Sample records for adult-generated granule cells

  1. Heterogeneous integration of adult-generated granule cells into the epileptic brain

    PubMed Central

    Murphy, Brian L.; Pun, Raymund Y.K.; Yin, Hulian; Faulkner, Christian R.; Loepke, Andreas W.; Danzer, Steve C.

    2011-01-01

    The functional impact of adult-generated granule cells in the epileptic brain is unclear, with data supporting both protective and maladaptive roles. These conflicting findings could be explained if new granule cells integrate heterogeneously, with some cells taking neutral or adaptive roles, while others contribute to recurrent circuitry supporting seizures. Here, we tested this hypothesis by completing detailed morphological characterizations of age- and experience-defined cohorts of adult-generated granule cells from transgenic mice. The majority of newborn cells exposed to an epileptogenic insult exhibited reductions in dendritic spine number, suggesting reduced excitatory input to these cells. A significant subset, however, exhibited higher spine numbers. These latter cells tended to have enlarged cell bodies, long basal dendrites or both. Moreover, cells with basal dendrites received significantly more recurrent mossy fiber input through their apical dendrites, indicating that these cells are robustly integrated into the pathological circuitry of the epileptic brain. These data imply that newborn cells play complex – and potentially conflicting – roles in epilepsy. PMID:21209195

  2. GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy.

    PubMed

    Koyama, Ryuta; Tao, Kentaro; Sasaki, Takuya; Ichikawa, Junya; Miyamoto, Daisuke; Muramatsu, Rieko; Matsuki, Norio; Ikegaya, Yuji

    2012-08-01

    Temporal lobe epilepsy (TLE) is accompanied by an abnormal location of granule cells in the dentate gyrus. Using a rat model of complex febrile seizures, which are thought to be a precipitating insult of TLE later in life, we report that aberrant migration of neonatal-generated granule cells results in granule cell ectopia that persists into adulthood. Febrile seizures induced an upregulation of GABA(A) receptors (GABA(A)-Rs) in neonatally generated granule cells, and hyperactivation of excitatory GABA(A)-Rs caused a reversal in the direction of granule cell migration. This abnormal migration was prevented by RNAi-mediated knockdown of the Na(+)K(+)2Cl(-) co-transporter (NKCC1), which regulates the excitatory action of GABA. NKCC1 inhibition with bumetanide after febrile seizures rescued the granule cell ectopia, susceptibility to limbic seizures and development of epilepsy. Thus, this work identifies a previously unknown pathogenic role of excitatory GABA(A)-R signaling and highlights NKCC1 as a potential therapeutic target for preventing granule cell ectopia and the development of epilepsy after febrile seizures.

  3. The effects of exercise and stress on the survival and maturation of adult-generated granule cells

    PubMed Central

    Snyder, Jason S.; Glover, Lucas R.; Sanzone, Kaitlin M.; Kamhi, J. Frances; Cameron, Heather A.

    2009-01-01

    Stress strongly inhibits proliferation of granule cell precursors in the dentate gyrus, while voluntary running has the opposite effect. Few studies, however, have examined the possible effects of these environmental manipulations on the maturation and survival of young granule cells. We examined number of surviving granule cells and the proportion of young neurons that were functionally mature, as defined by seizure-induced immediate-early gene expression, in 14 and 21 day-old granule cells in mice that were given access to a running wheel, restrained daily for 2 hours, or given no treatment during this period. Importantly, treatments began two days after BrdU injection, to isolate effects on survival from those on cell proliferation. We found a large increase in granule cell survival in running mice compared with controls at both time points. In addition, running increased the proportion of granule cells expressing the immediate-early gene Arc in response to seizures, suggesting that it speeds incorporation into circuits, i.e., functional maturation. Stressed mice showed no change in Arc expression, compared to control animals, but, surprisingly, showed a transient increase in survival of 14-day-old granule cells, which was gone 7 days later. Examination of cell proliferation, using the endogenous mitotic marker proliferating cell nuclear antigen (PCNA) showed an increase in cell proliferation after 12 days of running but not after 19 days of running. The number of proliferating cells was unchanged 24 hours after the 12th or 19th episode of daily restraint stress. These findings demonstrate that running has strong effects on survival and maturation of young granule cells as well as their birth and that stress can have positive but short-lived effects on granule cell survival. PMID:19156854

  4. NR2B-dependent plasticity of adult-born granule cells is necessary for context discrimination.

    PubMed

    Kheirbek, Mazen A; Tannenholz, Lindsay; Hen, René

    2012-06-20

    Adult-generated granule cells (GCs) in the dentate gyrus (DG) exhibit a period of heightened plasticity 4-6 weeks postmitosis. However, the functional contribution of this critical window of plasticity to hippocampal neurogenesis and behavior remains unknown. Here, we show that deletion of NR2B-containing NMDA receptors from adult-born GCs impairs a neurogenesis-dependent form of LTP in the DG and reduces dendritic complexity of adult-born GCs, but does not impact their survival. Mice in which the NR2B-containing NMDA receptor was deleted from adult-born GCs did not differ from controls in baseline anxiety-like behavior or discrimination of very different contexts, but were impaired in discrimination of highly similar contexts. These results indicate that NR2B-dependent plasticity of adult-born GCs is necessary for fine contextual discrimination and is consistent with their proposed role in pattern separation.

  5. GSK-3β Overexpression Alters the Dendritic Spines of Developmentally Generated Granule Neurons in the Mouse Hippocampal Dentate Gyrus.

    PubMed

    Pallas-Bazarra, Noemí; Kastanauskaite, Asta; Avila, Jesús; DeFelipe, Javier; Llorens-Martín, María

    2017-01-01

    The dentate gyrus (DG) plays a crucial role in hippocampal-related memory. The most abundant cellular type in the DG, namely granule neurons, are developmentally generated around postnatal day P6 in mice. Moreover, a unique feature of the DG is the occurrence of adult hippocampal neurogenesis, a process that gives rise to newborn granule neurons throughout life. Adult-born and developmentally generated granule neurons share some maturational aspects but differ in others, such as in their positioning within the granule cell layer. Adult hippocampal neurogenesis encompasses a series of plastic changes that modify the function of the hippocampal trisynaptic network. In this regard, it is known that glycogen synthase kinase 3β (GSK-3β) regulates both synaptic plasticity and memory. By using a transgenic mouse overexpressing GSK-3β in hippocampal neurons, we previously demonstrated that the overexpression of this kinase has deleterious effects on the maturation of newborn granule neurons. In the present study, we addressed the effects of GSK-3β overexpression on the morphology and number of dendritic spines of developmentally generated granule neurons. To this end, we performed intracellular injections of Lucifer Yellow in developmentally generated granule neurons of wild-type and GSK-3β-overexpressing mice and analyzed the number and morphologies of dendritic spines (namely, stubby, thin and mushroom). GSK-3β overexpression led to a general reduction in the number of dendritic spines. In addition, it caused a slight reduction in the percentage, head diameter and length of thin spines, whereas the head diameter of mushroom spines was increased.

  6. GSK-3β Overexpression Alters the Dendritic Spines of Developmentally Generated Granule Neurons in the Mouse Hippocampal Dentate Gyrus

    PubMed Central

    Pallas-Bazarra, Noemí; Kastanauskaite, Asta; Avila, Jesús; DeFelipe, Javier; Llorens-Martín, María

    2017-01-01

    The dentate gyrus (DG) plays a crucial role in hippocampal-related memory. The most abundant cellular type in the DG, namely granule neurons, are developmentally generated around postnatal day P6 in mice. Moreover, a unique feature of the DG is the occurrence of adult hippocampal neurogenesis, a process that gives rise to newborn granule neurons throughout life. Adult-born and developmentally generated granule neurons share some maturational aspects but differ in others, such as in their positioning within the granule cell layer. Adult hippocampal neurogenesis encompasses a series of plastic changes that modify the function of the hippocampal trisynaptic network. In this regard, it is known that glycogen synthase kinase 3β (GSK-3β) regulates both synaptic plasticity and memory. By using a transgenic mouse overexpressing GSK-3β in hippocampal neurons, we previously demonstrated that the overexpression of this kinase has deleterious effects on the maturation of newborn granule neurons. In the present study, we addressed the effects of GSK-3β overexpression on the morphology and number of dendritic spines of developmentally generated granule neurons. To this end, we performed intracellular injections of Lucifer Yellow in developmentally generated granule neurons of wild-type and GSK-3β-overexpressing mice and analyzed the number and morphologies of dendritic spines (namely, stubby, thin and mushroom). GSK-3β overexpression led to a general reduction in the number of dendritic spines. In addition, it caused a slight reduction in the percentage, head diameter and length of thin spines, whereas the head diameter of mushroom spines was increased. PMID:28344548

  7. Similar GABAergic inputs in dentate granule cells born during embryonic and adult neurogenesis.

    PubMed

    Laplagne, Diego A; Kamienkowski, Juan E; Espósito, M Soledad; Piatti, Verónica C; Zhao, Chunmei; Gage, Fred H; Schinder, Alejandro F

    2007-05-01

    Neurogenesis in the dentate gyrus of the hippocampus follows a unique temporal pattern that begins during embryonic development, peaks during the early postnatal stages and persists through adult life. We have recently shown that dentate granule cells born in early postnatal and adult mice acquire a remarkably similar afferent connectivity and firing behavior, suggesting that they constitute a homogeneous functional population [Laplagne et al. (2006)PLoS Biol., 4, e409]. Here we extend our previous study by comparing mature neurons born in the embryonic and adult hippocampus, with a focus on intrinsic membrane properties and gamma-aminobutyric acid (GABA)ergic synaptic inputs. For this purpose, dividing neuroblasts of the ventricular wall were retrovirally labeled with green fluorescent protein at embryonic day 15 (E15), and progenitor cells of the subgranular zone were labeled with red fluorescent protein in the same mice at postnatal day 42 (P42, adulthood). Electrophysiological properties of mature neurons born at either stage were then compared in the same brain slices. Evoked and spontaneous GABAergic postsynaptic responses of perisomatic and dendritic origin displayed similar characteristics in both neuronal populations. Miniature GABAergic inputs also showed similar functional properties and pharmacological profile. A comparative analysis of the present data with our previous observations rendered no significant differences among GABAergic inputs recorded from neurons born in the embryonic, early postnatal and adult mice. Yet, embryo-born neurons showed a reduced membrane excitability, suggesting a lower engagement in network activity. Our results demonstrate that granule cells of different age, location and degree of excitability receive GABAergic inputs of equivalent functional characteristics.

  8. Functional properties of granule cells with hilar basal dendrites in the epileptic dentate gyrus.

    PubMed

    Kelly, Tony; Beck, Heinz

    2017-01-01

    The maturation of adult-born granule cells and their functional integration into the network is thought to play a key role in the proper functioning of the dentate gyrus. In temporal lobe epilepsy, adult-born granule cells in the dentate gyrus develop abnormally and possess a hilar basal dendrite (HBD). Although morphological studies have shown that these HBDs have synapses, little is known about the functional properties of these HBDs or the intrinsic and network properties of the granule cells that possess these aberrant dendrites. We performed patch-clamp recordings of granule cells within the granule cell layer "normotopic" from sham-control and status epilepticus (SE) animals. Normotopic granule cells from SE animals possessed an HBD (SE + HBD + cells) or not (SE + HBD - cells). Apical and basal dendrites were stimulated using multiphoton uncaging of glutamate. Two-photon Ca 2+ imaging was used to measure Ca 2+ transients associated with back-propagating action potentials (bAPs). Near-synchronous synaptic input integrated linearly in apical dendrites from sham-control animals and was not significantly different in apical dendrites of SE + HBD - cells. The majority of HBDs integrated input linearly, similar to apical dendrites. However, 2 of 11 HBDs were capable of supralinear integration mediated by a dendritic spike. Furthermore, the bAP-evoked Ca 2+ transients were relatively well maintained along HBDs, compared with apical dendrites. This further suggests an enhanced electrogenesis in HBDs. In addition, the output of granule cells from epileptic tissue was enhanced, with both SE + HBD - and SE + HBD + cells displaying increased high-frequency (>100 Hz) burst-firing. Finally, both SE + HBD - and SE + HBD + cells received recurrent excitatory input that was capable of generating APs, especially in the absence of feedback inhibition. Taken together, these data suggest that the enhanced excitability of HBDs combined with the altered intrinsic and network

  9. Neuroligin-1 overexpression in newborn granule cells in vivo.

    PubMed

    Schnell, Eric; Bensen, Aesoon L; Washburn, Eric K; Westbrook, Gary L

    2012-01-01

    Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons.

  10. Clonal Analysis of Newborn Hippocampal Dentate Granule Cell Proliferation and Development in Temporal Lobe Epilepsy1,2,3

    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

  11. 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. Copyright © 2012 AlphaMed Press.

  12. Pten Knockdown in vivo Increases Excitatory Drive onto Dentate Granule Cells

    PubMed Central

    Luikart, Bryan W.; Schnell, Eric; Washburn, Eric K.; Bensen, AeSoon L.; Tovar, Kenneth R.; Westbrook, Gary L.

    2011-01-01

    Some cases of autism spectrum disorder (ASD) have mutations in the lipid phosphatase, Pten (phosphatase and tensin homolog on chromosome 10). Tissue specific deletion of Pten in the hippocampus and cortex of mice causes anatomical and behavioral abnormalities similar to human autism. However, the impact of reductions in Pten on synaptic and circuit function remains unexplored. We used in vivo stereotaxic injections of lentivirus expressing an shRNA to knockdown Pten in mouse neonatal and young adult dentate granule cells. We then assessed the morphology and synaptic physiology between two weeks and four months later. Confocal imaging of the hippocampus revealed a marked increase in granule cell size and an increase in dendritic spine density. The onset of morphological changes occurred earlier in neonatal mice than in young adults. We used whole-cell recordings from granule cells in acute slices to assess synaptic function following Pten knockdown. Consistent with the increase in dendritic spines, the frequency of excitatory miniature and spontaneous postsynaptic currents increased. However, there was little or no effect on inhibitory postsynaptic currents. Thus Pten knockdown results in an imbalance between excitatory and inhibitory synaptic activity. Because reductions in Pten affected mature granule cells as well as developing granule cells, we suggest that the disruption of circuit function by Pten hypofunction may be ongoing well beyond early development. PMID:21411674

  13. Reliable Genetic Labeling of Adult-Born Dentate Granule Cells Using Ascl1 CreERT2 and Glast CreERT2 Murine Lines.

    PubMed

    Yang, Sung M; Alvarez, Diego D; Schinder, Alejandro F

    2015-11-18

    Newly generated dentate granule cells (GCs) are relevant for input discrimination in the adult hippocampus. Yet, their precise contribution to information processing remains unclear. To address this question, it is essential to develop approaches to precisely label entire cohorts of adult-born GCs. In this work, we used genetically modified mice to allow conditional expression of tdTomato (Tom) in adult-born GCs and characterized their development and functional integration. Ascl1(CreERT2);CAG(floxStopTom) and Glast(CreERT2);CAG(floxStopTom) mice resulted in indelible expression of Tom in adult neural stem cells and their lineage upon tamoxifen induction. Whole-cell recordings were performed to measure intrinsic excitability, firing behavior, and afferent excitatory connectivity. Developing GCs were also staged by the expression of early and late neuronal markers. The slow development of adult-born GCs characterized here is consistent with previous reports using retroviral approaches that have revealed that a mature phenotype is typically achieved after 6-8 weeks. Our findings demonstrate that Ascl1(CreERT2) and Glast(CreERT2) mouse lines enable simple and reliable labeling of adult-born GC lineages within restricted time windows. Therefore, these mice greatly facilitate tagging new neurons and manipulating their activity, required for understanding adult neurogenesis in the context of network remodeling, learning, and behavior. Our study shows that Ascl1(CreERT2) and Glast(CreERT2) mice lines can be used to label large cohorts of adult-born dentate granule cells with excellent time resolution. Neurons labeled in this manner display developmental and functional profiles that are in full agreement with previous findings using thymidine analogs and retroviral labeling, thus providing an alternative approach to tackle fundamental questions on circuit remodeling. Because of the massive neuronal targeting and the simplicity of this method, genetic labeling will

  14. Distorted secretory granule composition in mast cells with multiple protease deficiency.

    PubMed

    Grujic, Mirjana; Calounova, Gabriela; Eriksson, Inger; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Tchougounova, Elena; Kjellén, Lena; Pejler, Gunnar

    2013-10-01

    Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.

  15. NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing

    PubMed Central

    Hsu, Hsiang-Ting; Viswanath, Dixita I.; Önfelt, Björn

    2016-01-01

    Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein- and integrin signal–dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector–target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific “bystander” killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells. PMID:27903610

  16. Active Dentate Granule Cells Encode Experience to Promote the Addition of Adult-Born Hippocampal Neurons

    PubMed Central

    Kirschen, Gregory W.; Shen, Jia; Wang, Jia; Man, Guoming; Wu, Song

    2017-01-01

    The continuous addition of new dentate granule cells (DGCs), which is regulated exquisitely by brain activity, renders the hippocampus plastic. However, how neural circuits encode experiences to affect the addition of adult-born neurons remains unknown. Here, we used endoscopic Ca2+ imaging to track the real-time activity of individual DGCs in freely behaving mice. For the first time, we found that active DGCs responded to a novel experience by increasing their Ca2+ event frequency preferentially. This elevated activity, which we found to be associated with object exploration, returned to baseline by 1 h in the same environment, but could be dishabituated via introduction to a novel environment. To transition seamlessly between environments, we next established a freely controllable virtual reality system for unrestrained mice. We again observed increased firing of active neurons in a virtual enriched environment. Interestingly, multiple novel virtual experiences increased the number of newborn neurons accumulatively compared with a single experience. Finally, optogenetic silencing of existing DGCs during novel environmental exploration perturbed experience-induced neuronal addition. Our study shows that the adult brain conveys novel, enriched experiences to increase the addition of adult-born hippocampal neurons by increasing the firing of active DGCs. SIGNIFICANCE STATEMENT Adult brains are constantly reshaping themselves from synapses to circuits as we encounter novel experiences from moment to moment. Importantly, this reshaping includes the addition of newborn hippocampal neurons. However, it remains largely unknown how our circuits encode experience-induced brain activity to govern the addition of new hippocampal neurons. By coupling in vivo Ca2+ imaging of dentate granule neurons with a novel, unrestrained virtual reality system for rodents, we discovered that a new experience increased firing of active dentate granule neurons rapidly and robustly

  17. Hilar Mossy Cell Degeneration Causes Transient Dentate Granule Cell Hyperexcitability and Impaired Pattern Separation

    PubMed Central

    Jinde, Seiichiro; Zsiros, Veronika; Jiang, Zhihong; Nakao, Kazuhito; Pickel, James; Kohno, Kenji; Belforte, Juan E.; Nakazawa, Kazu

    2012-01-01

    Summary Although excitatory mossy cells of the hippocampal hilar region are known to project both to dentate granule cells and to interneurons, it is as yet unclear whether mossy cell activity’s net effect on granule cells is excitatory or inhibitory. To explore their influence on dentate excitability and hippocampal function, we generated a conditional transgenic mouse line, using the Cre/loxP system, in which diphtheria toxin receptor was selectively expressed in mossy cells. One week after injecting toxin into this line, mossy cells throughout the longitudinal axis were degenerated extensively, theta wave power of dentate local field potentials increased during exploration, and deficits occurred in contextual discrimination. By contrast, we detected no epileptiform activity, spontaneous behavioral seizures, or mossy-fiber sprouting 5–6 weeks after mossy cell degeneration. These results indicate that the net effect of mossy cell excitation is to inhibit granule cell activity and enable dentate pattern separation. PMID:23259953

  18. Cerebellar granule cells encode the expectation of reward

    PubMed Central

    Wagner, Mark J; Kim, Tony Hyun; Savall, Joan; Schnitzer, Mark J; Luo, Liqun

    2017-01-01

    The human brain contains ~60 billion cerebellar granule cells1, which outnumber all other neurons combined. Classical theories posit that a large, diverse population of granule cells allows for highly detailed representations of sensorimotor context, enabling downstream Purkinje cells to sense fine contextual changes2–6. Although evidence suggests a role for cerebellum in cognition7–10, granule cells are known to encode only sensory11–13 and motor14 context. Using two-photon calcium imaging in behaving mice, here we show that granule cells convey information about the expectation of reward. Mice initiated voluntary forelimb movements for delayed water reward. Some granule cells responded preferentially to reward or reward omission, whereas others selectively encoded reward anticipation. Reward responses were not restricted to forelimb movement, as a Pavlovian task evoked similar responses. Compared to predictable rewards, unexpected rewards elicited markedly different granule cell activity despite identical stimuli and licking responses. In both tasks, reward signals were widespread throughout multiple cerebellar lobules. Tracking the same granule cells over several days of learning revealed that cells with reward-anticipating responses emerged from those that responded at the start of learning to reward delivery, whereas reward omission responses grew stronger as learning progressed. The discovery of predictive, non-sensorimotor encoding in granule cells is a major departure from current understanding of these neurons and dramatically enriches contextual information available to postsynaptic Purkinje cells, with important implications for cognitive processing in the cerebellum. PMID:28321129

  19. Colchicine induced intraneuronal free zinc accumulation and dentate granule cell degeneration.

    PubMed

    Choi, Bo Young; Lee, Bo Eun; Kim, Jin Hee; Kim, Hyun Jung; Sohn, Min; Song, Hong Ki; Chung, Tae Nyoung; Suh, Sang Won

    2014-08-01

    Colchicine has been discovered to inhibit many inflammatory processes such as gout, familial Mediterranean fever, pericarditis and Behcet disease. Other than these beneficial anti-inflammatory effects, colchicine blocks microtubule-assisted axonal transport, which results in the selective loss of dentate granule cells of the hippocampus. The mechanism of the colchicine-induced dentate granule cell death and depletion of mossy fiber terminals still remains unclear. In the present study, we hypothesized that colchicine-induced dentate granule cell death may be caused by accumulation of labile intracellular zinc. 10 μg kg(-1) of colchicine was injected into the adult rat hippocampus and then brain sections were evaluated at 1 day or 1 week later. Neuronal cell death was evaluated by H&E staining or Fluoro-Jade B. Zinc accumulation and vesicular zinc were detected by N-(6-methoxy-8-quinolyl)-para-toluene sulfonamide (TSQ) staining. To test whether an extracellular zinc chelator can prevent this process, CaEDTA was injected into the hippocampus over a 5 min period with colchicine. To test whether other microtubule toxins also produce similar effects as colchicine, vincristine was injected into the hippocampus. The present study found that colchicine injection induced intracellular zinc accumulation in the dentate granule cells and depleted vesicular zinc from mossy fiber terminals. Injection of a zinc chelator, CaEDTA, did not block the zinc accumulation and neuronal death. Vincristine also produced intracellular zinc accumulation and neuronal death. These results suggest that colchicine-induced dentate granule cell death is caused by blocking axonal zinc flow and accumulation of intracellular labile zinc.

  20. 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

  1. Functional Convergence of Neurons Generated in the Developing and Adult Hippocampus

    PubMed Central

    Piatti, Verónica C; Morgenstern, Nicolás A; Zhao, Chunmei; van Praag, Henriette; Gage, Fred H; Schinder, Alejandro F

    2006-01-01

    The dentate gyrus of the hippocampus contains neural progenitor cells (NPCs) that generate neurons throughout life. Developing neurons of the adult hippocampus have been described in depth. However, little is known about their functional properties as they become fully mature dentate granule cells (DGCs). To compare mature DGCs generated during development and adulthood, NPCs were labeled at both time points using retroviruses expressing different fluorescent proteins. Sequential electrophysiological recordings from neighboring neurons of different ages were carried out to quantitatively study their major synaptic inputs: excitatory projections from the entorhinal cortex and inhibitory afferents from local interneurons. Our results show that DGCs generated in the developing and adult hippocampus display a remarkably similar afferent connectivity with regard to both glutamate and GABA, the major neurotransmitters. We also demonstrate that adult-born neurons can fire action potentials in response to an excitatory drive, exhibiting a firing behavior comparable to that of neurons generated during development. We propose that neurons born in the developing and adult hippocampus constitute a functionally homogeneous neuronal population. These observations are critical to understanding the role of adult neurogenesis in hippocampal function. PMID:17121455

  2. Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition

    PubMed Central

    Duguid, Ian; Branco, Tiago; Chadderton, Paul; Arlt, Charlotte; Powell, Kate; Häusser, Michael

    2015-01-01

    Classical feed-forward inhibition involves an excitation–inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses. PMID:26432880

  3. Bmi1 overexpression in the cerebellar granule cell lineage of mice affects cell proliferation and survival without initiating medulloblastoma formation

    PubMed Central

    Behesti, Hourinaz; Bhagat, Heeta; Dubuc, Adrian M.; Taylor, Michael D.; Marino, Silvia

    2013-01-01

    SUMMARY BMI1 is a potent inducer of neural stem cell self-renewal and neural progenitor cell proliferation during development and in adult tissue homeostasis. It is overexpressed in numerous human cancers – including medulloblastomas, in which its functional role is unclear. We generated transgenic mouse lines with targeted overexpression of Bmi1 in the cerebellar granule cell lineage, a cell type that has been shown to act as a cell of origin for medulloblastomas. Overexpression of Bmi1 in granule cell progenitors (GCPs) led to a decrease in cerebellar size due to decreased GCP proliferation and repression of the expression of cyclin genes, whereas Bmi1 overexpression in postmitotic granule cells improved cell survival in response to stress by altering the expression of genes in the mitochondrial cell death pathway and of Myc and Lef-1. Although no medulloblastomas developed in ageing cohorts of transgenic mice, crosses with Trp53−/− mice resulted in a low incidence of medulloblastoma formation. Furthermore, analysis of a large collection of primary human medulloblastomas revealed that tumours with a BMI1high TP53low molecular profile are significantly enriched in Group 4 human medulloblastomas. Our data suggest that different levels and timing of Bmi1 overexpression yield distinct cellular outcomes within the same cellular lineage. Importantly, Bmi1 overexpression at the GCP stage does not induce tumour formation, suggesting that BMI1 overexpression in GCP-derived human medulloblastomas probably occurs during later stages of oncogenesis and might serve to enhance tumour cell survival. PMID:23065639

  4. 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

  5. From the Cover: Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells

    NASA Astrophysics Data System (ADS)

    Sapir, Tamar; Shternhall, Keren; Meivar-Levy, Irit; Blumenfeld, Tamar; Cohen, Hamutal; Skutelsky, Ehud; Eventov-Friedman, Smadar; Barshack, Iris; Goldberg, Iris; Pri-Chen, Sarah; Ben-Dor, Lya; Polak-Charcon, Sylvie; Karasik, Avraham; Shimon, Ilan; Mor, Eytan; Ferber, Sarah

    2005-05-01

    Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue. pancreas | transdifferentiation

  6. Cerebellar Granule Cell Replenishment Post-Injury by Adaptive Reprogramming of Nestin+ Progenitors

    PubMed Central

    Wojcinski, Alexandre; Lawton, Andrew K.; Bayin, N Sumru.; Lao, Zhimin; Stephen, Daniel N.; Joyner, Alexandra L.

    2017-01-01

    Regeneration of several organs involves adaptive reprogramming of progenitors, however, the intrinsic capacity of the developing brain to replenish lost cells remains largely unknown. In this study, we discovered that the developing cerebellum has unappreciated progenitor plasticity, since it undergoes near full growth and functional recovery following acute depletion of granule cells, the most plentiful neuron population in the brain. We demonstrate that following postnatal ablation of granule cell progenitors, Nestin-expressing progenitors (NEPs) specified during mid-embryogenesis to produce astroglia and interneurons, switch their fate and generate granule neurons in mice. Moreover, Hedgehog-signaling in two NEP populations is crucial not only for the compensatory replenishment of granule neurons but also to scale interneuron and astrocyte numbers. Thus we provide insights into the mechanisms underlying robustness of circuit formation in the cerebellum, and speculate that adaptive reprogramming of progenitors in other brain regions plays a greater role than appreciated in developmental regeneration. PMID:28805814

  7. Rabies Tracing of Birthdated Dentate Granule Cells in Rat Temporal Lobe Epilepsy

    PubMed Central

    Du, Xi; Zhang, Helen; Parent, Jack M.

    2017-01-01

    Objective To understand how monosynaptic inputs onto adult-born dentate granule cells (DGCs) are altered in experimental mesial temporal lobe epilepsy (mTLE) and whether their integration differs from early-born DGCs that are mature at the time of epileptogenesis. Methods A dual-virus tracing strategy combining retroviral birthdating with rabies virus-mediated putative retrograde trans-synaptic tracing was used to identify and compare presynaptic inputs onto adult- and early-born DGCs in the rat pilocarpine model of mTLE. Results Our results demonstrate that hilar ectopic DGCs preferentially synapse onto adult-born DGCs after pilocarpine-induced status epilepticus (SE) while normotopic DGCs synapse onto both adult- and early-born DGCs. We also find that parvalbumin+ and somatostatin+ interneuron inputs are greatly diminished onto early-born DGCs after SE. However, somatostatin+ interneuron inputs onto adult-born DGCs are maintained, likely due to preferential sprouting. Intriguingly, CA3 pyramidal cell backprojections that specifically target adult-born DGCs arise in the epileptic brain, while axons of interneurons and pyramidal cells in CA1 appear to sprout across the hippocampal fissure to preferentially synapse onto early-born DGCs. Interpretation These data support the presence of substantial hippocampal circuit remodeling after an epileptogenic insult that generates prominent excitatory monosynaptic inputs, both local recurrent and widespread feedback loops, involving DGCs. Both adult- and early-born DGCs are targets of new inputs from other DGCs as well as from CA3 and CA1 pyramidal cells after pilocarpine-treatment, changes that likely contribute to epileptogenesis in experimental mTLE. PMID:28470680

  8. 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.

  9. Semaphorin 5A inhibits synaptogenesis in early postnatal- and adult-born hippocampal dentate granule cells.

    PubMed

    Duan, Yuntao; Wang, Shih-Hsiu; Song, Juan; Mironova, Yevgeniya; Ming, Guo-li; Kolodkin, Alex L; Giger, Roman J

    2014-10-14

    Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain development is unknown. In this study, we show that mouse Sema5A negatively regulates synaptogenesis in early, developmentally born, hippocampal dentate granule cells (GCs). Sema5A is strongly expressed by GCs and regulates dendritic spine density in a cell-autonomous manner. In the adult mouse brain, newly born Sema5A-/- GCs show an increase in dendritic spine density and increased AMPA-type synaptic responses. Sema5A signals through PlexinA2 co-expressed by GCs, and the PlexinA2-RasGAP activity is necessary to suppress spinogenesis. Like Sema5A-/- mutants, PlexinA2-/- mice show an increase in GC glutamatergic synapses, and we show that Sema5A and PlexinA2 genetically interact with respect to GC spine phenotypes. Sema5A-/- mice display deficits in social interaction, a hallmark of autism-spectrum-disorders. These experiments identify novel intra-dendritic Sema5A/PlexinA2 interactions that inhibit excitatory synapse formation in developmentally born and adult-born GCs, and they provide support for SEMA5A contributions to autism-spectrum-disorders.

  10. Odontogenic Differentiation of Human Dental Pulp Stem Cells Stimulated by the Calcium Phosphate Porous Granules

    PubMed Central

    Nam, Sunyoung; Won, Jong-Eun; Kim, Cheol-Hwan; Kim, Hae-Won

    2011-01-01

    Effects of three-dimensional (3D) calcium phosphate (CaP) porous granules on the growth and odontogenic differentiation of human dental pulp stem cells (hDPSCs) were examined for dental tissue engineering. hDPSCs isolated from adult human dental pulps were cultured for 3-4 passages, and populated on porous granules. Cell growth on the culture dish showed an ongoing increase for up to 21 days, whereas the growth on the 3D granules decreased after 14 days. This reduction in proliferative potential on the 3D granules was more conspicuous under the osteogenic medium conditions, indicating that the 3D granules may induce the odontogenic differentiation of hDPSCs. Differentiation behavior on the 3D granules was confirmed by the increased alkaline phosphatase activity, up-regulation of odontoblast-specific genes, including dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) by quantitative polymerase chain reaction, and greater level of dentin sialoprotein synthesis by western blot. Moreover, the cellular mineralization, as assessed by Alizarin red S and calcium quantification, was significantly higher in the 3D CaP granules than in the culture dish. Taken all, the 3D CaP porous granules should be useful for dental tissue engineering in combination with hDPSCs by providing favorable 3D substrate conditions for cell growth and odontogenic development. PMID:21772958

  11. Second Harmonic Generation Mediated by Aligned Water in Starch Granules.

    PubMed

    Cisek, Richard; Tokarz, Danielle; Krouglov, Serguei; Steup, Martin; Emes, Michael J; Tetlow, Ian J; Barzda, Virginijus

    2014-12-26

    The origin of second harmonic generation (SHG) in starch granules was investigated using ab initio quantum mechanical modeling and experimentally examined using polarization-in, polarization-out (PIPO) second harmonic generation microscopy. Ab initio calculations revealed that the largest contribution to the SHG signal from A- and B-type allomorphs of starch originates from the anisotropic organization of hydroxide and hydrogen bonds mediated by aligned water found in the polymers. The hypothesis was experimentally tested by imaging maize starch granules under various hydration and heat treatment conditions that alter the hydrogen bond network. The highest SHG intensity was found in fully hydrated starch granules, and heat treatment diminished the SHG intensity. The PIPO SHG imaging showed that dried starch granules have a much higher nonlinear optical susceptibility component ratio than fully hydrated granules. In contrast, deuterated starch granules showed a smaller susceptibility component ratio demonstrating that SHG is highly sensitive to the organization of the hydroxyl and hydrogen bond network. The polarization SHG imaging results of potato starch granules, representing starch allomorph B, were compared to those of maize starch granules representing allomorph A. The results showed that the amount of aligned water was higher in the maize granules. Nonlinear microscopy of starch granules provides evidence that varying hydration conditions leads to significant changes in the nonlinear susceptibility ratio as well as the SHG intensity, supporting the hypothesis from ab initio calculations that the dominant contribution to SHG is due to the ordered hydroxide and hydrogen bond network.

  12. COUP-TFI mitotically regulates production and migration of dentate granule cells and modulates hippocampal Cxcr4 expression.

    PubMed

    Parisot, Joséphine; Flore, Gemma; Bertacchi, Michele; Studer, Michèle

    2017-06-01

    Development of the dentate gyrus (DG), the primary gateway for hippocampal inputs, spans embryonic and postnatal stages, and involves complex morphogenetic events. We have previously identified the nuclear receptor COUP-TFI as a novel transcriptional regulator in the postnatal organization and function of the hippocampus. Here, we dissect its role in DG morphogenesis by inactivating it in either granule cell progenitors or granule neurons. Loss of COUP-TFI function in progenitors leads to decreased granule cell proliferative activity, precocious differentiation and increased apoptosis, resulting in a severe DG growth defect in adult mice. COUP-TFI-deficient cells express high levels of the chemokine receptor Cxcr4 and migrate abnormally, forming heterotopic clusters of differentiated granule cells along their paths. Conversely, high COUP-TFI expression levels downregulate Cxcr4 expression, whereas increased Cxcr4 expression in wild-type hippocampal cells affects cell migration. Finally, loss of COUP-TFI in postmitotic cells leads to only minor and transient abnormalities, and to normal Cxcr4 expression. Together, our results indicate that COUP-TFI is required predominantly in DG progenitors for modulating expression of the Cxcr4 receptor during granule cell neurogenesis and migration. © 2017. Published by The Company of Biologists Ltd.

  13. Generation of functional human pancreatic β cells in vitro

    PubMed Central

    Pagliuca, Felicia W.; Millman, Jeffrey R.; Gürtler, Mads; Segel, Michael; Van Dervort, Alana; Ryu, Jennifer Hyoje; Peterson, Quinn P.; Greiner, Dale; Melton, Douglas A.

    2015-01-01

    Summary The generation of insulin-producing pancreatic β cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide β cells. Here we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem cell derived β cells (SC-β) express markers found in mature β cells, flux Ca2+ in response to glucose, package insulin into secretory granules and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice. PMID:25303535

  14. Time-lapse imaging reveals highly dynamic structural maturation of postnatally born dentate granule cells in organotypic entorhino-hippocampal slice cultures

    PubMed Central

    Radic, Tijana; Jungenitz, Tassilo; Singer, Mathias; Beining, Marcel; Cuntz, Hermann; Vlachos, Andreas; Deller, Thomas; Schwarzacher, Stephan W.

    2017-01-01

    Neurogenesis of hippocampal granule cells (GCs) persists throughout mammalian life and is important for learning and memory. How newborn GCs differentiate and mature into an existing circuit during this time period is not yet fully understood. We established a method to visualize postnatally generated GCs in organotypic entorhino-hippocampal slice cultures (OTCs) using retroviral (RV) GFP-labeling and performed time-lapse imaging to study their morphological development in vitro. Using anterograde tracing we could, furthermore, demonstrate that the postnatally generated GCs in OTCs, similar to adult born GCs, grow into an existing entorhino-dentate circuitry. RV-labeled GCs were identified and individual cells were followed for up to four weeks post injection. Postnatally born GCs exhibited highly dynamic structural changes, including dendritic growth spurts but also retraction of dendrites and phases of dendritic stabilization. In contrast, older, presumably prenatally born GCs labeled with an adeno-associated virus (AAV), were far less dynamic. We propose that the high degree of structural flexibility seen in our preparations is necessary for the integration of newborn granule cells into an already existing neuronal circuit of the dentate gyrus in which they have to compete for entorhinal input with cells generated and integrated earlier. PMID:28256620

  15. T2N as a new tool for robust electrophysiological modeling demonstrated for mature and adult-born dentate granule cells

    PubMed Central

    Mongiat, Lucas Alberto; Schwarzacher, Stephan Wolfgang

    2017-01-01

    Compartmental models are the theoretical tool of choice for understanding single neuron computations. However, many models are incomplete, built ad hoc and require tuning for each novel condition rendering them of limited usability. Here, we present T2N, a powerful interface to control NEURON with Matlab and TREES toolbox, which supports generating models stable over a broad range of reconstructed and synthetic morphologies. We illustrate this for a novel, highly detailed active model of dentate granule cells (GCs) replicating a wide palette of experiments from various labs. By implementing known differences in ion channel composition and morphology, our model reproduces data from mouse or rat, mature or adult-born GCs as well as pharmacological interventions and epileptic conditions. This work sets a new benchmark for detailed compartmental modeling. T2N is suitable for creating robust models useful for large-scale networks that could lead to novel predictions. We discuss possible T2N application in degeneracy studies. PMID:29165247

  16. Trajectory Analysis Unveils Reelin's Role in the Directed Migration of Granule Cells in the Dentate Gyrus.

    PubMed

    Wang, Shaobo; Brunne, Bianka; Zhao, Shanting; Chai, Xuejun; Li, Jiawei; Lau, Jeremie; Failla, Antonio Virgilio; Zobiak, Bernd; Sibbe, Mirjam; Westbrook, Gary L; Lutz, David; Frotscher, Michael

    2018-01-03

    Reelin controls neuronal migration and layer formation. Previous studies in reeler mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in reeler , but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated proopiomelanocortin-EGFP -expressing dentate granule cells in slice cultures from reeler , reeler -like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from reeler and reeler -like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in reeler slices cocultured to wild-type dentate gyrus showed that the reeler neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of reeler slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer. SIGNIFICANCE STATEMENT Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the

  17. Discovery of Transcription Factors Novel to Mouse Cerebellar Granule Cell Development Through Laser-Capture Microdissection.

    PubMed

    Zhang, Peter G Y; Yeung, Joanna; Gupta, Ishita; Ramirez, Miguel; Ha, Thomas; Swanson, Douglas J; Nagao-Sato, Sayaka; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Daub, Carsten O; Arner, Erik; de Hoon, Michiel; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide; Goldowitz, Dan

    2018-06-01

    Laser-capture microdissection was used to isolate external germinal layer tissue from three developmental periods of mouse cerebellar development: embryonic days 13, 15, and 18. The cerebellar granule cell-enriched mRNA library was generated with next-generation sequencing using the Helicos technology. Our objective was to discover transcriptional regulators that could be important for the development of cerebellar granule cells-the most numerous neuron in the central nervous system. Through differential expression analysis, we have identified 82 differentially expressed transcription factors (TFs) from a total of 1311 differentially expressed genes. In addition, with TF-binding sequence analysis, we have identified 46 TF candidates that could be key regulators responsible for the variation in the granule cell transcriptome between developmental stages. Altogether, we identified 125 potential TFs (82 from differential expression analysis, 46 from motif analysis with 3 overlaps in the two sets). From this gene set, 37 TFs are considered novel due to the lack of previous knowledge about their roles in cerebellar development. The results from transcriptome-wide analyses were validated with existing online databases, qRT-PCR, and in situ hybridization. This study provides an initial insight into the TFs of cerebellar granule cells that might be important for development and provide valuable information for further functional studies on these transcriptional regulators.

  18. Cell-type-specific expression of NFIX in the developing and adult cerebellum.

    PubMed

    Fraser, James; Essebier, Alexandra; Gronostajski, Richard M; Boden, Mikael; Wainwright, Brandon J; Harvey, Tracey J; Piper, Michael

    2017-07-01

    Transcription factors from the nuclear factor one (NFI) family have been shown to play a central role in regulating neural progenitor cell differentiation within the embryonic and post-natal brain. NFIA and NFIB, for instance, promote the differentiation and functional maturation of granule neurons within the cerebellum. Mice lacking Nfix exhibit delays in the development of neuronal and glial lineages within the cerebellum, but the cell-type-specific expression of this transcription factor remains undefined. Here, we examined the expression of NFIX, together with various cell-type-specific markers, within the developing and adult cerebellum using both chromogenic immunohistochemistry and co-immunofluorescence labelling and confocal microscopy. In embryos, NFIX was expressed by progenitor cells within the rhombic lip and ventricular zone. After birth, progenitor cells within the external granule layer, as well as migrating and mature granule neurons, expressed NFIX. Within the adult cerebellum, NFIX displayed a broad expression profile, and was evident within granule cells, Bergmann glia, and interneurons, but not within Purkinje neurons. Furthermore, transcriptomic profiling of cerebellar granule neuron progenitor cells showed that multiple splice variants of Nfix are expressed within this germinal zone of the post-natal brain. Collectively, these data suggest that NFIX plays a role in regulating progenitor cell biology within the embryonic and post-natal cerebellum, as well as an ongoing role within multiple neuronal and glial populations within the adult cerebellum.

  19. GABA regulates synaptic integration of newly generated neurons in the adult brain

    NASA Astrophysics Data System (ADS)

    Ge, Shaoyu; Goh, Eyleen L. K.; Sailor, Kurt A.; Kitabatake, Yasuji; Ming, Guo-Li; Song, Hongjun

    2006-02-01

    Adult neurogenesis, the birth and integration of new neurons from adult neural stem cells, is a striking form of structural plasticity and highlights the regenerative capacity of the adult mammalian brain. Accumulating evidence suggests that neuronal activity regulates adult neurogenesis and that new neurons contribute to specific brain functions. The mechanism that regulates the integration of newly generated neurons into the pre-existing functional circuitry in the adult brain is unknown. Here we show that newborn granule cells in the dentate gyrus of the adult hippocampus are tonically activated by ambient GABA (γ-aminobutyric acid) before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA, the major inhibitory neurotransmitter in the adult brain, initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic chloride ion content. Conversion of GABA-induced depolarization (excitation) into hyperpolarization (inhibition) in newborn neurons leads to marked defects in their synapse formation and dendritic development in vivo. Our study identifies an essential role for GABA in the synaptic integration of newly generated neurons in the adult brain, and suggests an unexpected mechanism for activity-dependent regulation of adult neurogenesis, in which newborn neurons may sense neuronal network activity through tonic and phasic GABA activation.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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. 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

  2. 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

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

    PubMed

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

    2008-03-01

    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).

  4. Gap Junction Modulation of Low-Frequency Oscillations in the Cerebellar Granule Cell Layer.

    PubMed

    Robinson, Jennifer Claire; Chapman, C Andrew; Courtemanche, Richard

    2017-08-01

    Local field potential (LFP) oscillations in the granule cell layer (GCL) of the cerebellar cortex have been identified previously in the awake rat and monkey during immobility. These low-frequency oscillations are thought to be generated through local circuit interactions between Golgi cells and granule cells within the GCL. Golgi cells display rhythmic firing and pacemaking properties, and also are electrically coupled through gap junctions within the GCL. Here, we tested if gap junctions in the rat cerebellar cortex contribute to the generation of LFP oscillations in the GCL. We recorded LFP oscillations under urethane anesthesia, and examined the effects of local infusion of gap junction blockers on 5-15 Hz oscillations. Local infusion of the gap junction blockers carbenoxolone and mefloquine resulted in significant decreases in the power of oscillations over a 30-min period, but the power of oscillations was unchanged in control experiments following vehicle injections. In addition, infusion of gap junction blockers had no significant effect on multi-unit activity, suggesting that the attenuation of low-frequency oscillations was likely due to reductions in electrical coupling rather than a decreased excitability within the granule cell layer. Our results indicate that electrical coupling among the Golgi cell networks in the cerebellar cortex contributes to the local circuit mechanisms that promote the occurrence of GCL LFP slow oscillations in the anesthetized rat.

  5. Constitutively polarized granules prime KHYG-1 NK cells.

    PubMed

    Suck, Garnet; Branch, Donald R; Aravena, Paola; Mathieson, Mark; Helke, Simone; Keating, Armand

    2006-09-01

    The major mechanism for NK cell lysis of tumor cells is granule-mediated cytotoxicity. Polarization of granules is a prelude to the release of their cytotoxic contents in response to target-cell binding. We describe the novel observation of constitutive granule polarization in the cytotoxic NK cell line, KHYG-1. Continuous degranulation of KHYG-1 cells, however, does not occur and still requires target-cell contact. Disruption of microtubules with colcemid is sufficient to disperse the granules in KHYG-1 and significantly decreases cytotoxicity. A similar effect is not obtained by inhibiting extracellular signal-related kinase 2 (ERK2), the most distal kinase investigated in the cytolytic pathway. Disruption of microtubules significantly down-regulates activation receptors, NKp44 and NKG2D, implicating them as potential microtubule-trafficking receptors. Such changes in upstream receptor expression may have caused deactivation of ERK2, since NKG2D cross-linking also leads to receptor down-regulation and diminished ERK phosphorylation. Thus, a functional role for NKG2D in KHYG-1 cytotoxicity is demonstrated. Moreover, the novel primed state may contribute to the high cytotoxicity exhibited by KHYG-1.

  6. Morphological Constraints on Cerebellar Granule Cell Combinatorial Diversity.

    PubMed

    Gilmer, Jesse I; Person, Abigail L

    2017-12-13

    Combinatorial expansion by the cerebellar granule cell layer (GCL) is fundamental to theories of cerebellar contributions to motor control and learning. Granule cells (GrCs) sample approximately four mossy fiber inputs and are thought to form a combinatorial code useful for pattern separation and learning. We constructed a spatially realistic model of the cerebellar GCL and examined how GCL architecture contributes to GrC combinatorial diversity. We found that GrC combinatorial diversity saturates quickly as mossy fiber input diversity increases, and that this saturation is in part a consequence of short dendrites, which limit access to diverse inputs and favor dense sampling of local inputs. This local sampling also produced GrCs that were combinatorially redundant, even when input diversity was extremely high. In addition, we found that mossy fiber clustering, which is a common anatomical pattern, also led to increased redundancy of GrC input combinations. We related this redundancy to hypothesized roles of temporal expansion of GrC information encoding in service of learned timing, and we show that GCL architecture produces GrC populations that support both temporal and combinatorial expansion. Finally, we used novel anatomical measurements from mice of either sex to inform modeling of sparse and filopodia-bearing mossy fibers, finding that these circuit features uniquely contribute to enhancing GrC diversification and redundancy. Our results complement information theoretic studies of granule layer structure and provide insight into the contributions of granule layer anatomical features to afferent mixing. SIGNIFICANCE STATEMENT Cerebellar granule cells are among the simplest neurons, with tiny somata and, on average, just four dendrites. These characteristics, along with their dense organization, inspired influential theoretical work on the granule cell layer as a combinatorial expander, where each granule cell represents a unique combination of inputs

  7. Rapid integration of young newborn dentate gyrus granule cells in the adult hippocampal circuitry.

    PubMed

    Ide, Yoko; Fujiyama, Fumino; Okamoto-Furuta, Keiko; Tamamaki, Nobuaki; Kaneko, Takeshi; Hisatsune, Tatsuhiro

    2008-12-01

    Newborn dentate gyrus granule cells (DGCs) are integrated into the hippocampal circuitry and contribute to the cognitive functions of learning and memory. The dendritic maturation of newborn DGCs in adult mice occurs by the first 3-4 weeks, but DGCs seem to receive a variety of neural inputs at both their dendrites and soma even shortly after their birth. However, few studies on the axonal maturation of newborn DGCs have focused on synaptic structure. Here, we investigated the potentiality of output and input in newborn DGCs, especially in the early period after terminal mitosis. We labeled nestin-positive progenitor cells by injecting GFP Cre-reporter adenovirus into Nestin-Cre mice, enabling us to trace the development of progenitor cells by their GFP expression. In addition to GABAergic input from interneurons, we observed that the young DGCs received axosomatic input from the medial septum as early as postinfection day 7 (PID 7). To evaluate the axonal maturation of the newborn DGCs compared with mature DCGs, we performed confocal and electron microscopic analyses. We observed that newborn DGCs projected their mossy fibers to the CA3 region, forming small terminals on hilar or CA3 interneurons and large boutons on CA3 pyramidal cells. These terminals expressed vesicular glutamate transporter 1, indicating they were glutamatergic terminals. Intriguingly, the terminals at PID 7 had already formed asymmetric synapses, similar to those of mature DGCs. Together, our findings suggest that newborn DGCs may form excitatory synapses on both interneurons and CA3 pyramidal cells within 7 days of their terminal mitosis.

  8. NAADP Activates Two-Pore Channels on T Cell Cytolytic Granules to Stimulate Exocytosis and Killing

    PubMed Central

    Davis, Lianne C.; Morgan, Anthony J.; Chen, Ji-Li; Snead, Charlotte M.; Bloor-Young, Duncan; Shenderov, Eugene; Stanton-Humphreys, Megan N.; Conway, Stuart J.; Churchill, Grant C.; Parrington, John; Cerundolo, Vincenzo; Galione, Antony

    2012-01-01

    Summary A cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Ca2+-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. Although inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum activates the store-operated Ca2+-influx pathway that is necessary for exocytosis, it is not a sufficient stimulus [1–4]. Here we identify the Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and its recently identified molecular target, two-pore channels (TPCs) [5–7], as being important for T cell receptor signaling in CTLs. We demonstrate that cytolytic granules are not only reservoirs of cytolytic proteins but are also the acidic Ca2+ stores mobilized by NAADP via TPC channels on the granules themselves, so that TPCs migrate to the immunological synapse upon CTL activation. Moreover, NAADP activates TPCs to drive exocytosis in a way that is not mimicked by global Ca2+ signals induced by IP3 or ionomycin, suggesting that critical, local Ca2+ nanodomains around TPCs stimulate granule exocytosis. Hence, by virtue of the NAADP/TPC pathway, cytolytic granules generate Ca2+ signals that lead to their own exocytosis and to cell killing. This study highlights a selective role for NAADP in stimulating exocytosis crucial for immune cell function and may impact on stimulus-secretion coupling in wider cellular contexts. PMID:23177477

  9. 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

  10. Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells

    PubMed Central

    Osorio, Nancy; Cathala, Laurence; Meisler, Miriam H; Crest, Marcel; Magistretti, Jacopo; Delmas, Patrick

    2010-01-01

    Cerebellar granule (CG) cells generate high-frequency action potentials that have been proposed to depend on the unique properties of their voltage-gated ion channels. To address the in vivo function of Nav1.6 channels in developing and mature CG cells, we combined the study of the developmental expression of Nav subunits with recording of acute cerebellar slices from young and adult granule-specific Scn8a KO mice. Nav1.2 accumulated rapidly at early-formed axon initial segments (AISs). In contrast, Nav1.6 was absent at early postnatal stages but accumulated at AISs of CG cells from P21 to P40. By P40–P65, both Nav1.6 and Nav1.2 co-localized at CG cell AISs. By comparing Na+ currents in mature CG cells (P66–P74) from wild-type and CG-specific Scn8a KO mice, we found that transient and resurgent Na+ currents were not modified in the absence of Nav1.6 whereas persistent Na+ current was strongly reduced. Action potentials in conditional Scn8a KO CG cells showed no alteration in threshold and overshoot, but had a faster repolarization phase and larger post-spike hyperpolarization. In addition, although Scn8a KO CG cells kept their ability to fire action potentials at very high frequency, they displayed increased interspike-interval variability and firing irregularity in response to sustained depolarization. We conclude that Nav1.6 channels at axon initial segments contribute to persistent Na+ current and ensure a high degree of temporal precision in repetitive firing of CG cells. PMID:20173079

  11. Olfactory granule cell development in normal and hyperthyroid rats.

    PubMed

    Brunjes, P C; Schwark, H D; Greenough, W T

    1982-10-01

    Dendritic development was examined in olfactory bulbs of both normal 7-, 14-, 21- and 60-day-old rats and littermates treated on postnatal days 1-4 with 1 microgram/g body weight of L-thyroxine sodium. Tissue was processed via the Golgi-Cox technique and subjected to quantitative analyses of mitral and internal layer granule cell development. These populations of granule cells were selected because their pattern of late proliferation suggested potentially greater susceptibility to postnatal hormonal alterations. Although neonatal hyperthyroidism induces widespread acceleration of maturation, including precocious chemosensitivity, granule cell development was unaffected relative to littermate controls. Both normal and hyperthyroid groups exhibited an inverted U-shaped pattern of cellular development, with rapid dendritic dendritic growth and expansion occurring during the earliest ages tested, but with loss of processes and dendritic field size occurring after day 21.

  12. Convergence of pontine and proprioceptive streams onto multimodal cerebellar granule cells

    PubMed Central

    Huang, Cheng-Chiu; Sugino, Ken; Shima, Yasuyuki; Guo, Caiying; Bai, Suxia; Mensh, Brett D; Nelson, Sacha B; Hantman, Adam W

    2013-01-01

    Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control. DOI: http://dx.doi.org/10.7554/eLife.00400.001 PMID:23467508

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

    PubMed

    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

    2016-10-17

    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 spines, the complexity of multisynaptic innervations and the degree of the perisynaptic astroglial ensheathment that controls synaptic homeostasis. These findings show a pivotal role of Reelin in GC synaptogenesis and provide a foundation for structural circuit alterations caused by Reelin deregulation that may occur in neurological and psychiatric disorders. © The Author 2016. Published by Oxford University Press.

  14. 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.

  15. Induction of stress granule-like structures in vesicular stomatitis virus-infected cells.

    PubMed

    Dinh, Phat X; Beura, Lalit K; Das, Phani B; Panda, Debasis; Das, Anshuman; Pattnaik, Asit K

    2013-01-01

    Previous studies from our laboratory revealed that cellular poly(C) binding protein 2 (PCBP2) downregulates vesicular stomatitis virus (VSV) gene expression. We show here that VSV infection induces the formation of granular structures in the cytoplasm containing cellular RNA-binding proteins, including PCBP2, T-cell-restricted intracellular antigen 1 (TIA1), and TIA1-related protein (TIAR). Depletion of TIA1 via small interfering RNAs (siRNAs), but not depletion of TIAR, results in enhanced VSV growth and gene expression. The VSV-induced granules appear to be similar to the stress granules (SGs) generated in cells triggered by heat shock or oxidative stress but do not contain some of the bona fide SG markers, such as eukaryotic initiation factor 3 (eIF3) or eIF4A, or the processing body (PB) markers, such as mRNA-decapping enzyme 1A (DCP1a), and thus may not represent canonical SGs or PBs. Our results revealed that the VSV-induced granules, called SG-like structures here, contain the viral replicative proteins and RNAs. The formation and maintenance of the SG-like structures required viral replication and ongoing protein synthesis, but an intact cytoskeletal network was not necessary. These results suggest that cells respond to VSV infection by aggregating the antiviral proteins, such as PCBP2 and TIA1, to form SG-like structures. The functional significance of these SG-like structures in VSV-infected cells is currently under investigation.

  16. Delayed coupling to feedback inhibition during a critical period for the integration of adult-born granule cells

    PubMed Central

    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.

    2014-01-01

    SUMMARY 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 (four-week-old) GCs can efficiently drive distal CA3 targets, but poorly activate proximal interneurons responsible for feedback inhibition (FBI). As new GCs transition towards 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

  17. 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

    2015-01-07

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. The biological significance of storage granules in rat parathyroid cells.

    PubMed

    Setoguti, T; Inoue, Y; Wild, P

    1995-10-01

    Both prosecretory and storage granules are concomitantly formed at the trans Golgi network including the innermost Golgi cisterna. Prosecretory granules develop into small secretory granules that release their contents by exocytosis finely regulated by a complex mechanism for maintaining calcium homeostasis. In the rat parathyroid cells, storage granules are large secretory granules storing parathyroid hormone for an emergency supply. The hormone is rapidly discharged by exocytosis when serum calcium concentration is decreased. The granules are constantly produced even under conditions of low serum calcium concentration in the regions of 8 mg/dl. The granule content is constantly hydrolyzed when not discharged, leading to a decreased core and finally to the formation of vacuolar bodies. The fate of the vacuolar bodies is unknown. Hypercalcemic conditions accelerate hydrolysis. The threshold value of calcium concentration required for the release of storage granule contents is between 8.0 and 7.5 mg/dl and that of calcium concentration for accelerating degradation of storage granules is about 11.5 mg/dl. Sympathetic stimulation causes storage granules to be discharged regardless of hypercalcemia or hypocalcemia. Parasympathetic stimulation accelerates hydrolysis. The degradation of storage granules seems to be closely associated with an intracellular regulatory mechanism for parathyroid hormone secretion.

  19. Cell-autonomous inactivation of the Reelin pathway impairs adult neurogenesis in the hippocampus

    PubMed Central

    Teixeira, Catia M.; Kron, Michelle M.; Masachs, Nuria; Zhang, Helen; Lagace, Diane C.; Martinez, Albert; Reillo, Isabel; Duan, Xin; Bosch, Carles; Pujadas, Lluis; Brunso, Lucas; Song, Hongjun; Eisch, Amelia J.; Borrell, Victor; Howell, Brian W.; Parent, Jack M.; Soriano, Eduardo

    2012-01-01

    Adult hippocampal neurogenesis is thought to be essential for learning and memory and has been implicated in the pathogenesis of several disorders. Although recent studies have identified key factors regulating neuroprogenitor proliferation in the adult hippocampus, the mechanisms that control the migration and integration of adult-born neurons into circuits are largely unknown. Reelin is an extracellular matrix protein that is vital for neuronal development. Activation of the Reelin cascade leads to phosphorylation of disabled-1 (Dab1), an adaptor protein required for Reelin signaling. Here we used transgenic mouse and retroviral reporters along with Reelin signaling gain- and loss-of-function studies to show that the Reelin pathway regulates migration and dendritic development of adult-generated hippocampal neurons. Whereas overexpression of Reelin accelerated dendritic maturation, inactivation of the Reelin signaling pathway specifically in adult neuroprogenitor cells resulted in aberrant migration, decreased dendrite development, formation of ectopic dendrites in the hilus and the establishment of aberrant circuits. Our findings support a cell-autonomous and critical role for the Reelin pathway in regulating dendritic development and the integration of adult-generated granule cells and point to this pathway as a key regulator of adult neurogenesis. Moreover, our data reveal a novel role of the Reelin cascade in adult brain function with potential implications for the pathogenesis of several neurological and psychiatric disorders. PMID:22933789

  20. Fast insulin secretion reflects exocytosis of docked granules in mouse pancreatic B-cells.

    PubMed

    Olofsson, Charlotta S; Göpel, Sven O; Barg, Sebastian; Galvanovskis, Juris; Ma, Xiaosong; Salehi, Albert; Rorsman, Patrik; Eliasson, Lena

    2002-05-01

    A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain approximately 9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10 min stimulation with high K+. This stimulus depolarized the cell to -10 mV, elevated cytosolic [Ca2+] ([Ca2+](i)) from a basal concentration of 130 nM to a peak of 1.3 microM and released 0.5 ng insulin/islet, corresponding to 200-300 granules/cell. The Ca2+ transient decayed towards the prestimulatory concentration within approximately 200 s, presumably reflecting Ca2+ channel inactivation. Renewed stimulation with high K+ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca2+. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.

  1. Simulating spinal border cells and cerebellar granule cells under locomotion--a case study of spinocerebellar information processing.

    PubMed

    Spanne, Anton; Geborek, Pontus; Bengtsson, Fredrik; Jörntell, Henrik

    2014-01-01

    The spinocerebellar systems are essential for the brain in the performance of coordinated movements, but our knowledge about the spinocerebellar interactions is very limited. Recently, several crucial pieces of information have been acquired for the spinal border cell (SBC) component of the ventral spinocerebellar tract (VSCT), as well as the effects of SBC mossy fiber activation in granule cells of the cerebellar cortex. SBCs receive monosynaptic input from the reticulospinal tract (RST), which is an important driving system under locomotion, and disynaptic inhibition from Ib muscle afferents. The patterns of activity of RST neurons and Ib afferents under locomotion are known. The activity of VSCT neurons under fictive locomotion, i.e. without sensory feedback, is also known, but there is little information on how these neurons behave under actual locomotion and for cerebellar granule cells receiving SBC input this is completely unknown. But the available information makes it possible to simulate the interactions between the spinal and cerebellar neuronal circuitries with a relatively large set of biological constraints. Using a model of the various neuronal elements and the network they compose, we simulated the modulation of the SBCs and their target granule cells under locomotion and hence generated testable predictions of their general pattern of modulation under this condition. This particular system offers a unique opportunity to simulate these interactions with a limited number of assumptions, which helps making the model biologically plausible. Similar principles of information processing may be expected to apply to all spinocerebellar systems.

  2. 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

  3. Changes in biochemical processes in cerebellar granule cells of mice exposed to methylmercury.

    PubMed

    Bellum, Sairam; Bawa, Bhupinder; Thuett, Kerry A; Stoica, Gheorghe; Abbott, Louise C

    2007-01-01

    At postnatal day 34, male and female C57BL/6J mice were exposed orally once a day to a total of five doses totaling 1.0 or 5.0 mg/kg of methylmercuric chloride or sterile deionized water in moistened rodent chow. Eleven days after the last dose cerebellar granule cells were acutely isolated to measure reactive oxygen species (ROS) levels and mitochondrial membrane potential using CM-H(2)DCFDA and TMRM dyes, respectively. For visualizing intracellular calcium ion distribution using transmission electron microscopy, mice were perfused 11 days after the last dose of methylmercury (MeHg) using the oxalate-pyroantimonate method. Cytosolic and mitochondrial protein fractions from acutely isolated granule cells were analyzed for cytochrome c content using Western blot analysis. Histochemistry (Fluoro-Jade dye) and immunohistochemistry (activated caspase 3) was performed on frozen serial cerebellar sections to label granule cell death and activation of caspase 3, respectively. Granule cells isolated from MeHg-treated mice showed elevated ROS levels and decreased mitochondrial membrane potential when compared to granule cells from control mice. Electron photomicrographs of MeHg-treated granule cells showed altered intracellular calcium ion homeostasis ([Ca(2+)](i)) when compared to control granule cells. However, in spite of these subcellular changes and moderate relocalization of cytochrome c into the cytosol, the concentrations of MeHg used in this study did not produce significant neuronal cell death/apoptosis at the time point examined, as evidenced by Fluoro-Jade and activated caspase 3 immunostaining, respectively. These results demonstrate that short-term in vivo exposure to total doses of 1.0 and 5.0 mg/kg MeHg through the most common exposure route (oral) can result in significant subcellular changes that are not accompanied by overt neuronal cell death.

  4. The life cycle of platelet granules.

    PubMed

    Sharda, Anish; Flaumenhaft, Robert

    2018-01-01

    Platelet granules are unique among secretory vesicles in both their content and their life cycle. Platelets contain three major granule types-dense granules, α-granules, and lysosomes-although other granule types have been reported. Dense granules and α-granules are the most well-studied and the most physiologically important. Platelet granules are formed in large, multilobulated cells, termed megakaryocytes, prior to transport into platelets. The biogenesis of dense granules and α-granules involves common but also distinct pathways. Both are formed from the trans -Golgi network and early endosomes and mature in multivesicular bodies, but the formation of dense granules requires trafficking machinery different from that of α-granules. Following formation in the megakaryocyte body, both granule types are transported through and mature in long proplatelet extensions prior to the release of nascent platelets into the bloodstream. Granules remain stored in circulating platelets until platelet activation triggers the exocytosis of their contents. Soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, located on both the granules and target membranes, provide the mechanical energy that enables membrane fusion during both granulogenesis and exocytosis. The function of these core fusion engines is controlled by SNARE regulators, which direct the site, timing, and extent to which these SNAREs interact and consequently the resulting membrane fusion. In this review, we assess new developments in the study of platelet granules, from their generation to their exocytosis.

  5. NMDA-receptor dependent synaptic activation of TRPC channels in olfactory bulb granule cells

    PubMed Central

    Stroh, Olga; Freichel, Marc; Kretz, Oliver; Birnbaumer, Lutz; Hartmann, Jana; Egger, Veronica

    2012-01-01

    TRPC channels are widely expressed throughout the nervous system including the olfactory bulb where their function is largely unknown. Here we describe their contribution to central synaptic processing at the reciprocal mitral and tufted cell - granule cell microcircuit, the most abundant synapse of the mammalian olfactory bulb. Suprathreshold activation of the synapse causes sodium action potentials in mouse granule cells and a subsequent long-lasting depolarization (LLD) linked to a global dendritic postsynaptic calcium signal recorded with two-photon laser scanning microscopy. These signals are not observed after action potentials evoked by current injection in the same cells. The LLD persists in the presence of group I metabotropic glutamate receptor antagonists but is entirely absent from granule cells deficient for the NMDA receptor subunit NR1. Moreover, both depolarization and Ca2+ rise are sensitive to the blockade of NMDA receptors. The LLD and the accompanying Ca2+ rise are also absent in granule cells from mice deficient for both TRPC channel subtypes 1 and 4, whereas the deletion of either TRPC1 or TRPC4 results in only a partial reduction of the LLD. Recordings from mitral cells in the absence of both subunits reveal a reduction of asynchronous neurotransmitter release from the granule cells during recurrent inhibition. We conclude that TRPC1 and TRPC4 can be activated downstream of NMDA receptor activation and contribute to slow synaptic transmission in the olfactory bulb, including the calcium dynamics required for asynchronous release from the granule cell spine. PMID:22539836

  6. Stimulation of microtubule-based transport by nucleation of microtubules on pigment granules

    PubMed Central

    Semenova, Irina; Gupta, Dipika; Usui, Takeo; Hayakawa, Ichiro; Cowan, Ann; Rodionov, Vladimir

    2017-01-01

    Microtubule (MT)-based transport can be regulated through changes in organization of MT transport tracks, but the mechanisms that regulate these changes are poorly understood. In Xenopus melanophores, aggregation of pigment granules in the cell center involves their capture by the tips of MTs growing toward the cell periphery, and granule aggregation signals facilitate capture by increasing the number of growing MT tips. This increase could be explained by stimulation of MT nucleation either on the centrosome or on the aggregate of pigment granules that gradually forms in the cell center. We blocked movement of pigment granules to the cell center and compared the MT-nucleation activity of the centrosome in the same cells in two signaling states. We found that granule aggregation signals did not stimulate MT nucleation on the centrosome but did increase MT nucleation activity of pigment granules. Elevation of MT-nucleation activity correlated with the recruitment to pigment granules of a major component of MT-nucleation templates, γ-tubulin, and was suppressed by γ-tubulin inhibitors. We conclude that generation of new MT transport tracks by concentration of the leading pigment granules provides a positive feedback loop that enhances delivery of trailing granules to the cell center. PMID:28381426

  7. Stochastic Seeding Coupled with mRNA Self-Recruitment Generates Heterogeneous Drosophila Germ Granules.

    PubMed

    Niepielko, Matthew G; Eagle, Whitby V I; Gavis, Elizabeth R

    2018-06-18

    The formation of ribonucleoprotein assemblies called germ granules is a conserved feature of germline development. In Drosophila, germ granules form at the posterior of the oocyte in a specialized cytoplasm called the germ plasm, which specifies germline fate during embryogenesis. mRNAs, including nanos (nos) and polar granule component (pgc), that function in germline development are localized to the germ plasm through their incorporation into germ granules, which deliver them to the primordial germ cells. Germ granules are nucleated by Oskar (Osk) protein and contain varying combinations and quantities of their constituent mRNAs, which are organized as spatially distinct, multi-copy homotypic clusters. The process that gives rise to such heterogeneous yet organized granules remains unknown. Here, we show that individual nos and pgc transcripts can populate the same nascent granule, and these first transcripts then act as seeds, recruiting additional like transcripts to form homotypic clusters. Within a granule, homotypic clusters grow independently of each other but depend on the simultaneous acquisition of additional Osk. Although granules can contain multiple clusters of a particular mRNA, granule mRNA content is dominated by cluster size. These results suggest that the accumulation of mRNAs in the germ plasm is controlled by the mRNAs themselves through their ability to form homotypic clusters; thus, RNA self-association drives germ granule mRNA localization. We propose that a stochastic seeding and self-recruitment mechanism enables granules to simultaneously incorporate many different mRNAs while ensuring that each becomes enriched to a functional threshold. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. The Prohormone VGF Regulates β Cell Function via Insulin Secretory Granule Biogenesis.

    PubMed

    Stephens, Samuel B; Edwards, Robert J; Sadahiro, Masato; Lin, Wei-Jye; Jiang, Cheng; Salton, Stephen R; Newgard, Christopher B

    2017-09-05

    The prohormone VGF is expressed in neuroendocrine and endocrine tissues and regulates nutrient and energy status both centrally and peripherally. We and others have shown that VGF-derived peptides have direct action on the islet β cell as secretagogues and cytoprotective agents; however, the endogenous function of VGF in the β cell has not been described. Here, we demonstrate that VGF regulates secretory granule formation. VGF loss-of-function studies in both isolated islets and conditional knockout mice reveal a profound decrease in stimulus-coupled insulin secretion. Moreover, VGF is necessary to facilitate efficient exit of granule cargo from the trans-Golgi network and proinsulin processing. It also functions to replenish insulin granule stores following nutrient stimulation. Our data support a model in which VGF operates at a critical node of granule biogenesis in the islet β cell to coordinate insulin biosynthesis with β cell secretory capacity. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. Turnover of pigment granules: cyclic catabolism and anabolism of ommochromes within epidermal cells.

    PubMed

    Insausti, T C; Casas, J

    2009-12-01

    Ommochromes are end products of the tryptophan metabolism in arthropods. While the anabolism of ommochromes has been well studied, the catabolism is totally unknown. In order to study it, we used the crab-spider Misumena vatia, which is able to change color reversibly in a few days, from yellow to white and back. Ommochromes is the only pigment class responsible for the body coloration in this animal. The aim of this study was to analyze the fine structure of the epidermal cells in bleaching spiders, in an attempt to correlate morphological changes with the fate of the pigment granules. Central to the process of bleaching is the lysis of the ommochrome granules. In the same cell, intact granules and granules in different degradation stages are found. The degradation begins with granule autolysis. Some components are extruded in the extracellular space and others are recycled via autophagy. Abundant glycogen appears associated to granulolysis. In a later stage of bleaching, ommochrome progranules, typical of white spiders, appear in the distal zone of the same epidermal cell. Catabolism and anabolism of pigment granules thus take place simultaneously in spider epidermal cells. A cyclic pathway of pigment granules formation and degradation, throughout a complete cycle of color change is proposed, together with an explanation for this turnover, involving photoprotection against UV by ommochromes metabolites. The presence of this turnover for melanins is discussed.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Miyagawa, Ryu; Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654; Mizuno, Rie

    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.more » 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.« less

  11. Environmental enrichment alters dentate granule cell morphology in oldest-old rat.

    PubMed

    Darmopil, Sanja; Petanjek, Zdravko; Mohammed, Abdul H; Bogdanović, Nenad

    2009-08-01

    The hippocampus of aged rats shows marked age-related morphological changes that could cause memory deficits. Experimental evidence has established that environmental enrichment attenuates memory deficits in aged rats. We therefore studied whether environmental enrichment produces morphological changes on the dentate granule cells of aged rats. Fifteen male Sprague-Dawley rats, 24 months of age, were randomly distributed in two groups that were housed under standard (n = 7) or enriched (n = 8) environmental conditions for 26 days. Quantitative data of dendritic morphology from dentate gyrus granule cells were obtained on Golgi-Cox stained sections. Environmental enrichment significantly increased the complexity and size of dendritic tree (total number of segments increased by 61% and length by 116%), and spine density (88% increase). There were large interindividual differences within the enriched group, indicating differential individual responses to environmental stimulation. Previous studies in young animals have shown changes produced by environmental enrichment in the morphology of dentate gyrus granule cells. The results of the present study show that environmental enrichment can also produce changes in dentate granule cell morphology in the senescent brain. In conclusion, the hippocampus retains its neuroplastic capacity during aging, and enriched environmental housing conditions can attenuate age-related dendritic regression and synaptic loss, thus preserving memory functions.

  12. MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

    PubMed

    Constantin, Lena; Wainwright, Brandon J

    2015-12-01

    MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

  13. Three-dimensional structural imaging of starch granules by second-harmonic generation circular dichroism.

    PubMed

    Zhuo, G-Y; Lee, H; Hsu, K-J; Huttunen, M J; Kauranen, M; Lin, Y-Y; Chu, S-W

    2014-03-01

    Chirality is one of the most fundamental and essential structural properties of biological molecules. Many important biological molecules including amino acids and polysaccharides are intrinsically chiral. Conventionally, chiral species can be distinguished by interaction with circularly polarized light, and circular dichroism is one of the best-known approaches for chirality detection. As a linear optical process, circular dichroism suffers from very low signal contrast and lack of spatial resolution in the axial direction. It has been demonstrated that by incorporating nonlinear interaction with circularly polarized excitation, second-harmonic generation circular dichroism can provide much higher signal contrast. However, previous circular dichroism and second-harmonic generation circular dichroism studies are mostly limited to probe chiralities at surfaces and interfaces. It is known that second-harmonic generation, as a second-order nonlinear optical effect, provides excellent optical sectioning capability when combined with a laser-scanning microscope. In this work, we combine the axial resolving power of second-harmonic generation and chiral sensitivity of second-harmonic generation circular dichroism to realize three-dimensional chiral detection in biological tissues. Within the point spread function of a tight focus, second-harmonic generation circular dichroism could arise from the macroscopic supramolecular packing as well as the microscopic intramolecular chirality, so our aim is to clarify the origins of second-harmonic generation circular dichroism response in complicated three-dimensional biological systems. The sample we use is starch granules whose second-harmonic generation-active molecules are amylopectin with both microscopic chirality due to its helical structure and macroscopic chirality due to its crystallized packing. We found that in a starch granule, the second-harmonic generation for right-handed circularly polarized excitation is

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

    PubMed

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

    2015-11-09

    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 (LKB1(Atoh1) CKO) to investigate the function of LKB1 in cerebellar development. The LKB1(Atoh1) CKO mice displayed motor dysfunction. In the LKB1(Atoh1) CKO cerebellum, the overall structure had a larger volume and more lobules. LKB1 inactivation led 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 LKB1(Atoh1) 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.

  15. 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

  16. Nitric oxide inhibits exocytosis of cytolytic granules from lymphokine-activated killer cells

    PubMed Central

    Ferlito, Marcella; Irani, Kaikobad; Faraday, Nauder; Lowenstein, Charles J.

    2006-01-01

    NO inhibits cytotoxic T lymphocyte killing of target cells, although the precise mechanism is unknown. We hypothesized that NO decreases exocytosis of cytotoxic granules from activated lymphocytes. We now show that NO inhibits lymphokine-activated killer cell killing of K562 target cells. Exogenous and endogenous NO decreases the release of granzyme B, granzyme A, and perforin: all contents of cytotoxic granules. NO inhibits the signal transduction cascade initiated by cross-linking of the T cell receptor that leads to granule exocytosis. In particular, we found that NO decreases the expression of Ras, a critical signaling component within the exocytic pathway. Ectopic expression of Ras prevents NO inhibition of exocytosis. Our data suggest that Ras mediates NO inhibition of lymphocyte cytotoxicity and emphasize that alterations in the cellular redox state may regulate the exocytic signaling pathway. PMID:16857739

  17. Spherical porous hydroxyapatite granules containing composites of magnetic and hydroxyapatite nanoparticles for the hyperthermia treatment of bone tumor.

    PubMed

    Kamitakahara, Masanobu; Ohtoshi, Naohiro; Kawashita, Masakazu; Ioku, Koji

    2016-05-01

    Spherical porous granules of hydroxyapatite (HA) containing magnetic nanoparticles would be suitable for the hyperthermia treatment of bone tumor, because porous HA granules act as a scaffold for bone regeneration, and magnetic nanoparticles generate sufficient heat to kill tumor cells under an alternating magnetic field. Although magnetic nanoparticles are promising heat generators, their small size makes them difficult to support in porous HA ceramics. We prepared micrometer-sized composites of magnetic and HA nanoparticles, and then supported them in porous HA granules composed of rod-like particles. The spherical porous HA granules containing the composites of magnetic and HA nanoparticle were successfully prepared using a hydrothermal process without changing the crystalline phase and heat generation properties of the magnetic nanoparticles. The obtained granules generated sufficient heat for killing tumor cells under an alternating magnetic field (300 Oe at 100 kHz). The obtained granules are expected to be useful for the hyperthermia treatment of bone tumors.

  18. 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

  19. Light scattering on PHA granules protects bacterial cells against the harmful effects of UV radiation.

    PubMed

    Slaninova, Eva; Sedlacek, Petr; Mravec, Filip; Mullerova, Lucie; Samek, Ota; Koller, Martin; Hesko, Ondrej; Kucera, Dan; Marova, Ivana; Obruca, Stanislav

    2018-02-01

    Numerous prokaryotes accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules. The primary function of PHA is the storage of carbon and energy. Nevertheless, there are numerous reports that the presence of PHA granules in microbial cells enhances their stress resistance and fitness when exposed to various stress factors. In this work, we studied the protective mechanism of PHA granules against UV irradiation employing Cupriavidus necator as a model bacterial strain. The PHA-accumulating wild type strain showed substantially higher UV radiation resistance than the PHA non-accumulating mutant. Furthermore, the differences in UV-Vis radiation interactions with both cell types were studied using various spectroscopic approaches (turbidimetry, absorption spectroscopy, and nephelometry). Our results clearly demonstrate that intracellular PHA granules efficiently scatter UV radiation, which provides a substantial UV-protective effect for bacterial cells and, moreover, decreases the intracellular level of reactive oxygen species in UV-challenged cells. The protective properties of the PHA granules are enhanced by the fact that granules specifically bind to DNA, which in turn provides shield-like protection of DNA as the most UV-sensitive molecule. To conclude, the UV-protective action of PHA granules adds considerable value to their primary storage function, which can be beneficial in numerous environments.

  20. 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. Copyright © 2014 by the Genetics Society of America.

  1. CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules

    PubMed Central

    Shamri, Revital; Melo, Rossana C. N.; Young, Kristen M.; Bivas-Benita, Maytal; Xenakis, Jason J.; Spencer, Lisa A.; Weller, Peter F.

    2012-01-01

    Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC50 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.—Shamri, R., Melo, R. C. N., Young, K. M., B.-B, M., Xenakis, J. J., Spencer, L. A., Weller, P. F. CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules. PMID:22294786

  2. A Role for Serglycin Proteoglycan in Mast Cell Apoptosis Induced by a Secretory Granule-mediated Pathway*

    PubMed Central

    Melo, Fabio Rabelo; Waern, Ida; Rönnberg, Elin; Åbrink, Magnus; Lee, David M.; Schlenner, Susan M.; Feyerabend, Thorsten B.; Rodewald, Hans-Reimer; Turk, Boris; Wernersson, Sara; Pejler, Gunnar

    2011-01-01

    Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin−/− cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis. PMID:21123167

  3. Morphologic Integration of Hilar Ectopic Granule Cells into Dentate Gyrus Circuitry in the Pilocarpine Model of Temporal Lobe Epilepsy

    PubMed Central

    Cameron, Michael C.; Zhan, Ren-Zhi; Nadler, J. Victor

    2014-01-01

    After pilocarpine-induced status epilepticus, many granule cells born into the postseizure environment migrate aberrantly into the dentate hilus. Hilar ectopic granule cells (HEGCs) are hyperexcitable and may therefore increase circuit excitability. This study determined the distribution of their axons and dendrites. HEGCs and normotopic granule cells were filled with biocytin during whole-cell patch clamp recording in hippocampal slices from pilocarpine-treated rats. The apical dendrite of 86% of the biocytin-labeled HEGCs extended to the outer edge of the dentate molecular layer. The total length and branching of HEGC apical dendrites that penetrated the molecular layer were significantly reduced compared with apical dendrites of normotopic granule cells. HEGCs were much more likely to have a hilar basal dendrite than normotopic granule cells. They were about as likely as normotopic granule cells to project to CA3 pyramidal cells within the slice, but were much more likely to send at least one recurrent mossy fiber into the molecular layer. HEGCs with burst capability had less well-branched apical dendrites than nonbursting HEGCs, their dendrites were more likely to be confined to the hilus, and some exhibited dendritic features similar to those of immature granule cells. HEGCs thus have many paths along which to receive synchronized activity from normotopic granule cells and to transmit their own hyperactivity to both normotopic granule cells and CA3 pyramidal cells. They may therefore contribute to the highly interconnected granule cell hubs that have been proposed as crucial to development of a hyperexcitable, potentially seizure-prone circuit. PMID:21455997

  4. The Low-Threshold Calcium Channel Cav3.2 Mediates Burst Firing of Mature Dentate Granule Cells

    PubMed Central

    Dumenieu, Mael; Senkov, Oleg; Mironov, Andrey; Bourinet, Emmanuel; Kreutz, Michael R; Dityatev, Alexander; Heine, Martin; Bikbaev, Arthur

    2018-01-01

    Abstract Mature granule cells are poorly excitable neurons that were recently shown to fire action potentials, preferentially in bursts. It is believed that the particularly pronounced short-term facilitation of mossy fiber synapses makes granule cell bursting a very effective means of properly transferring information to CA3. However, the mechanism underlying the unique bursting behavior of mature granule cells is currently unknown. Here, we show that Cav3.2 T-type channels at the axon initial segment are responsible for burst firing of mature granule cells in rats and mice. Accordingly, Cav3.2 knockout mice fire tonic spikes and exhibit impaired bursting, synaptic plasticity and dentate-to-CA3 communication. The data show that Cav3.2 channels are strong modulators of bursting and can be considered a critical molecular switch that enables effective information transfer from mature granule cells to the CA3 pyramids. PMID:29790938

  5. Somatic Arc protein expression in hippocampal granule cells is increased in response to environmental change but independent of task-specific learning.

    PubMed

    Cleland, J P; Willis, E F; Bartlett, P F; Vukovic, J

    2017-09-29

    Activated neurons express immediate-early genes, such as Arc. Expression of Arc in the hippocampal granule cell layer, an area crucial for spatial learning and memory, is increased during acquisition of spatial learning; however, it is unclear whether this effect is related to the task-specific learning process or to nonspecific aspects of the testing procedure (e.g. exposure to the testing apparatus and exploration of the environment). Herein, we show that Arc-positive cells numbers are increased to the same extent in the granule cell layer after both acquisition of a single spatial learning event in the active place avoidance task and exploration of the testing environment, as compared to naïve (i.e. caged) mice. Repeated exposure the testing apparatus and environment did not reduce Arc expression. Furthermore, Arc expression did not correlate with performance in both adult and aged animals, suggesting that exploration of the testing environment, rather than the specific acquisition of the active place avoidance task, induces Arc expression in the dentate granule cell layer. These findings thus suggest that Arc is an experience-induced immediate-early gene.

  6. Differential gene expression in dentate granule cells in mesial temporal lobe epilepsy with and without hippocampal sclerosis.

    PubMed

    Griffin, Nicole G; Wang, Yu; Hulette, Christine M; Halvorsen, Matt; Cronin, Kenneth D; Walley, Nicole M; Haglund, Michael M; Radtke, Rodney A; Skene, J H Pate; Sinha, Saurabh R; Heinzen, Erin L

    2016-03-01

    Hippocampal sclerosis is the most common neuropathologic finding in cases of medically intractable mesial temporal lobe epilepsy. In this study, we analyzed the gene expression profiles of dentate granule cells of patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis to show that next-generation sequencing methods can produce interpretable genomic data from RNA collected from small homogenous cell populations, and to shed light on the transcriptional changes associated with hippocampal sclerosis. RNA was extracted, and complementary DNA (cDNA) was prepared and amplified from dentate granule cells that had been harvested by laser capture microdissection from surgically resected hippocampi from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis. Sequencing libraries were sequenced, and the resulting sequencing reads were aligned to the reference genome. Differential expression analysis was used to ascertain expression differences between patients with and without hippocampal sclerosis. Greater than 90% of the RNA-Seq reads aligned to the reference. There was high concordance between transcriptional profiles obtained for duplicate samples. Principal component analysis revealed that the presence or absence of hippocampal sclerosis was the main determinant of the variance within the data. Among the genes up-regulated in the hippocampal sclerosis samples, there was significant enrichment for genes involved in oxidative phosphorylation. By analyzing the gene expression profiles of dentate granule cells from surgically resected hippocampal specimens from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis, we have demonstrated the utility of next-generation sequencing methods for producing biologically relevant results from small populations of homogeneous cells, and have provided insight on the transcriptional changes associated with this pathology. Wiley Periodicals, Inc. © 2016

  7. Endogenous neurotrophin-3 regulates short-term plasticity at lateral perforant path-granule cell synapses.

    PubMed

    Kokaia, M; Asztely, F; Olofsdotter, K; Sindreu, C B; Kullmann, D M; Lindvall, O

    1998-11-01

    In the adult brain, neurotrophin-3 (NT-3) is mainly localized in dentate granule cells, and its expression is decreased by various stimuli, e.g., seizure activity. We have examined the role of endogenous NT-3 for excitatory synaptic transmission at lateral perforant path-dentate granule cell synapses using hippocampal slices from NT-3 knock-out (+/-) and wild-type (+/+) mice. Paired-pulse facilitation (PPF) and also short-term synaptic plasticity induced by a brief, high-frequency train of afferent stimulation were reduced, but the expression of long-term potentiation was not affected in the NT-3+/- mice. Incubation of the slices with recombinant NT-3 reversed the deficit in PPF through a mechanism requiring de novo protein synthesis, implying that the impaired short-term plasticity does not result from a developmental alteration. No changes of overall presynaptic release probability, measured by the progressive block of NMDA receptor-mediated synaptic currents by MK-801, or desensitization of AMPA receptors were detected. Because NT-3 expression is reduced after focal seizures, impaired short-term facilitation may represent a protective response that limits the propagation of epileptiform activity from the entorhinal cortex to the hippocampus.

  8. Loss of protohaem IX farnesyltransferase in mature dentate granule cells impairs short-term facilitation at mossy fibre to CA3 pyramidal cell synapses.

    PubMed

    Booker, Sam A; Campbell, Graham R; Mysiak, Karolina S; Brophy, Peter J; Kind, Peter C; Mahad, Don J; Wyllie, David J A

    2017-03-15

    Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity. Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low-frequency dentate to CA3 glutamatergic synaptic transmission. High-frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase-deficient mice. Intact presynaptic mitochondrial function is critical for the short-term dynamics of mossy fibre to CA3 synaptic function. Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole-cell patch-clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV-deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy-fibre synapse because the amplitude, input-output relationship and 50 ms paired-pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short-term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired-pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect observed was not restricted to synapses

  9. High Plasticity of New Granule Cells in the Aging Hippocampus.

    PubMed

    Trinchero, Mariela F; Buttner, Karina A; Sulkes Cuevas, Jessica N; Temprana, Silvio G; Fontanet, Paula A; Monzón-Salinas, M Cristina; Ledda, Fernanda; Paratcha, Gustavo; Schinder, Alejandro F

    2017-10-31

    During aging, the brain undergoes changes that impair cognitive capacity and circuit plasticity, including a marked decrease in production of adult-born hippocampal neurons. It is unclear whether development and integration of those new neurons are also affected by age. Here, we show that adult-born granule cells (GCs) in aging mice are scarce and exhibit slow development, but they display a remarkable potential for structural plasticity. Retrovirally labeled 3-week-old GCs in middle-aged mice were small, underdeveloped, and disconnected. Neuronal development and integration were accelerated by voluntary exercise or environmental enrichment. Similar effects were observed via knockdown of Lrig1, an endogenous negative modulator of neurotrophin receptors. Consistently, blocking neurotrophin signaling by Lrig1 overexpression abolished the positive effects of exercise. These results demonstrate an unparalleled degree of plasticity in the aging brain mediated by neurotrophins, whereby new GCs remain immature until becoming rapidly recruited to the network by activity. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  10. Snapshot 3D tracking of insulin granules in live cells

    NASA Astrophysics Data System (ADS)

    Wang, Xiaolei; Huang, Xiang; Gdor, Itay; Daddysman, Matthew; Yi, Hannah; Selewa, Alan; Haunold, Theresa; Hereld, Mark; Scherer, Norbert F.

    2018-02-01

    Rapid and accurate volumetric imaging remains a challenge, yet has the potential to enhance understanding of cell function. We developed and used a multifocal microscope (MFM) for 3D snapshot imaging to allow 3D tracking of insulin granules labeled with mCherry in MIN6 cells. MFM employs a special diffractive optical element (DOE) to simultaneously image multiple focal planes. This simultaneous acquisition of information determines the 3D location of single objects at a speed only limited by the array detector's frame rate. We validated the accuracy of MFM imaging/tracking with fluorescence beads; the 3D positions and trajectories of single fluorescence beads can be determined accurately over a wide range of spatial and temporal scales. The 3D positions and trajectories of single insulin granules in a 3.2um deep volume were determined with imaging processing that combines 3D decovolution, shift correction, and finally tracking using the Imaris software package. We find that the motion of the granules is superdiffusive, but less so in 3D than 2D for cells grown on coverslip surfaces, suggesting an anisotropy in the cytoskeleton (e.g. microtubules and action).

  11. Long-term administration of scopolamine interferes with nerve cell proliferation, differentiation and migration in adult mouse hippocampal dentate gyrus, but it does not induce cell death

    PubMed Central

    Yan, Bing Chun; Park, Joon Ha; Chen, Bai Hui; Cho, Jeong-Hwi; Kim, In Hye; Ahn, Ji Hyeon; Lee, Jae-Chul; Hwang, In Koo; Cho, Jun Hwi; Lee, Yun Lyul; Kang, Il-Jun; Won, Moo-Ho

    2014-01-01

    Long-term administration of scopolamine, a muscarinic receptor antagonist, can inhibit the survival of newly generated cells, but its effect on the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus remain poorly understood. In this study, we used immunohistochemistry and western blot methods to weekly detect the biological behaviors of nerve cells in the hippocampal dentate gyrus of adult mice that received intraperitoneal administration of scopolamine for 4 weeks. Expression of neuronal nuclear antigen (NeuN; a neuronal marker) and Fluoro-Jade B (a marker for the localization of neuronal degeneration) was also detected. After scopolamine treatment, mouse hippocampal neurons did not die, and Ki-67 (a marker for proliferating cells)-immunoreactive cells were reduced in number and reached the lowest level at 4 weeks. Doublecortin (DCX; a marker for newly generated neurons)-immunoreactive cells were gradually shortened in length and reduced in number with time. After scopolamine treatment for 4 weeks, nearly all of the 5-bromo-2′-deoxyuridine (BrdU)-labeled newly generated cells were located in the subgranular zone of the dentate gyrus, but they did not migrate into the granule cell layer. Few mature BrdU/NeuN double-labeled cells were seen in the subgranular zone of the dentate gyrus. These findings suggest that long-term administration of scopolamine interferes with the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus, but it does not induce cell death. PMID:25422633

  12. Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

    PubMed Central

    Mouchacca, Pierre; Schmitt-Verhulst, Anne-Marie; Boyer, Claude

    2013-01-01

    To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo. PMID:23840635

  13. Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue.

    PubMed

    Sinha, Mithun; Sen, Chandan K; Singh, Kanhaiya; Das, Amitava; Ghatak, Subhadip; Rhea, Brian; Blackstone, Britani; Powell, Heather M; Khanna, Savita; Roy, Sashwati

    2018-03-05

    Inflammation, following injury, induces cellular plasticity as an inherent component of physiological tissue repair. The dominant fate of wound macrophages is unclear and debated. Here we show that two-thirds of all granulation tissue fibroblasts, otherwise known to be of mesenchymal origin, are derived from myeloid cells which are likely to be wound macrophages. Conversion of myeloid to fibroblast-like cells is impaired in diabetic wounds. In cross-talk between keratinocytes and myeloid cells, miR-21 packaged in extracellular vesicles (EV) is required for cell conversion. EV from wound fluid of healing chronic wound patients is rich in miR-21 and causes cell conversion more effectively compared to that by fluid from non-healing patients. Impaired conversion in diabetic wound tissue is rescued by targeted nanoparticle-based delivery of miR-21 to macrophages. This work introduces a paradigm wherein myeloid cells are recognized as a major source of fibroblast-like cells in the granulation tissue.

  14. Cytoplasmic RNA Granules in Somatic Maintenance.

    PubMed

    Moujaber, Ossama; Stochaj, Ursula

    2018-05-30

    Cytoplasmic RNA granules represent subcellular compartments that are enriched in protein-bound RNA species. RNA granules are produced by evolutionary divergent eukaryotes, including yeast, mammals, and plants. The functions of cytoplasmic RNA granules differ widely. They are dictated by the cell type and physiological state, which in turn is determined by intrinsic cell properties and environmental factors. RNA granules provide diverse cellular functions. However, all of the granules contribute to aspects of RNA metabolism. This is exemplified by transcription, RNA storage, silencing, and degradation, as well as mRNP remodeling and regulated translation. Several forms of cytoplasmic mRNA granules are linked to normal physiological processes. For instance, they may coordinate protein synthesis and thereby serve as posttranscriptional "operons". RNA granules also participate in cytoplasmic mRNA trafficking, a process particularly well understood for neurons. Many forms of RNA granules support the preservation of somatic cell performance under normal and stress conditions. On the other hand, severe insults or disease can cause the formation and persistence of RNA granules that contribute to cellular dysfunction, especially in the nervous system. Neurodegeneration and many other diseases linked to RNA granules are associated with aging. Nevertheless, information related to the impact of aging on the various types of RNA granules is presently very limited. This review concentrates on cytoplasmic RNA granules and their role in somatic cell maintenance. We summarize the current knowledge on different types of RNA granules in the cytoplasm, their assembly and function under normal, stress, or disease conditions. Specifically, we discuss processing bodies, neuronal granules, stress granules, and other less characterized cytoplasmic RNA granules. Our focus is primarily on mammalian and yeast models, because they have been critical to unravel the physiological role of various

  15. Preterm birth disrupts cerebellar development by affecting granule cell proliferation program and Bergmann glia.

    PubMed

    Iskusnykh, Igor Y; Buddington, Randal K; Chizhikov, Victor V

    2018-08-01

    Preterm birth is a leading cause of long-term motor and cognitive deficits. Clinical studies suggest that some of these deficits result from disruption of cerebellar development, but the mechanisms that mediate cerebellar abnormalities in preterm infants are largely unknown. Furthermore, it remains unclear whether preterm birth and precocious exposure to the ex-utero environment directly disrupt cerebellar development or indirectly by increasing the probability of cerebellar injury, including that resulting from clinical interventions and protocols associated with the care of preterm infants. In this study, we analyzed the cerebellum of preterm pigs delivered via c-section at 91% term and raised for 10 days, until term-equivalent age. The pigs did not receive any treatments known or suspected to affect cerebellar development and had no evidence of brain damage. Term pigs sacrificed at birth were used as controls. Immunohistochemical analysis revealed that preterm birth did not affect either size or numbers of Purkinje cells or molecular layer interneurons at term-equivalent age. The number of granule cell precursors and Bergmann glial fibers, however, were reduced in preterm pigs. Preterm pigs had reduced proliferation but not differentiation of granule cells. qRT-PCR analysis of laser capture microdissected external granule cell layer showed that preterm pigs had a reduced expression of Ccnd1 (Cyclin D1), Ccnb1 (Cyclin B1), granule cell master regulatory transcription factor Atoh1, and signaling molecule Jag1. In vitro rescue experiments identified Jag1 as a central granule cell gene affected by preterm birth. Thus, preterm birth and precocious exposure to the ex-utero environment disrupt cerebellum by modulating expression of key cerebellar developmental genes, predominantly affecting development of granule precursors and Bergmann glia. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Sensorimotor Representations in Cerebellar Granule Cells in Larval Zebrafish Are Dense, Spatially Organized, and Non-temporally Patterned.

    PubMed

    Knogler, Laura D; Markov, Daniil A; Dragomir, Elena I; Štih, Vilim; Portugues, Ruben

    2017-05-08

    A fundamental question in neurobiology is how animals integrate external sensory information from their environment with self-generated motor and sensory signals in order to guide motor behavior and adaptation. The cerebellum is a vertebrate hindbrain region where all of these signals converge and that has been implicated in the acquisition, coordination, and calibration of motor activity. Theories of cerebellar function postulate that granule cells encode a variety of sensorimotor signals in the cerebellar input layer. These models suggest that representations should be high-dimensional, sparse, and temporally patterned. However, in vivo physiological recordings addressing these points have been limited and in particular have been unable to measure the spatiotemporal dynamics of population-wide activity. In this study, we use both calcium imaging and electrophysiology in the awake larval zebrafish to investigate how cerebellar granule cells encode three types of sensory stimuli as well as stimulus-evoked motor behaviors. We find that a large fraction of all granule cells are active in response to these stimuli, such that representations are not sparse at the population level. We find instead that most responses belong to only one of a small number of distinct activity profiles, which are temporally homogeneous and anatomically clustered. We furthermore identify granule cells that are active during swimming behaviors and others that are multimodal for sensory and motor variables. When we pharmacologically change the threshold of a stimulus-evoked behavior, we observe correlated changes in these representations. Finally, electrophysiological data show no evidence for temporal patterning in the coding of different stimulus durations. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  17. Status epilepticus increases mature granule cells in the molecular layer of the dentate gyrus in rats★

    PubMed Central

    Liang, Zhaoliang; Gao, Fei; Wang, Fajun; Wang, Xiaochen; Song, Xinyu; Liu, Kejing; Zhan, Ren-Zhi

    2013-01-01

    Enhanced neurogenesis in the dentate gyrus of the hippocampus following seizure activity, especially status epilepticus, is associated with ectopic residence and aberrant integration of newborn granule cells. Hilar ectopic granule cells may be detrimental to the stability of dentate circuitry by means of their electrophysiological properties and synaptic connectivity. We hypothesized that status epilepticus also increases ectopic granule cells in the molecular layer. Status epilepticus was induced in male Sprague-Dawley rats by intraperitoneal injection of pilocarpine. Immunostaining showed that many doublecortin-positive cells were present in the molecular layer and the hilus 7 days after the induction of status epilepticus. At least 10 weeks after status epilepticus, the estimated number of cells positive for both prospero homeobox protein 1 and neuron-specific nuclear protein in the hilus was significantly increased. A similar trend was also found in the molecular layer. These findings indicate that status epilepticus can increase the numbers of mature and ectopic newborn granule cells in the molecular layer. PMID:25206705

  18. Wolfram syndrome 1 gene (WFS1) product localizes to secretory granules and determines granule acidification in pancreatic beta-cells.

    PubMed

    Hatanaka, Masayuki; Tanabe, Katsuya; Yanai, Akie; Ohta, Yasuharu; Kondo, Manabu; Akiyama, Masaru; Shinoda, Koh; Oka, Yoshitomo; Tanizawa, Yukio

    2011-04-01

    Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic β-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic β-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type β-cells, there was a 32% reduction in the intensity in WFS1-deficient β-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null β-cells relative to that in wild-type β-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of β-cell dysfunction in patients with Wolfram syndrome.

  19. Cerebellar GABAergic progenitors adopt an external granule cell-like phenotype in the absence of Ptf1a transcription factor expression.

    PubMed

    Pascual, Marta; Abasolo, Ibane; Mingorance-Le Meur, Ana; Martínez, Albert; Del Rio, José A; Wright, Christopher V E; Real, Francisco X; Soriano, Eduardo

    2007-03-20

    We report in this study that, in the cerebellum, the pancreatic transcription factor Ptf1a is required for the specific generation of Purkinje cells (PCs) and interneurons. Moreover, granule cell progenitors in the external GCL (EGL) appear to be unaffected by deletion of Ptf1a. Cell lineage analysis in Ptf1a(Cre/Cre) mice was used to establish that, in the absence of Ptf1a expression, ventricular zone progenitors, normally fated to produce PCs and interneurons, aberrantly migrate to the EGL and express typical markers of these cells, such as Math1, Reelin, and Zic1/2. Furthermore, these cells have a fine structure typical of EGL progenitors, indicating that they adopt an EGL-like cell phenotype. These findings indicate that Ptf1a is necessary for the specification and normal production of PCs and cerebellar interneurons. Moreover, our results suggest that Ptf1a is also required for the suppression of the granule cell specification program in cerebellar ventricular zone precursors.

  20. [Stem cells in adults].

    PubMed

    Borge, O J; Funderud, S

    2001-08-30

    We present a literature review of the plasticity observed by adult stem cells. We have reviewed the literature regarding stem cells from adults in order to summarise their ability to generate cells of other types than those of the tissue/organ from which they were isolated. Adult stem cells have recently been demonstrated to terminally differentiate into cells of other tissues than those from which they were originally isolated. For example, bone marrow cells have been shown to generate liver, nerve, heart and skeletal muscle cells in addition to their well-known ability to produce blood and mesenchymal cells. Most studies demonstrate a proof-of-principle in animal models; much more research is needed before adult stem cells can be utilised in human medicine. However, the published reports are encouraging and give reasons for a cautious optimism with regard to future clinical use.

  1. Mast cell granules modulate alveolar macrophage respiratory-burst activity and eicosanoid metabolism.

    PubMed

    Rock, M J; Despot, J; Lemanske, R F

    1990-10-01

    Alveolar macrophages (AMs) and mast cells reside in the airway, and both have been demonstrated to contribute independently to allergic inflammatory responses through the generation of respiratory-burst metabolites and the release of biologically active mediators, respectively. Since mast cell granules (MCGs) contain mediators that could potentially interact with the AM respiratory burst, we investigated the effects of isolated MCGs on this important inflammatory pathway of the AM. MCGs and AMs were obtained by peritoneal and tracheoalveolar lavage, respectively, of Sprague-Dawley rats. First, the overall respiratory-burst activity was measured by luminal-enhanced chemiluminescence (CL), and second, the individual oxygen species contributing to CL (superoxide anion [O2-], hydrogen peroxide [H2O2], and hypochlorous acid) were measured. MCGs alone enhanced AM CL responses to an equivalent degree compared to zymosan-stimulated AMs. However, AMs preincubated with MCGs followed by zymosan stimulation significantly and synergistically enhanced the CL responses. This enhanced CL was not due to an increased production of O2-, H2O2, or hypochlorous acid; in fact, there were decreased measured amounts of O2- and H2O2 from zymosan-stimulated AMs in the presence of MCGs, most likely caused by the content of granules of superoxide dismutase and peroxidase, respectively. The lipoxygenase inhibitor, nordihydroguaiaretic acid, completely abolished the enhanced CL of AM preincubated with MCGs and subsequently stimulated by zymosan, but O2- production was not affected by nordihydroguaiaretic acid. Taken together, these results suggest that derivatives of arachidonic acid metabolism, most likely those of the lipoxygenase pathway, are responsible for the enhanced AM CL response observed in the presence of MCGs. Thus, mast cell-macrophage interactions may be important within the airway in enhancing the generation of mediators that contribute to tissue inflammation and bronchospasm.

  2. Adult-onset deficiency in growth hormone and insulin-like growth factor-I decreases survival of dentate granule neurons: insights into the regulation of adult hippocampal neurogenesis.

    PubMed

    Lichtenwalner, Robin J; Forbes, M Elizabeth; Sonntag, William E; Riddle, David R

    2006-02-01

    Insulin-like growth factor-I (IGF-I), long thought to provide critical trophic support during development, also has emerged as a candidate for regulating ongoing neuronal production in adulthood. Whether and how IGF-I influences each phase of neurogenesis, however, remains unclear. In the current study, we used a selective model of growth hormone (GH) and plasma IGF-I deficiency to evaluate the role of GH and IGF-I in regulating cell proliferation, survival, and neuronal differentiation in the adult dentate gyrus. GH/IGF-I-deficient dwarf rats of the Lewis strain were made GH/IGF-I replete throughout development via twice daily injections of GH, and then GH/IGF-I deficiency was initiated in adulthood by removing animals from GH treatment. Bromodeoxyuridine (BrdU) labeling revealed no effect of GH/IGF-I deficiency on cell proliferation, but adult-onset depletion of GH and plasma IGF-I significantly reduced the survival of newly generated cells in the dentate gyrus. Colabeling for BrdU and markers of immature and mature neurons revealed a selective effect of GH/IGF-I deficiency on the survival of more mature new neurons. The number of BrdU-labeled cells expressing the immature neuronal marker TUC-4 did not differ between GH/IGF-I-deficient and -replete animals, but the number expressing only the marker of maturity NeuN was lower in depleted animals. Taken together, results from the present study suggest that, under conditions of short-term GH/IGF-I deficiency during adulthood, dentate granule cells continue to be produced, to commit to a neuronal fate, and to begin the process of neuronal maturation, whereas survival of the new neurons is impaired. Copyright 2005 Wiley-Liss, Inc.

  3. Mechanism of the formation of hollow spherical granules using a high shear granulator.

    PubMed

    Asada, Takumi; Nishikawa, Mitsunori; Ochiai, Yasushi; Noguchi, Shuji; Kimura, Shin-Ichiro; Iwao, Yasunori; Itai, Shigeru

    2018-05-30

    Recently, we have developed a novel granulation technology to manufacture hollow spherical granules (HSGs) for controlled-release formulations; however, the mechanism of the granulation is still unclear. The aim of this study is to determine the mechanism of the formation of the HSGs using a high shear granulator. Samples of granulated material were collected at various times during granulation and were investigated using scanning electron microscope and X-ray computed tomography. It was observed that the granulation proceeded by drug layering to the polymer, followed by formation of a hollow in the granule. In addition, it was also found that generation of a crack in the adhered drug layer and air flow into the granules might be involved in forming the hollow in the structure. Observation of the granulation of formulations with different types of drugs and polymers indicated that negative pressure in the granules occurred and the granules caved in when the hollow was formed. The hollow-forming speed and the shell density of the hollow granules depended on the particular drug and polymer. Taken together, the granulation mechanism of HSGs was determined and this information will be valuable for HSGs technology development. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Intrauterine Growth Restriction Affects Cerebellar Granule Cells in the Developing Guinea Pig Brain.

    PubMed

    Tolcos, Mary; McDougall, Annie; Shields, Amy; Chung, Yoonyoung; O'Dowd, Rachael; Turnley, Ann; Wallace, Megan; Rees, Sandra

    2018-01-01

    Intrauterine growth restriction (IUGR) can lead to adverse neurodevelopmental sequelae in postnatal life. However, the effects of IUGR on the cerebellum are still to be fully elucidated. A major determinant of growth and development of the cerebellum is proliferation and subsequent migration of cerebellar granule cells. Our objective was to determine whether IUGR, induced by chronic placental insufficiency (CPI) in guinea pigs, results in abnormal cerebellar development due to deficits suggestive of impaired granule cell proliferation and/or migration. CPI was induced by unilateral ligation of the uterine artery at mid-gestation, producing growth-restricted (GR) foetuses at 52 and 60 days of gestation (dg), and neonates at 1 week postnatal age (term approx. 67 dg). Controls were from sham-operated animals. In GR foetuses compared with controls at 52 dg, the external granular layer (EGL) width and internal granular layer (IGL) area were similar. In GR foetuses compared with controls at 60 dg: (a) the EGL width was greater (p < 0.005); (b) the IGL area was smaller (p < 0.005); (c) the density of Ki67-negative (postmitotic) granule cells in the EGL was greater (p < 0.01); (d) the somal area of Purkinje cells was reduced (p < 0.005), and (e) the linear density of Bergmann glia was similar. The EGL width in GR foetuses at 60 dg was comparable to that of 52 dg control and GR foetuses. The pattern of p27-immunoreactivity in the EGL was the inverse of Ki67-immunoreactivity at both foetal ages; there was no difference between control and GR foetuses at either age in the width of p27-immunoreactivity, or in the percentage of the EGL width that it occupied. In the molecular layer of GR neonates compared with controls there was an increase in the areal density of granule cells (p < 0.05) and in the percentage of migrating to total number of granule cells (p < 0.01) at 1 week but not at 60 dg (p > 0.05). Thus, we found no specific evidence that IUGR affects granule cell

  5. Properties of single NMDA receptor channels in human dentate gyrus granule cells

    PubMed Central

    Lieberman, David N; Mody, Istvan

    1999-01-01

    Cell-attached single-channel recordings of NMDA channels were carried out in human dentate gyrus granule cells acutely dissociated from slices prepared from hippocampi surgically removed for the treatment of temporal lobe epilepsy (TLE). The channels were activated by l-aspartate (250–500 nm) in the presence of saturating glycine (8 μm). The main conductance was 51 ± 3 pS. In ten of thirty granule cells, clear subconductance states were observed with a mean conductance of 42 ± 3 pS, representing 8 ± 2% of the total openings. The mean open times varied from cell to cell, possibly owing to differences in the epileptogenicity of the tissue of origin. The mean open time was 2.70 ± 0.95 ms (range, 1.24–4.78 ms). In 87% of the cells, three exponential components were required to fit the apparent open time distributions. In the remaining neurons, as in control rat granule cells, two exponentials were sufficient. Shut time distributions were fitted by five exponential components. The average numbers of openings in bursts (1.74 ± 0.09) and clusters (3.06 ± 0.26) were similar to values obtained in rodents. The mean burst (6.66 ± 0.9 ms), cluster (20.1 ± 3.3 ms) and supercluster lengths (116.7 ± 17.5 ms) were longer than those in control rat granule cells, but approached the values previously reported for TLE (kindled) rats. As in rat NMDA channels, adjacent open and shut intervals appeared to be inversely related to each other, but it was only the relative areas of the three open time constants that changed with adjacent shut time intervals. The long openings of human TLE NMDA channels resembled those produced by calcineurin inhibitors in control rat granule cells. Yet the calcineurin inhibitor FK-506 (500 nm) did not prolong the openings of human channels, consistent with a decreased calcineurin activity in human TLE. Many properties of the human NMDA channels resemble those recorded in rat hippocampal neurons. Both have similar slope conductances, five

  6. CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline

    PubMed Central

    Campbell, Anne C.; Updike, Dustin L.

    2015-01-01

    Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription. PMID:25968310

  7. CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline.

    PubMed

    Campbell, Anne C; Updike, Dustin L

    2015-05-15

    Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription. © 2015. Published by The Company of Biologists Ltd.

  8. Effects of adult dysthyroidism on the morphology of hippocampal granular cells in rats.

    PubMed

    Martí-Carbonell, Maria Assumpció; Garau, Adriana; Sala-Roca, Josefina; Balada, Ferran

    2012-01-01

    Thyroid hormones are essential for normal brain development and very important in the normal functioning of the brain. Thyroid hormones action in the adult brain has not been widely studied. The effects of adult hyperthyroidism are not as well understood as adult hypothyroidism, mainly in hippocampal granular cells. The purpose of the present study is to assess the consequences of adult hormone dysthyroidism (excess/deficiency of TH) on the morphology of dentate granule cells in the hippocampus by performing a quantitative study of dendritic arborizations and dendritic spines using Golgi impregnated material. Hypo-and hyperthyroidism were induced in rats by adding 0.02 percent methimazole and 1 percent L-thyroxine, respectively, to drinking water from 40 days of age. At 89 days, the animals' brains were removed and stained by a modified Golgi method and blood samples were collected in order to measure T4 serum levels. Neurons were selected and drawn using a camera lucida. Our results show that both methimazole and thyroxine treatment affect granule cell morphology. Treatments provoke alterations in the same direction, namely, reduction of certain dendritic-branching parameters that are more evident in the methimazole than in the thyroxine group. We also observe a decrease in spine density in both the methimazole and thyroxine groups.

  9. Clostridium perfringens epsilon toxin targets granule cells in the mouse cerebellum and stimulates glutamate release.

    PubMed

    Lonchamp, Etienne; Dupont, Jean-Luc; Wioland, Laetitia; Courjaret, Raphaël; Mbebi-Liegeois, Corinne; Jover, Emmanuel; Doussau, Frédéric; Popoff, Michel R; Bossu, Jean-Louis; de Barry, Jean; Poulain, Bernard

    2010-09-30

    Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca(2+) rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on

  10. Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

    PubMed Central

    Lonchamp, Etienne; Dupont, Jean-Luc; Wioland, Laetitia; Courjaret, Raphaël; Mbebi-Liegeois, Corinne; Jover, Emmanuel; Doussau, Frédéric; Popoff, Michel R.; Bossu, Jean-Louis; de Barry, Jean; Poulain, Bernard

    2010-01-01

    Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons

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

    PubMed

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

    2013-09-03

    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.

  12. Short-term block of Na+/K+-ATPase in neuro-glial cell cultures of cerebellum induces glutamate dependent damage of granule cells.

    PubMed

    Stelmashook, E V; Weih, M; Zorov, D; Victorov, I; Dirnagl, U; Isaev, N

    1999-07-30

    Granule cells in a dissociated neuro-glial cell culture of cerebellum when exposed to ouabain (10(-3) M) for 25 min apparently swell, increase their [Ca2+]i with obvious depolarization of the mitochondrial membrane. In 3 h after ouabain was omitted from the solution, 62 +/- 3% of granule cells had pycnotic nuclei. The supplement of a solution with competitive specific antagonist of NMDA receptors, L-2-amino-7-phosphonoheptanoate (10(-4) M, APH) together with ouabain prevented cells from swelling, mitochondrial deenergization, neuronal death and increase of [Ca2+]i. These data suggest that cellular Na+/K+-ATPase inactivation in neuro-glial cell cultures of cerebellum leads to glutamate (Glu) accumulation, hyperstimulation of glutamate receptors, higher Ca2+ and Na+ influxes into the cells through the channels activated by Glu. This process leads to cell swelling, mitochondrial deenergization and death of granule cells. Possibly, the decrease of Na+/K+-ATPase activity in brain cells can lead to the onset of at least some chronic neurological disorders.

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

    PubMed

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

    2016-10-31

    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.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro

    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 typesmore » of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.« less

  15. 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. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. 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

    2016-04-07

    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. Copyright © 2016 Huelgas-Morales et al.

  17. 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

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

    PubMed

    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; Wilson, Clive

    2016-10-01

    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.

  19. 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

  20. Proton sensitivity of rat cerebellar granule cell GABAA receptors: dependence on neuronal development

    PubMed Central

    Krishek, Belinda J; Smart, Trevor G

    2001-01-01

    The effect of GABAA receptor development in culture on the modulation of GABA-induced currents by external H+ was examined in cerebellar granule cells using whole-cell and single-channel recording. Equilibrium concentration-response curves revealed a lower potency for GABA between 11 and 12 days in vitro (DIV) resulting in a shift of the EC50 from 10.7 to 2.4 μM. For granule cells before 11 DIV, the peak GABA-activated current was inhibited at low external pH and enhanced at high pH with a pKa of 6.65. For the steady-state response, low pH was inhibitory with a pKa of 5.56. After 11 DIV, the peak GABA-activated current was largely pH insensitive; however, the steady-state current was potentiated at low pH with a pKa of 6.84. Single GABA-activated ion channels were recorded from outside-out patches of granule cell bodies. At pH 5.4-9.4, single GABA channels exhibited multiple conductance states occurring at 22-26, 16-17 and 12-14 pS. The conductance levels were not significantly altered over the time period of study, nor by changing the external H+ concentration. Two exponential functions were required to fit the open-time frequency histograms at both early (< 11 DIV) and late (> 11 DIV) development times at each H+ concentration. The short and long open time constants were unaffected either by the extracellular H+ concentration or by neuronal development. The distribution of all shut times was fitted by the sum of three exponentials designated as short, intermediate and long. At acidic pH, the long shut time constant decreased with development as did the relative contribution of these components to the overall distribution. This was concurrent with an increase in the mean probability of channel opening. In conclusion, this study demonstrates in cerebellar granule cells that external pH can either reduce, have no effect on, or enhance GABA-activated responses depending on the stage of development, possibly related to the subunit composition of the GABAA receptors

  1. Running reorganizes the circuitry of one-week-old adult-born hippocampal neurons.

    PubMed

    Sah, Nirnath; Peterson, Benjamin D; Lubejko, Susan T; Vivar, Carmen; van Praag, Henriette

    2017-09-07

    Adult hippocampal neurogenesis is an important form of structural and functional plasticity in the mature mammalian brain. The existing consensus is that GABA regulates the initial integration of adult-born neurons, similar to neuronal development during embryogenesis. Surprisingly, virus-based anatomical tracing revealed that very young, one-week-old, new granule cells in male C57Bl/6 mice receive input not only from GABAergic interneurons, but also from multiple glutamatergic cell types, including mature dentate granule cells, area CA1-3 pyramidal cells and mossy cells. Consistently, patch-clamp recordings from retrovirally labeled new granule cells at 7-8 days post retroviral injection (dpi) show that these cells respond to NMDA application with tonic currents, and that both electrical and optogenetic stimulation can evoke NMDA-mediated synaptic responses. Furthermore, new dentate granule cell number, morphology and excitatory synaptic inputs at 7 dpi are modified by voluntary wheel running. Overall, glutamatergic and GABAergic innervation of newly born neurons in the adult hippocampus develops concurrently, and excitatory input is reorganized by exercise.

  2. Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material.

    PubMed

    Harthoorn, L F; Diederen, J H; Oudejans, R C; Verstegen, M M; Vullings, H G; Van der Horst, D J

    2000-01-01

    The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.

  3. 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.; hide

    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.

  4. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Webb, Carol F., E-mail: carol-webb@omrf.org; Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK; Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights:more » • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.« less

  5. A novel two-chain IGF-II-derived peptide from purified β-cell granules.

    PubMed

    Buchanan, Christina M; Phillips, Anthony R J; Cooper, Garth J S

    2010-10-01

    Insulin-like growth factor II (IGF-II) is a potent mitogen that regulates prenatal growth and development in both humans and rodents. Its role in post-natal life is less clear although immunohistochemical studies have observed IGF-II-like immunoreactivity (IGF-II-LI) associated with insulin-producing pancreatic β-cells. Here we isolated secretory granules from a β-cell line, βTC6-F7, and characterized the nature of the IGF-II-LI located therein. Secretory granules were isolated from cultured mouse βTC6-F7 cells by ultracentrifugation. Granule protein content was separated by reversed-phase HPLC, and assayed for IGF-II (radioimmunoassay) prior to identification by gas-phase NH(2)-terminal sequencing and MALDI-TOF MS. Effects of glucose incorporation into muscle glycogen were determined by incubating with isolated rat soleus muscle strips. βTC6-F7 cells contained 60 ± 8 pmol of IGF-II-LI per 10⁶ cells compared to 340 ± 44 pmol insulin-LI per 10⁶ cells. IGF-II immunoreactive fractions were found to contain an IGF-II-like molecule with a molecular mass of 6847.6 Da. The protein was found to be a two-chain insulin-like product of Igf2 that corresponds to mouse des(37-40)IGF-II, which we termed 'vesiculin'. This molecule was also detectable in βTC6-F7 cells by intact-cell mass spectrometry. Mouse vesiculin evoked concentration-dependent stimulation of muscle glycogen synthesis ex vivo with an EC(50) value of 131 nM ± 1.35. Vesiculin, des(37-40)IGF-II, is a novel two-chain insulin-like hormone and the major "IGF-II-like" peptide found in purified mouse βTC6-F7 secretory granules. It stimulated ex vivo muscle glycogen synthesis with an efficacy greater than or equal to the intrinsic potency of IGF-II when compared to insulin derived from the same species. Copyright © 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

  6. New neurons generated from running are broadly recruited into neuronal activation associated with three different hippocampus-involved tasks

    PubMed Central

    Clark, Peter J.; Bhattacharya, Tushar K.; Miller, Daniel S.; Kohman, Rachel A.; DeYoung, Erin K.; Rhodes, Justin S.

    2012-01-01

    Running increases the formation of new neurons in the adult rodent hippocampus. However, the function of new neurons generated from running is currently unknown. One hypothesis is that new neurons from running contribute to enhanced cognitive function by increasing plasticity in the adult hippocampus. An alternative hypothesis is that new neurons generated from running incorporate into experience-specific hippocampal networks that only become active during running. The purpose of this experiment was to determine if new neurons generated from running are selectively activated by running, or can become recruited into granule cell activity occurring during performance on other behavioral tasks that engage the hippocampus. Therefore, the activation of new 5–6 week neurons was detected using BrdU, NeuN, and Zif268 triple-label immunohistochemistry in cohorts of female running and sedentary adult C57BL/6J mice following participation in one of three different tasks: the Morris water maze, novel environment exploration, or wheel running. Results showed that running and sedentary mice displayed a nearly equivalent proportion of new neurons that expressed Zif268 following each task. Since running approximately doubled the number of new neurons, the results demonstrated that running mice had a greater number of new neurons recruited into the Zif268 induction in the granule cell layer following each task than sedentary mice. The results suggest that new neurons incorporated into hippocampal circuitry from running are not just activated by wheel running itself, but rather become broadly recruited into granule cell layer activity during distinct behavioral experiences. PMID:22467337

  7. Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.

    PubMed

    Li, Jingsong; Luo, Ruihua; Hooi, Shing Chuan; Ruga, Pilar; Zhang, Jiping; Meda, Paolo; Li, GuoDong

    2005-03-22

    Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.

  8. Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD-transgenic mice producing human A{beta}{sub 1-42} and tau

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chakravarthy, Balu, E-mail: Balu.Chakravarthy@nrc-cnrc.gc.ca; Gaudet, Chantal; Menard, Michel

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer A{beta} and tau-induced neurofibrillary tangles play a key role in Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}{sub 1-42} and mutant tau protein together reduce the primary cilium length. Black-Right-Pointing-Pointer This shortening likely reduces cilium-dependent neurogenesis and memory function. Black-Right-Pointing-Pointer This provides a model of an A{beta}/tau targeting of a neuronal signaling organelle. -- Abstract: The hippocampal dentate gyrus is one of the two sites of continuous neurogenesis in adult rodents and humans. Virtually all dentate granule cells have a single immobile cilium with a microtubule spine or axoneme covered with a specialized cell membrane loaded with receptors such as the somatostatinmore » receptor 3 (SSTR3), and the p75 neurotrophin receptor (p75{sup NTR}). The signals from these receptors have been reported to stimulate neuroprogenitor proliferation and the post-mitotic maturation of newborn granule cells into functioning granule cells. We have found that in 6-24-months-old triple transgenic Alzheimer's disease model mice (3xTg-AD) producing both A{beta}{sub 1-42} and the mutant human tau protein tau{sub P301L,} the dentate granule cells still had immunostainable SSTR3- and p75{sup NTR}-bearing cilia but they were only half the length of the immunostained cilia in the corresponding wild-type mice. However, the immunostainable length of the granule cell cilia was not reduced either in 2xTg-AD mice accumulating large amounts of A{beta}{sub 1-42} or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of A{beta}{sub 1-42} and tau protein accumulation affects the levels of functionally important receptors in 3xTg-AD mice. These observations raise the important possibility that structural and functional changes in granule cell cilia might have a role in AD.« less

  9. 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.

  10. Alpha-SNAP functions in insulin exocytosis from mature, but not immature secretory granules in pancreatic beta cells.

    PubMed

    Nakamichi, Y; Nagamatsu, S

    1999-06-24

    To explore alpha-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic beta cells, we studied the effects of overexpression of adenovirus-mediated wild-type alpha-SNAP and C-terminally deleted alpha-SNAP mutant (1-285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing alpha-SNAP and mutant alpha-SNAP were pulse-chased. Exocytosis from immature and mature insulin secretory granules was measured as fractional (%) labeled-proinsulin release immediately after the pulse-labeling and percentage labeled-insulin release after a 3-h chase period, respectively. There was no difference in percentage labeled-proinsulin release between the control and alpha-SNAP or mutant alpha-SNAP-overexpressed islets. Although percentage labeled-insulin release after a 3-h chase period was significantly increased in alpha-SNAP-overexpressed islets, it was decreased in mutant alpha-SNAP-overexpressed islets. Thus, the results demonstrated that alpha-SNAP overexpression in rat islets primarily increased exocytosis from mature, but not immature insulin secretory granules. On the other hand, in MIN6 cells, alpha-SNAP overexpression scarcely affected glucose-stimulated insulin release; therefore, we examined the effect of mutant alpha-SNAP overexpression as the dominant-negative inhibitor on the newly synthesized proinsulin/insulin release using the same protocol as in the rat islet experiments. alpha-SNAP mutant (1-285) overexpression in MIN6 cells decreased the percentage labeled insulin release from mature secretory granules, but not percentage labeled proinsulin release from immature secretory granules. Thus, our data demonstrate that alpha-SNAP functions mainly in the mature insulin secretory granules in pancreatic beta cells. Copyright 1999 Academic Press.

  11. Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fujita-Yoshigaki, Junko; Katsumata, Osamu; Matsuki, Miwako

    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 proteinsmore » 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.« less

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

    PubMed

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

    2016-08-05

    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. Copyright © 2016. Published by Elsevier Inc.

  13. Physical Exercise Restores the Generation of Newborn Neurons in an Animal Model of Chronic Epilepsy

    PubMed Central

    Mendonça, Fabricio N.; Santos, Luiz E. C.; Rodrigues, Antônio M.; Gomes da Silva, Sérgio; Arida, Ricardo M.; da Silveira, Gilcélio A.; Scorza, Fulvio A.; Almeida, Antônio-Carlos G.

    2017-01-01

    Neurogenesis impairment is associated with the chronic phase of the epilepsy in humans and also observed in animal models. Recent studies with animal models have shown that physical exercise is capable of improving neurogenesis in adult subjects, alleviating cognitive impairment and depression. Here, we show that there is a reduction in the generation of newborn granule cells in the dentate gyrus of adult rats subjected to a chronic model of epilepsy during the postnatal period of brain development. We also show that the physical exercise was capable to restore the number of newborn granule cells in this animals to the level observed in the control group. Notably, a larger number of newborn granule cells exhibiting morphological characteristics indicative of correct targeting into the hippocampal circuitry and the absence of basal dendrite projections was also observed in the epileptic animals subjected to physical exercise compared to the epileptic animals. The results described here could represent a positive interference of the physical exercise on the neurogenesis process in subjects with chronic epilepsy. The results may also help to reinterpret the benefits of the physical exercise in alleviating symptoms of depression and cognitive dysfunction. PMID:28298884

  14. ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in Caenorhabditis elegans

    PubMed Central

    Andralojc, Karolina M.; Kelly, Ashley L.; Tanner, Paige C.

    2017-01-01

    Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3). The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules). ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline. PMID:28182654

  15. Killing effects of Huaier Granule combined with DC-CIK on nude mice transplanted with colon carcinoma cell line.

    PubMed

    Sun, Wen-Wen; Dou, Jin-Xia; Zhang, Lin; Qiao, Li-Kui; Shen, Na; Zhao, Qiang; Gao, Wen-Yuan

    2017-07-11

    This study aims to compare the efficacy of different treatments for nude mice transplanted with HT-29 colon carcinoma cell line. BalB/C nude mice were transplanted with HT-29 colon carcinoma cell line and randomly divided into four groups, with 5 mice in each group: blank control group, DC-CIK group, Huaier Granule group, and Huaier Granule group combined with DC-CIK group (combined treatment group). For DC-CIK group and combined treatment group, 1×106 DC-CIK cells were injected via the tail vein 4 days after transplantation. The injection was performed twice weekly for a total of 2 weeks. For Huaier Granule group and combined treatment group, Huaier Granule was administered at the dose of 20 g/60 g, by dissolving 20 g of Huaier granules in 600 ml of pure water. Intragastric administration of 0.2 ml of granules was performed once daily for 3 weeks. For the blank control group, equal volume of normal saline was given. Tumor size and body weight of nude mice were measured every 2 days during the 3-week treatment. The mice were sacrificed at the end of treatment to harvest tumors. Key genes of the signaling pathway were detected by RT-PCR. At the end of treatment, mice in combined treatment group, DC-CIK group and Huaier Granule group remained stable emotionally with normal mobility and water and food intake. However, in the blank control group, the mobility was restricted starting from the third week and the mice were on the verge of dying. The expression of PI3KR1, Akt, Wnt1, CTTNB1, Notch1, Notch2 and Notch3 genes were all downregulated significantly in the combined treatment group compared with DC-CIK group and Huaier Granule group (P<0.05). Therefore, the combined treatment of Huaier Granule combined with DC-CIK achieved the best effect in nude mice transplanted with HT-29 colon carcinoma cell line.

  16. Maternal dazap2 Regulates Germ Granules by Counteracting Dynein in Zebrafish Primordial Germ Cells.

    PubMed

    Forbes, Meredyth M; Rothhämel, Sophie; Jenny, Andreas; Marlow, Florence L

    2015-07-07

    Primordial germ cells (PGCs) are the stem cells of the germline. Generally, germline induction occurs via zygotic factors or the inheritance of maternal determinants called germ plasm (GP). GP is packaged into ribonucleoprotein complexes within oocytes and later promotes the germline fate in embryos. Once PGCs are specified by either mechanism, GP components localize to perinuclear granular-like structures. Although components of zebrafish PGC germ granules have been studied, the maternal factors regulating their assembly and contribution to germ cell development are unknown. Here, we show that the scaffold protein Dazap2 binds to Bucky ball, an essential regulator of oocyte polarity and GP assembly, and colocalizes with the GP in oocytes and in PGCs. Mutational analysis revealed a requirement for maternal Dazap2 (MDazap2) in germ-granule maintenance. Through molecular epistasis analyses, we show that MDazap2 is epistatic to Tdrd7 and maintains germ granules in the embryonic germline by counteracting Dynein activity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Excess influx of Zn(2+) into dentate granule cells affects object recognition memory via attenuated LTP.

    PubMed

    Suzuki, Miki; Fujise, Yuki; Tsuchiya, Yuka; Tamano, Haruna; Takeda, Atsushi

    2015-08-01

    The influx of extracellular Zn(2+) into dentate granule cells is nonessential for dentate gyrus long-term potentiation (LTP) and the physiological significance of extracellular Zn(2+) dynamics is unknown in the dentate gyrus. Excess increase in extracellular Zn(2+) in the hippocampal CA1, which is induced with excitation of zincergic neurons, induces memory deficit via excess influx of Zn(2+) into CA1 pyramidal cells. In the present study, it was examined whether extracellular Zn(2+) induces object recognition memory deficit via excess influx of Zn(2+) into dentate granule cells. KCl (100 mM, 2 µl) was locally injected into the dentate gyrus. The increase in intracellular Zn(2+) in dentate granule cells induced with high K(+) was blocked by co-injection of CaEDTA and CNQX, an extracellular Zn(2+) chelator and an AMPA receptor antagonist, respectively, suggesting that high K(+) increases the influx of Zn(2+) into dentate granule cells via AMPA receptor activation. Dentate gyrus LTP induction was attenuated 1 h after KCl injection into the dentate gyrus and also attenuated when KCl was injected 5 min after the induction. Memory deficit was induced when training of object recognition test was performed 1 h after KCl injection into the dentate gyrus and also induced when KCl was injected 5 min after the training. High K(+)-induced impairments of LTP and memory were rescued by co-injection of CaEDTA. These results indicate that excess influx of Zn(2+) into dentate granule cells via AMPA receptor activation affects object recognition memory via attenuated LTP induction. Even in the dentate gyrus where is scarcely innervated by zincergic neurons, it is likely that extracellular Zn(2+) homeostasis is strictly regulated for cognition. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. 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.

  19. Entorhinal Denervation Induces Homeostatic Synaptic Scaling of Excitatory Postsynapses of Dentate Granule Cells in Mouse Organotypic Slice Cultures

    PubMed Central

    Vlachos, Andreas; Becker, Denise; Jedlicka, Peter; Winkels, Raphael; Roeper, Jochen; Deller, Thomas

    2012-01-01

    Denervation-induced changes in excitatory synaptic strength were studied following entorhinal deafferentation of hippocampal granule cells in mature (≥3 weeks old) mouse organotypic entorhino-hippocampal slice cultures. Whole-cell patch-clamp recordings revealed an increase in excitatory synaptic strength in response to denervation during the first week after denervation. By the end of the second week synaptic strength had returned to baseline. Because these adaptations occurred in response to the loss of excitatory afferents, they appeared to be in line with a homeostatic adjustment of excitatory synaptic strength. To test whether denervation-induced changes in synaptic strength exploit similar mechanisms as homeostatic synaptic scaling following pharmacological activity blockade, we treated denervated cultures at 2 days post lesion for 2 days with tetrodotoxin. In these cultures, the effects of denervation and activity blockade were not additive, suggesting that similar mechanisms are involved. Finally, we investigated whether entorhinal denervation, which removes afferents from the distal dendrites of granule cells while leaving the associational afferents to the proximal dendrites of granule cells intact, results in a global or a local up-scaling of granule cell synapses. By using computational modeling and local electrical stimulations in Strontium (Sr2+)-containing bath solution, we found evidence for a lamina-specific increase in excitatory synaptic strength in the denervated outer molecular layer at 3–4 days post lesion. Taken together, our data show that entorhinal denervation results in homeostatic functional changes of excitatory postsynapses of denervated dentate granule cells in vitro. PMID:22403720

  20. Osseointegration of alumina bioceramic granules: A comparative experimental study

    NASA Astrophysics Data System (ADS)

    Rerikh, V. V.; Avetisyan, A. R.; Zaydman, A. M.; Anikin, K. A.; Bataev, V. A.; Nikulina, A. A.; Sadovoy, M. A.; Aronov, A. M.; Semantsova, E. S.

    2016-08-01

    To perform a comparative analysis of osseointegration of bioceramic alumina-based granules, hydroxyapatite-based granules, and deproteinized bone granules. The experiment was conducted on 52 adult male Kyoto-Wistar rats weighing 350 to 520 g. The animals were divided into five matched groups that differed only in the type of an implanted material. The granules were implanted in the lumbar vertebral bodies and in the distal right femur of each laboratory animal. Two months after surgery, the animals were euthanized, followed by tissue sampling for morphological studies. An examination of specimens from the groups with implanted alumina granules revealed the newly formed trabecular bone with remodeling signs. The bone tissue filled the intragranular space, tightly adhering to the granule surface. There was no connective tissue capsule on the border between bone tissue and alumina granules. Cylindrical bioceramic alumina-based granules with an open internal channel have a higher strength surpassing than that of analogs and the osseointegration ability close to that of hydroxyapatite and deproteinized bone granules.

  1. Glucokinase is an integral component of the insulin granules in glucose-responsive insulin secretory cells and does not translocate during glucose stimulation.

    PubMed

    Arden, Catherine; Harbottle, Andrew; Baltrusch, Simone; Tiedge, Markus; Agius, Loranne

    2004-09-01

    The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

  2. Loss of Melanin by Eye Retinal Pigment Epithelium Cells Is Associated with Its Oxidative Destruction in Melanolipofuscin Granules.

    PubMed

    Dontsov, A E; Sakina, N L; Ostrovsky, M A

    2017-08-01

    The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.

  3. Imaging of zymogen granules in fully wet cells: evidence for restricted mechanism of granule growth.

    PubMed

    Hammel, Ilan; Anaby, Debbie

    2007-09-01

    The introduction of wet SEM imaging technology permits electron microscopy of wet samples. Samples are placed in sealed specimen capsules and are insulated from the vacuum in the SEM chamber by an impermeable, electron-transparent membrane. The complete insulation of the sample from the vacuum allows direct imaging of fully hydrated, whole-mount tissue. In the current work, we demonstrate direct inspection of thick pancreatic tissue slices (above 400 mum). In the case of scanning of the pancreatic surface, the boundaries of intracellular features are seen directly. Thus no unfolding is required to ascertain the actual particle size distribution based on the sizes of the sections. This method enabled us to investigate the true granule size distribution and confirm early studies of improved conformity to a Poisson-like distribution, suggesting that the homotypic granule growth results from a mechanism, which favors the addition of a single unit granule to mature granules.

  4. Olfactory and cortical projections to bulbar and hippocampal adult-born neurons

    PubMed Central

    De La Rosa-Prieto, Carlos; De Moya-Pinilla, Miguel; Saiz-Sanchez, Daniel; Ubeda-banon, Isabel; Arzate, Dulce M.; Flores-Cuadrado, Alicia; Liberia, Teresa; Crespo, Carlos; Martinez-Marcos, Alino

    2015-01-01

    New neurons are continually generated in the subependymal layer of the lateral ventricles and the subgranular zone of dentate gyrus during adulthood. In the subventricular zone, neuroblasts migrate a long distance to the olfactory bulb where they differentiate into granule or periglomerular interneurons. In the hippocampus, neuroblasts migrate a short distance from the subgranular zone to the granule cell layer of the dentate gyrus to become granule neurons. In addition to the short-distance inputs, bulbar interneurons receive long-distance centrifugal afferents from olfactory-recipient structures. Similarly, dentate granule cells receive differential inputs from the medial and lateral entorhinal cortices through the perforant pathway. Little is known concerning these new inputs on the adult-born cells. In this work, we have characterized afferent inputs to 21-day old newly-born neurons. Mice were intraperitoneally injected with bromodeoxyuridine. Two weeks later, rhodamine-labeled dextran-amine was injected into the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral and medial entorhinal cortices. One week later, animals were perfused and immunofluorescences were carried out. The data show that projection neurons from the mentioned structures, establish putative synaptic contacts onto 21-day-old neurons in the olfactory bulb and dentate gyrus, in some cases even before they start to express specific subpopulation proteins. Long-distance afferents reach middle and outer one-third portions of the molecular layer of the dentate gyrus and granule and, interestingly, periglomerular layers of the olfactory bulb. In the olfactory bulb, these fibers appear to establish presumptive axo-somatic contacts onto newly-born granule and periglomerular cells. PMID:25698936

  5. Cellular stress induces cytoplasmic RNA granules in fission yeast.

    PubMed

    Nilsson, Daniel; Sunnerhagen, Per

    2011-01-01

    Severe stress causes plant and animal cells to form large cytoplasmic granules containing RNA and proteins. Here, we demonstrate the existence of stress-induced cytoplasmic RNA granules in Schizosaccharomyces pombe. Homologs to several known protein components of mammalian processing bodies and stress granules are found in fission yeast RNA granules. In contrast to mammalian cells, poly(A)-binding protein (Pabp) colocalizes in stress-induced granules with decapping protein. After glucose deprivation, protein kinase A (PKA) is required for accumulation of Pabp-positive granules and translational down-regulation. This is the first demonstration of a role for PKA in RNA granule formation. In mammals, the translation initiation protein eIF2α is a key regulator of formation of granules containing poly(A)-binding protein. In S. pombe, nonphosphorylatable eIF2α does not block but delays granule formation and subsequent clearance after exposure to hyperosmosis. At least two separate pathways in S. pombe appear to regulate stress-induced granules: pka1 mutants are fully proficient to form granules after hyperosmotic shock; conversely, eIF2α does not affect granule formation in glucose starvation. Further, we demonstrate a Pka1-dependent link between calcium perturbation and RNA granules, which has not been described earlier in any organism.

  6. Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity.

    PubMed

    Tuli, Amit; Thiery, Jerome; James, Ashley M; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B; Orange, Jordan S; Lieberman, Judy; Brenner, Michael B

    2013-12-01

    Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

  7. Protease nexin-1 promotes secretory granule biogenesis by preventing granule protein degradation.

    PubMed

    Kim, Taeyoon; Loh, Y Peng

    2006-02-01

    Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation.

  8. Protease Nexin-1 Promotes Secretory Granule Biogenesis by Preventing Granule Protein Degradation

    PubMed Central

    Kim, Taeyoon; Loh, Y. Peng

    2006-01-01

    Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation. PMID:16319172

  9. Extended-release mesalamine granules for ulcerative colitis.

    PubMed

    Love, Bryan L; Miller, April D

    2012-11-01

    To evaluate the efficacy and safety of extended-release mesalamine granules in the maintenance of remission in ulcerative colitis (UC). Literature was obtained through searches of MEDLINE (1990-June 2012) using the terms mesalamine granules, ulcerative colitis, Apriso, and Salofalk. Bibliographies from retrieved articles were searched for additional citations. All English-language articles reporting on use of extended-release mesalamine granules in humans identified through the search were evaluated and included. The preferred initial treatment for induction and maintenance of remission in mild to moderate UC is agents from the 5-aminosalicylate class (balsalazide, mesalamine, olsalazine, sulfasalazine). Mesalamine granules are available as an encapsulated product in the US and as a nonencapsulated formulation in Europe. Data evaluating encapsulated mesalamine granules for induction of remission are lacking; however, the European mesalamine granule formulation has been evaluated for induction of remission. Patients receiving mesalamine granules for induction achieved clinical and endoscopic remission more frequently than those receiving placebo. Two pivotal, randomized, double-blind, placebo-controlled, multicenter studies have evaluated encapsulated mesalamine granules for maintenance in 562 adults in remission from UC. In both studies, the proportion of patients who remained relapse-free at 6 months was higher for those receiving encapsulated mesalamine granules than placebo. Mesalamine granules are well tolerated, with headache, nausea, and upper respiratory infections being the most frequently reported adverse effects. Current evidence supports the use of extended-release mesalamine granules for maintenance of remission in mild to moderate UC. Further studies are necessary to examine the ideal dose and regimen of encapsulated mesalamine granules for induction of remission in UC.

  10. Lineage analysis of quiescent regenerative stem cells in the adult brain by genetic labelling reveals spatially restricted neurogenic niches in the olfactory bulb.

    PubMed

    Giachino, Claudio; Taylor, Verdon

    2009-07-01

    The subventricular zone (SVZ) of the lateral ventricles is the major neurogenic region in the adult mammalian brain, harbouring neural stem cells within defined niches. The identity of these stem cells and the factors regulating their fate are poorly understood. We have genetically mapped a population of Nestin-expressing cells during postnatal development to study their potential and fate in vivo. Taking advantage of the recombination characteristics of a nestin::CreER(T2) allele, we followed a subpopulation of neural stem cells and traced their fate in a largely unrecombined neurogenic niche. Perinatal nestin::CreER(T2)-expressing cells give rise to multiple glial cell types and neurons, as well as to stem cells of the adult SVZ. In the adult SVZ nestin::CreER(T2)-expressing neural stem cells give rise to several neuronal subtypes in the olfactory bulb (OB). We addressed whether the same population of neural stem cells play a role in SVZ regeneration. Following anti-mitotic treatment to eliminate rapidly dividing progenitors, relatively quiescent nestin::CreER(T2)-targeted cells are spared and contribute to SVZ regeneration, generating new proliferating precursors and neuroblasts. Finally, we have identified neurogenic progenitors clustered in ependymal-like niches within the rostral migratory stream (RMS) of the OB. These OB-RMS progenitors generate neuroblasts that, upon transplantation, graft, migrate and differentiate into granule and glomerular neurons. In summary, using conditional lineage tracing we have identified neonatal cells that are the source of neurogenic and regenerative neural stem cells in the adult SVZ and occupy a novel neurogenic niche in the OB.

  11. Large granulation cells on the surface of the giant star π1 Gruis

    NASA Astrophysics Data System (ADS)

    Paladini, C.; Baron, F.; Jorissen, A.; Le Bouquin, J.-B.; Freytag, B.; van Eck, S.; Wittkowski, M.; Hron, J.; Chiavassa, A.; Berger, J.-P.; Siopis, C.; Mayer, A.; Sadowski, G.; Kravchenko, K.; Shetye, S.; Kerschbaum, F.; Kluska, J.; Ramstedt, S.

    2018-01-01

    Convection plays a major part in many astrophysical processes, including energy transport, pulsation, dynamos and winds on evolved stars, in dust clouds and on brown dwarfs. Most of our knowledge about stellar convection has come from studying the Sun: about two million convective cells with typical sizes of around 2,000 kilometres across are present on the surface of the Sun—a phenomenon known as granulation. But on the surfaces of giant and supergiant stars there should be only a few large (several tens of thousands of times larger than those on the Sun) convective cells, owing to low surface gravity. Deriving the characteristic properties of convection (such as granule size and contrast) for the most evolved giant and supergiant stars is challenging because their photospheres are obscured by dust, which partially masks the convective patterns. These properties can be inferred from geometric model fitting, but this indirect method does not provide information about the physical origin of the convective cells. Here we report interferometric images of the surface of the evolved giant star π1 Gruis, of spectral type S5,7. Our images show a nearly circular, dust-free atmosphere, which is very compact and only weakly affected by molecular opacity. We find that the stellar surface has a complex convective pattern with an average intensity contrast of 12 per cent, which increases towards shorter wavelengths. We derive a characteristic horizontal granule size of about 1.2 × 1011 metres, which corresponds to 27 per cent of the diameter of the star. Our measurements fall along the scaling relations between granule size, effective temperature and surface gravity that are predicted by simulations of stellar surface convection.

  12. Large granulation cells on the surface of the giant star π1 Gruis.

    PubMed

    Paladini, C; Baron, F; Jorissen, A; Le Bouquin, J-B; Freytag, B; Van Eck, S; Wittkowski, M; Hron, J; Chiavassa, A; Berger, J-P; Siopis, C; Mayer, A; Sadowski, G; Kravchenko, K; Shetye, S; Kerschbaum, F; Kluska, J; Ramstedt, S

    2018-01-18

    Convection plays a major part in many astrophysical processes, including energy transport, pulsation, dynamos and winds on evolved stars, in dust clouds and on brown dwarfs. Most of our knowledge about stellar convection has come from studying the Sun: about two million convective cells with typical sizes of around 2,000 kilometres across are present on the surface of the Sun-a phenomenon known as granulation. But on the surfaces of giant and supergiant stars there should be only a few large (several tens of thousands of times larger than those on the Sun) convective cells, owing to low surface gravity. Deriving the characteristic properties of convection (such as granule size and contrast) for the most evolved giant and supergiant stars is challenging because their photospheres are obscured by dust, which partially masks the convective patterns. These properties can be inferred from geometric model fitting, but this indirect method does not provide information about the physical origin of the convective cells. Here we report interferometric images of the surface of the evolved giant star π 1 Gruis, of spectral type S5,7. Our images show a nearly circular, dust-free atmosphere, which is very compact and only weakly affected by molecular opacity. We find that the stellar surface has a complex convective pattern with an average intensity contrast of 12 per cent, which increases towards shorter wavelengths. We derive a characteristic horizontal granule size of about 1.2 × 10 11 metres, which corresponds to 27 per cent of the diameter of the star. Our measurements fall along the scaling relations between granule size, effective temperature and surface gravity that are predicted by simulations of stellar surface convection.

  13. Adult Olfactory Bulb Interneuron Phenotypes Identified by Targeting Embryonic and Postnatal Neural Progenitors

    PubMed Central

    Figueres-Oñate, Maria; López-Mascaraque, Laura

    2016-01-01

    Neurons are generated during embryonic development and in adulthood, although adult neurogenesis is restricted to two main brain regions, the hippocampus and olfactory bulb. The subventricular zone (SVZ) of the lateral ventricles generates neural stem/progenitor cells that continually provide the olfactory bulb (OB) with new granule or periglomerular neurons, cells that arrive from the SVZ via the rostral migratory stream. The continued neurogenesis and the adequate integration of these newly generated interneurons is essential to maintain homeostasis in the olfactory bulb, where the differentiation of these cells into specific neural cell types is strongly influenced by temporal cues. Therefore, identifying the critical features that control the generation of adult OB interneurons at either pre- or post-natal stages is important to understand the dynamic contribution of neural stem cells. Here, we used in utero and neonatal SVZ electroporation along with a transposase-mediated stable integration plasmid, in order to track interneurons and glial lineages in the OB. These plasmids are valuable tools to study the development of OB interneurons from embryonic and post-natal SVZ progenitors. Accordingly, we examined the location and identity of the adult progeny of embryonic and post-natally transfected progenitors by examining neurochemical markers in the adult OB. These data reveal the different cell types in the olfactory bulb that are generated in function of age and different electroporation conditions. PMID:27242400

  14. Early postnatal GFAP-expressing cells produce multilineage progeny in cerebrum and astrocytes in cerebellum of adult mice.

    PubMed

    Guo, Zhibao; Wang, Xijuan; Xiao, Jun; Wang, Yihui; Lu, Hong; Teng, Junfang; Wang, Wei

    2013-09-26

    Early postnatal GFAP-expressing cells are thought to be immature astrocytes. However, it is not clear if they possess multilineage capacity and if they can generate different lineages (astrocytes, neurons and oligodendrocytes) in the brain of adult mice. In order to identify the fate of astroglial cells in the postnatal brain, hGFAP-Cre-ER(T2) transgenic mice were crossed with the R26R Cre reporter mouse strains which exhibit constitutive expression of β-galactosidase (β-gal). Mice carrying the hGFAP-Cre-ER(T2)/R26R transgene were treated with Tamoxifen to induce Cre recombination in astroglial cells at postnatal (P) day 6 and Cre recombinase-expressing cells were identified by X-gal staining. Immunohistochemical staining was used to identify the type(s) of these reporter-tagged cells. Sixty days after recombination, X-gal-positive cells in different cerebral regions of the adult mice expressed the astroglial markers Blbp and GFAP, the neuronal marker NeuN, the oligodendrocyte precursor cell marker NG2 and the mature oligodendrocyte marker CC1. X-gal-positive cells in the cerebellum coexpressed the astroglial marker Blbp, but not the granule cell marker NeuN, Purkinje cell marker Calbindin or oligodendrocyte precursor cell marker NG2. Our genetic fate mapping data demonstrated that early postnatal GFAP-positive cells possessed multilineage potential and eventually differentiated into neurons, astrocytes, and oligodendrocyte precursor cells in the cerebrum and into astrocytes (including Bergmann glia) in the cerebellum of adult mice. © 2013 Elsevier B.V. All rights reserved.

  15. Heterogeneity of chromatoid bodies in adult pluripotent stem cells of planarian Dugesia japonica.

    PubMed

    Kashima, Makoto; Kumagai, Nobuyoshi; Agata, Kiyokazu; Shibata, Norito

    2016-02-01

    The robust regenerative ability of planarians is known to be dependent on adult pluripotent stem cells called neoblasts. One of the morphological features of neoblasts is cytoplasmic ribonucleoprotein granules (chromatoid bodies: CBs), which resemble germ granules present in germline cells in other animals. Previously, we showed by immuno-electron microscopic analysis that DjCBC-1, a planarian Me31B/Dhh1/DDX6 homologue, which is a component of ribonucleoprotein granules, was localized in CBs in the planarian Dugesia japonica. Also, recently it was reported using another planarian species that Y12 antibody recognizing symmetrical dimethylarginine (sDMA) specifically binds to CBs in which histone mRNA is co-localized. Here, we showed by double immunostaining and RNA interference (RNAi) that DjCBC-1-containing CBs and Y12-immunoreactive CBs are distinct structures, suggesting that CBs are composed of heterogeneous populations. We also found that the Y12-immunoreactive CBs specifically contained a cytoplasmic type of planarian PIWI protein (DjPiwiC). We revealed by RNAi experiments that Y12-immunoreactive CBs may have anti-transposable element activity involving the DjPiwiC protein in the neoblasts. © 2016 Japanese Society of Developmental Biologists.

  16. Facilitation of granule cell epileptiform activity by mossy fiber-released zinc in the pilocarpine model of temporal lobe epilepsy.

    PubMed

    Timofeeva, Olga; Nadler, J Victor

    2006-03-17

    Recurrent mossy fiber synapses in the dentate gyrus of epileptic brain facilitate the synchronous firing of granule cells and may promote seizure propagation. Mossy fiber terminals contain and release zinc. Released zinc inhibits the activation of NMDA receptors and may therefore oppose the development of granule cell epileptiform activity. Hippocampal slices from rats that had experienced pilocarpine-induced status epilepticus and developed a recurrent mossy fiber pathway were used to investigate this possibility. Actions of released zinc were inferred from the effects of chelation with 1 mM calcium disodium EDTA (CaEDTA). When granule cell population bursts were evoked by mossy fiber stimulation in the presence of 6 mM K(+) and 30 microM bicuculline, CaEDTA slowed the rate at which evoked bursting developed, but did not change the magnitude of the bursts once they had developed fully. The effects of CaEDTA were then studied on the pharmacologically isolated NMDA receptor- and AMPA/kainate receptor-mediated components of the fully developed bursts. CaEDTA increased the magnitude of NMDA receptor-mediated bursts and reduced the magnitude of AMPA/kainate receptor-mediated bursts. CaEDTA did not affect the granule cell bursts evoked in slices from untreated rats by stimulating the perforant path in the presence of bicuculline and 6 mM K(+). These results suggest that zinc released from the recurrent mossy fibers serves mainly to facilitate the recruitment of dentate granule cells into population bursts.

  17. Tonic Inhibitory Control of Dentate Gyrus Granule Cells by α5-Containing GABAA Receptors Reduces Memory Interference.

    PubMed

    Engin, Elif; Zarnowska, Ewa D; Benke, Dietmar; Tsvetkov, Evgeny; Sigal, Maksim; Keist, Ruth; Bolshakov, Vadim Y; Pearce, Robert A; Rudolph, Uwe

    2015-10-07

    Interference between similar or overlapping memories formed at different times poses an important challenge on the hippocampal declarative memory system. Difficulties in managing interference are at the core of disabling cognitive deficits in neuropsychiatric disorders. Computational models have suggested that, in the normal brain, the sparse activation of the dentate gyrus granule cells maintained by tonic inhibitory control enables pattern separation, an orthogonalization process that allows distinct representations of memories despite interference. To test this mechanistic hypothesis, we generated mice with significantly reduced expression of the α5-containing GABAA (α5-GABAARs) receptors selectively in the granule cells of the dentate gyrus (α5DGKO mice). α5DGKO mice had reduced tonic inhibition of the granule cells without any change in fast phasic inhibition and showed increased activation in the dentate gyrus when presented with novel stimuli. α5DGKO mice showed impairments in cognitive tasks characterized by high interference, without any deficiencies in low-interference tasks, suggesting specific impairment of pattern separation. Reduction of fast phasic inhibition in the dentate gyrus through granule cell-selective knock-out of α2-GABAARs or the knock-out of the α5-GABAARs in the downstream CA3 area did not detract from pattern separation abilities, which confirms the anatomical and molecular specificity of the findings. In addition to lending empirical support to computational hypotheses, our findings have implications for the treatment of interference-related cognitive symptoms in neuropsychiatric disorders, particularly considering the availability of pharmacological agents selectively targeting α5-GABAARs. Interference between similar memories poses a significant limitation on the hippocampal declarative memory system, and impaired interference management is a cognitive symptom in many disorders. Thus, understanding mechanisms of successful

  18. In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

    PubMed

    Ramachandran, Sarada Devi; Schirmer, Katharina; Münst, Bernhard; Heinz, Stefan; Ghafoory, Shahrouz; Wölfl, Stefan; Simon-Keller, Katja; Marx, Alexander; Øie, Cristina Ionica; Ebert, Matthias P; Walles, Heike; Braspenning, Joris; Breitkopf-Heinlein, Katja

    2015-01-01

    In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

  19. The presence of PHB granules in cytoplasm protects non-halophilic bacterial cells against the harmful impact of hypertonic environments.

    PubMed

    Obruca, Stanislav; Sedlacek, Petr; Mravec, Filip; Krzyzanek, Vladislav; Nebesarova, Jana; Samek, Ota; Kucera, Dan; Benesova, Pavla; Hrubanova, Kamila; Milerova, Miluse; Marova, Ivana

    2017-10-25

    Numerous prokaryotes accumulate polyhydroxybutyrate (PHB) intracellularly as a storage material. It has also been proposed that PHB accumulation improves bacterial stress resistance. Cupriavidus necator and its PHB non-accumulating mutant were employed to investigate the protective role of PHB under hypertonic conditions. The presence of PHB granules enhanced survival of the bacteria after exposure to hypertonic conditions. Surprisingly, when coping with such conditions, the bacteria did not utilize PHB to harvest carbon or energy, suggesting that, in the osmotic upshock of C. necator, the protective mechanism of PHB granules is not associated with their hydrolysis. The presence of PHB granules influenced the overall properties of the cells, since challenged PHB-free cells underwent massive plasmolysis accompanied by damage to the cell membrane and the leakage of cytoplasm content, while no such effects were observed in PHB containing bacteria. Moreover, PHB granules demonstrated "liquid-like" properties indicating that they can partially repair and stabilize cell membranes by plugging small gaps formed during plasmolysis. In addition, the level of dehydration and changes in intracellular pH in osmotically challenged cells were less pronounced for PHB-containing cultures, demonstrating the important role of PHB for bacterial survival under hyperosmotic conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Isolation of zymogen granules from rat pancreas.

    PubMed

    Rindler, Michael J

    2006-01-01

    This unit describes methods for preparing zymogen granules from rat pancreas. Zymogen granules are storage organelles in pancreatic acinar cells containing digestive enzymes that are released into the pancreatic duct. The protocols in this unit take advantage of the large size (up to 1 microm diameter) and high density (>1.20 g/cm(3) on sucrose gradients) of the granules as compared to other cellular organelles. They use a combination of differential sedimentation and density gradient separation to accomplish the purification. Similar procedures can be used to isolate zymogen granules from mouse pancreas and canine pancreas. A protocol for preparing zymogen granules from dog pancreas is also included.

  1. Shp2 Acts Downstream of SDF-1α/CXCR4 in Guiding Granule Cell Migration During Cerebellar Development

    PubMed Central

    Hagihara, Kazuki; Zhang, Eric E.; Ke, Yue-Hai; Liu, Guofa; Liu, Jan-Jan; Rao, Yi; Feng, Gen-Sheng

    2009-01-01

    Shp2 is a non-receptor protein tyrosine phosphatase containing two Src homology 2 (SH2) domains that is implicated in intracellular signaling events controlling cell proliferation, differentiation and migration. To examine the role of Shp2 in brain development, we created mice with Shp2 selectively deleted in neural stem/progenitor cells. Homozygous mutant mice exhibited early postnatal lethality with defects in neural stem cell self-renewal and neuronal/glial cell fate specification. Here we report a critical role of Shp2 in guiding neuronal cell migration in the cerebellum. In homozygous mutants, we observed reduced and less foliated cerebellum, ectopic presence of external granule cells and mispositioned Purkinje cells, a phenotype very similar to that of mutant mice lacking either SDF-1α or CXCR4. Consistently, Shp2-deficient granule cells failed to migrate toward SDF-1α in an in vitro cell migration assay, and SDF-1α treatment triggered a robust induction of tyrosyl phosphorylation on Shp2. Together, these results suggest that although Shp2 is involved in multiple signaling events during brain development, a prominent role of the phosphatase is to mediate SDF-1α/CXCR4 signal in guiding cerebellar granule cell migration. PMID:19635473

  2. A disynaptic feedback network activated by experience promotes the integration of new granule cells.

    PubMed

    Alvarez, Diego D; Giacomini, Damiana; Yang, Sung Min; Trinchero, Mariela F; Temprana, Silvio G; Büttner, Karina A; Beltramone, Natalia; Schinder, Alejandro F

    2016-10-28

    Experience shapes the development and connectivity of adult-born granule cells (GCs) through mechanisms that are poorly understood. We examined the remodeling of dentate gyrus microcircuits in mice in an enriched environment (EE). Short exposure to EE during early development of new GCs accelerated their functional integration. This effect was mimicked by in vivo chemogenetic activation of a limited population of mature GCs. Slice recordings showed that mature GCs recruit parvalbumin γ-aminobutyric acid-releasing interneurons (PV-INs) that feed back onto developing GCs. Accordingly, chemogenetic stimulation of PV-INs or direct depolarization of developing GCs accelerated GC integration, whereas inactivation of PV-INs prevented the effects of EE. Our results reveal a mechanism for dynamic remodeling in which experience activates dentate networks that "prime" young GCs through a disynaptic feedback loop mediated by PV-INs. Copyright © 2016, American Association for the Advancement of Science.

  3. Chromaffin granules in the rat adrenal medulla release their secretory content in a particulate fashion.

    PubMed

    Crivellato, Enrico; Belloni, Anna; Nico, Beatrice; Nussdorfer, Gastone G; Ribatti, Domenico

    2004-03-01

    Exocytosis is considered the main route of granule discharge in chromaffin cells. We recently provided ultrastructural evidence suggesting that piecemeal degranulation (PMD) occurs in mouse adrenal chromaffin cells. In the present study, we processed rat adrenal glands for transmission electron microscopy (TEM), and examined chromaffin cells for changes characteristic of PMD. Both adrenaline (A)- and noradrenaline (NA)-storing cells express ultrastructural features suggestive of a slow and particulate mode of granule discharge. In adrenaline-containing cells, some granules present enlarged dimensions accompanied by eroded or dissolved matrices. Likewise, a number of granules in NA-releasing cells show content reduction with variably expanded granule chambers. Dilated, empty granule containers are recognizable in the cytoplasm of both cell types. Characteristically, altered granules and empty containers are seen intermingled with normal, resting granules. In addition, chromaffin granules often show irregular profiles, with budding or tail-like projections of their limiting membranes. Thirty 150-nm-diameter membrane-bound vesicles with a moderately electron-dense or -lucent internal structure are observable in the cytoplasm of both cell types. These vesicles are seen among the granules and some of them are fused with the perigranule membranes in the process of attachment to or budding from the granules. These data add further support to the concept that PMD may be an alternative secretory pathway in adrenal chromaffin cells. Copyright 2004 Wiley-Liss, Inc.

  4. Formation of artificial granules for proving gelation as the main mechanism of aerobic granulation in biological wastewater treatment.

    PubMed

    Li, Yun; Yang, Shu-Fang; Zhang, Jian-Jun; Li, Xiao-Yan

    2014-01-01

    In this study, gelation-facilitated biofilm formation as a new mechanism is proposed for the phenomenon of aerobic granulation in biological wastewater treatment. To obtain an experimental proof for the gelation-based theory, the granulation process was simulated in a chemical system using latex particles for bacterial cells and organic polymers (alginate and peptone) for extracellular polymeric substances (EPS) in a solution with the addition of cations (Ca²⁺, Mg²⁺ and Fe³⁺). The results showed that at a low alginate content (70 mg g⁻¹ mixed liquid suspended solids (MLSS)) flocculation was observed in the suspension with loose flocs. At a higher alginate content (180 mg g⁻¹ MLSS), together with discharge of small flocs, formation of artificial gel granules was successfully achieved leading to granulation. The artificial granules show a morphological property similar to that of actual microbial granules. However, if the protein content increased, granulation became difficult with little gel formation. The experimental work demonstrates the importance of the bonding interactions between EPS functional groups and cations in gel formation and granulation. The laboratory results on the formation of artificial granules provide a sound proof for the theory of gelation-facilitated biofilm formation as the main mechanism for aerobic granulation in sludge suspensions.

  5. Roll compaction/dry granulation: comparison between roll mill and oscillating granulator in dry granulation.

    PubMed

    Sakwanichol, Jarunee; Puttipipatkhachorn, Satit; Ingenerf, Gernot; Kleinebudde, Peter

    2012-01-01

    Different experimental factorial designs were employed to evaluate granule properties obtained from oscillating granulator and roll mill. Four oscillating-granulator parameters were varied, i.e. rotor speed, oscillating angle, aperture of mesh screen and rotor type. Six roll-mill parameters that were throughput, speed ratio in both first and second stages, gap between roll pair in both stages and roll-surface texture were also investigated. Afterwards, the granule properties obtained from two milling types with similar median particle size were compared. All milling parameters in both milling types affected significantly the median particle size, size distribution and amount of fine particles (P < 0.05), except the rotor types of oscillating granulator on fines. Only three milling parameters influenced significantly the flowability (P < 0.05). These were the throughput and the gap size in the first stage of roll mill and the sieve size of oscillating granulator. In comparison between milling types, the differences of granule properties were not practically relevant. However, the roll mill had much higher capacity than the oscillating granulator about seven times, resulting in improving energy savings per unit of product. Consequently, the roll mill can be applied instead of oscillating granulator for roll compaction/dry granulation technique.

  6. Induction of c-Fos, Zif268, and Arc from acute bouts of voluntary wheel running in new and pre-existing adult mouse hippocampal granule neurons

    PubMed Central

    Clark, Peter J.; Bhattacharya, Tushar K.; Miller, Daniel S.; Rhodes, Justin S.

    2011-01-01

    The functional significance of newly formed granule neurons in the adult mammalian hippocampus remains a mystery. Recently, it was demonstrated that wheel running increases new neuron survival and c-Fos expression in new and pre-existing granule cells in an activity-dependent manner. It is currently unknown whether other immediate early genes (IEGs) become expressed in granule neurons from running. Further, it is unknown whether locomotor activity in home cages without wheels can influence neurogenesis and IEG expression similar to running. The purpose of this study was three fold: 1) to determine if Arc and Zif268 expression are also induced from wheel running in both pre-existing and newly formed neurons 2) to determine if neurogenesis and IEG induction is related to horizontal distance traveled in home cages without wheels and 3) to determine whether IEG induction is related to acute bouts of running or chronic effects. Adult C57BL/6J female mice were placed in cages with or without running wheels for 31 days. The first 10 days, mice received daily injections of 5-Bromo-2′-deoxyuridine (BrdU) to label dividing cells. On day 31, running and non-running animals were euthanized either 2 hours after peak activity, or during a period of relative inactivity. Immunohistochemistry was performed on hippocampal sections with antibodies against BrdU, mature neuron marker NeuN, c-Fos, Arc, and Zif268. Results demonstrate that Arc, Zif268, and c-Fos are induced from wheel running but not movement in cages without wheels. All IEGs were expressed in new neurons from running. Further, IEGs were induced acutely by running, as increased expression did not continue into the light cycle, a period of relative inactivity. The results suggest that robust movements, like running, are necessary to stimulate IEG expression and neurogenesis. Moreover, results suggest new neurons from running may be processing information about running behavior itself. PMID:21497182

  7. Imaging Ca2+-triggered exocytosis of single secretory granules on plasma membrane lawns from neuroendocrine cells.

    PubMed

    Lang, Thorsten

    2008-01-01

    This cell-free assay for exocytosis is particularly useful when spatial information about exocytotic sites and biochemical access to the plasma membrane within less than a minute is required. It is based on the study of plasma membrane lawns from secretory cells exhibiting secretory granules filled with neuropeptide Y-green fluorescent protein (NPY-GFP). The sample is prepared by subjecting NPY-GFP-expressing cells to a brief ultrasound pulse, leaving behind a basal, flat plasma membrane with fluorescent attached secretory organelles. These sheets can then be incubated in defined solutions with the benefit that complete solution changes can be achieved in less than 1 min. Individual secretory granules are monitored in the docked state and during exocytosis by video microscopy.

  8. Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest.

    PubMed

    Diederen, J H; Vullings, H G

    1995-03-01

    The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.

  9. Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation.

    PubMed

    Giordano, T; Brigatti, C; Podini, P; Bonifacio, E; Meldolesi, J; Malosio, M L

    2008-06-01

    We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli. The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media. Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin +/- high glucose release of chromogranin B predominated. Weak, Ca(2+)-dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca(2+) sensitivity of the specific granules. The unexpected complexity of the beta cell granule population in terms of heterogeneity, molecular plasticity and the differential discharge, could play an important role in physiological control of insulin release and possibly also in beta cell pathology.

  10. 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. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  11. PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

    PubMed

    Wahl, Andreas; Schuth, Nora; Pfeiffer, Daniel; Nussberger, Stephan; Jendrossek, Dieter

    2012-11-16

    Poly(3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes which allow survival of the cells in the absence of suitable carbon sources. Formation and subcellular localization of PHB granules was previously assumed to occur randomly in the cytoplasm of PHB accumulating bacteria. However, contradictionary results on subcellular localization of PHB granules in Ralstonia eutropha were published, recently. Here, we provide evidence by transmission electron microscopy that PHB granules are localized in close contact to the nucleoid region in R. eutropha during growth on nutrient broth. Binding of PHB granules to the nucleoid is mediated by PhaM, a PHB granule associated protein with phasin-like properties that is also able to bind to DNA and to phasin PhaP5. Over-expression of PhaM resulted in formation of many small PHB granules that were always attached to the nucleoid region. In contrast, PHB granules of ∆phaM strains became very large and distribution of granules to daughter cells was impaired. Association of PHB granules to the nucleoid region was prevented by over-expression of PhaP5 and clusters of several PHB granules were mainly localized near the cell poles. Subcellular localization of PHB granules is controlled in R. eutropha and depends on the presence and concentrations of at least two PHB granule associated proteins, PhaM and PhaP5.

  12. Exocyst sec5 regulates exocytosis of newcomer insulin granules underlying biphasic insulin secretion.

    PubMed

    Xie, Li; Zhu, Dan; Kang, Youhou; Liang, Tao; He, Yu; Gaisano, Herbert Y

    2013-01-01

    The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca(2+) channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.

  13. 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

  14. Docking is not a prerequisite but a temporal constraint for fusion of secretory granules.

    PubMed

    Kasai, Kazuo; Fujita, Takuji; Gomi, Hiroshi; Izumi, Tetsuro

    2008-07-01

    We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic beta cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.

  15. Correlation between loose density and compactibility of granules prepared by various granulation methods.

    PubMed

    Murakami, H; Yoneyama, T; Nakajima, K; Kobayashi, M

    2001-03-23

    The objectives of this study were to prepare the lactose granules by various granulation methods using polyethylene glycol 6000 (PEG 6000) as a binder and to evaluate the effects of granulation methods on the compressibility and compactibility of granules in tabletting. Lactose was granulated by seven granulation methods -- four wet granulations including wet massing granulation, wet high-speed mixer granulation, wet fluidized bed granulation and wet tumbling fluidized bed granulation; and three melt granulations including melt high-speed mixer granulation, melt fluidized bed granulation and melt tumbling fluidized bed granulation. The loose density, angle of repose, granule size distribution, mean diameter of granules, and the tensile strength and porosity of tablets were evaluated. The compactibilities of granules were varied by the granulation methods. However, the difference in compactibility of granules could not be explained due to the difference in compressibility, since there was no difference in Heckel plots due to granulation methods. Among their granule properties, the loose density of granules seemed to have a correlation with the tablet strength regardless of the granulation methods.

  16. Stimulation of insulin release by glucose is associated with an increase in the number of docked granules in the beta-cells of rat pancreatic islets.

    PubMed

    Straub, Susanne G; Shanmugam, Geetha; Sharp, Geoffrey W G

    2004-12-01

    Electron microscopy and quantitative stereological techniques were used to study the dynamics of the docked granule pool in the rat pancreatic beta-cell. The mean number of granules per beta-cell was 11,136. After equilibration in RPMI containing 5.6 mmol/l glucose, 6.4% of the granules (approximately 700) were docked at the plasma membrane (also measured as [means +/- SE] 4.3 +/- 0.6 docked granules per 10 microm of plasma membrane at the perimeter of the cell sections). After a 40-min exposure to 16.7 mmol/l glucose, 10.2% of the granules (approximately 1,060) were docked (6.4 +/- 0.8 granules per 10 microm of plasma membrane). Thus, the docked pool increased by 50% during stimulation with glucose. Islets were also exposed to 16.7 mmol/l glucose in the absence or presence of 10 micromol/l nitrendipine. In the absence and presence of nitrendipine, there were 6.1 +/- 0.7 and 6.3 +/- 0.6 granules per 10 microm of membrane, respectively. Thus, glucose increased granule docking independently of increased [Ca2+]i and exocytosis. The data suggest a limit to the number of docking sites. As the rate of docking exceeded the rate of exocytosis, docking is not rate limiting for insulin release. Only with extremely high release rates, glucose stimulation after a 4-h incubation with a high concentration of fatty acid-free BSA, was the docked granule pool reduced in size.

  17. Biogenesis of zinc storage granules in Drosophila melanogaster.

    PubMed

    Tejeda-Guzmán, Carlos; Rosas-Arellano, Abraham; Kroll, Thomas; Webb, Samuel M; Barajas-Aceves, Martha; Osorio, Beatriz; Missirlis, Fanis

    2018-03-19

    Membrane transporters and sequestration mechanisms concentrate metal ions differentially into discrete subcellular microenvironments for use in protein cofactors, signalling, storage or excretion. Here we identify zinc storage granules as the insect's major zinc reservoir in principal Malpighian tubule epithelial cells of Drosophila melanogaster The concerted action of Adaptor Protein-3, Rab32, HOPS and BLOC complexes as well as of the white-scarlet (ABCG2-like) and ZnT35C (ZnT2/ZnT3/ZnT8-like) transporters is required for zinc storage granule biogenesis. Due to lysosome-related organelle defects caused by mutations in the homologous human genes, patients with Hermansky-Pudlak syndrome may lack zinc granules in beta pancreatic cells, intestinal paneth cells and presynaptic vesicles of hippocampal mossy fibers. © 2018. Published by The Company of Biologists Ltd.

  18. Observing secretory granules with a multiangle evanescent wave microscope.

    PubMed Central

    Rohrbach, A

    2000-01-01

    In total internal reflection fluorescence microscopy (TIRFM), fluorophores near a surface can be excited with evanescent waves, which decay exponentially with distance from the interface. Penetration depths of evanescent waves from 60 nm to 300 nm were generated by varying the angle of incidence of a laser beam. With a novel telecentric multiangle evanescent wave microscope, we monitored and investigated both single secretory granules and pools of granules in bovine chromaffin cells. By measuring the fluorescence intensity as a function of penetration depth, it is possible through a Laplace transform to obtain the fluorophore distribution as a function of axial position. We discuss the extent to which it is possible to determine distances and diameters of granules with this microscopy technique by modeling the fluorescent volumes of spheres in evanescent fields. The anisotropic near-field detection of fluorophores and the influence of the detection point-spread function are considered. The diameters of isolated granules between 70 nm and 300 nm have been reconstructed, which is clearly beyond the resolution limit of a confocal microscope. Furthermore, the paper demonstrates how evanescent waves propagate along surfaces and scatter at objects with a higher refractive index. TIRFM will have a limited applicability for quantitative measurements when the parameters used to define evanescent waves are not optimally selected. PMID:10777760

  19. SoxC Transcription Factors Are Required for Neuronal Differentiation in Adult Hippocampal Neurogenesis

    PubMed Central

    Mu, Lifang; Berti, Lucia; Masserdotti, Giacomo; Covic, Marcela; Michaelidis, Theologos M.; Doberauer, Kathrin; Merz, Katharina; Rehfeld, Frederick; Haslinger, Anja; Wegner, Michael; Sock, Elisabeth; Lefebvre, Veronique; Couillard-Despres, Sebastien; Aigner, Ludwig; Berninger, Benedikt; Lie, D. Chichung

    2012-01-01

    Neural stem cells (NSCs) generate new hippocampal dentate granule neurons throughout adulthood. The genetic programs controlling neuronal differentiation of adult NSCs are only poorly understood. Here we show that, in the adult mouse hippocampus, expression of the SoxC transcription factors Sox4 and Sox11 is initiated around the time of neuronal commitment of adult NSCs and is maintained in immature neurons. Overexpression of Sox4 and Sox11 strongly promotes in vitro neurogenesis from adult NSCs, whereas ablation of Sox4/Sox11 prevents in vitro and in vivo neurogenesis from adult NSCs. Moreover, we demonstrate that SoxC transcription factors target the promoters of genes that are induced on neuronal differentiation of adult NSCs. Finally, we show that reprogramming of astroglia into neurons is dependent on the presence of SoxC factors. These data identify SoxC proteins as essential contributors to the genetic network controlling neuronal differentiation in adult neurogenesis and neuronal reprogramming of somatic cells. PMID:22378879

  20. PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha

    PubMed Central

    2012-01-01

    Background Poly(3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes which allow survival of the cells in the absence of suitable carbon sources. Formation and subcellular localization of PHB granules was previously assumed to occur randomly in the cytoplasm of PHB accumulating bacteria. However, contradictionary results on subcellular localization of PHB granules in Ralstonia eutropha were published, recently. Results Here, we provide evidence by transmission electron microscopy that PHB granules are localized in close contact to the nucleoid region in R. eutropha during growth on nutrient broth. Binding of PHB granules to the nucleoid is mediated by PhaM, a PHB granule associated protein with phasin-like properties that is also able to bind to DNA and to phasin PhaP5. Over-expression of PhaM resulted in formation of many small PHB granules that were always attached to the nucleoid region. In contrast, PHB granules of ∆phaM strains became very large and distribution of granules to daughter cells was impaired. Association of PHB granules to the nucleoid region was prevented by over-expression of PhaP5 and clusters of several PHB granules were mainly localized near the cell poles. Conclusion Subcellular localization of PHB granules is controlled in R. eutropha and depends on the presence and concentrations of at least two PHB granule associated proteins, PhaM and PhaP5. PMID:23157596

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.

    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 inhibitedmore » 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.« less

  2. Trim9 Deletion Alters the Morphogenesis of Developing and Adult-Born Hippocampal Neurons and Impairs Spatial Learning and Memory

    PubMed Central

    Winkle, Cortney C.; Olsen, Reid H. J.; Kim, Hyojin; Moy, Sheryl S.

    2016-01-01

    During hippocampal development, newly born neurons migrate to appropriate destinations, extend axons, and ramify dendritic arbors to establish functional circuitry. These developmental stages are recapitulated in the dentate gyrus of the adult hippocampus, where neurons are continuously generated and subsequently incorporate into existing, local circuitry. Here we demonstrate that the E3 ubiquitin ligase TRIM9 regulates these developmental stages in embryonic and adult-born mouse hippocampal neurons in vitro and in vivo. Embryonic hippocampal and adult-born dentate granule neurons lacking Trim9 exhibit several morphological defects, including excessive dendritic arborization. Although gross anatomy of the hippocampus was not detectably altered by Trim9 deletion, a significant number of Trim9−/− adult-born dentate neurons localized inappropriately. These morphological and localization defects of hippocampal neurons in Trim9−/− mice were associated with extreme deficits in spatial learning and memory, suggesting that TRIM9-directed neuronal morphogenesis may be involved in hippocampal-dependent behaviors. SIGNIFICANCE STATEMENT Appropriate generation and incorporation of adult-born neurons in the dentate gyrus are critical for spatial learning and memory and other hippocampal functions. Here we identify the brain-enriched E3 ubiquitin ligase TRIM9 as a novel regulator of embryonic and adult hippocampal neuron shape acquisition and hippocampal-dependent behaviors. Genetic deletion of Trim9 elevated dendritic arborization of hippocampal neurons in vitro and in vivo. Adult-born dentate granule cells lacking Trim9 similarly exhibited excessive dendritic arborization and mislocalization of cell bodies in vivo. These cellular defects were associated with severe deficits in spatial learning and memory. PMID:27147649

  3. Autophagy meets fused in sarcoma-positive stress granules.

    PubMed

    Matus, Soledad; Bosco, Daryl A; Hetz, Claudio

    2014-12-01

    Mutations in fused in sarcoma and/or translocated in liposarcoma (FUS, TLS or FUS) are linked to familial cases of amyotrophic lateral sclerosis (ALS). Mutant FUS selectively accumulates into discrete cytosolic structures known as stress granules under various stress conditions. In addition, mutant FUS expression can alter the dynamics and morphology of stress granules. Although the link between mutant FUS and stress granules is well established, the mechanisms modulating stress granule formation and disassembly in the context of ALS are poorly understood. In this issue of Neurobiology of Aging, Ryu et al. uncover the impact of autophagy on the potential toxicity of mutant FUS-positive stress granules. The authors provide evidence indicating that enhanced autophagy activity reduces the number of stress granules, which in the case of cells containing mutant FUS-positive stress granules, is neuroprotective. Overall, this study identifies an intersection between the proteostasis network and alterations in RNA metabolism in ALS through the dynamic assembly and disassembly of stress granules. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Neurite outgrowth of murine cerebellar granule cells can be enhanced by aniracetam with or without alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA).

    PubMed

    Fushiki, S; Matsumoto, K; Nagata, A

    1995-10-27

    To assess the neurotrophic effects of a nootropic drug, aniracetam, we studied neurite extension of mouse cerebellar granule cells in culture with low or with high K+ under different combinations of drugs and then immunohistochemically stained the cells with an antibody against L1, a neural cell adhesion molecule on cerebellar granule cells. Quantitative analyses using parameters of the total neurite length, maximal neurite length and number of branches disclosed that aniracetam, in the presence of high K+ and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), significantly enhanced neurite extension of cultured granule neurons. Aniracetam alone also stimulated neurite extension of cerebellar granule cells at a longer period of culture with low K+ showing a bell-shaped dose response curve with maximal effects at 10 microM. Aniracetam may influence remodeling of the neural network after injury.

  5. Generation of Functional Blood Vessels from a Single c-kit+ Adult Vascular Endothelial Stem Cell

    PubMed Central

    Fang, Shentong; Wei, Jing; Pentinmikko, Nalle; Leinonen, Hannele; Salven, Petri

    2012-01-01

    In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin−CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth. PMID:23091420

  6. Purification of a factor from the granules of a rat natural killer cell line (RNK) that reduces tumor cell growth and changes tumor morphology. Molecular identity with a granule serine protease (RNKP-1).

    PubMed

    Sayers, T J; Wiltrout, T A; Sowder, R; Munger, W L; Smyth, M J; Henderson, L E

    1992-01-01

    We have purified a protein from the granules of the rat NK leukemia cell line (RNK) that is cytostatic to a variety of tumor cells. This protein shows no species specificity because certain tumor cell lines of mouse, rat, and human origin were equally sensitive to its growth inhibitory effects. Treatment of sensitive cells resulted in a rounding of the cells followed by homotypic aggregation into large aggregates. The granule protein was distinct from cytolysin, Na-Cbz-Lys-thiobenzylester-esterase, or leukolexin. It had a molecular mass of 29 to 31 kDa, bound strongly to heparin, was inactivated by heating at 70 degrees C for 5 min or reduction, but was stable to trypsin treatment. By using molecular sieve chromatography, heparin agarose chromatography, and reverse phase HPLC, this protein was purified to homogeneity. The first 33 amino acids of the N-terminal amino acid sequence showed complete identity to the sequence predicted from a rat serine protease gene recently cloned and designated RNKP-1. Therefore we have purified a novel serine protease and demonstrated that it has effects on the growth and morphology of certain tumor cells. Other serine proteases that were structurally related and have substantial homology with RNKP-1 at the amino acid level showed neither growth inhibitory properties nor affected the morphology of the tumor target cells we used.

  7. Detection of component segregation in granules manufactured by high shear granulation with over-granulation conditions using near-infrared chemical imaging.

    PubMed

    Koide, Tatsuo; Nagato, Takuya; Kanou, Yoshiyuki; Matsui, Kou; Natsuyama, Susumu; Kawanishi, Toru; Hiyama, Yukio

    2013-01-30

    The objective of this study was to evaluate the high shear granulation process using near-infrared (NIR) chemical imaging technique and to make the findings available for pharmaceutical development. We prepared granules and tablets made under appropriate- and over-granulation conditions with high shear granulation and observed these granules and tablets using NIR chemical imaging system. We found an interesting phenomenon: lactose agglomeration and segregation of ingredients occurred in experimental tablets when over-granulation conditions, including greater impeller rotation speeds and longer granulation times, were employed. Granules prepared using over-granulation conditions were larger and had progressed to the consolidation stage; segregation between ethenzamide and lactose occurred within larger granules. The segregation observed here is not detectable using conventional analytical technologies such as high pressure liquid chromatography (HPLC) because the content of the granules remained uniform despite the segregation. Therefore, granule visualization using NIR chemical imaging is an effective method for investigating and evaluating the granulation process. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Iron and cell death in Parkinson's disease: a nuclear microscopic study into iron-rich granules in the parkinsonian substantia nigra of primate models

    NASA Astrophysics Data System (ADS)

    Thong, P. S. P.; Watt, F.; Ponraj, D.; Leong, S. K.; He, Y.; Lee, T. K. Y.

    1999-10-01

    Parkinson's disease is a degenerative brain disease characterised by a loss of cells in the substantia nigra (SN) region of the brain and accompanying biochemical changes such as inhibition of mitochondrial function, increased iron concentrations and decreased glutathione levels in the parkinsonian SN. Though the aetiology of the disease is still unknown, the observed biochemical changes point to the involvement of oxidative stress. In particular, iron is suspected to play a role by promoting free radical production, leading to oxidative stress and cell death. The increase in iron in the parkinsonian SN has been confirmed by several research groups, both in human post-mortem brains and in brain tissue from parkinsonian animal models. However, the question remains as to whether the observed increase in iron is a cause or a consequence of the SN cell death process. Our previous study using unilaterally 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-lesioned monkeys in a time sequence experiment has shown that the increase in bulk iron concentrations follow rather than precede dopaminergic cell death. However, changes in the localised iron concentrations, which may play a more direct role in SN cell death, may not be reflected at the bulk level. Indeed, we have observed iron-rich granules in parkinsonian SNs. From this time sequence study into the iron content of iron-rich granules in the SNs of an untreated control and unilaterally MPTP-lesioned parkinsonian models, we present the following observations: (1) Iron-rich granules are found in both control and parkinsonian SNs and are variable in size and iron content in any one model. (2) These iron-rich granules may be associated with neuromelanin granules found in the SN and are known to accumulate transition metal ions such as iron. (3) The early onset of bulk SN cell loss (35%) was accompanied by a significant elevation of iron in granules found in the MPTP-injected SN compared to the contra-lateral SN. This

  9. Solar granulation and statistical crystallography: A modeling approach using size-shape relations

    NASA Technical Reports Server (NTRS)

    Noever, D. A.

    1994-01-01

    The irregular polygonal pattern of solar granulation is analyzed for size-shape relations using statistical crystallography. In contrast to previous work which has assumed perfectly hexagonal patterns for granulation, more realistic accounting of cell (granule) shapes reveals a broader basis for quantitative analysis. Several features emerge as noteworthy: (1) a linear correlation between number of cell-sides and neighboring shapes (called Aboav-Weaire's law); (2) a linear correlation between both average cell area and perimeter and the number of cell-sides (called Lewis's law and a perimeter law, respectively) and (3) a linear correlation between cell area and squared perimeter (called convolution index). This statistical picture of granulation is consistent with a finding of no correlation in cell shapes beyond nearest neighbors. A comparative calculation between existing model predictions taken from luminosity data and the present analysis shows substantial agreements for cell-size distributions. A model for understanding grain lifetimes is proposed which links convective times to cell shape using crystallographic results.

  10. A New and Fast Technique to Generate Offspring after Germ Cells Transplantation in Adult Fish: The Nile Tilapia (Oreochromis niloticus) Model

    PubMed Central

    Lacerda, Samyra M. S. N.; Batlouni, Sergio R.; Costa, Guilherme M. J.; Segatelli, Tânia M.; Quirino, Bruno R.; Queiroz, Bruno M.; Kalapothakis, Evanguedes; França, Luiz R.

    2010-01-01

    Background Germ cell transplantation results in fertile recipients and is the only available approach to functionally investigate the spermatogonial stem cell biology in mammals and probably in other vertebrates. In the current study, we describe a novel non-surgical methodology for efficient spermatogonial transplantation into the testes of adult tilapia (O. niloticus), in which endogenous spermatogenesis had been depleted with the cytostatic drug busulfan. Methodology/Principal Findings Using two different tilapia strains, the production of fertile spermatozoa with donor characteristics was demonstrated in adult recipient, which also sired progeny with the donor genotype. Also, after cryopreservation tilapia spermatogonial cells were able to differentiate to spermatozoa in the testes of recipient fishes. These findings indicate that injecting germ cells directly into adult testis facilitates and enable fast generation of donor spermatogenesis and offspring compared to previously described methods. Conclusion Therefore, a new suitable methodology for biotechnological investigations in aquaculture was established, with a high potential to improve the production of commercially valuable fish, generate transgenic animals and preserve endangered fish species. PMID:20505774

  11. Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids.

    PubMed

    Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

    2016-05-25

    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.

  12. 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

  13. Exocytosis of Neutrophil Granule Subsets and Activation of Prolyl Isomerase 1 are required for Respiratory Burst Priming

    PubMed Central

    McLeish, Kenneth R.; Uriarte, Silvia M.; Tandon, Shweta; Creed, Timothy M.; Le, Junyi; Ward, Richard A.

    2013-01-01

    This study tested the hypothesis that priming the neutrophil respiratory burst requires both granule exocytosis and activation of the prolyl isomerase, Pin1. Fusion proteins containing the TAT cell permeability sequence and either the SNARE domain of syntaxin-4 or the N-terminal SNARE domain of SNAP-23 were used to examine the role of granule subsets in TNF-mediated respiratory burst priming using human neutrophils. Concentration-inhibition curves for exocytosis of individual granule subsets and for priming of fMLF-stimulated superoxide release and phagocytosis-stimulated H2O2 production were generated. Maximal inhibition of priming ranged from 72% to 88%. Linear regression lines for inhibition of priming versus inhibition of exocytosis did not differ from the line of identity for secretory vesicles and gelatinase granules, while the slopes or the y-intercepts were different from the line of identity for specific and azurophilic granules. Inhibition of Pin1 reduced priming by 56%, while exocytosis of secretory vesicles and specific granules was not affected. These findings indicate that exocytosis of secretory vesicles and gelatinase granules and activation of Pin1 are independent events required for TNF-mediated priming of neutrophil respiratory burst. PMID:23363774

  14. A Genomewide RNAi Screen for Genes That Affect the Stability, Distribution and Function of P Granules in Caenorhabditis elegans

    PubMed Central

    Updike, Dustin L.; Strome, Susan

    2009-01-01

    P granules are non-membrane-bound organelles found in the germ-line cytoplasm throughout Caenorhabditis elegans development. Like their “germ granule” counterparts in other animals, P granules are thought to act as determinants of the identity and special properties of germ cells, properties that include the unique ability to give rise to all tissues of future generations of an organism. Therefore, understanding how P granules work is critical to understanding how cellular immortality and totipotency are retained, gained, and lost. Here we report on a genomewide RNAi screen in C. elegans, which identified 173 genes that affect the stability, localization, and function of P granules. Many of these genes fall into specific classes with shared P-granule phenotypes, allowing us to better understand how cellular processes such as protein degradation, translation, splicing, nuclear transport, and mRNA homeostasis converge on P-granule assembly and function. One of the more striking phenotypes is caused by the depletion of CSR-1, an Argonaute associated with an endogenous siRNA pathway that functions in the germ line. We show that CSR-1 and two other endo-siRNA pathway members, the RNA-dependent RNA polymerase EGO-1 and the helicase DRH-3, act to antagonize RNA and P-granule accumulation in the germ line. Our findings strengthen the emerging view that germ granules are involved in numerous aspects of RNA metabolism, including an endo-siRNA pathway in germ cells. PMID:19805813

  15. Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles

    PubMed Central

    Woo, Sang Su; James, Declan J.; Martin, Thomas F. J.

    2017-01-01

    Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell–like RBL-2H3 cells provide direct evidence that Munc13–4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. PMID:28100639

  16. Identification of SNAREs that mediate zymogen granule exocytosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pickett, James A.; Campos-Toimil, Manuel; Thomas, Paul

    2007-08-03

    A secretagogue-stimulated pancreatic acinar cell releases digestive enzymes from its apical pole. We attempted to identify the SNAREs involved in zymogen granule exocytosis. Antibodies against syntaxins 2 and 3, SNAP-23 and VAMP 8, and the corresponding recombinant SNAREs, inhibited amylase secretion from streptolysin O-permeabilised acini; other anti-SNARE antibodies and SNAREs had no effect. Botulinum neurotoxin C, which cleaved syntaxin 2 and (to a lesser extent) syntaxin 3, but not syntaxins 4, 7 or 8, also inhibited exocytosis. We propose that syntaxin 2, SNAP-23 and VAMP 8 mediate primary granule-plasma membrane fusion. Syntaxin 3 may be involved in secondary granule-granule fusion.

  17. Physicochemical characteristics of insulin secretion granules

    PubMed Central

    Coore, H. G.; Hellman, B.; Pihl, E.; Täljedal, I.-B.

    1969-01-01

    β-Granules were prepared from micro-dissected pancreatic islets of obese–hyperglycaemic mice. This fraction contained 60% of the insulin, 30% of the cytochrome oxidase, 16% of the acid phosphatase activity and 20% of the protein present in whole islets. The isolated granules retained a heavy metal during fractionation. Optimum conditions for granule stability were low ionic strength and pH6, the granules being unexpectedly fragile at pH7·4. The stability of the granules was unaffected by sucrose in the concentration range 50–320mm, but 1% (w/v) sodium deoxycholate released all insulin. A solubilizing effect was also noted with ATP and citrate. Spinning through 1·6m-sucrose yielded a further purification in relation to mitochondria and acid-phosphatase-carrying particles but virtually no purification in relation to protein. Electron microscopy revealed that the major contaminants were rough-surfaced vesicles and membranes. A separation of granules from acid phosphatase was achieved by phase distribution in polyethylene glycol and dextran. The location of the enzyme to the interphase was so pronounced in systems buffered with lithium phosphate that the technique may be used for future purification of acid-phosphatase-carrying particles from the β-cells. ImagesPLATE 1 PMID:4887194

  18. Convergent evolution of germ granule nucleators: A hypothesis.

    PubMed

    Kulkarni, Arpita; Extavour, Cassandra G

    2017-10-01

    Germ cells have been considered "the ultimate stem cell" because they alone, during normal development of sexually reproducing organisms, are able to give rise to all organismal cell types. Morphological descriptions of a specialized cytoplasm termed 'germ plasm' and associated electron dense ribonucleoprotein (RNP) structures called 'germ granules' within germ cells date back as early as the 1800s. Both germ plasm and germ granules are implicated in germ line specification across metazoans. However, at a molecular level, little is currently understood about the molecular mechanisms that assemble these entities in germ cells. The discovery that in some animals, the gene products of a small number of lineage-specific genes initiate the assembly (also termed nucleation) of germ granules and/or germ plasm is the first step towards facilitating a better understanding of these complex biological processes. Here, we draw on research spanning over 100years that supports the hypothesis that these nucleator genes may have evolved convergently, allowing them to perform analogous roles across animal lineages. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Adult DRG Stem/Progenitor Cells Generate Pericytes in the Presence of Central Nervous System (CNS) Developmental Cues, and Schwann Cells in Response to CNS Demyelination.

    PubMed

    Vidal, Marie; Maniglier, Madlyne; Deboux, Cyrille; Bachelin, Corinne; Zujovic, Violetta; Baron-Van Evercooren, Anne

    2015-06-01

    It has been proposed that the adult dorsal root ganglia (DRG) harbor neural stem/progenitor cells (NPCs) derived from the neural crest. However, the thorough characterization of their stemness and differentiation plasticity was not addressed. In this study, we investigated adult DRG-NPC stem cell properties overtime, and their fate when ectopically grafted in the central nervous system. We compared them in vitro and in vivo to the well-characterized adult spinal cord-NPCs derived from the same donors. Using micro-dissection and neurosphere cultures, we demonstrate that adult DRG-NPCs have quasi unlimited self-expansion capacities without compromising their tissue specific molecular signature. Moreover, they differentiate into multiple peripheral lineages in vitro. After transplantation, adult DRG-NPCs generate pericytes in the developing forebrain but remyelinating Schwann cells in response to spinal cord demyelination. In addition, we show that axonal and endothelial/astrocytic factors as well astrocytes regulate the fate of adult DRG-NPCs in culture. Although the adult DRG-NPC multipotency is restricted to the neural crest lineage, their dual responsiveness to developmental and lesion cues highlights their impressive adaptive and repair potentials making them valuable targets for regenerative medicine. © 2015 AlphaMed Press.

  20. Studying melanin and lipofuscin in RPE cell culture models

    PubMed Central

    Boulton, Michael E

    2014-01-01

    The retinal pigment epithelium contains three major types of pigment granules; melanosomes, lipofuscin and melanolipofuscin. Melanosomes in the retinal pigment epithelium (RPE) are formed during embryogenesis and mature during early postnatal life while lipofuscin and melanolipofuscin granules accumulate as a function of age. The difficulty in studying the formation and consequences of melanosomes and lipofuscin granules in RPE cell culture is compounded by the fact that these pigment granules do not normally occur in established RPE cell lines and pigment granules are rapidly lost in adult human primary culture. This review will consider options available for overcoming these limitations and permitting the study of melanosomes and lipofuscin in cell culture and will briefly evaluate the advantages and disadvantages of the different protocols. PMID:25152361

  1. Drosophila germ granules are structured and contain homotypic mRNA clusters

    PubMed Central

    Trcek, Tatjana; Grosch, Markus; York, Andrew; Shroff, Hari; Lionnet, Timothée; Lehmann, Ruth

    2015-01-01

    Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. Here we show, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into ‘homotypic clusters' that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules. PMID:26242323

  2. Regulation by commensal bacteria of neurogenesis in the subventricular zone of adult mouse brain.

    PubMed

    Sawada, Naoki; Kotani, Takenori; Konno, Tasuku; Setiawan, Jajar; Nishigaito, Yuka; Saito, Yasuyuki; Murata, Yoji; Nibu, Ken-Ichi; Matozaki, Takashi

    2018-04-15

    In the mouse olfactory bulb (OB), interneurons such as granule cells and periglomerular cells are continuously replaced by adult-born neurons, which are generated in the subventricular zone (SVZ) of the brain. We have now investigated the role of commensal bacteria in regulation of such neuronal cell turnover in the adult mouse brain. Administration of mixture of antibiotics to specific pathogen-free (SPF) mice markedly attenuated the incorporation of bromodeoxyuridine (BrdU) into the SVZ cells. The treatment with antibiotics also reduced newly generated BrdU-positive neurons in the mouse OB. In addition, the incorporation of BrdU into the SVZ cells of germ-free (GF) mice was markedly reduced compared to that apparent for SPF mice. In contrast, the reduced incorporation of BrdU into the SVZ cells of GF mice was recovered by their co-housing with SPF mice, suggesting that commensal bacteria promote the incorporation of BrdU into the SVZ cells. Finally, we found that administration of ampicillin markedly attenuated the incorporation of BrdU into the SVZ cells of SPF mice. Our results thus suggest that ampicillin-sensitive commensal bacteria regulate the neurogenesis in the SVZ of adult mouse brain. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Morphometry of Hilar Ectopic Granule Cells in the Rat

    PubMed Central

    Pierce, Joseph P.; McCloskey, Daniel P.; Scharfman, Helen E.

    2014-01-01

    Granule cell (GC) neurogenesis in the dentate gyrus (DG) does not always proceed normally. After severe seizures (e.g., status epilepticus [SE]) and some other conditions, newborn GCs appear in the hilus. Hilar ectopic GCs (EGCs) can potentially provide insight into the effects of abnormal location and seizures on GC development. Additionally, hilar EGCs that develop after SE may contribute to epileptogenesis and cognitive impairments that follow SE. Thus, it is critical to understand how EGCs differ from normal GCs. Relatively little morphometric information is available on EGCs, especially those restricted to the hilus. This study quantitatively analyzed the structural morphology of hilar EGCs from adult male rats several months after pilocarpineinduced SE, when they are considered to have chronic epilepsy. Hilar EGCs were physiologically identified in slices, intracellularly labeled, processed for light microscopic reconstruction, and compared to GC layer GCs, from both the same post-SE tissue and the NeuroMorpho database (normal GCs). Consistently, hilar EGC and GC layer GCs had similar dendritic lengths and field sizes, and identifiable apical dendrites. However, hilar EGC dendrites were topologically more complex, with more branch points and tortuous dendritic paths. Three-dimensional analysis revealed that, remarkably, hilar EGC dendrites often extended along the longitudinal DG axis, suggesting increased capacity for septotemporal integration. Axonal reconstruction demonstrated that hilar EGCs contributed to mossy fiber sprouting. This combination of preserved and aberrant morphological features, potentially supporting convergent afferent input to EGCs and broad, divergent efferent output, could help explain why the hilar EGC population could impair DG function. PMID:21344409

  4. Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

    PubMed

    Mellor, J R; Wisden, W; Randall, A D

    2000-07-10

    Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden

  5. GABA-CREB signalling regulates maturation and survival of newly generated neurons in the adult hippocampus

    PubMed Central

    Jagasia, Ravi; Steib, Kathrin; Englberger, Elisabeth; Herold, Sabine; Faus-Kessler, Theresa; Saxe, Michael; Gage, Fred H.; Song, Hongjun; Lie, D. Chichung

    2009-01-01

    Survival and integration of new neurons in the hippocampal circuit are rate-limiting steps in adult hippocampal neurogenesis. Neuronal network activity is a major regulator of these processes, yet little is known about the respective downstream signalling pathways. Here, we investigate the role of CREB signalling in adult hippocampal neurogenesis. CREB is activated in new granule neurons during a distinct developmental period. Loss of CREB function in a cell-autonomous fashion impairs dendritic development, decreases the expression of the neurogenic transcription factor NeuroD and of the neuronal microtubule associated protein, DCX, and compromises the survival of newborn neurons. In addition, GABA-mediated excitation regulates CREB activation at early developmental stages. Importantly, developmental defects following loss of GABA-mediated excitation can be compensated by enhanced CREB signalling. These results indicate that CREB signalling is a central pathway in adult hippocampal neurogenesis, regulating the development and survival of new hippocampal neurons downstream of GABA-mediated excitation. PMID:19553437

  6. Differential Regulation of Cell Proliferation and Apoptosis by Melatonin Receptor Subtype-Signaling in the Adult Murine Brain.

    PubMed

    Fredrich, Michaela; Christ, Elmar; Korf, Horst-Werner

    2018-06-27


    Background/Aims: Zeitgeber time (ZT)-dependent changes in cell proliferation and apoptosis are regulated by melatonin receptor (MT)-mediated signaling in the adult hippocampus and hypothalamic-hypophyseal system. There are two G-protein-coupled MT-subtypes, MT1 and MT2. Therefore, the present study examined which MT-subtype is required for regulation of ZT-dependent changes in cell proliferation and/or apoptosis in the adult murine brain and pituitary. Adult melatonin-proficient (C3H) mice with targeted deletion of MT1 (MT1 KO) or MT2 (MT2 KO) were adapted to a 12-hour light, 12-hour dark photoperiod and sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 or activated caspase-3 served to quantify proliferating and apoptotic cells in the hippocampal subgranular zone (SGZ) and granule cell layer, the hypothalamic median eminence (ME), and the hypophyseal pars tuberalis. ZT-dependent changes in cell proliferation were found exclusively in the SGZ and ME of MT1 KO mice, while apoptosis showed no ZT-dependent changes in the regions analyzed, neither in MT1 nor in MT2 KO mice. Comparison with our previous studies in C3H mice with functional MTs and MT1/2 KO mice revealed that MT2-mediated signaling is required and sufficient for ZT-dependent changes in cell proliferation in the SGZ and ME, while ZT-dependent changes in apoptosis require signaling from both MT-subtypes. Our results indicate that generation and timing of ZT-dependent changes in cell proliferation and apoptosis by melatonin require different MT-subtype-constellations and emphasize the importance to shed light on the specific function of each receptor-subtype in different tissues and physiological conditions.
    . ©2018S. Karger AG, Basel.

  7. Retrograde monosynaptic tracing reveals the temporal evolution of inputs onto new neurons in the adult dentate gyrus and olfactory bulb

    PubMed Central

    Deshpande, Aditi; Bergami, Matteo; Ghanem, Alexander; Conzelmann, Karl-Klaus; Lepier, Alexandra; Götz, Magdalena; Berninger, Benedikt

    2013-01-01

    Identifying the connectome of adult-generated neurons is essential for understanding how the preexisting circuitry is refined by neurogenesis. Changes in the pattern of connectivity are likely to control the differentiation process of newly generated neurons and exert an important influence on their unique capacity to contribute to information processing. Using a monosynaptic rabies virus-based tracing technique, we studied the evolving presynaptic connectivity of adult-generated neurons in the dentate gyrus (DG) of the hippocampus and olfactory bulb (OB) during the first weeks of their life. In both neurogenic zones, adult-generated neurons first receive local connections from multiple types of GABAergic interneurons before long-range projections become established, such as those originating from cortical areas. Interestingly, despite fundamental similarities in the overall pattern of evolution of presynaptic connectivity, there were notable differences with regard to the development of cortical projections: although DG granule neuron input originating from the entorhinal cortex could be traced starting only from 3 to 5 wk on, newly generated neurons in the OB received input from the anterior olfactory nucleus and piriform cortex already by the second week. This early glutamatergic input onto newly generated interneurons in the OB was matched in time by the equally early innervations of DG granule neurons by glutamatergic mossy cells. The development of connectivity revealed by our study may suggest common principles for incorporating newly generated neurons into a preexisting circuit. PMID:23487772

  8. Subcellular TSC22D4 localization in cerebellum granule neurons of the mouse depends on development and differentiation.

    PubMed

    Canterini, Sonia; Bosco, Adriana; Carletti, Valentina; Fuso, Andrea; Curci, Armando; Mangia, Franco; Fiorenza, Maria Teresa

    2012-03-01

    We previously demonstrated that TSC22D4, a protein encoded by the TGF-β1-activated gene Tsc22d4 (Thg-1pit) and highly expressed in postnatal and adult mouse cerebellum with multiple post-translationally modified protein forms, moves to nucleus when in vitro differentiated cerebellum granule neurons (CGNs) are committed to apoptosis by hyperpolarizing KCl concentrations in the culture medium. We have now studied TSC22D4 cytoplasmic/nuclear localization in CGNs and Purkinje cells: (1) during CGN differentiation/maturation in vivo, (2) during CGN differentiation in vitro, and (3) by in vitro culturing ex vivo cerebellum slices under conditions favoring/inhibiting CGN/Purkinje cell differentiation. We show that TSC22D4 displays both nuclear and cytoplasmic localizations in undifferentiated, early postnatal cerebellum CGNs, irrespectively of CGN proliferation/migration from external to internal granule cell layer, and that it specifically accumulates in the somatodendritic and synaptic compartments when CGNs mature, as indicated by TSC22D4 abundance at the level of adult cerebellum glomeruli and apparent lack in CGN nuclei. These features were also observed in cerebellum slices cultured in vitro under conditions favoring/inhibiting CGN/Purkinje cell differentiation. In vitro TSC22D4 silencing with siRNAs blocked CGN differentiation and inhibited neurite elongation in N1E-115 neuroblastoma cells, pinpointing the relevance of this protein to CGN differentiation.

  9. Regulation of platelet granule exocytosis by S-nitrosylation

    PubMed Central

    Morrell, Craig N.; Matsushita, Kenji; Chiles, Kelly; Scharpf, Robert B.; Yamakuchi, Munekazu; Mason, Rebecca J. A.; Bergmeier, Wolfgang; Mankowski, Joseph L.; Baldwin, William M.; Faraday, Nauder; Lowenstein, Charles J.

    2005-01-01

    Nitric oxide (NO) regulates platelet activation by cGMP-dependent mechanisms and by mechanisms that are not completely defined. Platelet activation includes exocytosis of platelet granules, releasing mediators that regulate interactions between platelets, leukocytes, and endothelial cells. Exocytosis is mediated in part by N-ethylmaleimide-sensitive factor (NSF), an ATPase that disassembles complexes of soluble NSF attachment protein receptors. We now demonstrate that NO inhibits exocytosis of dense granules, lysosomal granules, and α-granules from human platelets by S-nitrosylation of NSF. Platelets lacking endothelial NO synthase show increased rolling on venules, increased thrombosis in arterioles, and increased exocytosis in vivo. Regulation of exocytosis is thus a mechanism by which NO regulates thrombosis. PMID:15738422

  10. Vascular endothelial growth factor receptor 1 (VEGFR1) tyrosine kinase signaling facilitates granulation tissue formation with recruitment of VEGFR1+ cells from bone marrow.

    PubMed

    Park, Keiichi; Amano, Hideki; Ito, Yoshiya; Mastui, Yoshio; Kamata, Mariko; Yamazaki, Yasuharu; Takeda, Akira; Shibuya, Masabumi; Majima, Masataka

    2018-06-01

    Vascular endothelial growth factor (VEGF)-A facilitates wound healing. VEGF-A binds to VEGF receptor 1 (VEGFR1) and VEGFR2 and induces wound healing through the receptor's tyrosine kinase (TK) domain. During blood flow recovery and lung regeneration, expression of VEGFR1 is elevated. However, the precise mechanism of wound healing, especially granulation formation on VEGFR1, is not well understood. We hypothesized that VEGFR1-TK signaling induces wound healing by promoting granulation tissue formation. A surgical sponge implantation model was made by implanting a sponge disk into dorsal subcutaneous tissue of mice. Granulation formation was estimated from the weight of the sponge and the granulation area from the immunohistochemical analysis of collagen I. The expression of fibroblast markers was estimated from the expression of transforming growth factor-beta (TGF-β) and cellular fibroblast growth factor-2 (FGF-2) using real-time PCR (polymerase chain reaction) and from the immunohistochemical analysis of S100A4. VEGFR1 TK knockout (TK -/- ) mice exhibited suppressed granulation tissue formation compared to that in wild-type (WT) mice. Expression of FGF-2, TGF-β, and VEGF-A was significantly suppressed in VEGFR1 TK -/- mice, and the accumulation of VEGFR1 + cells in granulation tissue was reduced in VEGFR1 TK -/- mice compared to that in WT mice. The numbers of VEGFR1 + cells and S100A4 + cells derived from bone marrow (BM) were higher in WT mice transplanted with green fluorescent protein (GFP) transgenic WT BM than in VEGFR1 TK -/- mice transplanted with GFP transgenic VEGFR1 TK -/- BM. These results indicated that VEGFR1-TK signaling induced the accumulation of BM-derived VEGFR1 + cells expressing F4/80 and S100A4 and contributed to granulation formation around the surgically implanted sponge area in a mouse model.

  11. PtdIns(4,5)P2 is not required for secretory granule docking.

    PubMed

    Omar-Hmeadi, Muhmmad; Gandasi, Nikhil R; Barg, Sebastian

    2018-06-01

    Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P 2 ) by recruitment of a 5'-phosphatase strongly inhibited Ca 2+ -dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by α-toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P 2 accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Trim9 Deletion Alters the Morphogenesis of Developing and Adult-Born Hippocampal Neurons and Impairs Spatial Learning and Memory.

    PubMed

    Winkle, Cortney C; Olsen, Reid H J; Kim, Hyojin; Moy, Sheryl S; Song, Juan; Gupton, Stephanie L

    2016-05-04

    During hippocampal development, newly born neurons migrate to appropriate destinations, extend axons, and ramify dendritic arbors to establish functional circuitry. These developmental stages are recapitulated in the dentate gyrus of the adult hippocampus, where neurons are continuously generated and subsequently incorporate into existing, local circuitry. Here we demonstrate that the E3 ubiquitin ligase TRIM9 regulates these developmental stages in embryonic and adult-born mouse hippocampal neurons in vitro and in vivo Embryonic hippocampal and adult-born dentate granule neurons lacking Trim9 exhibit several morphological defects, including excessive dendritic arborization. Although gross anatomy of the hippocampus was not detectably altered by Trim9 deletion, a significant number of Trim9(-/-) adult-born dentate neurons localized inappropriately. These morphological and localization defects of hippocampal neurons in Trim9(-/-) mice were associated with extreme deficits in spatial learning and memory, suggesting that TRIM9-directed neuronal morphogenesis may be involved in hippocampal-dependent behaviors. Appropriate generation and incorporation of adult-born neurons in the dentate gyrus are critical for spatial learning and memory and other hippocampal functions. Here we identify the brain-enriched E3 ubiquitin ligase TRIM9 as a novel regulator of embryonic and adult hippocampal neuron shape acquisition and hippocampal-dependent behaviors. Genetic deletion of Trim9 elevated dendritic arborization of hippocampal neurons in vitro and in vivo Adult-born dentate granule cells lacking Trim9 similarly exhibited excessive dendritic arborization and mislocalization of cell bodies in vivo These cellular defects were associated with severe deficits in spatial learning and memory. Copyright © 2016 the authors 0270-6474/16/364940-19$15.00/0.

  13. Clinical grade adult stem cell banking

    PubMed Central

    Thirumala, Sreedhar; Goebel, W Scott

    2009-01-01

    There has been a great deal of scientific interest recently generated by the potential therapeutic applications of adult stem cells in human care but there are several challenges regarding quality and safety in clinical applications and a number of these challenges relate to the processing and banking of these cells ex-vivo. As the number of clinical trials and the variety of adult cells used in regenerative therapy increases, safety remains a primary concern. This has inspired many nations to formulate guidelines and standards for the quality of stem cell collection, processing, testing, banking, packaging and distribution. Clinically applicable cryopreservation and banking of adult stem cells offers unique opportunities to advance the potential uses and widespread implementation of these cells in clinical applications. Most current cryopreservation protocols include animal serum proteins and potentially toxic cryoprotectant additives (CPAs) that prevent direct use of these cells in human therapeutic applications. Long term cryopreservation of adult stem cells under good manufacturing conditions using animal product free solutions is critical to the widespread clinical implementation of ex-vivo adult stem cell therapies. Furthermore, to avoid any potential cryoprotectant related complications, reduced CPA concentrations and efficient post-thaw washing to remove CPA are also desirable. The present review focuses on the current strategies and important aspects of adult stem cell banking for clinical applications. These include current good manufacturing practices (cGMPs), animal protein free freezing solutions, cryoprotectants, freezing & thawing protocols, viability assays, packaging and distribution. The importance and benefits of banking clinical grade adult stem cells are also discussed. PMID:20046678

  14. Generation of immortal cell lines from the adult pituitary: role of cAMP on differentiation of SOX2-expressing progenitor cells to mature gonadotropes.

    PubMed

    Kim, Ginah L; Wang, Xiaomei; Chalmers, Jennifer A; Thompson, David R; Dhillon, Sandeep S; Koletar, Margaret M; Belsham, Denise D

    2011-01-01

    The pituitary is a complex endocrine tissue composed of a number of unique cell types distinguished by the expression and secretion of specific hormones, which in turn control critical components of overall physiology. The basic function of these cells is understood; however, the molecular events involved in their hormonal regulation are not yet fully defined. While previously established cell lines have provided much insight into these regulatory mechanisms, the availability of representative cell lines from each cell lineage is limited, and currently none are derived from adult pituitary. We have therefore used retroviral transfer of SV40 T-antigen to mass immortalize primary pituitary cell culture from an adult mouse. We have generated 19 mixed cell cultures that contain cells from pituitary cell lineages, as determined by RT-PCR analysis and immunocytochemistry for specific hormones. Some lines expressed markers associated with multipotent adult progenitor cells or transit-amplifying cells, including SOX2, nestin, S100, and SOX9. The progenitor lines were exposed to an adenylate cyclase activator, forskolin, over 7 days and were induced to differentiate to a more mature gonadotrope cell, expressing significant levels of α-subunit, LHβ, and FSHβ mRNAs. Additionally, clonal populations of differentiated gonadotropes were exposed to 30 nM gonadotropin-releasing hormone and responded appropriately with a significant increase in α-subunit and LHβ transcription. Further, exposure of the lines to a pulse paradigm of GnRH, in combination with 17β-estradiol and dexamethasone, significantly increased GnRH receptor mRNA levels. This array of adult-derived pituitary cell models will be valuable for both studies of progenitor cell characteristics and modulation, and the molecular analysis of individual pituitary cell lineages.

  15. High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions.

    PubMed

    Rice, W G; Kinkade, J M; Parmley, R T

    1986-08-01

    Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA

  16. The effect of the chopper on granules from wet high-shear granulation using a PMA-1 granulator.

    PubMed

    Briens, Lauren; Logan, Ryan

    2011-12-01

    Chopper presence and then chopper speed was varied during wet high shear granulation of a placebo formulation using a PMA-1 granulator while also varying the impeller speed. The granules were extensively analyzed for differences due to the chopper. The effect of the chopper on the granules varied with impeller speed from no effect at a low impeller speed of 300 rpm to flow interruptions at an impeller speed of 700 rpm to minimal impact at very high impeller speeds as caking at the bowl perimeter obscured the effect of the chopper on the flow pattern. Differences in the granule flowability were minimal. However, it was concluded that the largest fraction of optimal granules would be obtained at an impeller speed of 700 rpm with the chopper at 1,000 rpm allowing balances between flow establishment, segregation, and centrifugal forces.

  17. Accumulation of PHA granules in Cupriavidus necator as seen by confocal fluorescence microscopy.

    PubMed

    Mravec, Filip; Obruca, Stanislav; Krzyzanek, Vladislav; Sedlacek, Petr; Hrubanova, Kamila; Samek, Ota; Kucera, Dan; Benesova, Pavla; Nebesarova, Jana

    2016-05-01

    Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting.

    PubMed

    Beuret, Nicole; Hasler, Franziska; Prescianotto-Baschong, Cristina; Birk, Julia; Rutishauser, Jonas; Spiess, Martin

    2017-01-26

    Aggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting. Here we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells. The same sequences - vasopressin and the glycopeptide - mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.

  19. Statistical properties of solar granulation from the SOUP instrument on Spacelab 2

    NASA Astrophysics Data System (ADS)

    Topka, K.; Title, A.; Tarbell, T.; Ferguson, S.; Shine, R.

    1988-11-01

    The Solar Optical Universal Polarimeter (SOUP) on Spacelab 2 collected movies of solar granulation completely free from atmospheric blurring, and are not degraded by pointint jitter (the pointing stability was 0.003 sec root mean square). The movies illustrate that the solar five minute oscillation has a major role in the appearance of solar granulation and that exploding granules are a common feature of the granule evolution. Using 3-D Fourier filtering techniques the oscillations were removed and it was demonstrated that the autocorrelation lifetime of granulation is a factor of two greater in magnetic field regions than in field-free quiet sun. Horizontal velocities were measured and flow patterns were observed on the scale of meso- and super granulation. In quiet regions the mean flow velocity is 370 m/s while in the magnetic regions it is about 125 m/s. It was also found that the root mean square (RMS) fluctuating horizonal velocity field is substantially greater in quiet sun than in strong magnetic field regions. By superimposing the location of exploding granules on the average flow maps it was found that they appear almost exclusively in the center of mesogranulation size flow cells. Because of the nonuniformity of the distribution of exploding granules, the evolution of the granulation pattern in mesogranule cell centers and boundaries differs fundamentally. It is clear from this study there is neither a typical granule nor a typical granule evolution.

  20. Statistical properties of solar granulation from the SOUP instrument on Spacelab 2

    NASA Technical Reports Server (NTRS)

    Topka, K.; Title, A.; Tarbell, T.; Ferguson, S.; Shine, R.

    1988-01-01

    The Solar Optical Universal Polarimeter (SOUP) on Spacelab 2 collected movies of solar granulation completely free from atmospheric blurring, and are not degraded by pointint jitter (the pointing stability was 0.003 sec root mean square). The movies illustrate that the solar five minute oscillation has a major role in the appearance of solar granulation and that exploding granules are a common feature of the granule evolution. Using 3-D Fourier filtering techniques the oscillations were removed and it was demonstrated that the autocorrelation lifetime of granulation is a factor of two greater in magnetic field regions than in field-free quiet sun. Horizontal velocities were measured and flow patterns were observed on the scale of meso- and super granulation. In quiet regions the mean flow velocity is 370 m/s while in the magnetic regions it is about 125 m/s. It was also found that the root mean square (RMS) fluctuating horizonal velocity field is substantially greater in quiet sun than in strong magnetic field regions. By superimposing the location of exploding granules on the average flow maps it was found that they appear almost exclusively in the center of mesogranulation size flow cells. Because of the nonuniformity of the distribution of exploding granules, the evolution of the granulation pattern in mesogranule cell centers and boundaries differs fundamentally. It is clear from this study there is neither a typical granule nor a typical granule evolution.

  1. Nucleotide and bivalent cation specificity of the insulin-granule proton translocase.

    PubMed Central

    Hutton, J C; Peshavaria, M

    1983-01-01

    1. The nucleotide and bivalent cation specificity of the proton translocase activity of insulin secretory granules was investigated by assessing the inhibitor-sensitive rates of nucleotide hydrolysis by these organelles in relation to their chemiosmotic properties. 2. The relative rates of nucleotide hydrolysis by freeze/thawed granule preparations were: Mg2+ATP (100%) greater than Mg2+GTP (55%) greater than Mg2+UTP (48%) greater than Mg2+ITP (44%) greater than Mg2+CTP (23%) greater than Mg2+TTP (20%), and by intact granules were: Mg2+ATP (100%) greater than Mg2+ITP (74%) greater than Mg2+GTP (60%) greater than Mg2+CTP (35%). Mg2+ATP, Mg2+GTP and Mg2+ITP hydrolyses were inhibited by tributyltin and stimulated, in intact granules, by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Mg2+CTP hydrolysis was not markedly affected by these compounds. Correspondingly, only Mg2+ATP, Mg2+GTP and Mg2+ITP produced large changes in the delta psi and delta mu H+ across the granule membrane. 3. The relative rates of maximal ATPase activity stimulated by bivalent cations in freeze/thawed granule preparations were: Mg2+ (100%) greater than Mn2+ (82%) greater than Ca2+ (40%) greater than Co2+ (36%) greater than Zn2+ (0%), and in intact granules were: Mg2+ (100%) greater than Mn2+ (85%) greater than Co2+ (61%) greater than Ca2+ (42%). Tributyltin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone affected Mg2+-, Mn2+- and Co2+-activated, but not Ca2+-activated, ATP hydrolysis. Correspondingly, only Mg2+, Mn2+ and Co2+ supported the generation of a delta psi and delta mu H+ across granule membranes in the presence of ATP. 4. The results were consistent with a single proton translocase that had its catalytic site exposed on the external face of the granule membrane. The indicated specificity (Mg2+ATP = Mn2+ATP greater than Co2+ATP greater than Mg2+GTP greater than Mg2+ITP) was similar to that of enzymes described in membrane fractions prepared from

  2. Dosage response mortality of Japanese beetle, masked chafer, and June beetle (Coleoptera: Scarabaeidae) adults when exposed to experimental and commercially available granules containing Metarhizium brunneum

    USDA-ARS?s Scientific Manuscript database

    Adult beetles of three different white grub species, Japanese beetle, Popillia japonica, June beetle, Phyllophaga spp., and masked chafer, Cyclocephala spp. were exposed to experimental and commercially available granules containing Metarhizium brunneum (Petch) strain F52, to determine susceptibilit...

  3. Rapid microwave fixation of rat mast cells. I. Localization of granule chymase with an ultrastructural postembedding immunogold technique.

    PubMed

    Login, G R; Galli, S J; Morgan, E; Arizono, N; Schwartz, L B; Dvorak, A M

    1987-11-01

    We defined the ultrastructural localization of chymase in rat peritoneal mast cells using standard aldehyde fixation and a newly described microwave fixation method (Login GR, Dvorak AM: Microwave energy fixation for electron microscopy. Am J Pathol 120: 230, 1985; Login GR, Stavinoha WB, Dvorak AM: Ultrafast microwave energy fixation for electron microscopy. J Histochem Cytochem 34:381, 1986) and postembedding immunogold labeling. Thin sections were exposed first to goat IgG anti-rat chymase and second to gold-conjugated rabbit Ig directed against goat IgG. By transmission electron microscopy, gold particles were localized to the matrix of cytoplasmic granules. Control sections treated with nonimmune sera did not exhibit labeling of mast cells. Thin sections treated simultaneously with purified rat mast cell chymase and anti-chymase antibody in competition studies, showed a marked reduction in granule staining. These findings demonstrate that a microwave fixation method can be used to rapidly fix cell suspensions for postembedding immunocytochemical studies.

  4. Differential Alterations in Excitatory and Inhibitory Networks Involving Dentate Granule Cells Following Chronic Treatment with Distinct Classes of NMDAR Antagonists in Hippocampal Slice Cultures

    DTIC Science & Technology

    2010-03-08

    1992; Jung and McNaughton, 1993); (2) low incidence of recurrent excitatory synapses between granule cells (Molnar and Nadler, 1999; Okazaki et al...neurons, dentate granule cells have a relatively more negative resting membrane potential and exhibit low-frequency firing (Staley et al., 1992; Jung ...inhibition plays a dual role in brain function and possibly seizure occurrence through balancing excitation and synchronizing neuronal firing. An

  5. Platelet granule release is associated with reactive oxygen species generation during platelet storage: A direct link between platelet pro-inflammatory and oxidation states.

    PubMed

    Ghasemzadeh, Mehran; Hosseini, Ehteramolsadat

    2017-08-01

    Upon platelet stimulation with agonists, reactive oxygen species (ROS) generation enhances platelet activation and granule release. Whether ROS generation during platelet storage could be directly correlated with the expression of proinflammatory molecules and granule release has been investigated in this study. PRP-platelet concentrates were subjected to flowcytometry analysis to assess the expression of platelet activation marker, P-selectin and CD40L during storage. Intracellular ROS generation was also detected in platelet by flowcytometry using dihydrorhodamine (DHR) 123. Through the dual staining, ROS production was analyzed in either P-selectin positive or negative populations. ROS formation in platelet population was significantly increased by either TRAP (a potent agonist that induces granule release) or PMA (a classic inducer of ROS generation), while the effects of each agonists on P-selectin expression and ROS generation in platelets were comparable. Platelet storage was also associated with the increasing levels of ROS (day 0 vs. day 5; p<0.001) while this increasing pattern was directly correlated with the either expressed P-selectin or CD40L. In addition, in 5 day-stored platelets, samples with ROS levels above 40% showed significantly higher levels of P-selectin and CD40L expression. P-selectin negative population of platelet did not show significant amount of ROS. Our data demonstrated decreased levels of important platelet pro-inflammatory molecules in stored platelets with lower levels of intraplatelet ROS. However, whether quenching of ROS generation during platelet storage can attenuate adverse transfusion reactions raised by platelet pro-inflammatory status is required to be further studied. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. High-shear granulation as a manufacturing method for cocrystal granules.

    PubMed

    Rehder, Sönke; Christensen, Niels Peter Aae; Rantanen, Jukka; Rades, Thomas; Leopold, Claudia S

    2013-11-01

    Cocrystal formation allows the tailoring of physicochemical as well as of mechanical properties of an API. However, there is a lack of large-scale manufacturing methods of cocrystals. Therefore, the objective of this work was to examine the suitability of high-shear wet granulation as a manufacturing method for cocrystal granules on a batch scale. Furthermore, the cocrystal granules were characterized regarding their mechanical properties as well as their dissolution behavior. High-shear wet granulation was found to be a feasible manufacturing method for cocrystal granules. Cocrystal formation depended on the exposure time of the solids to the granulation liquid (water), the amount of liquid, the impeller speed of the granulator, and on the excipients (hydroxyl propylcellulose, microcrystalline cellulose, calcium hydrogenphosphate) used in the formulation. Storage stability was strongly influenced by the excipients, since in presence of calcium hydrogenphosphate, the poorly water-soluble salt calcium tartrate monohydrate was formed at high relative humidity. Interestingly, compactability was increased by cocrystal formation compared to that of the reference granules (piracetam and the respective excipients). The drug release was slightly decreased by cocrystal formation, most likely due to the lower solubility of the cocrystal. In the presence of calcium hydrogenphosphate however, no influence of cocrystal formation on either compactability or on drug release were observed, compared with the reference tablets. It was concluded that high-shear wet granulation is a valuable, however complex, manufacturing method for cocrystals. Cocrystal formation may influence compactability and drug release and thus affect drug performance and should be investigated during pre-formulation. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Analysis of carbohydrate storage granules in the diazotrophic cyanobacterium Cyanothece sp. PCC 7822

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Welkie, David G.; Sherman, Debra M.; Chrisler, William B.

    The unicellular diazotrophic cyanobacteria of the genus Cyanothece demonstrate oscillations in nitrogenase activity and H 2 production when grown under 12h light-12h dark cycles. We established that Cyanothece sp. PCC 7822 allows for the construction of knock-out mutants and our objective was to improve the growth characteristics of this strain and to identify the nature of the intracellular storage granules. We report the physiological and morphological effects of reduction in nitrate and phosphate concentrations in BG-11 media on this strain. We developed a series of BG-11-derived growth media and monitored batch culture growth, nitrogenase activity and nitrogenase-mediated hydrogen production, culturemore » synchronicity, and intracellular storage content. Reduction in NaNO3 and K 2HPO 4 concentrations from 17.6 and 0.23 mM to 4.41 and 0.06 mM, respectively, improved growth characteristics such as cell size and uniformity, and enhanced the rate of cell division. Cells grown in this low NP BG-11 were less complex, a parameter that related to the composition of the intracellular storage granules. Cells grown in low NP BG-11 had less polyphosphate, fewer polyhydroxybutyrate granules and many smaller granules became evident. Biochemical analysis and transmission electron microscopy using the histocytochemical PATO technique demonstrated that these small granules contained glycogen. The glycogen levels and the number of granules per cell correlated nicely with a 2.3 to 3.3-fold change from the minimum at L0 to the maximum at D0. The differences in granule morphology and enzymes between Cyanothece ATCC 51142 and Cyanothece PCC 7822 provide insights into the formation of large starch-like granules in some cyanobacteria.« less

  8. A highly Ca2+-sensitive pool of granules is regulated by glucose and protein kinases in insulin-secreting INS-1 cells.

    PubMed

    Yang, Yan; Gillis, Kevin D

    2004-12-01

    We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca(2+) to directly measure the Ca(2+) sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca(2+) sensitivity of release in that a small proportion of granules makes up a highly Ca(2+)-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca(2+). A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca(2+) are not located close to Ca(2+) channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca(2+)-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca(2+), suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca(2+) sensor that is poised to respond to modest, global elevations of [Ca(2+)](i).

  9. Porous material based on spongy titanium granules: structure, mechanical properties, and osseointegration.

    PubMed

    Rubshtein, A P; Trakhtenberg, I Sh; Makarova, E B; Triphonova, E B; Bliznets, D G; Yakovenkova, L I; Vladimirov, A B

    2014-02-01

    A porous material has been produced by pressing spongy titanium granules with subsequent vacuum sintering. The material with porosity of more than 30% has an open system of interconnecting pores. The Young's modulus and 0.2% proof strength have been measured for the samples having 20-55% porosity. If the porosity is between 30 and 45%, the mechanical properties are determined by irregular shape of pores, which is due to spongy titanium granules. The experiment in vivo was performed on adult rabbits. Before surgery the implants were saturated with adherent autologous bone marrow cells. The implants were introduced into the defects formed in the condyles of tibias and femurs. Investigations of osseointegration of implants having 40% porosity showed that the whole system of pores was filled with mature bone tissue in 16 weeks after surgery. Neogenic bone tissue has an uneven surface formed by lacunas and craters indicative of active resorption and subsequent rearrangement (SEM examination). The bone tissue is pierced by neoformed vessels. Irregular-shaped pores with tortuous walls and numerous lateral channels going through the granules provide necessary conditions for the formation of functional bone tissue in the implant volume and the periimplant region. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Compressibility and compactibility of granules produced by wet and dry granulation.

    PubMed

    Bacher, C; Olsen, P M; Bertelsen, P; Sonnergaard, J M

    2008-06-24

    The bulk properties, compactibility and compressibility of granules produced by wet and dry granulation were compared applying a rotary tablet press, three different morphological forms of calcium carbonate and two particle sizes of sorbitol. Granules from both granulation methods possessed acceptable flow properties; however, the ground (Mikhart) and cubic (Scoralite) calcium carbonate demonstrated better die-filling abilities in the tablet press than the scalenhedral calcium carbonate (Sturcal). The wet processed granules showed in general larger compression properties. This was explained as these granules were mechanical stronger and had a higher initial porosity. In some cases, a large particle surface area of calcium carbonate and sorbitol resulted in a small, insignificant improvement of the consolidation characteristics. A correlation between the compression and compaction characteristics was demonstrated.

  11. Protein Mobility within Secretory Granules

    PubMed Central

    Weiss, Annita Ngatchou; Bittner, Mary A.; Holz, Ronald W.; Axelrod, Daniel

    2014-01-01

    We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10−10 cm2/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the

  12. Intrinsic neurophysiological properties of hilar ectopic and normotopic dentate granule cells in human temporal lobe epilepsy and a rat model.

    PubMed

    Althaus, A L; Sagher, O; Parent, J M; Murphy, G G

    2015-02-15

    Hilar ectopic dentate granule cells (DGCs) are a salient feature of aberrant plasticity in human temporal lobe epilepsy (TLE) and most rodent models of the disease. Recent evidence from rodent TLE models suggests that hilar ectopic DGCs contribute to hyperexcitability within the epileptic hippocampal network. Here we investigate the intrinsic excitability of DGCs from humans with TLE and the rat pilocarpine TLE model with the objective of comparing the neurophysiology of hilar ectopic DGCs to their normotopic counterparts in the granule cell layer (GCL). We recorded from 36 GCL and 7 hilar DGCs from human TLE tissue. Compared with GCL DGCs, hilar DGCs in patient tissue exhibited lower action potential (AP) firing rates, more depolarized AP threshold, and differed in single AP waveform, consistent with an overall decrease in excitability. To evaluate the intrinsic neurophysiology of hilar ectopic DGCs, we made recordings from retrovirus-birthdated, adult-born DGCs 2-4 mo after pilocarpine-induced status epilepticus or sham treatment in rats. Hilar DGCs from epileptic rats exhibited higher AP firing rates than normotopic DGCs from epileptic or control animals. They also displayed more depolarized resting membrane potential and wider AP waveforms, indicating an overall increase in excitability. The contrasting findings between disease and disease model may reflect differences between the late-stage disease tissue available from human surgical specimens and the earlier disease stage examined in the rat TLE model. These data represent the first neurophysiological characterization of ectopic DGCs from human hippocampus and prospectively birthdated ectopic DGCs in a rodent TLE model. Copyright © 2015 the American Physiological Society.

  13. New Insights into PhaM-PhaC-Mediated Localization of Polyhydroxybutyrate Granules in Ralstonia eutropha H16

    PubMed Central

    Bresan, Stephanie

    2017-01-01

    ABSTRACT The formation and localization of polyhydroxybutyrate (PHB) granules in Ralstonia eutropha are controlled by PhaM, which interacts both with the PHB synthase (PhaC) and with the bacterial nucleoid. Here, we studied the importance of proline and lysine residues of two C-terminal PAKKA motifs in PhaM for their importance in attaching PHB granules to DNA by in vitro and in vivo methods. Substitution of the lysine residues but not of the proline residues resulted in detachment of formed PHB granules from the nucleoid. Instead, formation of PHB granule clusters at polar regions of the rod-shaped cells and an unequal distribution of PHB granules to daughter cells were observed. The formation of PHB granules was studied by the expression of chromosomally anchored gene fusions of fluorescent proteins with PhaM and PhaC in different backgrounds. PhaM and PhaC fusions showed a distinct colocalization at formed PHB granules in the nucleoid region of the wild type. In a ΔphaC background, PhaM and the catalytically inactive PhaCC319A protein were not able to form fluorescent foci, indicating that correct positioning requires the formation of PHB. Furthermore, time-lapse experiments revealed that PhaC and PhaM proteins detach from formed PHB granules at later stages, resulting in a nonhomogeneous population of PHB granules. This could explain why growth of individual PHB granules stops under PHB-permissive conditions at a certain size. IMPORTANCE PHB granules are storage compounds for carbon and energy in many prokaryotes. Equal distribution of accumulated PHB granules during cell division is therefore important for optimal fitness of the daughter cells. In R. eutropha, PhaM is responsible for maximal activity of PHB synthase, for initiation of PHB granule formation at discrete regions in the cells, and for association of formed PHB granules with the nucleoid. Here we found that four lysine residues of C-terminal PhaM sequence motifs are essential for association of

  14. Membrane interactions between secretion granules and plasmalemma in three exocrine glands

    PubMed Central

    Tanaka, Y; De Camilli, P; Meldolesi, J

    1980-01-01

    Three types of membrane interactions were studied in three exocrine systems (the acinar cells of the rat parotid, rat lacrimal gland, and guinea pig pancrease) by freeze- fracture and thin-section electron microscopy: exocytosis, induced in vivo by specific pharmacological stimulations; the mutual apposition of secretory granule membranes in the intact cell; membrane appositions induced in vitro by centrifugation of the isolated granules. In all three glandular cells, the distribution of intramembrane particles (IMP) on the fracture faces of the luminal plasmagranule membrane particles (IMP) on the fracture faces of the lumenal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMP were rapidly clearly from the areas of granule- plasmalemma apposition in the parotid cells and, especially, in lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid they were less pronounced. Exocytotic openings were usually large and the fracture faces of their rims were covered with IMP. In contrast, in stimulated pancreatic acinar cells, the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrown necks, and no images which might correspond to early stages of membrane fusion were revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat, circular areas, also devoid of IMP. In thin sections, the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures. Isolated secretion granules were forced together in vitro by centrifugation. Under these conditions, increasing the centrifugal force from 1,600 to 50,000 g for 10 min resulted in a progressive, statistically

  15. Perinatal Asphyxia Reduces Dentate Granule Cells and Exacerbates Methamphetamine-Induced Hyperlocomotion in Adulthood

    PubMed Central

    Wakuda, Tomoyasu; Matsuzaki, Hideo; Suzuki, Katsuaki; Iwata, Yasuhide; Shinmura, Chie; Suda, Shiro; Iwata, Keiko; Yamamoto, Shigeyuki; Sugihara, Genichi; Tsuchiya, Kenji J.; Ueki, Takatoshi; Nakamura, Kazuhiko; Nakahara, Daiichiro; Takei, Nori; Mori, Norio

    2008-01-01

    Background Obstetric complications have been regarded as a risk factor for schizophrenia later in life. One of the mechanisms underlying the association is postulated to be a hypoxic process in the brain in the offspring around the time of birth. Hippocampus is one of the brain regions implicated in the late-onset dopaminergic dysfunction associated with hypoxic obstetric complications. Methodology/Principal Findings We used an animal model of perinatal asphyxia, in which rat pups were exposed to 15 min of intrauterine anoxia during Cesarean section birth. At 6 and 12 weeks after birth, the behavior of the pups was assessed using a methamphetamine-induced locomotion test. In addition, the histopathology of the hippocampus was examined by means of stereology. At 6 weeks, there was no change in the methamphetamine-induced locomotion. However, at 12 weeks of age, we found an elevation in methamphetamine-induced locomotor activity, which was associated with an increase of dopamine release in the nucleus accumbens. At the same age, we also found a reduction of the dentate granule cells of the hippocampus. Conclusions/Significance These results suggest that the dopaminergic dysregulation after perinatal asphyxia is associated with a reduction in hippocampal dentate granule cells, and this may partly contribute to the pathogenesis of schizophrenia. PMID:18985150

  16. Lack of effect of mossy fiber-released zinc on granule cell GABA(A) receptors in the pilocarpine model of epilepsy.

    PubMed

    Molnár, P; Nadler, J V

    2001-05-01

    The recurrent mossy fiber pathway of the dentate gyrus expands dramatically in the epileptic brain and serves as a mechanism for synchronization of granule cell epileptiform activity. It has been suggested that this pathway also promotes epileptiform activity by inhibiting GABA(A) receptor function through release of zinc. Hippocampal slices from pilocarpine-treated rats were used to evaluate this hypothesis. The rats had developed status epilepticus after pilocarpine administration, followed by robust recurrent mossy fiber growth. The ability of exogenously applied zinc to depress GABA(A) receptor function in dentate granule cells depended on removal of polyvalent anions from the superfusion medium. Under these conditions, 200 microM zinc reduced the amplitude of the current evoked by applying muscimol to the proximal portion of the granule cell dendrite (23%). It also reduced the mean amplitude (31%) and frequency (36%) of miniature inhibitory postsynaptic currents. Nevertheless, repetitive mossy fiber stimulation (10 Hz for 1 s, 100 Hz for 1 s, or 10 Hz for 5 min) at maximal intensity did not affect GABA(A) receptor-mediated currents evoked by photorelease of GABA onto the proximal portion of the dendrite, where recurrent mossy fiber synapses were located. These results could not be explained by stimulation-induced depletion of zinc from the recurrent mossy fiber boutons. Negative results were obtained even during exposure to conditions that promoted transmitter release and synchronized granule cell activity (6 mM [K(+)](o), nominally Mg(2+)-free medium, 33 degrees C). These results suggest that zinc released from the recurrent mossy fiber pathway did not reach a concentration at postsynaptic GABA(A) receptors sufficient to inhibit agonist-evoked activation.

  17. Differential timing of granule cell production during cerebellum development underlies generation of the foliation pattern.

    PubMed

    Legué, Emilie; Gottshall, Jackie L; Jaumouillé, Edouard; Roselló-Díez, Alberto; Shi, Wei; Barraza, Luis Humberto; Washington, Senna; Grant, Rachel L; Joyner, Alexandra L

    2016-09-08

    The mouse cerebellum (Cb) has a remarkably complex foliated three-dimensional (3D) structure, but a stereotypical cytoarchitecture and local circuitry. Little is known of the cellular behaviors and genes that function during development to determine the foliation pattern. In the anteroposterior axis the mammalian cerebellum is divided by lobules with distinct sizes, and the foliation pattern differs along the mediolateral axis defining a medial vermis and two lateral hemispheres. In the vermis, lobules are further grouped into four anteroposterior zones (anterior, central, posterior and nodular zones) based on genetic criteria, and each has distinct lobules. Since each cerebellar afferent group projects to particular lobules and zones, it is critical to understand how the 3D structure of the Cb is acquired. During cerebellar development, the production of granule cells (gcs), the most numerous cell type in the brain, is required for foliation. We hypothesized that the timing of gc accumulation is different in the four vermal zones during development and contributes to the distinct lobule morphologies. In order to test this idea, we used genetic inducible fate mapping to quantify accumulation of gcs in each lobule during the first two postnatal weeks in mice. The timing of gc production was found to be particular to each lobule, and delayed in the central zone lobules relative to the other zones. Quantification of gc proliferation and differentiation at three time-points in lobules representing different zones, revealed the delay involves a later onset of maximum differentiation and prolonged proliferation of gc progenitors in the central zone. Similar experiments in Engrailed mutants (En1 (-/+) ;En2 (-/-) ), which have a smaller Cb and altered foliation pattern preferentially outside the central zone, showed that gc production, proliferation and differentiation are altered such that the differences between zones are attenuated compared to wild-type mice. Our

  18. Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules

    PubMed Central

    Kroschwald, Sonja; Maharana, Shovamayee; Mateju, Daniel; Malinovska, Liliana; Nüske, Elisabeth; Poser, Ina; Richter, Doris; Alberti, Simon

    2015-01-01

    RNA-protein (RNP) granules have been proposed to assemble by forming solid RNA/protein aggregates or through phase separation into a liquid RNA/protein phase. Which model describes RNP granules in living cells is still unclear. In this study, we analyze P bodies in budding yeast and find that they have liquid-like properties. Surprisingly, yeast stress granules adopt a different material state, which is reminiscent of solid protein aggregates and controlled by protein disaggregases. By using an assay to ectopically nucleate RNP granules, we further establish that RNP granule formation does not depend on amyloid-like aggregation but rather involves many promiscuous interactions. Finally, we show that stress granules have different properties in mammalian cells, where they show liquid-like behavior. Thus, we propose that the material state of RNP granules is flexible and that the solid state of yeast stress granules is an adaptation to extreme environments, made possible by the presence of a powerful disaggregation machine. DOI: http://dx.doi.org/10.7554/eLife.06807.001 PMID:26238190

  19. Granule size control and targeting in pulsed spray fluid bed granulation.

    PubMed

    Ehlers, Henrik; Liu, Anchang; Räikkönen, Heikki; Hatara, Juha; Antikainen, Osmo; Airaksinen, Sari; Heinämäki, Jyrki; Lou, Honxiang; Yliruusi, Jouko

    2009-07-30

    The primary aim of the study was to investigate the effects of pulsed liquid feed on granule size. The secondary aim was to increase knowledge of this technique in granule size targeting. Pulsed liquid feed refers to the pump changing between on- and off-positions in sequences, called duty cycles. One duty cycle consists of one on- and off-period. The study was performed with a laboratory-scale top-spray fluid bed granulator with duty cycle length and atomization pressure as studied variables. The liquid feed rate, amount and inlet air temperature were constant. The granules were small, indicating that the powder has only undergone ordered mixing, nucleation and early growth. The effect of atomizing pressure on granule size depends on inlet air relative humidity, with premature binder evaporation as a reason. The duty cycle length was of critical importance to the end product attributes, by defining the extent of intermittent drying and rewetting. By varying only the duty cycle length, it was possible to control granule nucleation and growth, with a wider granule size target range in increased relative humidity. The present study confirms that pulsed liquid feed in fluid bed granulation is a useful tool in end product particle size targeting.

  20. 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 Central

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

    2011-01-01

    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. PMID:21402877

  1. The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

    PubMed Central

    Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

    1995-01-01

    The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7626033

  2. The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

    PubMed

    Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

    1995-07-15

    The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process.

  3. Reduced generation of lung tissue–resident memory T cells during infancy

    PubMed Central

    Zens, Kyra D.; Chen, Jun Kui; Wu, Felix L.; Cvetkovski, Filip

    2017-01-01

    Infants suffer disproportionately from respiratory infections and generate reduced vaccine responses compared with adults, although the underlying mechanisms remain unclear. In adult mice, lung-localized, tissue-resident memory T cells (TRMs) mediate optimal protection to respiratory pathogens, and we hypothesized that reduced protection in infancy could be due to impaired establishment of lung TRM. Using an infant mouse model, we demonstrate generation of lung-homing, virus-specific T effectors after influenza infection or live-attenuated vaccination, similar to adults. However, infection during infancy generated markedly fewer lung TRMs, and heterosubtypic protection was reduced compared with adults. Impaired TRM establishment was infant–T cell intrinsic, and infant effectors displayed distinct transcriptional profiles enriched for T-bet–regulated genes. Notably, mouse and human infant T cells exhibited increased T-bet expression after activation, and reduction of T-bet levels in infant mice enhanced lung TRM establishment. Our findings reveal that infant T cells are intrinsically programmed for short-term responses, and targeting key regulators could promote long-term, tissue-targeted protection at this critical life stage. PMID:28855242

  4. Granulation of fine powder

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Ching-Fong

    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 formmore » a dense compact with a higher density and more uniform pore size distribution.« less

  5. Incorporation of a circulating protein into megakaryocyte and platelet granules

    NASA Technical Reports Server (NTRS)

    Handagama, P. J.; George, J. N.; Shuman, M. A.; McEver, R. P.; Bainton, D. F.

    1987-01-01

    To determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for its uptake by electron microscopy and cytochemistry. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare cells. In megakaryocytes, 50% of alpha granules contained HRP between 75 min and 7 hr after injection. At 24 hr, 25% of the megakaryocyte granules were peroxidase-positive, less were positive by 48 hr, and there were none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. Platelet granules also contain HRP by 7 hr after injection, and they can secrete it in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. It is concluded that while some alpha granule proteins are synthesized by megakaryocytes, others may be acquired from plasma by endocytosis. In addition to providing evidence that some of the proteins of alpha granules may be of exogenous origin, this study has allowed the definition of a pathway whereby plasma proteins may be temporarily sequestered in megakaryocytes before reentering the circulation in platelets.

  6. Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles.

    PubMed

    Woo, Sang Su; James, Declan J; Martin, Thomas F J

    2017-03-15

    Munc13-4 is a Ca 2+ -dependent SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca 2+ -evoked secretion in various secretory cells. Studies in mast cell-like RBL-2H3 cells provide direct evidence that Munc13-4 with its two Ca 2+ -binding C2 domains functions as a Ca 2+ sensor for SG exocytosis. Unexpectedly, Ca 2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4 + /Rab7 + /Rab11 + endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4 + /Rab7 + SGs, followed by a merge with Rab11 + endosomes, and depended on Ca 2+ binding to Munc13-4. Munc13-4 promoted the Ca 2+ -stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca 2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. © 2017 Woo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  7. Granules harboring translationally active mRNAs provide a platform for P-body formation following stress.

    PubMed

    Lui, Jennifer; Castelli, Lydia M; Pizzinga, Mariavittoria; Simpson, Clare E; Hoyle, Nathaniel P; Bailey, Kathryn L; Campbell, Susan G; Ashe, Mark P

    2014-11-06

    The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Phleum pratense pollen starch granules induce humoral and cell-mediated immune responses in a rat model of allergy.

    PubMed

    Motta, A; Peltre, G; Dormans, J A M A; Withagen, C E T; Lacroix, G; Bois, F; Steerenberg, P A

    2004-02-01

    Timothy grass (Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma. We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats. Brown-Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 microm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay. An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells. It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma.

  9. 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.

  10. Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions.

    PubMed

    Nelson, Emily V; Schmidt, Kristina M; Deflubé, Laure R; Doğanay, Sultan; Banadyga, Logan; Olejnik, Judith; Hume, Adam J; Ryabchikova, Elena; Ebihara, Hideki; Kedersha, Nancy; Ha, Taekjip; Mühlberger, Elke

    2016-08-15

    A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2α phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions. Although progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by

  11. Starch granule formation and protein deposition in wheat (Triticum aestivum L.) starchy endosperm cells is altered by high temperature during grain fill

    NASA Astrophysics Data System (ADS)

    Hurkman, William J.; Wood, Delilah F.

    2010-06-01

    High temperatures during wheat grain fill decrease starch and protein levels, adversely affecting wheat yield and flour quality. To determine the effect of high temperature on starchy endosperm cell development, grain (Triticum aestivum L. 'Butte 86') was produced under a 24/17°C or 37/28°C day/night regimen imposed from flowering to maturity and starch and protein deposition examined using scanning electron microscopy. The high temperature regimen shortened the duration of grain fill from 40 to 18 days. Under the 37/28°C regimen, A- and B-type starch granules decreased in size. A-type starch granules also exhibited pitting, suggesting enhanced action of starch degradative enzymes. Under both temperature regimens, protein bodies originated early in development and coalesced during mid to late development to form a continuous protein matrix surrounding the starch granules. Under the 37/28°C regimen, the proportion of protein matrix increased in endosperm cells of mature grain. Taken together, the changes in starch granule number and size and in protein matrix amount provide clues for understanding how high temperature during grain fill can affect end use properties of wheat flour.

  12. JNK1 controls adult hippocampal neurogenesis and imposes cell-autonomous control of anxiety behaviour from the neurogenic niche

    PubMed Central

    Mohammad, H; Marchisella, F; Ortega-Martinez, S; Hollos, P; Eerola, K; Komulainen, E; Kulesskaya, N; Freemantle, E; Fagerholm, V; Savontous, E; Rauvala, H; Peterson, B D; van Praag, H; Coffey, E T

    2018-01-01

    Promoting adult hippocampal neurogenesis is expected to induce neuroplastic changes that improve mood and alleviate anxiety. However, the underlying mechanisms remain largely unknown and the hypothesis itself is controversial. Here we show that mice lacking Jnk1, or c-Jun N-terminal kinase (JNK) inhibitor-treated mice, display increased neurogenesis in adult hippocampus characterized by enhanced cell proliferation and survival, and increased maturation in the ventral region. Correspondingly, anxiety behaviour is reduced in a battery of tests, except when neurogenesis is prevented by AraC treatment. Using engineered retroviruses, we show that exclusive inhibition of JNK in adult-born granule cells alleviates anxiety and reduces depressive-like behaviour. These data validate the neurogenesis hypothesis of anxiety. Moreover, they establish a causal role for JNK in the hippocampal neurogenic niche and anxiety behaviour, and advocate targeting of JNK as an avenue for novel therapies against affective disorders. PMID:27843149

  13. Localization of SERBP1 in stress granules and nucleoli.

    PubMed

    Lee, Yu-Jen; Wei, Hung-Ming; Chen, Ling-Yun; Li, Chuan

    2014-01-01

    SERPINE1 mRNA-binding protein 1 (SERBP1) is an arginine-methylated RNA-binding protein whose modification affects protein interaction and intracellular localization. In the present study, we show that, under normal growth conditions without stress, SERBP1 interacts with arginine-methylated and stress granule-associated proteins such as heterogeneous nuclear ribonucleoprotein A1, fragile X mental retardation protein and fragile X mental retardation syndrome-related protein 1 in an RNA-dependent manner. We also show that, after arsenite treatment, a proportion of full-length SERBP1 protein co-localizes with the typical stress granule marker T-cell intracellular antigen-1 in the cytoplasmic stress granules. Truncated SERBP1 with an N-terminal, central RG or C-terminal deletion, or single-domain segments comprising the N-terminal, central or C-terminal region, were recruited to stress granules upon arsenite treatment but with reduced efficiency. In addition, upon arsenite treatment, the localization of SERBP1 changed from a diffuse cytoplasmic localization to nuclear-dominant (concentrated in the nucleolus) A similar distribution was observed when cells were treated with the methylation inhibitor adenosine periodate, and was also detected for N- or C-terminal domain deletions and all three single-domain fragments even without stress induction. We further demonstrate that adenosine periodate treatment delays the association/dissociation of SERBP1 with stress granules. Hypomethylation retains SERBP1 in the nucleus/nucleolus regardless of arsenite treatment. Our study indicates that arginine methylation is correlated with recruitment of SERBP to stress granules and nucleoli and its retention therein. To our knowledge, this is the first report of an RNA-binding protein that is shifted simultaneously to cytoplasmic stress granules and nucleoli, two ribonucleoprotein-enriched subcellular compartments, upon stress. © 2013 FEBS.

  14. The resident macrophages in murine pancreatic islets are constantly probing their local environment, capturing beta cell granules and blood particles.

    PubMed

    Zinselmeyer, Bernd H; Vomund, Anthony N; Saunders, Brian T; Johnson, Michael W; Carrero, Javier A; Unanue, Emil R

    2018-06-01

    We studied here the interactions between the resident macrophages of pancreatic islets with beta cells and the blood vasculature. We also examined the immunological consequences of such interactions. Islets were isolated from C57BL/6 mice expressing CX3C motif chemokine receptor 1-green fluorescent protein (CX3CR-GFP) and examined live by two-photon microscopy. Islets were also examined by electron microscopy to study the relationship of the intra-islet macrophages with the beta cells. In NOD.Rag1 -/- mice and young (non-diabetic) male mice, the acquisition of beta cell granules was tested functionally by probing with CD4 + T cells directed against insulin epitopes. Two-photon microscopy showed that the islet resident macrophages were in close contact with blood vessels and had extensive filopodial activity. Some filopodia had direct access to the vessel lumen and captured microparticles. Addition of glucose at high concentration reduced the degree of filopodia sampling of islets. This finding applied to in vivo injection of glucose or to in vitro cultures. Ultrastructural examination showed the close contacts of macrophages with beta cells. Such macrophages contained intact dense core granules. Functional studies in NOD mice indicated that the macrophages presented insulin peptides to insulin-reactive T cells. Presentation was increased after glucose challenge either ex vivo or after an in vivo pulse. In agreement with the morphological findings, presentation was not affected by insulin receptor blockade. Islet resident macrophages are highly active, sampling large areas of the islets and blood contents and capturing beta cell granules. After such interactions, macrophages present immunogenic insulin to specific autoreactive T cells.

  15. Origins of adult pigmentation: diversity in pigment stem cell lineages and implications for pattern evolution

    PubMed Central

    Spiewak, Jessica E.

    2014-01-01

    Summary Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage and mate choice and have played important roles in speciation. Here, we review recent studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post-embryonic, peripheral nerve associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow-orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns. PMID:25421288

  16. Influence of metronidazole particle properties on granules prepared in a high-shear mixer-granulator.

    PubMed

    Di Martino, Piera; Censi, Roberta; Malaj, Ledjan; Martelli, Sante; Joiris, Etienne; Barthélémy, Christine

    2007-02-01

    Metronidazole is a good example of high-dose drug substance with poor granulating and tableting properties. Tablets are generally produced by liquid granulation; however, the technological process failure is quite frequent. In order to verify how the metronidazole particle characteristics can influence granule properties, three metronidazole batches differing for crystal habit, mean particle size, BET surface area and wettability were selected, primarily designed according to their different elongation ratio: needle-shaped, stick-shaped, and isodimensional. In the presence of lactose monohydrate and pregelatinized maize starch, respectively as diluent and binder, they were included in a formula for wet granulation in a high-shear mixer-granulator. In order to render the process comparable as far as possible, all parameters and experimental conditions were maintained constant. Four granule batches were obtained: granules from placebo (G-placebo), granules from needle-shaped crystals (G-needle-shaped), granules from stick-shaped crystals (G-stick-shaped), and granules from isodimensional crystals (G-isodimensional). Different granule properties were considered, in particular concerning porosity, friability, loss on drying (LOD), and flowability. In order to study their tabletability and compressibility, the different granules obtained were then compressed in a rotary press. The best tabletability was obtained with the isodimensional batch, while the poorest was exhibited by the stick-shaped one. Differences in tabletability are in good accordance with compressibility results: to a better tabletability corresponds an important granule ability to undergo a volume reduction as a result of an applied pressure. In particular, it was proposed that the greatest compressibility of the G-isodimensional must be related to the greatest granule porosity percentage.

  17. Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study

    PubMed Central

    Toczylowska, Beata; Zieminska, Elzbieta; Goch, Grazyna; Milej, Daniel; Gerega, Anna; Liebert, Adam

    2014-01-01

    The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125μΜ ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 μM (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture. PMID:24688815

  18. New Class of Cargo Protein in Tetrahymena thermophila Dense Core Secretory Granules

    PubMed Central

    Haddad, Alex; Bowman, Grant R.; Turkewitz, Aaron P.

    2002-01-01

    Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules. PMID:12456006

  19. Progenitor cell domains in the developing and adult pancreas

    PubMed Central

    Kopp, Janel L; Dubois, Claire L; Hao, Ergeng; Thorel, Fabrizio; Herrera, Pedro L

    2011-01-01

    Unlike organs with defined stem cell compartments, such as the intestine, the pancreas has limited capacity to regenerate. The question of whether the adult pancreas harbors facultative stem/progenitor cells has been a prime subject of debate. Cumulative evidence from recent genetic lineage tracing studies, in which specific cell populations were marked and traced in adult mice, suggests that endocrine and acinar cells are no longer generated from progenitors in the adult pancreas. These studies further indicate that adult pancreatic ductal cells are not a source for endocrine cells following pancreatic injury, as previously suggested. Our own studies have shown that adult ductal cells reinitiate expression of some endocrine progenitor markers, including Ngn3, after injury by partial duct ligation (PDL), but that these cells do not undergo endocrine cell differentiation. Here, we present additional evidence that endocrine cells do not arise from ducts following β-cell ablation by streptozotocin or by a diphtheria toxin-expressing transgene or when β-cell ablation is combined with PDL. In this review, we discuss findings from recent lineage tracing studies of embryonic and adult pancreatic ductal cells. Based upon the combined evidence from these studies, we propose that multipotency is associated with a specific transcriptional signature. PMID:21558806

  20. Stem cells with potential to generate insulin producing cells in man.

    PubMed

    Zulewski, Henryk

    2006-10-14

    Replacement of insulin-producing cells represents an almost ideal treatment for patients with diabetes mellitus type 1. Transplantation of pancreatic islets of Langerhans--although successful in experienced centres--is limited by the lack of donor organs. Generation of insulin-producing cells from stem cells represents an attractive alternative. Stem cells with the potential to differentiate into insulin-producing cells include embryonic stem cells (ESC) as well as adult stem cells from various tissues including the pancreas, liver, central nervous system, bone marrow and adipose tissue. The use of human ESC is hampered by ethical concerns and the inability to create patient specific ESC with therapeutic cloning. Among adult stem cells mesenchymal stem cells appear to have a particular developmental plasticity ex vivo that include their ability to adopt a pancreatic endocrine phenotype. The present review summarises the current knowledge on the development of insulin-producing cells from stem cells with special emphasis on human mesenchymal stem cells isolated from the pancreas and adipose tissue.

  1. Stem cells with potential to generate insulin-producing cells in man.

    PubMed

    Zulewski, Henryk

    2007-03-02

    Replacement of insulin-producing cells represents an almost ideal treatment for patients with diabetes mellitus type 1. Transplantation of pancreatic islets of Langerhans--although successful in experienced centres--is limited by the lack of donor organs. Generation of insulin-producing cells from stem cells represents an attractive alternative. Stem cells with the potential to differentiate into insulin-producing cells include embryonic stem cells (ESC) as well as adult stem cells from various tissues including the pancreas, liver, central nervous system, bone marrow and adipose tissue. The use of human ESC is hampered by ethical concerns and the inability to create patient specific ESC with therapeutic cloning. Among adult stem cells mesenchymal stem cells appear to have a particular developmental plasticity ex vivo that include their ability to adopt a pancreatic endocrine phenotype. The present review summarises the current knowledge on the development of insulin-producing cells from stem cells with special emphasis on human mesenchymal stem cells isolated from the pancreas and adipose tissue.

  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. 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.

  4. Aspergillus oryzae AoSO is a novel component of stress granules upon heat stress in filamentous fungi.

    PubMed

    Huang, Hsiang-Ting; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2013-01-01

    Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP) granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi. The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.

  5. Unique processing during a period of high excitation/inhibition balance in adult-born neurons.

    PubMed

    Marín-Burgin, Antonia; Mongiat, Lucas A; Pardi, M Belén; Schinder, Alejandro F

    2012-03-09

    The adult dentate gyrus generates new granule cells (GCs) that develop over several weeks and integrate into the preexisting network. Although adult hippocampal neurogenesis has been implicated in learning and memory, the specific role of new GCs remains unclear. We examined whether immature adult-born neurons contribute to information encoding. By combining calcium imaging and electrophysiology in acute slices, we found that weak afferent activity recruits few mature GCs while activating a substantial proportion of the immature neurons. These different activation thresholds are dictated by an enhanced excitation/inhibition balance transiently expressed in immature GCs. Immature GCs exhibit low input specificity that switches with time toward a highly specific responsiveness. Therefore, activity patterns entering the dentate gyrus can undergo differential decoding by a heterogeneous population of GCs originated at different times.

  6. On the so-called membrane coating granules in keratinized lichen planus lesions of the buccal mucosa.

    PubMed

    El-Labban, N G; Wood, R D

    1982-11-01

    Serial sections of the so-called membrane-coating granules have been examined in keratinized oral epithelium of lichen planus lesions. As with 'granules' apparent in non-keratinized epithelium, it is found they do not represent specialized intra-cytoplasmic organelles, but are the result of sectioning at different areas, levels and planes through the plasma membrane of interdigitating cell processes. Such 'granules' appear mostly in the superficial, but not deep, part of the cytoplasm of the upper prickle cells. This is considered to be due to topographic differences between the upper and under surfaces of these cells and the presence of narrower intercellular spaces than those between deeper epithelial cells. Such arrangement often results in cell processes in sections appearing free in the superficial part of the cell below. The appearance of 'granules' arises when the plane of section is not at right angles to the two plasma membranes surrounding these processes.

  7. Distribution of binder in granules produced by means of twin screw granulation.

    PubMed

    Fonteyne, Margot; Fussell, Andrew Luke; Vercruysse, Jurgen; Vervaet, Chris; Remon, Jean Paul; Strachan, Clare; Rades, Thomas; De Beer, Thomas

    2014-02-28

    According to the quality by design principle processes may not remain black-boxes and full process understanding is required. The granule size distribution of granules produced via twin screw granulation is often found to be bimodal. The aim of this study was to gain a better understanding of binder distribution within granules produced via twin screw granulation in order to investigate if an inhomogeneous spread of binder is causing this bimodal size distribution. Theophylline-lactose-polyvinylpyrrolidone K30 (PVP) (30-67.5-2.5%, w/w) was used as a model formulation. The intra-granular distribution of PVP was evaluated by means of hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy. For the evaluated formulation, no PVP rich zones were detected when applying a lateral spatial resolution of 0.5 μm, indicating that PVP is homogenously distributed within the granules. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons.

    PubMed

    Bosch, Carles; Martínez, Albert; Masachs, Nuria; Teixeira, Cátia M; Fernaud, Isabel; Ulloa, Fausto; Pérez-Martínez, Esther; Lois, Carlos; Comella, Joan X; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2015-01-01

    The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM) and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM) allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs) in mice. 3D reconstruction of dendritic spines in GCs aged 3-4 and 8-9 weeks revealed two different stages of dendritic spine development and unexpected features of synapse formation, including vacant and branched dendritic spines and presynaptic terminals establishing synapses with up to 10 dendritic spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner.

  9. Can one generate stable hyaline cartilage from adult mesenchymal stem cells? A developmental approach.

    PubMed

    Hellingman, Catharine A; Koevoet, Wendy; van Osch, Gerjo J V M

    2012-11-01

    Chondrogenically differentiating bone marrow-derived mesenchymal stem cells (BMSCs) display signs of chondrocyte hypertrophy, such as production of collagen type X, MMP13 and alkaline phosphatase (ALPL). For cartilage reconstructions this is undesirable, as terminally differentiated cartilage produced by BMSCs mineralizes when implanted in vivo. Terminal differentiation is not restricted to BMSCs but is also encountered in chondrogenic differentiation of adipose-derived mesenchymal stem cells (MSCs) as well as embryonic stem cells, which by definition should be able to generate all types of tissues, including stable cartilage. Therefore, we propose that the currently used culture conditions may drive the cells towards terminal differentiation. In this manuscript we aim to review the literature, supplemented by our own data to answer the question, is it possible to generate stable hyaline cartilage from adult MSCs? We demonstrate that recently published methods for inhibiting terminal differentiation (through PTHrP, MMP13 or blocking phosphorylation of Smad1/5/8) result in cartilage formation with reduction of hypertrophic markers, although this does not reach the low level of stable chondrocytes. A set of hypertrophy markers should be included in future studies to characterize the phenotype more precisely. Finally, we used what is currently known in developmental biology about the differential development of hyaline and terminally differentiated cartilage to provide thought and insights to change current culture models for creating hyaline cartilage. Inhibiting terminal differentiation may not result in stable hyaline cartilage if the right balance of signals has not been created from the start of culture onwards. Copyright © 2011 John Wiley & Sons, Ltd.

  10. TIA-1 Self-Multimerization, Phase Separation, and Recruitment into Stress Granules Are Dynamically Regulated by Zn2.

    PubMed

    Rayman, Joseph B; Karl, Kevin A; Kandel, Eric R

    2018-01-02

    Stress granules are non-membranous structures that transiently form in the cytoplasm during cellular stress, where they promote translational repression of non-essential RNAs and modulate cell signaling by sequestering key signal transduction proteins. These and other functions of stress granules facilitate an adaptive cellular response to environmental adversity. A key component of stress granules is the prion-related RNA-binding protein, T cell intracellular antigen-1 (TIA-1). Here, we report that recombinant TIA-1 undergoes rapid multimerization and phase separation in the presence of divalent zinc, which can be reversed by the zinc chelator, TPEN. Similarly, the formation and maintenance of TIA-1-positive stress granules in arsenite-treated cells are inhibited by TPEN. In addition, Zn 2+ is released in cells treated with arsenite, before stress granule formation. These findings suggest that Zn 2+ is a physiological ligand of TIA-1, acting as a stress-inducible second messenger to promote multimerization of TIA-1 and subsequent localization into stress granules. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16.

    PubMed

    Pfeiffer, Daniel; Wahl, Andreas; Jendrossek, Dieter

    2011-11-01

    A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1. © 2011 Blackwell Publishing Ltd.

  12. Dynamin-related protein-1 controls fusion pore dynamics during platelet granule exocytosis.

    PubMed

    Koseoglu, Secil; Dilks, James R; Peters, Christian G; Fitch-Tewfik, Jennifer L; Fadel, Nathalie A; Jasuja, Reema; Italiano, Joseph E; Haynes, Christy L; Flaumenhaft, Robert

    2013-03-01

    Platelet granule exocytosis serves a central role in hemostasis and thrombosis. Recently, single-cell amperometry has shown that platelet membrane fusion during granule exocytosis results in the formation of a fusion pore that subsequently expands to enable the extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore expansion and collapse are not known. We identified dynamin-related protein-1 (Drp1) in platelets and found that an inhibitor of Drp1, mdivi-1, blocked exocytosis of both platelet dense and α-granules. We used single-cell amperometry to monitor serotonin release from individual dense granules and, thereby, measured the effect of Drp1 inhibition on fusion pore dynamics. Inhibition of Drp1 increased spike width and decreased prespike foot events, indicating that Drp1 influences fusion pore formation and expansion. Platelet-mediated thrombus formation in vivo after laser-induced injury of mouse cremaster arterioles was impaired after infusion of mdivi-1. These results demonstrate that inhibition of Drp1 disrupts platelet fusion pore dynamics and indicate that Drp1 can be targeted to control thrombus formation in vivo.

  13. HDAC6 regulates the dynamics of lytic granules in cytotoxic T lymphocytes

    PubMed Central

    Núñez-Andrade, Norman; Iborra, Salvador; Trullo, Antonio; Moreno-Gonzalo, Olga; Calvo, Enrique; Catalán, Elena; Menasche, Gaël; Sancho, David; Vázquez, Jesús; Yao, Tso-Pang

    2016-01-01

    HDAC6 is a tubulin deacetylase involved in many cellular functions related to cytoskeleton dynamics including cell migration and autophagy. In addition, HDAC6 affects antigen-dependent CD4+ T cell activation. In this study, we show that HDAC6 contributes to the cytotoxic function of CD8+ T cells. Immunization studies revealed defective cytotoxic activity in vivo in the absence of HDAC6. Adoptive transfer of wild-type or Hdac6-/- CD8+ T cells to Rag1-/- mice demonstrated specific impairment in CD8+ T cell responses against vaccinia infection. Mechanistically, HDAC6-deficient cytotoxic T lymphocytes (CTLs) showed defective in vitro cytolytic activity related to altered dynamics of lytic granules, inhibited kinesin 1 – dynactin mediated terminal transport of lytic granules to the immune synapse and deficient exocytosis, but not to target cell recognition, T cell receptor (TCR) activation or interferon (IFNγ) production. Our results establish HDAC6 as an effector of the immune cytotoxic response that acts by affecting the dynamics, transport and secretion of lytic granules by CTLs. PMID:26869226

  14. Linking local circuit inhibition to olfactory behavior: a critical role for granule cells in olfactory discrimination.

    PubMed

    Strowbridge, Ben W

    2010-02-11

    In this issue of Neuron, Abraham et al. report a direct connection between inhibitory function and olfactory behavior. Using molecular methods to alter glutamate receptor subunit composition in olfactory bulb granule cells, the authors found a selective modulation in the time required for difficult, but not simple, olfactory discrimination tasks. Copyright 2010 Elsevier Inc. All rights reserved.

  15. Live-cell imaging of multiple endogenous mRNAs permits the direct observation of RNA granule dynamics.

    PubMed

    Yatsuzuka, Kenji; Sato, Shin-Ichi; Pe, Kathleen Beverly; Katsuda, Yousuke; Takashima, Ippei; Watanabe, Mizuki; Uesugi, Motonari

    2018-06-08

    Here, we developed two pairs of high-contrast chemical probes and their RNA aptamers with distinct readout channels that permitted simultaneous live-cell imaging of endogenous β-actin and cortactin mRNAs. Application of this technology allowed the direct observation of the formation process of stress granules, protein-RNA assemblies essential for cellular response to the environment.

  16. The functional morphology of color changing in a spider: development of ommochrome pigment granules.

    PubMed

    Insausti, Teresita C; Casas, Jérôme

    2008-03-01

    Studies on the formation of ommochrome pigment granules are very few, despite their generalized occurrence as screening pigments in insect eyes. This is particularly true for ommochrome granules responsible for epidermal coloration. The aims of this study were to characterize the localization of major body pigments in a color changing mimetic spider, Misumena vatia (Thomisidae), and to describe the formation and location of ommochrome pigment granules responsible for the spider's color change from white to yellow. The unpigmented cuticula of this spider is transparent. Both the guanine localized in guanine cells in the opisthosoma and the uric acid localized in epidermis cells in the prosoma are responsible for the white coloration. The bright yellow color is due to the combination of ommochrome pigment granules and the white reflectance from coincident guanine and/or uric acid. The formation of ommochrome pigment granules in epidermis cells proceeds via three distinctive steps. Translucent, UV fluorescent, progranules (type I) are produced by a dense network of endoplasmic reticulum associated with numerous mitochondria and glycogen rosettes. These progranules are present in white spiders only, and regularly distributed in the cytoplasm. The merging of several progranules of type I into a transient state (progranule type II) leads to the formation of granules (type III) characterized by their lack of fluorescence, their spherical sections and their osmophilic-electron-dense contents. They are found in yellow spiders and in the red stripes on the body sides. Their color varies from yellow to red. Thus, white spiders contain only type I granules, yellow tinted spiders contain type II and III granules and bright yellow spiders contain only type III granules. We present a synthetic view of the ontogeny of ommochrome granules. We discuss the physiology of color changing and the nature of the chemical compounds in the different types of granules. Extended studies on the

  17. In Vivo Anomalous Diffusion and Weak Ergodicity Breaking of Lipid Granules

    NASA Astrophysics Data System (ADS)

    Jeon, Jae-Hyung; Tejedor, Vincent; Burov, Stas; Barkai, Eli; Selhuber-Unkel, Christine; Berg-Sørensen, Kirstine; Oddershede, Lene; Metzler, Ralf

    2011-01-01

    Combining extensive single particle tracking microscopy data of endogenous lipid granules in living fission yeast cells with analytical results we show evidence for anomalous diffusion and weak ergodicity breaking. Namely we demonstrate that at short times the granules perform subdiffusion according to the laws of continuous time random walk theory. The associated violation of ergodicity leads to a characteristic turnover between two scaling regimes of the time averaged mean squared displacement. At longer times the granule motion is consistent with fractional Brownian motion.

  18. Granule swelling and cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kato, Takayuki, E-mail: tkato@med.osaka-cu.ac.jp; Ikemoto, Masaru; Hato, Fumihiko

    2009-04-10

    Extracellular signal-regulated kinase and p38 have been shown to be cleaved in human neutrophils undergoing apoptosis induced by tumor necrosis factor-{alpha} and cycloheximide. However, the cleavage products of these molecules were undetected when apoptotic neutrophils were pretreated with phenylmethylsulfonyl fluoride or disrupted by nitrogen cavitation before preparation of cell lysates. The electron microscopy revealed that granules in apoptotic neutrophils were significantly swollen than those in control cells. These findings suggest that granule membrane may become destabilized during neutrophil apoptosis, leading to rapid proteolysis of these molecules by granule-derived serine proteases during preparation of cell lysates with the conventional lysis buffer.

  19. Measuring stellar granulation during planet transits

    NASA Astrophysics Data System (ADS)

    Chiavassa, A.; Caldas, A.; Selsis, F.; Leconte, J.; Von Paris, P.; Bordé, P.; Magic, Z.; Collet, R.; Asplund, M.

    2017-01-01

    Context. Stellar activity and convection-related surface structures might cause bias in planet detection and characterization that use these transits. Surface convection simulations help to quantify the granulation signal. Aims: We used realistic three-dimensional (3D) radiative hydrodynamical (RHD) simulations from the Stagger grid and synthetic images computed with the radiative transfer code Optim3D to model the transits of three prototype planets: a hot Jupiter, a hot Neptune, and a terrestrial planet. Methods: We computed intensity maps from RHD simulations of the Sun and a K-dwarf star at different wavelength bands from optical to far-infrared that cover the range of several ground- and space-based telescopes which observe exoplanet transits. We modeled the transit using synthetic stellar-disk images obtained with a spherical-tile imaging method and emulated the temporal variation of the granulation intensity generating random images covering a granulation time-series of 13.3 h. We measured the contribution of the stellar granulation on the light curves during the planet transit. Results: We identified two types of granulation noise that act simultaneously during the planet transit: (I) the intrinsic change in the granulation pattern with timescale (e.g., 10 min for solar-type stars assumed in this work) is smaller than the usual planet transit ( hours as in our prototype cases); and (II) the fact that the transiting planet occults isolated regions of the photosphere that differ in local surface brightness as a result of convective-related surface structures. First, we showed that our modeling approach returns granulation timescale fluctuations that are comparable with what has been observed for the Sun. Then, our statistical approach shows that the granulation pattern of solar and K-dwarf-type stars have a non-negligible effect of the light curve depth during the transit, and, consequentially on the determination of the planet transit parameters such as the

  20. Maintained LTP and Memory Are Lost by Zn2+ Influx into Dentate Granule Cells, but Not Ca2+ Influx.

    PubMed

    Takeda, Atsushi; Tamano, Haruna; Hisatsune, Marie; Murakami, Taku; Nakada, Hiroyuki; Fujii, Hiroaki

    2018-02-01

    The idea that maintained LTP and memory are lost by either increase in intracellular Zn 2+ in dentate granule cells or increase in intracellular Ca 2+ was examined to clarify significance of the increases induced by excess synapse excitation. Both maintained LTP and space memory were impaired by injection of high K + into the dentate gyrus, but rescued by co-injection of CaEDTA, which blocked high K + -induced increase in intracellular Zn 2+ but not high K + -induced increase in intracellular Ca 2+ . High K + -induced disturbances of LTP and intracellular Zn 2+ are rescued by co-injection of 6-cyano-7-nitroquinoxakine-2,3-dione, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, but not by co-injection of blockers of NMDA receptors, metabotropic glutamate receptors, and voltage-dependent calcium channels. Furthermore, AMPA impaired maintained LTP and the impairment was also rescued by co-injection of CaEDTA, which blocked increase in intracellular Zn 2+ , but not increase in intracellular Ca 2+ . NMDA and glucocorticoid, which induced Zn 2+ release from the internal stores, did not impair maintained LTP. The present study indicates that increase in Zn 2+ influx into dentate granule cells through AMPA receptors loses maintained LTP and memory. Regulation of Zn 2+ influx into dentate granule cells is more critical for not only memory acquisition but also memory retention than that of Ca 2+ influx.

  1. Localization of DNA and RNA in eosinophil secretory granules.

    PubMed

    Behzad, Ali R; Walker, David C; Abraham, Thomas; McDonough, John; Mahmudi-Azer, Salahadin; Chu, Fanny; Shaheen, Furquan; Hogg, James C; Paré, Peter D

    2010-01-01

    Although the accepted paradigm is that the proteins stored in eosinophil crystalloid granules are translated from messenger RNA transcribed in the cell nucleus, recent ultrastructural evidence suggests that protein synthesis may also take place within eosinophilic granules. We used 2 different methods to detect the presence of DNA and RNA in eosinophil secretory granules. Using bromodeoxyuridine, a thymidine analogue, and bromouridine, a uracil analogue, we labeled the DNA and RNA in eosinophils in vivo in rabbits. Immunoelectron microscopy to localize these molecules was performed on ultrathin sections of blood and bone marrow eosinophils using monoclonal anti-bromodeoxyuridine antibody with IgG as a control. The immunogold grain density was measured in each subcellular compartment within the eosinophils and analyzed using image analysis software. A combination of DNA/CD63 immunofluorescence staining and a fluorescently labeled molecular probe that stains RNA was used to examine the presence of DNA and RNA in the secretory granules of human blood eosinophils. The mean density of bromodeoxyuridine-labeled DNA and bromouridine-labeled RNA immunogold grains in the secretory granules of blood and bone marrow eosinophils were significantly higher (p < 0.0005) than cytoplasmic or background staining. We also demonstrated the existence of DNA and RNA in the CD63-positive secretory granules of human peripheral blood eosinophils by means of immunofluorescent staining and a fluorescently labeled molecular probe. These results provide evidence that eosinophil granules are the site of DNA and RNA synthesis and suggest the potential for a new role(s) for eosinophil-secretory granules. Copyright 2009 S. Karger AG, Basel.

  2. Ethanol impairs activation of retinoic acid receptors in cerebellar granule cells in a rodent model of fetal alcohol spectrum disorders.

    PubMed

    Kumar, Ambrish; Singh, Chandra K; DiPette, Donald D; Singh, Ugra S

    2010-05-01

    Ethanol is the main addictive and neurotoxic constituent of alcohol. Ethanol exposure during embryonic development causes dysfunction of the central nervous system (CNS) and leads to fetal alcohol spectrum disorders. The cerebellum is one of the CNS regions that are particularly vulnerable to ethanol toxic effects. Retinoic acid (RA) is a physiologically active metabolite of vitamin A that is locally synthesized in the cerebellum. Studies have shown that RA is required for neuronal development, but it remains unknown if ethanol impairs RA signaling and thus induces neuronal malformations. In this study, we tested the hypothesis that ethanol impairs the expression and activation of RA receptors in cerebellum and in cerebellar granule cells. The cerebellum of ethanol unexposed and exposed pups was used to study the expression of retinoic acid receptors (RARs or RXRs) by immunohistochemistry and by Western blot analysis. We also studied the effect of ethanol on expression of RA receptors in the cerebellar granule cells. Activation of RA receptors (DNA-binding activities) in response to high-dose ethanol was determined by electrophoretic mobility shift and supershift assays. Findings from these studies demonstrated that ethanol exposure reduced the expression of RARalpha/gamma while it increased the expression of RXRalpha/gamma in the cerebellum and in cerebellar granule neurons. Immuno-histological studies further strengthened the expression pattern of RA receptors in response to ethanol. The DNA-binding activity of RARs was reduced, while DNA-binding activity of RXRs was increased in response to ethanol exposure. For the first time, our studies have demonstrated that high-dose ethanol affects the expression and activation of RA receptors, which could impair the signaling events and induce harmful effects on the survival and differentiation of cerebellar granule cells. Taken together, these findings could provide insight into the treatment options for brain defects

  3. Experimental study on detection of electrostatic discharges generated by polymer granules inside a metal silo

    NASA Astrophysics Data System (ADS)

    Choi, Kwangseok; Mogami, Tomofumi; Suzuki, Teruo

    2014-04-01

    To detect electrostatic discharges generated by polymer granules within a metal silo, we developed a novel and simple electrostatic discharge detector that utilizes a photosensor. The novel detector consists of a photosensor module in a metal cylinder, an optical band-pass filter, a quartz glass, a power supply, an amplifier for the photosensor module, and a digital oscilloscope. In this study, we conducted experiments at a real pneumatic powder transport facility that includes a metal silo to evaluate the novel detector using polypropylene granules. To determine the performance of the novel detector, we observed the electrostatic discharge within the metal silo using a conventional image intensifier system. The results obtained from the experiments show that the novel detector worked well in this study. The signals obtained with the novel detector were identical to the electrostatic discharges obtained with the conventional image intensifier system. The greatest advantage of this novel detector is that it is effective even when placed under external lights. In addition, the influence of various optical band-pass filters on the performance of the novel detector was discussed. Our study confirmed that an optical band-pass filter with a center wavelength of λ 330 nm (λ1/2: 315-345 nm) was the best performer among the optical band-pass filters used in this study.

  4. Experimental study on detection of electrostatic discharges generated by polymer granules inside a metal silo.

    PubMed

    Choi, Kwangseok; Mogami, Tomofumi; Suzuki, Teruo

    2014-04-01

    To detect electrostatic discharges generated by polymer granules within a metal silo, we developed a novel and simple electrostatic discharge detector that utilizes a photosensor. The novel detector consists of a photosensor module in a metal cylinder, an optical band-pass filter, a quartz glass, a power supply, an amplifier for the photosensor module, and a digital oscilloscope. In this study, we conducted experiments at a real pneumatic powder transport facility that includes a metal silo to evaluate the novel detector using polypropylene granules. To determine the performance of the novel detector, we observed the electrostatic discharge within the metal silo using a conventional image intensifier system. The results obtained from the experiments show that the novel detector worked well in this study. The signals obtained with the novel detector were identical to the electrostatic discharges obtained with the conventional image intensifier system. The greatest advantage of this novel detector is that it is effective even when placed under external lights. In addition, the influence of various optical band-pass filters on the performance of the novel detector was discussed. Our study confirmed that an optical band-pass filter with a center wavelength of λ 330 nm (λ1/2: 315-345 nm) was the best performer among the optical band-pass filters used in this study.

  5. The effect of methylmercury exposure on behavior and cerebellar granule cell physiology in aged mice.

    PubMed

    Bellum, Sairam; Thuett, Kerry A; Bawa, Bhupinder; Abbott, Louise C

    2013-09-01

    Epidemiology studies have clearly documented that the central nervous system is highly susceptible to methylmercury toxicity, and exposure to this neurotoxicant in humans primarily results from consumption of contaminated fish. While the effects of methylmercury exposure have been studied in great detail, comparatively little is known about the effects of moderate to low dose methylmercury toxicity in the aging central nervous system. We examined the toxic effects of a moderate dose of methylmercury on the aging mouse cerebellum. Male and female C57BL/6 mice at 16-20 months of age were exposed to methylmercury by feeding a total dose of 5.0 mg kg(-1) body weight and assessed using four behavioral tests. Methylmercury-treated aged mice performed significantly worse in open field, footprint analysis and the vertical pole test compared with age-matched control mice. Isolated cerebellar granule cells from methylmercury-treated aged mice exhibited higher levels of reactive oxygen species and reduced mitochondrial membrane potentials, but no differences in basal intracellular calcium ion levels compared with age-matched control mice. When aged mice were exposed to a moderate dose of methylmercury, they exhibited a similar degree of impairment when compared with young adult mice exposed to the same moderate dose of methylmercury, as reported in earlier studies from this laboratory. Thus, at least in mice, exposure of the aged brain to moderate concentrations methylmercury does not pose greater risk compared with the young adult brain exposed to similar concentrations of methylmercury. Copyright © 2012 John Wiley & Sons, Ltd.

  6. Modification of drug release from acetaminophen granules by melt granulation technique - consideration of release kinetics.

    PubMed

    Uhumwangho, M U; Okor, R S

    2006-01-01

    Acetaminophen granules have been formed by a melt granulation process with the objective of retarding drug release for prolonged action formulations. The waxes used were goat wax, carnuba wax and glyceryl monostearate. In the melt granulation procedure, acetaminophen powder was triturated with the melted waxes and passed through a sieve of mesh 10 (aperture size 710 microm). The content of wax in resulting granules ranged from 10 to 40%w/w. Acetaminophen granules were also formed by the convectional method of wet granulation with starch mucilage (20%w/w). The granules were subjected to in-vitro drug release tests. The release data were subjected to analysis by three different well-established mathematical models (release kinetics) namely, - zero order flux, first order, and the Higuchi square root of time relationship. The convectional granules exhibited an initial zero order flux (first 55%) followed by a first order release profile (the remaining 45%). The pattern of drug release from the melt granulations was consistent with the first order kinetic and the Higuchi square root of time relationship, indicating a diffusion-controlled release mechanism. The first order release rate constant of the convectional granules was 1.95 +/- 0.02 h(-1). After melt granulation (wax content, 20%w/w) the rate constants dropped drastically to 0.130+/-0.001 h(-1) (goat wax), 0.120+/-0.003 h(-1) (carnuba wax), and 0.130+/-0.002 h(-1) (glyceryl monosterate) indicating that all three waxes were equivalent in retarding drug release from the melt granulations.

  7. Skin-derived neural precursors competitively generate functional myelin in adult demyelinated mice

    PubMed Central

    Mozafari, Sabah; Laterza, Cecilia; Roussel, Delphine; Bachelin, Corinne; Marteyn, Antoine; Deboux, Cyrille; Martino, Gianvito; Evercooren, Anne Baron-Van

    2015-01-01

    Induced pluripotent stem cell–derived (iPS-derived) neural precursor cells may represent the ideal autologous cell source for cell-based therapy to promote remyelination and neuroprotection in myelin diseases. So far, the therapeutic potential of reprogrammed cells has been evaluated in neonatal demyelinating models. However, the repair efficacy and safety of these cells has not been well addressed in the demyelinated adult CNS, which has decreased cell plasticity and scarring. Moreover, it is not clear if these induced pluripotent–derived cells have the same reparative capacity as physiologically committed CNS-derived precursors. Here, we performed a side-by-side comparison of CNS-derived and skin-derived neural precursors in culture and following engraftment in murine models of adult spinal cord demyelination. Grafted induced neural precursors exhibited a high capacity for survival, safe integration, migration, and timely differentiation into mature bona fide oligodendrocytes. Moreover, grafted skin–derived neural precursors generated compact myelin around host axons and restored nodes of Ranvier and conduction velocity as efficiently as CNS-derived precursors while outcompeting endogenous cells. Together, these results provide important insights into the biology of reprogrammed cells in adult demyelinating conditions and support use of these cells for regenerative biomedicine of myelin diseases that affect the adult CNS. PMID:26301815

  8. Application of tumbling melt granulation (TMG) method to prepare controlled-release fine granules.

    PubMed

    Maejima, T; Kubo, M; Osawa, T; Nakajima, K; Kobayashi, M

    1998-03-01

    The tumbling melt granulation (TMG) method was applied to prepare controlled-release fine granules of diltiazem hydrochloride (DH). The entire process, from the preparation of the cores by the adherence of DH to the sucrose crystal to the subsequent coating of the controlled-release layer, was performed without using any solvent. A mixture of meltable material, talc, and ethylcellulose was used for the controlled-release layer and controlled-release fine granules approximately 400 microns in diameter were obtained with excellent producibility. The dissolution rate of DH from these fine granules was similar to that of a once-a-day dosage form obtained in the market; further, the dependency of the dissolution profile on pH of the media was less. Thus, it was concluded that this TMG method was very useful for preparing not only controlled-release beads of granule size (usually 500 to 1400 microns) but also fine granules.

  9. A role for adult TLX-positive neural stem cells in learning and behaviour.

    PubMed

    Zhang, Chun-Li; Zou, Yuhua; He, Weimin; Gage, Fred H; Evans, Ronald M

    2008-02-21

    Neurogenesis persists in the adult brain and can be regulated by a plethora of external stimuli, such as learning, memory, exercise, environment and stress. Although newly generated neurons are able to migrate and preferentially incorporate into the neural network, how these cells are molecularly regulated and whether they are required for any normal brain function are unresolved questions. The adult neural stem cell pool is composed of orphan nuclear receptor TLX-positive cells. Here, using genetic approaches in mice, we demonstrate that TLX (also called NR2E1) regulates adult neural stem cell proliferation in a cell-autonomous manner by controlling a defined genetic network implicated in cell proliferation and growth. Consequently, specific removal of TLX from the adult mouse brain through inducible recombination results in a significant reduction of stem cell proliferation and a marked decrement in spatial learning. In contrast, the resulting suppression of adult neurogenesis does not affect contextual fear conditioning, locomotion or diurnal rhythmic activities, indicating a more selective contribution of newly generated neurons to specific cognitive functions.

  10. Registering parameters and granules of wave observations: IMAGE RPI success story

    NASA Astrophysics Data System (ADS)

    Galkin, I. A.; Charisi, A.; Fung, S. F.; Benson, R. F.; Reinisch, B. W.

    2015-12-01

    Modern metadata systems strive to help scientists locate data relevant to their research and then retrieve them quickly. Success of this mission depends on the organization and completeness of metadata. Each relevant data resource has to be registered; each content has to be described; each data file has to be accessible. Ultimately, data discoverability is about the practical ability to describe data content and location. Correspondingly, data registration has a "Parameter" level, at which content is specified by listing available observed properties (parameters), and a "Granule" level, at which download links are given to data records (granules). Until recently, both parameter- and granule-level data registrations were accomplished at NASA Virtual System Observatory easily by listing provided parameters and building Granule documents with URLs to the datafile locations, usually those at NASA CDAWeb data warehouse. With the introduction of the Virtual Wave Observatory (VWO), however, the parameter/granule concept faced a scalability challenge. The wave phenomenon content is rich with descriptors of the wave generation, propagation, interaction with propagation media, and observation processes. Additionally, the wave phenomenon content varies from record to record, reflecting changes in the constituent processes, making it necessary to generate granule documents at sub-minute resolution. We will present the first success story of registering 234,178 records of IMAGE Radio Plasma Imager (RPI) plasmagram data and Level 2 derived data products in ESPAS (near-Earth Space Data Infrastructure for e-Science), using the VWO-inspired wave ontology. The granules are arranged in overlapping display and numerical data collections. Display data include (a) auto-prospected plasmagrams of potential interest, (b) interesting plasmagrams annotated by human analysts or software, and (c) spectacular plasmagrams annotated by analysts as publication-quality examples of the RPI science

  11. Randomized, open-label, single-dose, crossover, relative bioavailability study in healthy adults, comparing the pharmacokinetics of rabeprazole granules administered using soft food or infant formula as dosing vehicle versus suspension.

    PubMed

    Thyssen, An; Solanki, Bhavna; Treem, William

    2012-07-01

    A sprinkle capsule formulation containing enteric-coated, delayed-release rabeprazole granules is being developed for the treatment of children with gastrointestinal reflux disease. The granules are designed to be mixed with vehicles that facilitate delivery to children, who may be unable to swallow solid formulations. The primary objective of this study-conducted on the sponsor's initiative-was to compare the bioavailability of rabeprazole granules when mixed with various dosing vehicles (small amount of soft food or infant formula) with that of a rabeprazole suspension with inactive vehicle granules (reference), to determine which dosing vehicle can be used to deliver rabeprazole in children. Tolerability was also assessed. This single-center, single-dose, randomized, open-label, 5-period crossover study was conducted in 35 healthy adult subjects. In a randomized sequence, fasting subjects received a single dose of 10-mg rabeprazole granules per treatment period, mixed with small amounts of 1 of 5 dosing vehicles (a strawberry-flavored suspension of rabeprazole granules with inactive vehicle granules reconstituted with water, yogurt [1 tablespoon], applesauce [1 tablespoon], or infant formula [5 mL], or a suspension of rabeprazole granules with inactive vehicle tablet reconstituted with water). Full plasma pharmacokinetic (PK) profiles of rabeprazole and its thioether metabolite were collected; concentrations were estimated via LC-MS/MS. PK properties were estimated using noncompartmental methods; 90% CIs around least squares mean test-to-reference ratios were calculated for C(max) and AUC values. All treatment-emergent adverse events (TEAEs) were recorded and assessed for severity (mild, moderate, or severe) and relationship to study drug. A total of 35 subjects were enrolled (mean age, 38 years; 54.3% female; 100% white; mean weight, 71.4 kg). Thirty-four subjects completed the study. Rabeprazole and rabeprazole thioether plasma PK properties were comparable

  12. Analysis of a mutant exhibiting conditional sorting to dense core secretory granules in Tetrahymena thermophila.

    PubMed

    Bowman, G R; Turkewitz, A P

    2001-12-01

    The formation of dense core granules (DCGs) requires both the sorting of granule contents from other secretory proteins and a postsorting maturation process. The Tetrahymena thermophila strain SB281 fails to synthesize DCGs, and previous analysis suggested that the defect lay at or near the sorting step. Because this strain represents one of the very few mutants in this pathway, we have undertaken a more complete study of the phenotype. Genetic epistasis analysis places the defect upstream of those in two other characterized Tetrahymena mutants. Using immunofluorescent detection of granule content proteins, as well as GFP tagging, we describe a novel cytoplasmic compartment to which granule contents can be sorted in growing SB281 cells. Cell fusion experiments indicate that this compartment is not a biosynthetic intermediate in DCG synthesis. Sorting in SB281 is strongly conditional with respect to growth. When cells are starved, the storage compartment is degraded and de novo synthesized granule proteins are rapidly secreted. The mutation in SB281 therefore appears to affect DCG synthesis at the level of both sorting and maturation.

  13. Analysis of a mutant exhibiting conditional sorting to dense core secretory granules in Tetrahymena thermophila.

    PubMed Central

    Bowman, G R; Turkewitz, A P

    2001-01-01

    The formation of dense core granules (DCGs) requires both the sorting of granule contents from other secretory proteins and a postsorting maturation process. The Tetrahymena thermophila strain SB281 fails to synthesize DCGs, and previous analysis suggested that the defect lay at or near the sorting step. Because this strain represents one of the very few mutants in this pathway, we have undertaken a more complete study of the phenotype. Genetic epistasis analysis places the defect upstream of those in two other characterized Tetrahymena mutants. Using immunofluorescent detection of granule content proteins, as well as GFP tagging, we describe a novel cytoplasmic compartment to which granule contents can be sorted in growing SB281 cells. Cell fusion experiments indicate that this compartment is not a biosynthetic intermediate in DCG synthesis. Sorting in SB281 is strongly conditional with respect to growth. When cells are starved, the storage compartment is degraded and de novo synthesized granule proteins are rapidly secreted. The mutation in SB281 therefore appears to affect DCG synthesis at the level of both sorting and maturation. PMID:11779800

  14. [In vitro generation of blood red cells from stem cells: a sketch of the future].

    PubMed

    Mazurier, Christelle; Douay, Luc

    2016-01-01

    Human adult pluripotent stem cells, stem cells of embryonic origin and induced pluripotent stem cells (iPS) provide cellular sources for new promising regenerative medicine approaches. Because these cells can be patient-specific, they allow considering a personalized medicine appropriate to the diagnosis of each. The generation of cultured red blood cells (cRBC) derived from stem cells is emblematic of personalized medicine. Indeed, these cells have the advantage of being selected according to a blood phenotype of interest and they may provide treatments to patients in situation of impossible transfusion (alloimmunized patients, rare phenotypes). Essential progresses have established proof of concept for this approach, still a concept some years ago. From adult stem cells, all steps of upstream research were successfully achieved, including the demonstration of the feasibility of injection into human. This leads us to believe that Red Blood Cells generated in vitro from stem cells will be the future players of blood transfusion. However, although theoretically ideal, these stem cells raise many biological challenges to overcome, although some tracks are identified. © Société de Biologie, 2016.

  15. Influence of granulating method on physical and mechanical properties, compression behavior, and compactibility of lactose and microcrystalline cellulose granules.

    PubMed

    Horisawa, E; Danjo, K; Sunada, H

    2000-06-01

    The physical and mechanical properties of lactose (LC) and microcrystalline cellulose (MCC) granules prepared by various granulating methods were determined, and their effects on the compression and strength of the tablets were examined. From the force-displacement curve obtained in a crushing test on a single granule, all LC granules appeared brittle, and MCC granules were somewhat plastically deformable. Inter-granular porosity epsilon inter clearly decreased with greater spherical granule shape for both materials. Decrease in intragranular porosity epsilon intra enhanced the crushing force of a single granule Fg. Agitating granulation brought about the most compactness and hardness of granules. In granule compression tests, the initial slope of Heckel plots K1 appeared closely related to ease of filling voids in a granule bed by the slippage or rolling of granules. The reciprocal of the slope in the succeeding step 1/K2 in compression of MCC granules indicated positive correlation to Fg, while in LC granules, no such obvious relation was evident. 1/K2 differed only slightly among granulating methods. Tensile strength of tablets Tt obtained by compression of various LC granules was low as a whole and was little influenced by granulating method. For MCC granules, which are plastically deformable, tablet strength greatly depended on granulation. Granules prepared by extruding or dry granulation gave strong tablets. Tablets prepared from granules made by the agitating method showed particularly low Tt. From stereomicroscopic observation, the contact area between granule particles in a tablet appeared smaller; this would explain the decrease in inter-granular bond formation.

  16. TDP-43 is directed to stress granules by sorbitol, a novel physiological osmotic and oxidative stressor.

    PubMed

    Dewey, Colleen M; Cenik, Basar; Sephton, Chantelle F; Dries, Daniel R; Mayer, Paul; Good, Shannon K; Johnson, Brett A; Herz, Joachim; Yu, Gang

    2011-03-01

    TDP-43, or TAR DNA-binding protein 43, is a pathological marker of a spectrum of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. TDP-43 is an RNA/DNA-binding protein implicated in transcriptional and posttranscriptional regulation. Recent work also suggests that TDP-43 associates with cytoplasmic stress granules, which are transient structures that form in response to stress. In this study, we establish sorbitol as a novel physiological stressor that directs TDP-43 to stress granules in Hek293T cells and primary cultured glia. We quantify the association of TDP-43 with stress granules over time and show that stress granule association and size are dependent on the glycine-rich region of TDP-43, which harbors the majority of pathogenic mutations. Moreover, we establish that cells harboring wild-type and mutant TDP-43 have distinct stress responses: mutant TDP-43 forms significantly larger stress granules, and is incorporated into stress granules earlier, than wild-type TDP-43; in striking contrast, wild-type TDP-43 forms more stress granules over time, but the granule size remains relatively unchanged. We propose that mutant TDP-43 alters stress granule dynamics, which may contribute to the progression of TDP-43 proteinopathies.

  17. The combined effect of wet granulation process parameters and dried granule moisture content on tablet quality attributes.

    PubMed

    Gabbott, Ian P; Al Husban, Farhan; Reynolds, Gavin K

    2016-09-01

    A pharmaceutical compound was used to study the effect of batch wet granulation process parameters in combination with the residual moisture content remaining after drying on granule and tablet quality attributes. The effect of three batch wet granulation process parameters was evaluated using a multivariate experimental design, with a novel constrained design space. Batches were characterised for moisture content, granule density, crushing strength, porosity, disintegration time and dissolution. Mechanisms of the effect of the process parameters on the granule and tablet quality attributes are proposed. Water quantity added during granulation showed a significant effect on granule density and tablet dissolution rate. Mixing time showed a significant effect on tablet crushing strength, and mixing speed showed a significant effect on the distribution of tablet crushing strengths obtained. The residual moisture content remaining after granule drying showed a significant effect on tablet crushing strength. The effect of moisture on tablet tensile strength has been reported before, but not in combination with granulation parameters and granule properties, and the impact on tablet dissolution was not assessed. Correlations between the energy input during granulation, the density of granules produced, and the quality attributes of the final tablets were also identified. Understanding the impact of the granulation and drying process parameters on granule and tablet properties provides a basis for process optimisation and scaling. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Glomerular and Mitral-Granule Cell Microcircuits Coordinate Temporal and Spatial Information Processing in the Olfactory Bulb.

    PubMed

    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.

  19. Granule fraction inhomogeneity of calcium carbonate/sorbitol in roller compacted granules.

    PubMed

    Bacher, C; Olsen, P M; Bertelsen, P; Sonnergaard, J M

    2008-02-12

    The granule fraction inhomogeneity of roller compacted granules was examined on mixtures of three different morphologic forms of calcium carbonate and three particle sizes of sorbitol. The granule fraction inhomogeneity was determined by the distribution of the calcium carbonate in each of the 10 size fractions between 0 and 2000 microm and by calculating the demixing potential. Significant inhomogeneous occurrence of calcium carbonate in the size fractions was demonstrated, depending mostly on the particles sizes of sorbitol but also on the morphological forms of calcium carbonate. The heterogeneous distribution of calcium carbonate was related to the decrease in compactibility of roller compacted granules in comparison to the ungranulated materials. This phenomenon was explained by a mechanism where fracturing of the ribbon during granulation occurred at the weakest interparticulate bonds (the calcium carbonate: calcium carbonate bonds) and consequently exposed the weakest areas of bond formation on the surface of the granules. Accordingly, the non-uniform allocation of the interparticulate attractive forces in a tablet would cause a lowering of the compactibility. Furthermore, the ability of the powder to agglomerate in the roller compactor was demonstrated to be related to the ability of the powder to be compacted into a tablet, thus the most compactable calcium carbonate and the smallest sized sorbitol improved the homogeneity by decreasing the demixing potential.

  20. 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

  1. Probing the role of nonmuscle tropomyosin isoforms in intracellular granule movement by microinjection of monoclonal antibodies

    PubMed Central

    1989-01-01

    Chicken embryo fibroblast (CEF) cells were microinjected with several different monoclonal antibodies that recognize certain nonmuscle isoforms of tropomyosin. Immediately after injection, cells were recorded with a time-lapse video imaging system; later analysis of the tapes revealed that particles in cells injected with one of these antibodies (CG1, specific for CEF tropomyosin isoforms 1 and 3) showed a dramatic decrease in instantaneous speed while moving, distance moved per saltation, and proportion of time spent in motion. Injection of Fab fragments of CG1 resulted in similar changes in the pattern of granule movement. This inhibition of granule movement by CG1 antibody was reversible; at 2.5 h after injection, granules in injected cells had already reached three-fourths of normal speed. The speed of granule movement in cells injected either with antibody specific for tropomyosin isoforms not present in CEF cells, or with CG1 antibody preabsorbed with tropomyosin, was not significantly different from the speed of granules in uninjected cells. When cells were injected with CG1 or Fab fragments of CG1, fixed, and counter-stained with rabbit antibodies to reveal the microtubule, microfilament, and intermediate filament systems, no obvious differences from the patterns normally seen in uninjected cells were observed. Examination of the ultrastructure of injected cells by EM confirmed the presence of apparently intact and normal microtubule, actin, and intermediate filament networks. These experiments suggest that tropomyosin may play an important role in the movement of vesicles and organelles in the cell cytoplasm. Also, we have shown previously that the CG1 determinant can undergo a motility-dependent change in reactivity, that may be important for the regulatory function of nonmuscle tropomyosin (Hegmann, T. E., J. L.-C. Lin, and J. J.-C. Lin. 1988. J. Cell Biol. 106:385-393). Therefore, in addition to postulated microtubule-based motors, microfilaments may

  2. Multi-species impurity granule injection and mass deposition projections in NSTX-U discharges

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lunsford, R.; Bortolon, A.; Roquemore, A. L.

    Here, by employing a neutral gas shielding (NGS) model to characterize impurity granule injection, the ablation rates for three different species of granule: lithium, boron, and carbon, are determined. Utilizing the duration of ablation events recorded on experiments performed at DIII-D to calibrate the NGS model, we quantify the ablation rate with respect to the plasma density profile. The species-specific granule shielding constant is then used to model granule ablation within NSTX-U discharges. Simulations of 300, 500 and 700 micron diameter granules injected at 50 m s –1 are presented for NSTX-U L-mode type plasmas, as well as H-mode dischargesmore » with low natural ELM frequency. Additionally, ablation calculations of 500 micron granules of each species are presented at velocities ranging from 50–150 m s –1. In H-mode discharges these simulations show that the majority of the injected granule is ablated within or just past the edge steep gradient region. At this radial position, the perturbation to the background plasma generated by the ablating granule can lead to conditions advantageous for the rapid triggering of ELM crashes.« less

  3. Multi-species impurity granule injection and mass deposition projections in NSTX-U discharges

    DOE PAGES

    Lunsford, R.; Bortolon, A.; Roquemore, A. L.; ...

    2017-05-16

    Here, by employing a neutral gas shielding (NGS) model to characterize impurity granule injection, the ablation rates for three different species of granule: lithium, boron, and carbon, are determined. Utilizing the duration of ablation events recorded on experiments performed at DIII-D to calibrate the NGS model, we quantify the ablation rate with respect to the plasma density profile. The species-specific granule shielding constant is then used to model granule ablation within NSTX-U discharges. Simulations of 300, 500 and 700 micron diameter granules injected at 50 m s –1 are presented for NSTX-U L-mode type plasmas, as well as H-mode dischargesmore » with low natural ELM frequency. Additionally, ablation calculations of 500 micron granules of each species are presented at velocities ranging from 50–150 m s –1. In H-mode discharges these simulations show that the majority of the injected granule is ablated within or just past the edge steep gradient region. At this radial position, the perturbation to the background plasma generated by the ablating granule can lead to conditions advantageous for the rapid triggering of ELM crashes.« less

  4. [Preparation and evaluation of taste masked orally disintegrating tablets with granules made by the wet granulation method].

    PubMed

    Kawano, Yayoi; Ito, Akihiko; Sasatsu, Masanaho; Machida, Yoshiharu; Onishi, Hiraku

    2010-12-01

    Using furosemide (FU) as a model drug, we examined the wet granulation method as a way to improve the taste masking and physical characteristics of orally disintegrating tablets (ODTs). In the wet granulation method, yogurt powder (YO) was used as a corrective and maltitol (MA) was used as a binding agent. The taste masked FU tablets were prepared using the direct compression method. Microcrystalline cellulose (Avicel® PH-302) and mannitol were added as excipients at a mixing ratio of 1/1 by weight. Based on the results of sensory test on taste, the prepared granules markedly improved the taste of FU, and a sufficient masking effect was obtained at the YO/FU ratio of 1 or more. Furthermore, it was found that the masking effect achieved by YO granules made with the wet granulation method was similar to or better than that produced by the granules made with dry granulation method. All types of tablets displayed sufficient hardness (over 3.5×10(-2) kN), and rapidly disintegrating tablets were obtained with YO granules produced at a mixing ratio of FU/YO=1/1, which disintegrated within 20 s. Disintegration time lengthened as the mixing ratio of YO to FU increased. In the mixing ratio of FU/YO=1/1, the hardness of tablets with granules made by the wet granulation method exceeded that of tablets with granules made by the dry granulation method, with minimal differences in disintegration time. The hardness and disintegration time of the tablets with granules made by the wet granulation method could be controlled by varying the compression force. In conclusion, YO was found to be a useful additive for masking unpleasant tastes. FU ODTs with improved taste, rapid disintegration and greater hardness could be prepared with YO-containing granules made by the wet granulation method using MA as a binding agent.

  5. Composition of the carbohydrate granules of the cyanobacterium, Cyanothece sp. strain ATCC 51142

    NASA Technical Reports Server (NTRS)

    Schneegurt, M. A.; Sherman, D. M.; Sherman, L. A.; Mitchell, C. A. (Principal Investigator)

    1997-01-01

    Cyanothece sp. strain ATCC 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that temporally separates O2-sensitive N2 fixation from oxygenic photosynthesis. The energy and reducing power needed for N2 fixation appears to be generated by an active respiratory apparatus that utilizes the contents of large interthylakoidal carbohydrate granules. We report here on the carbohydrate and protein composition of the granules of Cyanothece sp. strain ATCC 51142. The carbohydrate component is a glucose homopolymer with branches every nine residues and is chemically identical to glycogen. Granule-associated protein fractions showed temporal changes in the number of proteins and their abundance during the metabolic oscillations observed under diazotrophic conditions. There also were temporal changes in the protein pattern of the granule-depleted supernatant fractions from diazotrophic cultures. None of the granule-associated proteins crossreacted with antisera directed against several glycogen-metabolizing enzymes or nitrogenase, although these proteins were tentatively identified in supernatant fractions. It is suggested that the granule-associated proteins are structural proteins required to maintain a complex granule architecture.

  6. [A method for the primary culture of fibroblasts isolated from human airway granulation tissues].

    PubMed

    Chen, Nan; Zhang, Jie; Xu, Min; Wang, Yu-ling; Pei, Ying-hua

    2013-04-01

    To establish a feasible method to culture primary fibroblasts isolated from human airway granulation tissues, and therefore to provide experimental data for the investigation of the pathogenesis of benign airway stenosis. The granulation tissues were collected from 6 patients during routine bronchoscopy at our department of Beijing Tiantan Hospital from April to June 2011. Primary fibroblasts were obtained by culturing the explanted tissues. Cell growth was observed under inverted microscope. All of these 6 primary cultures were successful. Fibroblast-like cells were observed to migrate from the tissue pieces 3 d after inoculation. After 9-11 d of culture, cells reached to 90% confluence and could be sub-cultured. After passage, the cells were still in a typical elongated spindle-shape and grew well. The cells could be sub-cultured further when they formed a monolayer. Explant culture is a reliable method for culturing primary fibroblasts from human airway granulation tissues.

  7. Glomerular parietal epithelial cells of adult murine kidney undergo EMT to generate cells with traits of renal progenitors

    PubMed Central

    G, Swetha; Chandra, Vikash; Phadnis, Smruti; Bhonde, Ramesh

    2011-01-01

    Abstract Glomerular parietal epithelial cells (GPECs) are known to revert to embryonic phenotype in response to renal injury. However, the mechanism of de-differentiation in GPECs and the underlying cellular processes are not fully understood. In the present study, we show that cultured GPECs of adult murine kidney undergo epithelial-mesenchymal transition (EMT) to generate cells, which express CD24, CD44 and CD29 surface antigens. Characterization by qRT-PCR and immunostaining of these clonogenic cells demonstrate that they exhibit metastable phenotype with co-expression of both epithelial (cytokeratin-18) and mesenchymal (vimentin) markers. Transcript analysis by qRT-PCR revealed high expression of metanephric mesenchymal (Pax-2, WT-1, Six-1, Eya-1, GDNF) and uteric bud (Hoxb-7, C-Ret) genes in these cells, indicating their bipotent progenitor status. Incubation of GPECs with EMT blocker Prostaglandin E2, resulted in low expression of renal progenitor markers reflecting the correlation between EMT and acquired stemness in these cells. Additional in vitro renal commitment assays confirmed their functional staminality. When injected into E13.5 kidney rudiments, the cells incorporated into the developing kidney primordia and co-culture with E13.5 spinal cord resulted in branching and tubulogenesis in these cells. When implanted under renal capsule of unilaterally nephrectomized mice, these cells differentiated into immature glomeruli and vascular ducts. Our study demonstrates that EMT plays a major role in imparting plasticity to terminally differentiated GPECs by producing metastable cells with traits of kidney progenitors. The present study would improve our understanding on epithelial cell plasticity, furthering our knowledge of its role in renal repair and regeneration. PMID:19840197

  8. Stress granule formation via ATP depletion-triggered phase separation

    NASA Astrophysics Data System (ADS)

    Wurtz, Jean David; Lee, Chiu Fan

    2018-04-01

    Stress granules (SG) are droplets of proteins and RNA that form in the cell cytoplasm during stress conditions. We consider minimal models of stress granule formation based on the mechanism of phase separation regulated by ATP-driven chemical reactions. Motivated by experimental observations, we identify a minimal model of SG formation triggered by ATP depletion. Our analysis indicates that ATP is continuously hydrolysed to deter SG formation under normal conditions, and we provide specific predictions that can be tested experimentally.

  9. DAZL is essential for stress granule formation implicated in germ cell survival upon heat stress.

    PubMed

    Kim, Byunghyuk; Cooke, Howard J; Rhee, Kunsoo

    2012-02-01

    Mammalian male germ cells should be maintained below body temperature for proper development. Here, we investigated how male germ cells respond to heat stress. A short exposure of mouse testes to core body temperature induced phosphorylation of eIF2α and the formation of stress granules (SGs) in male germ cells. We observed that DAZL, a germ cell-specific translational regulator, was translocated to SGs upon heat stress. Furthermore, SG assembly activity was significantly diminished in the early male germ cells of Dazl-knockout mice. The DAZL-containing SGs played a protective role against heat stress-induced apoptosis by the sequestration of specific signaling molecules, such as RACK1, and the subsequent blockage of the apoptotic MAPK pathway. Based on these results, we propose that DAZL is an essential component of the SGs, which prevent male germ cells from undergoing apoptosis upon heat stress.

  10. Surface Modification of Porous Titanium Granules for Improving Bioactivity.

    PubMed

    Karaji, Zahra Gorgin; Houshmand, Behzad; Faghihi, Shahab

    The highly porous titanium granules are currently being used as bone substitute material and for bone tissue augmentation. However, they suffer from weak bone bonding ability. The aim of this study was to create a nanostructured surface oxide layer on irregularly shaped titanium granules to improve their bioactivity. This could be achieved using optimized electrochemical anodic oxidation (anodizing) and heat treatment processes. The anodizing process was done in an ethylene glycol-based electrolyte at an optimized condition of 60 V for 3 hours. The anodized granules were subsequently annealed at 450°C for 1 hour. Scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy (EDS), and x-ray diffraction (XRD) were used to characterize the surface structure and morphology of the granules. The in vitro bioactivity of the samples was evaluated by immersion of specimens in simulated body fluid (SBF) for 1, 2, and 3 weeks. The human osteoblastic sarcoma cell line, MG63, was used to evaluate cell viability on the samples using dimethylthiazol-diphenyl tetrazolium bromide (MTT) assay. The results demonstrated the formation of amorphous nanostructured titanium oxide after anodizing, which transformed to crystalline anatase and rutile phases upon heat treatment. After immersion in SBF, spherical aggregates of amorphous calcium phosphate were formed on the surface of the anodized sample, which turned into crystalline hydroxyapatite on the surface of the anodized annealed sample. No cytotoxicity was detected among the samples. It is suggested that anodic oxidation followed by heat treatment could be used as an effective surface treatment procedure to improve bioactivity of titanium granules implemented for bone tissue repair and augmentation.

  11. Early postnatal exposure to methylphenidate alters stress reactivity and increases hippocampal ectopic granule cells in adult rats

    PubMed Central

    Torres-Reveron, Annelyn; Gray, Jason D.; Melton, Jay T.; Punsoni, Michael; Tabori, Nora E.; Ward, Mary J.; Frys, Kelly; Iadecola, Costantino; Milner, Teresa A.

    2009-01-01

    To mimic clinical treatment with methylphenidate (MPH; Ritalin) for attention deficit/hyperactivity disorder (ADHD), rat pups were injected with MPH (5 mg/kg, I.P.) or placebo twice daily during their nocturnal active phase from postnatal day (PND) 7 to 35. Thirty-nine days after the last MPH administration (PND76), four litters of rats experienced stressful conditions during the 2003 New York City blackout. MPH-treated rats that endured the blackout lost more weight and regained it at a slower pace than controls (p<0.05; N=7–11/group). Furthermore, MPH-treated rats had elevated systolic arterial blood pressure (from 115.6 ± 1.2 to 126 ± 1.8 mmHg; p<0.05), assessed on PND130 by tail cuff plethysmography. Immunocytochemical studies of transmitter systems in the brain demonstrated rearrangements of catecholamine and neuropeptide Y fibers in select brain regions at PND135, which did not differ between blackout and control groups. However, MPH-treated rats that endured the blackout had more ectopic granule cells in the hilus of the dorsal hippocampal dentate gyrus compared to controls at PND 135 (p<0.05; N=6/group). These findings indicate that early postnatal exposure to high therapeutic doses of MPH can have long lasting effects on the plasticity of select brain regions and can induce changes in the reactivity to stress that persist into adulthood. PMID:19100815

  12. Granule Exocytosis Contributes to Priming and Activation of the Human Neutrophil Respiratory Burst

    PubMed Central

    Uriarte, Silvia M.; Rane, Madhavi J.; Luerman, Gregory C.; Barati, Michelle T.; Ward, Richard A.; Nauseef, William M.; McLeish, Kenneth R.

    2013-01-01

    The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT–SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT–SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT–SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT–SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT–SNAP-23 inhibited the increase in plasma membrane expression of gp91phox in TNF-α–primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase. PMID:21642540

  13. Distribution Profile of Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channels in α and β Cells of Pancreas: Dominant Localization in Secretory Granules and Common Error in Identification of Secretory Granule Membranes.

    PubMed

    Hur, Yong Suk; Yoo, Seung Hyun

    2015-01-01

    The α and β cells of pancreatic islet release important hormones in response to intracellular Ca increases that result from Ca releases through the inositol 1,4,5-trisphoshate receptor (IP3R)/Ca channels. Yet no systematic studies on distribution of IP3R/Ca channels have been done, prompting us to investigate the distribution of all 3 IP3R isoforms. Immunogold electron microscopy was performed to determine the presence and the relative concentrations of all 3 IP3R isoforms in 2 major organelles secretory granules (SGs) and the endoplasmic reticulum of α and β cells of rat pancreas. All 3 IP3R isoforms were present in SG membranes of both cells, and the IP3R concentrations in SGs were ∼2-fold higher than those in the endoplasmic reticulum. Moreover, large halos shown in the electron microscope images of insulin-containing SGs of β cells were gap spaces that resulted from separation of granule membranes from the surrounding cytoplasm. These results strongly suggest the important roles of SGs in IP3-induced, Ca-dependent regulatory secretory pathway in pancreas. Moreover, the accurate location of SG membranes of β cells was further confirmed by the location of another integral membrane protein synaptotagmin V and of membrane phospholipid PI(4,5)P2.

  14. Analysis of the release process of phenylpropanolamine hydrochloride from ethylcellulose matrix granules V. Release properties of ethylcellulose layered matrix granules.

    PubMed

    Fukui, Atsuko; Fujii, Ryuta; Yonezawa, Yorinobu; Sunada, Hisakazu

    2008-04-01

    In the pharmaceutical preparation of a controlled release drug, it is very important and necessary to understand the release properties. In previous papers, a combination of the square-root time law and cube-root law equations was confirmed to be a useful equation for qualitative treatment. It was also confirmed that the combination equation could analyze the release properties of layered granules as well as matrix granules. The drug release property from layered granules is different from that of matrix granules. A time lag occurs before release, and the entire release property of layered granules was analyzed using the combination of the square-root time law and cube-root law equations. It is considered that the analysis method is very useful and efficient for both matrix and layered granules. Comparing the granulation methods, it is easier to control the manufacturing process by tumbling granulation (method B) than by tumbling-fluidized bed granulation (method C). Ethylcellulose (EC) layered granulation by a fluidized bed granulator might be convenient for the preparation of controlled release dosage forms as compared with a tumbling granulator, because the layered granules prepared by the fluidized bed granulator can granulate and dry at the same time. The time required for drying by the fluidized bed granulator is shorter than that by the tumbling granulator, so the fluidized bed granulator is convenient for preparation of granules in handling and shorter processing time than the tumbling granulator. It was also suggested that the EC layered granules prepared by the fluidized bed granulator were suitable for a controlled release system as well as the EC matrix granules.

  15. Impaired neuronal maturation of hippocampal neural progenitor cells in mice lacking CRAF.

    PubMed

    Pfeiffer, Verena; Götz, Rudolf; Camarero, Guadelupe; Heinsen, Helmut; Blum, Robert; Rapp, Ulf Rüdiger

    2018-01-01

    RAF kinases are major constituents of the mitogen activated signaling pathway, regulating cell proliferation, differentiation and cell survival of many cell types, including neurons. In mammals, the family of RAF proteins consists of three members, ARAF, BRAF, and CRAF. Ablation of CRAF kinase in inbred mouse strains causes major developmental defects during fetal growth and embryonic or perinatal lethality. Heterozygous germline mutations in CRAF result in Noonan syndrome, which is characterized by neurocognitive impairment that may involve hippocampal physiology. The role of CRAF signaling during hippocampal development and generation of new postnatal hippocampal granule neurons has not been examined and may provide novel insight into the cause of hippocampal dysfunction in Noonan syndrome. In this study, by crossing CRAF-deficiency to CD-1 outbred mice, a CRAF mouse model was established which enabled us to investigate the interplay of neural progenitor proliferation and postmitotic differentiation during adult neurogenesis in the hippocampus. Albeit the general morphology of the hippocampus was unchanged, CRAF-deficient mice displayed smaller granule cell layer (GCL) volume at postnatal day 30 (P30). In CRAF-deficient mice a substantial number of abnormal, chromophilic, fast dividing cells were found in the subgranular zone (SGZ) and hilus of the dentate gyrus (DG), indicating that CRAF signaling contributes to hippocampal neural progenitor proliferation. CRAF-deficient neural progenitor cells showed an increased cell death rate and reduced neuronal maturation. These results indicate that CRAF function affects postmitotic neural cell differentiation and points to a critical role of CRAF-dependent growth factor signaling pathway in the postmitotic development of adult-born neurons.

  16. Impaired neuronal maturation of hippocampal neural progenitor cells in mice lacking CRAF

    PubMed Central

    Götz, Rudolf; Camarero, Guadelupe; Heinsen, Helmut; Blum, Robert; Rapp, Ulf Rüdiger

    2018-01-01

    RAF kinases are major constituents of the mitogen activated signaling pathway, regulating cell proliferation, differentiation and cell survival of many cell types, including neurons. In mammals, the family of RAF proteins consists of three members, ARAF, BRAF, and CRAF. Ablation of CRAF kinase in inbred mouse strains causes major developmental defects during fetal growth and embryonic or perinatal lethality. Heterozygous germline mutations in CRAF result in Noonan syndrome, which is characterized by neurocognitive impairment that may involve hippocampal physiology. The role of CRAF signaling during hippocampal development and generation of new postnatal hippocampal granule neurons has not been examined and may provide novel insight into the cause of hippocampal dysfunction in Noonan syndrome. In this study, by crossing CRAF-deficiency to CD-1 outbred mice, a CRAF mouse model was established which enabled us to investigate the interplay of neural progenitor proliferation and postmitotic differentiation during adult neurogenesis in the hippocampus. Albeit the general morphology of the hippocampus was unchanged, CRAF-deficient mice displayed smaller granule cell layer (GCL) volume at postnatal day 30 (P30). In CRAF-deficient mice a substantial number of abnormal, chromophilic, fast dividing cells were found in the subgranular zone (SGZ) and hilus of the dentate gyrus (DG), indicating that CRAF signaling contributes to hippocampal neural progenitor proliferation. CRAF-deficient neural progenitor cells showed an increased cell death rate and reduced neuronal maturation. These results indicate that CRAF function affects postmitotic neural cell differentiation and points to a critical role of CRAF-dependent growth factor signaling pathway in the postmitotic development of adult-born neurons. PMID:29590115

  17. Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bello, Oscar Daniel; Cappa, Andrea Isabel; Paola, Matilde de

    Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blotmore » analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs. - Highlights: • Rab3A has a similar migration pattern to cortical granules in mouse oocytes. • Rab3A can be a

  18. Independent transport and sorting of functionally distinct protein families in Tetrahymena thermophila dense core secretory granules.

    PubMed

    Rahaman, Abdur; Miao, Wei; Turkewitz, Aaron P

    2009-10-01

    Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, DeltaGRT1 DeltaGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in DeltaGRT1 DeltaGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from DeltaGRT1 DeltaGRT2 cells appear less adhesive than those from the wild type.

  19. Phenotypic properties and microbial diversity of methanogenic granules from a full-scale upflow anaerobic sludge bed reactor treating brewery wastewater.

    PubMed

    Díaz, Emiliano E; Stams, Alfons J M; Amils, Ricardo; Sanz, José L

    2006-07-01

    Methanogenic granules from an anaerobic bioreactor that treated wastewater of a beer brewery consisted of different morphological types of granules. In this study, the microbial compositions of the different granules were analyzed by molecular microbiological techniques: cloning, denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH), and scanning and transmission electron microscopy. We propose here that the different types of granules reflect the different stages in the life cycle of granules. Young granules were small, black, and compact and harbored active cells. Gray granules were the most abundant granules. These granules have a multilayer structure with channels and void areas. The core was composed of dead or starving cells with low activity. The brown granules, which were the largest granules, showed a loose and amorphous structure with big channels that resulted in fractured zones and corresponded to the older granules. Firmicutes (as determined by FISH) and Nitrospira and Deferribacteres (as determined by cloning and sequencing) were the predominant Bacteria. Remarkably, Firmicutes could not be detected in the brown granules. The methanogenic Archaea identified were Methanosaeta concilii (70 to 90% by FISH and cloning), Methanosarcina mazei, and Methanospirillum spp. The phenotypic appearance of the granules reflected the physiological condition of the granules. This may be valuable to easily select appropriate seed sludges to start up other reactors.

  20. Large-scale horizontal flows from SOUP observations of solar granulation

    NASA Technical Reports Server (NTRS)

    November, L. J.; Simon, G. W.; Tarbell, T. D.; Title, A. M.; Ferguson, S. H.

    1987-01-01

    Using high resolution time sequence photographs of solar granulation from the SOUP experiment on Spacelab 2, large scale horizontal flows were observed in the solar surface. The measurement method is based upon a local spatial cross correlation analysis. The horizontal motions have amplitudes in the range 300 to 1000 m/s. Radial outflow of granulation from a sunspot penumbra into surrounding photosphere is a striking new discovery. Both the supergranulation pattern and cellular structures having the scale of mesogranulation are seen. The vertical flows that are inferred by continuity of mass from these observed horizontal flows have larger upflow amplitudes in cell centers than downflow amplitudes at cell boundaries.

  1. Large-scale horizontal flows from SOUP observations of solar granulation

    NASA Astrophysics Data System (ADS)

    November, L. J.; Simon, G. W.; Tarbell, T. D.; Title, A. M.; Ferguson, S. H.

    1987-09-01

    Using high-resolution time-sequence photographs of solar granulation from the SOUP experiment on Spacelab 2 the authors observed large-scale horizontal flows in the solar surface. The measurement method is based upon a local spatial cross correlation analysis. The horizontal motions have amplitudes in the range 300 to 1000 m/s. Radial outflow of granulation from a sunspot penumbra into the surrounding photosphere is a striking new discovery. Both the supergranulation pattern and cellular structures having the scale of mesogranulation are seen. The vertical flows that are inferred by continuity of mass from these observed horizontal flows have larger upflow amplitudes in cell centers than downflow amplitudes at cell boundaries.

  2. Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo

    PubMed Central

    Ren, Wenwen; Lewandowski, Brian C.; Watson, Jaime; Aihara, Eitaro; Iwatsuki, Ken; Bachmanov, Alexander A.; Margolskee, Robert F.; Jiang, Peihua

    2014-01-01

    Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5+) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5+ or Lgr6+ cells from taste tissue can generate continuously expanding 3D structures (“organoids”). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2’-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5+ cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6+ cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5+ or Lgr6+ cells, validating the use of this model for the study of taste cell generation. PMID:25368147

  3. Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo.

    PubMed

    Ren, Wenwen; Lewandowski, Brian C; Watson, Jaime; Aihara, Eitaro; Iwatsuki, Ken; Bachmanov, Alexander A; Margolskee, Robert F; Jiang, Peihua

    2014-11-18

    Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5(+)) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5(+) or Lgr6(+) cells from taste tissue can generate continuously expanding 3D structures ("organoids"). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2'-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5(+) cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6(+) cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5(+) or Lgr6(+) cells, validating the use of this model for the study of taste cell generation.

  4. Generation of Mast Cells from Mouse Fetus: Analysis of Differentiation and Functionality, and Transcriptome Profiling Using Next Generation Sequencer

    PubMed Central

    Fukuishi, Nobuyuki; Igawa, Yuusuke; Kunimi, Tomoyo; Hamano, Hirofumi; Toyota, Masao; Takahashi, Hironobu; Kenmoku, Hiromichi; Yagi, Yasuyuki; Matsui, Nobuaki; Akagi, Masaaki

    2013-01-01

    While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy. PMID:23573287

  5. Common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cameron, R.S.; Cameron, P.L.; Castle, J.D.

    1986-10-01

    A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues - pancreatic, lacrimal, and submandibular - from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pImore » and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.« less

  6. Continuous melt granulation: Influence of process and formulation parameters upon granule and tablet properties.

    PubMed

    Monteyne, Tinne; Vancoillie, Jochem; Remon, Jean-Paul; Vervaet, Chris; De Beer, Thomas

    2016-10-01

    The pharmaceutical industry has a growing interest in alternative manufacturing models allowing automation and continuous production in order to improve process efficiency and reduce costs. Implementing a switch from batch to continuous processing requires fundamental process understanding and the implementation of quality-by-design (QbD) principles. The aim of this study was to examine the relationship between formulation-parameters (type binder, binder concentration, drug-binder miscibility), process-parameters (screw speed, powder feed rate and granulation temperature), granule properties (size, size distribution, shape, friability, true density, flowability) and tablet properties (tensile strength, friability, dissolution rate) of four different drug-binder formulations using Design of experiments (DOE). Two binders (polyethylene glycol (PEG) and Soluplus®) with a different solid state, semi-crystalline vs amorphous respectively, were combined with two model-drugs, metoprolol tartrate (MPT) and caffeine anhydrous (CAF), both having a contrasting miscibility with the binders. This research revealed that the granule properties of miscible drug-binder systems depended on the powder feed rate and barrel filling degree of the granulator whereas the granule properties of immiscible systems were mainly influenced by binder concentration. Using an amorphous binder, the tablet tensile strength depended on the granule size. In contrast, granule friability was more important for tablet quality using a brittle binder. However, this was not the case for caffeine-containing blends, since these phenomena were dominated by the enhanced compression properties of caffeine Form I, which was formed during granulation. Hence, it is important to gain knowledge about formulation behavior during processing since this influences the effect of process parameters onto the granule and tablet properties. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons

    PubMed Central

    Bosch, Carles; Martínez, Albert; Masachs, Nuria; Teixeira, Cátia M.; Fernaud, Isabel; Ulloa, Fausto; Pérez-Martínez, Esther; Lois, Carlos; Comella, Joan X.; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2015-01-01

    The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM) and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM) allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs) in mice. 3D reconstruction of dendritic spines in GCs aged 3–4 and 8–9 weeks revealed two different stages of dendritic spine development and unexpected features of synapse formation, including vacant and branched dendritic spines and presynaptic terminals establishing synapses with up to 10 dendritic spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner. PMID:26052271

  8. Adult pituitary stem cells: from pituitary plasticity to adenoma development.

    PubMed

    Florio, Tullio

    2011-01-01

    The pituitary needs high plasticity of the hormone-producing cell compartment to generate the continuously changing hormonal signals that govern the key physiological processes it is involved in, as well as homeostatic cell turnover. However, the underlying mechanisms are still poorly understood. It was proposed that adult stem cells direct the generation of newborn cells with a hormonal phenotype according to the physiological requirements. However, only in recent years adult pituitary stem cells have begun to be phenotypically characterized in several studies that identified multiple stem/progenitor cell candidates. Also considering the incompletely defined features of this cell subpopulation, some discrepancies among the different reports are clearly apparent and long-term self-renewal remains to be unequivocally demonstrated. Here, all the recently published evidence is analyzed, trying, when possible, to reconcile the results of the different studies. Finally, with the perspective of shedding light on pituitary tumorigenesis and the development of potentially new pharmacological approaches directed against these cells, very recent evidence on the presence of putative cancer stem cells in human pituitary adenomas is discussed. Copyright © 2011 S. Karger AG, Basel.

  9. Gastroretentive extended-release floating granules prepared using a novel fluidized hot melt granulation (FHMG) technique.

    PubMed

    Zhai, H; Jones, D S; McCoy, C P; Madi, A M; Tian, Y; Andrews, G P

    2014-10-06

    The objective of this work was to investigate the feasibility of using a novel granulation technique, namely, fluidized hot melt granulation (FHMG), to prepare gastroretentive extended-release floating granules. In this study we have utilized FHMG, a solvent free process in which granulation is achieved with the aid of low melting point materials, using Compritol 888 ATO and Gelucire 50/13 as meltable binders, in place of conventional liquid binders. The physicochemical properties, morphology, floating properties, and drug release of the manufactured granules were investigated. Granules prepared by this method were spherical in shape and showed good flowability. The floating granules exhibited sustained release exceeding 10 h. Granule buoyancy (floating time and strength) and drug release properties were significantly influenced by formulation variables such as excipient type and concentration, and the physical characteristics (particle size, hydrophilicity) of the excipients. Drug release rate was increased by increasing the concentration of hydroxypropyl cellulose (HPC) and Gelucire 50/13, or by decreasing the particle size of HPC. Floating strength was improved through the incorporation of sodium bicarbonate and citric acid. Furthermore, floating strength was influenced by the concentration of HPC within the formulation. Granules prepared in this way show good physical characteristics, floating ability, and drug release properties when placed in simulated gastric fluid. Moreover, the drug release and floating properties can be controlled by modification of the ratio or physical characteristics of the excipients used in the formulation.

  10. Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

    PubMed Central

    Tan, Kah Yong; Eminli, Sarah; Hettmer, Simone; Hochedlinger, Konrad; Wagers, Amy J.

    2011-01-01

    Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that “epigenetic memory” significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle. PMID:22028872

  11. Pro-hormone Secretogranin II Regulates Dense Core Secretory Granule Biogenesis in Catecholaminergic Cells*

    PubMed Central

    Courel, Maïté; Soler-Jover, Alex; Rodriguez-Flores, Juan L.; Mahata, Sushil K.; Elias, Salah; Montero-Hadjadje, Maïté; Anouar, Youssef; Giuly, Richard J.; O'Connor, Daniel T.; Taupenot, Laurent

    2010-01-01

    Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H+-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network. PMID:20061385

  12. Pro-hormone secretogranin II regulates dense core secretory granule biogenesis in catecholaminergic cells.

    PubMed

    Courel, Maïté; Soler-Jover, Alex; Rodriguez-Flores, Juan L; Mahata, Sushil K; Elias, Salah; Montero-Hadjadje, Maïté; Anouar, Youssef; Giuly, Richard J; O'Connor, Daniel T; Taupenot, Laurent

    2010-03-26

    Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H(+)-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network.

  13. Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Somanath, Sangeeta; Partridge, Christopher J.; Marshall, Catriona

    Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation.more » These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation. - Highlights: • Snapin mediates granule docking. • Snapin binds SNAP-25. • SNARE complex forms downstream.« less

  14. Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells.

    PubMed

    Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

    2014-02-18

    In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca(2+)] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.

  15. Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

    PubMed Central

    Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

    2014-01-01

    In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells. PMID:24535122

  16. RNA granules: the good, the bad and the ugly

    PubMed Central

    Thomas, María Gabriela; Loschi, Mariela; Desbats, María Andrea; Boccaccio, Graciela Lidia

    2010-01-01

    Processing bodies (PBs) and Stress granules (SGs) are the founding members of a new class of RNA granules, known as mRNA silencing foci, as they harbor transcripts circumstantially excluded from the translationally active pool. PBs and SGs are able to release mRNAs thus allowing their translation. PBs are constitutive, but respond to stimuli that affect mRNA translation and decay, whereas SGs are specifically induced upon cellular stress, which triggers a global translational silencing by several pathways, including phosphorylation of the key translation initiation factor elF2alpha, and tRNA cleavage among others. PBs and SGs with different composition may coexist in a single cell. These macromolecular aggregates are highly conserved through evolution, from unicellular organisms to vertebrate neurons. Their dynamics is regulated by several signaling pathways, and depends on microfilaments and microtubules, and the cognate molecular motors myosin, dynein, and kinesin. SGs share features with aggresomes and related aggregates of unfolded proteins frequently present in neurodegenerative diseases, and may play a role in the pathology. Virus infections may induce or impair SG formation. Besides being important for mRNA regulation upon stress, SGs modulate the signaling balancing apoptosis and cell survival. Finally, the formation of nuclear stress bodies (nSBs), which share components with SGs, and the assembly of additional cytosolic aggregates containing RNA—the UV granules and the Ire1 foci—, all them induced by specific cell damage factors, contribute to cell survival. PMID:20813183

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rowe, Alexander M.; Brundage, Kathleen M.; Center for Immunopathology and Microbial Pathogenesis, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506

    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 hypothesizedmore » 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.« less

  18. Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles.

    PubMed

    Lei, Lei; Spradling, Allan C

    2013-05-21

    Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.

  19. Plant RNA Regulatory Network and RNA Granules in Virus Infection.

    PubMed

    Mäkinen, Kristiina; Lõhmus, Andres; Pollari, Maija

    2017-01-01

    Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual

  20. 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. © 2013 John Wiley & Sons Ltd.

  1. AMPK is activated early in cerebellar granule cells undergoing apoptosis and influences VADC1 phosphorylation status and activity.

    PubMed

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

    2017-09-01

    The neurodegeneration of cerebellar granule cells, after low potassium induced apoptosis, is known to be temporally divided into an early and a late phase. Voltage-dependent anion channel-1 (VDAC1) protein, changing from the closed inactive state to the active open state, is central to the switch between the early and late phase. It is also known that: (i) VDAC1 can undergo phosphorylation events and (ii) AMP-activated protein kinase (AMPK), the sensor of cellular stress, may have a role in neuronal homeostasis. In the view of this, the involvement of AMPK activation and its correlation with VDAC1 status and activity has been investigated in the course of cerebellar granule cells apoptosis. The results reported in this study show that an increased level of the phosphorylated, active, isoform of AMPK occurs in the early phase, peaks at 3 h and guarantees an increase in the phosphorylation status of VDCA1, resulting in a reduced activity of this latter. However this situation is transient in nature, since, in the late phase, AMPK activation decreases as well as the level of phosphorylated VDAC1. In a less phosphorylated status, VDAC1 fully recovers its gating activity and drives cells along the death route.

  2. Homologs of PROTEIN TARGETING TO STARCH Control Starch Granule Initiation in Arabidopsis Leaves[OPEN

    PubMed Central

    David, Laure C.; Abt, Melanie; Lu, Kuan-Jen

    2017-01-01

    The molecular mechanism that initiates the synthesis of starch granules is poorly understood. Here, we discovered two plastidial proteins involved in granule initiation in Arabidopsis thaliana leaves. Both contain coiled coils and a family-48 carbohydrate binding module (CBM48) and are homologs of the PROTEIN TARGETING TO STARCH (PTST) protein; thus, we named them PTST2 and PTST3. Chloroplasts in mesophyll cells typically contain five to seven granules, but remarkably, most chloroplasts in ptst2 mutants contained zero or one large granule. Chloroplasts in ptst3 had a slight reduction in granule number compared with the wild type, while those of the ptst2 ptst3 double mutant contained even fewer granules than ptst2. The ptst2 granules were larger but similar in morphology to wild-type granules, but those of the double mutant had an aberrant morphology. Immunoprecipitation showed that PTST2 interacts with STARCH SYNTHASE4 (SS4), which influences granule initiation and morphology. Overexpression of PTST2 resulted in chloroplasts containing many small granules, an effect that was dependent on the presence of SS4. Furthermore, isothermal titration calorimetry revealed that the CBM48 domain of PTST2, which is essential for its function, interacts with long maltooligosaccharides. We propose that PTST2 and PTST3 are critical during granule initiation, as they bind and deliver suitable maltooligosaccharide primers to SS4. PMID:28684429

  3. Ablation of Mouse Adult Neurogenesis Alters Olfactory Bulb Structure and Olfactory Fear Conditioning

    PubMed Central

    Valley, Matthew T.; Mullen, Tanner R.; Schultz, Lucy C.; Sagdullaev, Botir T.; Firestein, Stuart

    2009-01-01

    Adult neurogenesis replenishes olfactory bulb (OB) interneurons throughout the life of most mammals, yet during this constant flux it remains unclear how the OB maintains a constant structure and function. In the mouse OB, we investigated the dynamics of turnover and its impact on olfactory function by ablating adult neurogenesis with an x-ray lesion to the sub-ventricular zone (SVZ). Regardless of the magnitude of the lesion to the SVZ, we found no change in the survival of young adult born granule cells (GCs) born after the lesion, and a gradual decrease in the population of GCs born before the lesion. After a lesion producing a 96% reduction of incoming adult born GCs to the OB, we found a diminished behavioral fear response to conditioned odor cues but not to audio cues. Interestingly, despite this behavioral deficit and gradual anatomical changes, we found no electrophysiological changes in the GC population assayed in vivo through dendro-dendritic synaptic plasticity and odor-evoked local field potential oscillations. These data indicate that turnover in the granule cell layer is generally decoupled from the rate of adult neurogenesis, and that OB adult neurogenesis plays a role in a wide behavioral system extending beyond the OB. PMID:20582278

  4. SAMHD1 Inhibits LINE-1 Retrotransposition by Promoting Stress Granule Formation

    PubMed Central

    Xu, Fengwen; Mei, Shan; Le Duff, Yann; Yin, Lijuan; Pang, Xiaojing; Cen, Shan; Jin, Qi; Liang, Chen; Guo, Fei

    2015-01-01

    The SAM domain and HD domain containing protein 1 (SAMHD1) inhibits retroviruses, DNA viruses and long interspersed element 1 (LINE-1). Given that in dividing cells, SAMHD1 loses its antiviral function yet still potently restricts LINE-1, we propose that, instead of blocking viral DNA synthesis by virtue of its dNTP triphosphohydrolase activity, SAMHD1 may exploit a different mechanism to control LINE-1. Here, we report a new activity of SAMHD1 in promoting cellular stress granule assembly, which correlates with increased phosphorylation of eIF2α and diminished eIF4A/eIF4G interaction. This function of SAMHD1 enhances sequestration of LINE-1 RNP in stress granules and consequent blockade to LINE-1 retrotransposition. In support of this new mechanism of action, depletion of stress granule marker proteins G3BP1 or TIA1 abrogates stress granule formation and overcomes SAMHD1 inhibition of LINE-1. Together, these data reveal a new mechanism for SAMHD1 to control LINE-1 by activating cellular stress granule pathway. PMID:26134849

  5. Comparison of low-shear and high-shear granulation processes: effect on implantable calcium phosphate granule properties.

    PubMed

    Chevalier, E; Viana, M; Cazalbou, S; Chulia, D

    2009-10-01

    Calcium phosphate porous ceramics present a great interest not only as complex bone defect fillers but also as drug delivery systems. Most of the methods described in the literature to fabricate pellets are based on compaction, casting into spherical molds, or on processes such as liquid immiscibility or foaming. Despite wet granulation is used in a wide range of applications in pharmaceuticals, food, detergents, fertilizers, and minerals, it is not applied in the biomaterial field to produce granules. In this study physicochemical and in vitro drug delivery properties of implantable calcium phosphate granules, produced by two wet agglomeration processes, were compared. Pellets obtained by high shear granulation (granulation in a Mi-Pro apparatus) were shown to be more spherical and less friable than granules elaborated by low shear process (granulation in a Kenwood apparatus). Although Mi-Pro pellets had a slightly lower porosity compared to Kenwood granules, ibuprofen loading efficiency and dissolution profiles were not statistically different and the release mechanism was mainly controlled by diffusion, in both cases. Mi-Pro pellets appeared to be better candidates as bone defect fillers and local drug delivery systems as far as they were more spherical and less friable than Kenwood agglomerates.

  6. Lysine acetyltransferase NuA4 and acetyl-CoA regulate glucose-deprived stress granule formation in Saccharomyces cerevisiae

    PubMed Central

    Huard, Sylvain; Morettin, Alan; Fullerton, Morgan D.; Côté, Jocelyn

    2017-01-01

    Eukaryotic cells form stress granules under a variety of stresses, however the signaling pathways regulating their formation remain largely unknown. We have determined that the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 is required for stress granule formation upon glucose deprivation but not heat stress. Further, the Tip60 complex, the human homolog of the NuA4 complex, is required for stress granule formation in cancer cell lines. Surprisingly, the impact of NuA4 on glucose-deprived stress granule formation is partially mediated through regulation of acetyl-CoA levels, which are elevated in NuA4 mutants. While elevated acetyl-CoA levels suppress the formation of glucose-deprived stress granules, decreased acetyl-CoA levels enhance stress granule formation upon glucose deprivation. Further our work suggests that NuA4 regulates acetyl-CoA levels through the Acetyl-CoA carboxylase Acc1. Altogether this work establishes both NuA4 and the metabolite acetyl-CoA as critical signaling pathways regulating the formation of glucose-deprived stress granules. PMID:28231279

  7. Impact of screw configuration on the particle size distribution of granules produced by twin screw granulation.

    PubMed

    Vercruysse, J; Burggraeve, A; Fonteyne, M; Cappuyns, P; Delaet, U; Van Assche, I; De Beer, T; Remon, J P; Vervaet, C

    2015-02-01

    Twin screw granulation (TSG) has been reported by different research groups as an attractive technology for continuous wet granulation. However, in contrast to fluidized bed granulation, granules produced via this technique typically have a wide and multimodal particle size distribution (PSD), resulting in suboptimal flow properties. The aim of the current study was to evaluate the impact of granulator screw configuration on the PSD of granules produced by TSG. Experiments were performed using a 25 mm co-rotating twin screw granulator, being part of the ConsiGma™-25 system (a fully continuous from-powder-to-tablet manufacturing line from GEA Pharma Systems). Besides the screw elements conventionally used for TSG (conveying and kneading elements), alternative designs of screw elements (tooth-mixing-elements (TME), screw mixing elements (SME) and cutters) were investigated using an α-lactose monohydrate formulation granulated with distilled water. Granulation with only conveying elements resulted in wide and multimodal PSD. Using kneading elements, the width of the PSD could be partially narrowed and the liquid distribution was more homogeneous. However, still a significant fraction of oversized agglomerates was obtained. Implementing additional kneading elements or cutters in the final section of the screw configuration was not beneficial. Furthermore, granulation with only TME or SME had limited impact on the width of the PSD. Promising results were obtained by combining kneading elements with SME, as for these configurations the PSD was narrower and shifted to the size fractions suitable for tableting. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

    PubMed Central

    Fukada, So-ichiro; Yamaguchi, Masahiko; Kokubo, Hiroki; Ogawa, Ryo; Uezumi, Akiyoshi; Yoneda, Tomohiro; Matev, Miroslav M.; Motohashi, Norio; Ito, Takahito; Zolkiewska, Anna; Johnson, Randy L.; Saga, Yumiko; Miyagoe-Suzuki, Yuko; Tsujikawa, Kazutake; Takeda, Shin’ichi; Yamamoto, Hiroshi

    2011-01-01

    Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7+ MyoD– undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis. PMID:21989910

  9. Intracellular production of hydrogels and synthetic RNA granules by multivalent enhancers

    PubMed Central

    Nakamura, Hideki; Lee, Albert A.; Afshar, Ali Sobhi; Watanabe, Shigeki; Rho, Elmer; Razavi, Shiva; Suarez, Allison; Lin, Yu-Chun; Tanigawa, Makoto; Huang, Brian; DeRose, Robert; Bobb, Diana; Hong, William; Gabelli, Sandra B.; Goutsias, John; Inoue, Takanari

    2018-01-01

    Non-membrane bound, hydrogel-like entities, such as RNA granules, nucleate essential cellular functions through their unique physico-chemical properties. However, these intracellular hydrogels have not been as extensively studied as their extracellular counterparts, primarily due to technical challenges in probing these materials in situ. Here, by taking advantage of a chemically inducible dimerization paradigm, we developed iPOLYMER, a strategy for rapid induction of protein-based hydrogels inside living cells. A series of biochemical and biophysical characterizations, in conjunction with computational modeling, revealed that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that behaves as a size-dependent molecular sieve. We studied several properties of the gel and functionalized it with RNA binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. Therefore, we here demonstrate that iPOLYMER presents a unique and powerful approach to synthetically reconstitute hydrogel-like structures including RNA granules in intact cells. PMID:29115293

  10. Adult T-Cell Leukemia/Lymphoma

    MedlinePlus

    ... Adult T-Cell Leukemia/Lymphoma Adult T-Cell Leukemia/Lymphoma Adult T-cell A type of white ... immune responses by destroying harmful substances or cells. leukemia Disease generally characterized by the overproduction of abnormal ...

  11. Dendrites of cerebellar granule cells correctly recognize their target axons for synaptogenesis in vitro.

    PubMed

    Ito, Shoko; Takeichi, Masatoshi

    2009-08-04

    Neural circuits are generated by precisely ordered synaptic connections among neurons, and this process is thought to rely on the ability of neurons to recognize specific partners. However, it is also known that neurons promiscuously form synapses with nonspecific partners, in particular when cultured in vitro, causing controversies about neural recognition mechanisms. Here we reexamined whether neurons can or cannot select particular partners in vitro. In the cerebellum, granule cell (GC) dendrites form synaptic connections specifically with mossy fibers, but not with climbing fibers. We cocultured GC neurons with pontine or inferior olivary axons, the major sources for mossy and climbing fibers, respectively, as well as with hippocampal axons as a control. The GC neurons formed synapses with pontine axons predominantly at the distal ends of their dendrites, reproducing the characteristic morphology of their synapses observed in vivo, whereas they failed to do so when combined with other axons. In the latter case, synaptic proteins could accumulate between axons and dendrites, but these synapses were randomly distributed throughout the contact sites, and also their synaptic vesicle recycling was anomalous. These observations suggest that GC dendrites can select their authentic partners for synaptogenesis even in vitro, forming the synapses with a GC-specific nature only with them.

  12. Generation of enteroendocrine cell diversity in midgut stem cell lineages

    PubMed Central

    Beehler-Evans, Ryan; Micchelli, Craig A.

    2015-01-01

    The endocrine system mediates long-range peptide hormone signaling to broadcast changes in metabolic status to distant target tissues via the circulatory system. In many animals, the diffuse endocrine system of the gut is the largest endocrine tissue, with the full spectrum of endocrine cell subtypes not yet fully characterized. Here, we combine molecular mapping, lineage tracing and genetic analysis in the adult fruit fly to gain new insight into the cellular and molecular mechanisms governing enteroendocrine cell diversity. Neuropeptide hormone distribution was used as a basis to generate a high-resolution cellular map of the diffuse endocrine system. Our studies show that cell diversity is seen at two distinct levels: regional and local. We find that class I and class II enteroendocrine cells can be distinguished locally by combinatorial expression of secreted neuropeptide hormones. Cell lineage tracing studies demonstrate that class I and class II cells arise from a common stem cell lineage and that peptide profiles are a stable feature of enteroendocrine cell identity during homeostasis and following challenge with the enteric pathogen Pseudomonas entomophila. Genetic analysis shows that Notch signaling controls the establishment of class II cells in the lineage, but is insufficient to reprogram extant class I cells into class II enteroendocrine cells. Thus, one mechanism by which secretory cell diversity is achieved in the diffuse endocrine system is through cell-cell signaling interactions within individual adult stem cell lineages. PMID:25670792

  13. Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin.

    PubMed

    Johnson, Adam S; García, Dana M

    2007-12-19

    Inside bluegill (Lepomis macrochirus) retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog) is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion. Carbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II) failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion. A rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms), our evidence does not support a significant role for PKC.

  14. Investigation of Physicochemical Drug Properties to Prepare Fine Globular Granules Composed of Only Drug Substance in Fluidized Bed Rotor Granulation.

    PubMed

    Mise, Ryohei; Iwao, Yasunori; Kimura, Shin-Ichiro; Osugi, Yukiko; Noguchi, Shuji; Itai, Shigeru

    2015-01-01

    The effect of some drug properties (wettability and particle size distribution) on granule properties (mean particle size, particle size distribution, sphericity, and granule strength) were investigated in a high (>97%) drug-loading formulation using fluidized bed rotor granulation. Three drugs: acetaminophen (APAP); ibuprofen (IBU); and ethenzamide (ETZ) were used as model drugs based on their differences in wettability and particle size distribution. Granules with mean particle sizes of 100-200 µm and a narrow particle size distribution (PSD) could be prepared regardless of the drug used. IBU and ETZ granules showed a higher sphericity than APAP granules, while APAP and ETZ granules exhibited higher granule strength than IBU. The relationship between drug and granule properties suggested that the wettability and the PSD of the drugs were critical parameters affecting sphericity and granule strength, respectively. Furthermore, the dissolution profiles of granules prepared with poorly water-soluble drugs (IBU and ETZ) showed a rapid release (80% release in 20 min) because of the improved wettability with granulation. The present study demonstrated for the first time that fluidized bed rotor granulation can prepare high drug-loaded (>97%) globular granules with a mean particle size of less than 200 µm and the relationship between physicochemical drug properties and the properties of the granules obtained could be readily determined, indicating the potential for further application of this methodology to various drugs.

  15. Granules and Golgi vesicles with differential reactivity to ACTH antiserum in the corticotroph of the rat anterior pituitary.

    PubMed

    Weber, E; Voigt, K H; Martin, R

    1978-05-01

    Slices of unembedded rat anterior pituitaries, fixed with a periodate-lysine-paraformaldehyde (PLP) fixative, were incubated with guinea pig antiserum to ACTH and stained with a peroxidase-conjugated IgG fraction of anti-guinea pig gamma-globulin serum from rabbits. The fine structure of the stained cells was identical to that of the ACTH-secreting cell, as described by Siperstein and coworkers. Immunoreactive granules were mainly located at the periphery of the cell. Numerous granules of the inner cytoplasm and also the Golgi complex were nonreactive to the antiserum. The differential labeling for granules and Golgi apparatus peptide.

  16. Generation of functional organs from stem cells.

    PubMed

    Liu, Yunying; Yang, Ru; He, Zuping; Gao, Wei-Qiang

    2013-01-01

    We are now well entering the exciting era of stem cells. Potential stem cell therapy holds great promise for the treatment of many diseases such as stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral-sclerosis, myocardial infarction, muscular dystrophy, diabetes, and etc. It is generally believed that transplantation of specific stem cells into the injured tissue to replace the lost cells is an effective way to repair the tissue. In fact, organ transplantation has been successfully practiced in clinics for liver or kidney failure. However, the severe shortage of donor organs has been a major obstacle for the expansion of organ transplantation programs. Toward that direction, generation of transplantable organs using stem cells is a desirable approach for organ replacement and would be of great interest for both basic and clinical scientists. Here we review recent progress in the field of organ generation using various methods including single adult tissue stem cells, a blastocyst complementation system, tissue decellularization/recellularization and a combination of stem cells and tissue engineering.

  17. PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse.

    PubMed

    Huang, Yu; Chen, Zhiying; Jang, Joon Hee; Baig, Mirza S; Bertolet, Grant; Schroeder, Casey; Huang, Shengjian; Hu, Qian; Zhao, Yong; Lewis, Dorothy E; Qin, Lidong; Zhu, Michael Xi; Liu, Dongfang

    2018-04-18

    The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. We sought to determine the effect of PD-1 signaling on NK cells. PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  18. Chromogranin A: a new proposal for trafficking, processing and induction of granule biogenesis.

    PubMed

    Koshimizu, Hisatsugu; Kim, Taeyoon; Cawley, Niamh X; Loh, Y Peng

    2010-02-25

    Chromogranin A (CgA), a member of the granin family serves several important cell biological roles in (neuro)endocrine cells which are summarized in this review. CgA is a "prohormone" that is synthesized at the rough endoplasmic reticulum and transported into the cisternae of this organelle via its signal peptide. It is then trafficked to the Golgi complex and then to the trans-Golgi network (TGN) where CgA aggregates at low pH in the presence of calcium. The CgA aggregates provide the physical driving force to induce budding of the TGN membrane resulting in dense core granule (DCG) formation. Within the granule, a small amount of the CgA is processed to bioactive peptides, including a predicted C-terminal peptide, serpinin. Upon stimulation, DCGs undergo exocytosis and CgA and its derived peptides are released. Serpinin, acting extracellularly is able to signal the increase in transcription of a serine protease inhibitor, protease nexin-1 (PN-1) that protects DCG proteins against degradation in the Golgi complex, which then enhances DCG biogenesis to replenish those that were released. Thus CgA and its derived peptide, serpinin, plays a significant role in granule formation and regulation of granule biogenesis, respectively, in (neuro) endocrine cells. Published by Elsevier B.V.

  19. Comparative evaluation of different calcium phosphate-based bone graft granules - an in vitro study with osteoblast-like cells.

    PubMed

    Bernhardt, Anne; Lode, Anja; Peters, Fabian; Gelinsky, Michael

    2013-04-01

    Granule-shaped calcium phosphate-based bone graft materials are often required for bone regeneration especially in implant dentistry. Two newly developed bone graft materials are Ceracell(®) , an open-celled highly porous bioceramic from β-tricalcium phosphate (β-TCP) under addition of bioglass and Osseolive(®) , an open porous glass ceramic with the general formula Ca2 KNa(PO4 )2 . The goal of this study was to characterize different modifications of the two bone graft materials in vitro in comparison to already established ceramic bone grafts Cerasorb M(®) , NanoBone(®) and BONIT Matrix(®) . Adhesion and proliferation of SaOS-2 osteoblast-like cells were evaluated quantitatively by determining DNA content and lactate dehydrogenase (LDH) activity and qualitatively by scanning electron microscopy (SEM). In addition, MTT cell-vitality staining was applied to confirm the attachment of viable cells to the different materials. Osteogenic differentiation was evaluated by measurement of alkaline phosphatase (ALP) activity as well as gene expression analysis of osteogenic markers using reverse transcriptase PCR. DNA content and LDH activity revealed good cell attachment and proliferation for Ceracell and Cerasorb M. When pre-incubated with cell-culture medium, also Osseolive showed good cell attachment and proliferation. Attachment and proliferation of osteoblast-like cells on NanoBone and BONIT Matrix was very low, even after pre-incubation with cell-culture medium. Specific ALP activity on Ceracell(®) , Osseolive (®) and Cerasorb M(®) increased with time and expression of bone-related genes ALP, osteonectin, osteopontin and bone sialoprotein II was demonstrated. Ceracell as well as Osseolive granules support proliferation and osteogenic differentiation in vitro and may be promising candidates for in vivo applications. © 2011 John Wiley & Sons A/S.

  20. Signaling mechanisms regulating adult neural stem cells and neurogenesis

    PubMed Central

    Faigle, Roland; Song, Hongjun

    2012-01-01

    Background Adult neurogenesis occurs throughout life in discrete regions of the mammalian brain and is tightly regulated via both extrinsic environmental influences and intrinsic genetic factors. In recent years, several crucial signaling pathways have been identified in regulating self-renewal, proliferation, and differentiation of neural stem cells, as well as migration and functional integration of developing neurons in the adult brain. Scope of review Here we review our current understanding of signaling mechanisms, including Wnt, notch, sonic hedgehog, growth and neurotrophic factors, bone morphogenetic proteins, neurotransmitters, transcription factors, and epigenetic modulators, and crosstalk between these signaling pathways in the regulation of adult neurogenesis. We also highlight emerging principles in the vastly growing field of adult neural stem cell biology and neural plasticity. Major conclusions Recent methodological advances have enabled the field to identify signaling mechanisms that fine-tune and coordinate neurogenesis in the adult brain, leading to a better characterization of both cell-intrinsic and environmental cues defining the neurogenic niche. Significant questions related to niche cell identity and underlying regulatory mechanisms remain to be fully addressed and will be the focus of future studies. General significance A full understanding of the role and function of individual signaling pathways in regulating neural stem cells and generation and integration of newborn neurons in the adult brain may lead to targeted new therapies for neurological diseases in humans. PMID:22982587

  1. Effects of artemisinin sustained-release granules on mixed alga growth and microcystins production and release.

    PubMed

    Ni, Lixiao; Li, Danye; Hu, Shuzhen; Wang, Peifang; Li, Shiyin; Li, Yiping; Li, Yong; Acharya, Kumud

    2015-12-01

    To safely and effectively apply artemisinin sustained-release granules to control and prevent algal water-blooms, the effects of artemisinin and its sustained-release granules on freshwater alga (Scenedesmus obliquus (S. obliquus) and Microcystis aeruginosa (M. aeruginosa)), as well as the production and release of microcystins (MCs) were studied. The results showed that artemisinin sustained-release granules inhibited the growth of M. aeruginosa (above 95% IR) and S. obliquus (about 90% IR), with M. aeruginosa more sensitive. The artemisinin sustained-release granules had a longer inhibition effect on growth of pure algae and algal coexistence than direct artemisinin dosing. The artemisinin sustained-release granules could decrease the production and release of algal toxins due to the continued stress of artemisinin released from artemisinin sustained-release granules. There was no increase in the total amount of MC-LR in the algal cell culture medium.

  2. Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions

    NASA Astrophysics Data System (ADS)

    Nakamura, Hideki; Lee, Albert A.; Afshar, Ali Sobhi; Watanabe, Shigeki; Rho, Elmer; Razavi, Shiva; Suarez, Allister; Lin, Yu-Chun; Tanigawa, Makoto; Huang, Brian; Derose, Robert; Bobb, Diana; Hong, William; Gabelli, Sandra B.; Goutsias, John; Inoue, Takanari

    2018-01-01

    Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.

  3. Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures.

    PubMed

    Connor, J A; Tseng, H Y; Hockberger, P E

    1987-05-01

    Digital imaging of the Ca indicator fura-2 has been used to study the responses of developing granule cells in culture to depolarization and transmitter action. Unstimulated cells bathed in Krebs saline exhibited cytoplasmic Ca ion concentrations, [Ca2+], that were generally in the 30-60 nM range. Exposure of cells to high-potassium (25 mM) saline depolarized the membrane potential and produced an immediate rise in [Ca2+] that recovered within 2-3 min in normal saline. The response grew progressively larger over the first 20 d in culture. Transient increases in [Ca2+] to levels greater than 1 microM were observed after 12-14 d in vitro, at which time the cells displayed intense electrical activity when exposed to high K. At this stage, the increases were attenuated by blocking action potential activity with TTX. In TTX-treated or immature cells, in which the transient phase of the Ca change was relatively small, a second exposure to high K typically produced a much larger Ca response that the initial exposure. The duration of this facilitation of the response persisted for periods longer than 5 min. Application of the neurotransmitter GABA induced a transient increase in membrane conductance, with a reversal potential near resting potential (approx. -60 mV), and caused an intracellular Ca2+ increase that outlasted the exposure to GABA by several minutes. Glutamate, or kainate, induced an increase in membrane conductance but with a reversal potential more positive than spike threshold. These agents also elevated intracellular Ca2+, but unlike the case with GABA, this Ca response reversed rapidly upon removal of the transmitter. The facilitatory effect of repeated exposures to high-K saline, as well as the persistent Ca elevation following a brief GABA application, suggests that granule cells possess the capability of displaying activity-dependent changes in Ca levels in culture.

  4. Characterization of Pu-238 Heat Source Granule Containment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Richardson, Paul Dean II; Sanchez, Joey Leo; Wall, Angelique Dinorah

    The Milliwatt Radioisotopic Themoelectric Generator (RTG) provides power for permissive-action links. Essentially these are nuclear batteries that convert thermal energy to electrical energy using a doped silicon-germanium thermopile. The thermal energy is provided by a heat source made of 238Pu, in the form of 238PuO 2 granules. The granules are contained by 3 layers of encapsulation. A thin T-111 liner surrounds the 238PuO 2 granules and protects the second layer (strength member) from exposure to the fuel granules. An outer layer of Hastalloy-C protects the T-111 from oxygen embrittlement. The T-111 strength member is considered the critical component in thismore » 238PuO 2 containment system. Any compromise in the strength member seen during destructive testing required by the RTG surveillance program is characterized. The T-111 strength member is characterized through Scanning Electron Microscopy (SEM), and Metallography. SEM is used in the Secondary Electron mode to reveal possible grain boundary deformation and/or cracking in the region of the strength member weld. Deformation and cracking uncovered by SEM are further characterized by Metallography. Metallography sections are mounted and polished, observed using optical microscopy, then documented in the form of microphotographs. SEM mat further be used to examine polished Metallography mounts to characterize elements using the SEM mode of Energy Dispersive X-ray spectroscopy (EDS).« less

  5. Breakage and drying behaviour of granules in a continuous fluid bed dryer: Influence of process parameters and wet granule transfer.

    PubMed

    De Leersnyder, F; Vanhoorne, V; Bekaert, H; Vercruysse, J; Ghijs, M; Bostijn, N; Verstraeten, M; Cappuyns, P; Van Assche, I; Vander Heyden, Y; Ziemons, E; Remon, J P; Nopens, I; Vervaet, C; De Beer, T

    2018-03-30

    Although twin screw granulation has already been widely studied in recent years, only few studies addressed the subsequent continuous drying which is required after wet granulation and still suffers from a lack of detailed understanding. The latter is important for optimisation and control and, hence, a cost-effective practical implementation. Therefore, the aim of the current study is to increase understanding of the drying kinetics and the breakage and attrition phenomena during fluid bed drying after continuous twin screw granulation. Experiments were performed on a continuous manufacturing line consisting of a twin-screw granulator, a six-segmented fluid bed dryer, a mill, a lubricant blender and a tablet press. Granulation parameters were fixed in order to only examine the effect of drying parameters (filling time, drying time, air flow, drying air temperature) on the size distribution and moisture content of granules (both of the entire granulate and of size fractions). The wet granules were transferred either gravimetrically or pneumatically from the granulator exit to the fluid bed dryer. After a certain drying time, the moisture content reached an equilibrium. This drying time was found to depend on the applied airflow, drying air temperature and filling time. The moisture content of the granules decreased with an increasing drying time, airflow and drying temperature. Although smaller granules dried faster, the multimodal particle size distribution of the granules did not compromise uniform drying of the granules when the target moisture content was achieved. Extensive breakage of granules was observed during drying. Especially wet granules were prone to breakage and attrition during pneumatic transport, either in the wet transfer line or in the dry transfer line. Breakage and attrition of granules during transport and drying should be anticipated early on during process and formulation development by performing integrated experiments on the granulator

  6. Selective vulnerability of dentate granule cells prior to amyloid deposition in PDAPP mice: digital morphometric analyses.

    PubMed

    Wu, Chi-Cheng; Chawla, Faisal; Games, Dora; Rydel, Russell E; Freedman, Stephen; Schenk, Dale; Young, Warren G; Morrison, John H; Bloom, Floyd E

    2004-05-04

    Increasing evidence from mouse models of Alzheimer's disease shows that overexpression of a mutant form of the amyloid precursor protein (APP) and its product, beta-amyloid peptide, initiate pathological changes before amyloid deposition. To evaluate the cytological basis for one of these early changes, namely reduced volume of the dentate gyrus (DG), we have used high-throughput diOlistic cell loading and 3D neuronal reconstruction to investigate potential dendritic pathology of granule cells (GCs) in 90-day-old PDAPP mice. Labeled GCs from fixed hippocampal slices were selected randomly and imaged digitally by using confocal laser-scanning microscopy. The dendritic complexity of GCs was quantified according to subordinate morphological parameters, including soma position within the granule cell layer (superficial versus deep) and topographic location within the DG (dorsal versus ventral blade) along the anterior-posterior hippocampal axis. Initial analysis, which included all sampled GC types, revealed a 12% reduction of total dendritic length in PDAPP mice compared with littermate controls. Further analysis, performed with refined subgroups, found that superficially located GCs in the dorsal blade were profoundly altered, exhibiting a 23% loss in total dendritic length, whereas neurons in the ventral blade were unaffected. Superficial GCs were particularly vulnerable (a 32% reduction) in the posterior region of the DG. Furthermore, the dendritic reductions of this select group were uniformly localized within middle-to-outer portions of the dentate molecular layer. We conclude that substantial dendritic pathology is evident in 90-day-old PDAPP mice for a spatially defined subset of GCs well before amyloid accumulation occurs.

  7. Selective vulnerability of dentate granule cells prior to amyloid deposition in PDAPP mice: Digital morphometric analyses

    PubMed Central

    Wu, Chi-Cheng; Chawla, Faisal; Games, Dora; Rydel, Russell E.; Freedman, Stephen; Schenk, Dale; Young, Warren G.; Morrison, John H.; Bloom, Floyd E.

    2004-01-01

    Increasing evidence from mouse models of Alzheimer's disease shows that overexpression of a mutant form of the amyloid precursor protein (APP) and its product, β-amyloid peptide, initiate pathological changes before amyloid deposition. To evaluate the cytological basis for one of these early changes, namely reduced volume of the dentate gyrus (DG), we have used high-throughput diOlistic cell loading and 3D neuronal reconstruction to investigate potential dendritic pathology of granule cells (GCs) in 90-day-old PDAPP mice. Labeled GCs from fixed hippocampal slices were selected randomly and imaged digitally by using confocal laser-scanning microscopy. The dendritic complexity of GCs was quantified according to subordinate morphological parameters, including soma position within the granule cell layer (superficial versus deep) and topographic location within the DG (dorsal versus ventral blade) along the anterior-posterior hippocampal axis. Initial analysis, which included all sampled GC types, revealed a 12% reduction of total dendritic length in PDAPP mice compared with littermate controls. Further analysis, performed with refined subgroups, found that superficially located GCs in the dorsal blade were profoundly altered, exhibiting a 23% loss in total dendritic length, whereas neurons in the ventral blade were unaffected. Superficial GCs were particularly vulnerable (a 32% reduction) in the posterior region of the DG. Furthermore, the dendritic reductions of this select group were uniformly localized within middle-to-outer portions of the dentate molecular layer. We conclude that substantial dendritic pathology is evident in 90-day-old PDAPP mice for a spatially defined subset of GCs well before amyloid accumulation occurs. PMID:15118092

  8. Segregation of large granules from close-packed cluster of small granules due to buoyancy.

    PubMed

    Yang, Xian-qing; Zhou, Kun; Qiu, Kang; Zhao, Yue-min

    2006-03-01

    Segregation of large granules in a vibrofluidized granular bed with inhomogeneous granular number density distribution is studied by an event-driven algorithm. Simulation results show that the mean vertical position of large granules decreases with the increase of the density ration of the large granules to the small ones. This conclusion is consistent with the explanation that the net pressure due to the small surrounding particle impacts balances the large granular weight, and indict that the upward movement of the large granules is driven by the buoyancy. The values of temperature, density, and pressure of the systems are also computed by changing the conditions such as heating temperature on the bottom and restitution coefficient of particles. These results indicate that the segregation of large granules also happen in the systems with density inversion or even close-packed cluster of particles floating on a low-density fluid, due to the buoyancy. An equation of state is proposed to explain the buoyancy.

  9. Comparative transcriptomic analysis of endothelial progenitor cells derived from umbilical cord blood and adult peripheral blood: Implications for the generation of induced pluripotent stem cells.

    PubMed

    Gao, Xiugong; Yourick, Jeffrey J; Sprando, Robert L

    2017-12-01

    Induced pluripotent stem cells (iPSCs) offer the potential to generate tissues with ethnic diversity enabling toxicity testing on selected populations. Recently, it has been reported that endothelial progenitor cells (EPCs) derived from umbilical cord blood (CB) or adult peripheral blood (PB) afford a practical and efficient cellular substrate for iPSC generation. However, differences between EPCs from different blood sources have rarely been studied. In the current study, we derived EPCs from blood mononuclear cells (MNCs) and reprogrammed EPCs into iPSCs. We also explored differences between CB-EPCs and PB-EPCs at the molecular and cellular levels through a combination of transcriptomic analysis and cell biology techniques. EPC colonies in CB-MNCs emerged 5-7days earlier, were 3-fold higher in number, and consistently larger in size than in PB-MNCs. Similarly, iPSC colonies generated from CB-EPCs was 2.5-fold higher in number than from PB-EPCs, indicating CB-EPCs have a higher reprogramming efficiency than PB-EPCs. Transcriptomic analysis using microarrays found a total of 1133 genes differentially expressed in CB-EPCs compared with PB-EPCs, with 675 genes upregulated and 458 downregulated. Several canonical pathways were impacted, among which the human embryonic stem cell pluripotency pathway was of particular interest. The differences in the gene expression pattern between CB-EPCs and PB-EPCs provide a molecular basis for the discrepancies seen in their derivation and reprogramming efficiencies, and highlight the advantages of using CB as the cellular source for the generation of iPSCs and their derivative tissues for ethnic-related toxicological applications. Published by Elsevier B.V.

  10. Investigation of the effect of impeller speed on granules formed using a PMA-1 high shear granulator.

    PubMed

    Logan, R; Briens, L

    2012-11-01

    Impeller speed was varied from 300 to 1500 rpm during the wet high shear granulation of a placebo formulation using a new vertical shaft PharmaMATRIX-1 granulator. The resulting granules were extensively analysed for differences caused by the varying impeller speed with emphasis on flowability. Microscopy showed that initial granules were formed primarily from microcrystalline cellulose at all tested impeller speeds. At low impeller speed of 300 rpm in the "bumpy" flow regime, forces from the impeller were insufficient to incorporate all the components of the formulation into the granules and to promote granule growth to a size that significantly improved flowability. The "roping" flow regime at higher impeller speeds promoted granule growth to a median particle size of at least 100 µm that improved the flowability of the mixture. Particle size distribution measurements and advanced indicators based on avalanching behavior, however, showed that an impeller speed of 700 rpm produced the largest fraction of optimal granules with the best flowability potential. This impeller speed allowed good development of "roping" flow for sufficient mixing, collision rates and kinetic energy for collisions while minimizing excessive centrifugal forces that promote buildup around the bowl perimeter.

  11. Costus root granules improve ulcerative colitis through regulation of TGF-β mediation of the PI3K/AKT signaling pathway

    PubMed Central

    Wang, Xiaohong; Li, Dan; Zhang, Yong; Wu, Shuang; Tang, Fang

    2018-01-01

    Ulcerative colitis is a chronic nonspecific inflammatory disease that occurs in the colon and rectum. Costus root is a type of traditional Chinese medicine that exhibits antibacterial properties and serves an inhibitory role in the regeneration of gut bacteria. However, the molecular mechanisms underlying Costus root-mediated improvements in ulcerative colitis remain unclear. A complex formula of Costus root granules was created and investigated in the present study for its therapeutic effects in a rat model of ulcerative colitis. Ingredient dissolution into a traditional water decoction was used as a control. The potential mechanism mediated by Costus root granules was also analyzed in colonic epithelial cells isolated from the experimental rats. The results of the present study demonstrated that Costus root granule treatment inhibited inflammation in colonic tissue. Costus root granule treatment also suppressed the apoptosis of colonic epithelial cells isolated from the rat model of ulcerative colitis. Analyses of the underlying mechanisms of these effects indicated that the administration of Costus root granules increased transforming growth factor β expression, which activated the phosphoinositide 3-kinase/RAC-α serine/threonine-protein kinase signaling pathway in colonic epithelial cells. Notably, the administration of Costus root granules improved stomachache, diarrhea and hematochezia in and increased the body weight of, the ulcerative colitis rats. In conclusion, these results indicate that Costus root granules markedly ameliorate inflammation of the colonic epithelium, decrease the apoptosis of colonic epithelial cells and improve colonic function, which suggests that Costus root granules are an efficient agent for the treatment of ulcerative colitis. PMID:29731832

  12. SOLAR X-RAY JETS, TYPE-II SPICULES, GRANULE-SIZE EMERGING BIPOLES, AND THE GENESIS OF THE HELIOSPHERE

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Moore, Ronald L.; Sterling, Alphonse C.; Cirtain, Jonathan W.

    2011-04-10

    From Hinode observations of solar X-ray jets, Type-II spicules, and granule-size emerging bipolar magnetic fields in quiet regions and coronal holes, we advocate a scenario for powering coronal heating and the solar wind. In this scenario, Type-II spicules and Alfven waves are generated by the granule-size emerging bipoles (EBs) in the manner of the generation of X-ray jets by larger magnetic bipoles. From observations and this scenario, we estimate that Type-II spicules and their co-generated Alfven waves carry into the corona an area-average flux of mechanical energy of {approx}7 x 10{sup 5} erg cm{sup -2} s{sup -1}. This is enoughmore » to power the corona and solar wind in quiet regions and coronal holes, and therefore indicates that the granule-size EBs are the main engines that generate and sustain the entire heliosphere.« less

  13. The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma membrane.

    PubMed

    Wang, Hao; Ishizaki, Ray; Xu, Jun; Kasai, Kazuo; Kobayashi, Eri; Gomi, Hiroshi; Izumi, Tetsuro

    2013-02-01

    Granuphilin, an effector of the small GTPase Rab27a, mediates the stable attachment (docking) of insulin granules to the plasma membrane and inhibits subsequent fusion of docked granules, possibly through interaction with a fusion-inhibitory Munc18-1/syntaxin complex. However, phenotypes of insulin exocytosis differ considerably between Rab27a- and granuphilin-deficient pancreatic β cells, suggesting that other Rab27a effectors function in those cells. We found that one of the putative Rab27a effector family proteins, exophilin7/JFC1/Slp1, is expressed in β cells; however, unlike granuphilin, exophilin7 overexpressed in the β-cell line MIN6 failed to show granule-docking or fusion-inhibitory activity. Furthermore, exophilin7 has no affinities to either Munc18-1 or Munc18-1-interacting syntaxin-1a, in contrast to granuphilin. Although β cells of exophilin7-knockout mice show no apparent abnormalities in intracellular distribution or in ordinary glucose-induced exocytosis of insulin granules, they do show impaired fusion in response to some stronger stimuli, specifically from granules that have not been docked to the plasma membrane. Exophilin7 appears to mediate the fusion of undocked granules through the affinity of its C2A domain toward the plasma membrane phospholipids. These findings indicate that the two Rab27a effectors, granuphilin and exophilin7, differentially regulate the exocytosis of either stably or minimally docked granules, respectively.

  14. Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome

    PubMed Central

    Sharda, Anish; Kim, Sarah H.; Jasuja, Reema; Gopal, Srila; Flaumenhaft, Robert; Furie, Barbara C.

    2015-01-01

    Protein disulfide isomerase (PDI), secreted from platelets and endothelial cells after injury, is required for thrombus formation. The effect of platelet and endothelial cell granule contents on PDI-mediated thrombus formation was studied by intravital microscopy using a mouse model of Hermansky-Pudlak syndrome in which platelet dense granules are absent. Platelet deposition and fibrin generation were nearly absent, and extracellular PDI was significantly reduced in HPS6−/− mice after vascular injury. HPS6−/− platelets displayed impaired PDI secretion and impaired exocytosis of α granules, lysosomes, and T granules due to decreased sensitivity to thrombin, but these defects could be corrected by addition of subthreshold amounts of adenosine 5′-diphosphate (ADP). Human Hermansky-Pudlak syndrome platelets demonstrated similar characteristics. Infusion of wild-type platelets rescued thrombus formation in HPS6−/− mice. Human umbilical vein endothelial cells in which the HPS6 gene was silenced displayed impaired PDI secretion and exocytosis of Weibel-Palade bodies. Defective thrombus formation in Hermansky-Pudlak syndrome, associated with impaired exocytosis of residual granules in endothelial cells and platelets, the latter due to deficiency of ADP, is characterized by a defect in T granule secretion, a deficiency in extracellular PDI secretion, and impaired fibrin generation and platelet aggregation. Hermansky-Pudlak syndrome is an example of a hereditary disease whereby impaired PDI secretion contributes to a bleeding phenotype. PMID:25593336

  15. Distinct Molecular Events during Secretory Granule Biogenesis Revealed by Sensitivities to Brefeldin A

    PubMed Central

    Fernandez, Carlos J.; Haugwitz, Michael; Eaton, Benjamin; Moore, Hsiao-Ping H.

    1997-01-01

    The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse–chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20°C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from ISGs is

  16. Distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin A.

    PubMed

    Fernandez, C J; Haugwitz, M; Eaton, B; Moore, H P

    1997-11-01

    The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse-chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20 degrees C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from

  17. ZIO impregnation and cytochemical localization of thiamine pyrophosphatase and acid phosphatase activities in small granule-containing (SGC) cells of rat superior cervical ganglia.

    PubMed

    Chau, Y P; Lu, K S

    1994-10-01

    Cytochemical relationship between Golgi complex and dense-cored granules (DCGs) of small granule-containing (SGC) cells in rat superior cervical ganglia was examined in electron microscopy by zinc-iodide-osmium tetroxide (ZIO) method and by enzyme cytochemistry for thiamine pyrophosphatase (TPPase) and acid phosphatase (ACPase). After ZIO impregnation, all the saccules of Golgi apparatus and some of tubular rough endoplasmic reticulum (rER) were stained. DCGs in periphery of SGC cells were not stained, but varying degrees of dense deposits occurred in the DCGs in vicinity of Golgi trans-saccules. Both TPPase and ACPase activities were localized in one or two stacked layers of saccules on the trans side of the Golgi complex. No reaction products were demonstrated in the DCGs. From these results, we suggest that the DCGs of SGC cells in rat superior cervical ganglia are derived from the Golgi complex, and that lysosomal cleavage of protein contents in the DCGs may occur in the trans Golgi saccules.

  18. Identification of the Haloarchaeal Phasin (PhaP) That Functions in Polyhydroxyalkanoate Accumulation and Granule Formation in Haloferax mediterranei

    PubMed Central

    Cai, Shuangfeng; Cai, Lei; Liu, Hailong; Liu, Xiaoqing; Han, Jing; Zhou, Jian

    2012-01-01

    The polyhydroxyalkanoate (PHA) granule-associated proteins (PGAPs) are important for PHA synthesis and granule formation, but currently little is known about the haloarchaeal PGAPs. This study focused on the identification and functional analysis of the PGAPs in the haloarchaeon Haloferax mediterranei. These PGAPs were visualized with two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization–tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). The most abundant protein on the granules was identified as a hypothetical protein, designated PhaP. A genome-wide analysis revealed that the phaP gene is located upstream of the previously identified phaEC genes. Through an integrative approach of gene knockout/complementation and fermentation analyses, we demonstrated that this PhaP is involved in PHA accumulation. The ΔphaP mutant was defective in both PHA biosynthesis and cell growth compared to the wild-type strain. Additionally, transmission electron microscopy results indicated that the number of PHA granules in the ΔphaP mutant cells was significantly lower, and in most of the ΔphaP cells only a single large granule was observed. These results demonstrated that the H. mediterranei PhaP was the predominant structure protein (phasin) on the PHA granules involved in PHA accumulation and granule formation. In addition, BLASTp and phylogenetic results indicate that this type of PhaP is exclusively conserved in haloarchaea, implying that it is a representative of the haloarchaeal type PHA phasin. PMID:22247127

  19. Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.

    PubMed

    Ohara-Imaizumi, Mica; Nakamichi, Yoko; Tanaka, Toshiaki; Katsuta, Hidenori; Ishida, Hitoshi; Nagamatsu, Shinya

    2002-04-01

    The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic beta-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH 7.4, but not at approximately pH 5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy (5 or 10 s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50 mM KCl and 22 mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin-pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

  20. Maternal Forced Swimming Reduces Cell Proliferation in the Postnatal Dentate Gyrus of Mouse Offspring

    PubMed Central

    Wasinski, Frederick; Estrela, Gabriel R.; Arakaki, Aline M.; Bader, Michael; Alenina, Natalia; Klempin, Friederike; Araújo, Ronaldo C.

    2016-01-01

    Physical exercise positively affects the metabolism and induces proliferation of precursor cells in the adult brain. Maternal exercise likewise provokes adaptations early in the offspring. Using a high-intensity swimming protocol that comprises forced swim training before and during pregnancy, we determined the effect of maternal swimming on the mouse offspring's neurogenesis. Our data demonstrate decreased proliferation in sublayers of the postnatal dentate gyrus in offspring of swimming mother at postnatal day (P) 8 accompanied with decreased survival of newly generated cells 4 weeks later. The reduction in cell numbers was predominantly seen in the hilus and molecular layer. At P35, the reduced amount of cells was also reflected by a decrease in the population of newly generated immature and mature neurons of the granule cell layer. Our data suggest that forced maternal swimming at high-intensity has a negative effect on the neurogenic niche development in postnatal offspring. PMID:27621701

  1. Induced adult stem (iAS) cells and induced transit amplifying progenitor (iTAP) cells-a possible alternative to induced pluripotent stem (iPS) cells?

    PubMed

    Heng, Boon Chin; Richards, Mark; Ge, Zigang; Shu, Yimin

    2010-02-01

    The successful derivation of iPSC lines effectively demonstrates that it is possible to reset the 'developmental clock' of somatic cells all the way back to the initial embryonic state. Hence, it is plausible that this clock may instead be turned back half-way to a less immature developmental stage that is more directly applicable to clinical therapeutic applications or for in vitro pharmacology/toxicology screening assays. Such a suitable developmental state is postulated to be either the putative transit amplifying progenitor stage or adult stem cell stage. It is hypothetically possible to reprogram mature and terminally differentiated somatic cells back to the adult stem cell or transit amplifying progenitor stage, in a manner similar to the derivation of iPSC. It is proposed that the terminology 'Induced Adult Stem Cells' (iASC) or 'Induced Transit Amplifying Progenitor Cells' (iTAPC) be used to described such reprogrammed somatic cells. Of particular interest, is the possibility of resetting the developmental clock of mature differentiated somatic cells of the mesenchymal lineage, explanted from adipose tissue, bone marrow and cartilage. The putative adult stem cell sub-population from which these cells are derived, commonly referred to as 'mesenchymal stem cells', are highly versatile and hold much therapeutic promise in regenerative medicine, as attested to by numerous human clinical trials and animal studies. Perhaps it may be appropriate to term such reprogrammed cells as 'Induced Mesenchymal Stem Cells' (iMSC) or as 'Induced Mesenchumal Progenitor Cells' (iMPC). Given that cells from the same organ/tissue will share some commonalities in gene expression, we hypothesize that the generation of iASC or iTAPC would be more efficient as compared to iPSC generation, since a common epigenetic program must exist between the reprogrammed cells, adult stem cell or progenitor cell types and terminally differentiated cell types from the same organ/tissue.

  2. 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

  3. Bioequivalence of the 4-mg Oral Granules and Chewable Tablet Formulations of Montelukast.

    PubMed

    Knorr, Barbara; Hartford, Alan; Li, Xiujiang Susie; Yang, Amy Yifan; Noonan, Gertrude; Migoya, Elizabeth

    2010-06-01

    PURPOSE: The primary objective of the studies was to demonstrate bioequivalence between the oral granules formulation and chewable tablet of montelukast in the fasted state. Effect of food on the pharmacokinetics of the oral granules was also evaluated. METHODS: The Formulation Biocomparison Study (Study 1) and the Final Market Image Study (Study 2) each used an open-label, randomized, 3-period crossover design where healthy adult subjects (N = 24 and 30, respectively) received montelukast as a single 4-mg dose of the oral granules formulation and a 4-mg chewable tablet fasted, and a single 4-mg dose of the oral granules formulation with food (on 2 teaspoons of applesauce [Study 1] or after consumption of a high-fat breakfast [Study 2]). The formulations were to be considered bioequivalent if the 90% confidence intervals (CIs) for geometric mean ratios (GMRs) (oral granules/chewable tablet) for the AUC(0-infinity) and C(max) of montelukast were within the prespecified comparability bounds of (0.80, 1.25). For the food-effect assessment in Study 1, comparability bounds were prespecified as (0.50, 2.00) only for the 90% CI of the GMR (oral granules fed/oral granules fasted) for the AUC(0-infinity) of montelukast; the 90% CI of the GMR for the C(max) of montelukast, however, also was computed. In Study 2, 90% CIs of the GMRs (oral granules fed/oral granules fasted) for the AUC(0-infinity) and C(max) of montelukast were computed; comparability bounds were not prespecified. RESULTS: Comparing the exposure of the formulations, the 90% CIs of the GMRs for AUC(0-infinity) and C(max) were within the prespecified bound of (0.80, 1.25). For AUC(0-infinity), the GMRs (90% CI) for Study 1 and Study 2 were 1.01 (0.92, 1.11) and 0.95 (0.91, 0.99), respectively. For C(max), respective values were 0.99 (0.86, 1.13) and 0.92 (0.84, 1.01). When the oral granules formulation was administered with food, 90% CIs of the GMRs for both AUC(0-infinity) and C(max) in both studies were

  4. Bioequivalence of the 4-mg Oral Granules and Chewable Tablet Formulations of Montelukast

    PubMed Central

    Knorr, Barbara; Hartford, Alan; Li, Xiujiang (Susie); Yang, Amy Yifan; Noonan, Gertrude; Migoya, Elizabeth

    2010-01-01

    Purpose The primary objective of the studies was to demonstrate bioequivalence between the oral granules formulation and chewable tablet of montelukast in the fasted state. Effect of food on the pharmacokinetics of the oral granules was also evaluated. Methods The Formulation Biocomparison Study (Study 1) and the Final Market Image Study (Study 2) each used an open-label, randomized, 3-period crossover design where healthy adult subjects (N = 24 and 30, respectively) received montelukast as a single 4-mg dose of the oral granules formulation and a 4-mg chewable tablet fasted, and a single 4-mg dose of the oral granules formulation with food (on 2 teaspoons of applesauce [Study 1] or after consumption of a high-fat breakfast [Study 2]). The formulations were to be considered bioequivalent if the 90% confidence intervals (CIs) for geometric mean ratios (GMRs) (oral granules/chewable tablet) for the AUC0-∞ and Cmax of montelukast were within the prespecified comparability bounds of (0.80, 1.25). For the food-effect assessment in Study 1, comparability bounds were prespecified as (0.50, 2.00) only for the 90% CI of the GMR (oral granules fed/oral granules fasted) for the AUC0-∞ of montelukast; the 90% CI of the GMR for the Cmax of montelukast, however, also was computed. In Study 2, 90% CIs of the GMRs (oral granules fed/oral granules fasted) for the AUC0-∞ and Cmax of montelukast were computed; comparability bounds were not prespecified. Results Comparing the exposure of the formulations, the 90% CIs of the GMRs for AUC0-∞ and Cmax were within the prespecified bound of (0.80, 1.25). For AUC0-∞, the GMRs (90% CI) for Study 1 and Study 2 were 1.01 (0.92, 1.11) and 0.95 (0.91, 0.99), respectively. For Cmax, respective values were 0.99 (0.86, 1.13) and 0.92 (0.84, 1.01). When the oral granules formulation was administered with food, 90% CIs of the GMRs for both AUC0-∞ and Cmax in both studies were contained within the interval of (0.50, 2.00). Conclusions The

  5. Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

    PubMed Central

    Wang, Tongguang; Choi, Elliot; Monaco, Maria Chiara G.; Campanac, Emilie; Medynets, Marie; Do, Thao; Rao, Prashant; Johnson, Kory R.; Elkahloun, Abdel G.; Von Geldern, Gloria; Johnson, Tory; Subramaniam, Sriram; Hoffman, Dax; Major, Eugene; Nath, Avindra

    2013-01-01

    Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine. PMID:24303066

  6. Pur-alpha regulates cytoplasmic stress granule dynamics and ameliorates FUS toxicity

    PubMed Central

    Daigle, J Gavin; Krishnamurthy, Karthik; Ramesh, Nandini; Casci, Ian; Monaghan, John; McAvoy, Kevin; Godfrey, Earl W; Daniel, Dianne C.; Johnson, Edward M.; Monahan, Zach; Shewmaker, Frank; Pasinelli, Piera; Pandey, Udai Bhan

    2016-01-01

    Amyotrophic lateral Sclerosis is characterized by progressive loss of motor neurons in the brain and spinal cord. Mutations in several genes, including FUS, TDP43, Matrin 3, hnRNPA2 and other RNA binding proteins, have been linked to ALS pathology. Recently, Pur-alpha a DNA/RNA binding protein was found to bind to C9orf72 repeat expansions and could possibly play a role in the pathogenesis of ALS. When overexpressed, Pur-alpha mitigates toxicities associated with Fragile X tumor ataxia syndrome (FXTAS) and C9orf72 repeat expansion diseases in Drosophila and mammalian cell culture models. However, the function of Pur-alpha in regulating ALS pathogenesis has not been fully understood. We identified Pur-alpha as a novel component of cytoplasmic stress granules (SGs) in ALS patient cells carrying disease-causing mutations in FUS. When cells were challenged with stress, we observed that Pur-alpha co-localized with mutant FUS in ALS patient cells and became trapped in constitutive SGs. We also found that FUS physically interacted with Pur-alpha in mammalian neuronal cells. Interestingly, shRNA mediated knock down of endogenous Pur-alpha significantly reduced formation of cytoplasmic stress granules in mammalian cells suggesting that Pur-alpha is essential for the formation of SGs. Furthermore, ectopic expression of Pur-alpha blocked cytoplasmic mislocalization of mutant FUS and strongly suppressed toxicity associated with mutant FUS expression in primary motor neurons. Our data emphasizes the importance of stress granules in ALS pathogenesis and identifies Pur-alpha as a novel regulator of SG dynamics. PMID:26728149

  7. Visualization and understanding of the granulation liquid mixing and distribution during continuous twin screw granulation using NIR chemical imaging.

    PubMed

    Vercruysse, Jurgen; Toiviainen, Maunu; Fonteyne, Margot; Helkimo, Niko; Ketolainen, Jarkko; Juuti, Mikko; Delaet, Urbain; Van Assche, Ivo; Remon, Jean Paul; Vervaet, Chris; De Beer, Thomas

    2014-04-01

    Over the last decade, there has been increased interest in the application of twin screw granulation as a continuous wet granulation technique for pharmaceutical drug formulations. However, the mixing of granulation liquid and powder material during the short residence time inside the screw chamber and the atypical particle size distribution (PSD) of granules produced by twin screw granulation is not yet fully understood. Therefore, this study aims at visualizing the granulation liquid mixing and distribution during continuous twin screw granulation using NIR chemical imaging. In first instance, the residence time of material inside the barrel was investigated as function of screw speed and moisture content followed by the visualization of the granulation liquid distribution as function of different formulation and process parameters (liquid feed rate, liquid addition method, screw configuration, moisture content and barrel filling degree). The link between moisture uniformity and granule size distributions was also studied. For residence time analysis, increased screw speed and lower moisture content resulted to a shorter mean residence time and narrower residence time distribution. Besides, the distribution of granulation liquid was more homogenous at higher moisture content and with more kneading zones on the granulator screws. After optimization of the screw configuration, a two-level full factorial experimental design was performed to evaluate the influence of moisture content, screw speed and powder feed rate on the mixing efficiency of the powder and liquid phase. From these results, it was concluded that only increasing the moisture content significantly improved the granulation liquid distribution. This study demonstrates that NIR chemical imaging is a fast and adequate measurement tool for allowing process visualization and hence for providing better process understanding of a continuous twin screw granulation system. Copyright © 2013 Elsevier B.V. All

  8. Upgrading of automobile shredder residue via innovative granulation process 'ReGran'.

    PubMed

    Holthaus, Philip; Kappes, Moritz; Krumm, Wolfgang

    2017-01-01

    Stricter regulatory requirements concerning end-of-life vehicles and rising disposal costs necessitate new ways for automobile shredder residue utilisation. The shredder granulate and fibres, produced by the VW-SICON-Process, have a high energy content of more than 20 MJ kg -1 , which makes energy recovery an interesting possibility. Shredder fibres have a low bulk density of 60 kg m -3 , which prevents efficient storing and utilisation as a refuse-derived fuel. By mixing fibres with plastic-rich shredder granulate and heating the mixture, defined granules can be produced. With this 'ReGran' process, the bulk density can be enhanced by a factor of seven by embedding shredder fibres in the partially melted plastic mass. A minimum of 26-33 wt% granulate is necessary to create enough melted plastic. The process temperature should be between 240 °C and 250 °C to assure fast melting while preventing extensive outgassing. A rotational frequency of the mixing tool of 1000 r min -1 during heating and mixing ensures a homogenous composition of the granules. During cooling, lower rotational frequencies generate bigger granules with particles sizes of up to 60 mm at 300 r min -1 . To keep outgassing to a minimum, it is suggested to melt shredder granulate first and then add shredder fibres. Adding coal, wood or tyre fluff as a third component reduces chlorine levels to less than 1 wt%. The best results can be achieved with tyre fluff. In combination with the VW-SICON-Process, ReGran produces a solid recovered fuel or 'design fuel' tailored to the requirements of specific thermal processes.

  9. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreasesmore » significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.« less

  10. The cerebellar Golgi cell and spatiotemporal organization of granular layer activity

    PubMed Central

    D'Angelo, Egidio; Solinas, Sergio; Mapelli, Jonathan; Gandolfi, Daniela; Mapelli, Lisa; Prestori, Francesca

    2013-01-01

    The cerebellar granular layer has been suggested to perform a complex spatiotemporal reconfiguration of incoming mossy fiber signals. Central to this role is the inhibitory action exerted by Golgi cells over granule cells: Golgi cells inhibit granule cells through both feedforward and feedback inhibitory loops and generate a broad lateral inhibition that extends beyond the afferent synaptic field. This characteristic connectivity has recently been investigated in great detail and been correlated with specific functional properties of these neurons. These include theta-frequency pacemaking, network entrainment into coherent oscillations and phase resetting. Important advances have also been made in terms of determining the membrane and synaptic properties of the neuron, and clarifying the mechanisms of activation by input bursts. Moreover, voltage sensitive dye imaging and multi-electrode array (MEA) recordings, combined with mathematical simulations based on realistic computational models, have improved our understanding of the impact of Golgi cell activity on granular layer circuit computations. These investigations have highlighted the critical role of Golgi cells in: generating dense clusters of granule cell activity organized in center-surround structures, implementing combinatorial operations on multiple mossy fiber inputs, regulating transmission gain, and cut-off frequency, controlling spike timing and burst transmission, and determining the sign, intensity and duration of long-term synaptic plasticity at the mossy fiber-granule cell relay. This review considers recent advances in the field, highlighting the functional implications of Golgi cells for granular layer network computation and indicating new challenges for cerebellar research. PMID:23730271

  11. Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin

    PubMed Central

    Johnson, Adam S; García, Dana M

    2007-01-01

    Background Inside bluegill (Lepomis macrochirus) retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog) is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion. Results Carbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II) failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion. Conclusion A rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms), our evidence does not support a significant role for PKC. PMID:18093324

  12. Chromogranin A deficiency in transgenic mice leads to aberrant chromaffin granule biogenesis.

    PubMed

    Kim, Taeyoon; Zhang, Chun-fa; Sun, Ziqing; Wu, Heling; Loh, Y Peng

    2005-07-27

    The biogenesis of dense-core secretory granules (DCGs), organelles responsible for the storage and secretion of neurotransmitters and neuropeptides in chromaffin cells, is poorly understood. Chromogranin A (CgA), which binds catecholamines for storage in the lumen of chromaffin granules, has been shown to be involved in DCG biogenesis in neuroendocrine PC12 cells. Here, we report that downregulation of CgA expression in vivo by expressing antisense RNA against CgA in transgenic mice led to a significant reduction in DCG formation in adrenal chromaffin cells. The number of DCGs formed in CgA antisense transgenic mice was directly correlated with the amount of CgA present in adrenal medulla. In addition, DCGs showed an increase in size, with enlargement in the volume around the dense core, a phenomenon that occurs to maintain constant "free" catecholamine concentration in the lumen of these granules. The extent of DCG swelling was inversely correlated with the number of DCGs formed, as well as the amount of CgA present in the adrenal glands of CgA antisense transgenic mice. These data indicate an essential role of CgA in regulating chromaffin DCG biogenesis and catecholamine storage in vivo.

  13. Prompt triggering of edge localized modes through lithium granule injection on EAST

    NASA Astrophysics Data System (ADS)

    Lunsford, Robert; Sun, Z.; Hu, J. S.; Xu, W.; Zuo, G. Z.; Gong, X. Z.; Wan, B. N.; Li, J. G.; Huang, M.; Maingi, R.; Diallo, A.; Tritz, K.; the EAST Team

    2017-10-01

    We report successful triggering of edge localized mode (ELMs) in EAST with Lithium (Li) micropellets, and the observed dependence of ELM triggering efficiency on granule size. ELM control is essential for successful ITER operation throughout the entire campaign, relying on magnetic perturbations for ELM suppression and ELM frequency enhancement via pellet injection. To separate the task of fueling from ELM pacing, we initiate the prompt generation of ELMs via impurity granule injection. Lithium granules ranging in size from 200 - 1000 microns are mechanically injected into upper-single null EAST long pulse H-mode discharges. The injections are monitored for their effect on high Z impurity accumulation and to assess the pressure perturbation required for reliable ELM triggering. We have determined that granules of diameter larger than 600 microns (corresponding to 5.2 x 1018 Li atoms) are successful at triggering ELMs more than 90% of the time. The triggering efficiency drops precipitously to less than 40% as the granule size is reduced to 400 microns (1.5 x 1018 Li atoms), indicating a triggering threshold has been crossed. Using this information an optimal impurity granule size which will regularly trigger a prompt ELM in these EAST discharges is determined. Coupling these results with alternate discharge scenarios on EAST and similar experiments performed on DIII-D provides the possibility of extrapolation to future devices.

  14. Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins

    PubMed Central

    Thomson, Travis; Liu, Niankun; Arkov, Alexey; Lehmann, Ruth; Lasko, Paul

    2008-01-01

    Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components we isolated proteins that associate in early embryos with Vasa (VAS) and Tudor (TUD), two known polar granule associated molecules. We identified Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) as components of both VAS and TUD complexes and confirmed their localization to polar granules by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. Our recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly. PMID:18590813

  15. Crystallization and initial X-ray analysis of polyhydroxyalkanoate granule-associated protein from Aeromonas hydrophila

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhao, Minglian; Li, Zhenguo; Zheng, Wei

    The phasin PhaP{sub Ah} from A. hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were discovered in PHA-accumulating bacteria. They play a crucial role as a structural protein during initial PHA-granule formation and granule growth and also serve as interfaces for granule stabilization in vivo. The phasin PhaP{sub Ah} from Aeromonas hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Single crystals were cryocooled for X-ray diffraction analysis. The phasin crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 80.8, b = 108.9, c = 134.4 Å.

  16. Mitotic events in cerebellar granule progenitor cells that expand cerebellar surface area are critical for normal cerebellar cortical lamination in mice.

    PubMed

    Chang, Joshua C; Leung, Mark; Gokozan, Hamza Numan; Gygli, Patrick Edwin; Catacutan, Fay Patsy; Czeisler, Catherine; Otero, José Javier

    2015-03-01

    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 stereologic principles. We demonstrate that, during the proliferative phase of the external granular 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 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 intermitotic times for β events on a per-cell basis in postnatal 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 stereologic studies.

  17. Granule mobility, fusion frequency and insulin secretion are differentially affected by insulinotropic stimuli.

    PubMed

    Schumacher, Kirstin; Matz, Magnus; Brüning, Dennis; Baumann, Knut; Rustenbeck, Ingo

    2015-05-01

    The pre-exocytotic behavior of insulin granules was studied against the background of the entirety of submembrane granules in MIN6 cells, and the characteristics were compared with the macroscopic secretion pattern and the cytosolic Ca(2+) concentration of MIN6 pseudo-islets at 22°C, 32°C and 37°C. The mobility of granules labeled by insulin-EGFP and the fusion events were assessed by TIRF microscopy utilizing an observer-independent algorithm. In the z-dimension, 40 mm K(+) or 30 mm glucose increased the granule turnover. The effect of high K(+) was quickly reversible. The increase by glucose was more sustained and modified the efficacy of a subsequent K(+) stimulus. The effect size of glucose increased with physiological temperature whereas that of high K(+) did not. The mobility in the x/y-dimension and the fusion rates were little affected by the stimuli, in contrast to secretion. Fusion and secretion, however, had the same temperature dependence. Granules that appeared and fused within one image sequence had significantly larger caging diameters than pre-existent granules that underwent fusion. These in turn had a different mobility than residence-matched non-fusing granules. In conclusion, delivery to the membrane, tethering and fusion of granules are differently affected by insulinotropic stimuli. Fusion rates and secretion do not appear to be tightly coupled. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Characterization by X-ray tomography of granulated alumina powder during in situ die compaction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cottrino, Sandrine; Jorand, Yves, E-mail: yves.jorand@insa-lyon.fr; Maire, Eric

    2013-07-15

    Compaction process, the aim of which being to obtain green bodies with low porosity and small size, is often used before sintering treatment. Prior to die filling, the ceramic powder is generally granulated to improve flowability. However during compaction, density heterogeneity and critical size defects may appear due to intergranule and granule-die wall frictions. In this work, the influence of granule formulation on the compact morphology has been studied. To do so, a compaction setup was installed inside an X-ray tomography equipment so that the evolution of the compact morphology could be analysed during the whole compaction process. We havemore » demonstrated that high humidity rate and the addition of binder in the granule formulation increase density heterogeneity and generate larger defects. - Highlights: • An original compaction set up was installed inside an X-Ray tomography equipment. • The compaction process of granulated ceramic powder is imaged. • The compact green microstructure is quantified and related to the compaction stages. • The most detrimental defects of dry-pressed parts are caused by hollow granules. • Formulations without binder allow a reduction of the number of large defects.« less

  19. Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation

    PubMed Central

    Malinova, Irina

    2017-01-01

    An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5–7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology. PMID:29155859

  20. Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation.

    PubMed

    Malinova, Irina; Fettke, Joerg

    2017-01-01

    An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.

  1. THE THIRD SIGNATURE OF GRANULATION IN BRIGHT-GIANT AND SUPERGIANT STARS

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gray, David F.; Pugh, Teznie, E-mail: dfgray@uwo.ca

    2012-04-15

    We investigated third-signature granulation plots for 18 bright giants and supergiants and one giant of spectral classes G0 to M3. These plots reveal the net granulation velocities, averaged over the stellar disk, as a function of depth. Supergiants show significant differences from the 'standard' shape seen for lower-luminosity stars. Most notable is a striking reversal of slope seen for three of the nine supergiants, i.e., stronger lines are more blueshifted than weaker lines, opposite the solar case. Changes in the third-signature plot of {alpha} Sco (M1.5 Iab) with time imply granulation cells that penetrate only the lower portion of themore » photosphere. For those stars showing the standard shape, we derive scaling factors relative to the Sun that serve as a first-order measure of the strength of the granulation relative to the Sun. For G-type stars, the third-signature scale of the bright giants and supergiants is approximately 1.5 times as strong as in dwarfs, but for K stars, there in no discernible difference between higher-luminosity stars and dwarfs. Classical macroturbulence, a measure of the velocity dispersion of the granulation, increases with the third-signature-plot scale factors, but at different rates for different luminosity classes.« less

  2. Amyloid β-Mediated Zn2+ Influx into Dentate Granule Cells Transiently Induces a Short-Term Cognitive Deficit

    PubMed Central

    Takeda, Atsushi; Nakamura, Masatoshi; Fujii, Hiroaki; Uematsu, Chihiro; Minamino, Tatsuya; Adlard, Paul A.; Bush, Ashley I.; Tamano, Haruna

    2014-01-01

    We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn2+ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn2+ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn2+ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn2+ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn2+ levels in the dentate granule cell layer in ACSF without Zn2+, but in ACSF containing Zn2+. The increase in intracellular Zn2+ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn2+ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn2+ may play a key role for transiently Aβ-induced cognition deficits. PMID:25536033

  3. Amyloid β-mediated Zn2+ influx into dentate granule cells transiently induces a short-term cognitive deficit.

    PubMed

    Takeda, Atsushi; Nakamura, Masatoshi; Fujii, Hiroaki; Uematsu, Chihiro; Minamino, Tatsuya; Adlard, Paul A; Bush, Ashley I; Tamano, Haruna

    2014-01-01

    We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn2+ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn2+ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn2+ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn2+ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn2+ levels in the dentate granule cell layer in ACSF without Zn2+, but in ACSF containing Zn2+. The increase in intracellular Zn2+ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn2+ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn2+ may play a key role for transiently Aβ-induced cognition deficits.

  4. [Drying characteristics and apparent change of sludge granules during drying].

    PubMed

    Ma, Xue-Wen; Weng, Huan-Xin; Zhang, Jin-Jun

    2011-08-01

    Three different weight grades of sludge granules (2.5, 5, 10 g) were dried at constant temperature of 100, 200, 300, 400 and 500 degrees C, respectively. Then characteristics of weight loss and change of apparent form during sludge drying were analyzed. Results showed that there were three stages during sludge drying at 100-200 degrees C: acceleration phase, constant-rate phase, and falling-rate phase. At 300-500 degrees C, there were no constant-rate phase, but due to lots of cracks generated at sludge surface, average drying rates were still high. There was a quadratic nonlinear relationship between average drying rate and drying temperature. At 100-200 degrees C, drying processes of different weight grade sludge granules were similar. At 300-500 degrees C, drying processes of same weight grade of sludge granules were similar. Little organic matter decomposed till sludge burning at 100-300 degrees C, while some organic matter began to decompose at the beginning of sludge drying at 400-500 degrees C.

  5. Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells.

    PubMed

    Rajaei, Bahareh; Shamsara, Mehdi; Amirabad, Leila Mohammadi; Massumi, Mohammad; Sanati, Mohammad Hossein

    2017-10-01

    Human-induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient- and disease-specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin-producing pancreatic β-cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five-step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose-responsive insulin-producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut-tube endoderm, posterior foregut, pancreatic endoderm, and endocrine precursor. Each stage of differentiation were characterized by stage-specific markers. The produced cells exhibited many properties of functional β-cells, including expression of critical β-cells transcription factors, the potency to secrete C-peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin-secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs-derived glucose-responsive insulin-secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. J. Cell. Physiol. 232: 2616-2625, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. α-Synuclein binds the KATP channel at insulin-secretory granules and inhibits insulin secretion

    PubMed Central

    Geng, Xuehui; Lou, Haiyan; Wang, Jian; Li, Lehong; Swanson, Alexandra L.; Sun, Ming; Beers-Stolz, Donna; Watkins, Simon; Perez, Ruth G.

    2011-01-01

    α-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic β-cells, α-synuclein might therefore play a similar role by interacting with organelles involved in insulin secretion. We tested for α-synuclein localizing to insulin-secretory granules and characterized its role in glucose-stimulated insulin secretion. Immunohistochemistry and fluorescent sulfonylureas were used to test for α-synuclein localization to insulin granules in β-cells, immunoprecipitation with Western blot analysis for interaction between α-synuclein and KATP channels, and ELISA assays for the effect of altering α-synuclein expression up or down on insulin secretion in INS1 cells or mouse islets, respectively. Differences in cellular phenotype between α-synuclein knockout and wild-type β-cells were found by using confocal microscopy to image the fluorescent insulin biosensor Ins-C-emGFP and by using transmission electron microscopy. The results show that anti-α-synuclein antibodies labeled secretory organelles within β-cells. Anti-α-synuclein antibodies colocalized with KATP channel, anti-insulin, and anti-C-peptide antibodies. α-Synuclein coimmunoprecipitated in complexes with KATP channels. Expression of α-synuclein downregulated insulin secretion at 2.8 mM glucose with little effect following 16.7 mM glucose stimulation. α-Synuclein knockout islets upregulated insulin secretion at 2.8 and 8.4 mM but not 16.7 mM glucose, consistent with the depleted insulin granule density at the β-cell surface membranes observed in these islets. These findings demonstrate that α-synuclein interacts with KATP channels and insulin-secretory granules and functionally acts as a brake on secretion that glucose stimulation can override. α-Synuclein might play similar roles in diabetes as it does in other degenerative diseases, including Alzheimer's and Parkinson's diseases. PMID:20858756

  7. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy.

    PubMed

    Pareja, Eugenia; Gómez-Lechón, María José; Tolosa, Laia

    2017-01-01

    Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.

  9. Chronic corticosterone administration reduces dendritic complexity in mature, but not young granule cells in the rat dentate gyrus.

    PubMed

    Yau, Suk-Yu; Li, Ang; Tong, Jian-Bin; Bostrom, Crystal; Christie, Brian R; Lee, Tatia M C; So, Kwok-Fai

    2016-09-21

    Our previous work has shown that exposure to the stress hormone corticosterone (40 mg/kg CORT) for two weeks induces dendritic atrophy of pyramidal neurons in the hippocampal CA3 region and behavioral deficits. However, it is unclear whether this treatment also affects the dentate gyrus (DG), a subregion of the hippocampus comprising a heterogeneous population of young and mature neurons. We examined the effect of CORT treatment on the dendritic complexity of mature and young granule cells in the DG. We utilized a Golgi staining method to investigate the dendritic morphology and spine density of young neurons in the inner granular cell layer (GCL) and mature neurons in the outer GCL in response to CORT application. The expressions of glucocorticoid receptors during neuronal maturation were examined using Western blot analysis in a primary hippocampal neuronal culture. Sholl analysis revealed that CORT treatment decreased the number of intersections and shortened the dendritic length in mature, but not young, granule cells. However, the spine density of mature and young neurons was not affected. Western blot analysis showed a progressive increase in the protein levels of glucocorticoid receptors (GRs) in the cultured primary hippocampal neurons during neuronal maturation. These data suggest that mature neurons are likely more vulnerable to chronic exposure to CORT; this may be due to their higher expression of GRs when compared to younger DG neurons.

  10. Dogs cloned from adult somatic cells.

    PubMed

    Lee, Byeong Chun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hossein, M Shamim; Shamim, M Hossein; Kim, Jung Ju; Kang, Sung Keun; Schatten, Gerald; Hwang, Woo Suk

    2005-08-04

    Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

  11. Oscillating behavior of carbohydrate granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC 51142

    NASA Technical Reports Server (NTRS)

    Schneegurt, M. A.; Sherman, D. M.; Nayar, S.; Sherman, L. A.; Mitchell, C. A. (Principal Investigator)

    1994-01-01

    It has been shown that some aerobic, unicellular, diazotrophic cyanobacteria temporally separate photosynthetic O2 evolution and oxygen-sensitive N2 fixation. Cyanothece sp. ATCC strain 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that fixes N2 during discrete periods of its cell cycle. When the bacteria are maintained under diurnal light-dark cycles, N2 fixation occurs in the dark. Similar cycling is observed in continuous light, implicating a circadian rhythm. Under N2-fixing conditions, large inclusion granules form between the thylakoid membranes. Maximum granulation, as observed by electron microscopy, occurs before the onset of N2 fixation, and the granules decrease in number during the period of N2 fixation. The granules can be purified from cell homogenates by differential centrifugation. Biochemical analyses of the granules indicate that these structures are primarily carbohydrate, with some protein. Further analyses of the carbohydrate have shown that it is a glucose polymer with some characteristics of glycogen. It is proposed that N2 fixation is driven by energy and reducing power stored in these inclusion granules. Cyanothece sp. strain ATCC 51142 represents an excellent experimental organism for the study of the protective mechanisms of nitrogenase, metabolic events in cyanobacteria under normal and stress conditions, the partitioning of resources between growth and storage, and biological rhythms.

  12. Shear Resistance Variations in Experimentally Sheared Mudstone Granules: A Possible Shear-Thinning and Thixotropic Mechanism

    NASA Astrophysics Data System (ADS)

    Hu, Wei; Xu, Qiang; Wang, Gonghui; Scaringi, Gianvito; Mcsaveney, Mauri; Hicher, Pierre-Yves

    2017-11-01

    We present results of ring shear frictional resistance for mudstone granules of different size obtained from a landslide shear zone. Little rate dependency of shear resistance was observed in sand-sized granules in any wet or dry test, while saturated gravel-sized granules exhibited significant and abrupt reversible rate-weakening (from μ = 0.6 to 0.05) at about 2 mm/s. Repeating resistance variations occurred also under constant shear displacement rate. Mudstone granules generate mud as they are crushed and softened. Shear-thinning and thixotropic behavior of the mud can explain the observed behavior: with the viscosity decreasing, the mud can flow through the coarser soil pores and migrate out from the shear zone. This brings new granules into contact which produces new mud. Thus, the process can start over. Similarities between experimental shear zones and those of some landslides in mudstone suggest that the observed behavior may play a role in some landslide kinematics.

  13. Bioequivalence among three methods of administering pantoprazole granules in healthy subjects.

    PubMed

    Tammara, Brinda; Weisel, Kathy; Katz, Arie; Meng, Xu

    2009-11-01

    The bioequivalence among three methods of administering pantoprazole granules was studied in healthy subjects. In this randomized, open-label, three-period, crossover study, 25 healthy adults received a single 40-mg dose of pantoprazole granules with applesauce orally, with apple juice orally, and with apple juice administered via a nasogastric tube. Subjects were randomly assigned to one of six treatment sequences. Blood samples were collected within 2 hours before treatment administration on study day 1 and at 0.33, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 16, and 24 hours after treatment administration. Plasma pantoprazole concentrations were analyzed by a validated liquid chromatography-tandem mass spectrometry method. The plasma pantoprazole concentration-time data for each subject were analyzed using noncompartmental methods. The 90% confidence intervals (CIs) for the test:reference geometric mean ratio were calculated for the peak pantoprazole concentration (C(max) ) and area under the concentration-time curve (AUC). Of the 25 subjects enrolled, 100% completed the study. The mean C(max) and AUC values were similar for the three administration methods. The 90% CIs for the ratios of the geometric means of the granules in apple juice orally (92.4-112.5%) and in apple juice administered through a nasogastric tube (102.7-125.2%), relative to the granules administered with applesauce orally, were essentially within the bioequivalent limits of 80-125%. No serious adverse events or study discontinuations occurred. Three methods of administering pantoprazole delayed-release granules for oral suspension-with apple juice orally, with applesauce orally, and with apple juice through a nasogastric tube--were bioequivalent in healthy subjects.

  14. Translation suppression promotes stress granule formation and cell survival in response to cold shock

    PubMed Central

    Hofmann, Sarah; Cherkasova, Valeria; Bankhead, Peter; Bukau, Bernd; Stoecklin, Georg

    2012-01-01

    Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock–induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2α phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia. PMID:22875991

  15. Reprint of: Chromogranin A: a new proposal for trafficking, processing and induction of granule biogenesis.

    PubMed

    Koshimizu, Hisatsugu; Kim, Taeyoon; Cawley, Niamh X; Loh, Y Peng

    2010-11-30

    Chromogranin A (CgA), a member of the granin family serves several important cell biological roles in (neuro)endocrine cells which are summarized in this review. CgA is a "prohormone" that is synthesized at the rough endoplasmic reticulum and transported into the cisternae of this organelle via its signal peptide. It is then trafficked to the Golgi complex and then to the trans-Golgi network (TGN) where CgA aggregates at low pH in the presence of calcium. The CgA aggregates provide the physical driving force to induce budding of the TGN membrane resulting in dense core granule (DCG) formation. Within the granule, a small amount of the CgA is processed to bioactive peptides, including a predicted C-terminal peptide, serpinin. Upon stimulation, DCGs undergo exocytosis and CgA and its derived peptides are released. Serpinin, acting extracellularly is able to signal the increase in transcription of a serine protease inhibitor, protease nexin-1 (PN-1) that protects DCG proteins against degradation in the Golgi complex, which then enhances DCG biogenesis to replenish those that were released. Thus CgA and its derived peptide, serpinin, plays a significant role in granule formation and regulation of granule biogenesis, respectively, in (neuro) endocrine cells. Copyright © 2010. Published by Elsevier B.V.

  16. Reprint of: Chromogranin A: A new proposal for trafficking, processing and induction of granule biogenesis☆

    PubMed Central

    Koshimizu, Hisatsugu; Kim, Taeyoon; Cawley, Niamh X.; Loh, Y. Peng

    2014-01-01

    Chromogranin A (CgA), a member of the granin family serves several important cell biological roles in (neuro) endocrine cells which are summarized in this review. CgA is a “prohormone” that is synthesized at the rough endoplasmic reticulum and transported into the cisternae of this organelle via its signal peptide. It is then trafficked to the Golgi complex and then to the trans-Golgi network (TGN) where CgA aggregates at low pH in the presence of calcium. The CgA aggregates provide the physical driving force to induce budding of the TGN membrane resulting in dense core granule (DCG) formation. Within the granule, a small amount of the CgA is processed to bioactive peptides, including a predicted C-terminal peptide, serpinin. Upon stimulation, DCGs undergo exocytosis and CgA and its derived peptides are released. Serpinin, acting extracellularly is able to signal the increase in transcription of a serine protease inhibitor, protease nexin-1 (PN-1) that protects DCG proteins against degradation in the Golgi complex, which then enhances DCG biogenesis to replenish those that were released. Thus CgA and its derived peptide, serpinin, plays a significant role in granule formation and regulation of granule biogenesis, respectively, in (neuro) endocrine cells. PMID:20920534

  17. Early natural stimulation through environmental enrichment accelerates neuronal development in the mouse dentate gyrus.

    PubMed

    Liu, Na; He, Shan; Yu, Xiang

    2012-01-01

    The dentate gyrus is the primary afferent into the hippocampal formation, with important functions in learning and memory. Granule cells, the principle neuronal type in the dentate gyrus, are mostly formed postnatally, in a process that continues into adulthood. External stimuli, including environmental enrichment, voluntary exercise and learning, have been shown to significantly accelerate the generation and maturation of dentate granule cells in adult rodents. Whether, and to what extent, such environmental stimuli regulate the development and maturation of dentate granule cells during early postnatal development is largely unknown. Furthermore, whether natural stimuli affect the synaptic properties of granule cells had been investigated neither in newborn neurons of the adult nor during early development. To examine the effect of natural sensory stimulation on the dentate gyrus, we reared newborn mice in an enriched environment (EE). Using immunohistochemistry, we showed that dentate granule cells from EE-reared mice exhibited earlier morphological maturation, manifested as faster peaking of doublecortin expression and elevated expression of mature neuronal markers (including NeuN, calbindin and MAP2) at the end of the second postnatal week. Also at the end of the second postnatal week, we found increased density of dendritic spines across the entire dentate gyrus, together with elevated levels of postsynaptic scaffold (post-synaptic density 95) and receptor proteins (GluR2 and GABA(A)Rγ2) of excitatory and inhibitory synapses. Furthermore, dentate granule cells of P14 EE-reared mice had lower input resistances and increased glutamatergic and GABAergic synaptic inputs. Together, our results demonstrate that EE-rearing promotes morphological and electrophysiological maturation of dentate granule cells, underscoring the importance of natural environmental stimulation on development of the dentate gyrus.

  18. Starch synthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion

    PubMed Central

    Crumpton-Taylor, Matilda; Pike, Marilyn; Lu, Kuan-Jen; Hylton, Christopher M; Feil, Regina; Eicke, Simona; Lunn, John E; Zeeman, Samuel C; Smith, Alison M

    2013-01-01

    Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants. Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume. We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape. SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules. PMID:23952675

  19. Requirements for the identification of dense-core granules.

    PubMed

    Meldolesi, Jacopo; Chieregatti, Evelina; Luisa Malosio, Maria

    2004-01-01

    Dense-core granules (DCGs), cytoplasmic organelles competent for regulated exocytosis, show considerable heterogeneity depending upon the specificity of their expressing cells--primarily neurons and neurosecretory cells. DCGs have been mainly identified by detecting their cargo molecules, often members of the granin family, and using conventional electron microscopy and immunocytochemistry. However, by a critical analysis of the various stages of DCG "life" within neurosecretory cells, we have highlighted several specific molecular and functional properties that are common to all these organelles. We propose that these properties be considered as strict requirements for the identification of DCGs.

  20. No evidence for β cell neogenesis in murine adult pancreas

    PubMed Central

    Xiao, Xiangwei; Chen, Zean; Shiota, Chiyo; Prasadan, Krishna; Guo, Ping; El-Gohary, Yousef; Paredes, Jose; Welsh, Carey; Wiersch, John; Gittes, George K.

    2013-01-01

    Whether facultative β cell progenitors exist in the adult pancreas is a major unsolved question. To date, lineage-tracing studies have provided conflicting results. To track β cell neogenesis in vivo, we generated transgenic mice that transiently coexpress mTomato and GFP in a time-sensitive, nonconditional Cre-mediated manner, so that insulin-producing cells express GFP under control of the insulin promoter, while all other cells express mTomato (INSCremTmG mice). Newly differentiated β cells were detected by flow cytometry and fluorescence microscopy, taking advantage of their transient coexpression of GFP and mTomato fluorescent proteins. We found that β cell neogenesis predominantly occurs during embryogenesis, decreases dramatically shortly after birth, and is completely absent in adults across various models of β cell loss, β cell growth and regeneration, and inflammation. Moreover, we demonstrated upregulation of neurogenin 3 (NGN3) in both proliferating ducts and preexisting β cells in the ligated pancreatic tail after pancreatic ductal ligation. These results are consistent with some recent reports, but argue against the widely held belief that NGN3 marks cells undergoing endocrine neogenesis in the pancreas. Our data suggest that β cell neogenesis in the adult pancreas occurs rarely, if ever, under either normal or pathological conditions. PMID:23619362

  1. Use of a continuous twin screw granulation and drying system during formulation development and process optimization.

    PubMed

    Vercruysse, J; Peeters, E; Fonteyne, M; Cappuyns, P; Delaet, U; Van Assche, I; De Beer, T; Remon, J P; Vervaet, C

    2015-01-01

    Since small scale is key for successful introduction of continuous techniques in the pharmaceutical industry to allow its use during formulation development and process optimization, it is essential to determine whether the product quality is similar when small quantities of materials are processed compared to the continuous processing of larger quantities. Therefore, the aim of this study was to investigate whether material processed in a single cell of the six-segmented fluid bed dryer of the ConsiGma™-25 system (a continuous twin screw granulation and drying system introduced by GEA Pharma Systems, Collette™, Wommelgem, Belgium) is predictive of granule and tablet quality during full-scale manufacturing when all drying cells are filled. Furthermore, the performance of the ConsiGma™-1 system (a mobile laboratory unit) was evaluated and compared to the ConsiGma™-25 system. A premix of two active ingredients, powdered cellulose, maize starch, pregelatinized starch and sodium starch glycolate was granulated with distilled water. After drying and milling (1000 μm, 800 rpm), granules were blended with magnesium stearate and compressed using a Modul™ P tablet press (tablet weight: 430 mg, main compression force: 12 kN). Single cell experiments using the ConsiGma™-25 system and ConsiGma™-1 system were performed in triplicate. Additionally, a 1h continuous run using the ConsiGma™-25 system was executed. Process outcomes (torque, barrel wall temperature, product temperature during drying) and granule (residual moisture content, particle size distribution, bulk and tapped density, hausner ratio, friability) as well as tablet (hardness, friability, disintegration time and dissolution) quality attributes were evaluated. By performing a 1h continuous run, it was detected that a stabilization period was needed for torque and barrel wall temperature due to initial layering of the screws and the screw chamber walls with material. Consequently, slightly deviating

  2. THE SYNTHESIS OF THE STARCH GRANULE.

    PubMed

    Smith, A. M.; Denyer, K.; Martin, C.

    1997-06-01

    This review describes and discusses the implications of recent discoveries about how starch polymers are synthesized and organized to form a starch granule. Three issues are highlighted. 1. The role and importance of ADPglucose pyrophosphorylase in the generation of ADPglucose as the substrate for polymer synthesis. 2. The contributions of isoforms of starch-branching enzyme, starch synthase, and debranching enzyme to the synthesis and ordered packing of amylopectin molecules. 3. The requirements for and regulation of the synthesis of amylose.

  3. Pharmaceutical production of tableting granules in an ultra-small-scale high-shear granulator as a pre-formulation study.

    PubMed

    Ogawa, Tatsuya; Uchino, Tomohiro; Takahashi, Daisuke; Izumi, Tsuyoshi; Otsuka, Makoto

    2012-11-01

    In some of drug developments, the amount of bulk drug powder to use in early stages is limited and it is not easy to supply a sufficient drug amount for conventional preparation methods. Therefore, an ultra-small-scale high-shear granulator (less than 5 g) (USG) was developed and applied to small-scale granulation as a pre-formulation. The sample powder consisted of 66.5% lactose, 28.5% microcrystalline cellulose and 5.0% hydroxypropylcellulose. The granules were obtained to agitate 5 g of the sample powder with 1.0 mL of water at 300 rpm for 5 min after pre-powder mixing for 3 min by the USG and the manual hand (HM) methods. The granules were evaluated by the 10% and 90% accumulated particle size and the recoveries of the granules and the powder solid. Median particle size for the USG and the HM methods was 159.2 ± 2.3 and 270.9 ± 14.9 µm, respectively. The USG method had a narrower particle size distribution than those by the HM method. The recovery of the granules by USG was significantly larger than that by the HM method. Characteristics of all of the granules indicated that the USG method could produce higher quality granules within a shorter time than the HM methods.

  4. Evaluation of beta-lactose, PVP K12 and PVP K90 as excipients to prepare piroxicam granules using two wet granulation techniques.

    PubMed

    Albertini, Beatrice; Cavallari, Cristina; Passerini, Nadia; González-Rodríguez, M L; Rodriguez, Lorenzo

    2003-11-01

    The present investigation aimed at evaluating the use of different excipients, beta-lactose and polyvinylpyrrolidone of two molecular weights (PVP K12 and PVP K90), in the production of improved release piroxicam granules, by wet granulation using both water and steam as granulation liquid. The formulations examined were: piroxicam (Px)/beta-lactose; Px/PVP K12 and Px/PVP K90, each one at a 1:9 weight ratio. The most significant difference between beta-lactose and PVP is that, using the first excipient, both steam and water granules were produced while, when PVP were employed, only steam granules were obtained. Image analysis revealed that beta-lactose steam granules had a larger surface area with respect to water granules, whereas lower values of this parameter were observed in PVP-s granules, confirming the Scanning Electron Microscopy micrographs and the fractal analysis results. As regards the enhancement of the dissolution profiles, the best result was obtained using beta-lactose steam granules followed by PVP K12 ones, even if the reactive dimension values indicated that during the dissolution process PVP K12 granules modified the surface more than beta-lactose granules. As regards PVP K90, this excipient was the one less influencing the granule morphology and the dissolution behaviour. Differential Scanning Calorimetry analysis suggested the partial amorphisation of the drug in the granules containing the three excipients. This result was then confirmed by X-ray powder diffraction analysis. Therefore, beta-lactose and PVP K12 could be proposed as useful excipients to enhance the dissolution rate of Px from granules prepared using the steam granulation technique.

  5. Reversal of hippocampal neuronal maturation by serotonergic antidepressants

    PubMed Central

    Kobayashi, Katsunori; Ikeda, Yumiko; Sakai, Atsushi; Yamasaki, Nobuyuki; Haneda, Eisuke; Miyakawa, Tsuyoshi; Suzuki, Hidenori

    2010-01-01

    Serotonergic antidepressant drugs have been commonly used to treat mood and anxiety disorders, and increasing evidence suggests potential use of these drugs beyond current antidepressant therapeutics. Facilitation of adult neurogenesis in the hippocampal dentate gyrus has been suggested to be a candidate mechanism of action of antidepressant drugs, but this mechanism may be only one of the broad effects of antidepressants. Here we show a distinct unique action of the serotonergic antidepressant fluoxetine in transforming the phenotype of mature dentate granule cells. Chronic treatments of adult mice with fluoxetine strongly reduced expression of the mature granule cell marker calbindin. The fluoxetine treatment induced active somatic membrane properties resembling immature granule cells and markedly reduced synaptic facilitation that characterizes the mature dentate-to-CA3 signal transmission. These changes cannot be explained simply by an increase in newly generated immature neurons, but best characterized as “dematuration” of mature granule cells. This granule cell dematuration developed along with increases in the efficacy of serotonin in 5-HT4 receptor-dependent neuromodulation and was attenuated in mice lacking the 5-HT4 receptor. Our results suggest that serotonergic antidepressants can reverse the established state of neuronal maturation in the adult hippocampus, and up-regulation of 5-HT4 receptor-mediated signaling may play a critical role in this distinct action of antidepressants. Such reversal of neuronal maturation could affect proper functioning of the mature hippocampal circuit, but may also cause some beneficial effects by reinstating neuronal functions that are lost during development. PMID:20404165

  6. Ca2+ influx does not trigger glucose-induced traffic of the insulin granules and alteration of their distribution.

    PubMed

    Niki, Ichiro; Niwa, Tae; Yu, Wei; Budzko, Dorota; Miki, Takashi; Senda, Takao

    2003-11-01

    This study investigated mechanisms by which glucose increases readily releasable secretory granules via acting on preexocytotic steps, i.e., intracellular granule movement and granule access to the plasma membrane using a pancreatic beta-cell line, MIN6. Glucose-induced activation of the movement occurred at a substimulatory concentration with regard to insulin output. Glucose activation of the movement was inhibited by pretreatment with thapsigargin plus acetylcholine to suppress intracellular Ca2+ mobilization. Inhibitors of calmodulin and myosin light chain kinase also suppressed glucose activation of the movement. Simultaneous addition of glucose with Ca2+ channel blockers or the ATP-sensitive K+ channel opener diazoxide failed to suppress the traffic activation, and addition of these substances on top of glucose stimulation resulted in a further increase. Although stimulatory glucose had minimal changes in the intracellular granule distribution, inhibition of Ca2+ influx revealed increases by glucose of the granules in the cell periphery. In contrast, high K+ depolarization decreased the peripheral granules. Glucose-induced granule margination was abolished when the protein kinase C activity was downregulated. These findings indicate that preexocytotic control of insulin release is regulated by distinct mechanisms from Ca2+ influx, which triggers insulin exocytosis. The nature of the regulation by glucose may explain a part of potentiating effects of the hexose independent of the closure of the ATP-sensitive K+ channel.

  7. Effects of Extracts from Tiaozhi Granule and Its Components on Expression of Scavenger Receptor Class B Type I

    PubMed Central

    Yu, Xiao; Zhao, Xiao-Dong; Bao, Rong-Qi; Yu, Jia-Yu; Zhang, Guo-Xing

    2016-01-01

    Sera from the rats with different drug treatments (atorvastatin, Tiaozhi granule, or its extracts) were collected. LO-2 cells or HepG2 cells were pretreated with different sera as the following groups randomly: (1) blank control group, (2) positive control group (atorvastatin group), (3) Tiaozhi granule water extract groups, (4) Tiaozhi granule alcohol extract groups, and (5) alcohol extracts for each component: Pollen Typhae Angustifoliae, Curcuma longa L., and Rhizoma Alismatis. LO-2 cells were cotransfected with plasmid carrying SR-BI and pRL-TK promoter genes. Promoter activity was measured by the luciferase reporter gene assay. The mRNA and protein expressions of SR-BI were examined using real-time PCR and western blot analyses. Our results show that promoter activity and mRNA and protein expression levels of the SR-BI were significantly upregulated by Tiaozhi granules alcohol or water extracts in a dose-dependent manner. Pollen Typhae Angustifoliae alcohol extract with a high dosage could also increase SR-BI activity and expression, but not the extracts from Curcuma longa L. and Rhizoma Alismatis. Both Tiaozhi granule alcohol and water extracts can upregulate SR-BI gene expression. Among the components, Pollen Typhae Angustifoliae are important for the regulatory effect coordinating with Curcuma longa L. and Rhizoma Alismatis. PMID:28050195

  8. Characterization of Pu-238 heat source granule containment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Richardson Ii, P D; Thronas, D L; Romero, J P

    2008-01-01

    The Milliwatt Radioisotopic Thermoelectric Generator (RTG) provides power for permissive-action links. These nuclear batteries convert thermal energy to electrical energy using a doped silicon-germanium thermopile. The thermal energy is provided by a heat source made of {sup 238}Pu, in the form of {sup 238}PuO{sub 2} granules. The granules are contained in 3 layers of encapsulation. A thin T-111 liner surrounds the {sup 238}PuO{sub 2} granules and protects the second layer (strength member) from exposure to the fuel granules. The T-111 strength member contains the fuel under impact condition. An outer clad of Hastelloy-C protects the T-111 from oxygen embrittlement. Themore » T-111 strength member is considered the critical component in this {sup 238}PuO{sub 2} containment system. Any compromise in the strength member is something that needs to be characterized. Consequently, the T-111 strength member is characterized upon it's decommissioning through Scanning Electron Microscopy (SEM), and Metallography. SEM is used in Secondary Electron mode to reveal possible grain boundary deformation and/or cracking in the region of the strength member weld. Deformation and cracking uncovered by SEM are further characterized by Metallography. Metallography sections are mounted and polished, observed using optical microscopy, then documented in the form of photomicrographs. SEM may further be used to examine polished Metallography mounts to characterize elements using the SEM mode of Energy Dispersive X-ray Spectroscopy (EDS). This paper describes the characterization of the metallurgical condition of decommissioned RTG heat sources.« less

  9. Adult neurogenesis in the hedgehog (Erinaceus concolor) and mole (Talpa europaea).

    PubMed

    Bartkowska, K; Turlejski, K; Grabiec, M; Ghazaryan, A; Yavruoyan, E; Djavadian, R L

    2010-01-01

    We investigated adult neurogenesis in two species of mammals belonging to the superorder Laurasiatheria, the southern white-breasted hedgehog (order Erinaceomorpha, species Erinaceus concolor) from Armenia and the European mole (order Soricomorpha, species Talpa europaea) from Poland. Neurogenesis in the brain of these species was examined immunohistochemically, using the endogenous markers doublecortin (DCX) and Ki-67, which are highly conserved among species. We found that in both the hedgehog and mole, like in the majority of earlier investigated mammals, neurogenesis continues in the subventricular zone (SVZ) of the lateral ventricles and in the dentate gyrus (DG). In the DG of both species, DCX-expressing cells and Ki-67-labeled cells were present in the subgranular and granular layers. In the mole, a strong bundle of DCX-labeled processes, presumably axons of granule cells, was observed in the center of the hilus. Proliferating cells (expressing Ki-67) were identified in the SVZ of lateral ventricles of both species, but neuronal precursor cells (expressing DCX) were also observed in the olfactory bulb (OB). In both species, the vast majority of cells expressing DCX in the OB were granule cells with radially orientated dendrites, although some periglomerular cells surrounding the glomeruli were also labeled. In addition, this paper is the first to show DCX-labeled fibers in the anterior commissure of the hedgehog and mole. These fibers must be axons of new neurons making interhemispheric connections between the two OB or piriform (olfactory) cortices. DCX-expressing neurons were observed in the striatum and piriform cortex of both hedgehog and mole. We postulate that in both species a fraction of cells newly generated in the SVZ migrates along the rostral migratory stream to the piriform cortex. This pattern of migration resembles that of the 'second-wave neurons' generated during embryonal development of the neocortex rather than the pattern observed during

  10. Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells.

    PubMed

    Weber, Marlen; Apostolova, Galina; Widera, Darius; Mittelbronn, Michel; Dechant, Georg; Kaltschmidt, Barbara; Rohrer, Hermann

    2015-02-01

    Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage. © 2014 AlphaMed Press.

  11. Microtubule-dependent association of AKAP350A and CCAR1 with RNA stress granules

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kolobova, Elena; Efimov, Andrey; Kaverina, Irina

    Recent investigations have highlighted the importance of subcellular localization of mRNAs to cell function. While AKAP350A, a multifunctional scaffolding protein, localizes to the Golgi apparatus and centrosomes, we have now identified a cytosolic pool of AKAP350A. Analysis of AKAP350A scaffolded complexes revealed two novel interacting proteins, CCAR1 and caprin-1. CCAR1, caprin-1 and AKAP350A along with G3BP, a stress granule marker, relocate to RNA stress granules after arsenite treatment. Stress also caused loss of AKAP350 from the Golgi and fragmentation of the Golgi apparatus. Disruption of microtubules with nocodazole altered stress granule formation and changed their morphology by preventing fusion ofmore » stress granules. In the presence of nocodazole, arsenite induced smaller granules with the vast majority of AKAP350A and CCAR1 separated from G3BP-containing granules. Similar to nocodazole treatment, reduction of AKAP350A or CCAR1 expression also altered the size and number of G3BP-containing stress granules induced by arsenite treatment. A limited set of 69 mRNA transcripts was immunoisolated with AKAP350A even in the absence of stress, suggesting the association of AKAP350A with mRNA transcripts. These results provide the first evidence for the microtubule dependent association of AKAP350A and CCAR1 with RNA stress granules.« less

  12. Qiwei granules alleviates podocyte lesion in kidney of diabetic KK-Ay mice.

    PubMed

    Zhou, Jingxin; Sun, Wen; Yoshitomi, Hisae; Li, Linyi; Qin, Lingling; Guo, Xiangyu; Wu, Lili; Zhang, Yan; Wu, Xinli; Xu, Tunhai; Gao, Ming; Liu, Tonghua

    2015-03-31

    Chinese medicine comprised of all natural herbs is widespread used in the treatment of diabetic nephropathy (DN). Podocyte contributes to the integrity of glomerular filtration barrier whose injury plays an important role in the initiation and progression of DN. Our study aimed to investigate the effect of Qiwei granules on podocyte lesion in diabetic KK-A(y) mice kidney and its underlying mechanism. Twelve-week-old male KK-A(y) mice were randomly divided in vehicle group and Qiwei granules group, while C57BL/6J mice were used as normal control. The mice were gavage with 1.37 g/kg/day Qiwei granules or water for 10 weeks. We measured water, food intake and body weight (BW) and fasting blood glucose (FBG) every 2 weeks, and urine protein every 4 weeks. At the end of the experiment, all surviving mice were sacrificed. The kidney weight and serum renal parameters were measured, and the renal morphology was observed. To search the underlying mechanism, we examined the podocyte positive marker, slit diaphragm protein expression and some involved cell signal pathway. Qiwei granules treatment significantly improved the metabolic parameters, alleviated the urinary protein, and protected renal function in KK-A(y) mice. In addition, the glomerular injuries and podocyte lesions were mitigated with Qiwei granules treatment. Furthermore, Qiwei granules increased expression of nephrin, CD2AP, and integrin alpha3beta1 in the podocytes of KK-A(y) mice. Qiwei granules improved the phosphoration of Akt and inhibited cleaved caspase-3 protein expression. These finding suggest that Qiwei granules protects the podocyte from the development of DN via improving slit diaphragm (SD) molecules expression and likely activating Akt signaling pathway in KK-A(y) mice.

  13. The Role of Astrocytes in the Generation, Migration, and Integration of New Neurons in the Adult Olfactory Bulb

    PubMed Central

    Gengatharan, Archana; Bammann, Rodrigo R.; Saghatelyan, Armen

    2016-01-01

    In mammals, new neurons in the adult olfactory bulb originate from a pool of neural stem cells in the subventricular zone of the lateral ventricles. Adult-born cells play an important role in odor information processing by adjusting the neuronal network to changing environmental conditions. Olfactory bulb neurogenesis is supported by several non-neuronal cells. In this review, we focus on the role of astroglial cells in the generation, migration, integration, and survival of new neurons in the adult forebrain. In the subventricular zone, neural stem cells with astrocytic properties display regional and temporal specificity when generating different neuronal subtypes. Non-neurogenic astrocytes contribute to the establishment and maintenance of the neurogenic niche. Neuroblast chains migrate through the rostral migratory stream ensheathed by astrocytic processes. Astrocytes play an important regulatory role in neuroblast migration and also assist in the development of a vasculature scaffold in the migratory stream that is essential for neuroblast migration in the postnatal brain. In the olfactory bulb, astrocytes help to modulate the network through a complex release of cytokines, regulate blood flow, and provide metabolic support, which may promote the integration and survival of new neurons. Astrocytes thus play a pivotal role in various processes of adult olfactory bulb neurogenesis, and it is likely that many other functions of these glial cells will emerge in the near future. PMID:27092050

  14. Stem cell sources for clinical islet transplantation in type 1 diabetes: embryonic and adult stem cells.

    PubMed

    Miszta-Lane, Helena; Mirbolooki, Mohammadreza; James Shapiro, A M; Lakey, Jonathan R T

    2006-01-01

    Lifelong immunosuppressive therapy and inadequate sources of transplantable islets have led the islet transplantation benefits to less than 0.5% of type 1 diabetics. Whereas the potential risk of infection by animal endogenous viruses limits the uses of islet xeno-transplantation, deriving islets from stem cells seems to be able to overcome the current problems of islet shortages and immune compatibility. Both embryonic (derived from the inner cell mass of blastocysts) and adult stem cells (derived from adult tissues) have shown controversial results in secreting insulin in vitro and normalizing hyperglycemia in vivo. ESCs research is thought to have much greater developmental potential than adult stem cells; however it is still in the basic research phase. Existing ESC lines are not believed to be identical or ideal for generating islets or beta-cells and additional ESC lines have to be established. Research with ESCs derived from humans is controversial because it requires the destruction of a human embryo and/or therapeutic cloning, which some believe is a slippery slope to reproductive cloning. On the other hand, adult stem cells are already in some degree specialized, recipients may receive their own stem cells. They are flexible but they have shown mixed degree of availability. Adult stem cells are not pluripotent. They may not exist for all organs. They are difficult to purify and they cannot be maintained well outside the body. In order to draw the future avenues in this field, existent discrepancies between the results need to be clarified. In this study, we will review the different aspects and challenges of using embryonic or adult stem cells in clinical islet transplantation for the treatment of type 1 diabetes.

  15. The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

    PubMed Central

    Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

    2014-01-01

    Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity. PMID:23545413

  16. Live Imaging of Adult Neural Stem Cells in Rodents

    PubMed Central

    Ortega, Felipe; Costa, Marcos R.

    2016-01-01

    The generation of cells of the neural lineage within the brain is not restricted to early development. New neurons, oligodendrocytes, and astrocytes are produced in the adult brain throughout the entire murine life. However, despite the extensive research performed in the field of adult neurogenesis during the past years, fundamental questions regarding the cell biology of adult neural stem cells (aNSCs) remain to be uncovered. For instance, it is crucial to elucidate whether a single aNSC is capable of differentiating into all three different macroglial cell types in vivo or these distinct progenies constitute entirely separate lineages. Similarly, the cell cycle length, the time and mode of division (symmetric vs. asymmetric) that these cells undergo within their lineage progression are interesting questions under current investigation. In this sense, live imaging constitutes a valuable ally in the search of reliable answers to the previous questions. In spite of the current limitations of technology new approaches are being developed and outstanding amount of knowledge is being piled up providing interesting insights in the behavior of aNSCs. Here, we will review the state of the art of live imaging as well as the alternative models that currently offer new answers to critical questions. PMID:27013941

  17. Joint Effects of Granule Size and Degree of Substitution on Octenylsuccinated Sweet Potato Starch Granules As Pickering Emulsion Stabilizers.

    PubMed

    Li, Jinfeng; Ye, Fayin; Lei, Lin; Zhou, Yun; Zhao, Guohua

    2018-05-02

    The granules of sweet potato starch were size fractionated into three portions with significantly different median diameters ( D 50 ) of 6.67 (small-sized), 11.54 (medium-sized), and 16.96 μm (large-sized), respectively. Each portion was hydrophobized at the mass-based degrees of substitution (DS m ) of approximately 0.0095 (low), 0.0160 (medium), and 0.0230 (high). The Pickering emulsion-stabilizing capacities of modified granules were tested, and the resultant emulsions were characterized. The joint effects of granule size and DS m on emulsifying capacity (EC) were investigated by response surface methodology. For small-, medium-, and large-sized fractions, their highest emulsifying capacities are comparable but, respectively, encountered at high (0.0225), medium (0.0158), and low (0.0095) DS m levels. The emulsion droplet size increased with granule size, and the number of freely scattered granules in emulsions decreased with DS m . In addition, the term of surface density of the octenyl succinic group (SD -OSG ) was first proposed for modified starch granules, and it was proved better than DS m in interpreting the emulsifying capacities of starch granules with varying sizes. The present results implied that, as the particulate stabilizers, the optimal DS m of modified starch granules is size specific.

  18. The application of a generativity model for older adults.

    PubMed

    Ehlman, Katie; Ligon, Mary

    2012-01-01

    Generativity is a concept first introduced by Erik Erikson as a part of his psychosocial theory which outlines eight stages of development in the human life. Generativity versus stagnation is the main developmental concern of middle adulthood; however, generativity is also recognized as an important theme in the lives of older adults. Building on the work of Erikson, McAdams and de St. Aubin (1992) developed a model explaining the generative process. The aims of this article are: (a) to explore the relationship between generativity and older adults as it appears in research literature; and (b) to examine McAdam's model and use it to explain the role of generativity in older adults who share life stories with gerontology students through an oral history project.

  19. The influence of granulation on super disintegrant performance.

    PubMed

    Zhao, Na; Augsburger, Larry L

    2006-02-01

    The purpose of this study is to identify the causes of efficiency loss of super disintegrants following granulation or reworking. Two processes, precompression and prewetting, were proposed to simulate the processes during dry and wet granulation, respectively. The disintegration efficiency of the resulting disintegrant granules was tested in model formulations composed of dicalcium phosphate and lactose with the unprocessed disintegrants as controls. No significant difference was shown in the intrinsic swelling and the water uptake abilities of all super disintegrants following dry granulation. However, a significant decrease was observed for both Primojel and Polyplasdone XL10 in the rate of water being absorbed into the tablet matrix following wet granulation, but not for Ac-Di-Sol. United States Pharmacopeia (USP) disintegration testing without disc revealed a significant increase in disintegration time for tablets formulated with dry granulated Primojel and Polyplasdone XL10 and all wet granulated disintegrants. The increase in particle size following granulation appears to be the cause of the loss in disintegration efficiency. In conclusion, Ac-Di-Sol is less affected by both precompression and prewetting. The efficiency of Primojel and Polyplasdone XL10 is highly dependent on their particle size. Descreasing the particle size tends to increase their efficiency. Due to the size increase following granulation, a higher addition level of super disintegrant is required to ensure fast and uniform disintegration of tablets prepared by granulation.

  20. Native granule associated short chain length polyhydroxyalkanoate synthase from a marine derived Bacillus sp. NQ-11/A2.

    PubMed

    Prabhu, Nimali N; Santimano, Maria Celisa; Mavinkurve, Suneela; Bhosle, Saroj N; Garg, Sandeep

    2010-01-01

    A rapidly growing marine derived Bacillus sp. strain NQ-11/A2, identified as Bacillus megaterium, accumulated 61% polyhydroxyalkanoate by weight. Diverse carbon sources served as substrates for the accumulation of short chain length polyhydroxyalkanoate. Three to nine granules either single or attached as buds could be isolated intact from each cell. Maximum activity of polyhydroxyalkanoate synthase was associated with the granules. Granule-bound polyhydroxyalkanoate synthase had a K(m) of 7.1 x 10(-5) M for DL-beta-hydroxybutyryl-CoA. Temperature and pH optima for maximum activity were 30 degrees C and 7.0, respectively. Sodium ions were required for granule-bound polyhydroxyalkanoate synthase activity and inhibited by potassium. Granule-bound polyhydroxyalkanoate synthase was apparently covalently bound to the polyhydroxyalkanoate-core of the granules and affected by the chaotropic reagent urea. Detergents inhibited the granule-bound polyhydroxyalkanoate synthase drastically whilst glycerol and bovine serum albumin stabilized the synthase.

  1. The morphology of solar granulations and dark networks

    NASA Astrophysics Data System (ADS)

    Graves, J. Elon; Pierce, A. Keith

    1986-08-01

    Solar granules are classified into four groups based on shape and splitting by sharp rifts crossing them. Grains are classified as: single granules varying in size from 1/8 to 3 in., single granules embayed by a broad dark area or possessing a central darkening, single granules split by very narrow rifts which are significantly narrower than the intergranular lanes, and complexes of granules displaying a daisy pattern. The formation and growth of 'white-light dark networks' are also discussed

  2. 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.

  3. Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

    PubMed Central

    Smith, Jarrett; Calidas, Deepika; Schmidt, Helen; Lu, Tu; Rasoloson, Dominique; Seydoux, Geraldine

    2016-01-01

    RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings suggest that MEX-5 interferes with MEG-3’s access to RNA, thus locally suppressing MEG-3 phase separation to drive P granule asymmetry. Regulated access to RNA, combined with RNA-induced phase separation of key scaffolding proteins, may be a general mechanism for controlling the formation of RNA granules in space and time. DOI: http://dx.doi.org/10.7554/eLife.21337.001 PMID:27914198

  4. Similar Responses of Intestinal T Cells From Untreated Children and Adults With Celiac Disease to Deamidated Gluten Epitopes.

    PubMed

    Ráki, Melinda; Dahal-Koirala, Shiva; Yu, Hao; Korponay-Szabó, Ilma R; Gyimesi, Judit; Castillejo, Gemma; Jahnsen, Jørgen; Qiao, Shuo-Wang; Sollid, Ludvig M

    2017-09-01

    Celiac disease is a chronic small intestinal inflammatory disorder mediated by an immune response to gluten peptides in genetically susceptible individuals. Celiac disease is often diagnosed in early childhood, but some patients receive a diagnosis late in life. It is uncertain whether pediatric celiac disease is distinct from adult celiac disease. It has been proposed that gluten-reactive T cells in children recognize deamidated and native gluten epitopes, whereas T cells from adults only recognize deamidated gluten peptides. We studied the repertoire of gluten epitopes recognized by T cells from children and adults. We examined T-cell responses against gluten by generating T-cell lines and T-cell clones from intestinal biopsies of adults and children and tested proliferative response to various gluten peptides. We analyzed T cells from 14 children (2-5 years old) at high risk for celiac disease who were followed for celiac disease development. We also analyzed T cells from 6 adults (26-55 years old) with untreated celiac disease. All children and adults were positive for HLA-DQ2.5. Biopsies were incubated with gluten digested with chymotrypsin (modified or unmodified by the enzyme transglutaminase 2) or the peptic-tryptic digest of gliadin (in native and deamidated forms) before T-cell collection. Levels of T-cell responses were higher to deamidated gluten than to native gluten in children and adults. T cells from children and adults each reacted to multiple gluten epitopes. Several T-cell clones were cross-reactive, especially clones that recognized epitopes from γ-and ω-gliadin. About half of the generated T-cell clones from children and adults reacted to unknown epitopes. T-cell responses to different gluten peptides appear to be similar between adults and children at the time of diagnosis of celiac disease. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

  5. Fabrication of dicalcium phosphate dihydrate-coated β-TCP granules and evaluation of their osteoconductivity using experimental rats.

    PubMed

    Shariff, Khairul Anuar; Tsuru, Kanji; Ishikawa, Kunio

    2017-06-01

    β-Tricalcium phosphate (β-TCP) has attracted much attention as an artificial bone substitute owing to its biocompatibility and osteoconductivity. In this study, osteoconductivity of β-TCP bone substitute was enhanced without using growth factors or cells. Dicalcium phosphate dihydrate (DCPD), which is known to possess the highest solubility among calcium phosphates, was coated on β-TCP granules by exposing their surface with acidic calcium phosphate solution. The amount of coated DCPD was regulated by changing the reaction time between β-TCP granules and acidic calcium phosphate solution. Histomorphometry analysis obtained from histological results revealed that the approximately 10mol% DCPD-coated β-TCP granules showed the largest new bone formation compared to DCPD-free β-TCP granules, approximately 2.5mol% DCPD-coated β-TCP granules, or approximately 27mol% DCPD-coated β-TCP granules after 2 and 4weeks of implantation. Based on this finding, we demonstrate that the osteoconductivity of β-TCP granules could be improved by coating their surface with an appropriate amount of DCPD. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Preparation of orally disintegrating tablets with taste-masking function: masking effect in granules prepared with correctives using the dry granulation method and evaluation of tablets prepared using the taste-masked granules.

    PubMed

    Kawano, Yayoi; Ito, Akihiko; Sasatsu, Masanaho; Machida, Yoshiharu

    2010-01-01

    We investigated several methods of taste masking in the preparation of orally disintegrating tablets (ODTs), using furosemide (FU) as a model drug. Four types of FU preparations were prepared: granules with maltitol (MA), granules with yogurt powder (YO), a physical mixture of FU and MA, and a physical mixture of FU and YO. All taste-masking granules were prepared using the dry granulation method. The taste of each type of preparation was evaluated. All four preparations markedly improved the taste of the FU tablets, but the mixing ratios of the correctives did not affect the masking effect. No difference in masking effect was found between MA and YO in the physical mixtures, but the masking effect in the granules with YO was superior to that of the granules with MA. Taste-masked FU tablets were prepared using the direct compression method; crystalline cellulose (Avicel PH-302) and mannitol were added as excipients at the mixing ratio of 1/1. All four types of tablets displayed sufficient hardness, but MA-containing tablets were harder than YO-containing tablets. The hardness of the tablets prepared from YO granules increased as the YO content increased. The most rapidly disintegrating tablets were those of YO granules prepared at a mixing ratio of FU/YO=1/1, which disintegrated within 20 s, followed by the tablets of MA granules prepared at a mixing ratio of FU/MA=1/1. The disintegration times of the tablets made from physical mixtures, in contrast, were longer than 200 s. Disintegration time lengthened as the mixing ratio of YO or MA increased. The hardness and disintegration time of these tablets could be controlled by varying the compression pressure. We found that YO is more useful than MA in masking unpleasant tastes and confirmed that orally disintegrating tablets with taste-masking function can be prepared using granules of YO prepared using the dry granulation method as a new corrective.

  7. Bi-Parental Care Contributes to Sexually Dimorphic Neural Cell Genesis in the Adult Mammalian Brain

    PubMed Central

    Mak, Gloria K.; Antle, Michael C.; Dyck, Richard H.; Weiss, Samuel

    2013-01-01

    Early life events can modulate brain development to produce persistent physiological and behavioural phenotypes that are transmissible across generations. However, whether neural precursor cells are altered by early life events, to produce persistent and transmissible behavioural changes, is unknown. Here, we show that bi-parental care, in early life, increases neural cell genesis in the adult rodent brain in a sexually dimorphic manner. Bi-parentally raised male mice display enhanced adult dentate gyrus neurogenesis, which improves hippocampal neurogenesis-dependent learning and memory. Female mice display enhanced adult white matter oligodendrocyte production, which increases proficiency in bilateral motor coordination and preference for social investigation. Surprisingly, single parent-raised male and female offspring, whose fathers and mothers received bi-parental care, respectively, display a similar enhancement in adult neural cell genesis and phenotypic behaviour. Therefore, neural plasticity and behavioural effects due to bi-parental care persist throughout life and are transmitted to the next generation. PMID:23650527

  8. CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

    PubMed Central

    Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

    2010-01-01

    Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

  9. Vanillin Inhibits Translation and Induces Messenger Ribonucleoprotein (mRNP) Granule Formation in Saccharomyces cerevisiae: Application and Validation of High-Content, Image-Based Profiling

    PubMed Central

    Suga, Yohei; Izawa, Shingo; Ohya, Yoshikazu

    2013-01-01

    Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells. PMID:23637899

  10. Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice

    PubMed Central

    Nilforoushzadeh, Mohammadali; Rahimi Jameh, Elham; Jaffary, Fariba; Abolhasani, Ehsan; Keshtmand, Gelavizh; Zarkob, Hajar; Mohammadi, Parvaneh; Aghdami, Nasser

    2017-01-01

    Objective Dermal papilla and hair epithelial stem cells regulate hair formation and the growth cycle. Damage to or loss of these cells can cause hair loss. Although several studies claim to reconstitute hairs using rodent cells in an animal model, additional research is needed to develop a stable human hair follicle reconstitution protocol. In this study, we have evaluated hair induction by injecting adult cultured human dermal papilla cells and a mixture of hair epithelial and dermal papilla cells in a mouse model. Materials and Methods In this experimental study, discarded human scalp skins were used to obtain dermal papilla and hair epithelial cells. After separation, cells were cultured and assessed for their characteristics. We randomly allocated 15 C57BL/6 nude mice into three groups that received injections in their dorsal skin. The first group received cultured dermal papilla cells, the second group received a mixture of cultured epithelial and dermal papilla cells, and the third group (control) received a placebo [phosphate-buffered saline (PBS-)]. Results Histopathologic examination of the injection sites showed evidence of hair growth in samples that received cells compared with the control group. However, the group that received epithelial and dermal papilla cells had visible evidence of hair growth. PKH tracing confirmed the presence of transplanted cells in the new hair. Conclusion Our data showed that injection of a combination of adult human cultured dermal papilla and epithelial cells could induce hair growth in nude mice. This study emphasized that the combination of human adult cultured dermal papilla and epithelial cells could induce new hair in nude mice. PMID:28670518

  11. Effect of Environmental Chemical Exposures on Adult Human Cardiac Progenitor Cell Viability and Differentiation

    EPA Science Inventory

    Cell biology has revealed that the adult heart is not a terminally differentiated organ but is capable of generating new cardiomyocytes (CMs) from cardiac stem cells (CSC) and/or progenitor cells (CPC) throughout life. The impact that environmental chemical exposures have on adul...

  12. FUS/TLS assembles into stress granules and is a prosurvival factor during hyperosmolar stress.

    PubMed

    Sama, Reddy Ranjith K; Ward, Catherine L; Kaushansky, Laura J; Lemay, Nathan; Ishigaki, Shinsuke; Urano, Fumihiko; Bosco, Daryl A

    2013-11-01

    FUsed in Sarcoma/Translocated in LipoSarcoma (FUS/TLS or FUS) has been linked to several biological processes involving DNA and RNA processing, and has been associated with multiple diseases, including myxoid liposarcoma and amyotrophic lateral sclerosis (ALS). ALS-associated mutations cause FUS to associate with stalled translational complexes called stress granules under conditions of stress. However, little is known regarding the normal role of endogenous (non-disease linked) FUS in cellular stress response. Here, we demonstrate that endogenous FUS exerts a robust response to hyperosmolar stress induced by sorbitol. Hyperosmolar stress causes an immediate re-distribution of nuclear FUS to the cytoplasm, where it incorporates into stress granules. The redistribution of FUS to the cytoplasm is modulated by methyltransferase activity, whereas the inhibition of methyltransferase activity does not affect the incorporation of FUS into stress granules. The response to hyperosmolar stress is specific, since endogenous FUS does not redistribute to the cytoplasm in response to sodium arsenite, hydrogen peroxide, thapsigargin, or heat shock, all of which induce stress granule assembly. Intriguingly, cells with reduced expression of FUS exhibit a loss of cell viability in response to sorbitol, indicating a prosurvival role for endogenous FUS in the cellular response to hyperosmolar stress. Copyright © 2013 Wiley Periodicals, Inc.

  13. FUS/TLS assembles into stress granules and is a prosurvival factor during hyperosmolar stress

    PubMed Central

    Sama, Reddy Ranjith K; Ward, Catherine L.; Kaushansky, Laura J.; Lemay, Nathan; Ishigaki, Shinsuke; Urano, Fumihiko; Bosco, Daryl A.

    2014-01-01

    FUsed in Sarcoma/Translocated in LipoSarcoma (FUS/TLS or FUS) has been linked to several biological processes involving DNA and RNA processing, and has been associated with multiple diseases, including myxoid liposarcoma and amyotrophic lateral sclerosis (ALS). ALS-associated mutations cause FUS to associate with stalled translational complexes called stress granules under conditions of stress. However, little is known regarding the normal role of endogenous (non-disease linked) FUS in cellular stress response. Here, we demonstrate that endogenous FUS exerts a robust response to hyperosmolar stress induced by sorbitol. Hyperosmolar stress causes an immediate re-distribution of nuclear FUS to the cytoplasm, where it incorporates into stress granules. The redistribution of FUS to the cytoplasm is modulated by methyltransferase activity, whereas the inhibition of methyltransferase activity does not affect the incorporation of FUS into stress granules. The response to hyperosmolar stress is specific, since endogenous FUS does not redistribute to the cytoplasm in response to sodium arsenite, hydrogen peroxide, thapsigargin, or heat shock, all of which induce stress granule assembly. Intriguingly, cells with reduced expression of FUS exhibit a loss of cell viability in response to sorbitol, indicating a prosurvival role for endogenous FUS in the cellular response to hyperosmolar stress. PMID:23625794

  14. Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice

    PubMed Central

    Bucks, Stephanie A; Cox, Brandon C; Vlosich, Brittany A; Manning, James P; Nguyen, Tot B; Stone, Jennifer S

    2017-01-01

    Vestibular hair cells in the inner ear encode head movements and mediate the sense of balance. These cells undergo cell death and replacement (turnover) throughout life in non-mammalian vertebrates. However, there is no definitive evidence that this process occurs in mammals. We used fate-mapping and other methods to demonstrate that utricular type II vestibular hair cells undergo turnover in adult mice under normal conditions. We found that supporting cells phagocytose both type I and II hair cells. Plp1-CreERT2-expressing supporting cells replace type II hair cells. Type I hair cells are not restored by Plp1-CreERT2-expressing supporting cells or by Atoh1-CreERTM-expressing type II hair cells. Destruction of hair cells causes supporting cells to generate 6 times as many type II hair cells compared to normal conditions. These findings expand our understanding of sensorineural plasticity in adult vestibular organs and further elucidate the roles that supporting cells serve during homeostasis and after injury. DOI: http://dx.doi.org/10.7554/eLife.18128.001 PMID:28263708

  15. Biofilm formation and granule properties in anaerobic digestion at high salinity.

    PubMed

    Gagliano, M C; Ismail, S B; Stams, A J M; Plugge, C M; Temmink, H; Van Lier, J B

    2017-09-15

    For the anaerobic biological treatment of saline wastewater, Anaerobic Digestion (AD) is currently a possibility, even though elevated salt concentrations can be a major obstacle. Anaerobic consortia and especially methanogenic archaea are very sensitive to fluctuations in salinity. When working with Upflow Sludge Blanket Reactor (UASB) technology, in which the microorganisms are aggregated and retained in the system as a granular biofilm, high sodium concentration negatively affects aggregation and consequently process performances. In this research, we analysed the structure of the biofilm and granules formed during the anaerobic treatment of high salinity (at 10 and 20 g/L of sodium) synthetic wastewater at lab scale. The acclimated inoculum was able to accomplish high rates of organics removal at all the salinity levels tested. 16S rRNA gene clonal analysis and Fluorescence In Situ Hybridization (FISH) analyses identified the acetoclastic Methanosaeta harundinacea as the key player involved acetate degradation and microbial attachment/granulation. When additional calcium (1 g/L) was added to overcome the negative effect of sodium on microbial aggregation, during the biofilm formation process microbial attachment and acetate degradation decreased. The same result was observed on granules formation: while calcium had a positive effect on granules strength when added to UASB reactors, Methanosaeta filaments were not present and the degradation of the partially acidified substrate was negatively influenced. This research demonstrated the possibility to get granulation at high salinity, bringing to the forefront the importance of a selection towards Methanosaeta cells growing in filamentous form to obtain strong and healthy granules. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Probing the spatial dependence of the emission spectrum of single human retinal lipofuscin granules using near-field scanning optical microscopy.

    PubMed

    Haralampus-Grynaviski, N M; Lamb, L E; Simon, J D; Krogmeier, J R; Dunn, R C; Pawlak, A; Rózanowska, M; Sarna, T; Burke, J M

    2001-08-01

    The emission spectra of single lipofuscin granules are examined using spectrally resolved confocal microscopy and near-field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distribution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale approximately 150 nm.

  17. Fuel cell generator

    DOEpatents

    Makiel, Joseph M.

    1985-01-01

    A high temperature solid electrolyte fuel cell generator comprising a housing means defining a plurality of chambers including a generator chamber and a combustion products chamber, a porous barrier separating the generator and combustion product chambers, a plurality of elongated annular fuel cells each having a closed end and an open end with the open ends disposed within the combustion product chamber, the cells extending from the open end through the porous barrier and into the generator chamber, a conduit for each cell, each conduit extending into a portion of each cell disposed within the generator chamber, each conduit having means for discharging a first gaseous reactant within each fuel cell, exhaust means for exhausting the combustion product chamber, manifolding means for supplying the first gaseous reactant to the conduits with the manifolding means disposed within the combustion product chamber between the porous barrier and the exhaust means and the manifolding means further comprising support and bypass means for providing support of the manifolding means within the housing while allowing combustion products from the first and a second gaseous reactant to flow past the manifolding means to the exhaust means, and means for flowing the second gaseous reactant into the generator chamber.

  18. Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell–mediated cytotoxicity

    PubMed Central

    Tuli, Amit; Thiery, Jerome; James, Ashley M.; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B.; Orange, Jordan S.; Lieberman, Judy; Brenner, Michael B.

    2013-01-01

    Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity. PMID:24088571

  19. A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic β cells

    PubMed Central

    Kalwat, Michael A.; Yoder, Stephanie M.; Wang, Zhanxiang; Thurmond, Debbie C.

    2012-01-01

    Human islet studies implicate an important signaling role for the Cdc42 effector protein p21-activated kinase (PAK1) in the sustained/second-phase of insulin secretion. Because human islets from type 2 diabetic donors lack ~80% of normal PAK1 protein levels, the mechanistic requirement for PAK1 signaling in islet function was interrogated. Similar to MIN6 β cells, human islets elicited glucose-stimulated PAK1 activation that was sensitive to the PAK1 inhibitor, IPA3. Given that sustained insulin secretion has been correlated with glucose-induced filamentous actin (F-actin) remodeling, we tested the hypothesis that a Cdc42-activated PAK1 signaling cascade is required to elicit F-actin remodeling to mobilize granules to the cell surface. Live-cell imaging captured the glucose-induced cortical F-actin remodeling in MIN6 β cells; IPA3-mediated inhibition of PAK1 abolished this remodeling. IPA3 also ablated glucose-stimulated insulin granule accumulation at the plasma membrane, consistent with its role in sustained/second-phase insulin release. Both IPA3 and a selective inhibitor of the Cdc42 GTPase, ML-141, blunted the glucose-stimulated activation of Raf-1, suggesting Raf-1 to be downstream of Cdc42→PAK1. IPA3 also inhibited MEK1/2 activation, implicating the MEK1/2→ERK1/2 cascade to occur downstream of PAK1. Importantly, PD0325901, a new selective inhibitor of MEK1/2→ERK1/2 activation, impaired F-actin remodeling and the sustained/amplification pathway of insulin release. Taken together, these data suggest that glucose-mediated activation of Cdc42 leads to activation of PAK1 and prompts activation of its downstream targets Raf-1, MEK1/2 and ERK1/2 to elicit F-actin remodeling and recruitment of insulin granules to the plasma membrane to support the sustained phase of insulin release. PMID:23246867

  20. Inhibitory effect of mitomycin C on proliferation of primary cultured fibroblasts from human airway granulation tissues.

    PubMed

    Chen, Nan; Zhang, Jie; Xu, Min; Wang, Yu Ling; Pei, Ying Hua

    2013-01-01

    Airway granulation tissue and scar formation pose a challenge because of the high incidence of recurrence after treatment. As an emerging treatment modality, topical application of mitomycin C has potential value in delaying the recurrence of airway obstruction. Several animal and clinical studies have already proven its feasibility and efficacy. However, the ideal dosage has still not been determined. To establish a novel method for culturing primary fibroblasts isolated from human airway granulation tissue, and to investigate the dose-effect of mitomycin C on the fibroblast proliferation in vitro, so as to provide an experimental reference for clinical practitioners. Granulation tissues were collected during the routine bronchoscopy at our department. The primary fibroblasts were obtained by culturing the explanted tissues. The cells were treated with different concentrations of mitomycin C (0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml) for 5 min followed by additional 48-hour culture before an MTT assay was performed to measure cell viability. MTT assay showed that mitomycin C reduced cell viability at all tested concentrations. The inhibitory ratios were 10.26, 26.77, 32.88, 64.91 and 80.45% for cells treated with mitomycin C at 0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml, respectively. Explant culture is a reliable method for culturing primary fibroblasts from human airway granulation tissue, and mitomycin C can inhibit proliferation of the fibroblasts in vitro. Copyright © 2013 S. Karger AG, Basel.

  1. Chromogranin A: A New Proposal for Trafficking, Processing and Induction of Granule Biogenesis

    PubMed Central

    Koshimizu, Hisatsugu; Kim, Taeyoon; Cawley, Niamh X.; Loh, Y. Peng

    2009-01-01

    Chromogranin A (CgA), a member of the granin family serves several important cell biological roles in (neuro)endocrine cells which are summarized in this review. CgA is a “prohormone” that is synthesized at the rough endoplasmic reticulum and transported into the cisternae of this organelle via its signal peptide. It is then trafficked to the Golgi complex and then to the trans-Golgi network (TGN) where CgA aggregates at low pH in the presence of calcium. The CgA aggregates provide the physical driving force to induce budding of the TGN membrane resulting in dense core granule (DCG) formation. Within the granule, a small amount of the CgA is processed to bioactive peptides, including a predicted C-terminal peptide, serpinin. Upon stimulation, DCGs undergo exocytosis and CgA and its derived peptides are released. Serpinin, acting extracellularly is able to signal the increase in transcription of a serine protease inhibitor, protease nexin-1 (PN-1) that protects DCG proteins against degradation in the Golgi complex, which then enhances DCG biogenesis to replenish those that were released. Thus CgA and its derived peptide, serpinin, plays a significant role in the formation and regulation, respectively, of granule biogenesis in (neuro)endocrine cells. PMID:20006653

  2. Key role of T cell defects in age-related vulnerability to West Nile virus.

    PubMed

    Brien, James D; Uhrlaub, Jennifer L; Hirsch, Alec; Wiley, Clayton A; Nikolich-Zugich, Janko

    2009-11-23

    West Nile virus (WNV) infection causes a life-threatening meningoencephalitis that becomes increasingly more prevalent over the age of 50 and is 40-50x more prevalent in people over the age of 70, compared with adults under the age of 40. In a mouse model of age-related vulnerability to WNV, we demonstrate that death correlates with increased viral titers in the brain and that this loss of virus control with age was the result of defects in the CD4 and CD8 T cell response against WNV. Specific age-related defects in T cell responses against dominant WNV epitopes were detected at the level of cytokine and lytic granule production, each of which are essential for resistance against WNV, and in the ability to generate multifunctional anti-WNV effector T cells, which are believed to be critical for robust antiviral immunity. In contrast, at the peak of the response, old and adult T cells exhibited superimposable peptide sensitivity. Most importantly, although the adult CD4 or CD8 T cells readily protected immunodeficient mice upon adoptive transfer, old T cells of either subset were unable to provide WNV-specific protection. Consistent with a profound qualitative and quantitative defect in T cell immunity, old brains contained at least 12x fewer total effector CD8 T cells compared with adult mice at the peak of brain infection. These findings identify potential targets for immunomodulation and treatment to combat lethal WNV infection in the elderly.

  3. Survival of adult neurons lacking cholesterol synthesis in vivo

    PubMed Central

    Fünfschilling, Ursula; Saher, Gesine; Xiao, Le; Möbius, Wiebke; Nave, Klaus-Armin

    2007-01-01

    Background Cholesterol, an essential component of all mammalian plasma membranes, is highly enriched in the brain. Both during development and in the adult, brain cholesterol is derived from local cholesterol synthesis and not taken up from the circulation. However, the contribution of neurons and glial cells to total brain cholesterol metabolism is unknown. Results Using conditional gene inactivation in the mouse, we disrupted the squalene synthase gene (fdft1), which is critical for cholesterol synthesis, in cerebellar granule cells and some precerebellar nuclei. Mutant mice showed no histological signs of neuronal degeneration, displayed ultrastructurally normal synapses, and exhibited normal motor coordination. This revealed that these adult neurons do not require cell-autonomous cholesterol synthesis for survival or function. Conclusion We conclude that at least some adult neurons no longer require endogenous cholesterol synthesis and can fully meet their cholesterol needs by uptake from their surrounding. Glia are a likely source of cholesterol in the central nervous system. PMID:17199885

  4. Survival of adult neurons lacking cholesterol synthesis in vivo.

    PubMed

    Fünfschilling, Ursula; Saher, Gesine; Xiao, Le; Möbius, Wiebke; Nave, Klaus-Armin

    2007-01-02

    Cholesterol, an essential component of all mammalian plasma membranes, is highly enriched in the brain. Both during development and in the adult, brain cholesterol is derived from local cholesterol synthesis and not taken up from the circulation. However, the contribution of neurons and glial cells to total brain cholesterol metabolism is unknown. Using conditional gene inactivation in the mouse, we disrupted the squalene synthase gene (fdft1), which is critical for cholesterol synthesis, in cerebellar granule cells and some precerebellar nuclei. Mutant mice showed no histological signs of neuronal degeneration, displayed ultrastructurally normal synapses, and exhibited normal motor coordination. This revealed that these adult neurons do not require cell-autonomous cholesterol synthesis for survival or function. We conclude that at least some adult neurons no longer require endogenous cholesterol synthesis and can fully meet their cholesterol needs by uptake from their surrounding. Glia are a likely source of cholesterol in the central nervous system.

  5. Electron microprobe analysis of human labial gland secretory granules in cystic fibrosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Izutsu, K.; Johnson, D.; Schubert, M.

    1985-06-01

    X-ray microanalysis of freeze-dried labial gland cryosections revealed that Na concentration was doubled and the Ca/S concentration ratio was decreased in secretory granules of labial glands from patients with cystic fibrosis (CF) when compared with glands from normal subjects. Other results suggested that the decrease in the Ca/S concentration ratio resulted from an increase in S concentration. These findings imply that mucous granules in labial saliva showed a CF-related increase in Na and S content, and such changes would be expected to affect the rheology of the mucus after exocytosis. In contrast with a previous study in human parotid glands,more » no evidence was found for CF-related changes in cytoplasmic or nuclear Na, K, and Ca concentrations. Significant elemental differences were found between secretory granules and nuclei and cytoplasm of control cells.« less

  6. A system to investigate the remediation of organic vapors using microwave-induced plasma with fluidized carbon granules

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dawson, Elizabeth A.; Parkes, Gareth M. B.; Bond, Gary

    This article describes a system to investigate the parameters for the remediation of organic vapors using microwave-induced plasma on fluidized carbon granules. The system is based on a single mode microwave apparatus with a variable power (2.45 GHz) generator. Carbon granules are fluidized in a silica tube situated in the sample section of a waveguide incorporating two additional ports to allow plasma intensity monitoring using a light sensor and imaging with a digital camera. A fluoroptic probe is used for in situ measurement of the carbon granule temperature, while the effluent gas temperature is measured with a thermocouple situated inmore » the silica tube outside the cavity. Data acquisition and control software allow experiments using a variety of microwave power regimes while simultaneously recording the light intensity of any plasma generated within the carbon bed, together with its temperature. Evaluation using two different granular activated carbons and ethyl acetate, introduced as a vapor into the fluidizing air stream at a concentration of 1 ppm, yielded results which indicated that significant destruction of ethyl acetate, as monitored using a mass spectrometer, was achieved only with the carbon granules showing high plasma activity under pulsed microwave conditions. The system is therefore suitable for comparison of the relative microwave activities of various activated carbon granules and their performance in microwave remediation and regeneration.« less

  7. Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

    PubMed Central

    Mezzina, Mariela P.

    2016-01-01

    Phasins are the major polyhydroxyalkanoate (PHA) granule-associated proteins. They promote bacterial growth and PHA synthesis and affect the number, size, and distribution of the granules. These proteins can be classified in 4 families with distinctive characteristics. Low-resolution structural studies and in silico predictions were performed in order to elucidate the structure of different phasins. Most of these proteins share some common structural features, such as a preponderant α-helix composition, the presence of disordered regions that provide flexibility to the protein, and coiled-coil interacting regions that form oligomerization domains. Due to their amphiphilic nature, these proteins play an important structural function, forming an interphase between the hydrophobic content of PHA granules and the hydrophilic cytoplasm content. Phasins have been observed to affect both PHA accumulation and utilization. Apart from their role as granule structural proteins, phasins have a remarkable variety of additional functions. Different phasins have been determined to (i) activate PHA depolymerization, (ii) increase the expression and activity of PHA synthases, (iii) participate in PHA granule segregation, and (iv) have both in vivo and in vitro chaperone activities. These properties suggest that phasins might play an active role in PHA-related stress protection and fitness enhancement. Due to their granule binding capacity and structural flexibility, several biotechnological applications have been developed using different phasins, increasing the interest in the study of these remarkable proteins. PMID:27287326

  8. 54. View from southeast of KinneyOsborne slag granulator at right ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    54. View from southeast of Kinney-Osborne slag granulator at right and stack at left that vented steam generated when water was sprayed on hot slag to solidify and fracture it. - Sloss-Sheffield Steel & Iron, First Avenue North Viaduct at Thirty-second Street, Birmingham, Jefferson County, AL

  9. Maintaining sufficient nanos is a critical function for polar granule component in the specification of primordial germ cells.

    PubMed

    Deshpande, Girish; Spady, Emma; Goodhouse, Joe; Schedl, Paul

    2012-11-01

    Primordial germ cells (PGC) are the precursors of germline stem cells. In Drosophila, PGC specification is thought to require transcriptional quiescence and three genes, polar granule component (pgc), nanos (nos), and germ cell less (gcl) function to downregulate Pol II transcription. While it is not understood how nos or gcl represses transcription, pgc does so by inhibiting the transcription elongation factor b (P-TEFb), which is responsible for phosphorylating Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of the largest Pol II subunit. In the studies reported here, we demonstrate that nos are a critical regulatory target of pgc. We show that a substantial fraction of the PGCs in pgc embryos have greatly reduced levels of Nos protein and exhibit phenotypes characteristic of nos PGCs. Lastly, restoring germ cell-specific expression of Nos is sufficient to ameliorate the pgc phenotype.

  10. Adult bone marrow-derived stem cells for organ regeneration and repair.

    PubMed

    Tögel, Florian; Westenfelder, Christof

    2007-12-01

    Stem cells have been recognized as a potential tool for the development of innovative therapeutic strategies. There are in general two types of stem cells, embryonic and adult stem cells. While embryonic stem cell therapy has been riddled with problems of allogeneic rejection and ethical concerns, adult stem cells have long been used in the treatment of hematological malignancies. With the recognition of additional, potentially therapeutic characteristics, bone marrow-derived stem cells have become a tool in regenerative medicine. The bone marrow is an ideal source of stem cells because it is easily accessible and harbors two types of stem cells. Hematopoietic stem cells give rise to all blood cell types and have been shown to exhibit plasticity, while multipotent marrow stromal cells are the source of osteocytes, chondrocytes, and fat cells and have been shown to support and generate a large number of different cell types. This review describes the general characteristics of these stem cell populations and their current and potential future applications in regenerative medicine. 2007 Wiley-Liss, Inc

  11. Why do gelatinized starch granules not dissolve completely? Roles for amylose, protein, and lipid in granule "ghost" integrity.

    PubMed

    Debet, Martine R; Gidley, Michael J

    2007-06-13

    After gelatinization in water, starch granules persist in swollen hydrated forms known as ghosts. Three potential mechanisms for ghost formation are tested. Proteins and lipids on the granule surface are found to be a determinant of ghost robustness, but not ghost formation. Proteins inside pre-made maize or wheat starch ghosts are degraded extensively by proteases without any apparent change in ghost properties, making an internal protein cross-linking mechanism unlikely. Waxy maize mutants with a range of amylose contents have ghost integrities that correlate with (low) apparent amylose levels. It is hypothesized that ghost formation is due to cross-linking of polysaccharide chains within swollen granules, most likely involving double helices formed from polymer chains that become free to move following heat-induced granule swelling. The size and robustness of granule ghosts is proposed to be determined by the relative rates of swelling and cross-linking, modulated by surface non-polysaccharide components.

  12. In-vivo RGB marking and multicolour single-cell tracking in the adult brain

    PubMed Central

    Gomez-Nicola, Diego; Riecken, Kristoffer; Fehse, Boris; Perry, V. Hugh

    2014-01-01

    In neuroscience it is a technical challenge to identify and follow the temporal and spatial distribution of cells as they differentiate. We hypothesised that RGB marking, the tagging of individual cells with unique hues resulting from simultaneous expression of the three basic colours red, green and blue, provides a convenient toolbox for the study of the CNS anatomy at the single-cell level. Using γ-retroviral and lentiviral vector sets we describe for the first time the in-vivo multicolour RGB marking of neurons in the adult brain. RGB marking also enabled us to track the spatial and temporal fate of neural stem cells in the adult brain. The application of different viral envelopes and promoters provided a useful approach to track the generation of neurons vs. glial cells at the neurogenic niche, allowing the identification of the prominent generation of new astrocytes to the striatum. Multicolour RGB marking could serve as a universal and reproducible method to study and manipulate the CNS at the single-cell level, in both health and disease. PMID:25531807

  13. Effect of the granule size in porous silicon on the photosensitization efficiency of molecular oxygen on the surface of silicon nanocrystals

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Demin, V. A.; Konstantinova, E. A., E-mail: liza35@mail.ru; Gongal'skii, M. B.

    2009-03-15

    Photoluminescence is used to study the effect of the granule size in porous silicon on the generation efficiency of the excited state of molecular oxygen ({sup 1}O{sub 2}) on the surface of silicon nanocrystals. The generation efficiency is found to increase as the granule size becomes smaller than 100 nm, which can be explained by a change in the conditions of exciton diffusion along a network of silicon nanocrystals.

  14. Overexpression of Lin28b in Neural Stem Cells is Insufficient for Brain Tumor Formation, but Induces Pathological Lobulation of the Developing Cerebellum.

    PubMed

    Wefers, Annika K; Lindner, Sven; Schulte, Johannes H; Schüller, Ulrich

    2017-02-01

    LIN28B is a homologue of the RNA-binding protein LIN28A and regulates gene expression during development and carcinogenesis. It is strongly upregulated in a variety of brain tumors, such as medulloblastoma, embryonal tumor with multilayered rosettes (ETMR), atypical teratoid/rhabdoid tumor (AT/RT), or glioblastoma, but the effect of an in vivo overexpression of LIN28B on the developing central nervous system is unknown. We generated transgenic mice that either overexpressed Lin28b in Math1-positive cerebellar granule neuron precursors or in a broad range of Nestin-positive neural precursors. Sections of the cerebellar vermis from adult Math1-Cre::lsl-Lin28b mice had an additional subfissure in lobule IV. Vermes from p0 and p7 Nestin-Cre::lsl-Lin28b mice appeared normal, but we found a pronounced vermal hypersublobulation at p15 and p21 in these mice. Also, the external granule cell layer (EGL) was thicker at p15 than in controls, contained more proliferating cells, and persisted up to p21. Consistently, some Pax6- and NeuN-positive cells were present in the EGL of Nestin-Cre::lsl-Lin28b mice even at p21, and we detected more NeuN-positive granule neuron precursors in the molecular layer (ML) as compared to control. Finally, we found some residual Pax2-positive precursors of inhibitory interneurons in the ML of Nestin-Cre::lsl-Lin28b mice at p21, which have already disappeared in controls. We conclude that while overexpression of LIN28B in Nestin-positive cells does not lead to tumor formation, it results in a protracted development of granule cells and inhibitory interneurons and leads to a hypersublobulation of the cerebellar vermis.

  15. 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. Copyright © 2016 by The American Association of Immunologists, Inc.

  16. Biomass conversion inhibitors furfural and 5-hydroxymethylfurfural induce formation of messenger RNP granules and attenuate translation activity in Saccharomyces cerevisiae.

    PubMed

    Iwaki, Aya; Kawai, Takao; Yamamoto, Yosuke; Izawa, Shingo

    2013-03-01

    Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.

  17. Biomass Conversion Inhibitors Furfural and 5-Hydroxymethylfurfural Induce Formation of Messenger RNP Granules and Attenuate Translation Activity in Saccharomyces cerevisiae

    PubMed Central

    Iwaki, Aya; Kawai, Takao; Yamamoto, Yosuke

    2013-01-01

    Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates. PMID:23275506

  18. In vitro utilization of amylopectin and high-amylose maize (Amylomaize) starch granules by human colonic bacteria.

    PubMed

    Wang, X; Conway, P L; Brown, I L; Evans, A J

    1999-11-01

    It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M(r)) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch.

  19. In Vitro Utilization of Amylopectin and High-Amylose Maize (Amylomaize) Starch Granules by Human Colonic Bacteria

    PubMed Central

    Wang, Xin; Conway, Patricia Lynne; Brown, Ian Lewis; Evans, Anthony John

    1999-01-01

    It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (Mr) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch. PMID:10543795

  20. Oligodendrocyte ablation affects the coordinated interaction between granule and Purkinje neurons during cerebellum development

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Collin, Ludovic; Doretto, Sandrine; Department of Psychiatry and Human Behavior, University of California Irvine, 3226 Gillespie Neuroscience Research Facility, Irvine CA 92697

    2007-08-01

    Oligodendrocytes (OLs) are the glial cells of the central nervous system (CNS) classically known to be devoted to the formation of myelin sheaths around most axons of the vertebrate brain. We have addressed the role of these cells during cerebellar development, by ablating OLs in vivo. Previous analyses had indicated that OL ablation during the first six postnatal days results into a striking cerebellar phenotype, whose major features are a strong reduction of granule neurons and aberrant Purkinje cells development. These two cell types are highly interconnected during cerebellar development through the production of molecules that help their proliferation, differentiationmore » and maintenance. In this article, we present data showing that OL ablation has major effects on the physiology of Purkinje (PC) and granule cells (GC). In particular, OL ablation results into a reduction of sonic hedgehog (Shh), Brain Derived Neurotrophic Factor (BDNF), and Reelin (Rln) expression. These results indicate that absence of OLs profoundly alters the normal cerebellar developmental program.« less

  1. Glucose recruits K(ATP) channels via non-insulin-containing dense-core granules.

    PubMed

    Yang, Shao-Nian; Wenna, Nancy Dekki; Yu, Jia; Yang, Guang; Qiu, Hua; Yu, Lina; Juntti-Berggren, Lisa; Köhler, Martin; Berggren, Per-Olof

    2007-09-01

    beta cells rely on adenosine triphosphate-sensitive potassium (K(ATP)) channels to initiate and end glucose-stimulated insulin secretion through changes in membrane potential. These channels may also act as a constituent of the exocytotic machinery to mediate insulin release independent of their electrical function. However, the molecular mechanisms whereby the beta cell plasma membrane maintains an appropriate number of K(ATP) channels are not known. We now show that glucose increases K(ATP) current amplitude by increasing the number of K(ATP) channels in the beta cell plasma membrane. The effect was blocked by inhibition of protein kinase A (PKA) as well as by depletion of extracellular or intracellular Ca(2+). Furthermore, glucose promoted recruitment of the potassium inward rectifier 6.2 to the plasma membrane, and intracellular K(ATP) channels localized in chromogranin-positive/insulin-negative dense-core granules. Our data suggest that glucose can recruit K(ATP) channels to the beta cell plasma membrane via non-insulin-containing dense-core granules in a Ca(2+)- and PKA-dependent manner.

  2. Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins.

    PubMed

    Mezzina, Mariela P; Pettinari, M Julia

    2016-09-01

    Phasins are the major polyhydroxyalkanoate (PHA) granule-associated proteins. They promote bacterial growth and PHA synthesis and affect the number, size, and distribution of the granules. These proteins can be classified in 4 families with distinctive characteristics. Low-resolution structural studies and in silico predictions were performed in order to elucidate the structure of different phasins. Most of these proteins share some common structural features, such as a preponderant α-helix composition, the presence of disordered regions that provide flexibility to the protein, and coiled-coil interacting regions that form oligomerization domains. Due to their amphiphilic nature, these proteins play an important structural function, forming an interphase between the hydrophobic content of PHA granules and the hydrophilic cytoplasm content. Phasins have been observed to affect both PHA accumulation and utilization. Apart from their role as granule structural proteins, phasins have a remarkable variety of additional functions. Different phasins have been determined to (i) activate PHA depolymerization, (ii) increase the expression and activity of PHA synthases, (iii) participate in PHA granule segregation, and (iv) have both in vivo and in vitro chaperone activities. These properties suggest that phasins might play an active role in PHA-related stress protection and fitness enhancement. Due to their granule binding capacity and structural flexibility, several biotechnological applications have been developed using different phasins, increasing the interest in the study of these remarkable proteins. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. PHYSICAL PROPERTIES OF LARGE AND SMALL GRANULES IN SOLAR QUIET REGIONS

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yu Daren; Xie Zongxia; Hu Qinghua

    The normal mode observations of seven quiet regions obtained by the Hinode spacecraft are analyzed to study the physical properties of granules. An artificial intelligence technique is introduced to automatically find the spatial distribution of granules in feature spaces. In this work, we investigate the dependence of granular continuum intensity, mean Doppler velocity, and magnetic fields on granular diameter. We recognized 71,538 granules by an automatic segmentation technique and then extracted five properties: diameter, continuum intensity, Doppler velocity, and longitudinal and transverse magnetic flux density to describe the granules. To automatically explore the intrinsic structures of the granules in themore » five-dimensional parameter space, the X-means clustering algorithm and one-rule classifier are introduced to define the rules for classifying the granules. It is found that diameter is a dominating parameter in classifying the granules and two families of granules are derived: small granules with diameters smaller than 1.''44, and large granules with diameters larger than 1.''44. Based on statistical analysis of the detected granules, the following results are derived: (1) the averages of diameter, continuum intensity, and Doppler velocity in the upward direction of large granules are larger than those of small granules; (2) the averages of absolute longitudinal, transverse, and unsigned flux density of large granules are smaller than those of small granules; (3) for small granules, the average of continuum intensity increases with their diameters, while the averages of Doppler velocity, transverse, absolute longitudinal, and unsigned magnetic flux density decrease with their diameters. However, the mean properties of large granules are stable; (4) the intensity distributions of all granules and small granules do not satisfy Gaussian distribution, while that of large granules almost agrees with normal distribution with a peak at 1.04 I{sub 0}.« less

  4. Enhancement of ibuprofen dissolution via wet granulation with beta-cyclodextrin.

    PubMed

    Ghorab, M K; Adeyeye, M C

    2001-08-01

    The purpose was to investigate the effect of wet granulation with beta-cyclodextrin (betaCD) on the enhancement of ibuprofen (IBU) dissolution. The effect of the granulation variables on the physical properties as well as the dissolution of tablets prepared from these granules was also examined. Granulation was performed using three granulating solvents: water, ethanol (95 vol%), and isopropanol. Granules were either oven-dried for 2 h or air-dried for 3 days. The granules or respective physical mixtures were compressed into tablets. Powder X-ray diffraction showed that oven-dried granulation resulted in less amorphous entities thatfacilitated IBU-betaCD complexation in solution and enhanced the dissolution of the corresponding tablets compared to the physical mixture with or without oven drying. In contrast, air-dried granulation did not cause any differences in the X-ray diffraction pattern (crystallinity) or the dissolution compared to the physical mixture without drying. Isopropanol and water, as granulating solvents, enhanced the dissolution of the oven-dried batches more than ethanol. The Differential scanning calorimetry (DSC) and Thermogravimetric analysis (TGA) data showed that tablets prepared from oven-dried granules, but not air-dried granules, had lower AH values and percent loss in weight, respectively, than those prepared from the physical mixture as a result of the expulsion of the water molecules from the betaCD cavity and enhancement of the complexation in solution. These results showed that oven-dried granulation of IBU and betaCD provided faster IBU dissolution than the physical mixture; air-dried granulation did not substantially affect the dissolution of IBU.

  5. Normal neutrophil differentiation and secondary granule gene expression in the EML and MPRO cell lines.

    PubMed

    Lawson, N D; Krause, D S; Berliner, N

    1998-11-01

    The EML and MPRO cell lines express a dominant negative retinoic acid receptor alpha that causes a block at specific stages of myelopoiesis. The EML cell line is multipotent and gives rise to erythroid, lymphoid, and myeloid lineages depending on the presence of appropriate cytokines. The MPRO cell line is promyelocytic and undergoes neutrophilic differentiation when induced with all-trans retinoic acid in the presence of granulocyte/macrophage colony-stimulating factor. Previous studies have shown that both of these cell lines undergo morphological differentiation into neutrophils. In this study, we show that unlike other models of neutrophil differentiation such as NB4 and HL60, both EML and MPRO cell lines undergo complete, normal granulocytic differentiation programs. Similar to HL60, MPRO and EML induce expression of CD11b/CD18 and also exhibit downregulation of CD34 on differentiation. In contrast to HL60 and NB4, EML and MPRO cell lines coordinately upregulate secondary granule transcripts for lactoferrin and neutrophil gelatinase. Furthermore, we have confirmed previous observations that serum can induce a low level of differentiation in MPRO cells and that it is possible to grow these cells in serum-free medium, thereby eliminating this effect. Based on these studies, it appears that these lines can serve as a model for normal retinoic acid-induced neutrophil differentiation and provide insight into the role of the retinoic acid-responsive pathway in normal and leukemic myelopoiesis.

  6. Identification and characterization of putative stem cells in the adult pig ovary.

    PubMed

    Bui, Hong-Thuy; Van Thuan, Nguyen; Kwon, Deug-Nam; Choi, Yun-Jung; Kang, Min-Hee; Han, Jae-Woong; Kim, Teoan; Kim, Jin-Hoi

    2014-06-01

    Recently, the concept of 'neo-oogenesis' has received increasing attention, since it was shown that adult mammals have a renewable source of eggs. The purpose of this study was to elucidate the origin of these eggs and to confirm whether neo-oogenesis continues throughout life in the ovaries of the adult mammal. Adult female pigs were utilized to isolate, identify and characterize, including their proliferation and differentiation capabilities, putative stem cells (PSCs) from the ovary. PSCs were found to comprise a heterogeneous population based on c-kit expression and cell size, and also express stem and germ cell markers. Analysis of PSC molecular progression during establishment showed that these cells undergo cytoplasmic-to-nuclear translocation of Oct4 in a manner reminiscent of gonadal primordial germ cells (PGCs). Hence, cells with the characteristics of early PGCs are present or are generated in the adult pig ovary. Furthermore, the in vitro establishment of porcine PSCs required the presence of ovarian cell-derived extracellular regulatory factors, which are also likely to direct stem cell niche interactions in vivo. In conclusion, the present work supports a crucial role for c-kit and kit ligand/stem cell factor in stimulating the growth, proliferation and nuclear reprogramming of porcine PSCs, and further suggests that porcine PSCs might be the culture equivalent of early PGCs. © 2014. Published by The Company of Biologists Ltd.

  7. Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein.

    PubMed

    Fitzgerald, Kerry D; Semler, Bert L

    2013-09-01

    Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein

    PubMed Central

    Fitzgerald, Kerry D.; Semler, Bert L.

    2013-01-01

    Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. PMID:23830997

  9. Decrement of GABAA receptor-mediated inhibitory postsynaptic currents in dentate granule cells in epileptic hippocampus.

    PubMed

    Isokawa, M

    1996-05-01

    1. Inhibitory postsynaptic currents (IPSCs) were studied in hippocampal dentate granule cells (DGCs) in the pilocarpine model and human temporal lobe epilepsy, with the use of the whole cell patch-clamp recording technique in slice preparations. 2. In the pilocarpine model, hippocampal slices were prepared from rats that were allowed to experience spontaneous seizures for 2 mo. Human hippocampal specimens were obtained from epileptic patients who underwent surgical treatment for medically intractable seizures. 3. IPSCs were generated by single perforant path stimulation and recorded at a membrane potential (Vm) of 0 mV near the reversal potential of glutamate excitatory postsynaptic currents in the voltage-clamp recording. IPSCs were pharmacologically identified as gamma-aminobutyric acid-A (GABAA) IPSCs by 10 microM bicuculline methiodide. 4. During low-frequency stimulation, IPSCs were not different in amplitude among non-seizure-experienced rat hippocampi, human nonsclerotic hippocampi, seizure-experienced rat hippocampi, and human sclerotic hippocampi. In the last two groups of DGCs, current-clamp recordings indicated the presence of prolonged excitatory postsynaptic potentials (EPSPs) mediated by the N-methyl-D-aspartate (NMDA) receptor. 5. High-frequency stimulation, administered at Vm = -30 mV to activate NMDA currents, reduced GABAA IPSC amplitude specifically in seizure-experienced rat hippocampi (t = 2.5, P < 0.03) and human sclerotic hippocampi (t = 7.7, P < 0.01). This reduction was blocked by an NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV) (50 microM). The time for GABAA IPSCs to recover to their original amplitude was also shortened by the application of APV. 6. I conclude that, when intensively activated, NMDA receptor-mediated excitatory transmission may interact with GABAergic synaptic inhibition in DGCs in seizure-experienced hippocampus to transiently reduce GABA(A) receptor-channel function. Such interactions may contribute to

  10. Perspectives for computational modeling of cell replacement for neurological disorders

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aimone, James B.; Weick, Jason P.

    In mathematical modeling of anatomically-constrained neural networks we provide significant insights regarding the response of networks to neurological disorders or injury. Furthermore, a logical extension of these models is to incorporate treatment regimens to investigate network responses to intervention. The addition of nascent neurons from stem cell precursors into damaged or diseased tissue has been used as a successful therapeutic tool in recent decades. Interestingly, models have been developed to examine the incorporation of new neurons into intact adult structures, particularly the dentate granule neurons of the hippocampus. These studies suggest that the unique properties of maturing neurons, can impactmore » circuit behavior in unanticipated ways. In this perspective, we review the current status of models used to examine damaged CNS structures with particular focus on cortical damage due to stroke. Secondly, we suggest that computational modeling of cell replacement therapies can be made feasible by implementing approaches taken by current models of adult neurogenesis. The development of these models is critical for generating hypotheses regarding transplant therapies and improving outcomes by tailoring transplants to desired effects.« less

  11. Perspectives for computational modeling of cell replacement for neurological disorders

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aimone, James B.; Weick, Jason P.

    Mathematical modeling of anatomically-constrained neural networks has provided significant insights regarding the response of networks to neurological disorders or injury. A logical extension of these models is to incorporate treatment regimens to investigate network responses to intervention. The addition of nascent neurons from stem cell precursors into damaged or diseased tissue has been used as a successful therapeutic tool in recent decades. Interestingly, models have been developed to examine the incorporation of new neurons into intact adult structures, particularly the dentate granule neurons of the hippocampus. These studies suggest that the unique properties of maturing neurons, can impact circuit behaviormore » in unanticipated ways. In this perspective, we review the current status of models used to examine damaged CNS structures with particular focus on cortical damage due to stroke. Secondly, we suggest that computational modeling of cell replacement therapies can be made feasible by implementing approaches taken by current models of adult neurogenesis. The development of these models is critical for generating hypotheses regarding transplant therapies and improving outcomes by tailoring transplants to desired effects.« less

  12. Early healing in alveolar sockets grafted with titanium granules. An experimental study in a dog model.

    PubMed

    Arruda, Thiago; Sukekava, Flávia; de Souza, André B; Rasmusson, Lars; Araújo, Maurício G

    2013-07-01

    The aim of the present study was to evaluate the effect of the placement of titanium granules in fresh extraction sockets on early bone formation. The mesial roots of the third maxillary premolars of five adult beagle dogs were removed. On one side of the maxilla (Test group) the fresh extraction socket was grafted with titanium granules, while the contra-lateral socket was left non-grafted (Control group). After 1 month of healing, the dogs were euthanized and biopsies were obtained. The healing tissues were described, and histometric measurements were performed to obtain the percentage area occupied by connective tissue, new mineralized bone, bone marrow, and biomaterial particles. After 1 month of healing the findings from the histological examination revealed the titanium graft to be well incorporated into the provisional connective tissue or newly formed woven bone. The histometric measurements showed, however, that less mineralized bone was formed in the Test group than in the Control group. The present study suggests that the use of titanium granules in fresh extraction sockets was conducive to new bone formation. The graft of titanium granules seems, however, to delay the early phase of the healing process. Copyright © 2012 Wiley Periodicals, Inc.

  13. Conversion of adult endothelium to immunocompetent haematopoietic stem cells.

    PubMed

    Lis, Raphael; Karrasch, Charles C; Poulos, Michael G; Kunar, Balvir; Redmond, David; Duran, Jose G Barcia; Badwe, Chaitanya R; Schachterle, William; Ginsberg, Michael; Xiang, Jenny; Tabrizi, Arash Rafii; Shido, Koji; Rosenwaks, Zev; Elemento, Olivier; Speck, Nancy A; Butler, Jason M; Scandura, Joseph M; Rafii, Shahin

    2017-05-25

    Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1 + FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.

  14. [Protective effect and mechanism of compound Ginkgo biloba granules on oxidative stress injury of HUVEC].

    PubMed

    Li, Qi; Chen, Xi; Kan, Xiao-Xi; Li, Yu-Jie; Yang, Qing; Wang, Ya-Jie; Chen, Ying; Weng, Xiao-Gang; Cai, Wei-Yan; Huang, He-Fei; Zhu, Xiao-Xin

    2016-02-01

    To reveal the protective and anti-apoptosis effect of compound Ginkgo biloba granules on oxidative stress injury of human umbilical vein endothelial cells (HUVEC). Negative control group, H2O2 model group and 4 drug pretreatment groups (80, 160, 320, 640 mg• L⁻¹) were established. The cell proliferation, morphological changes in each group after oxidative stress injury was detected by MTT assay and through microscope observation respectively. The content of LDH, MDA, SOD and NO and SOD activity in supernatant were detected to judge the protection effect of the drugs on endothelial cells. The protective effect on HUVEC apoptosis was analyzed by Caspase-3 activity test and Annexin V-FITC/PI staining. Western blot was used to observe the expression of apoptosis-related proteins Bcl-2 and Bax. Results showed that 1 200 μmol• L⁻¹ H2O2 can induce oxidative stress injury in endothelial cells and reduce the cell survival rate; cell proliferation inhibition degree is positively correlated with the effect time of H2O2. Besides, 80, 160, 320 640 mg•L⁻¹ compound Ginkgo biloba granules can protect HUVEC from oxidative stress injury, recover the normal proliferation level of cells, improve their state, prohibit cell apoptosis, and can up-regulate and down-regulate the expression level of Bcl-2 and Bax respectively. In conclusion, compound G. biloba granules can protect HUVEC from the oxidative stress injury induced by H2O2, its mechanism may be correlated with inhibition of the mitochondrial apoptotic pathway in HUVEC. Copyright© by the Chinese Pharmaceutical Association.

  15. Impact of fill-level in twin-screw granulation on critical quality attributes of granules and tablets.

    PubMed

    Meier, Robin; Moll, Klaus-Peter; Krumme, Markus; Kleinebudde, Peter

    2017-06-01

    In a previous study a change of the fill-level in the barrel exerted a huge influence on the twin-screw granulation (TSG) process of a high drug loaded, simplified formulation. The present work investigated this influence systematically. The specific feed load (SFL) indicating the mass per revolution as surrogate parameter for the fill-level was applied and the correlation to the real volumetric fill level of an extruder could be demonstrated by a newly developed method. A design of experiments was conducted to examine the combined influence of SFL and screw speed on the process and on critical quality attributes of granules and tablets. The same formulation was granulated at constant liquid level with the same screw configuration and led to distinctively different results by only changing the fill-level and the screw speed. The power consumption of the extruder increased at higher SFLs with hardly any influence of screw speed. At low SFL the median residence time was mainly fill-level dependent and at higher SFL mainly screw speed dependent. Optimal values for the product characteristics were found at medium values for the SFL. Granule size distributions shifted from mono-modal and narrow shape to broader and even bimodal distributions of larger median granule sizes, when exceeding or falling below a certain fill-level. Deviating from the optimum fill-level, tensile strength of tablets decreased by about 25% and disintegration times of tablets increased for more than one third. At low fill-levels, material accumulation in front of the kneading zone was detected by pressure measurements and was assumed to be responsible for the unfavored product performance. At high fill-levels, granule consolidation due to higher propensity of contact with the result of higher material temperature was accounted for inferior product performance. The fill-level was found to be an important factor in assessment and development of twin-screw granulation processes as it impacted

  16. Clarithromycin highly-loaded gastro-floating fine granules prepared by high-shear melt granulation can enhance the efficacy of Helicobacter pylori eradication.

    PubMed

    Aoki, Hajime; Iwao, Yasunori; Mizoguchi, Midori; Noguchi, Shuji; Itai, Shigeru

    2015-05-01

    In an effort to develop a new gastro-retentive drug delivery system (GRDDS) without a large amount of additives, 75% clarithromycin (CAM) loaded fine granules were prepared with three different hydrophobic binders by high-shear melt granulation and their properties were evaluated. Granules containing the higher hydrophobic binder showed sustained drug release and were able to float over 24h. The synchrotron X-ray CT measurement indicated that both the high hydrophobicity of the binder and the void space inside the granules might be involved in their buoyancy. In an in vivo experiment, the floating granules more effectively eradicated Helicobacter pylori than a CAM suspension by remaining in the stomach for a longer period. In short, CAM highly-loaded gastro-floating fine granules can enhance the eradication efficiency of H. pylori compared with CAM alone. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Defense function of pigment granules in the ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus (Rhizopodea) and Climacostomum virens (Ciliata).

    PubMed

    Terazima, Masayo Noda; Harumoto, Terue

    2004-08-01

    The defense function of pigment granules in the red ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus and Climacostomum virens, was investigated by (1) comparing normally-pigmented and albino mutant cells of B. japonicum as the prey of these predators and (2) comparing resistance of the predators to blepharismin, the toxic pigment contained in the pigment granules of B. japonicum. Normally pigmented cells which contained more blepharismin than albino cells were less vulnerable to A. proteus than albino cells, but not to C. virens. C. virens was more resistant than A. proteus to the lethal effect of blepharismin. The results indicate that pigment granules of B. japonicum function as defense organelles against A. proteus but not against C. virens and suggest that successful defense against a predator depends on the susceptibility of the predator to blepharismin.

  18. Effect of different excipients on the physical characteristics of granules and tablets with carbamazepine prepared with polyethylene glycol 6000 by fluidized hot-melt granulation (FHMG).

    PubMed

    Kraciuk, Radosław; Sznitowska, Malgorzata

    2011-12-01

    The objective of this study was to investigate the properties of granules and tablets with carbamazepine which were prepared employing a fluidized hot-melt granulation (FHMG) technique. The FHMG process was carried out at 65°C. Macrogol 6000 (PEG 6000) was used as a binder at the content 10% (w/w) of the granulated mass. Granules containing up to 70% (w/w) of the drug and 20-90% (w/w) of a filler (lactose, mannitol, calcium hydrogen phosphate (Di-Cafos), pregelatinized starch, and microcrystalline cellulose (MCC)) were produced. When the drug content was 30% (w/w), the yield of the process was satisfying (>95%) and flowability of the granules was better than placebo granules or drug-loaded granules prepared by wet granulation. Type of a filler had strong impact on physical properties of granules, and size distribution of the particles was the most homogenous when lactose or Di-Cafos were used. The FHMG technique enabled preparation of granules with better compressability compared with the wet-granulated product or with non-granulated powders. Tablets with shorter disintegration time than 10 min were obtained with 2.0% crospovidone added as a disintegrant. In comparison to tablets prepared from the wet-granulated mass, employment of the FHMG method resulted in tablets with faster dissolution of carbamazepine (more than 80% of the drug released within 15 min). This was achieved with mannitol or lactose/MCC, as fillers.

  19. Bioequivalence of two formulations of montelukast sodium 4 mg oral granules in healthy adults

    PubMed Central

    2014-01-01

    Montelukast is an effective and well-tolerated treatment for the prophylaxis and chronic treatment of asthma, acute prevention of exercise-induced bronchoconstriction and symptomatic relief of seasonal allergic rhinitis and perennial allergic rhinitis. The aim of the study was to compare bioavailability, and characterise the pharmacokinetic profile and safety of Sandoz generic montelukast 4 mg oral granules relative to Singulair® mini (Merck, Sharp & Dohme). An open-label, randomised, single-dose, two-treatment, two-period, two-sequence, two-way crossover bioequivalence study was conducted in healthy male volunteers aged 18–55 years, under fasting conditions. The duration of the clinical part of the trial was ≈ 11 days. Montelukast levels in plasma were quantified using a validated liquid chromatography tandem mass spectrometry method, and pharmacokinetic parameters calculated from the drug concentration–time profile using a non-compartmental model. A total of 40 subjects completed both study periods. The ratio test/reference of geometric least squares means was calculated for both formulations of montelukast for the In-transformed pharmacokinetic parameters; the 90% confidence intervals (CIs) were within the pre-defined limits of 80.00–125.00%: 92.2% (90% CI: 87.42–97.30%) for Cmax, 98.1% (90% CI: 94.49–101.81%) for AUC0–t and 97.6% (90% CI: 94.14–101.27%) for AUC0–∞. Two study subjects each reported one mild adverse event: dyspepsia (possibly related to study medication) and throat pain (not considered related to study medication). Sandoz montelukast 4 mg oral granules are bioequivalent to Singulair® 4 mg mini oral granules, with a similar safety profile. This suggests that these two preparations can be considered interchangeable in clinical practice. PMID:25250173

  20. Biomaterials differentially regulate Src kinases and phosphoinositide 3-kinase-γ in polymorphonuclear leukocyte primary and tertiary granule release

    PubMed Central

    Cohen, Hannah Caitlin; Frost, Dustin C.; Lieberthal, Tyler Jacob; Li, Lingjun; Kao, W. John

    2018-01-01

    In the foreign body response, infiltrating PMNs exocytose granule subsets to influence subsequent downstream inflammatory and wound healing events. In previous studies, we found that PMNs cultured on poly(ethylene glycol) (PEG)-containing hydrogels (i.e., PEG and gelatin + PEG hydrogels) had enhanced primary granule release, yet similar tertiary granule release compared with PMNs cultured on polydimethylsiloxane or tissue culture polystyrene. PMN primary granules contain microbicidal proteins and proteases, which can potentially injure bystander cells, degrade the extracellular matrix, and promote inflammation. Here, we sought to understand the mechanism of the enhanced primary granule release from PMNs on PEG hydrogels. We found that primary granule release from PMNs on PEG hydrogels was adhesion mediated and involved Src family kinases and PI3K-γ. The addition of gelatin to PEG hydrogels did not further enhance PMN primary granule release. Using stable-isotope dimethyl labeling-based shotgun proteomics, we identified many serum proteins – including Ig gamma constant chain region proteins and alpha-1-acid glycoprotein 1 – that were absorbed/adsorbed in higher quantities on PEG hydrogels than on TCPS, and may be involved in mediating PMN primary granule release. Ultimately, this mechanistic knowledge can be used to direct inflammation and wound healing following biomaterial implantation to promote a more favorable healing response. PMID:25736495