, but the molecular mechanisms of anti-allergic effects by polyphenols remain poorly understood. In the present study, we show that the release of granular cargo molecules, contained in distinct subsets of granules of mast cells, is specifically mediated by two sets of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and that various polyphenols differentially inhibit the formation of those SNARE complexes. Expression analysis of RBL-2H3 cells for 11 SNARE genes and a lipid mixing assay of 24 possible combinations of reconstituted SNAREs indicated that the only two active SNARE complexes involved in mast cell degranulation are Syn (syntaxin) 4/SNAP (23 kDa synaptosome-associated protein)-23/VAMP (vesicle-associated membrane protein) 2 and Syn4/SNAP-23/VAMP8. Various polyphenols selectively or commonly interfered with ternary complex formation of these two SNARE complexes, thereby stopping membrane fusion between granules and plasma membrane. This led to the differential effect of polyphenols on degranulation of three distinct subsets of granules. These results suggest the possibility that formation of a variety of SNARE complexes in numerous cell types is controlled by polyphenols which, in turn, might regulate corresponding membrane trafficking. PMID:23252429

  1. Erythropoietin Receptor Antagonist Suppressed Ectopic Hemoglobin Synthesis in Xenografts of HeLa Cells to Promote Their Destruction.

    PubMed

    Yasuda, Yoshiko; Fujita, Mitsugu; Koike, Eiji; Obata, Koshiro; Shiota, Mitsuru; Kotani, Yasushi; Musha, Terunaga; Tsuji-Kawahara, Sachiyo; Satou, Takao; Masuda, Seiji; Okano, Junko; Yamasaki, Harufumi; Okumoto, Katsumi; Uesugi, Tadao; Nakao, Shinichi; Hoshiai, Hiroshi; Mandai, Masaki

    2015-01-01

    The aim of this study is to explore a cause-oriented therapy for patients with uterine cervical cancer that expresses erythropoietin (Epo) and its receptor (EpoR). Epo, by binding to EpoR, stimulates the proliferation and differentiation of erythroid progenitor cells into hemoglobin-containing red blood cells. In this study, we report that the HeLa cells in the xenografts expressed ε, γ, and α globins as well as myoglobin (Mb) to produce tetrameric α2ε2 and α2γ2 and monomeric Mb, most of which were significantly suppressed with an EpoR antagonist EMP9. Western blotting revealed that the EMP9 treatment inhibited the AKT-pAKT, MAPKs-pMAPKs, and STAT5-pSTAT5 signaling pathways. Moreover, the treatment induced apoptosis and suppression of the growth and inhibited the survival through disruption of the harmonized hemoprotein syntheses in the tumor cells concomitant with destruction of vascular nets in the xenografts. Furthermore, macrophages and natural killer (NK) cells with intense HIF-1α expression recruited significantly more in the degenerating foci of the xenografts. These findings were associated with the enhanced expressions of nNOS in the tumor cells and iNOS in macrophages and NK cells in the tumor sites. The treated tumor cells exhibited a substantial number of perforations on the cell surface, which indicates that the tumors were damaged by both the nNOS-induced nitric oxide (NO) production in the tumor cells as well as the iNOS-induced NO production in the innate immune cells. Taken together, these data suggest that HeLa cells constitutively acquire ε, γ and Mb synthetic capacity for their survival. Therefore, EMP9 treatment might be a cause-oriented and effective therapy for patients with squamous cell carcinoma of the uterine cervix. PMID:25874769

  2. Inhibitory effect of fangchinoline on excitatory amino acids-induced neurotoxicity in cultured rat cerebellar granule cells.

    PubMed

    Kim, S D; Oh, S K; Kim, H S; Seong, Y H

    2001-04-01

    Glutamate receptors-mediated excitotoxicity is believed to play a role in the pathophysiology of neurodegenerative diseases. The present study was performed to evaluate the inhibitory effect of fangchinoline, a bis-benzylisoquinoline alkaloid, which has a characteristic as a Ca2+ channel blocker, on excitatory amino acids (EAAs)-induced neurotoxicity in cultured rat cerebellar granule neuron. Fangchinoline (1 and 5 microM) inhibited glutamate (1 mM), N-methyl-D-aspartate (NMDA; 1 mM) and kainate (100 microM)-induced neuronal cell death which was measured by trypan blue exclusion test. Fangchinoline (1 and 5 microM) inhibited glutamate release into medium induced by NMDA (1 mM) and kainate (100 microM), which was measured by HPLC. And fangchinoline (5 microM) inhibited glutamate (1 mM)-induced elevation of intracellular calcium concentration. These results suggest that inhibition of Ca2+ influx by fangchinoline may contribute to the beneficial effects on neurodegenerative effect of glutamate in pathophysiological conditions. PMID:11339637

  3. A realistic bi-hemispheric model of the cerebellum uncovers the purpose of the abundant granule cells during motor control.

    PubMed

    Pinzon-Morales, Ruben-Dario; Hirata, Yutaka

    2015-01-01

    The cerebellar granule cells (GCs) have been proposed to perform lossless, adaptive spatio-temporal coding of incoming sensory/motor information required by downstream cerebellar circuits to support motor learning, motor coordination, and cognition. Here we use a physio-anatomically inspired bi-hemispheric cerebellar neuronal network (biCNN) to selectively enable/disable the output of GCs and evaluate the behavioral and neural consequences during three different control scenarios. The control scenarios are a simple direct current motor (1 degree of freedom: DOF), an unstable two-wheel balancing robot (2 DOFs), and a simulation model of a quadcopter (6 DOFs). Results showed that adequate control was maintained with a relatively small number of GCs (< 200) in all the control scenarios. However, the minimum number of GCs required to successfully govern each control plant increased with their complexity (i.e., DOFs). It was also shown that increasing the number of GCs resulted in higher robustness against changes in the initialization parameters of the biCNN model (i.e., synaptic connections and synaptic weights). Therefore, we suggest that the abundant GCs in the cerebellar cortex provide the computational power during the large repertoire of motor activities and motor plants the cerebellum is involved with, and bring robustness against changes in the cerebellar microcircuit (e.g., neuronal connections).

  4. A realistic bi-hemispheric model of the cerebellum uncovers the purpose of the abundant granule cells during motor control.

    PubMed

    Pinzon-Morales, Ruben-Dario; Hirata, Yutaka

    2015-01-01

    The cerebellar granule cells (GCs) have been proposed to perform lossless, adaptive spatio-temporal coding of incoming sensory/motor information required by downstream cerebellar circuits to support motor learning, motor coordination, and cognition. Here we use a physio-anatomically inspired bi-hemispheric cerebellar neuronal network (biCNN) to selectively enable/disable the output of GCs and evaluate the behavioral and neural consequences during three different control scenarios. The control scenarios are a simple direct current motor (1 degree of freedom: DOF), an unstable two-wheel balancing robot (2 DOFs), and a simulation model of a quadcopter (6 DOFs). Results showed that adequate control was maintained with a relatively small number of GCs (< 200) in all the control scenarios. However, the minimum number of GCs required to successfully govern each control plant increased with their complexity (i.e., DOFs). It was also shown that increasing the number of GCs resulted in higher robustness against changes in the initialization parameters of the biCNN model (i.e., synaptic connections and synaptic weights). Therefore, we suggest that the abundant GCs in the cerebellar cortex provide the computational power during the large repertoire of motor activities and motor plants the cerebellum is involved with, and bring robustness against changes in the cerebellar microcircuit (e.g., neuronal connections). PMID:25983678

  5. The GABAA antagonist DPP-4-PIOL selectively antagonises tonic over phasic GABAergic currents in dentate gyrus granule cells.

    PubMed

    Boddum, Kim; Frølund, Bente; Kristiansen, Uffe

    2014-11-01

    GABAA receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABAA receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABAA receptors. The two inhibitory current types are mediated by different subpopulations of GABAA receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate that phasic and tonic GABAA receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent inhibiting phasic currents compared to tonic currents (IC50 values: 101 vs. 427 nM). Conversely, DPP-4-PIOL was estimated to be more than 20 times as potent inhibiting tonic current compared to phasic current (IC50 values: 0.87 vs. 21.3 nM). Consequently, we were able to impose a pronounced reduction in tonic GABA mediated current (>70 %) by concentrations of DPP-4-PIOL, at which no significant effect on the phasic current was seen. Our findings demonstrate that selective inhibition of GABA mediated tonic current is possible, when targeting a subpopulation of GABAA receptors located extrasynaptically using the antagonist, DPP-4-PIOL. PMID:25103229

  6. Neurotensinergic Excitation of Dentate Gyrus Granule Cells via Gαq-Coupled Inhibition of TASK-3 Channels.

    PubMed

    Zhang, Haopeng; Dong, Hailong; Cilz, Nicholas I; Kurada, Lalitha; Hu, Binqi; Wada, Etsuko; Bayliss, Douglas A; Porter, James E; Lei, Saobo

    2016-03-01

    Neurotensin (NT) is a 13-amino acid peptide and serves as a neuromodulator in the brain. Whereas NT has been implicated in learning and memory, the underlying cellular and molecular mechanisms are ill-defined. Because the dentate gyrus receives profound innervation of fibers containing NT and expresses high density of NT receptors, we examined the effects of NT on the excitability of dentate gyrus granule cells (GCs). Our results showed that NT concentration dependently increased action potential (AP) firing frequency of the GCs by the activation of NTS1 receptors resulting in the depolarization of the GCs. NT-induced enhancement of AP firing frequency was not caused indirectly by releasing glutamate, GABA, acetylcholine, or dopamine, but due to the inhibition of TASK-3 K(+) channels. NT-mediated excitation of the GCs was G protein dependent, but independent of phospholipase C, intracellular Ca(2+) release, and protein kinase C. Immunoprecipitation experiment demonstrates that the activation of NTS1 receptors induced the association of Gαq/11 and TASK-3 channels suggesting a direct coupling of Gαq/11 to TASK-3 channels. Endogenously released NT facilitated the excitability of the GCs contributing to the induction of long-term potentiation at the perforant path-GC synapses. Our results provide a cellular mechanism that helps to explain the roles of NT in learning and memory. PMID:25405940

  7. Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.

    PubMed

    Stone, Scellig S D; Teixeira, Cátia M; Zaslavsky, Kirill; Wheeler, Anne L; Martinez-Canabal, Alonso; Wang, Afra H; Sakaguchi, Masanori; Lozano, Andres M; Frankland, Paul W

    2011-12-01

    In the hippocampus, the production of dentate granule cells (DGCs) persists into adulthood. As adult-generated neurons are thought to contribute to hippocampal memory processing, promoting adult neurogenesis therefore offers the potential for restoring mnemonic function in the aged or diseased brain. Within this regenerative context, one key issue is whether developmentally generated and adult-generated DGCs represent functionally equivalent or distinct neuronal populations. To address this, we labeled separate cohorts of developmentally generated and adult-generated DGCs and used immunohistochemical approaches to compare their integration into circuits supporting hippocampus-dependent memory in intact mice. First, in the water maze task, rates of integration of adult-generated DGCs were regulated by maturation, with maximal integration not occurring until DGCs were five or more weeks in age. Second, these rates of integration were equivalent for embryonically, postnatally, and adult-generated DGCs. Third, these findings generalized to another hippocampus-dependent task, contextual fear conditioning. Together, these experiments indicate that developmentally generated and adult-generated DGCs are integrated into hippocampal memory networks at similar rates, and suggest a functional equivalence between DGCs generated at different developmental stages. PMID:20824726

  8. Effects of prenatal stress and exercise on dentate granule cells maturation and spatial memory in adolescent mice.

    PubMed

    Bustamante, Carlos; Bilbao, Pamela; Contreras, William; Martínez, Mauricio; Mendoza, Antonio; Reyes, Alvaro; Pascual, Rodrigo

    2010-11-01

    Exposure to prenatal stress (PS) increases the risk of developing neurobehavioral disturbances later in life. Previous work has shown that exercise can exert beneficial effects on brain damage; however, it is unknown whether voluntary wheel running (VWR) can ameliorate the neurobehavioral impairments induced by PS in adolescent offspring. Pregnant CF-1 mice were randomly assigned to control (n=5) or stressed (n=5) groups. Pregnant dams were subjected to restraint stress between gestational days 14 and 21 (G14-21), whereas controls remained undisturbed in their home cages. On postnatal day 21 (P21), male pups were randomly assigned to the following experimental groups: control (n=5), stressed (n=5), and stressed mice+daily submitted to VWR (n=4). At P52, all groups were behaviorally evaluated in the Morris water maze. Animals were then sacrificed, and Golgi-impregnated granule cells were morphometrically analyzed. The results indicate that PS produced significant behavioral and neuronal impairments in adolescent offspring and that VWR significantly offset these deleterious effects.

  9. A realistic bi-hemispheric model of the cerebellum uncovers the purpose of the abundant granule cells during motor control

    PubMed Central

    Pinzon-Morales, Ruben-Dario; Hirata, Yutaka

    2015-01-01

    The cerebellar granule cells (GCs) have been proposed to perform lossless, adaptive spatio-temporal coding of incoming sensory/motor information required by downstream cerebellar circuits to support motor learning, motor coordination, and cognition. Here we use a physio-anatomically inspired bi-hemispheric cerebellar neuronal network (biCNN) to selectively enable/disable the output of GCs and evaluate the behavioral and neural consequences during three different control scenarios. The control scenarios are a simple direct current motor (1 degree of freedom: DOF), an unstable two-wheel balancing robot (2 DOFs), and a simulation model of a quadcopter (6 DOFs). Results showed that adequate control was maintained with a relatively small number of GCs (< 200) in all the control scenarios. However, the minimum number of GCs required to successfully govern each control plant increased with their complexity (i.e., DOFs). It was also shown that increasing the number of GCs resulted in higher robustness against changes in the initialization parameters of the biCNN model (i.e., synaptic connections and synaptic weights). Therefore, we suggest that the abundant GCs in the cerebellar cortex provide the computational power during the large repertoire of motor activities and motor plants the cerebellum is involved with, and bring robustness against changes in the cerebellar microcircuit (e.g., neuronal connections). PMID:25983678

  10. Naringin attenuates granule cell dispersion in the dentate gyrus in a mouse model of temporal lobe epilepsy.

    PubMed

    Jang, Hannah; Jeong, Kyoung Hoon; Kim, Sang Ryong

    2016-07-01

    Morphological abnormalities of the dentate gyrus (DG) are an important phenotype in the hippocampus of patients with temporal lobe epilepsy. We recently reported that naringin, a bioflavonoid in grapefruit and citrus fruits, exerts beneficial effects in the kainic acid (KA) mouse model of epilepsy. We found that naringin treatment reduced seizure activities and decreased autophagic stress and neuroinflammation in the hippocampus following in vivo lesion with KA. However, it remains unclear whether naringin may also attenuate seizure-induced morphological changes in the DG, collectively known as granule cell dispersion (GCD). To clarify whether naringin treatment reduces GCD, we evaluated the effects of intraperitoneal injection of naringin on GCD and activation of mammalian target of rapamycin complex 1 (mTORC1), an important regulator of GCD, following intrahippocampal injection of KA. Our results showed that naringin treatment significantly reduced KA-induced GCD and mTORC1 activation, which was confirmed by assessing the phosphorylated form of the mTORC1 substrate, 4E-BP1, in the hippocampus. These results suggest that naringin treatment may help prevent epilepsy-induced hippocampal injury by inhibiting mTORC1 activation and thereby reducing GCD in the hippocampus in vivo.

  11. Stable Ectopic Expression of ST6GALNAC5 Induces Autocrine MET Activation and Anchorage-Independence in MDCK Cells.

    PubMed

    Chu, Chia; Bottaro, Donald P; Betenbaugh, Michael J; Shiloach, Joseph

    2016-01-01

    The epithelial-mesenchymal transition (EMT) is a complex cancer progression that can boost the metastatic potential of transformed cells by inducing migration, loss of cell adhesion, and promoting proliferation under anchorage-independent conditions. A DNA microarray analysis was performed comparing parental anchorage-dependent MDCK cells and anchorage-independent MDCK cells that were engineered to express human siat7e (ST6GALNAC5). The comparison identified several genes involved in the EMT process that were differentially expressed between the anchorage-dependent and the anchorage-independent MDCK cell lines. The hepatocyte growth factor gene (hgf) was found to be over-expressed in the engineered MDCK-siat7e cells at both transcription and protein expression levels. Phosphorylation analysis of the MET receptor tyrosine kinase confirmed the activation of an autocrine loop of the HGF/ MET signaling pathway in the MDCK-siat7e cells. When MET activities were suppressed by using the small-molecular inhibitor drug PF-02341066 (Crizotinib), the anchorage-independent MDCK-siat7e cells reverted to the cellular morphology of the parental anchorage-dependent MDCK cells. These observations indicate that the MET receptor plays a central role in the growth properties of the MDCK cells and its phosphorylation status is likely dependent on sialylation. Further investigation of the downstream signaling targets in the MET network showed that the degree of MDCK cell adhesion correlated with secretion levels of a matrix metalloproteinase, MMP1, suggesting a role of metalloproteinases in the EMT process. These results demonstrate that in addition to its application in biotechnology processes, MDCK-siat7e may serve as a model cell for metastasis studies to decipher the sequence of events leading up to the activation of EMT. PMID:26848584

  12. Stable Ectopic Expression of ST6GALNAC5 Induces Autocrine MET Activation and Anchorage-Independence in MDCK Cells

    PubMed Central

    Chu, Chia; Bottaro, Donald P.; Betenbaugh, Michael J.; Shiloach, Joseph

    2016-01-01

    The epithelial-mesenchymal transition (EMT) is a complex cancer progression that can boost the metastatic potential of transformed cells by inducing migration, loss of cell adhesion, and promoting proliferation under anchorage-independent conditions. A DNA microarray analysis was performed comparing parental anchorage-dependent MDCK cells and anchorage-independent MDCK cells that were engineered to express human siat7e (ST6GALNAC5). The comparison identified several genes involved in the EMT process that were differentially expressed between the anchorage-dependent and the anchorage-independent MDCK cell lines. The hepatocyte growth factor gene (hgf) was found to be over-expressed in the engineered MDCK-siat7e cells at both transcription and protein expression levels. Phosphorylation analysis of the MET receptor tyrosine kinase confirmed the activation of an autocrine loop of the HGF/ MET signaling pathway in the MDCK-siat7e cells. When MET activities were suppressed by using the small-molecular inhibitor drug PF-02341066 (Crizotinib), the anchorage-independent MDCK-siat7e cells reverted to the cellular morphology of the parental anchorage-dependent MDCK cells. These observations indicate that the MET receptor plays a central role in the growth properties of the MDCK cells and its phosphorylation status is likely dependent on sialylation. Further investigation of the downstream signaling targets in the MET network showed that the degree of MDCK cell adhesion correlated with secretion levels of a matrix metalloproteinase, MMP1, suggesting a role of metalloproteinases in the EMT process. These results demonstrate that in addition to its application in biotechnology processes, MDCK-siat7e may serve as a model cell for metastasis studies to decipher the sequence of events leading up to the activation of EMT. PMID:26848584

  13. ONTOGENY OF PROTEINS ASSOCIATED WITH NEURITE GROWTH AND SYNAPTOGENESIS IN CEREBELLAR GRANULE CELLS IN VITRO.

    EPA Science Inventory

    In vitro techniques may be useful in screening for effects of developmental neurotoxicants. Previously, we characterized changes in biochemical markers associated with neuronal development in a PC12 cell model of differentiation and growth. The current research extended these stu...

  14. Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

    SciTech Connect

    Jung, Eun Joo; Kim, Deok Ryong

    2011-01-21

    Research highlights: {yields} We established TrkA-inducible U2OS cells stably expressing GFP-H2AX proteins. {yields} GFP-H2AX was colocalized with TrkA in the cytoplasm. {yields} {gamma}H2AX production was significantly increased upon activation of TrkA and suppressed by TrkA inhibitor or JNK inhibitor. {yields} Ectopic expression of H2AX promoted TrkA-mediated cell death through the modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. -- Abstract: We previously reported that TrkA overexpression causes accumulation of {gamma}H2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we established TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and {gamma}H2AX in TrkA-induced cell death. {gamma}H2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.

  15. Ectopic bone formation using an injectable biphasic calcium phosphate/Si-HPMC hydrogel composite loaded with undifferentiated bone marrow stromal cells.

    PubMed

    Trojani, Christophe; Boukhechba, Florian; Scimeca, Jean-Claude; Vandenbos, Fanny; Michiels, Jean-François; Daculsi, Guy; Boileau, Pascal; Weiss, Pierre; Carle, Georges F; Rochet, Nathalie

    2006-06-01

    We have used a new synthetic injectable composite constituted of hydroxyapatite/tricalcium phosphate (HA/TCP) particles in suspension in a self-hardening Si-hydroxypropylmethylcellulose (HPMC) hydrogel. The aim of this study was to evaluate in vivo the biocompatibility and the new bone formation efficacy of this scaffold loaded with undifferentiated bone marrow stromal cells (BMSCs). This biomaterial was mixed extemporaneously with BMSCs prepared from C57BL/6 mice, injected in subcutaneous and intramuscular sites and retrieved 4 and 8 weeks after implantation. Dissection of the implants revealed a hard consistency and the absence of a fibrous capsule reflecting a good integration into the host tissues. Histological analysis showed mineralized woven bone in the granule inter-space with numerous active osteoclasts attached to the particles as assessed by the presence of multinucleated cells positively stained for TRAP activity and for the a3 subunit of the V-ATPase. Small vessels were homogenously distributed in the whole implants. Similar results were obtained in SC and IM sites and no bone formation was observed in the control groups when cell-free and particle-free transplants were injected. These results indicate that this injectable biphasic calcium phosphate-hydrogel composite mixed with undifferentiated BMSCs is a new promising osteoinductive bone substitute. It also provides with an original in vivo model of osteoclast differentiation and function. PMID:16510180

  16. Beneficial effect of Boswellia serrata gum resin on spatial learning and the dendritic tree of dentate gyrus granule cells in aged rats

    PubMed Central

    Hosseini-Sharifabad, Mohammad; Kamali-Ardakani, Razieh; Hosseini-Sharifabad, Ali

    2016-01-01

    Objective: The hippocampal formation, particularly the dentate gyrus (DG), shows age-related morphological changes that could cause memory decline. It is indicated that Boswellia resins attenuates memory deficits and the major component of Boswellia serrata (Bs) gum resin, beta boswellic acid increased neurite outgrowth and branching in hippocampal neurons. This study was designed to investigate the effect of Boswellia treatment on spatial learning performance and the morphology of dentate granule cells in aged rats. Materials and Methods: Sixteen male Wistar rats (24 months old) were divided into experimental and control groups. Experimental group was intragastrically administered with the aqueous extract of Bs (100 mg/kg/d for 8 weeks) and control group received a similar volume of water. Spatial learning performance of rats was tested using Morris water maze task. At the end of experiment, the brain was removed and the right hippocampus was serially sectioned for morphometric analysis. The Cavalieri principle was employed to estimate the volume of the DG. A quantitative Golgi study was used to analyze the dendritic trees of dentate granule cells. Results: Chronic treatment with Bs improved spatial learning capability during the three acquisition days. Comparisons also revealed that Bs-treated aged rat had greater DG with increased dendritic complexity in the dentate granule cells than control rats. Hippocampal granule cells of Bs-treated aged rats had more dendritic segments, larger arbors, more numerical branching density and more dendritic spines in comparison to control animals. Conclusion: This study provided a neuro-anatomical basis for memory improvement due to chronic treatment with Bs. PMID:27222832

  17. Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart

    SciTech Connect

    Serakinci, Nedime . E-mail: nserakinci@health.sdu.dk; Christensen, Rikke; Graakjaer, Jesper; Cairney, Claire J.; Keith, W. Nicol; Alsner, Jan; Saretzki, Gabriele; Kolvraa, Steen

    2007-03-10

    During the past several years increasing evidence indicating that the proliferation capacity of mammalian cells is highly radiosensitive, regardless of the species and the tissue of origin of the cells, has accumulated. It has also been shown that normal bone marrow cells of mice have a similar radiosensitivity to other mammalian cells so far tested. In this study, we investigated the genetic effects of ionizing radiation (2.5-15 Gy) on normal human mesenchymal stem cells and their telomerised counterpart hMSC-telo1. We evaluated overall genomic integrity, DNA damage/repair by applying a fluorescence-detected alkaline DNA unwinding assay together with Western blot analyses for phosphorylated H2AX and Q-FISH was applied for investigation of telomeric damage. Our results indicate that hMSC and TERT-immortalized hMSCs can cope with relatively high doses of {gamma}-rays and that overall DNA repair is similar in the two cell lines. The telomeres were extensively destroyed after irradiation in both cell types suggesting that telomere caps are especially sensitive to radiation. The TERT-immortalized hMSCs showed higher stability at telomeric regions than primary hMSCs indicating that cells with long telomeres and high telomerase activity have the advantage of re-establishing the telomeric caps.

  18. Short-time survival and engraftment of bone marrow stromal cells in an ectopic model of bone regeneration.

    PubMed

    Giannoni, Paolo; Scaglione, Silvia; Daga, Antonio; Ilengo, Cristina; Cilli, Michele; Quarto, Rodolfo

    2010-02-01

    In tissue-engineered applications bone marrow stromal cells (BMSCs) are combined with scaffolds to target bone regeneration; animal models have been devised and the cells' long-term engraftment has been widely studied. However, in regenerated bone, the cell number is severely reduced with respect to the initially seeded BMSCs. This reflects the natural low cellularity of bone but underlines the selectivity of the differentiation processes. In this respect, we evaluated the short-term survival of BMSCs, transduced with the luciferase gene, after implantation of cell-seeded scaffolds in a nude mouse model. Cell proliferation/survival was assessed by bioluminescence imaging: light production was decreased by 30% on the first day, reaching a 50% loss within 48 h. Less than 5% of the initial signal remained after 2 months in vivo. Apoptotic BMSCs were detected within the first 2 days of implantation. Interestingly, the initial frequency of clonogenic progenitors matched the percentage of in vivo surviving cells. Cytokines and inflammation may contribute to the apoptotic onset at the implant milieu. However, preculturing cells with tumor necrosis factor alpha enhanced survival, allowing detection of 8.1% of the seeded BMSCs 2 months after implantation. Thus culturing conditions may reduce the apoptotic overload of seeded osteoprogenitors, strengthening the constructs' osteogenic potential.

  19. Mefenamic acid bi-directionally modulates the transient outward K{sup +} current in rat cerebellar granule cells

    SciTech Connect

    Zhang Man; Shi Wenjie; Fei Xiaowei; Liu Yarong; Zeng Ximin; Mei Yanai

    2008-02-01

    The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on ion channels has been widely studied in several cell models, but less is known about their modulatory mechanisms. In this report, the effect of mefenamic acid on voltage-activated transient outward K{sup +} current (I{sub A}) in cultured rat cerebellar granule cells was investigated. At a concentration of 5 {mu}M to 100 {mu}M, mefenamic acid reversibly inhibited I{sub A} in a dose-dependent manner. However, mefenamic acid at a concentration of 1 {mu}M significantly increased the amplitude of I{sub A} to 113 {+-} 1.5% of the control. At more than 10 {mu}M, mefenamic acid inhibited the amplitude of I{sub A} without any effect on activation or inactivation. In addition, a higher concentration of mefenamic acid induced a significant acceleration of recovery from inactivation with an increase of the peak amplitude elicited by the second test pulse. Intracellular application of mefenamic acid could significantly increase the amplitude of I{sub A}, but had no effect on the inhibition induced by extracellular mefenamic acid, implying that mefenamic acid may exert its effect from both inside and outside the ion channel. Furthermore, the activation of current induced by intracellular application of mefenamic acid was mimicked by other cyclooxygenase inhibitors and arachidonic acid. Our data demonstrate that mefenamic acid is able to bi-directionally modulate I{sub A} channels in neurons at different concentrations and by different methods of application, and two different mechanisms may be involved.

  20. Suppression of cell expansion by ectopic expression of the Arabidopsis SUPERMAN gene in transgenic petunia and tobacco.

    PubMed

    Kater, M M; Franken, J; van Aelst, A; Angenent, G C

    2000-08-01

    Molecular and genetic analyses have shown that the Arabidopsis thaliana gene SUPERMAN (SUP) has at least two functions in Arabidopsis flower development. SUP is necessary to control the correct distribution of cells with either a stamen or carpel fate, and is essential for proper outgrowth of the ovule outer integument. Both these functions indicate a role for SUP in cell proliferation. To study the function of the Arabidopsis SUP gene in more detail, we over-expressed the SUP gene in petunia and tobacco in a tissue-specific manner. The petunia FLORAL BINDING PROTEIN 1 (FBP1) gene promoter was used to restrict the expression of SUP to petals and stamens. The development of petals and stamens was severely affected in both petunia and tobacco plants over-expressing SUP. Petals remained small and did not unfold, resulting in closed flowers. Stamen filaments were thin and very short. Detailed analysis of these floral organs from the petunia transformants showed that cell expansion was dramatically reduced without affecting cell division. These results reveal a novel activity for SUP as a regulator of cell expansion. PMID:10929133

  1. Effect of heart failure on catecholamine granule morphology and storage in chromaffin cells.

    PubMed

    Mahata, Sushil K; Zheng, Hong; Mahata, Sumana; Liu, Xuefei; Patel, Kaushik P

    2016-09-01

    One of the key mechanisms involved in sympathoexcitation in chronic heart failure (HF) is the activation of the adrenal glands. Impact of the elevated catecholamines on the hemodynamic parameters has been previously demonstrated. However, studies linking the structural effects of such overactivation with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not been previously reported. In this study, HF was induced in male Sprague-Dawley rats by ligation of the left coronary artery. Five weeks after surgery, cardiac function was assessed by ventricular hemodynamics. HF rats showed increased adrenal weight and adrenal catecholamine levels (norepinephrine, epinephrine and dopamine) compared with sham-operated rats. Rats with HF demonstrated increased small synaptic and dense core vesicle in splanchnic-adrenal synapses indicating trans-synaptic activation of catecholamine biosynthetic enzymes, increased endoplasmic reticulum and Golgi lumen width to meet the demand of increased catecholamine synthesis and release, and more mitochondria with dilated cristae and glycogen to accommodate for the increased energy demand for the increased biogenesis and exocytosis of catecholamines from the adrenal medulla. These findings suggest that increased trans-synaptic activation of the chromaffin cells within the adrenal medulla may lead to increased catecholamines in the circulation which in turn contributes to the enhanced neurohumoral drive, providing a unique mechanistic insight for enhanced catecholamine levels in plasma commonly observed in chronic HF condition. PMID:27402067

  2. Effect of heart failure on catecholamine granule morphology and storage in chromaffin cells

    PubMed Central

    Mahata, Sushil K; Zheng, Hong; Mahata, Sumana; Liu, Xuefei

    2016-01-01

    One of the key mechanisms involved in sympathoexcitation in chronic heart failure (HF) is the activation of the adrenal glands. Impact of the elevated catecholamines on the hemodynamic parameters has been previously demonstrated. However, studies linking the structural effects of such overactivation with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not been previously reported. In this study, HF was induced in male Sprague-Dawley rats by ligation of the left coronary artery. Five weeks after surgery, cardiac function was assessed by ventricular hemodynamics. HF rats showed increased adrenal weight and adrenal catecholamine levels (norepinephrine, epinephrine and dopamine) compared with sham-operated rats. Rats with HF demonstrated increased small synaptic and dense core vesicle in splanchnic–adrenal synapses indicating trans-synaptic activation of catecholamine biosynthetic enzymes, increased endoplasmic reticulum and Golgi lumen width to meet the demand of increased catecholamine synthesis and release, and more mitochondria with dilated cristae and glycogen to accommodate for the increased energy demand for the increased biogenesis and exocytosis of catecholamines from the adrenal medulla. These findings suggest that increased trans-synaptic activation of the chromaffin cells within the adrenal medulla may lead to increased catecholamines in the circulation which in turn contributes to the enhanced neurohumoral drive, providing a unique mechanistic insight for enhanced catecholamine levels in plasma commonly observed in chronic HF condition. PMID:27402067

  3. AtMYB41 activates ectopic suberin synthesis and assembly in multiple plant species and cell types

    PubMed Central

    Kosma, Dylan K; Murmu, Jhadeswar; Razeq, Fakhria M; Santos, Patricia; Bourgault, Richard; Molina, Isabel; Rowland, Owen

    2014-01-01

    Suberin is a lipid and phenolic cell wall heteropolymer found in the roots and other organs of all vascular plants. Suberin plays a critical role in plant water relations and in protecting plants from biotic and abiotic stresses. Here we describe a transcription factor, AtMYB41 (At4g28110), that can activate the steps necessary for aliphatic suberin synthesis and deposition of cell wall-associated suberin-like lamellae in both Arabidopsis thaliana and Nicotiana benthamiana. Overexpression of AtMYB41 increased the abundance of suberin biosynthetic gene transcripts by orders of magnitude and resulted in the accumulation of up to 22 times more suberin-type than cutin-type aliphatic monomers in leaves. Overexpression of AtMYB41 also resulted in elevated amounts of monolignols in leaves and an increase in the accumulation of phenylpropanoid and lignin biosynthetic gene transcripts. Surprisingly, ultrastructural data indicated that overexpression led to the formation of suberin-like lamellae in both epidermal and mesophyll cells of leaves. We further implicate AtMYB41 in the production of aliphatic suberin under abiotic stress conditions. These results provide insight into the molecular-genetic mechanisms of the biosynthesis and deposition of a ubiquitous cell wall-associated plant structure and will serve as a basis for discovering the transcriptional network behind one of the most abundant lipid-based polymers in nature. PMID:25060192

  4. Running rewires the neuronal network of adult-born dentate granule cells.

    PubMed

    Vivar, Carmen; Peterson, Benjamin D; van Praag, Henriette

    2016-05-01

    Exercise improves cognition in humans and animals. Running increases neurogenesis in the dentate gyrus of the hippocampus, a brain area important for learning and memory. It is unclear how running modifies the circuitry of new dentate gyrus neurons to support their role in memory function. Here we combine retroviral labeling with rabies virus mediated trans-synaptic retrograde tracing to define and quantify new neuron afferent inputs in young adult male C57Bl/6 mice, housed with or without a running wheel for one month. Exercise resulted in a shift in new neuron networks that may promote sparse encoding and pattern separation. Neurogenesis increased in the dorsal, but not the ventral, dentate gyrus by three-fold, whereas afferent traced cell labeling doubled in number. Regional analysis indicated that running differentially affected specific inputs. Within the hippocampus the ratio of innervation from inhibitory interneurons and glutamatergic mossy cells to new neurons was reduced. Distal traced cells were located in sub-cortical and cortical regions, including perirhinal, entorhinal and sensory cortices. Innervation from entorhinal cortex (EC) was augmented, in proportion to the running-induced enhancement of adult neurogenesis. Within EC afferent input and short-term synaptic plasticity from lateral entorhinal cortex, considered to convey contextual information to the hippocampus was increased. Furthermore, running upregulated innervation from regions important for spatial memory and theta rhythm generation, including caudo-medial entorhinal cortex and subcortical medial septum, supra- and medial mammillary nuclei. Altogether, running may facilitate contextual, spatial and temporal information encoding by increasing adult hippocampal neurogenesis and by reorganization of new neuron circuitry. PMID:26589333

  5. Fluoro-jade identification of cerebellar granule cell and purkinje cell death in the alpha1A calcium ion channel mutant mouse, leaner.

    PubMed

    Frank, T C; Nunley, M C; Sons, H D; Ramon, R; Abbott, L C

    2003-01-01

    Cell death is a critical component of normal nervous system development; too little or too much results in abnormal development and function of the nervous system. The leaner mouse exhibits excessive, abnormal cerebellar granule cell and Purkinje cell death during postnatal development, which is a consequence of a mutated calcium ion channel subunit, alpha(1A). Previous studies have shown that leaner cerebellar Purkinje cells die in a specific pattern that appears to be influenced by functional and anatomical boundaries of the cerebellum. However, the mechanism of Purkinje cell death and the specific timing of the spatial pattern of cell death remain unclear. By double labeling both leaner and wild-type cerebella with Fluoro-Jade and terminal deoxynucleotide transferase-mediated, deoxyuridine triphosphate nick-end labeling or Fluoro-Jade and tyrosine hydroxylase immunohistochemistry we demonstrated that the relatively new stain, Fluoro-Jade, will label neurons that are dying secondary to a genetic mutation. Then, by staining leaner and wild-type cerebella between postnatal days 20 and 80 with Fluoro-Jade, we were able to show that Purkinje cell death begins at approximately postnatal day 25, peaks in the vermis about postnatal day 40 and in the hemispheres at postnatal day 50 and persists at a low level at postnatal day 80. In addition, we showed that there is a significant difference in the amount of cerebellar Purkinje cell death between rostral and caudal divisions of the leaner cerebellum, and that there is little to no Purkinje cell death in the wild type cerebellum at the ages we examined. This is the first report of the use of Fluoro-Jade to identify dying neurons in a genetic model for neuronal cell death. By using Fluoro-Jade, we have specifically defined the temporospatial pattern of postnatal Purkinje cell death in the leaner mouse. This information can be used to gain insight into the dynamic mechanisms controlling Purkinje cell death in the leaner

  6. Single channel properties of P2X ATP receptors in outside-out patches from rat hippocampal granule cells

    PubMed Central

    Wong, Adrian YC; Burnstock, Geoffrey; Gibb, Alasdair J

    2000-01-01

    The single channel properties of P2X ATP receptors were investigated in outside-out patches from hippocampal granule cells in brain slices from 12-day-old rats. The results demonstrate that functional P2X ATP receptors are expressed in hippocampal granule cells and, combined with previously published information on the P2X subunits expressed in the hippocampus, suggest that the receptors may be heteromers of the P2X4 and P2X6 subunits or P2X1, P2X2, P2X4 and P2X6 subunits. Two distinct types of P2X channel openings were observed. A flickery P2X receptor channel was observed in three patches with a mean chord conductance of 32 ± 6 pS, a mean open time of 1.0 ± 0.3 ms and a mean burst length of 11 ± 5 ms at a membrane potential of −60 mV. A large conductance P2X receptor was observed in 19 out of 98 patches with a mean conductance of 56 ± 1.8 pS, a linear current-voltage relationship between −80 and +60 mV with a reversal potential around 0 mV, a mean open time of 2.6 ± 0.2 ms and a mean burst length of 8.8 ± 1.8 ms at −60 mV. At an ATP concentration of 1 mm, these channels exhibited a low steady-state open probability (Popen, 0.07 ± 0.008; n = 15), little apparent desensitisation and were also activated by α,β-methylene ATP (α,β-meATP, 40 μm; Popen, 0.007 ± 0.0002; conductance, 57 ± 1.1 pS; n = 3). No decrease in the single channel conductance was observed on increasing the free extracellular calcium concentration from 0.3 to 0.85 mm. Channel closed time distributions were fitted with five exponential components with time constants (and relative areas) of 90 μs (20%), 0.77 ms (32%), 10 ms (15%), 90 ms (18%) and 403 ms (15%) at 1 mm ATP. Of these, the first two components are suggested to represent gaps within single activations of the receptor based on the lack of agonist concentration dependence of these two shut time components between 1 μm and 1 mm ATP. Suramin (40 μm) significantly increased the single channel conductance (19 ± 7%; n = 5

  7. Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus

    PubMed Central

    Kitahara, Yosuke; Ohta, Keisuke; Hasuo, Hiroshi; Shuto, Takahide; Kuroiwa, Mahomi; Sotogaku, Naoki; Togo, Akinobu; Nakamura, Kei-ichiro; Nishi, Akinori

    2016-01-01

    A selective serotonin reuptake inhibitor is the most commonly prescribed antidepressant for the treatment of major depression. However, the mechanisms underlying the actions of selective serotonin reuptake inhibitors are not fully understood. In the dentate gyrus, chronic fluoxetine treatment induces increased excitability of mature granule cells (GCs) as well as neurogenesis. The major input to the dentate gyrus is the perforant path axons (boutons) from the entorhinal cortex (layer II). Through voltage-sensitive dye imaging, we found that the excitatory neurotransmission of the perforant path synapse onto the GCs in the middle molecular layer of the mouse dentate gyrus (perforant path-GC synapse) is enhanced after chronic fluoxetine treatment (15 mg/kg/day, 14 days). Therefore, we further examined whether chronic fluoxetine treatment affects the morphology of the perforant path-GC synapse, using FIB/SEM (focused ion beam/scanning electron microscopy). A three-dimensional reconstruction of dendritic spines revealed the appearance of extremely large-sized spines after chronic fluoxetine treatment. The large-sized spines had a postsynaptic density with a large volume. However, chronic fluoxetine treatment did not affect spine density. The presynaptic boutons that were in contact with the large-sized spines were large in volume, and the volumes of the mitochondria and synaptic vesicles inside the boutons were correlated with the size of the boutons. Thus, the large-sized perforant path-GC synapse induced by chronic fluoxetine treatment contains synaptic components that correlate with the synapse size and that may be involved in enhanced glutamatergic neurotransmission. PMID:26788851

  8. Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus.

    PubMed

    Kitahara, Yosuke; Ohta, Keisuke; Hasuo, Hiroshi; Shuto, Takahide; Kuroiwa, Mahomi; Sotogaku, Naoki; Togo, Akinobu; Nakamura, Kei-ichiro; Nishi, Akinori

    2016-01-01

    A selective serotonin reuptake inhibitor is the most commonly prescribed antidepressant for the treatment of major depression. However, the mechanisms underlying the actions of selective serotonin reuptake inhibitors are not fully understood. In the dentate gyrus, chronic fluoxetine treatment induces increased excitability of mature granule cells (GCs) as well as neurogenesis. The major input to the dentate gyrus is the perforant path axons (boutons) from the entorhinal cortex (layer II). Through voltage-sensitive dye imaging, we found that the excitatory neurotransmission of the perforant path synapse onto the GCs in the middle molecular layer of the mouse dentate gyrus (perforant path-GC synapse) is enhanced after chronic fluoxetine treatment (15 mg/kg/day, 14 days). Therefore, we further examined whether chronic fluoxetine treatment affects the morphology of the perforant path-GC synapse, using FIB/SEM (focused ion beam/scanning electron microscopy). A three-dimensional reconstruction of dendritic spines revealed the appearance of extremely large-sized spines after chronic fluoxetine treatment. The large-sized spines had a postsynaptic density with a large volume. However, chronic fluoxetine treatment did not affect spine density. The presynaptic boutons that were in contact with the large-sized spines were large in volume, and the volumes of the mitochondria and synaptic vesicles inside the boutons were correlated with the size of the boutons. Thus, the large-sized perforant path-GC synapse induced by chronic fluoxetine treatment contains synaptic components that correlate with the synapse size and that may be involved in enhanced glutamatergic neurotransmission. PMID:26788851

  9. Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus.

    PubMed

    Kitahara, Yosuke; Ohta, Keisuke; Hasuo, Hiroshi; Shuto, Takahide; Kuroiwa, Mahomi; Sotogaku, Naoki; Togo, Akinobu; Nakamura, Kei-ichiro; Nishi, Akinori

    2016-01-01

    A selective serotonin reuptake inhibitor is the most commonly prescribed antidepressant for the treatment of major depression. However, the mechanisms underlying the actions of selective serotonin reuptake inhibitors are not fully understood. In the dentate gyrus, chronic fluoxetine treatment induces increased excitability of mature granule cells (GCs) as well as neurogenesis. The major input to the dentate gyrus is the perforant path axons (boutons) from the entorhinal cortex (layer II). Through voltage-sensitive dye imaging, we found that the excitatory neurotransmission of the perforant path synapse onto the GCs in the middle molecular layer of the mouse dentate gyrus (perforant path-GC synapse) is enhanced after chronic fluoxetine treatment (15 mg/kg/day, 14 days). Therefore, we further examined whether chronic fluoxetine treatment affects the morphology of the perforant path-GC synapse, using FIB/SEM (focused ion beam/scanning electron microscopy). A three-dimensional reconstruction of dendritic spines revealed the appearance of extremely large-sized spines after chronic fluoxetine treatment. The large-sized spines had a postsynaptic density with a large volume. However, chronic fluoxetine treatment did not affect spine density. The presynaptic boutons that were in contact with the large-sized spines were large in volume, and the volumes of the mitochondria and synaptic vesicles inside the boutons were correlated with the size of the boutons. Thus, the large-sized perforant path-GC synapse induced by chronic fluoxetine treatment contains synaptic components that correlate with the synapse size and that may be involved in enhanced glutamatergic neurotransmission.

  10. Synaptic commitment: developmentally regulated reciprocal changes in hippocampal granule cell NMDA and AMPA receptors over the lifespan.

    PubMed

    Yang, Zhiyong; Krause, Michael; Rao, Geeta; McNaughton, Bruce L; Barnes, C A

    2008-06-01

    Synaptic transmission in hippocampal field CA1 is largely N-methyl-d-aspartate receptor (NMDA(R)) dependent during the early postnatal period. It becomes increasingly mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) receptors until an adult ratio of AMPA to NMDA receptors is achieved. It is shown here that increases in the AMPA receptor (AMPA(R))-mediated field potential response continue over the life span of the F-344 rat at the perforant path-granule cell synapse in the dentate gyrus. In contrast, the NMDA(R)-dependent component of the response decreases with age between 1 and 27 mo, leading to an increase of AMPA(R)/NMDA(R) ratio with age. One possible explanation of this age difference is that the AMPA(R)/NMDA(R) ratio can be modified by experience. To test the idea that the changed ratio is caused by the old rats' longer lives, an intensive 10-mo period of enrichment treatment was given to a group of animals, beginning at 3 mo of age. Compared with animals housed in standard cages, the enrichment treatment did not alter the glutamatergic response ratio measured with field potential recording methods. These data provide support for the conclusion that the observed change with age is developmentally regulated rather than experience dependent. Given the role of the NMDA(R) in synaptic plasticity, these changes suggest a progressive commitment of perforant path synapses to particular weights over the life span. One possible implication of this effect includes preservation of selected memories, ultimately at the expense of a reduced capacity to store new information.

  11. Effect of neurotrophin-3 precursor on glutamate-induced calcium homeostasis deregulation in rat cerebellum granule cells.

    PubMed

    Safina, Dina R; Surin, Alexander M; Pinelis, Vsevolod G; Kostrov, Sergey V

    2015-12-01

    Neurotrophin-3 (NT-3) belongs to the family of highly conserved dimeric growth factors that controls the differentiation and activity of various neuronal populations. Mammals contain both the mature (NT-3) and the precursor (pro-NT-3) forms of neurotrophin. Members of the neurotrophin family are involved in the regulation of calcium homeostasis in neurons; however, the role of NT-3 and pro-NT-3 in this process remains unclear. The current study explores the effects of NT-3 and pro-NT-3 on disturbed calcium homeostasis and decline of mitochondrial potential induced by a neurotoxic concentration of glutamate (Glu; 100 µM) in the primary culture of rat cerebellar granule cells. In this Glu excitotoxicity model, mature NT-3 had no effect on the induced changes in Ca²⁺ homeostasis. In contrast, pro-NT-3 decreased the period of delayed calcium deregulation (DCD) and concurrent strong mitochondrial depolarization. According to the amplitude of the increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]i ) and Fura-2 fluorescence quenching by Mn²⁺ within the first 20 sec of exposure to Glu, pro-NT-3 had no effect on the initial rate of Ca²⁺ entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca²⁺]i rise was 1.2-fold greater in the presence of pro-NT-3 than in the presence of Glu alone (1.67 ±  0.07 and 1.39 ± 0.04, respectively, P < 0.05). The Glu-induced changes in Са²⁺ homeostasis in the presence of pro-NT-3 likely are due to the decreased rate of Са²⁺ removal from the cytosol during the DCD latency period.

  12. Ectopic expression of anti-HIV-1 shRNAs protects CD8{sup +} T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation

    SciTech Connect

    Kamata, Masakazu; Kim, Patrick Y.; Ng, Hwee L.; Ringpis, Gene-Errol E.; Kranz, Emiko; Chan, Joshua; O'Connor, Sean; Yang, Otto O.; Chen, Irvin S.Y.

    2015-07-31

    Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8{sup +} T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8{sup +} T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8{sup +} T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24{sup Gag} in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8{sup +} T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8{sup +} T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8{sup +} T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance. - Highlights: • Ectopic expression of CD4ζ CAR in CD8{sup +} T cells renders them susceptible to HIV-1 infection. • Co-expression of two anti-HIV-1 shRNAs protects CD4ζ CAR-modified CD8{sup +} T cells from HIV-1 infection. • Protecting CD4ζ CAR-modified CD8{sup +} T cells from HIV-1 infection suppresses its cytopathic effect.

  13. Continuous electricity generation by a graphite granule baffled air-cathode microbial fuel cell.

    PubMed

    Feng, Yujie; Lee, He; Wang, Xin; Liu, Yaolan; He, Weihua

    2010-01-01

    A baffled air-cathode microbial fuel cell (BAFMFC) was designed and operated under continuous flow. With glucose fed as substrate, an average voltage of 652 mV was obtained under the external resistance of 1000 Omega (30 degrees C). The maximum power density was 15.2 W/m(3) with the chemical oxygen demand (COD) removal rate of 88.0%. The overall resistance was 13.7 Omega while ohmic internal resistance was 10.8 Omega. Average COD removal rate was 69.7-88.0%, when COD loading varied from 4.11 kg COD/(m(3)NACd) to 16.0 kg COD/(m(3)NACd). The liquid from corn stover steam explosion process (COD=7160+/-50mg/L) was treated by BAFMFC, and the maximum power density was 10.7 W/m(3) with the average COD removal rate was 89.1%. The present study indicated BAFMFC can be comparable to the traditional anaerobic baffled reactor in COD removal rate for high-concentration wastewater and have an advantage in energy harvest from wastewater.

  14. Ectopic Osteogenesis of Macroscopic Tissue Constructs Assembled from Human Mesenchymal Stem Cell-Laden Microcarriers through In Vitro Perfusion Culture

    PubMed Central

    Chen, Maiqin; Zhou, Min; Ye, Zhaoyang; Zhou, Yan; Tan, Wen-Song

    2014-01-01

    We had previously demonstrated the feasibility of preparing a centimeter-sized bone tissue construct by following a modular approach. In the present study, the objectives were to evaluate osteogenesis and tissue formation of human amniotic mesenchymal stem cells-laden CultiSpher S microcarriers during in vitro perfusion culture and after subcutaneous implantation. Microtissues were prepared in dynamic culture using spinner flasks in 28 days. In comparison with 1-week perfusion culture, microtissues became more obviously fused, demonstrating significantly higher cellularity, metabolic activity, ALP activity and calcium content while maintaining cell viability after 2-week perfusion. After subcutaneous implantation in nude mice for 6 and 12 weeks, all explants showed tight contexture, suggesting profound tissue remodeling in vivo. In addition, 12-week implantation resulted in slightly better tissue properties. However, in vitro perfusion culture time exerted great influence on the properties of corresponding explants. Degradation of microcarriers was more pronounced in the explants of 2-week perfused macrotissues compared to those of 1-week perfusion and directly implanted microtissues. Moreover, more blood vessel infiltration and bone matrix deposition with homogeneous spatial distribution were found in the explants of 2-week perfused macrotissues. Taken together, in vitro perfusion culture time is critical in engineering bone tissue replacements using such a modular approach, which holds great promise for bone regeneration. PMID:25275528

  15. Granulation of fine powder

    DOEpatents

    Chen, Ching-Fong

    2016-08-09

    A mixture of fine powder including thorium oxide was converted to granulated powder by forming a first-green-body and heat treating the first-green-body at a high temperature to strengthen the first-green-body followed by granulation by crushing or milling the heat-treated first-green-body. The granulated powder was achieved by screening through a combination of sieves to achieve the desired granule size distribution. The granulated powder relies on the thermal bonding to maintain its shape and structure. The granulated powder contains no organic binder and can be stored in a radioactive or other extreme environment. The granulated powder was pressed and sintered to form a dense compact with a higher density and more uniform pore size distribution.

  16. 2-Arachidonoylglycerol elicits neuroprotective effects on excitotoxically lesioned dentate gyrus granule cells via abnormal-cannabidiol-sensitive receptors on microglial cells.

    PubMed

    Kreutz, Susanne; Koch, Marco; Böttger, Charlotte; Ghadban, Chalid; Korf, Horst-Werner; Dehghani, Faramarz

    2009-02-01

    Endocannabinoids like 2-arachidonoylglycerol (2-AG) exert neuroprotective effects after brain injuries. According to current concepts, these neuroprotective effects are due to interactions between 2-AG and cannabinoid (CB)1 receptors on neurons. Moreover, 2-AG modulates migration and proliferation of microglial cells which are rapidly activated after brain lesion. This effect is mediated via CB2- and abnormal-cannabidiol (abn-CBD)-sensitive receptors. In the present study, we investigated whether the abn-CBD-sensitive receptor on microglial cells contributes to 2-AG-mediated neuroprotection in organotypic hippocampal slice cultures (OHSCs) after excitotoxic lesion induced by NMDA (50 microM) application for 4 h. This lesion caused neuronal damage and accumulation of microglial cells within the granule cell layer. To analyze the role of abn-CBD-sensitive receptors for neuroprotection and microglial cell accumulation, two agonists of the abn-CBD-sensitive receptor, abn-CBD or 2-AG, two antagonists, 1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen1-yl]-benzene (O-1918) or cannabidiol (CBD), and the CB1 receptor antagonist AM251, were applied to NMDA-lesioned OHSC. Propidium iodide (PI) labeling was used as a marker of degenerating neurons and isolectin B(4) (IB(4)) as a marker of microglial cells. Application of both, abn-CBD or 2-AG to lesioned OHSC significantly decreased the number of IB(4)(+) microglial cells and PI(+) neurons in the dentate gyrus. In contrast to AM251, application of O-1918 or CBD antagonized these effects. When microglial cells were depleted by preincubation of OHSC with the bisphosphonate clodronate (100 microg/mL) for 5 days before excitotoxic lesion, 2-AG and abn-CBD lost their neuroprotective effects. We therefore propose that the endocannabinoid 2-AG exerts its neuroprotective effects via activation of abn-CBD-sensitive receptors on microglial cells.

  17. Impaired reelin processing and secretion by Cajal-Retzius cells contributes to granule cell dispersion in a mouse model of temporal lobe epilepsy.

    PubMed

    Duveau, Venceslas; Madhusudan, Amrita; Caleo, Matteo; Knuesel, Irene; Fritschy, Jean-Marc

    2011-09-01

    Cajal-Retzius cells play a crucial role during ontogeny in regulating cortical lamination via release of reelin. In adult brain, they comprise small calretinin-positive interneurons located in the marginal zone of the cerebral cortex and in the hippocampal fissure. Alterations of reelin signaling or expression have been involved in major neurological disorders, and they underlie granule cell dispersion (GCD) in mesial temporal lobe epilepsy (TLE). Here, we investigated in a mouse model of TLE the contribution of Cajal-Retzius cells to reelin production in epileptic hippocampus and the molecular mechanisms underlying GCD. Following unilateral intrahippocampal Kainic acid injection in adult mice to induce an epileptic focus, we observed that Cajal-Retzius cells gradually became strongly immunopositive for reelin, due to intracellular accumulation. This phenotype resembled the morphology of Cajal-Retzius cells in reeler Orleans (reln (orl/orl) ) mice, which express a secretion-deficient 310-kDa reelin fragment. The possibility that GCD might result from abnormal reelin processing in Cajal-Retzius cells, leading to a lack of reelin secretion, was confirmed by KA injection in reln (orl/+) mice, which induced severe GCD. Furthermore, Western blot analysis in KA-treated wildtype mice revealed increased production of ∼300-kDa reelin fragments, confirming abnormal proteolytic processing. This effect was not seen upon treatment with Botulinum neurotoxin E (BoNT/E), which prevents GCD in KA-lesioned hippocampus by chronic blockade of synaptic transmission. Furthermore, BoNT/E blocked upregulation of TrkB in Cajal-Retzius cells, suggesting that production of truncated reelin in KA-treated hippocampus is activity-dependent and regulated by BDNF. Altogether, these data reveal that GCD results from abnormal reelin processing in Cajal-Retzius cells under the control of BDNF. Our findings highlight the critical role played by Cajal-Retzius cells for hippocampal neuronal

  18. Label-free distinguishing between neurons and glial cells based on two-photon excited fluorescence signal of neuron perinuclear granules

    NASA Astrophysics Data System (ADS)

    Du, Huiping; Jiang, Liwei; Wang, Xingfu; Liu, Gaoqiang; Wang, Shu; Zheng, Liqin; Li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Chen, Jianxin

    2016-08-01

    Neurons and glial cells are two critical cell types of brain tissue. Their accurate identification is important for the diagnosis of psychiatric disorders such as depression and schizophrenia. In this paper, distinguishing between neurons and glial cells by using the two-photon excited fluorescence (TPEF) signals of intracellular intrinsic sources was performed. TPEF microscopy combined with TUJ-1 and GFAP immunostaining and quantitative image analysis demonstrated that the perinuclear granules of neurons in the TPEF images of brain tissue and the primary cultured cortical cells were a unique characteristic of neurons compared to glial cells which can become a quantitative feature to distinguish neurons from glial cells. With the development of miniaturized TPEF microscope (‘two-photon fiberscopes’) imaging devices, TPEF microscopy can be developed into an effective diagnostic and monitoring tool for psychiatric disorders such as depression and schizophrenia.

  19. Impairment of stress granule assembly via inhibition of the eIF2alpha phosphorylation sensitizes glioma cells to chemotherapeutic agents.

    PubMed

    Vilas-Boas, Fabrício de Almeida Souza; da Silva, Aristóbolo Mendes; de Sousa, Lirlândia Pires; Lima, Kátia Maciel; Vago, Juliana Priscila; Bittencourt, Lucas Felipe Fernandes; Dantas, Arthur Estanislau; Gomes, Dawidson Assis; Vilela, Márcia Carvalho; Teixeira, Mauro Martins; Barcelos, Lucíola Silva

    2016-04-01

    Malignant gliomas are a lethal type of brain tumors that poorly respond to chemotherapeutic drugs. Several therapy resistance mechanisms have been characterized. However, the response to stress through mRNA translational control has not been evaluated for this type of tumor. A potential target would involve the alpha subunit of eukaryotic translation initiation factor (eIF2α) that leads to assembly of stress granules (SG) which are cytoplasmic granules mainly composed by RNA binding proteins and untranslated mRNAs. We assessed whether glioma cells are capable of assembling SG after exposure to different classes of chemotherapeutic agents through evaluation of the effects of interfering in this process by impairing the eIF2α signaling. C6 and U87MG cells were exposed to bortezomib, cisplatin, or etoposide. Forced expression of a dominant negative mutant of eIF2α (eIF2α(DN)) was employed to block this pathway. We observed that exposure to drugs stimulated SG assembly. This was reduced in eIF2α(DN)-transfected cells and this strategy enhanced chemotherapeutically-induced cell death for all drugs. Our data suggest that SG assembly occurs in glioma cells in response to chemotherapeutic drugs in an eIF2α-dependent manner and this response is relevant for drug resistance. Interfering with eIF2α signaling pathway may be a potential strategy for new co-adjuvant therapies to treat gliomas. PMID:26732083

  20. The natural scorpion peptide, BmK NT1 activates voltage-gated sodium channels and produces neurotoxicity in primary cultured cerebellar granule cells.

    PubMed

    Zou, Xiaohan; He, Yuwei; Qiao, Jinping; Zhang, Chunlei; Cao, Zhengyu

    2016-01-01

    The scorpion Buthus martensii Karsch has been used in Traditional Chinese Medicine to treat neuronal diseases such as neuropathic pain, paralysis and epilepsy for thousands of years. Studies have demonstrated that scorpion venom is the primary active component. Although scorpion venom can effectively attenuate pain in the clinic, it also produces neurotoxic response. In this study, toxicity guided purification led to identify a mammalian toxin termed BmK NT1 comprising of 65 amino acid residues and an amidated C-terminus, a mature peptide encoded by the nucleotide sequence (GenBank No. AF464898). In contract to the recombinant product of the same nucleotide sequence, BmK AGAP, which displayed analgesic and anti-tumor effect, intravenous injection (i.v.) of BmK NT1 produced acute toxicity in mice with an LD50 value of 1.36 mg/kg. In primary cultured cerebellar granule cells, BmK NT1 produced a concentration-dependent cell death with an IC50 value of 0.65 μM (0.41-1.03 μM, 95% Confidence Intervals, 95% CI) which was abolished by TTX, a voltage-gated sodium channel (VGSC) blocker. We also demonstrated that BmK NT1 produced modest sodium influx in cerebellar granule cell cultures with an EC50 value of 2.19 μM (0.76-6.40 μM, 95% CI), an effect similar to VGSC agonist, veratridine. The sodium influx response was abolished by TTX suggesting that BmK NT1-induced sodium influx is solely through activation of VGSC. Considered these data together, we demonstrated that BmK NT1 activated VGSC and produced neurotoxicity in cerebellar granule cell cultures. PMID:26598793

  1. Impairment of granulation tissue formation after menopause.

    PubMed

    Gniadecki, R; Wyrwas, B; Kabala, A; Matecka, J

    1996-04-01

    Formation of connective tissue is an essential step in the process of wound healing. After menopause an atrophy of connective tissues in skin, bone, and reproductive organs takes place. Using a dead-space wound healing model we measured collagen synthesis and deposition, and cell replication in the granulation tissue of 18 premenopausal and 23 peri- and postmenopausal women not receiving any hormonal therapy. In the postmenopausal group collagen synthesis and deposition and cell number in the granulation tissue were diminished. These results document the impairment of the granulation tissue formation after menopause.

  2. Clinical study of ectopic pregnancy.

    PubMed

    Chhabra, S; Aher, K; Jaiswal, M

    1992-01-01

    Ectopic pregnancy remains a leading cause of maternal mortality and accounts for a sizeable proportion of infertility and ectopic recurrence. The possibility that a woman is experiencing an ectopic pregnancy must be considered when evaluating a woman, especially a sterilized woman, who has a possible pregnancy, amenorrhea, abdominal pain, or abnormal bleeding; studies have found that one in six pregnancies occurring after tubal sterilization are ectopic. The authors present a clinical study of 82 cases of ectopic pregnancy admitted to the department of Obstetrics and Gynecology of Mahatma Gandhi Institute of Medical Sciences, Sevagram. Cases of ectopic pregnancy represent 0.99% of total obstetric admissions, of whom 69.51% were diagnosed as such on admission. 40.24% of the women were older than 30 years, while 34.14% were elderly beyond third parity. 70.73% of the women presented before missing their second period. Patients presented with multiple complaints, but the most common was abdominal pain reported by 61.70%. 78.04% were admitted with an acute abdomen, but shock was present in only 7.14% of cases. The main surgical treatment modality was salpingectomy among 59.75%. There was no maternal mortality through postoperative morbidity in the form of paralytic ileus, although fever did occur in some women.

  3. YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog - dependent cerebellar granule neuron progenitor cells and medulloblastoma cells

    PubMed Central

    Dey, Abhinav; Robitaille, Mélanie; Remke, Marc; Maier, Caroline; Malhotra, Anshu; Gregorieff, Alex; Wrana, Jeffrey L; Taylor, Michael D; Angers, Stéphane; Kenney, Anna Marie

    2016-01-01

    Post-natal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for the SHH-associated subgroup of medulloblastoma (MB), is driven by Sonic Hedgehog (Shh) and Insulin-like Growth Factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is up-regulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh-subgroup of MB in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and medulloblastoma cells and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas. PMID:26725322

  4. Methylmercury disrupts the balance between phosphorylated and non-phosphorylated cofilin in primary cultures of mice cerebellar granule cells A proteomic study

    SciTech Connect

    Vendrell, Iolanda; Carrascal, Montserrat; Abian, Joaquin

    2010-01-01

    Methylmercury is an environmental contaminant that is particularly toxic to the developing central nervous system; cerebellar granule neurons are especially vulnerable. Here, primary cultures of cerebellar granule cells (CGCs) were continuously exposed to methylmercury for up to 16 days in vitro (div). LC50 values were 508 +- 199, 345 +- 47, and 243 +- 45 nM after exposure for 6, 11, and 16 div, respectively. Proteins from cultured mouse CGCs were separated by 2DE. Seventy-one protein spots were identified by MALDI-TOF PMF and MALDI-TOF/TOF sequencing. Prolonged exposure to a subcytotoxic concentration of methylmercury significantly increased non-phosphorylated cofilin both in cell protein extracts (1.4-fold; p < 0.01) and in mitochondrial-enriched fractions (1.7-fold; p < 0.01). The decrease in P-cofilin induced by methylmercury was concentration-dependent and occurred after different exposure times. The percentage of P-cofilin relative to total cofilin significantly decreased to 49 +- 13% vs. control cells after exposure to 300 nM methylmercury for 5 div. The balance between the phosphorylated and non-phosphorylated form of cofilin regulates actin dynamics and facilitates actin filament turnover. Filamentous actin dynamics and reorganization are responsible of neuron shape change, migration, polarity formation, regulation of synaptic structures and function, and cell apoptosis. An alteration of the complex regulation of the cofilin phosphorylation/dephosphorylation pathway could be envisaged as an underlying mechanism compatible with reported signs of methylmercury-induced neurotoxicity.

  5. Synaptic and extrasynaptic plasticity in glutamatergic circuits involving dentate granule cells following chronic N-methyl-d-aspartate receptor inhibition

    PubMed Central

    He, Shuijin; Shao, Li-Rong; Wang, Yu

    2013-01-01

    Chronic global N-methyl-d-aspartate receptor (NMDAR) blockade leads to changes in glutamatergic transmission. The impact of more subunit-selective NMDAR inhibition on glutamatergic circuits remains incomplete. To this end, organotypic hippocampal slice cultures were treated for 17–21 days with the high-affinity competitive antagonist d-aminophosphonovaleric acid (d-APV), the allosteric GluN2B-selective antagonist Ro25-6981, or the newer competitive GluN2A-preferring antagonist NVP-AAM077. Electrophysiological recordings from dentate granule cells revealed that chronic d-APV treatment increased, whereas chronic Ro25-6981 reduced, epileptiform event-associated large-amplitude spontaneous excitatory postsynaptic currents (sEPSC) compared with all other treatment groups, consistent with opposite effects on glutamatergic networks. Presynaptically, chronic d-APV or Ro25-6981 increased small-amplitude sEPSCs and AMPA/kainate receptor-mediated miniature EPSCs (mEPSCAMPAR) frequency. Chronic d-APV or NVP-AAM077, but not Ro25-6981, increased putative vGlut1-positive glutamatergic synapses. Postsynaptically, chronic d-APV dramatically increased mEPSCAMPAR and profoundly decreased NMDAR-mediated mEPSC (mEPSCNMDAR) measures, suggesting increased AMPAR/NMDAR ratio. Ro25-6981 decreased mEPSCAMPAR charge transfer and modestly decreased mEPSCNMDAR frequency and decay, suggesting downward scaling of AMPAR and NMDAR function without dramatically altering AMPAR/NMDAR ratio. Extrasynaptically, threo-β-benzyloxyaspartate-enhanced “tonic” NMDAR current amplitude and activated channel number estimates were significantly increased only by chronic Ro25-6981. For intrinsic excitability, action potential threshold was slightly more negative following chronic d-APV or NVP-AAM077. The predominant pro-excitatory effects of chronic d-APV are consistent with increased glutamatergic transmission and network excitability. The minor effects of chronic NVP-AAM077 on action potential threshold

  6. Nano-granulization of gadolinia-doped ceria electrolyte surface by aerosol-assisted chemical vapor deposition for low-temperature solid oxide fuel cells

    NASA Astrophysics Data System (ADS)

    Kim, Jun Woo; Jang, Dong Young; Kim, Manjin; Choi, Hyung Jong; Shim, Joon Hyung

    2016-01-01

    We have fabricated nano-scale gadolinia-doped ceria (GDC) at the electrode-electrolyte boundary by aerosol-assisted chemical vapor deposition (AACVD) for high-performance solid oxide fuel cells (SOFCs) working at low temperatures below 500 °C. In AACVD, temperature is the key factor affecting the grain size. We have confirmed that by nano-granulizing the electrolyte surface using optimized AACVD, the power output of the SOFC is 50% higher than that of the bare GDC SOFC. From the impedance analysis, significant enhancement of the cathodic oxygen reduction reaction is identified from the AACVD-GDC nano-grain surface treatment.

  7. Ectopic expression of anti-HIV-1 shRNAs protects CD8+ T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation

    PubMed Central

    Kamata, Masakazu; Kim, Patrick Y.; Ng, Hwee L.; Ringpis, Gene-Errol E.; Kranz, Emiko; Chan, Joshua; O'Connor, Sean; Yang, Otto O.; Chen, Irvin S.Y.

    2015-01-01

    Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8+ T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8+ T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8+ T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24Gag in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8+ T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8+ T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8+ T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance. PMID:25998390

  8. Ectopic expression of anti-HIV-1 shRNAs protects CD8(+) T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation.

    PubMed

    Kamata, Masakazu; Kim, Patrick Y; Ng, Hwee L; Ringpis, Gene-Errol E; Kranz, Emiko; Chan, Joshua; O'Connor, Sean; Yang, Otto O; Chen, Irvin S Y

    2015-07-31

    Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8(+) T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8(+) T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8(+) T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24(Gag) in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8(+) T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8(+) T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8(+) T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance. PMID:25998390

  9. [Ectopic hamartomatous thymoma: a case report and review of the literature].

    PubMed

    de Saint Aubain Somerhausen, Nicolas; Richard, Christine; Priollet, Dominique; Dequanter, Didier

    2004-04-01

    We report a case of a rare tumor of the head and neck: ectopic hamartomatous thymoma. The tumor involved the sus-clavicular area of a 48 year-old male. Ectopic hamartomatous thymoma exclusively occurs in the sus-scapular region. Histologically, it is characterized by the association of spindle-cell fascicles, epithelial solid or cystic structures and foci of mature adipose tIssue. Spindle cells express diffusely cytokeratins. The differential diagnosis of ectopic hamartomatous thymoma includes mixed tumor of salivary glands, biphasic synovial sarcoma and sarcomatoid carcinoma. Ectopic hamartomatous thymoma is a benign tumor which does not recur after complete excision.

  10. Sniff-Like Patterned Input Results in Long-Term Plasticity at the Rat Olfactory Bulb Mitral and Tufted Cell to Granule Cell Synapse

    PubMed Central

    Perez de los Cobos Pallares, Fernando; Loebel, Alex; Lukas, Michael

    2016-01-01

    During odor sensing the activity of principal neurons of the mammalian olfactory bulb, the mitral and tufted cells (MTCs), occurs in repetitive bursts that are synchronized to respiration, reminiscent of hippocampal theta-gamma coupling. Axonless granule cells (GCs) mediate self- and lateral inhibitory interactions between the excitatory MTCs via reciprocal dendrodendritic synapses. We have explored long-term plasticity at this synapse by using a theta burst stimulation (TBS) protocol and variations thereof. GCs were excited via glomerular stimulation in acute brain slices. We find that TBS induces exclusively long-term depression in the majority of experiments, whereas single bursts (“single-sniff paradigm”) can elicit both long-term potentiation and depression. Statistical analysis predicts that the mechanism underlying this bidirectional plasticity involves the proportional addition or removal of presynaptic release sites. Gamma stimulation with the same number of APs as in TBS was less efficient in inducing plasticity. Both TBS- and “single-sniff paradigm”-induced plasticity depend on NMDA receptor activation. Since the onset of plasticity is very rapid and requires little extra activity, we propose that these forms of plasticity might play a role already during an ongoing search for odor sources. Our results imply that components of both short-term and long-term olfactory memory may be encoded at this synapse. PMID:27747107

  11. Protection of NMDA-induced neuronal cell damage by methanol extract of zizyphi spinosi semen in cultured rat cerebellar granule cells.

    PubMed

    Park, Jeong Hee; Lee, Hyun Joo; Koh, Sang Bum; Ban, Ju Yeon; Seong, Yeon Hee

    2004-11-01

    Zizypus is one of the herbs widely used in Korea and China due to the CNS calming effect. The present study aims to investigate the effect of the methanol extract of Zizyphi Spinosi Semen (ZSS), the seeds of Zizyphus jujuba Mill var. spinosa, on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in cultured rat cerebellar granule neuron. ZSS, over a concentration range of 0.05-5 microg/ml, inhibited NMDA (1 mM)-induced neuronal cell death, which was measured by a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. ZSS (0.5 microg/ml) inhibited glutamate release into medium induced by NMDA (1mM), which was measured by HPLC. Pretreatment of ZSS (0.5 microg/ml) inhibited NMDA (1mM)-induced elevation of cytosolic calcium concentration ([Ca(2+)](c)), which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that ZSS prevents NMDA-induced neuronal cell damage in vitro. PMID:15374605

  12. Perinatal exposure to low-dose methylmercury induces dysfunction of motor coordination with decreases in synaptophysin expression in the cerebellar granule cells of rats.

    PubMed

    Fujimura, Masatake; Cheng, Jinping; Zhao, Wenchang

    2012-06-29

    Methylmercury (MeHg) is an environmental pollutant that is toxic to the developing central nervous system (CNS) in children, even at low exposure levels. Perinatal exposure to MeHg is known to induce neurological symptoms with neuropathological changes in the CNS. However, the relationship between the neurological symptoms and neuropathological changes induced in offspring as a result of exposure to low-dose MeHg is not well defined. In the present study, neurobehavioral analyses revealed that exposure to a low level of MeHg (5 ppm in drinking water) during developmental caused a significant deficit in the motor coordination of rats in the rotating rod test. In contrast, general neuropathological findings, including neuronal cell death and the subsequent nerve inflammation, were not observed in the region of the cerebellum responsible for regulating motor coordination. Surprisingly, the expression of synaptophysin (SPP), a marker protein for synaptic formation, significantly decreased in cerebellar granule cells. These results showed that perinatal exposure to low-dose MeHg causes neurobehavioral impairment without general neuropathological changes in rats. We demonstrated for the first time that exposure to low-dose MeHg during development induces the dysfunction of motor coordination due to changes of synaptic homeostasis in cerebellar granule cells.

  13. Mitotic Events in Cerebellar Granule Progenitor Cells that Expand Cerebellar Surface Area Are Critical for Normal Cerebellar Cortical Lamination in Mice

    PubMed Central

    Chang, Joshua C.; Leung, Mark; Gokozan, Hamza Numan; Gygli, Patrick Edwin; Catacutan, Fay Patsy; Czeisler, Catherine; Otero, José Javier

    2015-01-01

    Late embryonic and postnatal cerebellar folial surface area expansion promotes cerebellar cortical cytoarchitectural lamination. We developed a streamlined sampling scheme to generate unbiased estimates of murine cerebellar surface area and volume using stereological principles. We demonstrate that during the proliferative phase of the external granule layer (EGL) and folial surface area expansion, EGL thickness does not change and thus is a topological proxy for progenitor self-renewal. The topological constraints indicate that during proliferative phases, migration out of the EGL is balanced by self-renewal. Progenitor self-renewal must, therefore, include mitotic events yielding either 2 cells in the same layer to increase surface area (β-events) and mitotic events yielding 2 cells, with 1 cell in a superficial layer and 1 cell in a deeper layer (α-events). As the cerebellum grows, therefore, β-events lie upstream of α-events. Using a mathematical model constrained by the measurements of volume and surface area, we could quantify inter-mitotic times for β-events on a per-cell basis in post-natal mouse cerebellum. Furthermore, we found that loss of CCNA2, which decreases EGL proliferation and secondarily induces cerebellar cortical dyslamination, shows preserved α-type events. Thus, CCNA2-null cerebellar granule progenitor cells are capable of self-renewal of the EGL stem cell niche; this is concordant with prior findings of extensive apoptosis in CCNA2-null mice. Similar methodologies may provide another layer of depth to the interpretation of results from stereological studies. PMID:25668568

  14. The type 3 adenylyl cyclase is required for the survival and maturation of newly generated granule cells in the olfactory bulb.

    PubMed

    Luo, Jie; Chen, Xuanmao; Pan, Yung-Wei; Lu, Song; Xia, Zhengui; Storm, Daniel R

    2015-01-01

    The type 3 adenylyl cyclase (AC3) is localized to olfactory cilia in the main olfactory epithelium (MOE) and primary cilia in the adult mouse brain. Although AC3 has been strongly implicated in odor perception and olfactory sensory neuron (OSN) targeting, its role in granule cells (GCs), the most abundant interneurons in the main olfactory bulb (MOB), remains largely unknown. Here, we report that the deletion of AC3 leads to a significant reduction in the size of the MOB as well as the level of adult neurogenesis. The cell proliferation and cell cycle in the subventricular zone (SVZ), however, are not suppressed in AC3-/- mice. Furthermore, AC3 deletion elevates the apoptosis of GCs and disrupts the maturation of newly formed GCs. Collectively, our results identify a fundamental role for AC3 in the development of adult-born GCs in the MOB.

  15. Selective isolation of ammonia-oxidizing bacteria from autotrophic nitrifying granules by applying cell-sorting and sub-culturing of microcolonies.

    PubMed

    Fujitani, Hirotsugu; Kumagai, Asami; Ushiki, Norisuke; Momiuchi, Kengo; Tsuneda, Satoshi

    2015-01-01

    Nitrification is a key process in the biogeochemical nitrogen cycle and biological wastewater treatment that consists of two stepwise reactions, ammonia oxidation by ammonia-oxidizing bacteria (AOB) or archaea followed by nitrite oxidation by nitrite-oxidizing bacteria. One of the representatives of the AOB group is Nitrosomonas mobilis species. Although a few pure strains of this species have been isolated so far, approaches to their preservation in pure culture have not been established. Here, we report isolation of novel members of the N. mobilis species from autotrophic nitrifying granules used for ammonia-rich wastewater treatment. We developed an isolation method focusing on microcolonies formation of nitrifying bacteria. Two kinds of distinctive light scattering signatures in a cell-sorting system enabled to separate microcolonies from single cells and heterogeneous aggregates within granule samples. Inoculation of a pure microcolony into 96-well microtiter plates led to successful sub-culturing and increased probability of isolation. Obtained strain Ms1 is cultivated in the liquid culture with relatively high ammonia or nitrite concentration, not extremely slow growing. Considering environmental clones that were closely related to N. mobilis and detected in various environments, the availability of this novel strain would facilitate to reveal this member's ecophysiology in a variety of habitats. PMID:26528282

  16. EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization.

    PubMed

    Pérez-Quintero, Luis-Alberto; Roncagalli, Romain; Guo, Huaijian; Latour, Sylvain; Davidson, Dominique; Veillette, André

    2014-04-01

    Ewing's sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase Cγ, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation.

  17. Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids

    PubMed Central

    Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

    2016-01-01

    Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only. PMID:27222167

  18. Heterochronic bilateral ectopic pregnancy after ovulation induction*

    PubMed Central

    Zhu, Bo; Xu, Gu-feng; Liu, Yi-feng; Qu, Fan; Yao, Wei-miao; Zhu, Yi-min; Gao, Hui-juan; Zhang, Dan

    2014-01-01

    Ectopic pregnancy is identified with the widely-applied assisted reproductive technology (ART). Bilateral ectopic pregnancy is a rare form of ectopic pregnancy which is difficult to be diagnosed at the pre-operation stage. In this paper, we presented an unusual case of heterochronic bilateral ectopic pregnancy after stimulated intrauterine insemination (IUI), where there has been a delay of 22 d between the diagnoses of the two ectopic pregnancies. Literature was reviewed on the occurrence of bilateral ectopic pregnancy during the past four years in the MEDLINE database. We found 16 cases of bilateral ectopic pregnancy reported since 2008, and analyzed the characteristics of those cases of bilateral ectopic pregnancy. We emphasize that ovulation induction and other ARTs may increase the risk of bilateral ectopic pregnancy. Because of the difficulty in identification of bilateral ectopic pregnancy by ultrasonography, the clinician should be aware that the treatment of one ectopic pregnancy does not preclude the occurrence of a second ectopic pregnancy in the same patient and should pay attention to the intra-operation inspection of both side fallopian tubes in any ectopic pregnancy case. PMID:25091994

  19. MEMBRANE-COATING GRANULES OF KERATINIZING EPITHELIA.

    PubMed

    MATOLTSY, A G; PARAKKAL, P F

    1965-02-01

    The purpose of this study has been to obtain information on the development of the envelop of horny cells that resists the action of keratinolytic agents. Toward this end the epidermis, oral mucosa, and tongue epithelium of various vertebrates, as well as the isolated envelopes of horny cells, were examined by electron microscopy. It was found that small cytoplasmic granules (1,000 to 5,000 A) that develop within differentiating epithelial cells move toward the cell periphery, and after fusion with the plasma membrane, empty their contents into the intercellular spaces. The content of the granules spreads over the cell surfaces, and subsequently a thickened and coated cell envelope is formed that resists the action of keratinolytic agent. The membrane-coating granule is regarded as a specific differentiation product of the keratinizing epithelium. It contains numerous inner membranes and is assumed to engage in synthetic activities such as, perhaps, the formation of polysaccharides.

  20. Granule proteases of hematopoietic cells, a family of versatile inflammatory mediators - an update on their cleavage specificity, in vivo substrates, and evolution.

    PubMed

    Hellman, Lars; Thorpe, Michael

    2014-01-01

    Cells from several of the hematopoietic cell lineages including mast cells, basophils, neutrophils, cytotoxic T cells, and natural killer (NK) cells store proteases at very high levels within their cytoplasmic granules. In mast cells, these proteases can account for up to 35% of the total cellular protein, and the absolute majority of these belong to the chymotrypsin-related serine protease family. A number of very diverse functions have been identified for these proteases, including apoptosis induction, blood pressure regulation, inactivation of insect and snake toxins, intestinal parasite expulsion, killing of bacteria and fungi, induction, mobilization, or degradation of cytokines, and the degradation of connective tissue components. A very broad spectrum of primary cleavage specificities has also been observed, including chymase, tryptase, asp-ase, elastase, and met-ase specificities, which highlights the large flexibility in the active site of these proteases. Mast cells primarily express chymases and tryptases with chymotryptic or tryptic primary cleavage specificities, respectively. Neutrophils have several enzymes with chymase, elastase, and tryptase specificities. T cells and NK cells express between 5 and 14 different granzymes, depending on the species, and these enzymes have tryptase, asp-ase, chymase, and met-ase specificities. This review focuses on the appearance of these proteases during vertebrate evolution, their primary and extended cleavage specificities, and their potential in vivo substrates. The in vivo substrates and functions are a particular challenging issue because several of these enzymes have a relatively broad specificity and may therefore cleave a wide range of different substrates. PMID:23969467

  1. Pdcd4 Is Involved in the Formation of Stress Granule in Response to Oxidized Low-Density Lipoprotein or High-Fat Diet

    PubMed Central

    Bai, Yang; Dong, Zhaojing; Shang, Qianwen; Zhao, Hui; Wang, Liyang; Guo, Chun; Gao, Fei; Zhang, Lining; Wang, Qun

    2016-01-01

    Stress granules (SGs) in response to various stresses have been reported in many diseases. We previously reported the implication of programmed cell death 4 (Pdcd4) in obesity-induced stress responses, but the possible link between Pdcd4 and SGs remains lacking. In this study we showed that oxidized low-density lipoprotein (ox-LDL) or high-fat diet (HFD) induced SG formation in mouse macrophages and liver tissues, and Pdcd4 deficiency in mice remarkably reduced its formation. In response to ox-LDL, either endogenous or ectopic Pdcd4 displayed granule-like expression and co-localized with SG markers including T-cell-restricted intracellular antigen-1, fragile X mental retardation-related protein 1, and eukaryotic initiation factor 4A. Ectopic expression of truncated Pdcd4 that depleted specific RNA-binding motif significantly disrupted the SG formation, suggesting the direct involvement of Pdcd4 in ox-LDL-induced SGs through its RNA-binding activity. Additionally, Pdcd4 deficiency drove AKT activation and suppression of eIF2α phosphorylation, thereby contributing to the resistance to ox-LDL or HFD-induced SG formation. Collectively, our data suggest that Pdcd4 as a crucial regulator in SGs induced by ox-LDL or HFD maybe a potential target for mitigating SG-associated stress responses in obesity and related diseases. PMID:27454120

  2. Slow recovery from inactivation regulates the availability of voltage-dependent Na(+) channels in hippocampal granule cells, hilar neurons and basket cells.

    PubMed

    Ellerkmann, R K; Riazanski, V; Elger, C E; Urban, B W; Beck, H

    2001-04-15

    1. Fundamental to the understanding of CNS function is the question of how individual neurons integrate multiple synaptic inputs into an output consisting of a sequence of action potentials carrying information coded as spike frequency. The availability for activation of neuronal Na(+) channels is critical for this process and is regulated both by fast and slow inactivation processes. Here, we have investigated slow inactivation processes in detail in hippocampal neurons. 2. Slow inactivation was induced by prolonged (10-300 s) step depolarisations to -10 mV at room temperature. In isolated hippocampal dentate granule cells (DGCs), recovery from this inactivation was biexponential, with time constants for the two phases of slow inactivation tau(slow,1) and tau(slow,2) ranging from 1 to 10 s and 20 to 50 s, respectively. Both (slow,1) and tau(slow,2) were related to the duration of prior depolarisation by a power law function of the form tau(t) = a (t/a)b, where t is the duration of the depolarisation, a is a constant kinetic setpoint and b is a scaling power. This analysis yielded values of a = 0.034 s and b = 0.62 for tau(slow,1) and a = 24 s and b = 0.30 for tau(slow,2) in the rat. 3. When a train of action potential-like depolarisations of different frequencies (50, 100, 200 Hz) was used to induce inactivation, a similar relationship was found between the frequency of depolarisation and both tau(slow,1) and tau(slow,2) (a = 0.58 s, b = 0.39 for tau(slow,1) and a = 3.77 s and b = 0.42 for tau(slow,2)). 4. Using nucleated patches from rat hippocampal slices, we have addressed possible cell specific differences in slow inactivation. In fast-spiking basket cells a similar scaling relationship can be found (a = 3.54 s and b = 0.39) as in nucleated patches from DGCs (a = 2.3 s and b = 0.48) and non-fast-spiking hilar neurons (a = 2.57 s and b = 0.49). 5. Likewise, comparison of human and rat granule cells showed that properties of ultra-slow recovery from inactivation

  3. High frequency burst firing of granule cells ensures transmission at the parallel fiber to purkinje cell synapse at the cost of temporal coding.

    PubMed

    van Beugen, Boeke J; Gao, Zhenyu; Boele, Henk-Jan; Hoebeek, Freek; De Zeeuw, Chris I

    2013-01-01

    Cerebellar granule cells (GrCs) convey information from mossy fibers (MFs) to Purkinje cells (PCs) via their parallel fibers (PFs). MF to GrC signaling allows transmission of frequencies up to 1 kHz and GrCs themselves can also fire bursts of action potentials with instantaneous frequencies up to 1 kHz. So far, in the scientific literature no evidence has been shown that these high-frequency bursts also exist in awake, behaving animals. More so, it remains to be shown whether such high-frequency bursts can transmit temporally coded information from MFs to PCs and/or whether these patterns of activity contribute to the spatiotemporal filtering properties of the GrC layer. Here, we show that, upon sensory stimulation in both un-anesthetized rabbits and mice, GrCs can show bursts that consist of tens of spikes at instantaneous frequencies over 800 Hz. In vitro recordings from individual GrC-PC pairs following high-frequency stimulation revealed an overall low initial release probability of ~0.17. Nevertheless, high-frequency burst activity induced a short-lived facilitation to ensure signaling within the first few spikes, which was rapidly followed by a reduction in transmitter release. The facilitation rate among individual GrC-PC pairs was heterogeneously distributed and could be classified as either "reluctant" or "responsive" according to their release characteristics. Despite the variety of efficacy at individual connections, grouped activity in GrCs resulted in a linear relationship between PC response and PF burst duration at frequencies up to 300 Hz allowing rate coding to persist at the network level. Together, these findings support the hypothesis that the cerebellar granular layer acts as a spatiotemporal filter between MF input and PC output (D'Angelo and De Zeeuw, 2009). PMID:23734102

  4. High frequency burst firing of granule cells ensures transmission at the parallel fiber to purkinje cell synapse at the cost of temporal coding.

    PubMed

    van Beugen, Boeke J; Gao, Zhenyu; Boele, Henk-Jan; Hoebeek, Freek; De Zeeuw, Chris I

    2013-01-01

    Cerebellar granule cells (GrCs) convey information from mossy fibers (MFs) to Purkinje cells (PCs) via their parallel fibers (PFs). MF to GrC signaling allows transmission of frequencies up to 1 kHz and GrCs themselves can also fire bursts of action potentials with instantaneous frequencies up to 1 kHz. So far, in the scientific literature no evidence has been shown that these high-frequency bursts also exist in awake, behaving animals. More so, it remains to be shown whether such high-frequency bursts can transmit temporally coded information from MFs to PCs and/or whether these patterns of activity contribute to the spatiotemporal filtering properties of the GrC layer. Here, we show that, upon sensory stimulation in both un-anesthetized rabbits and mice, GrCs can show bursts that consist of tens of spikes at instantaneous frequencies over 800 Hz. In vitro recordings from individual GrC-PC pairs following high-frequency stimulation revealed an overall low initial release probability of ~0.17. Nevertheless, high-frequency burst activity induced a short-lived facilitation to ensure signaling within the first few spikes, which was rapidly followed by a reduction in transmitter release. The facilitation rate among individual GrC-PC pairs was heterogeneously distributed and could be classified as either "reluctant" or "responsive" according to their release characteristics. Despite the variety of efficacy at individual connections, grouped activity in GrCs resulted in a linear relationship between PC response and PF burst duration at frequencies up to 300 Hz allowing rate coding to persist at the network level. Together, these findings support the hypothesis that the cerebellar granular layer acts as a spatiotemporal filter between MF input and PC output (D'Angelo and De Zeeuw, 2009).

  5. [Cushing syndrome due to ectopic ACTH secretion].

    PubMed

    Mendonça, B B; Madureira, G; Bloise, W; Albergaria, A; Halpern, A; Liberman, B; Villares, S M; Batista, M C; Avancini, V F; Nitterdorfi, C T

    1989-01-01

    The authors studied 8 patients (4 males and 4 females) with Cushing's syndrome due to ectopic ACTH secretion. Chronological age ranged from 15 to 45 years and duration of the disease ranged from 3 to 48 months. All patients presented typical signs of Cushing's syndrome, blood hypertension, and four of them had hyperpigmentation of the skin. Five patients had fasting hyperglycemia and all patients but one had serum hypokalemia (serum K = 2.2 to 3.9mEq/l). The circadian rhythm of cortisol was absent in all patients and basal cortisol levels were elevated in all patients but one. Basal ACTH levels evaluated in 7 patients were elevated in 6 (29 to 1050 pg/ml-MRC). One patient presented normal depression of urinary 17-OH after two days of dexamethasone and normal increase of urinary 17-OH and serum 11-dexycortisol after methyrapone. Four patients had carcinoid tumor (3 thymic and 1 bronchial), two had pancreatic islets cell tumors, one had bilateral pheochromocytoma and medular carcinoma of the thyroid, and one had oat cell carcinoma of the lung and medular carcinoma of the thyroid. Thoracic X-rays identified the ectopic ACTH secretion tumor in four cases, all confirmed by CT scan. Abdominal CT showed a difuse enlargement of the adrenals in seven cases and bilateral nodules in one case (pheochromocytomas). Six patients died within 3 years of the diagnosis. The authors concluded that clinical and hormonal findings could mislead the findings of ACTH ectopic secretion and Cushing's disease, and suggest that thoracic X-rays and CT scans of the skull, thorax, and abdome should be done in all cases of Cushing's syndrome. PMID:2559451

  6. Ectopic microRNA-150-5p transcription sensitizes glucocorticoid therapy response in MM1S multiple myeloma cells but fails to overcome hormone therapy resistance in MM1R cells.

    PubMed

    Palagani, Ajay; Op de Beeck, Ken; Naulaerts, Stefan; Diddens, Jolien; Sekhar Chirumamilla, Chandra; Van Camp, Guy; Laukens, Kris; Heyninck, Karen; Gerlo, Sarah; Mestdagh, Pieter; Vandesompele, Joke; Berghe, Wim Vanden

    2014-01-01

    Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC

  7. Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells

    PubMed Central

    Palagani, Ajay; Op de Beeck, Ken; Naulaerts, Stefan; Diddens, Jolien; Sekhar Chirumamilla, Chandra; Van Camp, Guy; Laukens, Kris; Heyninck, Karen; Gerlo, Sarah; Mestdagh, Pieter; Vandesompele, Joke; Berghe, Wim Vanden

    2014-01-01

    Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC

  8. [Vital reactions in Pacchioni granulations].

    PubMed

    Földes, V; Mojzes, L; Antal, A

    1987-01-01

    By means of histological methods the authors examined the blood and fluid circulatory disturbances associated with cranial and cerebral injuries. The presence of vital reactions was studied by means of the combined histological study of the dura mater, pacchionian granulations and the central nervous system. Samples for histological study were taken from 115 cadavers who had suffered cranial injuries, from 15 individuals who died from destructive cerebral apoplexy caused by a disease and from 30 individuals who died of natural causes. The authors applied a special fixation and sampling technique and, using various histological reactions, the following vital reactions were observed: the appearance of blood-cell elements in the granulation, a moderate fibrin degradation product and hemoglobin phagocytosis, and occasionally lipid phagocytosis. The authors worked out a method that was shown to be highly effective in the more precise determination of the induction time of cerebral apoplexy caused by a disease and that of traumatic injury of the brain.

  9. Isolation Rearing Reduces Neuronal Excitability in Dentate Gyrus Granule Cells of Adolescent C57BL/6J Mice: Role of GABAergic Tonic Currents and Neurosteroids.

    PubMed

    Talani, Giuseppe; Biggio, Francesca; Licheri, Valentina; Locci, Valentina; Biggio, Giovanni; Sanna, Enrico

    2016-01-01

    Early-life exposure to stress, by impacting on a brain still under development, is considered a critical factor for the increased vulnerability to psychiatric disorders and abuse of psychotropic substances during adulthood. As previously reported, rearing C57BL/6J weanling mice in social isolation (SI) from their peers for several weeks, a model of prolonged stress, is associated with a decreased plasma and brain levels of neuroactive steroids such as 3α,5α-THP, with a parallel up-regulation of extrasynaptic GABAA receptors (GABAAR) in dentate gyrus (DG) granule cells compared to group-housed (GH) mice. In the present study, together with the SI-induced decrease in plasma concentration of both progesterone and 3α,5α-THP, and an increase in THIP-stimulated GABAergic tonic currents, patch-clamp analysis of DG granule cells revealed a significant decrease in membrane input resistance and action potential (AP) firing rate, in SI compared to GH mice, suggesting that SI exerts an inhibitory action on neuronal excitability of these neurons. Voltage-clamp recordings of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) revealed a SI-associated decrease in frequency as well as a shift from paired-pulse (PP) depression to PP facilitation (PPF) of evoked EPSCs, indicative of a reduced probability of glutamate release. Daily administration of progesterone during isolation reverted the changes in plasma 3α,5α-THP as well as in GABAergic tonic currents and neuronal excitability caused by SI, but it had only a limited effect on the changes in the probability of presynaptic glutamate release. Overall, the results obtained in this work, together with those previously published, indicate that exposure of mice to SI during adolescence reduces neuronal excitability of DG granule cells, an effect that may be linked to the increased GABAergic tonic currents as a consequence of the sustained decrease in plasma and hippocampal levels of neurosteroids. All these

  10. Isolation Rearing Reduces Neuronal Excitability in Dentate Gyrus Granule Cells of Adolescent C57BL/6J Mice: Role of GABAergic Tonic Currents and Neurosteroids

    PubMed Central

    Talani, Giuseppe; Biggio, Francesca; Licheri, Valentina; Locci, Valentina; Biggio, Giovanni; Sanna, Enrico

    2016-01-01

    Early-life exposure to stress, by impacting on a brain still under development, is considered a critical factor for the increased vulnerability to psychiatric disorders and abuse of psychotropic substances during adulthood. As previously reported, rearing C57BL/6J weanling mice in social isolation (SI) from their peers for several weeks, a model of prolonged stress, is associated with a decreased plasma and brain levels of neuroactive steroids such as 3α,5α-THP, with a parallel up-regulation of extrasynaptic GABAA receptors (GABAAR) in dentate gyrus (DG) granule cells compared to group-housed (GH) mice. In the present study, together with the SI-induced decrease in plasma concentration of both progesterone and 3α,5α-THP, and an increase in THIP-stimulated GABAergic tonic currents, patch-clamp analysis of DG granule cells revealed a significant decrease in membrane input resistance and action potential (AP) firing rate, in SI compared to GH mice, suggesting that SI exerts an inhibitory action on neuronal excitability of these neurons. Voltage-clamp recordings of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) revealed a SI-associated decrease in frequency as well as a shift from paired-pulse (PP) depression to PP facilitation (PPF) of evoked EPSCs, indicative of a reduced probability of glutamate release. Daily administration of progesterone during isolation reverted the changes in plasma 3α,5α-THP as well as in GABAergic tonic currents and neuronal excitability caused by SI, but it had only a limited effect on the changes in the probability of presynaptic glutamate release. Overall, the results obtained in this work, together with those previously published, indicate that exposure of mice to SI during adolescence reduces neuronal excitability of DG granule cells, an effect that may be linked to the increased GABAergic tonic currents as a consequence of the sustained decrease in plasma and hippocampal levels of neurosteroids. All these

  11. Cognitive performances and locomotor activity following dentate granule cell damage in rats: role of lesion extent and type of memory tested.

    PubMed

    Jeltsch, H; Bertrand, F; Lazarus, C; Cassel, J C

    2001-07-01

    Intradentate injection of colchicine is one of the techniques used to destroy granule cells. This study compared the behavioral effects of various amounts of colchicine (1.0, 3.0, and 6.0 microg; Col 1, Col 3, and Col 6, respectively) injected into the dentate gyrus of adult Long-Evans male rats. Starting 10 days after lesion surgery, behavioral testing assessed home-cage and open-field locomotion, alternation in a T-maze, water-maze, and radial-maze learning according to protocols placing emphasis on reference, and working memory. All of these tasks are sensitive to hippocampal disruption. Histological verifications showed that the extent of the lesions depends on the dose of colchicine (index of dentate gyrus shrinkage: -33% in Col 1, -54% in Col 3, and -67% in Col 6 rats). Colchicine dose-dependently increased nocturnal home cage activity (an effect found 10 days but not 5 months after surgery), but had no significant effect on open-field locomotion or T-maze alternation. A dose-dependent reference memory impairment was found during the acquisition of spatial navigation in the water maze; Col 3 and Col 6 rats were more impaired than Col 1 rats. During the probe trial (platform removed), control rats spent a longer distance swimming over the platform area than all rats with colchicine lesions. In the working memory version of the test, all rats with colchicine lesions showed significant deficits. The deficits were larger in Col 3 and Col 6 rats compared to Col 1 rats. The lesions had no effect on swimming speed. In the radial-maze test, there was also a dose-dependent working memory impairment. However, reference memory was disrupted in a manner that did not differ among the three groups of lesioned rats. Our data are in line with the view that the dentate gyrus plays an important role in the acquisition of new information and is an integral neural substrate for spatial reference and spatial working memory. They also suggest that damage to granule cells might

  12. Isolation Rearing Reduces Neuronal Excitability in Dentate Gyrus Granule Cells of Adolescent C57BL/6J Mice: Role of GABAergic Tonic Currents and Neurosteroids.

    PubMed

    Talani, Giuseppe; Biggio, Francesca; Licheri, Valentina; Locci, Valentina; Biggio, Giovanni; Sanna, Enrico

    2016-01-01

    Early-life exposure to stress, by impacting on a brain still under development, is considered a critical factor for the increased vulnerability to psychiatric disorders and abuse of psychotropic substances during adulthood. As previously reported, rearing C57BL/6J weanling mice in social isolation (SI) from their peers for several weeks, a model of prolonged stress, is associated with a decreased plasma and brain levels of neuroactive steroids such as 3α,5α-THP, with a parallel up-regulation of extrasynaptic GABAA receptors (GABAAR) in dentate gyrus (DG) granule cells compared to group-housed (GH) mice. In the present study, together with the SI-induced decrease in plasma concentration of both progesterone and 3α,5α-THP, and an increase in THIP-stimulated GABAergic tonic currents, patch-clamp analysis of DG granule cells revealed a significant decrease in membrane input resistance and action potential (AP) firing rate, in SI compared to GH mice, suggesting that SI exerts an inhibitory action on neuronal excitability of these neurons. Voltage-clamp recordings of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) revealed a SI-associated decrease in frequency as well as a shift from paired-pulse (PP) depression to PP facilitation (PPF) of evoked EPSCs, indicative of a reduced probability of glutamate release. Daily administration of progesterone during isolation reverted the changes in plasma 3α,5α-THP as well as in GABAergic tonic currents and neuronal excitability caused by SI, but it had only a limited effect on the changes in the probability of presynaptic glutamate release. Overall, the results obtained in this work, together with those previously published, indicate that exposure of mice to SI during adolescence reduces neuronal excitability of DG granule cells, an effect that may be linked to the increased GABAergic tonic currents as a consequence of the sustained decrease in plasma and hippocampal levels of neurosteroids. All these

  13. A comparison of artificial solar granules with real solar granules

    NASA Astrophysics Data System (ADS)

    Woehl, H.; Nordlund, A.

    1985-06-01

    The properties of computer-generated images of solar granules were compared with data from the literature and with observations of granules from 1975 and 1979. The lifetimes, shapes, and dimensions of the granules were estimated, and the results are discussed. No significant differences were found between the artificial images and the observed granules. The ratios of width to length among the artificial granules are given in a table.

  14. Regulation of action potential delays via voltage-gated potassium Kv1.1 channels in dentate granule cells during hippocampal epilepsy

    PubMed Central

    Kirchheim, Florian; Tinnes, Stefanie; Haas, Carola A.; Stegen, Michael; Wolfart, Jakob

    2013-01-01

    Action potential (AP) responses of dentate gyrus granule (DG) cells have to be tightly regulated to maintain hippocampal function. However, which ion channels control the response delay of DG cells is not known. In some neuron types, spike latency is influenced by a dendrotoxin (DTX)-sensitive delay current (ID) mediated by unidentified combinations of voltage-gated K+ (Kv) channels of the Kv1 family Kv1.1–6. In DG cells, the ID has not been characterized and its molecular basis is unknown. The response phenotype of mature DG cells is usually considered homogenous but intrinsic plasticity likely occurs in particular in conditions of hyperexcitability, for example during temporal lobe epilepsy (TLE). In this study, we examined response delays of DG cells and underlying ion channel molecules by employing a combination of gramicidin-perforated patch-clamp recordings in acute brain slices and single-cell reverse transcriptase quantitative polymerase chain reaction (SC RT-qPCR) experiments. An in vivo mouse model of TLE consisting of intrahippocampal kainate (KA) injection was used to examine epilepsy-related plasticity. Response delays of DG cells were DTX-sensitive and strongly increased in KA-injected hippocampi; Kv1.1 mRNA was elevated 10-fold, and the response delays correlated with Kv1.1 mRNA abundance on the single cell level. Other Kv1 subunits did not show overt changes in mRNA levels. Kv1.1 immunolabeling was enhanced in KA DG cells. The biophysical properties of ID and a delay heterogeneity within the DG cell population was characterized. Using organotypic hippocampal slice cultures (OHCs), where KA incubation also induced ID upregulation, the homeostatic reversibility and neuroprotective potential for DG cells were tested. In summary, the AP timing of DG cells is effectively controlled via scaling of Kv1.1 subunit transcription. With this antiepileptic mechanism, DG cells delay their responses during hyperexcitation. PMID:24367293

  15. Aberrant, ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

    SciTech Connect

    Ahluwalia, Amrita; Jones, Michael K.; Szabo, Sandor; Tarnawski, Andrzej S.

    2013-08-09

    Highlights: •Malignant colonic epithelial cells express VEGF and its receptors. •Cultured colon cancer cells secrete VEGF into the medium. •Inhibition of VEGF receptor significantly decreases colon cancer cell proliferation. •VEGF is critical for colon cancer cell growth. -- Abstract: Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy. Material and methods: We examined and quantified expression of VEGF, VEGF-R1 and VEGF-R2 in three different human colonic tissue arrays containing sections of adenocarcinoma (n = 43) and normal mucosa (n = 41). In human colon cancer cell lines HCT116 and HT29 and normal colon cell lines NCM356 and NCM460, we examined expression of VEGF, VEGF-R1 and VEGF-R2 mRNA and protein, VEGF production and secretion into the culture medium; and, the effect of a potent, selective inhibitor of VEGF receptors, AL-993, on cell proliferation. Results: Human colorectal cancer specimens had strong expression of VEGF in cancer cells and also expressed VEGF-R1 and VEGF-R2.In vitro studies showed that human colon cancer cell lines, HCT116 and HT29, but not normal colonic cell lines, express VEGF, VEGF-R1 and VEGF-R2 and secrete VEGF into the medium up to a concentration 2000 pg/ml within 48 h. Furthermore, we showed that inhibition of VEGF receptors using a specific VEGF-R inhibitor significantly reduced proliferation (by >50%) of cultured colon cancer cell lines. Conclusions: Our findings support the contention that VEGF generated by colon cancer cells stimulates their growth directly through an autocrine mechanism that is

  16. Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components

    PubMed Central

    Toyooka, Kiminori; Okamoto, Takashi; Minamikawa, Takao

    2001-01-01

    α-Amylase is expressed in cotyledons of germinated Vigna mungo seeds and is responsible for the degradation of starch that is stored in the starch granule (SG). Immunocytochemical analysis of the cotyledon cells with anti–α-amylase antibody showed that α-amylase is transported to protein storage vacuole (PSV) and lytic vacuole (LV), which is converted from PSV by hydrolysis of storage proteins. To observe the insertion/degradation processes of SG into/in the inside of vacuoles, ultrastructural analyses of the cotyledon cells were conducted. The results revealed that SG is inserted into LV through autophagic function of LV and subsequently degraded by vacuolar α-amylase. The autophagy for SG was structurally similar to micropexophagy detected in yeast cells. In addition to the autophagic process for SG, autophagosome-mediated autophagy for cytoplasm and mitochondria was detected in the cotyledon cells. When the embryo axes were removed from seeds and the detached cotyledons were incubated, the autophagosome-mediated autophagy was observed, but the autophagic process for the degradation of SG was not detected, suggesting that these two autophagic processes were mediated by different cellular mechanisms. The two distinct autophagic processes were thought to be involved in the breakdown of SG and cell components in the cells of germinated cotyledon. PMID:11524437

  17. Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components.

    PubMed

    Toyooka, K; Okamoto, T; Minamikawa, T

    2001-09-01

    alpha-Amylase is expressed in cotyledons of germinated Vigna mungo seeds and is responsible for the degradation of starch that is stored in the starch granule (SG). Immunocytochemical analysis of the cotyledon cells with anti-alpha-amylase antibody showed that alpha-amylase is transported to protein storage vacuole (PSV) and lytic vacuole (LV), which is converted from PSV by hydrolysis of storage proteins. To observe the insertion/degradation processes of SG into/in the inside of vacuoles, ultrastructural analyses of the cotyledon cells were conducted. The results revealed that SG is inserted into LV through autophagic function of LV and subsequently degraded by vacuolar alpha-amylase. The autophagy for SG was structurally similar to micropexophagy detected in yeast cells. In addition to the autophagic process for SG, autophagosome-mediated autophagy for cytoplasm and mitochondria was detected in the cotyledon cells. When the embryo axes were removed from seeds and the detached cotyledons were incubated, the autophagosome-mediated autophagy was observed, but the autophagic process for the degradation of SG was not detected, suggesting that these two autophagic processes were mediated by different cellular mechanisms. The two distinct autophagic processes were thought to be involved in the breakdown of SG and cell components in the cells of germinated cotyledon.

  18. Humanized mice with ectopic artificial liver tissues

    PubMed Central

    Chen, Alice A.; Thomas, David K.; Ong, Luvena L.; Schwartz, Robert E.; Golub, Todd R.; Bhatia, Sangeeta N.

    2011-01-01

    “Humanized” mice offer a window into aspects of human physiology that are otherwise inaccessible. The best available methods for liver humanization rely on cell transplantation into immunodeficient mice with liver injury but these methods have not gained widespread use due to the duration and variability of hepatocyte repopulation. In light of the significant progress that has been achieved in clinical cell transplantation through tissue engineering, we sought to develop a humanized mouse model based on the facile and ectopic implantation of a tissue-engineered human liver. These human ectopic artificial livers (HEALs) stabilize the function of cryopreserved primary human hepatocytes through juxtacrine and paracrine signals in polymeric scaffolds. In contrast to current methods, HEALs can be efficiently established in immunocompetent mice with normal liver function. Mice transplanted with HEALs exhibit humanized liver functions persistent for weeks, including synthesis of human proteins, human drug metabolism, drug–drug interaction, and drug-induced liver injury. Here, mice with HEALs are used to predict the disproportionate metabolism and toxicity of “major” human metabolites using multiple routes of administration and monitoring. These advances may enable manufacturing of reproducible in vivo models for diverse drug development and research applications. PMID:21746904

  19. Protective effects of fangchinoline and tetrandrine on hydrogen peroxide-induced oxidative neuronal cell damage in cultured rat cerebellar granule cells.

    PubMed

    Koh, Sang Bum; Ban, Ju Yeon; Lee, Bo Young; Seong, Yeon Hee

    2003-06-01

    The present study was performed to examine the neuroprotective effects of fangchinoline (FAN) and tetrandrine (TET), bis-benzylisoquinoline alkaloids, which exhibit the characteristics of Ca 2+ channel blockers, on H2O2 -induced neurotoxicity using cultured rat cerebellar granule neurons. H2O2 produced a concentration-dependent reduction of cell viability, which was blocked by (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,10-imine (MK-801), an N-methyl- D-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca 2+ channel blocker, and NG-nitro- L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. Pretreatment with FAN and TET over a concentration range of 0.1 to 10 microM significantly decreased the H2O2 -induced neuronal cell death as assessed by a trypan blue exclusion test, a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the number of apoptotic nuclei. In addition, FAN and TET inhibited the H2O2 -induced elevation of glutamate release into the medium, elevation of the cytosolic free Ca 2+ concentration ([Ca 2+] c ), and generation of reactive oxygen species (ROS). These results suggest that FAN and TET may mitigate the harmful effects of H2O2 -induced neuronal cell death by interfering with the increase of [Ca 2+] c, and then by inhibiting glutamate release and generation of ROS. Abbreviations. AP5:D(-)-2-amino-5-phosphonopentanoic acid DMSO:dimethyl sulfoxide FAN:fangchinoline H 2 DCF-DA:2',7'-dichlorodihydrofluorescin diacetate MK-801:(5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,20-imine MTT:3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide L-NAME: NG-Nitro- L-arginine methyl ester NMDA: N-methyl- D-aspartate TET:tetrandrine PMID:12865967

  20. Novel Tools to Analyze the Function of Salmonella Effectors Show That SvpB Ectopic Expression Induces Cell Cycle Arrest in Tumor Cells

    PubMed Central

    Mesa-Pereira, Beatriz; Medina, Carlos; Camacho, Eva María; Flores, Amando; Santero, Eduardo

    2