Sample records for cell secretory granules

  1. Biogenesis and Transport of Secretory Granules to Release Site in Neuroendocrine Cells

    Microsoft Academic Search

    Joshua J. Park; Hisatsugu Koshimizu; Y. Peng Loh

    2009-01-01

    Biogenesis and post-Golgi transport of peptidergic secretory granules to the release site are crucial for secretion of neuropeptides\\u000a from neuroendocrine cells. Recent studies have uncovered multilevel molecular mechanisms for the regulation of secretory granule\\u000a biogenesis. Insulinoma-associated protein 2 (ICA512\\/IA-2), polypyrimidine-tract binding protein, and chromogranin A have been\\u000a identified to regulate secretory granule biogenesis at the transcriptional, posttranscriptional, and posttranslational levels,

  2. Biogenesis and transport of secretory granules to release site in neuroendocrine cells.

    PubMed

    Park, Joshua J; Koshimizu, Hisatsugu; Loh, Y Peng

    2009-02-01

    Biogenesis and post-Golgi transport of peptidergic secretory granules to the release site are crucial for secretion of neuropeptides from neuroendocrine cells. Recent studies have uncovered multilevel molecular mechanisms for the regulation of secretory granule biogenesis. Insulinoma-associated protein 2 (ICA512/IA-2), polypyrimidine-tract binding protein, and chromogranin A have been identified to regulate secretory granule biogenesis at the transcriptional, posttranscriptional, and posttranslational levels, respectively, by increasing granule protein levels, which in turn drives granule formation after stimulation. Post-Golgi transport of secretory granules is microtubule-based and mediated by transmembrane carboxypeptidase E (CPE). The cytoplasmic tail of CPE anchors secretory granules to the microtubule motors, kinesin-2 and -3, or dynein, via interaction with the adaptor, dynactin, to mediate anterograde and retrograde transport, respectively. PMID:18607778

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

    SciTech Connect

    Baconnais, S.; Delavoie, F. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France)]|[INSERM UMRS 514, IFR 53, CHU Maison Blanche, 45, rue Cognac-Jay, 51092 Reims Cedex (France); Zahm, J.M.; Milliot, M.; Castillon, N. [INSERM UMRS 514, IFR 53, CHU Maison Blanche, 45, rue Cognac-Jay, 51092 Reims Cedex (France); Terryn, C. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France); Banchet, V. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France); Michel, J. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France); Danos, O. [Genethon, CNRS UMR 8115, 1bis rue de l'Internationale, Evry (France); Merten, M. [INSERM EMI 0014, Faculte de Medecine, 9, Avenue de la Foret de Haye, BP 184, 54505 Vandoeuvre Les Nancy cedex, (France); Chinet, T. [Laboratoire de Biologie et Pharmacologie des Epitheliums Respiratoires, UFR Paris Ile de France Ouest, Boulogne-Billancourt (France); Zierold, K. [Max Planck Institute of Molekular Physiology, Laboratory for Analytical Microscopy, Otto-Hahn-Strasse 11, D-44227 Dortmund (Germany); Bonnet, N. [INSERM UMRS 514, IFR 53, CHU Maison Blanche, 45, rue Cognac-Jay, 51092 Reims Cedex (France); Puchelle, E. [INSERM UMRS 514, IFR 53, CHU Maison Blanche, 45, rue Cognac-Jay, 51092 Reims Cedex (France)], E-Mail: edith.puchelle@univ-reims.fr; Balossier, G. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France)

    2005-10-01

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

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

    PubMed

    Baconnais, S; Delavoie, F; Zahm, J M; Milliot, M; Terryn, C; Castillon, N; Banchet, V; Michel, J; Danos, O; Merten, M; Chinet, T; Zierold, K; Bonnet, N; Puchelle, E; Balossier, G

    2005-10-01

    The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na(+) absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na(+), Mg(2+), P, S and Cl(-)) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR(inh)-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF. PMID:16051214

  5. Serous cells in the parotid glands of two species of tamarins: polarized secretory granules.

    PubMed

    Tandler, Bernard

    2008-10-01

    The parotid glands of two species of tamarins were examined by electron microscopy. Endpiece cells are typical in appearance, with an extensive rough endoplasmic reticulum, prominent Golgi apparatuses, and numerous serous granules. In the saddleback tamarin, the secretory granules contain a dense spherule pressed against the inner aspect of the limiting membrane, leading to a surface bulge. During the course of merocrine secretion (a form of exocytosis), such morphologically polarized granules approach the luminal plasma membranes with the bulge in the vanguard. It is these protuberances that fuse with the plasmalemma. In contrast, although serous granules in the cotton top tamarin contain a spherule, they lack surface bulges and their docking on luminal membranes seems to be a random event with respect to their surface morphology. Moreover, certain other types of cells in a taxonomically wide spectrum of species have granules with a less obvious structural polarity, as well as cells whose granules lack morphological polarity but have a functional polarity that comes into play during exocytosis of such secretory granules. PMID:18780306

  6. Avian minor salivary glands: an ultrastructural study of the secretory granules in mucous and seromucous cells.

    PubMed

    Olmedo, L A; Samar, M E; Avila, R E; de Crosa, M G; Dettin, L

    2000-01-01

    Ultrastructural descriptions in birds are scarce thus, in this study we have characterized the secretory granules of mucous and seromucous cells from the palatine and lingual salivary glands of birds with different diets. The samples were taken from the tongue and palatine mucosa of chicken (Gallus gallus), quail (Coturnix coturnix), chimango (Milvago chimango) and white heron (Egretta thula). The samples were processed for observation by transmission electron microscopy (TEM) employing 4% Karnovsky solution for fixation. The most noteworthy finding was the heterogeneous ultrastructural appearance of the secretory granules. Differences in substructure were found between the four species, between the palatine and lingual glands in the same species and even within the same acinus and the same cell. At variance with other authors, these differences cannot be attributed to the type of fixative solution used taking into account that all the samples were processed in the same way. Previous histochemical studies have shown the presence of sulfated and non sulfated glycoconjugates in these glands which can be associated to the maturation of the granules. These granules are probably representative of peculiar storage of the secretory products that would give rise to a heterogeneous and complex ultrastructural pattern of granules in the mucosa and seromucosa cells of these avian species. PMID:15211928

  7. Low-voltage transmission electron microscopy reveals SV40 viral particles within secretory granules in pancreatic cells.

    PubMed

    Bendayan, Moise; Gingras, Diane; Ziv, Ehud; Haviv, Yosef S

    2008-09-01

    Novel approach in low voltage transmission electron microscopy (TEM) has revealed the presence of SV40 viral like particles in the secretory zymogen granules and in spherical membrane-bound dense bodies of SV40 infected pancreatic cells. The presence of SV40 antigen in these cellular compartments was confirmed by immunocytochemistry of the VP1 antigen. Visualization of the viral particles was only possible by examining ultrathin tissue sections with low-voltage TEM that significantly enhances imaging contrast. Results indicate that following infection of the cell entry and trafficking of the viral particles are present in unique cellular compartments such as ER, dense bodies, and secretory granules. PMID:18512738

  8. Proteomics Analysis of Insulin Secretory Granules

    Microsoft Academic Search

    Yannick Brunner; Yohann Coute; Mariella Iezzi; Michelangelo Foti; Mitsonuri Fukuda; Denis F. Hochstrasser; Claes B. Wollheim; Jean-Charles Sanchez

    2007-01-01

    Insulin secretory granules (ISGs) are cytoplasmic or- ganelles of pancreatic -cells. They are responsible for the storage and secretion of insulin. To date, only about 30 different proteins have been clearly described to be associated with these organelles. However, data from two-dimensional gel electrophoresis analyses suggested that almost 150 different polypeptides might be present within ISGs. The elucidation of the

  9. Dense-Core Secretory Granule Biogenesis

    NSDL National Science Digital Library

    Taeyoon Kim (National Institutes of Health Section on Cellular Neurobiology, National Institute of Child Health and Human Development)

    2006-04-01

    The dense-core secretory granule is a key organelle for secretion of hormones and neuropeptides in endocrine cells and neurons, in response to stimulation. Cholesterol and granins are critical for the assembly of these organelles at the trans-Golgi network, and their biogenesis is regulated quantitatively by posttranscriptional and posttranslational mechanisms.

  10. Regulation of secretory granule size by the precise generation and fusion of unit granules

    PubMed Central

    Hammel, Ilan; Lagunoff, David; Galli, Stephen J.

    2010-01-01

    Morphometric evidence derived from studies of mast cells, pancreatic acinar cells, and other cell types supports a model in which the post Golgi processes that generate mature secretory granules can be resolved into three steps: 1) fusion of small, Golgi-derived progranules to produce immature secretory granules which have a highly constrained volume; 2) transformation of such immature granules into mature secretory granules, a process often associated with a reduction in the maturing granule's volume, as well as changes in the appearance of its content; and 3) fusion of secretory granules of the smallest size, termed “unit granules”, forming granules whose volumes are multiples of the unit granule's volume. Mutations which perturb this process can cause significant pathology. For example, CHS/Lyst mutations result in giant secretory granules in a number of cell types in humans with the Chediak-Higashi syndrome and in “beige” (Lystbg/Lystbg) mice. Analysis of the secretory granules of mast cells and pancreatic acinar cells in Lyst-deficient beige mice suggests that beige mouse secretory granules retain the ability to fuse randomly with other secretory granules no matter what the size of the fusion partners. By contrast, in normal mice, the pattern of granule-granule fusion occurs exclusively by the addition of unit granules, either to each other or to larger granules. The normal pattern of fusion is termed unit addition and the fusion evident in cells with CHS/Lyst mutations is called random addition. The proposed model of secretory granule formation has several implications. For example, in neurosecretory cells, the secretion of small amounts of cargo in granules constrained to a very narrow size increases the precision of the information conveyed by secretion. By contrast, in pancreatic acinar cells and mast cells, large granules composed of multiple unit granules permit the cells to store large amounts of material without requiring the amount of membrane necessary to package the same amount of cargo into small granules. In addition, the formation of mature secretory granules that are multimers of unit granules provides a mechanism for mixing in large granules the contents of unit granules which differ in their content of cargo. PMID:20406331

  11. SORCS1 is necessary for normal insulin secretory granule biogenesis in metabolically stressed ? cells

    PubMed Central

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

    2014-01-01

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

  12. Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells.

    PubMed Central

    Zimmerberg, J; Curran, M; Cohen, F S; Brodwick, M

    1987-01-01

    Mast cells show dramatic morphological changes when undergoing exocytosis. We have investigated whether the first of those morphological changes, swelling of the secretory granule, precedes--and therefore possibly initiates--secretion or whether it occurs after fusion of the granule and plasma membranes. We used cell membrane capacitance to detect the moment when granule and plasma membrane become continuous. We measured large capacitance increases, often preceded by transients in capacitance. The rise-times of the capacitance increases were half-maximal at 2-59 msec. We observed cells with high-resolution video microscopy while these measurements were done. The capacitance increase always preceded the granular swelling that leads to exocytosis. To rule out the possibility that fusion was induced by a mechanical stress imparted by the internal pressure of a taut granule, we performed control experiments using cells in which vesicles were shrunken with hyperosmotic solutions. With these flaccid granules, again, the capacitance rise always preceded the swelling of the granules. We conclude that swelling cannot be the driving force for membrane fusion in this system. Images PMID:3470745

  13. Mefloquine, an anti-malaria agent, causes reactive oxygen species-dependent cell death in mast cells via a secretory granule-mediated pathway

    PubMed Central

    Paivandy, Aida; Calounova, Gabriela; Zarnegar, Behdad; Öhrvik, Helena; Melo, Fabio R; Pejler, Gunnar

    2014-01-01

    Mast cells are known to have a detrimental impact on a variety of pathological conditions. There is therefore an urgent need of developing strategies that limit their harmful effects. The aim of this study was to accomplish this by developing a means of inducing mast cell apoptosis. The strategy was to identify novel compounds that induce mast cell apoptosis by permeabilization of their secretory lysosomes (granules). As a candidate, we assessed mefloquine, an anti-malarial drug that has been proposed to have lysosome-permeabilizing activity. Mefloquine was added to mast cells and administered in vivo, followed by assessment of the extent and mechanisms of mast cell death. Mefloquine was cytotoxic to murine and human mast cells. Mefloquine induced apoptotic cell death of wild-type mast cells whereas cells lacking the granule compounds serglycin proteoglycan or tryptase were shown to undergo necrotic cell death, the latter finding indicating a role of the mast cell granules in mefloquine-induced cell death. In support of this, mefloquine was shown to cause compromised granule integrity and to induce leakage of granule components into the cytosol. Mefloquine-induced cell death was refractory to caspase inhibitors but was completely abrogated by reactive oxygen species inhibition. These findings identify mefloquine as a novel anti-mast cell agent, which induces mast cell death through a granule-mediated pathway. Mefloquine may thus become useful in therapy aiming at limiting harmful effects of mast cells. PMID:25505612

  14. Age-dependent labeling and imaging of insulin secretory granules.

    PubMed

    Ivanova, Anna; Kalaidzidis, Yannis; Dirkx, Ronald; Sarov, Mihail; Gerlach, Michael; Schroth-Diez, Britta; Müller, Andreas; Liu, Yanmei; Andree, Cordula; Mulligan, Bernard; Münster, Carla; Kurth, Thomas; Bickle, Marc; Speier, Stephan; Anastassiadis, Konstantinos; Solimena, Michele

    2013-11-01

    Insulin is stored within the secretory granules of pancreatic ?-cells, and impairment of its release is the hallmark of type 2 diabetes. Preferential exocytosis of newly synthesized insulin suggests that granule aging is a key factor influencing insulin secretion. Here, we illustrate a technology that enables the study of granule aging in insulinoma cells and ?-cells of knock-in mice through the conditional and unequivocal labeling of insulin fused to the SNAP tag. This approach, which overcomes the limits encountered with previous strategies based on radiolabeling or fluorescence timer proteins, allowed us to formally demonstrate the preferential release of newly synthesized insulin and reveal that the motility of cortical granules significantly changes over time. Exploitation of this approach may enable the identification of molecular signatures associated with granule aging and unravel possible alterations of granule turnover in diabetic ?-cells. Furthermore, the method is of general interest for the study of membrane traffic and aging. PMID:23929935

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

    SciTech Connect

    Fujita-Yoshigaki, Junko [Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587 (Japan)]. E-mail: yoshigaki.junko@nihon-u.ac.jp; Katsumata, Osamu [Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587 (Japan); Matsuki, Miwako [Department of Pathology, Tokyo Dental College, Chiba 261-8502 (Japan); Yoshigaki, Tomoyoshi [Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587 (Japan); Furuyama, Shunsuke [Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587 (Japan); Sugiya, Hiroshi [Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587 (Japan)

    2006-05-26

    Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.

  16. Signaling from the secretory granule to the nucleus

    PubMed Central

    Rajagopal, Chitra; Mains, Richard E.; Eipper, Betty A.

    2014-01-01

    Neurons and endocrine cells use a complex array of signaling molecules to communicate with each other and with various targets. The majority of these signaling molecules are stored in specialized organelles awaiting release on demand: 40–60 nm vesicles carry conventional or small molecule neurotransmitters, and 200–400 nm granules contain bioactive peptides. The supply of small molecule neurotransmitters is tightly regulated by local feedback of synthetic rates and transport processes at sites of release. The larger granules that contain bioactive peptides present the secretory cell with special challenges, since the peptide precursors are inserted into the lumen of the secretory pathway in the cell soma and undergo biosynthetic processing while being transported to distant sites for eventual secretion. One solution to this dilemma in information handling has been to employ proteolytic cleavage of secretory granule membrane proteins to produce cytosolic fragments that can signal to the nucleus, affecting gene expression. The use of regulated intramembrane proteolysis to signal from secretory granules to the nucleus is compared to its much better understood role in relaying information from the endoplasmic reticulum by SREBP and ATF6 and from the plasma membrane by Cadherins, Notch and ErbB4. PMID:22681236

  17. Patch clamp studies of single intact secretory granules.

    PubMed Central

    Oberhauser, A F; Fernandez, J M

    1993-01-01

    The membrane of secretory granules is involved in the molecular events that cause exocytotic fusion. Several of the proteins that have been purified from the membrane of secretory granules form ion channels when they are reconstituted in lipid bilayers and, therefore, have been thought to form part of the molecular structure of the exocytotic fusion pore. We have used the patch clamp technique to study ion conductances in single isolated secretory granules from beige mouse mast cells. We found that the membrane of the intact granule had a conductance of < 50 pS. No abrupt changes in current corresponding to the opening and closing of ion channels were observed, even under conditions where exocytotic fusion occurred. However, mechanical tension or a large voltage pulse caused the breakdown of the granule membrane resulting in the abrupt opening of a pore with an ion conductance of about 1 nS that fluctuated rapidly and could expand to an immeasurably large conductance or close completely. Surprisingly, the behavior of these pores resembled the pattern of conductance changes of exocytotic fusion pores observed in degranulating beige mast cells. This similarity supports the view that the earliest fusion pore is formed upon the breakdown of a bilayer such as that formed during hemifusion. Images FIGURE 1 FIGURE 6 PMID:7507717

  18. Rab5 is a novel regulator of mast cell secretory granules: impact on size, cargo, and exocytosis.

    PubMed

    Azouz, Nurit P; Zur, Neta; Efergan, Adi; Ohbayashi, Norihiko; Fukuda, Mitsunori; Amihai, Dina; Hammel, Ilan; Rothenberg, Marc E; Sagi-Eisenberg, Ronit

    2014-05-01

    Secretion of inflammatory mediators prestored in mast cells secretory granules (SGs) enhances immune responses such as in allergy and host defense. However, the mechanisms underlying the biogenesis of the SGs remain largely unresolved. By combining high-resolution live cell imaging and quantitative morphometric analyses, we show that the small GTPase Rab5 controls the SG size and cargo composition by a VAMP8-dependent fusion mechanism. Knockdown of the endogenous Rab5, or expression of constitutively negative mutants, significantly reduces the size of SGs and increases their number. Conversely, expression of constitutively active Rab5 mutants induces few, but giant, SGs. Both the small and giant SGs maintain their exocytosis competence. Finally, we show that Rab5-mediated fusion between Golgi-derived SGs and early endosomes precedes the maturation of the SGs, as reflected by the recruitment of Rab27B, and allows the incorporation of cargo, such as CD63, that traffics through endosomes. Collectively, our results assign Rab5 a key role in mediating mast cell SG fusion during biogenesis, thereby controlling the amount and composition of the SGs content and maintaining the communication between new and pre-existing SGs. PMID:24696234

  19. Statistical analysis of the quantal basis of secretory granule formation.

    PubMed

    K?epelová-Dror, Marika; Hammel, Ilan; Meilijson, Isaac

    2014-01-01

    The size distribution of vesicles exocytosed from secretory cells displays quantal nature, vesicle volume is periodic multi-modal, suggesting that these heterogeneous vesicles are aggregate sums of a variable number of homogeneous basic granules. Whether heterogeneity is a lumping-together artifact of the measurement or an inherent intra-cell feature of the vesicles is an unresolved question. Recent empirical evidence will be provided for the quantal nature of intra-cell vesicle volume, supporting the controversial paradigm of homotypic fusion: basic cytoplasmic granules fuse with each other to create heterogeneously sized vesicles. An EM-algorithm-based method is presented for the conversion of multi-modal to quantal data that provides as by-product estimates of means and variances of basic granule packaging. PMID:24185612

  20. T-cell clones from a type-1 diabetes patient respond to insulin secretory granule proteins

    Microsoft Academic Search

    Bart O. Roep; Susan D. Arden; René R. P. de Vries; John C. Hutton

    1990-01-01

    T LYMPHOCYTES reactive to pancreatic beta-cells are thought to have a central role in the autoimmune process leading to type 1 (insulin-dependent) diabetes1-6, but the molecular targets of these T cells have not yet been defined. As identification of such antigens may enable measures to be developed to prevent the disease, we have characterized an antigen that is recognized by

  1. Real-time imaging of the dynamics of secretory granules in growth cones

    PubMed Central

    Abney, JR; Meliza, CD; Cutler, B; Kingma, M; Lochner, JE; Scalettar, BA

    1999-01-01

    Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer. PMID:10545386

  2. An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

    PubMed

    Rosa, P; Weiss, U; Pepperkok, R; Ansorge, W; Niehrs, C; Stelzer, E H; Huttner, W B

    1989-07-01

    We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles. PMID:2663878

  3. Separation of rat pituitary secretory granules by continuous flow electrophoresis

    NASA Technical Reports Server (NTRS)

    Hayes, Daniel; Exton, Carrie; Salada, Thomas; Shellenberger, Kathy; Waddle, Jenny; Hymer, W. C.

    1990-01-01

    The separation of growth hormone-containing cytoplasmic secretory granules from the rat pituitary gland by continuous flow electrophoresis is described. The results are consistent with the hypothesis that granule subpopulations can be separated due to differences in surface charge; these, in turn, may be related to the oligomeric state of the hormone.

  4. Receptor-Mediated Targeting of Hormones to Secretory Granules

    Microsoft Academic Search

    Y. Peng Loh; Christopher R Snell; David R Cool

    1997-01-01

    Peptide hormones, neuropeptides, and other molecules such as the granins are specifically packaged into granules of the regulated secretory pathway and released in a calcium-dependent manner upon stimulation. Many of these molecules are synthesized as larger precursors (prohormones) that are processed to biologically active products within the granules. It has now become apparent that prohormones, proneuropeptides, and the granins contain

  5. A novel regulatory mechanism for trimeric GTP-binding proteins in the membrane and secretory granule fractions of human and rodent beta cells.

    PubMed Central

    Kowluru, A; Seavey, S E; Rhodes, C J; Metz, S A

    1996-01-01

    Recently we described roles for heterotrimeric and low-molecular-mass GTP-binding proteins in insulin release from normal rat islets. During these studies, we observed that a protein with an apparent molecular mass (37 kDa) similar to that of the beta subunit of trimeric GTP-binding proteins underwent phosphorylation in each of five classes of insulin-secreting cells. Incubation of the beta cell total membrane fraction or the isolated secretory granule fraction (but not the cytosolic fraction) with [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of this protein, which was selectively immunoprecipitated by an anti-serum directed against the common beta subunit of trimeric G-proteins. Disruption of the alpha beta gamma trimer (by pretreatment with either fluoroaluminate or guanosine 5'(-)[gamma-thio]triphosphate) prevented beta subunit phosphorylation. Based on differential sensitivities to pH, heat and the histidine-selective reagent diethyl pyrocarbonate (and reversal of the latter by hydroxylamine), the phosphorylated amino acid was presumptively identified as histidine. Incubation of pure beta subunit alone or in combination with the exogenous purified alpha subunit of transducin did not result in the phosphorylation of the beta subunit, but addition of the islet cell membrane fraction did support this event, suggesting that membrane localization (or a membrane-associated factor) is required for beta subunit phosphorylation. Incubation of phosphorylated beta subunit with G alpha.GDP accelerated the dephosphorylation of the beta subunit, accompanied by the formation of G alpha-GTP. Immunoblotting detected multiple alpha subunits (of Gi, G(o) and Gq) and at least one beta subunit in the secretory granule fraction of normal rat islets and insulinoma cells. These data describe a potential alternative mechanism for the activation of GTP-binding proteins in beta cells which contrasts with the classical receptor-agonist mechanism: G beta undergoes transient phosphorylation at a histidine residue by a GTP-specific protein kinase; this phosphate, in turn, may be transferred via a classical Ping-Pong mechanism to G alpha.GDP (inactive), yielding the active configuration G alpha.GTP in secretory granules (a strategic location to modulate exocytosis). PMID:8546716

  6. Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

    PubMed Central

    1994-01-01

    Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3- like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis. PMID:8294505

  7. Compartmentalization of pancreatic secretory zymogen granules as revealed by low-voltage transmission electron microscopy.

    PubMed

    Bendayan, Moise; Londono, Irene; Paransky, Eugene

    2011-10-01

    Low-voltage (5-kV) transmission electron microscopy revealed a novel aspect of the pancreatic acinar cell secretory granules not previously detected by conventional (80-kV) transmission electron microscopy. Examination of ultra-thin (30-nm) sections of non-osmicated, stain-free pancreatic tissue sections by low-voltage electron microscopy revealed the existence of granules with non-homogeneous matrix and sub-compartments having circular or oval profiles of different electron densities and sizes. Such partition is completely masked when observing tissues after postfixation with osmium tetroxide by low-voltage transmission electron microscopy at 5 kV and/or when thicker sections (70 nm) are examined at 80 kV. This morphological partition reflects an internal compartmentalization of the granule content that was previously predicted by morphological, physiological, and biochemical means. It corresponds to the segregation of the different secretory proteins inside the granule as demonstrated by high-resolution immunocytochemistry and reflects a well-organized aggregation of the secretory proteins at the time of granule formation in the trans-Golgi. Such partition of the granule matrix undergoes changes under experimental conditions known to alter the secretory process such as stimulation of secretion or diabetes. PMID:21832147

  8. Compartmentalization of Pancreatic Secretory Zymogen Granules as Revealed by Low-Voltage Transmission Electron Microscopy

    PubMed Central

    Bendayan, Moise; Londono, Irene; Paransky, Eugene

    2011-01-01

    Low-voltage (5-kV) transmission electron microscopy revealed a novel aspect of the pancreatic acinar cell secretory granules not previously detected by conventional (80-kV) transmission electron microscopy. Examination of ultra-thin (30-nm) sections of non-osmicated, stain-free pancreatic tissue sections by low-voltage electron microscopy revealed the existence of granules with non-homogeneous matrix and sub-compartments having circular or oval profiles of different electron densities and sizes. Such partition is completely masked when observing tissues after postfixation with osmium tetroxide by low-voltage transmission electron microscopy at 5 kV and/or when thicker sections (70 nm) are examined at 80 kV. This morphological partition reflects an internal compartmentalization of the granule content that was previously predicted by morphological, physiological, and biochemical means. It corresponds to the segregation of the different secretory proteins inside the granule as demonstrated by high-resolution immunocytochemistry and reflects a well-organized aggregation of the secretory proteins at the time of granule formation in the trans-Golgi. Such partition of the granule matrix undergoes changes under experimental conditions known to alter the secretory process such as stimulation of secretion or diabetes. PMID:21832147

  9. Degradation of human anaphylatoxin C3a by rat peritoneal mast cells: a role for the secretory granule enzyme chymase and heparin proteoglycan

    SciTech Connect

    Gervasoni, J.E. Jr.; Conrad, D.H.; Hugli, T.E.; Schwartz, L.B.; Ruddy, S.

    1986-01-01

    Purified human C3a was iodinated (/sup 125/I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of /sup 125/I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Once /sup 125/I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of /sup 125/I precipitable by TCA and the appearance of /sup 125/-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of /sup 125/I-C3a. Treatment of RMC bearing /sup 125/I-C3a with bis (sulfosuccinimidyl) suberate (BS/sup 3/) covalently cross-linked the /sup 125/I-C3a to chymase, the predominant enzyme found in the secretory granules. Indirect immunofluorescence of RMC by using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on /sup 125/I-C3a, but together they resulted in prompt degradation of the /sup 125/I-C3a.

  10. Electron microprobe analysis of human labial gland secretory granules in cystic fibrosis

    SciTech Connect

    Izutsu, K.; Johnson, D.; Schubert, M.; Wang, E.; Ramsey, B.; Tamarin, A.; Truelove, E.; Ensign, W.; Young, 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, 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.

  11. Effects of fasting and refeeding on secretory granules of the mouse gallbladder epithelium. A quantitative electron microscopic study.

    PubMed

    Wahlin, T; Bloom, G D; Carlsöö, B; Rhodin, L

    1976-03-01

    Mouse gallbladder epithelial cells were studied with the electron microscope during fasting and refeeding. Morphometric data were obtained from randomly selected epithelial cells of normal starved (12, 24, and 48 hr) and refed (12 hr) mice. Deprivation of food significantly diminishes the volume density of the mucinous secretory granules by about 70% after 48 hr of fasting. Upon refeeding, this secretory granule parameter increases significantly ( 2.5 times). Stereological measurements were also performed on nuclei, mitochondria, and lysosomes, but no major morphometric changes were observed in these organelles. The findings suggest that a basal secretion of mucin granules occur in the mouse gallbladder, irrespective of the animal's nutritional state and that this discharge during starvation exceeds the formation of new granule material. The findings are discussed in relation to effects of fasting and refeeding on other secretory cell systems. PMID:765187

  12. Trafficking\\/sorting and granule biogenesis in the ? -cell

    Microsoft Academic Search

    Miguel Molinete; Jean-Claude Irminger; Sharon A. Tooze; Philippe A. Halban

    2000-01-01

    Proinsulin is packaged into nascent (immature, clathrin-coated) secretory granules in the trans-Golgi network (TGN) of the ? -cell along with other granular constituents including the proinsulin conversion enzymes. It is assumed that such packaging is dependent on an active sorting process, separating granular proteins from other secretory or membrane proteins, but the mechanism remains elusive. As granules mature, the clathrin

  13. Porosome: The Universal Secretory Portal in Cells

    NASA Astrophysics Data System (ADS)

    Jena, Bhanu

    2012-10-01

    In the past 50 years it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the ``all or none'' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. Moreover, it makes no sense for mammalian cells to possess such `all or none' mechanism of cell secretion, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such as the secretion apparatus of Toxoplasma gondii, the contractile vacuoles in paramecium, or the various types of secretory structures in bacteria. Therefore, in 1993 in a News and Views article in Nature, E. Neher wrote ``It seems terribly wasteful that, during the release of hormones and neurotransmitters from a cell, the membrane of a vesicle should merge with the plasma membrane to be retrieved for recycling only seconds or minutes later.'' This conundrum in the molecular mechanism of cell secretion was finally resolved in 1997 following discovery of the ``Porosome,'' the universal secretory machinery in cells. Porosomes are supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release inravesicular contents to the outside during cell secretion. In the past decade, the composition of the porosome, its structure and dynamics at nm resolution and in real time, and its functional reconstitution into artificial lipid membrane, have all been elucidated. Since porosomes in exocrine and neuroendocrine cells measure 100-180 nm, and only 20-45% increase in porosome diameter is demonstrated following the docking and fusion of 0.2-1.2 ?m in diameter secretory vesicles, it is concluded that secretory vesicles ``transiently'' dock and fuse, rather than completely merge at the base of the porosome complex to release their contents to the outside. In agreement, it has been demonstrated that ``secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells''; that ``single synaptic vesicles fuse transiently and successively without loss of identity''; and that``zymogen granule (the secretory vesicle in exocrine pancreas) exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity.'' In this presentation, the discovery of the porosome, resulting in a paradigm shift in our understanding of cell secretion will be briefly discussed.

  14. Co-localization of islet amyloid polypeptide and insulin in the B cell secretory granules of the human pancreatic islets

    Microsoft Academic Search

    A. Lukinius; E. Wilander; G. T. Westermark; U. Engström; P. Westermark

    1989-01-01

    Summary  Islet amyloid polypeptide is a novel 37 amino-acid-residues polypeptide which has been isolated from amyloid deposits in an insulinoma, and in human and cat islets of Langerhans. The molecule has 46% homology with the calcitonin gene-related peptide. Light microscopy examination of the pancreas shows that islet amyloid polypeptide immunoreactivity is restricted to the islet B cells. The present study utilized

  15. Signaling from the Secretory Granule to the Nucleus: Uhmk1 and PAM

    PubMed Central

    Francone, Victor P.; Ifrim, Marius F.; Rajagopal, Chitra; Leddy, Christopher J.; Wang, Yanping; Carson, John H.; Mains, Richard E.; Eipper, Betty A.

    2010-01-01

    Neurons and endocrine cells package peptides in secretory granules (large dense-core vesicles) for storage and stimulated release. Studies of peptidylglycine ?-amidating monooxygenase (PAM), an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain (CD) of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by U2AF homology motif kinase 1 (Uhmk1) and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble CD fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously overexpressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wild-type mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels whereas expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent. PMID:20573687

  16. Increased biogenesis of glucagon-containing secretory granules and glucagon secretion in BIG3-knockout mice

    PubMed Central

    Li, Hongyu; Liu, Tao; Lim, Joy; Gounko, Natalia V.; Hong, Wanjin; Han, Weiping

    2015-01-01

    Objective Although both insulin and glucagon are intimately involved in the regulation of glucose homeostasis, the intrinsic control of glucagon secretion, including the biogenesis and exocytosis of glucagon-containing granules, is far less understood compared with that of insulin. As Brefeldin A-inhibited guanine nucleotide exchange protein 3 (BIG3) is a negative regulator of insulin-granule biogenesis and insulin secretion, we investigated whether BIG3 plays any role in alpha-cells and glucagon secretion. Methods We examined the expression of BIG3 in islet cells by immuno-fluorescence and confocal microscopy, and measured glucagon production and secretion in BIG3-depleted and wild-type mice, islets and cells. Results BIG3 is highly expressed in pancreatic alpha-cells in addition to beta-cells, but is absent in delta-cells. Depletion of BIG3 in alpha-cells leads to elevated glucagon production and secretion. Consistently, BIG3-knockout (BKO) mice display increased glucagon release under hypoglycemic conditions. Conclusions Together with our previous studies, the current data reveal a conserved role for BIG3 in regulating alpha- and beta-cell functions. We propose that BIG3 negatively regulates hormone production at the secretory granule biogenesis stage and that such regulatory mechanism may be used in secretory pathways of other endocrine cells. PMID:25737957

  17. 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 ISGs is impaired by BFA, proteolytic processing of POMC within this organelle proceeds unaffected. The finding that BFA impairs constitutive secretion from both the TGN and ISGs also suggests that these secretory processes may be related in mechanism. Finally, our data indicate that the unusually high levels of unregulated secretion often associated with endocrine tumors may result, at least in part, from inefficient storage of secretory products at the level of ISGs. PMID:9362061

  18. The Arf family G protein Arl1 is required for secretory granule biogenesis in Drosophila

    PubMed Central

    Torres, Isabel L.; Rosa-Ferreira, Cláudia; Munro, Sean

    2014-01-01

    ABSTRACT The small G protein Arf like 1 (Arl1) is found at the Golgi complex, and its GTP-bound form recruits several effectors to the Golgi including GRIP-domain-containing coiled-coil proteins, and the Arf1 exchange factors Big1 and Big2. To investigate the role of Arl1, we have characterised a loss-of-function mutant of the Drosophila Arl1 orthologue. The gene is essential, and examination of clones of cells lacking Arl1 shows that it is required for recruitment of three of the four GRIP domain golgins to the Golgi, with Drosophila GCC185 being less dependent on Arl1. At a functional level, Arl1 is essential for formation of secretory granules in the larval salivary gland. When Arl1 is missing, Golgi are still present but there is a dispersal of adaptor protein 1 (AP-1), a clathrin adaptor that requires Arf1 for its membrane recruitment and which is known to be required for secretory granule biogenesis. Arl1 does not appear to be required for AP-1 recruitment in all tissues, suggesting that it is crucially required to enhance Arf1 activation at the trans-Golgi in particular tissues. PMID:24610947

  19. [Histochemical properties of secretory granules and fine structure of terminal portion in the Japanese Macaque labial gland].

    PubMed

    Tsutusumi, T; Kurabuchi, S; Aiyama, S

    1989-06-01

    The terminal portion of the Japanese macaque (Macaca fuscata) labial gland was examined ultrastructurally and histochemically. The results obtained are as follows. 1) The Japanese macaque, Macaca fuscata has the upper and the lower labial glands, which can be described as a compound tubulo-acinar gland. 2) The terminal portion of the labial gland appears to be consisted of the mucous acini and the demilunes when examined with the light microscope. 3) The secretory granules containing in the glandular cells of the mucous acini and the demilunes are negative with napthol yellow S, ninhydrin-Schiff and DMAB nitrite. 4) The secretory granules containing in the glandular cells of mucous acini stain intensely with PAS, alcian blue (pH1.0, 2.5, 3.5), colloidal iron and PA-methenamine silver, while those of demilunes are negative with alcian blue (pH1.0). The glandular cells of demilune with the PA-methenamine silver method shows weak positive granules and positive granules which are limited to the hallo of them. 5) In the mucous acini the mucous granules are ejected from glandular cells by the process of exocytosis. 6) The myoepithelial cell can be seen in the terminal portion. This cell surrounds the acini with long processes. These findings suggest that the glandular cells of the demilune have the granules containing mucopoly saccharides and a small quantity of protein in addition to the mucous granules, although the terminal portion of the Japanese macaque labial gland is nearly composed of mucous cells. PMID:2637414

  20. Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena

    PubMed Central

    Briguglio, Joseph S.; Kumar, Santosh

    2013-01-01

    Secretory granules, such as neuronal dense core vesicles, are specialized for storing cargo at high concentration and releasing it via regulated exocytosis in response to extracellular stimuli. Here, we used expression profiling to identify new components of the machinery for sorting proteins into mucocysts, secretory granule-like vesicles in the ciliate Tetrahymena thermophila. We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis. In particular, the delivery of nonaggregated, but not aggregated, cargo proteins requires classical receptors of the sortilin/VPS10 family, which indicates that dual mechanisms are involved in sorting to this secretory compartment. In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation. Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes. PMID:24189272

  1. RNA Granules in Germ Cells

    PubMed Central

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

    2011-01-01

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

  2. A functioning artificial secretory cell

    PubMed Central

    Simonsson, Lisa; Kurczy, Michael E.; Trouillon, Raphaël; Hook, Fredrik; Cans, Ann-Sofie

    2012-01-01

    We present an amperometric study of content release from individual vesicles in an artificial secretory cell designed with the minimal components required to carry out exocytosis. Here, the membranes of the cell and vesicles are substituted for protein-free giant and large unilamellar vesicles respectively. In replacement of the SNARE-complex, the cell model was equipped with an analog composed of complimentary DNA constructs. The DNA constructs hybridize in a zipper-like fashion to bring about docking of the artificial secretory vesicles and following the addition of Ca2+ artificial exocytosis was completed. Exocytotic events recorded from the artificial cell closely approximate exocytosis in live cells. The results together with simulations of vesicular release demonstrate that the molecular flux in this model is attenuated and we suggest that this is the result of restricted diffusion through a semi-stable fusion pore or a partitioning of the signalling molecule out of the fused vesicle membrane. PMID:23139869

  3. CEREBELLAR GRANULE CELLS IN VITRO

    PubMed Central

    Seil, Fredrick J.; Herndon, Robert M.

    1970-01-01

    The behavior of granule cells in mature cerebellar cultures derived from newborn mice was studied by light and electron microscopy. Many granule cells remained in the explants as an external granular layer. These cells were differentiated, as evidenced by formation of bundles of parallel fibers and by development of synapses between granule cell axons and Purkinje cell branchlet spines, and between Golgi cell axons and granule cell dendrites. Although the over-all architecture of the cerebellar explants after 18–33 days in vitro was similar to that of the newborn mouse, the evident differentiation of the granule cells suggested that interneuronal relationships resemble those of the mature cerebellum in vivo. PMID:5513604

  4. Role of Secretory Carrier Membrane Protein SCAMP2 in Granule Exocytosis

    PubMed Central

    Liu, Lixia; Guo, Zhenheng; Tieu, Quyen; Castle, Anna; Castle, David

    2002-01-01

    In secretory carrier membrane proteins (SCAMPs), the most conserved structural segment is between transmembrane spans 2 and 3, facing the cytosol. A synthetic peptide, CWYRPIYKAFR (E peptide), from this segment of SCAMP2 potently inhibits exocytosis in permeabilized neuroendocrine (PC12) cells. E peptide blocked discharge of 35S-labeled secretogranin with the same structural selectivity and potency as observed for hexosaminidase secretion in mast cells. SCAMPs 1 and 2 are concentrated primarily on intracellular membranes in PC12 cells. Both, however, are found on plasma membranes, but neither is present on large dense-core vesicles. Yet, large dense-core vesicles marked by secretogranin attach to plasma membranes at foci containing SCAMP2 along with syntaxin1 and complexin at putative cell-surface docking/fusion sites. Regulated overexpression of SCAMP2 with point mutations in its E peptide but not of normal SCAMP2 caused dose-dependent inhibition of depolarization-induced secretion. The SCAMP2 mutants also inhibited secretion stimulated by elevated calcium. Inhibition was largely overcome by adding lysophosphatidylcholine to the medium at concentrations that do not otherwise affect secretion. Although overexpression of normal or mutant SCAMP2 slightly inhibits endocytosis, this effect does not appear to be related to the specific effect of the mutant SCAMP on stimulated exocytosis. Thus, SCAMP2 not only colocalizes with fusion sites but also appears to have an essential function in granule exocytosis through actions mediated by its E peptide–containing domain. PMID:12475951

  5. A Nibbling Mechanism for Clathrin-mediated Retrieval of Secretory Granule Membrane after Exocytosis*

    PubMed Central

    Bittner, Mary A.; Aikman, Rachel L.; Holz, Ronald W.

    2013-01-01

    Clathrin-mediated endocytosis is the major pathway for recycling of granule membrane components after strong stimulation and high exocytotic rates. It resembles “classical” receptor-mediated endocytosis but has a trigger that is unique to secretion, the sudden appearance of the secretory granule membrane in the plasma membrane. The spatial localization, the relationship to individual fusion events, the nature of the cargo, and the timing and nature of the nucleation events are unknown. Furthermore, a size mismatch between chromaffin granules (?300-nm diameter) and typical clathrin-coated vesicles (?90 nm) makes it unlikely that clathrin-mediated endocytosis internalizes as a unit the entire fused granule membrane. We have used a combination of total internal reflection fluorescence microscopy of transiently expressed proteins and time-resolved quantitative confocal imaging of endogenous proteins along with a fluid-phase marker to address these issues. We demonstrate that the fused granule membrane remains a distinct entity and serves as a nucleation site for clathrin- and dynamin-mediated endocytosis that internalizes granule membrane components in small increments. PMID:23386611

  6. A Dynamic Analysis of Secretory Granules Containing Proteins Involved In Learning

    NASA Astrophysics Data System (ADS)

    Prahl, Louis; Simon, Alex; Jacobs, Conor; Fulwiler, Audrey; Hilken, Lindsay; Scalettar, Bethe; Lochner, Janis

    2010-10-01

    Formation and encoding of long-term memories requires a series of structural changes at synapses, or sites of neuronal communication, in the hippocampus; these changes are mediated by neuromodulatory proteins and serve to strengthen synapses to improve communication. Two prominent neuromodulators, tissue plasminogen activator (tPA) and brain-derived neurotrophic factor (BDNF), are copackaged into secretory granules (SGs) in the body of nerve cells and are transported to distal synapses by motor proteins. At synapses, particularly presynaptic sites, the fate of tPA and BDNF is largely unknown. Motivated by this, and by recent data implicating presynaptic BDNF in early phases of learning, we used fluorescence microscopy to elucidate dynamic properties of presynaptic tPA and BDNF. We find that presynaptic SGs containing tPA and/or BDNF undergo Brownian and anomalous diffusive motion that, in 75% of cases, is so slow that it typically would be classified as immobility. These results suggest that tPA and BDNF are retained at presynaptic sites to facilitate their corelease and role in learning.

  7. Salmeterol restores secretory functions in cystic fibrosis airway submucosal gland serous cells.

    PubMed

    Delavoie, Franck; Molinari, Michael; Milliot, Magali; Zahm, Jean-Marie; Coraux, Christelle; Michel, Jean; Balossier, Gérard

    2009-04-01

    The activity of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) can be mediated by surface G protein-coupled receptors such as the beta(2)-adrenergic receptor. In this study, we explored the effect of a long-acting beta(2)-adrenergic agonist, salmeterol, on the CFTR-dependent secretory capacity of a human CF tracheal gland serous cell line (CF-KM4), homozygous for the delF508 mutation. We showed that, compared with the untreated CF serous cells, a 24-hour pre-incubation period with 200 nM salmeterol induced an 83% increase in delF508-CFTR-mediated chloride efflux. The restoration of the bioelectric properties is associated with increased apical surface pool of delF508-CFTR. Salmeterol induced a decrease in ion concentration and an increase in the level of hydration of the mucus packaged inside the CF secretory granules. The effects of salmeterol are not associated with a persistent production of cAMP. Western blotting on isolated secretory granules demonstrated immunoreactivity for CFTR and lysozyme. In parallel, we measured by atomic force microscopy an increased size of secretory granules isolated from CF serous cells compared with non-CF serous cells (MM39 cell line) and showed that salmeterol was able to restore a CF cell granule size similar to that of non-CF cells. To demonstrate that the salmeterol effect was a CFTR-dependent mechanism, we showed that the incubation of salmeterol-treated CF serous cells with CFTR-inh172 suppressed the restoration of normal secretory functions. The capacity of salmeterol to restore the secretory capacity of glandular serous cells suggests that it could also improve the airway mucociliary clearance in patients with CF. PMID:18931328

  8. Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

    PubMed Central

    Hammel, Ilan; Shoichetman, Tanya; Amihai, Dina; Galli, Stephen J.; Skutelsky, Ehud

    2013-01-01

    We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky's fixative and OsO4 and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower adivin gold binding density (by approximately 50%, P<0.001). The latter result provides additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild type mice. In both wild type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild type cells (P < 0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in the bm/bm cells. PMID:20127366

  9. The stealthy nano-machine behind mast cell granule size distribution.

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2015-01-01

    The classical model of mast cell secretory granule formation suggests that newly synthesized secretory mediators, transported from the rough endoplasmic reticulum to the Golgi complex, undergo post-transitional modification and are packaged for secretion by condensation within membrane-bound granules of unit size. These unit granules may fuse with other granules to form larger granules that reside in the cytoplasm until secreted. A novel stochastic model for mast cell granule growth and elimination (G&E) as well as inventory management is presented. Resorting to a statistical mechanics approach in which SNAP (Soluble NSF Attachment Protein) REceptor (SNARE) components are viewed as interacting particles, the G&E model provides a simple 'nano-machine' of SNARE self-aggregation that can perform granule growth and secretion. Granule stock is maintained as a buffer to meet uncertainty in demand by the extracellular environment and to serve as source of supply during the lead time to produce granules of adaptive content. Experimental work, mathematical calculations, statistical modeling and a rationale for the emergence of nearly last-in, first out inventory management, are discussed. PMID:24629227

  10. Secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways

    PubMed Central

    1985-01-01

    The mouse pituitary cell line, AtT-20, packages the adrenocorticotropic hormone (ACTH) in secretory vesicles and releases it when the cell is stimulated with secretagogues. These cells have the capacity, after transfection with the appropriate DNA, to package heterologous peptide hormones into the regulated secretory vesicles (Moore, H. P. H., M. D. Walker, F. Lee, and R. B. Kelly, 1983, Cell, 35:531-538). To test if other secreted proteins prefer a different route to the surface, we have transfected AtT-20 cells with DNAs coding for a fragment of a membrane protein, the vesicular stomatitis virus G protein from which the membrane spanning domain has been deleted (Rose, J. K., and J. E. Bergmann, 1982, Cell, 17:813-819). We found that the secreted vesicular stomatitis virus G proteins were not transported to the regulated secretory vesicles. Instead they preferentially exited the cell by the constitutive pathway previously found in these cells (Gumbiner, B., and R. B. Kelly, 1982, Cell, 28:51-59). In contrast, human growth hormone transfected into the cells by the same procedure was transported to the regulated pathway with a similar efficiency as the endogenous hormone ACTH. Transport of the secreted G protein to the regulated pathway, if it occurs at all, is at least 30-fold less efficient than peptide hormones. We conclude that the transport machinery in AtT-20 cells must selectively recognize different secreted proteins and sort them into distinct secretory pathways. PMID:2997234

  11. Phosphatidylinositol kinase in rat mast cell granules

    SciTech Connect

    Kurosawa, M.; Parker, C.W.

    1986-01-01

    Intact granules were isolated from sonicated purified rat serosal mast cells on a Percoll gradient. The granules were shown to contain a highly active phosphatidylinositol kinase that catalyzes the formation of diphosphoinositide from endogenous phosphatidylinositol in the granule membrane. The enzyme requires ATP and Mg/sup 2 +/ or Mn/sup 2 +/ for activity; Ca/sup 2 +/, fluoride and cyclic AMP are inhibitory. The K/sub m/ for ATP is 25 ..mu..M. The initial reaction is rapid, but the response ceases within a few minutes. A comparison of the rate of phosphorylation of intact and broken membrane granules suggests that the phosphorylation occurs on the outer (cytoplasmic) surface of the granules.

  12. In vitro conditions modify immunoassayability of bovine pituitary prolactin and growth hormone: insights into their secretory granule storage forms

    SciTech Connect

    Lorenson, M.Y.

    1985-04-01

    The amount of immunoassayable intracellular bovine (b) PRL and GH varies depending on treatment conditions. The present studies were designed to characterize the mechanisms involved and to compare immunoassayability of both hormones under similar conditions. Pituitary homogenate and secretory granule hormones displayed both time- and temperature-dependent increases when incubated at pH 10.5 with reduced glutathione. Changes in immunoassayability seem to reflect conversion from poorly immunoactive tissue hormone oligomers to monomeric hormone. The data indicate that oligomeric bPRL is stabilized primarily by intermolecular disulfide bonds, although it is also susceptible to urea, SDS, and EDTA; granule thiols may also influence the conversion to monomer. The storage form of bGH appears to be stabilized differently. Maneuvers demonstrated in these studies to influence immunoassayability correlate very well with their previously established effects on hormone release and secretion, strengthening the likelihood that a functional link exists between assayability and secretion.

  13. Rab27A and its effector MyRIP link secretory granules to F-actin and control their motion towards release sites

    PubMed Central

    Desnos, Claire; Schonn, Jean-Sébastien; Huet, Sébastien; Tran, Viet Samuel; El-Amraoui, Aziz; Raposo, Graça; Fanget, Isabelle; Chapuis, Catherine; Ménasché, Gaël; de Saint Basile, Genevičve; Petit, Christine; Cribier, Sophie; Henry, Jean-Pierre; Darchen, François

    2003-01-01

    The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex. PMID:14610058

  14. Hilar mossy cell circuitry controlling dentate granule cell excitability

    PubMed Central

    Jinde, Seiichiro; Zsiros, Veronika; Nakazawa, Kazu

    2013-01-01

    Glutamatergic hilar mossy cells of the dentate gyrus can either excite or inhibit distant granule cells, depending on whether their direct excitatory projections to granule cells or their projections to local inhibitory interneurons dominate. However, it remains controversial whether the net effect of mossy cell loss is granule cell excitation or inhibition. Clarifying this controversy has particular relevance to temporal lobe epilepsy, which is marked by dentate granule cell hyperexcitability and extensive loss of dentate hilar mossy cells. Two diametrically opposed hypotheses have been advanced to explain this granule cell hyperexcitability—the “dormant basket cell” and the “irritable mossy cell” hypotheses. The “dormant basket cell” hypothesis proposes that mossy cells normally exert a net inhibitory effect on granule cells and therefore their loss causes dentate granule cell hyperexcitability. The “irritable mossy cell” hypothesis takes the opposite view that mossy cells normally excite granule cells and that the surviving mossy cells in epilepsy increase their activity, causing granule cell excitation. The inability to eliminate mossy cells selectively has made it difficult to test these two opposing hypotheses. To this end, we developed a transgenic toxin-mediated, mossy cell-ablation mouse line. Using these mutants, we demonstrated that the extensive elimination of hilar mossy cells causes granule cell hyperexcitability, although the mossy cell loss observed appeared insufficient to cause clinical epilepsy. In this review, we focus on this topic and also suggest that different interneuron populations may mediate mossy cell-induced translamellar lateral inhibition and intralamellar recurrent inhibition. These unique local circuits in the dentate hilar region may be centrally involved in the functional organization of the dentate gyrus. PMID:23407806

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

    PubMed Central

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

    2013-01-01

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

  16. Tracheobronchial epithelium of the sheep: IV. Lectin histochemical characterization of secretory epithelial cells.

    PubMed

    Mariassy, A T; Plopper, C G; St George, J A; Wilson, D W

    1988-09-01

    Conventional histochemical characterization of the mucus secretory apparatus is often difficult to reconcile with the biochemical analysis of respiratory secretions. This study was designed to examine the secretory glycoconjugates in airways using lectins with biochemically defined affinities for main sugar residues of mucus. We used five biotinylated lectins--DBA (Dolichos biflorus) and SBA (Glycine max) for N-acetyl galactosamine (galNAc), BSA I (Bandeiraea simplicifolia) and PNA (Arachis hypogea) for galactose (gal), and UEA I (Ulex europeus)--for detection of fucose (fuc) in HgCl2-fixed, paraffin-embedded, serially sectioned trachea, lobar and segmental bronchi and bronchioles of nine sheep. Lectins selectively localized the carbohydrate residues in luminal secretions, on epithelial cell surfaces, and in secretory cells. In proximal airways, the major carbohydrate residues in luminal secretions, cell surfaces, goblet cells, and glands were fuc and gal-NAc. PNA reacted mainly with apical granules of less than 10% of goblet cells, and gal residues were only detected in some of the mucous cells and on basolateral cell surfaces. Distal airways contained sparse secretion in the lumen, mucous cells contained weakly reactive fuc and gal-NAc, and the epithelial surfaces of Clara cells contained gal. Sugars abundant in the airway secretions were also the major component of cells in glands. We conclude that there is a correlation between specific sugar residues in secretory cells, glycocalyx, and luminal secretions in proximal and distal airways. This suggests that lectins may be used to obtain information about airway secretory cell composition from respiratory secretions. PMID:3189886

  17. Young Dentate Granule Cells Mediate Pattern Separation, whereas Old Granule Cells Facilitate Pattern Completion

    E-print Network

    Nakashiba, Toshiaki

    Adult-born granule cells (GCs), a minor population of cells in the hippocampal dentate gyrus, are highly active during the first few weeks after functional integration into the neuronal network, distinguishing them from ...

  18. Stability of proICA512/IA-2 and Its Targeting to Insulin Secretory Granules Require ?4-Sheet-Mediated Dimerization of Its Ectodomain in the Endoplasmic Reticulum.

    PubMed

    Torkko, Juha M; Primo, M Evangelina; Dirkx, Ronald; Friedrich, Anne; Viehrig, Antje; Vergari, Elisa; Borgonovo, Barbara; Sönmez, Anke; Wegbrod, Carolin; Lachnit, Martina; Münster, Carla; Sica, Mauricio P; Ermácora, Mario R; Solimena, Michele

    2015-03-15

    The type 1 diabetes autoantigen ICA512/IA-2/RPTPN is a receptor protein tyrosine phosphatase of the insulin secretory granules (SGs) which regulates the size of granule stores, possibly via cleavage/signaling of its cytosolic tail. The role of its extracellular region remains unknown. Structural studies indicated that ?2- or ?4-strands in the mature ectodomain (ME ICA512) form dimers in vitro. Here we show that ME ICA512 prompts proICA512 dimerization in the endoplasmic reticulum. Perturbation of ME ICA512 ?2-strand N-glycosylation upon S508A replacement allows for proICA512 dimerization, O-glycosylation, targeting to granules, and conversion, which are instead precluded upon G553D replacement in the ME ICA512 ?4-strand. S508A/G553D and N506A/G553D double mutants dimerize but remain in the endoplasmic reticulum. Removal of the N-terminal fragment (ICA512-NTF) preceding ME ICA512 allows an ICA512-?NTF G553D mutant to exit the endoplasmic reticulum, and ICA512-?NTF is constitutively delivered to the cell surface. The signal for SG sorting is located within the NTF RESP18 homology domain (RESP18-HD), whereas soluble NTF is retained in the endoplasmic reticulum. Hence, we propose that the ME ICA512 ?2-strand fosters proICA512 dimerization until NTF prevents N506 glycosylation. Removal of this constraint allows for proICA512 ?4-strand-induced dimerization, exit from the endoplasmic reticulum, O-glycosylation, and RESP18-HD-mediated targeting to granules. PMID:25561468

  19. Essential Role of the Disulfide-bonded Loop of Chromogranin B for Sorting to Secretory Granules Is Revealed by Expression of a Deletion Mutant in the Absence of Endogenous Granin Synthesis

    PubMed Central

    Krömer, Andreas; Glombik, Michael M.; Huttner, Wieland B.; Gerdes, Hans-Hermann

    1998-01-01

    Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH2-terminal disulfide– bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (?cys-hCgB) lacking the 22–amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, ?cys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with ?cys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus–mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules. PMID:9508767

  20. 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.

  1. Vesicle associated membrane protein 8 (VAMP8)-mediated zymogen granule exocytosis is dependent on endosomal trafficking via the constitutive-like secretory pathway.

    PubMed

    Messenger, Scott W; Falkowski, Michelle A; Thomas, Diana D H; Jones, Elaina K; Hong, Wanjin; Gaisano, Herbert Y; Giasano, Herbert Y; Boulis, Nicholas M; Groblewski, Guy E

    2014-10-01

    Acinar cell zymogen granules (ZG) express 2 isoforms of the vesicle-associated membrane protein family (VAMP2 and -8) thought to regulate exocytosis. Expression of tetanus toxin to cleave VAMP2 in VAMP8 knock-out (-/-) acini confirmed that VAMP2 and -8 are the primary VAMPs for regulated exocytosis, each contributing ?50% of the response. Analysis of VAMP8(-/-) acini indicated that although stimulated secretion was significantly reduced, a compensatory increase in constitutive secretion maintained total secretion equivalent to wild type (WT). Using a perifusion system to follow secretion over time revealed VAMP2 mediates an early rapid phase peaking and falling within 2-3 min, whereas VAMP8 controls a second prolonged phase that peaks at 4 min and slowly declines over 20 min to support the protracted secretory response. VAMP8(-/-) acini show increased expression of the endosomal proteins Ti-VAMP7 (2-fold) and Rab11a (4-fold) and their redistribution from endosomes to ZGs. Expression of GDP-trapped Rab11a-S25N inhibited secretion exclusively from the VAMP8 but not the VAMP2 pathway. VAMP8(-/-) acini also showed a >90% decrease in the early endosomal proteins Rab5/D52/EEA1, which control anterograde trafficking in the constitutive-like secretory pathway. In WT acini, short term (14-16 h) culture also results in a >90% decrease in Rab5/D52/EEA1 and a complete loss of the VAMP8 pathway, whereas VAMP2-secretion remains intact. Remarkably, rescue of Rab5/D52/EEA1 expression restored the VAMP8 pathway. Expressed D52 shows extensive colocalization with Rab11a and VAMP8 and partially copurifies with ZG fractions. These results indicate that robust trafficking within the constitutive-like secretory pathway is required for VAMP8- but not VAMP2-mediated ZG exocytosis. PMID:25138214

  2. Monoclonal Antibodies as Probes for Unique Antigens in Secretory Cells of Mixed Exocrine Organs

    NASA Astrophysics Data System (ADS)

    Basbaum, C. B.; Mann, J. K.; Chow, A. W.; Finkbeiner, W. E.

    1984-07-01

    In the past, it has been difficult to identify the secretory product and control mechanisms associated with individual cell types making up mixed exocrine organs. This report establishes the feasibility of using immunological methods to characterize both the biochemical constituents and regulatory mechanisms associated with secretory cells in the trachea. Monoclonal antibodies directed against components of tracheal mucus were produced by immunizing mice with dialyzed, desiccated secretions harvested from tracheal organ culture. An immunofluorescence assay revealed that of the total 337 hybridomas screened, 100 produced antibodies recognizing goblet cell granules; 64, gland cell granules; and 3, antigen confined to the ciliated apical surface of the epithelium. The tracheal goblet cell antibody described in this report was strongly cross-reactive with intestinal goblet cells, as well as with a subpopulation of submandibular gland cells, but not with cells of Brunner's glands or the ciliated cell apical membrane. The serous cell antibody was not cross-reactive with goblet, Brunner's gland, or submandibular cells, or the ciliated cell apical membrane. The antibody directed against the apical membrane of ciliated cells did not cross-react with gland or goblet cells or the apical membrane of epithelial cells in the duodenum. Monoclonal antibodies, therefore, represent probes by which products unique to specific cells or parts of cells in the trachea can be distinguished. The antibodies, when used in enzyme immunoassays, can be used to quantitatively monitor secretion by individual cell types under a variety of physiological and pathological conditions. They also provide the means for purification and characterization of cell-specific products by immunoaffinity chromatography.

  3. Single granule cells excite Golgi cells and evoke feedback inhibition in the cochlear nucleus.

    PubMed

    Yaeger, Daniel B; Trussell, Laurence O

    2015-03-18

    In cerebellum-like circuits, synapses from thousands of granule cells converge onto principal cells. This fact, combined with theoretical considerations, has led to the concept that granule cells encode afferent input as a population and that spiking in individual granule cells is relatively unimportant. However, granule cells also provide excitatory input to Golgi cells, each of which provide inhibition to hundreds of granule cells. We investigated whether spiking in individual granule cells could recruit Golgi cells and thereby trigger widespread inhibition in slices of mouse cochlear nucleus. Using paired whole-cell patch-clamp recordings, trains of action potentials at 100 Hz in single granule cells was sufficient to evoke spikes in Golgi cells in ?40% of paired granule-to-Golgi cell recordings. High-frequency spiking in single granule cells evoked IPSCs in ?5% of neighboring granule cells, indicating that bursts of activity in single granule cells can recruit feedback inhibition from Golgi cells. Moreover, IPSPs mediated by single Golgi cell action potentials paused granule cell firing, suggesting that inhibitory events recruited by activity in single granule cells were able to control granule cell firing. These results suggest a previously unappreciated relationship between population coding and bursting in single granule cells by which spiking in a small number of granule cells may have an impact on the activity of a much larger number of granule cells. PMID:25788690

  4. Ethanol effects on dentate granule cell LTP

    Microsoft Academic Search

    M. J. Wayner; D. L. Armstrong; C. F. Phelix

    2003-01-01

    In previous studies we identified a lateral hypothalamic area (LHA) sensitive to ethanol, <5.0 mM, when the perifornical region\\u000a of the area is perfused with different concentrations of ethanol. Some of these perifornical neurons contain angiotensin (Ang)\\u000a and project directly to the dentate gyrus where angiotensin is released and inhibits LTP in medial perforant path-dentate\\u000a granule cell synapses. The AT1

  5. Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway

    Microsoft Academic Search

    P. Rosa; RAYMOND W. H. LEE; ANTONIA ZANINI; PIETRO De CAMILLI; WIELAND B. HUTTNER

    1985-01-01

    We report on the biochemical and immunological properties as well as on the cellular and subcellular distribution of two proteins, called secretogranins I and II. These proteins specifically occur in a wide variety of endocrine and neuronal cells that package and sort regulatory peptides into secretory granules. Both secretogranins take the same intracellular route as the peptides and are also

  6. Cell-specific analysis of tracheobronchial secretory cells and secretions

    SciTech Connect

    Finkbeiner, W.E.

    1989-01-01

    In these studies, two methods (cell culture and monoclonal antibody production) that allowed cell-specific analysis of tracheobronchial secretion were used. Bovine tracheal submucosal gland cells were isolated, placed into culture and serially propagated. In culture, the cells maintained features of serous cells. The cells incorporated {sup 35}S into high molecular weight molecules. {beta}-adrenergic agonists stimulated release of radiolabeled molecules and elevations in intracellular cAMP levels, responses that could be blocked by the {beta}-adrenergic antagonist propranolol. Cyclic AMP appeared to be involved in the stimulus-secretion coupling events in serous cells since the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine potentiated the effects of isoproterenol on the secretory response and the elevation of intracellular cAMP levels. Furthermore, cAMP analogues elicited a secretory response in the absence of cAMP. The phosphorylation state of several cytosolic and particulate phosphoproteins was altered by cAMP-activated kinase activity. Monoclonal antibodies were produced against human airway secretions.

  7. Cytoplasmic calcium stimulates exocytosis in a plant secretory cell.

    PubMed

    Tester, M; Zorec, R

    1992-09-01

    Although exocytosis is likely to occur in plant cells, the control of this process is the subject of speculation, as no direct measurements of vesicle fusion to the plasma membrane have been made. We used the patch clamp technique to monitor the secretory activity of single aleurone protoplasts by measuring membrane capacitance (C(m)), while dialyzing the cytosol with different Ca(2+) containing solutions. Secretory activity increased with [Ca(2+)](i) approximately 1 muM. This demonstrates directly the existence of exocytosis in plant cells, and suggests that both plant and animal cells share common mechanisms (cytosolic Ca(2+)) for the control of exocytotic secretion. PMID:19431846

  8. Cytoplasmic calcium stimulates exocytosis in a plant secretory cell

    PubMed Central

    Tester, Mark; Zorec, Robert

    1992-01-01

    Although exocytosis is likely to occur in plant cells, the control of this process is the subject of speculation, as no direct measurements of vesicle fusion to the plasma membrane have been made. We used the patch clamp technique to monitor the secretory activity of single aleurone protoplasts by measuring membrane capacitance (Cm), while dialyzing the cytosol with different Ca2+ containing solutions. Secretory activity increased with [Ca2+]i ? 1 ?M. This demonstrates directly the existence of exocytosis in plant cells, and suggests that both plant and animal cells share common mechanisms (cytosolic Ca2+) for the control of exocytotic secretion. PMID:19431846

  9. Tumor protein D52 controls trafficking of an apical endolysosomal secretory pathway in pancreatic acinar cells

    PubMed Central

    Messenger, Scott W.; Thomas, Diana D. H.; Falkowski, Michelle A.; Byrne, Jennifer A.; Gorelick, Fred S.

    2013-01-01

    Zymogen granule (ZG) formation in acinar cells involves zymogen cargo sorting from trans-Golgi into immature secretory granules (ISGs). ISG maturation progresses by removal of lysosomal membrane and select content proteins, which enter endosomal intermediates prior to their apical exocytosis. Constitutive and stimulated secretion through this mechanism is termed the constitutive-like and minor-regulated pathways, respectively. However, the molecular components that control membrane trafficking within these endosomal compartments are largely unknown. We show that tumor protein D52 is highly expressed in endosomal compartments following pancreatic acinar cell stimulation and regulates apical exocytosis of an apically directed endolysosomal compartment. Secretion from the endolysosomal compartment was detected by cell-surface antigen labeling of lysosome-associated membrane protein LAMP1, which is absent from ZGs, and had incomplete overlap with surface labeling of synaptotagmin 1, a marker of ZG exocytosis. Although culturing (16–18 h) of isolated acinar cells is accompanied by a loss of secretory responsiveness, the levels of SNARE proteins necessary for ZG exocytosis were preserved. However, levels of endolysosomal proteins D52, EEA1, Rab5, and LAMP1 markedly decreased with culture. When D52 levels were restored by adenoviral delivery, the levels of these regulatory proteins and secretion of both LAMP1 (endolysosomal) and amylase was strongly enhanced. These secretory effects were absent in alanine and aspartate substitutions of serine 136, the major D52 phosphorylation site, and were inhibited by brefeldin A, which does not directly affect the ZG compartment. Our results indicate that D52 directly regulates apical endolysosomal secretion and are consistent with previous studies, suggesting that this pathway indirectly regulates ZG secretion of digestive enzymes. PMID:23868405

  10. Abnormal Acidification of Melanoma Cells Induces Tyrosinase Retention in the Early Secretory Pathway*

    E-print Network

    Hebert, Daniel N.

    Abnormal Acidification of Melanoma Cells Induces Tyrosinase Retention in the Early Secretory, Pennsylvania 19107 In tyrosinase-positive amelanotic melanoma cells, in- active tyrosinase accumulates-ATPase)-mediated proton transport in melanoma cells disrupts tyrosinase trafficking through the secretory pathway

  11. The process of granule exocytosis in non-stimulated atrial granular cells of the snail, Achatina achatina: an ultrastructural, histochemical and immunocytochemical study.

    PubMed

    Bystrova, Olga A; Shabelnikov, Sergej V; Martynova, Marina G

    2014-01-01

    Abundant secretory granular cells (GCs) in the Giant African land snail atrium harbor a range of bioactive substances and undergo rapid total degranulation in response to stimulation of the cardiac nerve or stressful influences. Here we have analyzed exocytotic events in the non-stimulated GCs. It was shown that the GCs contain three major distinct types of granules that differ histochemically, immunocytochemically and ultrastructurally, each performing specific functions. The type I granules characteristically filled with electron-lucent homogeneous materials exhibit intense immunoreactivity for bioactive proteins and therefore are considered to be storage granules. Histochemistry using vital staining with Acridine Orange and Gomori acid phosphatase technique has revealed lysosomal-related nature of the electron-dense type II granules. Digestion remnants appearing as fine filamentous materials fill the type III granules. Only the type III granules fuse together and with the plasma membrane form degranulation channels and surface pores, through which the debris is removed from the cell. The finding of granules exhibiting intermediate ultrastructural, histochemical and immunocytochemical features suggests that the major granule types represent most stable states along a granule empting continuum. Thus, under physiological conditions, the GCs continuously produce secretory proteins and so maintain readiness for stress-response, but use protein degradation machinery to prevent massive release of these bioactive substances into hemolymph. PMID:23706530

  12. Oxytocin stimulates secretory processes in lactating rabbit mammary epithelial cells

    PubMed Central

    Lollivier, Vanessa; Marnet, Pierre-Guy; Delpal, Serge; Rainteau, Dominique; Achard, Caroline; Rabot, Aline; Ollivier-Bousquet, Michčle

    2006-01-01

    Oxytocin plays a major role in lactation mainly by its action on milk ejection via the contraction of myoepithelial cells. The effect of oxytocin on milk production and the presence of oxytocin receptors on different epithelial cells suggest that this hormone may play a role in mammary epithelial cells. To determine precisely the various roles of oxytocin, we studied localization of oxytocin receptors in lactating rabbit and rat mammary tissue and the influence of oxytocin on secretory processes in lactating rabbit mammary epithelial cells. Immunolocalization of oxytocin receptors on mammary epithelial cells by immunofluorescence and in mammary tissue by immunogold in addition to in situ hybridization showed that lactating rat and rabbit mammary epithelial cells expressed oxytocin receptors. Moreover, oxytocin bound specifically to epithelial cells. To determine whether oxytocin had an effect on lactating rabbit mammary epithelial cells, isolated mammary fragments were incubated in the presence or absence of 10?6 i.u. ml?1 of oxytocin. After 1 min of incubation with oxytocin, the morphology of epithelial cells and the localization of caseins and proteins associated with the secretory traffic suggested a striking acceleration of the transport leading to exocytosis, whereas the contraction of myoepithelial cells was only detectable after 7 min. Addition of 10?8 g ml?1 of atosiban before the addition of oxytocin prevented the oxytocin effect on secretory processes and on myoepithelial cell contraction. Addition of 10?6 i.u. ml?1 of vasopressin to the incubation medium did not mimic the stimulating effect of oxytocin on secretory traffic. These results show that lactating rabbit and rat mammary epithelial cells express oxytocin receptors and that oxytocin binds to these receptors. They strongly suggest that oxytocin has a dual effect on lactating mammary tissue: an acceleration of the intracellular transfer of caseins in mammary epithelial cells followed by the contraction of myoepithelial cells. PMID:16166151

  13. Modulating zymogen granule formation in pancreatic AR42J cells.

    PubMed

    Rinn, Cornelia; Aroso, Miguel; Prüssing, Judith; Islinger, Markus; Schrader, Michael

    2012-09-10

    Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. To investigate ZG biogenesis, cargo sorting and packaging, suitable cellular model systems are required. Here, we demonstrate that granule formation in pancreatic AR42J cells, an acinar model system, can be modulated by altering the growth conditions in cell culture. We find that cultivation of AR42J cells in Panserin™ 401, a serum-free medium, enhances the induction of granule formation in the presence or absence of dexamethasone when compared to standard conditions including serum. Biochemical and morphological studies revealed an increase in ZG markers on the mRNA and protein level, as well as in granule size compared to standard conditions. Our data indicate that this effect is related to pronounced differentiation of AR42J cells. To address if enhanced expression of ZG proteins promotes granule formation, we expressed several zymogens and ZG membrane proteins in unstimulated AR42J cells and in constitutively secreting COS-7 cells. Neither single expression nor co-expression was sufficient to initiate granule formation in AR42J cells or the formation of granule-like structures in COS-7 cells as described for neuroendocrine cargo proteins. The importance of our findings for granule formation in exocrine cells is discussed. PMID:22683857

  14. Secretory and basal cells of the epithelium of the tubular glands in the male Mullerian gland of the caecilian Uraeotyphlus narayani (Amphibia: Gymnophiona).

    PubMed

    George, Jancy M; Smita, Matthew; Kadalmani, Balamuthu; Girija, Ramankutty; Oommen, Oommen V; Akbarsha, Mohammad A

    2004-12-01

    Caecilians are exceptional among the vertebrates in that males retain the Mullerian duct as a functional glandular structure. The Mullerian gland on each side is formed from a large number of tubular glands connecting to a central duct, which either connects to the urogenital duct or opens directly into the cloaca. The Mullerian gland is believed to secrete a substance to be added to the sperm during ejaculation. Thus, the Mullerian gland could function as a male accessory reproductive gland. Recently, we described the male Mullerian gland of Uraeotyphlus narayani using light and transmission electron microscopy (TEM) and histochemistry. The present TEM study reports that the secretory cells of both the tubular and basal portions of the tubular glands of the male Mullerian gland of this caecilian produce secretion granules in the same manner as do other glandular epithelial cells. The secretion granules are released in the form of structured granules into the lumen of the tubular glands, and such granules are traceable to the lumen of the central duct of the Mullerian gland. This is comparable to the situation prevailing in the epididymal epithelium of several reptiles. In the secretory cells of the basal portion of the tubular glands, mitochondria are intimately associated with fabrication of the secretion granules. The structural and functional organization of the epithelium of the basal portion of the tubular glands is complicated by the presence of basal cells. This study suggests the origin of the basal cells from peritubular tissue leukocytes. The study also indicates a role for the basal cells in acquiring secretion granules from the neighboring secretory cells and processing them into lipofuscin material in the context of regression of the Mullerian gland during the period of reproductive quiescence. In these respects the basal cells match those in the epithelial lining of the epididymis of amniotes. PMID:15487004

  15. The fine structural localization of acid phosphatase in the prolactin cell of the teleost pituitary following the stimulation and inhibition of secretory activity.

    PubMed

    Hopkins, C R

    1969-01-01

    Fine structural studies have been made upon the prolactin cells of Poecilia latipinna when the fish is subjected to a series of changes in its environmental salinity. Under the regimen employed it is shown that the secretory mechanism of the cell is initially stimulated in fresh water and then inhibited in salt water. The latter event is accompanied by the appearance of demonstrable acid phosphatase activity within the Golgi complex and by the formation of a variety of acid phosphatase-positive bodies. The way in which this acid hydrolase activity is concerned with the removal of surplus secretory granules is described and discussed. PMID:18631492

  16. The External Granule Layer of the Developing Chick Cerebellum Generates Granule Cells and Cells of the Isthmus and Rostral

    E-print Network

    Tabin, Cliff

    The External Granule Layer of the Developing Chick Cerebellum Generates Granule Cells and Cells a retroviral library to mark clones in the chick embryo at Hamberger­Hamilton stages 10­12. RRL clones; Alder et al., 1996). Likewise, retrovirally la- beled clones originating from the chick rostral rhombic

  17. Mitochondria distinguish granule-stored from de novo synthesized TNF secretion in human mast cells

    PubMed Central

    Zhang, Bodi; Weng, Zuyi; Sismanopoulos, Nikolaos; Asadi, Sharhzad; Therianou, Anastasia; Alysandratos, Konstantinos-Dionysios; Angelidou, Asimenia; Shirihai, Orian; Theoharides, Theoharis C.

    2013-01-01

    Background Mast cells are immune cells derived from hematopoietic precursors that mature in the tissue microenvironment. Mast cells are critical for allergic, immune and inflammatory processes, many of which involve TNF. Mast cells uniquely store TNF in their secretory granules. Upon stimulation, mast cells rapidly (30 min) secrete beta-hexosaminidase (beta-hex) and granule-stored TNF through degranulation, but also increase TNF mRNA and release de novo synthesized TNF 24 hr later. Regulation of these two distinct pathways is poorly understood. Methods human LAD2 leukemic mast cells are stimulated by substance P (SP). TNF secretion and gene expression were measured by ELISA and Real-Time PCR. Live cell mitochondrial dynamics was observed under Confocal Microscopy. Cell energy consumption were measured in term of oxygen consumption rate. Results Here we show that granule-stored TNF is preformed and its secretion from LAD2 mast cells stimulated by SP exhibits (a) higher energy consumption and is inhibited by the mitochondrial ATP pump blocker oligomycin, (b) rapid increase of intracellular calcium levels, and (c) reversible mitochondrial translocation, from a perinuclear distribution to the cell surface, as compared to de novo synthesized TNF release induced by lipopolysaccharide (LPS). This mitochondrial translocation is confirmed using primary human umbilical cord blood-derived mast cells (hCBMCs) stimulated by an allergic trigger (IgE/streptavidin). Conclusion These findings indicate that unique mitochondrial functions distinguish granule-stored from newly synthesized TNF release from human mast cells, thus permitting the versatile involvement of mast cells in different biological processes. PMID:22555146

  18. Approaches to imaging unfolded secretory protein stress in living cells

    PubMed Central

    Lajoie, Patrick; Fazio, Elena N.; Snapp, Erik L.

    2014-01-01

    The endoplasmic reticulum (ER) is the point of entry of proteins into the secretory pathway. Nascent peptides interact with the ER quality control machinery that ensures correct folding of the nascent proteins. Failure to properly fold proteins can lead to loss of protein function and cytotoxic aggregation of misfolded proteins that can lead to cell death. To cope with increases in the ER unfolded secretory protein burden, cells have evolved the Unfolded Protein Response (UPR). The UPR is the primary signaling pathway that monitors the state of the ER folding environment. When the unfolded protein burden overwhelms the capacity of the ER quality control machinery, a state termed ER stress, sensor proteins detect accumulation of misfolded peptides and trigger the UPR transcriptional response. The UPR, which is conserved from yeast to mammals, consists of an ensemble of complex signaling pathways that aims at adapting the ER to the new misfolded protein load. To determine how different factors impact the ER folding environment, various tools and assays have been developed. In this review, we discuss recent advances in live cell imaging reporters and model systems that enable researchers to monitor changes in the unfolded secretory protein burden and activation of the UPR and its associated signaling pathways. PMID:25419521

  19. Biochemical analysis of secretory trafficking in mammalian cells.

    PubMed

    Roboti, Peristera; Witkos, Tomasz M; Lowe, Martin

    2013-01-01

    Protein trafficking within the secretory pathway of mammalian cells is amenable to analysis by biochemical methods. This can be achieved by monitoring posttranslational modifications that occur naturally within the secretory pathway, or by measuring the delivery of cargo to the cell surface or extracellular medium. These approaches can be combined with additional manipulations such as specific temperature blocks that permit analysis of distinct trafficking steps. Biochemical analysis is advantageous in that it permits both a sensitive and quantitative measure of trafficking along the pathway. The methods discussed in this chapter permit the analysis of trafficking of both endogenous cargo proteins and ectopically expressed model cargos, which can be followed using either Western blotting or metabolic pulse-chase approaches. These methods are relatively straightforward and suitable for use in most modern cell biology laboratories. In addition to the well-established methods that we describe here in detail, we also refer to the development of more recent tailored approaches that add further to the arsenal of tools that can be used to assess trafficking in the secretory pathway. PMID:24295302

  20. How helminths use excretory secretory fractions to modulate dendritic cells

    PubMed Central

    White, Rhiannon R.; Artavanis-Tsakonas, Katerina

    2012-01-01

    It is well known that helminth parasites have immunomodulatory effects on their hosts. They characteristically cause a skew toward TH2 immunity, stimulate Treg cells while simultaneously inhibiting TH1 and TH17 responses. Additionally, they induce eosinophilia and extensive IgE release. The exact mechanism of how the worms achieve this effect have yet to be fully elucidated; however, parasite-derived secretions and their interaction with antigen presenting cells have been centrally implicated. Herein, we will review the effects of helminth excretory-secretory fractions on dendritic cells and discuss how this interaction is crucial in shaping the host response. PMID:23221477

  1. Reelin deficiency causes granule cell dispersion in epilepsy

    Microsoft Academic Search

    Carola A. Haas; Michael Frotscher

    2010-01-01

    Cortical migration defects are often associated with epilepsy. In mesial temporal lobe epilepsy (MTLE), granule cell dispersion\\u000a (GCD), a migration defect of dentate granule cells, is frequently observed. Little is known how GCD develops and to which\\u000a extent it contributes to the development of seizure activity. Since the reelin-deficient reeler mouse mutant shows a similar migration defect of dentate cells,

  2. Exocytosis in secretory cells of rat lacrimal gland. Peroxidase release from lobules and isolated cells upon cholinergic stimulation.

    PubMed

    Herzog, V; Sies, H; Miller, F

    1976-09-01

    Release of peroxidase from secretory cells of rat lacrimal gland upon cholinergic stimulation was studied in vitro with single lobules and isolated cells (lacrimocytes). Isolated lobules, kept in Eagle's medium, remain structurally intact and reaction product of peroxidase is confined to cisternae of rough endoplasmic reticulum, elements of the Golgi apparatus, and all secretory granules. Morphologically, exocytosis occurs by membrane fusion and discharge of granule content. The highest rate of peroxidase released from lobules is observed at 10(-4) M carbamylcholine. The specific activity of peroxidase released into the medium is fourfold higher as compared to the lobules. Release of peroxidase is suppressed by atropine when added before or after the addition of carbamylcholine. At 4 degrees C, no peroxidase release occurs upon cholinergic stimulation. The exocytotic release of peroxidase is dependent on energy supply, as indicated by substantial inhibition (at 37 degrees C) under anoxic conditions or in the presence of dinitrophenol, KCN, or carboxyatractyloside. Furthermore, the process is sensitive to colchicine and vinblastine. Isolated lacrimocytes, consiting of 95% secretory acinar cells, are prepared by digestion with collagenase, hyaluronidase, and trypsin. They retain the characteristic polarity of secretory cells in situ, and localization of peroxidase is the same as in lobules. Since isolated lacrimocytes respond to cholinergic stimulation in the same way as lobules, the receptors are not damaged by the isolation procedure and appear to be associated directly with the exocrine cell. Oxygen uptake by isolated lacrimocytes is about 14 nmol O2 X min-1 X 10(-6) cells; it is about doubled by uncoupling with dinitrophenol. Oxygen uptake rises by 20-30% above the resting rate upon cholinergic stimulation. This additional uptake is suppressed by atropine or by added cholinesterase, indicating that continuous receptor occupancy may be required for the energy demand by exocytosis. On the basis of the specific activity of peroxidase in the medium, the energy demand resulting from cholinergic stimulation is estimated to be 0.08 mumol ATP (or energy-rich phosphate bonds) per microgram of protein released from the lacrimocytes. PMID:956271

  3. Exocytosis in secretory cells of rat lacrimal gland. Peroxidase release from lobules and isolated cells upon cholinergic stimulation

    PubMed Central

    1976-01-01

    Release of peroxidase from secretory cells of rat lacrimal gland upon cholinergic stimulation was studied in vitro with single lobules and isolated cells (lacrimocytes). Isolated lobules, kept in Eagle's medium, remain structurally intact and reaction product of peroxidase is confined to cisternae of rough endoplasmic reticulum, elements of the Golgi apparatus, and all secretory granules. Morphologically, exocytosis occurs by membrane fusion and discharge of granule content. The highest rate of peroxidase released from lobules is observed at 10(- 4) M carbamylcholine. The specific activity of peroxidase released into the medium is fourfold higher as compared to the lobules. Release of peroxidase is suppressed by atropine when added before or after the addition of carbamylcholine. At 4 degrees C, no peroxidase release occurs upon cholinergic stimulation. The exocytotic release of peroxidase is dependent on energy supply, as indicated by substantial inhibition (at 37 degrees C) under anoxic conditions or in the presence of dinitrophenol, KCN, or carboxyatractyloside. Furthermore, the process is sensitive to colchicine and vinblastine. Isolated lacrimocytes, consiting of 95% secretory acinar cells, are prepared by digestion with collagenase, hyaluronidase, and trypsin. They retain the characteristic polarity of secretory cells in situ, and localization of peroxidase is the same as in lobules. Since isolated lacrimocytes respond to cholinergic stimulation in the same way as lobules, the receptors are not damaged by the isolation procedure and appear to be associated directly with the exocrine cell. Oxygen uptake by isolated lacrimocytes is about 14 nmol O2 X min-1 X 10(-6) cells; it is about doubled by uncoupling with dinitrophenol. Oxygen uptake rises by 20-30% above the resting rate upon cholinergic stimulation. This additional uptake is suppressed by atropine or by added cholinesterase, indicating that continuous receptor occupancy may be required for the energy demand by exocytosis. On the basis of the specific activity of peroxidase in the medium, the energy demand resulting from cholinergic stimulation is estimated to be 0.08 mumol ATP (or energy-rich phosphate bonds) per microgram of protein released from the lacrimocytes. PMID:956271

  4. Densely Granulated Murine NK Cells Eradicate Large Solid Tumors

    PubMed Central

    Liu, Rebecca B.; Engels, Boris; Arina, Ainhoa; Schreiber, Karin; Hyjek, Elizabeth; Schietinger, Andrea; Binder, David C.; Butz, Eric; Krausz, Thomas; Rowley, Donald A.; Jabri, Bana; Schreiber, Hans

    2013-01-01

    NK cells inhibit early stages of tumor formation, recurrence and metastasis. Here we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to IL15 released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphological feature of being densely loaded with PAS-positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL15 receptor ? on cancer cells was needed to efficiently induce granulated NK cells and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors. PMID:22374983

  5. UNUSUAL EOSINOPHILIC GRANULE CELL PROLIFERATION IN COHO SALMON (ONCHORHYNCHUS KISUTCH)

    EPA Science Inventory

    Proliferative lesions comprised of eosinophilic granule cells (EGCs) extended throughout the gastrointestinal tract of several mature, spawning coho salmon Oncorhynchus kisutch (Walbaum). istological examination of the tumour showed extensive proliferation and infiltration of EGC...

  6. Isolating stromal stem cells from periodontal granulation tissues

    Microsoft Academic Search

    Tzu-Yuan Hung; Hsiang-Chun Lin; Ying-Jen Chan; Kuo Yuan

    Stem cell therapy is a promising area in regenerative medicine. Periodontal granulation tissues are often discarded during\\u000a conventional surgery. If stromal stem cells can be isolated from these tissues, they can be used for subsequent surgery on\\u000a the same patient. Fifteen human periodontal granulation tissue samples were obtained from intrabony defects during surgery.\\u000a Immunohistochemistry (IHC) was carried out on five

  7. UVC-Induced Stress Granules in Mammalian Cells

    PubMed Central

    Moutaoufik, Mohamed Taha; El Fatimy, Rachid; Nassour, Hassan; Gareau, Cristina; Lang, Jérôme; Tanguay, Robert M.; Mazroui, Rachid; Khandjian, Edouard W.

    2014-01-01

    Stress granules (SGs) are well characterized cytoplasmic RNA bodies that form under various stress conditions. We have observed that exposure of mammalian cells in culture to low doses of UVC induces the formation of discrete cytoplasmic RNA granules that were detected by immunofluorescence staining using antibodies to RNA-binding proteins. UVC-induced cytoplasmic granules are not Processing Bodies (P-bodies) and are bone fide SGs as they contain TIA-1, TIA-1/R, Caprin1, FMRP, G3BP1, PABP1, well known markers, and mRNA. Concomitant with the accumulation of the granules in the cytoplasm, cells enter a quiescent state, as they are arrested in G1 phase of the cell cycle in order to repair DNA damages induced by UVC irradiation. This blockage persists as long as the granules are present. A tight correlation between their decay and re-entry into S-phase was observed. However the kinetics of their formation, their low number per cell, their absence of fusion into larger granules, their persistence over 48 hours and their slow decay, all differ from classical SGs induced by arsenite or heat treatment. The induction of these SGs does not correlate with major translation inhibition nor with phosphorylation of the ? subunit of eukaryotic translation initiation factor 2 (eIF2?). We propose that a restricted subset of mRNAs coding for proteins implicated in cell cycling are removed from the translational apparatus and are sequestered in a repressed form in SGs. PMID:25409157

  8. Granule lattice protein 1 (Grl1p), an acidic, calcium-binding protein in Tetrahymena thermophila dense-core secretory granules, influences granule size, shape, content organization, and release but not protein sorting or condensation

    Microsoft Academic Search

    N. Doane Chilcoat; Sharon M. Melia; Alex Haddad; Aaron P. Turkewitz

    1996-01-01

    The electron-dense cores of regulated secre- tory granules in the ciliate Tetrahymena thermophila are crystal lattices composed of multiple proteins. Granule synthesis involves a series of steps beginning with protein sorting, followed by the condensation and precise geometric assembly of the granule cargo. These steps may to various degrees be determined by the cargo proteins themselves. A prominent group of

  9. Event-driven simulation of cerebellar granule cells.

    PubMed

    Carrillo, Richard R; Ros, Eduardo; Tolu, Silvia; Nieus, Thierry; D'Angelo, Egidio

    2008-01-01

    Around half of the neurons of a human brain are granule cells (approximately 10(11)granule neurons) [Kandel, E.R., Schwartz, J.H., Jessell, T.M., 2000. Principles of Neural Science. McGraw-Hill Professional Publishing, New York]. In order to study in detail the functional role of the intrinsic features of this cell we have developed a pre-compiled behavioural model based on the simplified granule-cell model of Bezzi et al. [Bezzi, M., Nieus, T., Arleo, A., D'Angelo, E., Coenen, O.J.-M.D., 2004. Information transfer at the mossy fiber-granule cell synapse of the cerebellum. 34th Annual Meeting. Society for Neuroscience, San Diego, CA, USA]. We can use an efficient event-driven simulation scheme based on lookup tables (EDLUT) [Ros, E., Carrillo, R.R., Ortigosa, E.M., Barbour, B., Ags, R., 2006. Event-driven simulation scheme for spiking neural networks using lookup tables to characterize neuronal dynamics. Neural Computation 18 (12), 2959-2993]. For this purpose it is necessary to compile into tables the data obtained through a massive numerical calculation of the simplified cell model. This allows network simulations requiring minimal numerical calculation. There are three major features that are considered functionally relevant in the simplified granule cell model: bursting, subthreshold oscillations and resonance. In this work we describe how the cell model is compiled into tables keeping these key properties of the neuron model. PMID:18616981

  10. Assessing the secretory capacity of pancreatic acinar cells.

    PubMed

    Geron, Erez; Schejter, Eyal D; Shilo, Ben-Zion

    2014-01-01

    Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex vivo culture will represent the in vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion. PMID:25226212

  11. Light microscopic characterization of glycoconjugates in secretory cells of the carp ( Cyprinus carpio ) gill epithelium

    Microsoft Academic Search

    J. Hidalgo; A. Velasco; I. Sánchez Aguayo; P. Amores

    1987-01-01

    Secretory products of granular and mucous cells in the gill epithelium of the carp, Cyprinus carpio, were distinguished by their cytochemical reactions with peroxidase-labelled lectins and with the galactose oxidase (GO)-Schiff reagents. Secretory products of granular cells reacted with lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Dolichos biflorus (DBA), Glycine max (SAB), and Lotus tetragonolobus (LTA). They also reacted

  12. Acid-induced secretory cell metaplasia in hamster bronchi

    SciTech Connect

    Christensen, T.G.; Lucey, E.C.; Breuer, R.; Snider, G.L.

    1988-02-01

    Hamsters were exposed to an intratracheal instillation of 0.5 ml of 0.08 N nitric, hydrochloric, or sulfuric acid to determine their airway epithelial response. Three weeks after exposure, the left intrapulmonary bronchi in Alcian blue/PAS-strained paraffin sections were evaluated for the amount of secretory product in the airway epithelium as a measure of secretory cell metaplasia (SCM). Compared to saline-treated control animals, all three acids caused statistically significant SCM. In addition to the bronchial lesion, all three acids caused similar interstitial fibrosis, bronchiolectasis, and bronchiolization of alveoli that varied in individual animals from mild to severe. In a separate experiment to study the persistence of the SCM, hamsters treated with a single instillation of 0.1 N nitric acid showed significant SCM 3, 7, and 17 weeks after exposure. There was a high correlation (r = 0.96) between a subjective assessment of SCM and objective assessment using a digital image-analysis system. We conclude that protons induce SCM independently of the associated anion; the SCM persists at least 17 weeks. Sulfuric acid is an atmospheric pollutant and nitric acid may form locally on the mucosa of lungs exposed to nitrogen dioxide. These acids may contribute to the development of maintenance of the SCM seen in the conducting airways of humans with chronic obstructive pulmonary disease.

  13. Secretogranins I and I1: Two Tyrosine-Sulfated Secretory Proteins Common to a Variety of Cells Secreting Peptides by the Regulated Pathway

    E-print Network

    Patrizla Rosa; Annette Hille; Raymond W. H. Lee; Antonia Zanini; Pietro De Camilli; Wieland B. Huttner

    ABSTRACT We report on the biochemical and immunological properties as well as on the cellular and subcellular distribution of two proteins, called secretogranins I and II. These proteins specifically occur in a wide variety of endocrine and neuronal cells that package and sort regulatory peptides into secretory granules. Both secretogranins take the same intracellular route as the peptides and are also sorted into secretory granules. Secretogranins I and II are biochemically and immunologically distinct proteins and differ from chromogranin A. Yet, these three proteins are similar to each other in many respects and therefore constitute one class of proteins. A remarkable feature of this protein class is a very acidic pl, brought about by a high content of acidic amino acids as well as by phosphorylation on serine and sulfation on tyrosine and O-linked carbohydrate. As a result, this class of proteins has a high net negative charge even at the acidic pH of the trans Golgi cisternae. We discuss the possibility that this property of the proteins may point to a role in the packaging of regulatory peptides into secretory granules. The intracellular process by which peptide hormones and neuropeptides are secreted has been intensively studied, and

  14. Sparse incomplete representations: a potential role of olfactory granule cells.

    PubMed

    Koulakov, Alexei A; Rinberg, Dmitry

    2011-10-01

    Mitral/tufted cells of the olfactory bulb receive odorant information from receptor neurons and transmit this information to the cortex. Studies in awake behaving animals have found that sustained responses of mitral cells to odorants are rare, suggesting sparse combinatorial representation of the odorants. Careful alignment of mitral cell firing with the phase of the respiration cycle revealed brief transient activity in the larger population of mitral cells, which respond to odorants during a small fraction of the respiration cycle. Responses of these cells are therefore temporally sparse. Here, we propose a mathematical model for the olfactory bulb network that can reproduce both combinatorially and temporally sparse mitral cell codes. We argue that sparse codes emerge as a result of the balance between mitral cells' excitatory inputs and inhibition provided by the granule cells. Our model suggests functional significance for the dendrodendritic synapses mediating interactions between mitral and granule cells. PMID:21982374

  15. Sparse incomplete representations: a potential role of olfactory granule cells

    PubMed Central

    Koulakov, Alexei A.; Rinberg, Dmitry

    2011-01-01

    Mitral/tufted cells of the olfactory bulb receive odorant information from receptor neurons and transmit this information to the cortex. Studies in awake behaving animals have found that sustained responses of mitral cells to odorants are rare, suggesting sparse combinatorial representation of the odorants. Careful alignment of mitral cell firing with the phase of the respiration cycle revealed brief transient activity in the larger population of mitral cells, which respond to odorants during a small fraction of the respiration cycle. Responses of these cells are therefore temporally sparse. Here, we propose a mathematical model for the olfactory bulb network that can reproduce both combinatorially and temporally sparse mitral cell codes. We argue that sparse codes emerge as a result of the balance between mitral cells' excitatory inputs and inhibition provided by the granule cells. Our model suggests functional significance for the dendrodendritic synapses mediating interactions between mitral and granule cells. PMID:21982374

  16. Influence of external calcium ions on labelled calcium efflux from pancreatic beta-cells and insulin granules in mice.

    PubMed Central

    Abrahamsson, H; Gylfe, E; Hellman, B

    1981-01-01

    Addition of Ca2+ to a glucose-free perifusion medium stimulated the efflux of 45Ca from prelabelled pancreatic islets isolated from ob/ob-mice. This effect differed from that of glucose in being transient, markedly stimulated by previous exposure to a Ca2+-deficient medium and resulting in mobilization also of substantial amounts of 45Ca incorporated in the absence of glucose. The glucose action on 45Ca efflux reflected the balance between inhibitory and stimulatory components which differed with respect to their chronological order and sensitivity to glucose. The magnitude of the stimulatory phase was related linearly to the extracellular concentration of Ca2+ up to 2.40 mM. The efflux of 45Ca from isolated secretory granules was stimulated by Mg-ATP. The latter made the 45Ca efflux from the granules sensitive to Ca2+; significant stimulation being seen when increasing the medium Ca2+ from 0.1 to 10 microM. The results support the concept of an efficient Ca/Ca exchange mechanism in the depolarized beta-cells, emphasizing a role for the secretory granules in this process. PMID:7021800

  17. Mast Cell Mediators: Their Differential Release and the Secretory Pathways Involved

    PubMed Central

    Moon, Tae Chul; Befus, A. Dean; Kulka, Marianna

    2014-01-01

    Mast cells (MC) are widely distributed throughout the body and are common at mucosal surfaces, a major host–environment interface. MC are functionally and phenotypically heterogeneous depending on the microenvironment in which they mature. Although MC have been classically viewed as effector cells of IgE-mediated allergic diseases, they are also recognized as important in host defense, innate and acquired immunity, homeostatic responses, and immunoregulation. MC activation can induce release of pre-formed mediators such as histamine from their granules, as well as release of de novo synthesized lipid mediators, cytokines, and chemokines that play diverse roles, not only in allergic reactions but also in numerous physiological and pathophysiological responses. Indeed, MC release their mediators in a discriminating and chronological manner, depending upon the stimuli involved and their signaling cascades (e.g., IgE-mediated or Toll-like receptor-mediated). However, the precise mechanisms underlying differential mediator release in response to these stimuli are poorly known. This review summarizes our knowledge of MC mediators and will focus on what is known about the discriminatory release of these mediators dependent upon diverse stimuli, MC phenotypes, and species of origin, as well as on the intracellular synthesis, storage, and secretory processes involved. PMID:25452755

  18. Minireview in press The Cerebellum On the Induction of Postsynaptic Granule Cell Purkinje Neuron

    E-print Network

    Paris-Sud XI, Université de

    1 Minireview in press The Cerebellum On the Induction of Postsynaptic Granule Cell Purkinje Neuron of erroneously activated cerebellar granule cell (CGN) to Purkinje neuron (PN) synapses is the most important

  19. Expression of the secretory leukoprotease inhibitor gene in epithelial cells.

    PubMed Central

    Abe, T; Kobayashi, N; Yoshimura, K; Trapnell, B C; Kim, H; Hubbard, R C; Brewer, M T; Thompson, R C; Crystal, R G

    1991-01-01

    The secretory leukoprotease inhibitor (SLPI) gene codes for a 12-kD protein that within the lung protects the airway epithelium from neutrophil elastase. Screening of 228 alleles in 114 individuals for sequence differences by RNase protection of genomic DNA revealed no detectable polymorphisms in SLPI gene exons II-IV. SLPI gene expression in the lung was demonstrated by identifying SLPI mRNA transcripts in bronchial epithelial cells freshly isolated from normals. Cell lines derived from mucosal surfaces (HS-24 bronchial squamous cell carcinoma, HeLa cervical carcinoma) actively transcribe the SLPI gene and contain SLPI mRNA transcripts, while lung fibroblasts demonstrate no evidence of SLPI gene expression. SLPI mRNA transcripts appear to be relatively stable, with mRNA levels only mildly affected by inhibition of RNA synthesis. Chromatin DNA of HS-24 cells demonstrates two DNase I hypersensitivity sites within the 5' flanking region of exon I of the SLPI gene, whereas fibroblast chromatin has no DNase I accessible sites in the same region. Further analysis of the 5' flanking region demonstrated two contiguous transcription start sites, CAAT and TATA boxes, and several potential regions of known DNA binding proteins. Overall, the SLPI gene appears to be a relatively nonpolymorphic, stable gene that is constitutively expressed at specific tissue sites, but has the potential to be modulated at both the transcriptional and posttranscriptional levels. Images PMID:1674946

  20. Dynamic functions of GABA signaling during granule cell maturation

    PubMed Central

    Dieni, Cristina V.; Chancey, Jessica H.; Overstreet-Wadiche, Linda S.

    2013-01-01

    The dentate gyrus is one of the few areas of the brain where new neurons are generated throughout life. Neural activity influences multiple stages of neurogenesis, thereby allowing experience to regulate the production of new neurons. It is now well established that GABAA receptor-mediated signaling plays a pivotal role in mediating activity-dependent regulation of adult neurogenesis. GABA first acts as a trophic signal that depolarizes progenitors and early post mitotic granule cells, enabling network activity to control molecular cascades essential for proliferation, survival and growth. Following the development of glutamatergic synaptic inputs, GABA signaling switches from excitatory to inhibitory. Thereafter robust synaptic inhibition enforces low spiking probability of granule cells in response to cortical excitatory inputs and maintains the sparse activity patterns characteristic of this brain region. Here we review these dynamic functions of GABA across granule cell maturation, focusing on the potential role of specific interneuron circuits at progressive developmental stages. We further highlight questions that remain unanswered about GABA signaling in granule cell development and excitability. PMID:23316139

  1. Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of reserpine treated guinea pigs

    Microsoft Academic Search

    Inderbir Singh

    1967-01-01

    The individual granules of enterochromaffin cells of normal and reserpine treated guinea pigs have been studied by staining slides of the duodenum first by an argentaffin method and subsequently by an argyrophile method. Some argentaffin cells can be shown to contain not only argentaffin granules, but also granules that are purely argyrophile. The relative number of such argentaffin cells is

  2. THE JOURNAL OF COMPARATIVE NEUROLOGY 369~345-360 ( 1996) Ultrastructural Study of the Granule Cell

    E-print Network

    Ryugo, David K.

    cortex. The granule cell domain also receives projections from the cuneate and trigeminal nuclei, which inputs. The granule cell areas receive projections from neurons in higher auditory nuclei, includingTHE JOURNAL OF COMPARATIVE NEUROLOGY 369~345-360 ( 1996) Ultrastructural Study of the Granule Cell

  3. Protein 7B2 is essential for the targeting and activation of PC2 into the regulated secretory pathway of rMTC 6-23 cells.

    PubMed

    Barbero, P; Kitabgi, P

    1999-04-13

    Among the prohormone convertases, PC2 is unique in that it specifically binds to the neuroendocrine-specific protein 7B2 in the endoplasmic reticulum (ER) and is activated late in the regulated secretory pathway of neuroendocrine cells. Several roles, sometimes contradictory, have been suggested for 7B2 with regard to PC2 cellular fate. Thus, 7B2 was proposed to act as a PC2 chaperone in the ER, or to facilitate 7B2 transport from the ER to the trans-Golgi network and to be necessary for proPC2 activation, or to inhibit PC2 enzymatic activity until the latter reaches the secretory granules. To gain insight into the function of 7B2, we sought to block its expression in PC2-expressing endocrine cells using antisense strategies. We have previously shown that the endocrine rMTC 6-23 cell line expresses PC2 and that the enzyme is responsible for the processing of pro-neurotensin/neuromedin N (proNT/NN). Here, we show that rMTC 6-23 cells express 7B2 and that the protein was coordinately induced with PC2 and proNT/NN by dexamethasone. Stable transfection of rMTC 6-23 cells with 7B2 antisense cDNA led to a marked reduction (>90%) in 7B2 levels. ProPC2 was expressed to normal levels and cleaved to yield a PC2 form that was constitutively released, was not stored within secretory granules and was unable to process proNT/NN. We conclude that 7B2 is essential for the sorting and activation of PC2 into the regulated secretory pathway of endocrine cells. PMID:10198237

  4. L E T T E R S secretory progenitor cells revert to stem cells

    E-print Network

    van Oudenaarden, Alexander

    L E T T E R S Dll1+ secretory progenitor cells revert to stem cells upon crypt damage Johan H. van,6 , Nick Barker3 , Alexander van Oudenaarden2 and Hans Clevers1,8 Lgr5+ intestinal stem cells generate is expressed by a subset of immediate stem cell daughters. Lineage tracing in Dll1GFP­ires­CreERT2 knock

  5. Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathways.

    PubMed

    Chanat, E; Weiss, U; Huttner, W B; Tooze, S A

    1993-05-01

    The role of the single, highly conserved disulfide bond in chromogranin B (secretogranin I) on the sorting of this regulated secretory protein to secretory granules was investigated in the neuroendocrine cell line PC12. Treatment of PC12 cells with dithiothreitol (DTT), a membrane permeable thiol reducing agent known to prevent disulfide bond formation in intact cells, resulted in the secretion of newly synthesized chromogranin B, but only slightly decreased the intracellular storage of newly synthesized secretogranin II, a regulated secretory protein devoid of cysteines. The secretion of newly synthesized chromogranin B in the presence of DTT occurred with similar kinetics to those of a heparan sulfate proteoglycan, a known marker of the constitutive secretory pathway in PC12 cells. Analysis of the various secretory vesicles derived from the trans-Golgi network (TGN) indicated that DTT treatment diverted newly synthesized chromogranin B to constitutive secretory vesicles, whereas the packaging of secretogranin II into immature secretory granules was unaffected by the reducing agent. The chromogranin B molecules diverted to constitutive secretory vesicles, in contrast to those stored in secretory granules, were found to contain free sulfhydryl residues. The effect of DTT on chromogranin B occurred in the TGN rather than in the endoplasmic reticulum. We conclude that the sorting of CgB in the TGN to secretory granules is dependent upon the integrity of its single disulfide bond. PMID:8491204

  6. Neuroligin-1 Overexpression in Newborn Granule Cells In Vivo

    PubMed Central

    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. PMID:23110172

  7. Newborn granule cells in the ageing dentate gyrus

    PubMed Central

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

    2008-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-05-01

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

  9. Modulation of dendritic cell function by Trichomonas vaginalis-derived secretory products

    PubMed Central

    Song, Min-Ji; Lee, Jong-Joo; Nam, Young Hee; Kim, Tae-Gyun; Chung, Youn Wook; Kim, Mikyoung; Choi, Ye-Eun; Shin, Myeong Heon; Kim, Hyoung-Pyo

    2015-01-01

    Trichomoniasis caused by the parasitic protozoan Trichomonas vaginalis is the most common sexually transmitted disease in the world. Dendritic cells are antigen presenting cells that initiate immune responses by directing the activation and differentiation of naďve T cells. In this study, we analyzed the effect of Trichomonas vaginalis-derived Secretory Products on the differentiation and function of dendritic cells. Differentiation of bone marrow-derived dendritic cells in the presence of T. vaginalis-derived Secretory Products resulted in inhibition of lipopolysaccharide-induced maturation of dendritic cells, down-regulation of IL-12, and up-regulation of IL-10. The protein components of T. vaginalis-derived Secretory Products were shown to be responsible for altered function of bone marrow-derived dendritic cells. Chromatin immunoprecipitation assay demonstrated that IL-12 expression was regulated at the chromatin level in T. vaginalis-derived Secretory Productstreated dendritic cells. Our results demonstrated that T. vaginalis-derived Secretory Products modulate the maturation and cytokine production of dendritic cells leading to immune tolerance. [BMB Reports 2015; 48(2): 103-108] PMID:24965578

  10. Estradiol increases cAMP in the oviductal secretory cells through a nongenomic mechanism.

    PubMed

    Oróstica, María L; Lopez, John; Rojas, Israel; Rocco, Jocelyn; Díaz, Patricia; Reuquén, Patricia; Cardenas, Hugo; Parada-Bustamante, Alexis; Orihuela, Pedro A

    2014-09-01

    In the rat oviduct, estradiol (E2) accelerates egg transport by a nongenomic action that requires previous conversion of E2 to methoxyestrogens via catechol-O-methyltranferase (COMT) and activation of estrogen receptor (ER) with subsequent production of cAMP and inositol triphosphate (IP3). However, the role of the different oviductal cellular phenotypes on this E2 nongenomic pathway remains undetermined. The aim of this study was to investigate the effect of E2 on the levels of cAMP and IP3 in primary cultures of secretory and smooth muscle cells from rat oviducts and determine the mechanism by which E2 increases cAMP in the secretory cells. In the secretory cells, E2 increased cAMP but not IP3, while in the smooth muscle cells E2 decreased cAMP and increased IP3. Suppression of protein synthesis by actinomycin D did not prevent the E2-induced cAMP increase, but this was blocked by the ER antagonist ICI 182?780 and the inhibitors of COMT OR 486, G protein-? inhibitory (G?i) protein pertussis toxin and adenylyl cyclase (AC) SQ 22536. Expression of the mRNA for the enzymes that metabolizes estrogens, Comt, Cyp1a1, and Cyp1b1 was found in the secretory cells, but this was not affected by E2. Finally, confocal immunofluorescence analysis showed that E2 induced colocalization between ESR1 (ER?) and G?i in extranuclear regions of the secretory cells. We conclude that E2 differentially regulates cAMP and IP3 in the secretory and smooth muscle cells of the rat oviduct. In the secretory cells, E2 increases cAMP via a nongenomic action that requires activation of COMT and ER, coupling between ESR1 and G?i, and stimulation of AC. PMID:25038866

  11. Discovery and progress in our understanding of the regulated secretory pathway in neuroendocrine cells

    PubMed Central

    Morvan, Joëlle

    2008-01-01

    In this review we start with a historical perspective beginning with the early morphological work done almost 50 years ago. The importance of these pioneering studies is underscored by our brief summary of the key questions addressed by subsequent research into the mechanism of secretion. We then highlight important advances in our understanding of the formation and maturation of neuroendocrine secretory granules, first using in vitro reconstitution systems, then most recently biochemical approaches, and finally genetic manipulations in vitro and in vivo. PMID:18197413

  12. An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse.

    PubMed

    Bertrand, Florie; Müller, Sabina; Roh, Kyung-Ho; Laurent, Camille; Dupré, Loďc; Valitutti, Salvatore

    2013-04-01

    It is presently assumed that lethal hit delivery by cytotoxic T lymphocytes (CTLs) is mechanistically linked to centrosome polarization toward target cells, leading to dedicated release of lytic granules within a confined secretory domain. Here we provide three lines of evidence showing that this mechanism might not apply as a general paradigm for lethal hit delivery. First, in CTLs stimulated with immobilized peptide-MHC complexes, lytic granules and microtubule organizing center localization into synaptic areas are spatio-temporally dissociated, as detected by total internal reflection fluorescence microscopy. Second, in many CTL/target cell conjugates, lytic granule secretion precedes microtubule polarization and can be detected during the first minute after cell-cell contact. Third, inhibition of microtubule organizing center and centrosome polarization impairs neither lytic granule release at the CTL synapse nor killing efficiency. Our results broaden current views of CTL biology by revealing an extremely rapid step of lytic granule secretion and by showing that microtubule organizing center polarization is dispensable for efficient lethal hit delivery. PMID:23536289

  13. An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

    PubMed Central

    Bertrand, Florie; Müller, Sabina; Roh, Kyung-Ho; Laurent, Camille; Dupré, Loďc; Valitutti, Salvatore

    2013-01-01

    It is presently assumed that lethal hit delivery by cytotoxic T lymphocytes (CTLs) is mechanistically linked to centrosome polarization toward target cells, leading to dedicated release of lytic granules within a confined secretory domain. Here we provide three lines of evidence showing that this mechanism might not apply as a general paradigm for lethal hit delivery. First, in CTLs stimulated with immobilized peptide–MHC complexes, lytic granules and microtubule organizing center localization into synaptic areas are spatio-temporally dissociated, as detected by total internal reflection fluorescence microscopy. Second, in many CTL/target cell conjugates, lytic granule secretion precedes microtubule polarization and can be detected during the first minute after cell–cell contact. Third, inhibition of microtubule organizing center and centrosome polarization impairs neither lytic granule release at the CTL synapse nor killing efficiency. Our results broaden current views of CTL biology by revealing an extremely rapid step of lytic granule secretion and by showing that microtubule organizing center polarization is dispensable for efficient lethal hit delivery. PMID:23536289

  14. Proteomics of Dense Core Secretory Vesicles Reveal Distinct Protein Categories for Secretion of Neuroeffectors for Cell-Cell Communication

    PubMed Central

    Wegrzyn, Jill L.; Bark, Steven J.; Funkelstein, Lydiane; Mosier, Charles; Yap, Angel; Kazemi-Esfarjani, Parasa; La Spada, Albert; Sigurdson, Christina; O’Connor, Daniel T.; Hook, Vivian

    2010-01-01

    Regulated secretion of neurotransmitters and neurohumoural factors from dense core secretory vesicles provides essential neuroeffectors for cell-cell communication in the nervous and endocrine systems. This study provides comprehensive proteomic characterization of the categories of proteins in chromaffin dense core secretory vesicles that participate in cell-cell communication from the adrenal medulla. Proteomic studies were conducted by nano-HPLC Chip MS/MS tandem mass spectrometry. Results demonstrate that these secretory vesicles contain proteins of distinct functional categories consisting of neuropeptides and neurohumoural factors, protease systems, neurotransmitter enzymes and transporters, receptors, enzymes for biochemical processes, reduction/oxidation regulation, ATPases, protein folding, lipid biochemistry, signal transduction, exocytosis, calcium regulation, as well as structural and cell adhesion proteins. The secretory vesicle proteomic data identified 371 distinct proteins in the soluble fraction and 384 distinct membrane proteins, for a total of 686 distinct secretory vesicle proteins. Notably, these proteomic analyses illustrate the presence of several neurological disease-related proteins in these secretory vesicles, including huntingtin interacting protein, cystatin C, ataxin 7, and prion protein. Overall, these findings demonstrate that multiple protein categories participate in dense core secretory vesicles for production, storage, and secretion of bioactive neuroeffectors for cell-cell communication in health and disease. PMID:20695487

  15. [Quantitative determination of cell composition of human granulation tissue by fluorescence activated cell sorting (FAC)].

    PubMed

    Koschnick, M; Rösken, F; Keller, J; Busser, F; Hanselmann, R; Koch, B; Wirbel, R; Mutschler, W

    1998-01-01

    Granulation tissue of normal and non-healing human wounds showed a similar distribution pattern of the cell populations investigated by FACS analysis, whereas only non-healing wounds revealed a reduced density of cells and intercellular matrix. Thus, supporting the thesis that impaired wound healing is not caused by changes of the cellular distribution pattern of granulation tissue, but possibly by lessened cell function. PMID:14518209

  16. Acute ethanol exposure inhibits silencing of cerebellar Golgi cell firing induced by granule cell axon input.

    PubMed

    Botta, Paolo; Zucca, Aya; Valenzuela, C Fernando

    2014-01-01

    Golgi cells (GoCs) are specialized interneurons that provide inhibitory input to granule cells in the cerebellar cortex. GoCs are pacemaker neurons that spontaneously fire action potentials, triggering spontaneous inhibitory postsynaptic currents in granule cells and also contributing to the generation tonic GABAA receptor-mediated currents in granule cells. In turn, granule cell axons provide feedback glutamatergic input to GoCs. It has been shown that high frequency stimulation of granule cell axons induces a transient pause in GoC firing in a type 2-metabotropic glutamate receptor (mGluR2)-dependent manner. Here, we investigated the effect ethanol on the pause of GoC firing induced by high frequency stimulation of granule cell axons. GoC electrophysiological recordings were performed in parasagittal cerebellar vermis slices from postnatal day 23 to 26 rats. Loose-patch cell-attached recordings revealed that ethanol (40 mM) reversibly decreases the pause duration. An antagonist of mGluR2 reduced the pause duration but did not affect the effect of ethanol. Whole-cell voltage-clamp recordings showed that currents evoked by an mGluR2 agonist were not significantly affected by ethanol. Perforated-patch experiments in which hyperpolarizing and depolarizing currents were injected into GoCs demonstrated that there is an inverse relationship between spontaneous firing and pause duration. Slight inhibition of the Na(+)/K(+) pump mimicked the effect of ethanol on pause duration. In conclusion, ethanol reduces the granule cell axon-mediated feedback mechanism by reducing the input responsiveness of GoCs. This would result in a transient increase of GABAA receptor-mediated inhibition of granule cells, limiting information flow at the input stage of the cerebellar cortex. PMID:24567705

  17. Automated insulin granule segmentation from electron photomicrographs of rat pancreatic ?-cells

    NASA Astrophysics Data System (ADS)

    McClanahan, Timothy P.; Straub, Susanne G.; Sharp, Geoffrey W. G.; Loew, Murray

    2005-04-01

    Increased blood glucose stimulates pancreatic ?-cells and induces an exocytotic release of insulin. The ?-cell, which contains ~10^4 insulin-containing granules, releases only a few percent of the granules during a given stimulus such as a meal. The temporal response function to a square wave increase in the concentration of glucose is characteristically biphasic. It is not known whether the granules exhibit random or directed migration patterns as a function of phase. Directed migration would suggest the development of an intracellular gradient directing the path and velocity of insulin granule movement. Our ongoing research investigates this process using manual morphometric analysis of electron micrographs of rat pancreatic ?-cells. This is a tedious and time-consuming stereological process. Consequently, we have developed an automated algorithm for accurately segmenting and deriving granule counts, areas, and measuring distance to the plasma membrane. The method is a data-driven image processing approach that implements Mahalanobis classifiers to hierarchically classify pixel candidates and subsequently pixel aggregates as insulin granules. Granule cores and halos are classified independently and fused by intersecting the convex difference of granule halos with core candidates. Once fused, total and individual granule areas and distance metrics to the ?-cell plasma membrane are obtained. This algorithm provides a rapid and accurate method for the determination of granule numbers, location, and potential gradients in the pancreatic ?-cell under different experimental conditions.

  18. 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

  19. Association between endocrine pancreatic secretory granules and in-vitro-assembled microtubules is dependent upon microtubule-associated proteins

    E-print Network

    Suprenant, Kathy A.; Dentler, William L., Jr

    1982-04-01

    -microtubule/actin attachments . Sherline et al . (49) studied the associations between microtu- THE JOURNAL OF CELL BIOLOGY " VOLUME 93 APRIL 1982 164-174 ©The Rockefeller University Press " 0021-9525/82/04/0164/11 $1 .00 o n Septem ber 10, 2014 jcb.rupress.org D ow nloaded...M in GTPand l mM in MgSO4. The tubulin-containing fractions were pooled and made 10% in dimethyl sulfoxide (DMSO) (24), and microtubules were assembled by warming the preparations to 37°C. Microtubules were pelleted, resuspended at a concentration of 10-15 mg...

  20. Loss-of-function mutations in ABCA1 and enhanced ?-cell secretory capacity in young adults.

    PubMed

    Rickels, Michael R; Goeser, Eugen S; Fuller, Carissa; Lord, Christine; Bowler, Anne M; Doliba, Nicolai M; Hegele, Robert A; Cuchel, Marina

    2015-01-01

    Loss-of-function mutations affecting the cholesterol transporter ATP-binding cassette transporter subfamily A member 1 (ABCA1) impair cellular cholesterol efflux and are associated with reduced HDL-cholesterol (HDL-C) levels. ABCA1 may also be important in regulating ?-cell cholesterol homeostasis and insulin secretion. We sought to determine whether loss-of-function ABCA1 mutations affect ?-cell secretory capacity in humans by performing glucose-potentiated arginine tests in three subjects homozygous for ABCA1 mutations (age 25 ± 11 years), eight heterozygous subjects (28 ± 7 years), and eight normal control subjects pair-matched to the heterozygous carriers. To account for any effect of low HDL-C on insulin secretion, we studied nine subjects with isolated low HDL-C with no ABCA1 mutations (age 26 ± 6 years) and nine pair-matched control subjects. Homozygotes for ABCA1 mutations exhibited enhanced oral glucose tolerance and dramatically increased ?-cell secretory capacity that was also greater in ABCA1 heterozygous subjects than in control subjects, with no differences in insulin sensitivity. Isolated low HDL-C subjects also demonstrated an increase in ?-cell secretory capacity but in contrast to those with ABCA1 mutations, exhibited impaired insulin sensitivity, supporting ?-cell compensation for increased insulin demand. These data indicate that loss-of-function mutations in ABCA1 in young adults may be associated with enhanced ?-cell secretory capacity and normal insulin sensitivity and support the importance of cellular cholesterol homeostasis in regulating ?-cell insulin secretion. PMID:25125487

  1. Clostridium difficile toxin B inhibits the secretory response of human mast cell line-1 (HMC-1) cells stimulated with high free-Ca˛? and GTP?S.

    PubMed

    Balletta, Andrea; Lorenz, Dorothea; Rummel, Andreas; Gerhard, Ralf; Bigalke, Hans; Wegner, Florian

    2015-02-01

    Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho, Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency. Inactivation of Rho-family GTPases by this toxin in our experimental conditions induced morphological changes and reduction of electron-dense mast cell-specific granules in human mast cell line-1 (HMC-1) cells, but not cell death or permeabilisation of plasma-membranes. Previously reported patch-clamp dialysis experiments revealed that high intracellular free-Ca(2+) and GTP?S concentrations are capable of inducing exocytosis as indicated by significant membrane capacitance (Cm) increases in HMC-1 cells. In this study, we investigated the direct effects of TcdB upon HMC-1 cell "stimulated" Cm increase, as well as on "constitutive" secretion of hexosaminidase and interleukin-16 (IL-16). Compared to untreated control cells, HMC-1 cells incubated with TcdB for 3-24h exhibited a significant reduction of the mean absolute and relative Cm increase in response to free-Ca(2+) and GTP?S suggesting an inhibition of secretory processes by TcdB. In conclusion, the HMC-1 cell line represents a suitable model for the study of direct effects of C. difficile toxins on human mast cell secretory activity. PMID:25497110

  2. Selective delivery of secretory cargo in Golgi-derived carriers of nonepithelial cells.

    PubMed

    Rustom, Amin; Bajohrs, Mark; Kaether, Christoph; Keller, Patrick; Toomre, Derek; Corbeil, Denis; Gerdes, Hans-Hermann

    2002-04-01

    In epithelial cells, soluble cargo proteins destined for basolateral or apical secretion are packaged into distinct trans-Golgi network-derived transport carriers. Similar carriers, termed basolateral- and apical-like, have been observed in nonepithelial cells using ectopically expressed membrane marker proteins. Whether these cells are capable of selectively packaging secretory proteins into distinct carriers is still an open question. Here, we have addressed this issue by analyzing the packaging and transport of secretory human chromogranin B fusion proteins using a green fluorescent protein-based high-resolution, dual-color imaging technique. We were able to show that these secretory markers were selectively packaged at the Golgi into tubular/vesicular-like transport carriers containing basolateral membrane markers, resulting in extensive cotransport. In contrast, deletion mutants of the human chromogranin B fusion proteins lacking an N-terminal loop structure were efficiently transported in both basolateral- and apical-like carriers, the latter displaying a spherical morphology. Similarly, in polarized epithelial cells, the human chromogranin B fusion protein was secreted basolaterally and the loop-deleted analogue into both the basolateral and apical medium. These findings suggest that nonepithelial cells, like their epithelial counterparts, possess a sorting machinery capable of selective packaging of secretory cargo into distinct types of carriers. PMID:11929609

  3. Developmental regulation of granule size and numbers in larval salivary glands of drosophila by steroid hormone ecdysone.

    PubMed

    Farkas, R; Suáková, G

    1999-01-01

    The size and number of secretory granules in late larval salivary glands of Drosophila melanogaster have been related to interecdysial and early metamorphic development represented by well-known puffs in polytene chromosomes. Interecdysial period (puff stage 1 (PS1)) is characterized by presence of numerous small granules (11,000 per cell). The transition from PSI to early metamorphic phase (PS2 and upwards), induced by rapid elevation in endogenous steroid hormone ecdysone, is accompanied by continuous growth of granule diameter with concomitant reduction in their number per cell. In the PS4, just prior to secretion, approximately 3000 mature granules occur per cell. The mature state is associated with the change from hyperbolic to Gaussian distribution of granule number over their size range. Similar changes in secretory granule parameters were observed in interecdysial salivary glands explanted from 3rd instar larvae and cultured in vitro in medium containing 5x10(-6) m ecdysone. PMID:10736190

  4. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells.

    PubMed

    Nyegaard, Steffen; Novakovic, Valerie A; Rasmussen, Jan T; Gilbert, Gary E

    2013-01-01

    Secretory phospholipase A2 (sPLA2) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2's do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50-60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2. PMID:24194865

  5. Lactadherin Inhibits Secretory Phospholipase A2 Activity on Pre-Apoptotic Leukemia Cells

    PubMed Central

    Nyegaard, Steffen; Novakovic, Valerie A.; Rasmussen, Jan T.; Gilbert, Gary E.

    2013-01-01

    Secretory phospholipase A2 (sPLA2) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2’s do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50–60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2. PMID:24194865

  6. In vitro viability and secretory capacity of human luteinized granulosa cells after gonadotropin-releasing

    E-print Network

    Terasaki, Mark

    protocol. Intervention(s): In vitro fertilization cycles. Main Outcome Measure(s): Proportion of apoptosisIn vitro viability and secretory capacity of human luteinized granulosa cells after gonadotropin-based fertility center. Patient(s): A subset of patients who underwent a randomized trial involving GnRH agonist

  7. Calcium transients in cerebellar granule cell presynaptic terminals.

    PubMed Central

    Regehr, W G; Atluri, P P

    1995-01-01

    Calcium ions act presynaptically to modulate synaptic strength and to trigger neurotransmitter release. Here we detect stimulus-evoked changes in residual free calcium ([Ca2+]i) in rat cerebellar granule cell presynaptic terminals. Granule cell axons, known as parallel fibers, and their associated boutons, were labeled with several calcium indicators. When parallel fibers were extracellularly activated with stimulus trains, calcium accumulated in the terminals, producing changes in the fluorescence of the indicators. During the stimulus train, the fluorescence change per pulse became progressively smaller with the high affinity indicators Fura-2 and calcium green-2 but remained constant with the low affinity dyes BTC and furaptra. In addition, fluorescence transients of high affinity dyes were slower than those of low affinity indicators, which appear to accurately report the time course of calcium transients. Simulations show that differences in the observed transients can be explained by the different affinities and off rates of the fluorophores. The return of [Ca2+]i to resting levels can be approximated by an exponential decay with a time constant of 150 ms. On the basis of the degree of saturation in the response of high affinity dyes observed during trains, we estimate that each action potential increases [Ca2+]i in the terminal by several hundred nanomolar. These findings indicate that in these terminals [Ca2+]i transients are much larger and faster than those observed in larger boutons, such as those at the neuromuscular junction. Such rapid [Ca2+]i dynamics may be found in many of the terminals in the mammalian brain that are similar in size to parallel fiber boutons. Images FIGURE 1 PMID:7612860

  8. Ultrastructure and cytochemistry of lipid granules in the many-celled magnetotactic prokaryote, 'Candidatus Magnetoglobus multicellularis'.

    PubMed

    Silva, Karen Tavares; Abreu, Fernanda; Keim, Carolina N; Farina, Marcos; Lins, Ulysses

    2008-12-01

    Conspicuous cytoplasmic granules are reported in a magnetotactic multicellular prokaryote named 'Candidatus Magnetoglobus multicellularis'. Unfortunately, this microorganism, which consists of an assembly of gram-negative bacterial cells, cannot yet be cultivated, limiting the biochemical analysis of the granules and preventing in vitro studies with starvation/excess of nutrients. In this scenario, light and electron microscopy techniques were used to partially address the nature of the granules. Besides magnetosomes, three types of inclusions were observed: small (mean diameter=124 nm) polyhydroxyalkanoate-like (PHA) granules, large (diameters ranging from 0.11 to 2.5 microm) non-PHA lipid granules, and rare phosphorus-rich granules, which probably correspond to polyphosphate bodies. The PHA granules were rounded in projection, non-reactive with OsO(4), and suffered the typical plastic deformation of PHAs after freeze fracturing. The nature of the large granules, consisting of round globular structures (mean diameter=0.76 microm), was classified as non-PHA based on the following data: (a) multilayered structure in freeze-fracture electron microscopy, typical of non-PHA lipids; (b) Nile blue fluorescence imaging detected non-PHA lipids; (c) imidazole buffered osmium tetroxide and ruthenium red cytochemistry stained the globules, which appeared as electron-dense granules instead of electron lucent as PHAs do. Most likely, 'Candidatus Magnetoglobus multicellularis' stores carbon mainly as unusual lipid granules, together with smaller amounts of PHAs. PMID:18599298

  9. The Regulatory Connection between the Activity of Granule Cell NMDA Receptors and Dendritic Differentiation of Cerebellar Purkinje Cells

    Microsoft Academic Search

    Hirokazu Hirai; Thomas Launey

    2000-01-01

    It is known that cerebellar granule cells are powerful inducers for the differentiation of Purkinje cells. However, the detailed mech- anism of this regulation has not yet been clarified. Here, using cerebellar neuronal culture, we show that the activation of NMDA receptors expressed by granule cells triggers the signaling path- way for the dendritic differentiation of Purkinje cells. This signal

  10. Neuroendocrinelike (small granule) epithelial cells of the lung.

    PubMed Central

    DiAugustine, R P; Sonstegard, K S

    1984-01-01

    The presence of neuroendocrinelike epithelial cells in the lung of numerous species has been demonstrated by light and electron microscopy. Histochemical methods used to identify these cells have included staining with silver, amine-type fluorescence (APUD cell), periodic acid Schiff (PAS)-lead hematoxylin, and immunohistochemical localization of neuron-specific enolase. Cytoplasmic dense core vesicles (70-200 nm in diameter) have served as the major ultrastructural characteristic. Lung neuroendocrinelike cells have been shown to occur in fetal and adult mammals as solitary-type cells or as distinct organoids known as neuroepithelial bodies ( NEBs ). Although the frequency of both populations is considered low, solitary-type cells with dense-core granules can be found in as high as 5% of epithelial cells in the cricoid region of the guinea-pig larynx. The solitary cells can be found throughout the airways of mammals, whereas the NEBs are confined to the intrapulmonary airways. Unmyelinated fibers have been traced from the lamina propria and into the NEB, where they ramified between the component cells of the NEB. The function of lung neuroendocrinelike cells is not known, but morphological and cytochemical studies suggest that the NEBs are intrapulmonary chemoreceptors that can respond to changes in airway gas composition. Hypoxia or hypercapnia has been shown to decrease the amine cytofluorescence in these organoids and apparently to increase the exocytosis of dense core vesicles from the basal region of the cell. Immunohistochemical studies have suggested that some lung epithelial cells may contain a known neuropeptide(s), but further investigation is needed to confirm the presence of such compounds in lung neuroendocrinelike cells and their physiochemical properties. Apparent hyperplasia of lung neuroendocrinelike cells can occur readily in hamsters treated with diethylnitrosamine. It has been postulated that human lung tumors with endocrinelike properties, namely, bronchial carcinoids and lung small cell carcinomas, may originate from lung neuroendocrinelike cells. However, a more plausible explanation, based on cytokinetic studies of epithelial neuroendocrinelike cells in the lung and other organs, is that these cells originate from a nonneuroendocrine population. Interaction of such a progenitor cell population with selected carcinogens may lead to stimulation of the rate of normal differentiation or, alternately, to selection of an abnormal route of differentiation that possesses a neuroendocrine phenotype. Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. FIGURE 11. FIGURE 12. FIGURE 13. FIGURE 14. FIGURE 15. PMID:6376101

  11. Dentate Granule Cell Neurogenesis Is Increased by Seizures and Contributes to Aberrant Network Reorganization in the Adult Rat Hippocampus

    Microsoft Academic Search

    Jack M. Parent; Timothy W. Yu; Rebecca T. Leibowitz; Daniel H. Geschwind; Robert S. Sloviter; Daniel H. Lowenstein

    The dentate granule cell layer of the rodent hippocampal formation has the distinctive property of ongoing neurogen- esis that continues throughout adult life. In both human temporal lobe epilepsy and rodent models of limbic epilepsy, this same neuronal population undergoes extensive remod- eling, including reorganization of mossy fibers, dispersion of the granule cell layer, and the appearance of granule cells

  12. Isolation of secretory cells from plant glandular trichomes and their use in biosynthetic studies of monoterpenes and other gland products.

    PubMed

    Gershenzon, J; McCaskill, D; Rajaonarivony, J I; Mihaliak, C; Karp, F; Croteau, R

    1992-01-01

    The natural products that accumulate in or exude from plant glandular trichomes are biosynthesized by secretory cells located at the apex of the trichome. To investigate the formation of glandular trichome constituents in several species of mints (Lamiaceae), a new procedure was developed for isolating large numbers of highly purified secretory cells. In this method, the leaf surface is gently abraded with glass beads in a way that fragments the glandular trichomes and yields clusters of intact secretory cells. The isolated, intact secretory cells and cell-free preparations derived from them are very active in monoterpene biosynthesis and provide useful starting materials for the purification of several key enzymes of monoterpene metabolism. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of whole cell and cell-free systems for biosynthetic studies of plant natural products found in glandular trichomes. PMID:1595887

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

    PubMed

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

    2015-03-19

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The ?7(?)nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional ?7(?)nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2-3 week-old Wistar rats, and 2-9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of ?7(?)nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric ?7(?)nicotinic receptor modulator, which were blocked by a specific ?7(?)nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic ?7(?)nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating ?7(?)nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain. PMID:25553616

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

    PubMed Central

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

    2015-01-01

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The ?7?nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional ?7?nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2–3 week-old Wistar rats, and 2–9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of ?7?nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric ?7?nicotinic receptor modulator, which were blocked by a specific ?7?nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic ?7?nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating ?7?nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain. PMID:25553616

  15. Original article Membrane-bound iron-rich granules in fat cells and

    E-print Network

    Boyer, Edmond

    honeybee (Apis mellifera L.) H. Raes W. Bohyn P.H. De Rycke F. Jacobs ! Labo/'afo/y!o/'Zoop/tys/o/ot! State administered Pb is accumulated in the iron rich granules as well as in the spherocrystals. Apis mellifera; accepted 18 May 1989) Summary — Previous studies have found iron-containing granules in fat cells

  16. Effect of oral acetylcysteine on tobacco smoke-induced secretory cell hyperplasia.

    PubMed

    Jeffery, P K; Rogers, D F; Ayers, M M

    1985-01-01

    The present investigation explores whether N-acetylcysteine (NAC) inhibits the secretory cell hyperplasia known to occur experimentally in specific pathogen-free (SPF) bronchitic rats. The animals were divided into 4 groups: no tobacco smoke (TS), no drug, no TS but NAC (1040 mg/kg body weight), TS but no drug, and TS plus NAC. NAC-treated animals showed no ill effects, TS exposed animals showed an initial fall in weight gain which never fully recovered (P less than 0.01): NAC did not protect. TS caused a significant increase (62-421%) in secretory cell number at all airway levels distal to the upper trachea (P less than 0.01) and NAC significantly inhibited it (P less than 0.01-0.05) in all, mostly in secretory cells containing acidic glycoprotein. TS exposure also induced a significant rise in epithelial cell concentration and of ciliated, mucous and especially basal cell number (P less than 0.001). NAC inhibited the mucous cell increase (P less than 0.001) and had 3 effects on the peak of dividing cells: it was (a) delayed until 3 days (b) greatly reduced in size and (c) prolonged at a lower level until its return to control values at 10 days of TS exposure. PMID:3862604

  17. 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

  18. Paneth cell granule depletion in the human small intestine under infective and nutritional stress.

    PubMed

    Kelly, P; Feakins, R; Domizio, P; Murphy, J; Bevins, C; Wilson, J; McPhail, G; Poulsom, R; Dhaliwal, W

    2004-02-01

    Paneth cells are important contributors to the intestinal antimicrobial barrier through synthesis and release of antimicrobial peptides and proteins. Animal studies indicate that Paneth cell numbers, location and granule morphology are altered by infection and zinc status. We examined human tissue to determine whether Paneth cell numbers, distribution or granule morphology are altered in infective, inflammatory and nutritional disorders. Archival sections from infective disorders (giardiasis, cryptosporidiosis, HIV, helminth infection) were compared with active inflammatory conditions (coeliac, Crohn's and graft-versus-host diseases) and histologically normal tissues. A subset of tissues was studied by electron microscopy and TUNEL staining for apoptosis. Human defensin-5 (HD5) peptide and mRNA was analysed by immunohistochemistry, in situ hybridization and quantitative reverse transcription polymerase chain reaction. Sections from a tropical population cohort study were then analysed to determine the relationship of granule depletion to infection, nutritional status and plasma zinc concentration. In HIV-related cryptosporidiosis, but not other disorders, Paneth cells were reduced in number and markedly depleted of granules. Paneth cell granule depletion was associated with reduced HD5 immunoreactivity, but this was not due to apoptosis and there was no reduction in mRNA transcripts. In the tropical population studied, depletion of granules was associated with reduced body mass index, reduced plasma zinc levels and HIV infection. Paneth cell granules in human small intestine may be depleted in response to infective and nutritional stress. We postulate that this is one mechanism through which zinc status influences host susceptibility to intestinal infection. PMID:14738460

  19. NR5A nuclear receptor Hr39 controls three-cell secretory unit formation in Drosophila female reproductive glands

    PubMed Central

    Sun, Jianjun; Spradling, Allan C.

    2012-01-01

    Summary Background Secretions within the adult female reproductive tract mediate sperm survival, storage, activation and selection. Drosophila female reproductive gland secretory cells reside within the adult spermathecae and parovaria, but their development remains poorly characterized. Results With cell-lineage tracing, we found that precursor cells down-regulate lozenge and divide sterotypically to generate three-cell secretory units during pupal development. The NR5A-class nuclear hormone receptor Hr39 is essential for precursor cell division and secretory unit formation. Moreover, ectopic Hr39 in multiple tissues generates reproductive gland-like primordia. Rarely, in male genital discs these primordia can develop into sperm-filled testicular spermathecae. Conclusion Drosophila spermathecae provide a powerful model for studying gland development. Hr39 functions as a master regulator of a program that may have been conserved throughout animal evolution for the production of female reproductive glands and other secretory tissues. PMID:22560612

  20. Activation of NOTCH1 or NOTCH3 Signaling Skews Human Airway Basal Cell Differentiation toward a Secretory Pathway

    PubMed Central

    Gomi, Kazunori; Arbelaez, Vanessa; Crystal, Ronald G.; Walters, Matthew S.

    2015-01-01

    Airway basal cells (BC) function as stem/progenitor cells capable of differentiating into the luminal ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. The objective of this study was to define the role of Notch signaling in regulating human airway BC differentiation into a pseudostratified mucociliated epithelium. Notch inhibition with ?-secretase inhibitors demonstrated Notch activation is essential for BC differentiation into secretory and ciliated cells, but more so for the secretory lineage. Sustained cell autonomous ligand independent Notch activation via lentivirus expression of the intracellular domain of each Notch receptor (NICD1-4) demonstrated that the NOTCH2 and 4 pathways have little effect on BC differentiation into secretory and ciliated cells, while activation of the NOTCH1 or 3 pathways has a major influence, with persistent expression of NICD1 or 3 resulting in a skewing toward secretory cell differentiation with a parallel decrease in ciliated cell differentiation. These observations provide insights into the control of the balance of BC differentiation into the secretory vs ciliated cell lineage, a balance that is critical for maintaining the normal function of the airway epithelium in barrier defense against the inhaled environment. PMID:25700162

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

    SciTech Connect

    Miyagawa, Ryu [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan) [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan); Mizuno, Rie [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan)] [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Watanabe, Kazunori, E-mail: watanabe@ric.u-tokyo.ac.jp [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan)] [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Ijiri, Kenichi [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan) [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan)

    2012-02-03

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

  2. Central nervous system neurons acquire mast cell products via transgranulation

    E-print Network

    Silver, Rae

    of the granule and plasma membranes (mast cell and neuron); by capture of insoluble granule remnants and or in association with small vesicles and the trans-Golgi network. Capture of granule remnants is the most., 2005). When released in the CNS, mast cell secretory products can alter the function of both neural

  3. 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. PMID:19631357

  4. Distinct Determinants of Sparse Activation during Granule Cell Maturation

    PubMed Central

    Dieni, Cristina V.; Nietz, Angela K.; Panichi, Roberto

    2013-01-01

    Adult neurogenesis continually produces a small population of immature granule cells (GCs) within the dentate gyrus. The physiological properties of immature GCs distinguish them from the more numerous mature GCs and potentially enables distinct network functions. To test how the changing properties of developing GCs affect spiking behavior, we examined synaptic responses of mature and immature GCs in hippocampal slices from adult mice. Whereas synaptic inhibition restricted GC spiking at most stages of maturation, the relative influence of inhibition, excitatory synaptic drive, and intrinsic excitability shifted over the course of maturation. Mature GCs received profuse afferent innervation such that spiking was suppressed primarily by inhibition, whereas immature GC spiking was also limited by the strength of excitatory drive. Although the input resistance was a reliable indicator of maturation, it did not determine spiking probability at immature stages. Our results confirm the existence of a transient period during GC maturation when perforant path stimulation can generate a high probability of spiking, but also reveal that immature GC excitability is tempered by functional synaptic inhibition and reduced excitatory innervation, likely maintaining the sparse population activity observed in vivo. PMID:24305810

  5. Expression differences in mitochondrial and secretory chaperonin 60 (Cpn60) in pancreatic acinar cells

    PubMed Central

    Li, Y.; Gingras, D.; Londońo, I.; Bendayan, M.

    2003-01-01

    In pancreatic acinar cells, chaperonin Cpn60 is present in all the cellular compartments involved in protein secretion as well as in mitochondria. To better understand the role Cpn60 plays in pancreatic secretion, we have evaluated its changes under experimental conditions known to alter pancreatic secretion. Quantitative protein A–gold immunocytochemistry was used to reveal Cpn60 in pancreatic acinar cells. Cpn60 immunolabelings in cellular compartments involved in secretion were found to decrease in acute pancreatitis as well as upon stimulation of secretion and in starvation conditions. A major increase in Cpn60 was recorded in diabetic condition. This was normalized by insulin treatment. Although in certain situations changes in secretory enzymes and in Cpn60 correlate well, in others, nonparallel secretion seemed to take place. In contrast, expression of mitochondrial Cpn60 in acinar cells appeared to remain stable in all conditions except starvation, where its levels decreased. Expression of Cpn60 in the secretory pathway and in mitochondria thus appears to behave differently, and Cpn60 in the secretory pathway must be important for quality control and integrity of secretion. PMID:14984062

  6. Characterization of the neuroprotective activity of rasagiline in cerebellar granule cells

    Microsoft Academic Search

    Dafna Bonneh-Barkay; Noam Ziv; John P. M. Finberg

    2005-01-01

    Rasagiline (N-propargyl-1-R-aminoindan) is a new selective inhibitor of MAO-B which is in development for the treatment of Parkinson's disease. The aim of the present study was to evaluate the neuroprotective properties of rasagiline and characterize the mechanism by which it exerts its neuroprotective effect in cerebellar granule cells. Cerebellar granule cells were prepared from 7 to 8 days postnatal Sprague-Dawley

  7. Cysteamine depletes prolactin (PRL) but does not alter the structure of PRL-containing granules in the anterior pituitary

    SciTech Connect

    Weinstein, L.A.; Landis, D.M.; Sagar, S.M.; Millard, W.J.; Martin, J.B.

    1984-10-01

    Cysteamine causes a profound depletion of PRL in the anterior pituitary and in the systemic circulation, as measured by RIA and bioassay. However, electron microscopic study of PRL-containing cells in rat anterior pituitary does not reveal changes in secretory granule or cytoplasmic structure during the interval of depressed PRL content and of subsequent recovery to normal levels. In contrast to the results obtained by RIA, PRL-like immunoreactivity as detected by immunocyto-chemistry is present and similar to that of control preparations after cysteamine administration. We suggest that cysteamine alters PRL structure in secretory granules, probably by interacting with the disulfide bonds of PRL, thereby altering bioactivity and immunoreactivity. The presence of cysteamine-altered PRL in secretory granules does not seem to trigger degradation of granules by the lysosomal system.

  8. Hilar Mossy Cells Provide the First Glutamatergic Synapses to Adult-Born Dentate Granule Cells

    PubMed Central

    Chancey, Jessica H.; Poulsen, David J.

    2014-01-01

    Adult-generated granule cells (GCs) in the dentate gyrus must establish synapses with preexisting neurons to participate in network activity. To determine the source of early glutamatergic synapses on newborn GCs in adult mice, we examined synaptic currents at the developmental stage when NMDA receptor-mediated silent synapses are first established. We show that hilar mossy cells provide initial glutamatergic synapses as well as disynaptic GABAergic input to adult-generated dentate GCs. PMID:24501373

  9. Nitric oxide as a secretory product of mammalian cells

    Microsoft Academic Search

    CARL NATHAN

    1992-01-01

    Evolution has resorted to nitric oxide (NO), a tiny, reactive radical gas, to mediate both ser- voregulatory and cytotoxic functions. This article reviews how different forms of nitric oxide synthase help confer specificity and diversity on the effects of this remarkable signaling molecule.- Nathan, C. Nitric oxide as a secre- tory product of mammalian cells. FASEBJ. 6: 3051-3064; 1992.

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

    PubMed Central

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

    2013-01-01

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

  11. 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

  12. Trafficking along the secretory pathway in Drosophila cell line and tissues: a light and electron microscopy approach.

    PubMed

    Zacharogianni, Margarita; Rabouille, Catherine

    2013-01-01

    In the past, Drosophila has been used for molecular and developmental biology studies that have led to many important conceptual advances. In the last decade, this model organism has also been utilized to address cell biology issues, in particular those related to membrane traffic through the secretory pathway. This has confirmed that the functional organization of the secretory pathway is conserved and it allowed further integrating secretion to signaling and development. Furthermore, Drosophila tissue culture S2 cells have been the basis of many RNAi screens, some addressing aspects of the functional organization of the secretory pathway and others identifying proteins of the secretory pathway in seemingly unrelated processes. Taken together, studying the protein trafficking and the organization of the secretory pathway both in S2 cells and in tissues has become important. Here, we review light and electron microscopy techniques applied to Drosophila that allow gaining insight into the secretory pathway, and can easily be extended to other cell biology-related fields. PMID:24295299

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

    PubMed Central

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

    2012-01-01

    In the developing nervous system synaptic refinement, typified by the neuromuscular junction where supernumerary connections are eliminated by axon retraction leaving the postsynaptic target innervated by a single dominant input, critically regulates neuronal circuit formation. Whether such competition-based pruning continues in established circuits of mature animals remains unknown. This question is particularly relevant in the context of adult neurogenesis where newborn cells must integrate into preexisting circuits, and thus, potentially compete with functionally mature synapses to gain access to their postsynaptic targets. The hippocampus plays an important role in memory formation/retrieval and the dentate gyrus (DG) subfield exhibits continued neurogenesis into adulthood. Therefore, this region contains both mature granule cells (old GCs) and immature recently born GCs that are generated throughout adult life (young GCs), providing a neurogenic niche model to examine the role of competition in synaptic refinement. Recent work from an independent group in developing animals indicated that embryonically/early postnatal generated GCs placed at a competitive disadvantage by selective expression of tetanus toxin (TeTX) to prevent synaptic release rapidly retracted their axons, and that this retraction was driven by competition from newborn GCs lacking TeTX. In contrast, following 3–6 months of selective TeTX expression in old GCs of adult mice we did not observe any evidence of axon retraction. Indeed ultrastructural analyses indicated that the terminals of silenced GCs even maintained synaptic contact with their postsynaptic targets. Furthermore, we did not detect any significant differences in the electrophysiological properties between old GCs in control and TeTX conditions. Thus, our data demonstrate a remarkable stability in the face of a relatively prolonged period of altered synaptic competition between two populations of neurons within the adult brain. PMID:23162435

  14. 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. PMID:17564908

  15. Rapid Signaling Actions of Environmental Estrogens in Developing Granule Cell Neurons Are Mediated by Estrogen Receptor ?

    PubMed Central

    Le, Hoa H.; Belcher, Scott M.

    2010-01-01

    Estrogenic endocrine disrupting chemicals (EDCs) constitute a diverse group of man-made chemicals and natural compounds derived from plants and microbial metabolism. Estrogen-like actions are mediated via the nuclear hormone receptor activity of estrogen receptor (ER)? and ER? and rapid regulation of intracellular signaling cascades. Previous study defined cerebellar granule cell neurons as estrogen responsive and that granule cell precursor viability was developmentally sensitive to estrogens. In this study experiments using Western blot analysis and pharmacological approaches have characterized the receptor and signaling modes of action of selective and nonselective estrogen ligands in developing cerebellar granule cells. Estrogen treatments were found to briefly increase ERK1/2-phosphorylation and then cause prolonged depression of ERK1/2 activity. The sensitivity of granule cell precursors to estrogen-induced cell death was found to require the integrated activation of membrane and intracellular ER signaling pathways. The sensitivity of granule cells to selective and nonselective ER agonists and a variety of estrogenic and nonestrogenic EDCs was also examined. The ER? selective agonist DPN, but not the ER? selective agonist 4,4?,4?-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol or other ER?-specific ligands, stimulated cell death. Only EDCs with selective or nonselective ER? activities like daidzein, equol, diethylstilbestrol, and bisphenol A were observed to induce E2-like neurotoxicity supporting the conclusion that estrogen sensitivity in granule cells is mediated via ER?. The presented results also demonstrate the utility of estrogen sensitive developing granule cells as an in vitro assay for elucidating rapid estrogen-signaling mechanisms and to detect EDCs that act at ER? to rapidly regulate intracellular signaling. PMID:20926581

  16. Streptozotocin-resistant BRIN-BD11 cells possess wide spectrum of toxin tolerance and enhanced insulin-secretory capacity

    Microsoft Academic Search

    Hui-Kang Liu; Jane T. McCluskey; Neville H. McClenghan; Peter R. Flatt

    2007-01-01

    Since streptozotocin (STZ) exhibits beta-cell toxicity, mediated through diverse mechanisms, multiple toxin resistance can\\u000a be expected in insulin-secretory cells rendered STZ-resistant. RINm5F, but not all cell lines surviving STZ treatment, possess\\u000a higher insulin content than native parental cells and additional tolerance against alloxan. To understand the impact of STZ\\u000a tolerant cell selection on toxin resistance and insulin-secretory function, STZ-resistant BRIN-BD11

  17. A micromethod for the assay of cellular secretory physiology: Application to rabbit parietal cells

    SciTech Connect

    Adrian, T.E.; Goldenring, J.R.; Oddsdottir, M.; Zdon, M.J.; Zucker, K.A.; Lewis, J.J.; Modlin, I.M. (Yale Univ. School of Medicine, West Haven, CT (USA))

    1989-11-01

    A micromethod for investigating secretory physiology in isolated cells was evaluated. The method utilized a specially designed polycarbonate incubation chamber to provide constant oxygenation to cells incubating in a 96-well microtiter plate. Cells were rapidly separated from media by vacuum filtration. Isolated parietal cells were utilized to demonstrate the versatility of the method for assay of intracellular accumulation of ({sup 14}C)-aminopyrine, secretion of intrinsic factor into the medium, and assay of intracellular cAMP. Histamine stimulated the uptake of ({sup 14}C)aminopyrine and intrinsic factor secretion in a sustained and linear fashion. At the end of the 2-h period uptake of aminopyrine and secretion of intrinsic factor were increased 17- and 5-fold, respectively. This response to histamine was accompanied by a rapid and sustained 3-fold rise in intracellular cyclic AMP. In contrast, carbamylcholine caused a transient increase in ({sup 14}C)aminopyrine accumulation and intrinsic factor secretion which was most pronounced during the first 10 min and had almost ceased by 30 min. Carbamylcholine had no effect on intracellular cAMP levels. This new method, which can handle 400 replicates using parietal cells from the fundic mucosa of a single rabbit, is suitable for studying the time course of intracellular events which accompany general secretory processes.

  18. Streptozotocin-resistant BRIN-BD11 cells possess wide spectrum of toxin tolerance and enhanced insulin-secretory capacity.

    PubMed

    Liu, Hui-Kang; McCluskey, Jane T; McClenghan, Neville H; Flatt, Peter R

    2007-08-01

    Since streptozotocin (STZ) exhibits beta-cell toxicity, mediated through diverse mechanisms, multiple toxin resistance can be expected in insulin-secretory cells rendered STZ-resistant. RINm5F, but not all cell lines surviving STZ treatment, possess higher insulin content than native parental cells and additional tolerance against alloxan. To understand the impact of STZ tolerant cell selection on toxin resistance and insulin-secretory function, STZ-resistant BRIN-BD11 cells were generated by iterative acute exposure to 20 mM STZ. These cells, denoted BRINst cells, exhibited resistance to toxic challenges from STZ, H(2)O(2), and ninhydrin. Insulin content and both glucose and arginine-stimulated insulin secretion were significantly enhanced in BRINst cells. The toxin-resistance of BRINst cells was gradually lost during continuous cultivation without STZ challenge. However, enhanced insulin secretory capacity at high passage in BRINst cells persisted. Although total SOD activity was decreased, catalase activity was increased and appeared to be important for the ninhydrin and STZ resistance of BRINst cells. This was associated with reductions of both STZ- and ninhydrin-induced DNA damage, although DNA repair was abolished. Further characterization of cells exhibiting multiple toxin tolerance and an enhanced insulin secretory function could provide useful lessons for understanding of beta-cell survival. PMID:17992598

  19. Secretory prostate apoptosis response (Par)-4 sensitizes multicellular spheroids (MCS) of glioblastoma multiforme cells to tamoxifen-induced cell death

    PubMed Central

    Jagtap, Jayashree C.; Parveen, D.; Shah, Reecha D.; Desai, Aarti; Bhosale, Dipali; Chugh, Ashish; Ranade, Deepak; Karnik, Swapnil; Khedkar, Bhushan; Mathur, Aaishwarya; Natesh, Kumar; Chandrika, Goparaju; Shastry, Padma

    2014-01-01

    Glioblastoma multiforme (GBM) is the most malignant form of brain tumor and is associated with resistance to conventional therapy and poor patient survival. Prostate apoptosis response (Par)-4, a tumor suppressor, is expressed as both an intracellular and secretory/extracellular protein. Though secretory Par-4 induces apoptosis in cancer cells, its potential in drug-resistant tumors remains to be fully explored. Multicellular spheroids (MCS) of cancer cells often acquire multi-drug resistance and serve as ideal experimental models. We investigated the role of Par-4 in Tamoxifen (TAM)-induced cell death in MCS of human cell lines and primary cultures of GBM tumors. TCGA and REMBRANT data analysis revealed that low levels of Par-4 correlated with low survival period (21.85 ± 19.30 days) in GBM but not in astrocytomas (59.13 ± 47.26 days) and oligodendrogliomas (58.04 ± 59.80 days) suggesting low PAWR expression as a predictive risk factor in GBM. Consistently, MCS of human cell lines and primary cultures displayed low Par-4 expression, high level of chemo-resistance genes and were resistant to TAM-induced cytotoxicity. In monolayer cells, TAM-induced cytotoxicity was associated with enhanced expression of Par-4 and was alleviated by silencing of Par-4 using specific siRNA. TAM effectively induced secretory Par-4 in conditioned medium (CM) of cells cultured as monolayer but not in MCS. Moreover, MCS were rendered sensitive to TAM-induced cell death by exposure to conditioned medium (CM)-containing Par-4 (derived from TAM-treated monolayer cells). Also TAM reduced the expression of Akt and PKC? in GBM cells cultured as monolayer but not in MCS. Importantly, combination of TAM with inhibitors to PI3K inhibitor (LY294002) or PKC? resulted in secretion of Par-4 and cell death in MCS. Since membrane GRP78 is overexpressed in most cancer cells but not normal cells, and secretory Par-4 induces apoptosis by binding to membrane GRP78, secretory Par-4 is an attractive candidate for potentially overcoming therapy-resistance not only in malignant glioma but in broad spectrum of cancers. PMID:25685660

  20. Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells.

    PubMed

    Ohashi, Ryo; Sakata, Shin-ichi; Naito, Asami; Hirashima, Naohide; Tanaka, Masahiko

    2014-04-01

    Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca(2+) release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single-cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain-derived neurotrophic factor (BDNF) in the culture medium. The ryanodine-induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells. PMID:24123915

  1. Role of granule-cell transmission in memory trace of cerebellum-dependent optokinetic motor learning

    PubMed Central

    Wada, Norio; Funabiki, Kazuo; Nakanishi, Shigetada

    2014-01-01

    Adaptation of the optokinetic response (OKR) is an eye movement enhanced by repeated motion of a surrounding visual field and represents a prototype of cerebellum-dependent motor learning. Purkinje cells and vestibular nuclei (VN) receive optokinetic and retinal slip signals via the mossy fiber-granule cell pathway and climbing-fiber projections, respectively. To explore the neural circuits and mechanisms responsible for OKR adaptation, we adopted the reversible neurotransmission-blocking (RNB) technique, in which granule-cell transmission to Purkinje cells was selectively and reversibly blocked by doxycycline-dependent expression of transmission-blocking tetanus toxin in granule cells. Blockade of granule-cell inputs abolished both short-term and long-term OKR adaptation induced by repeated OKR training, but normal levels of both responses were immediately evoked in the pretrained RNB mice by OKR retraining once granule-cell transmission had recovered. Importantly, eye movement elicited by electrical stimulation of the cerebellar focculus was elevated by long-term but not by short-term OKR training in adaptive OKR-negative RNB mice. Furthermore, when the flocculus of adaptive OKR-negative RNB mice was electrically excited in-phase with OKR stimulation, these mice exhibited long-term adaptive OKR. These results indicate that convergent information to the VN was critical for acquisition and storage of long-term OKR adaptation with conjunctive action of Purkinje cells for OKR expression. Interestingly, in contrast to conditioned eyeblink memory, the expression of once acquired adaptive long-term OKR was not abrogated by blockade of granule-cell transmission, suggesting that distinct forms of neural plasticity would operate in different forms of cerebellum-dependent motor learning. PMID:24706878

  2. Transport of Fragile X Mental Retardation Protein via Granules in Neurites of PC12 Cells

    PubMed Central

    De Diego Otero, Yolanda; Severijnen, Lies-Anne; van Cappellen, Gert; Schrier, Mariëtte; Oostra, Ben; Willemsen, Rob

    2002-01-01

    Lack of fragile X mental retardation protein (FMRP) causes fragile X syndrome, a common form of inherited mental retardation. FMRP is an RNA binding protein thought to be involved in translation efficiency and/or trafficking of certain mRNAs. Recently, a subset of mRNAs to which FMRP binds with high affinity has been identified. These FMRP-associated mRNAs contain an intramolecular G-quartet structure. In neurons, dendritic mRNAs are involved in local synthesis of proteins in response to synaptic activity, and this represents a mechanism for synaptic plasticity. To determine the role of FMRP in dendritic mRNA transport, we have generated a stably FMR1-enhanced green fluorescent protein (EGFP)-transfected PC12 cell line with an inducible expression system (Tet-On) for regulated expression of the FMRP-GFP fusion protein. After doxycycline induction, FMRP-GFP was localized in granules in the neurites of PC12 cells. By using time-lapse microscopy, the trafficking of FMRP-GFP granules into the neurites of living PC12 cells was demonstrated. Motile FMRP-GFP granules displayed two types of movements: oscillatory (bidirectional) and unidirectional anterograde. The average velocity of the granules was 0.19 ?m/s with a maximum speed of 0.71 ?m/s. In addition, we showed that the movement of FMRP-GFP labeled granules into the neurites was microtubule dependent. Colocalization studies further showed that the FMRP-GFP labeled granules also contained RNA, ribosomal subunits, kinesin heavy chain, and FXR1P molecules. This report is the first example of trafficking of RNA-containing granules with FMRP as a core constituent in living PC12 cells. PMID:12417734

  3. Serpinin: A Novel Chromogranin A-Derived, Secreted Peptide Up-Regulates Protease Nexin-1 Expression and Granule Biogenesis in Endocrine Cells

    PubMed Central

    Koshimizu, Hisatsugu; Cawley, Niamh X.; Kim, Taeyoon; Yergey, Alfred L.

    2011-01-01

    Previously we demonstrated that chromogranin A (CgA) promoted secretory granule biogenesis in endocrine cells by stabilizing and preventing granule protein degradation in the Golgi, through up-regulation of expression of the protease inhibitor, protease nexin-1 (PN-1). However, the mechanism by which CgA signals the increase of PN-1 expression is unknown. Here we identified a 2.9-kDa CgA-C-terminus peptide, which we named serpinin, in conditioned media from AtT-20 cells, a corticotroph cell line, which up-regulated PN-1 mRNA expression. Serpinin was secreted from AtT-20 cells upon high potassium stimulation and increased PN-1 mRNA transcription in these cells, in an actinomycin D-inhibitable manner. CgA itself and other CgA-derived peptides, when added to AtT-20 cell media, had no effect on PN-1 expression. Treatment of AtT-20 cells with 10 nm serpinin elevated cAMP levels and PN-1 mRNA expression, and this effect was inhibited by a protein kinase A inhibitor, 6–22 amide. Serpinin and a cAMP analog, 8-bromo-cAMP, promoted the translocation of the transcription factor Sp1 into the nucleus, which is known to drive PN-1 expression. Additionally, an Sp1 inhibitor, mithramycin A inhibited the serpinin-induced PN-1 mRNA up-regulation. Furthermore, a luciferase reporter assay demonstrated serpinin-induced up-regulation of PN-1 promoter activity in an Sp1-dependent manner. When added to CgB-transfected 6T3 cells, a mutant AtT20 cell line, serpinin induced granule biogenesis as evidenced by the presence of CgB puncta accumulation in the processes and tips. Our findings taken together show that serpinin, a novel CgA-derived peptide, is secreted upon stimulation of corticotrophs and plays an important autocrine role in up-regulating PN-1-dependent granule biogenesis via a cAMP-protein kinase A-Sp1 pathway to replenish released granules. PMID:21436258

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

    PubMed Central

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

    2014-01-01

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

  5. Demonstration of bone marrow derived cells in synovial lining by means of giant intracellular granules as genetic markers

    Microsoft Academic Search

    J C Edwards; D A Willoughby

    1982-01-01

    Beige mice carry a gene (bg) which codes for the presence of giant intracellular granules in a variety of cell types. Bone marrow from beige mice was transplanted into irradiated normal mice. Giant granules similar to those seen in beige mouse synovial cells were observed subsequently in the synovial lining cells of marrow recipients, indicating an influx of bone marrow

  6. Granulysin, a new human cytolytic granule-associated protein with possible involvement in cell-mediated cytotoxicity

    Microsoft Academic Search

    Susan V. Peńa; Alan M. Krensky

    1997-01-01

    A primary process by which cytotoxic T lymphocytes (CTL) and natural killer (NK) cells lyse target cells involves the regulated exocytosis of granules present in the cytoplasm of the effector. These granules contain proteins, such as perforin and the granzymes, that play a direct role in the killing process. The localization of a human T and NK cell-specific protein, granulysin

  7. Bone morphogenetic protein- and mating-dependent secretory cell growth and migration in the Drosophila accessory gland

    PubMed Central

    Leiblich, Aaron; Marsden, Luke; Gandy, Carina; Corrigan, Laura; Jenkins, Rachel; Hamdy, Freddie; Wilson, Clive

    2012-01-01

    The paired male accessory glands of Drosophila melanogaster enhance sperm function, stimulate egg production, and reduce female receptivity to other males by releasing a complex mixture of glycoproteins from a secretory epithelium into seminal fluid. A small subpopulation of about 40 specialized secretory cells, called secondary cells, resides at the distal tip of each gland. We show that these cells grow via mechanisms promoted by mating. If aging males mate repeatedly, a subset of these cells delaminates from and migrates along the apical surface of the glandular epithelium toward the proximal end of the gland. Remarkably, these secretory cells can transfer to females with sperm during mating. The frequency of this event increases with age, so that more than 50% of triple-mated, 18-d-old males transfer secondary cells to females. Bone morphogenetic protein signaling specifically in secondary cells is needed to drive all of these processes and is required for the accessory gland to produce its normal effects on female postmating behavior in multiply mated males. We conclude that secondary cells are secretory cells with unusual migratory properties that can allow them to be transferred to females, and that these properties are a consequence of signaling that is required for secondary cells to maintain their normal reproductive functions as males age and mate. PMID:23129615

  8. Colloidal gold granules as markers for cell surface receptors in the scanning electron microscope

    Microsoft Academic Search

    M. Horisberger; Jaqueline Rosset; H. Bauer

    1975-01-01

    Summary  A rapid method has been developed to visualize cell surface receptors in the SEM. Thus mannan at the surface ofCandida utilis cells was localized by stabilized colloidal gold granules coated with either antimannan antibodies or Con A.

  9. TWIK-1 contributes to the intrinsic excitability of dentate granule cells in mouse hippocampus.

    PubMed

    Yarishkin, Oleg; Lee, Da; Kim, Eunju; Cho, Chang-Hoon; Choi, Jae; Lee, C; Hwang, Eun; Park, Jae-Yong

    2014-11-19

    BackgroundTwo-pore domain K+ (K2P) channels have been shown to modulate neuronal excitability. However, physiological function of TWIK-1, the first identified member of the mammalian K2P channel family, in neuronal cells is largely unknown.ResultsWe found that TWIK-1 proteins were expressed and localized mainly in the soma and proximal dendrites of dentate gyrus granule cells (DGGCs) rather than in distal dendrites or mossy fibers. Gene silencing demonstrates that the outwardly rectifying K+ current density was reduced in TWIK-1-deficient granule cells. TWIK-1 deficiency caused a depolarizing shift in the resting membrane potential (RMP) of DGGCs and enhanced their firing rate in response to depolarizing current injections. Through perforant path stimulation, TWIK-1 deficient granule cells showed altered signal input-output properties with larger EPSP amplitude values and increased spiking compared to control DGGCs. In addition, supra-maximal perforant path stimulation evoked a graded burst discharge in 44% of TWIK-1-deficient cells, which implies impairment of EPSP-spike coupling.ConclusionsThese results showed that TWIK-1 is functionally expressed in DGGCs and contributes to the intrinsic excitability of these cells. The TWIK-1 channel is involved in establishing the RMP of DGGCs; it attenuates sub-threshold depolarization of the cells during neuronal activity, and contributes to EPSP-spike coupling in perforant path-to-granule cell synaptic transmission. PMID:25406588

  10. Quantitative subcellular study of apical pole membranes from chicken oxyntic cells in resting and HCl secretory state

    Microsoft Academic Search

    Cecilia S. Koenig; Miguel Bronfman

    1987-01-01

    Vertebrate oxyntic cells, responsible for gastric HCI production, undergo a remarkable mor- phological reorganization in relation to their secretory cycle. In resting state, the luminal surface of the cells is smooth; a peculiar system of endocellular mem- branes, the tubular system, occupies the luminal cyto- plasm. Actin filaments frame a cortical network be- tween the tubular system and the luminal

  11. Increased Secretory Leukocyte Protease Inhibitor (SLPI) Production by Highly Metastatic Mouse Breast Cancer Cells

    PubMed Central

    Sayers, Kevin T.; Brooks, Alan D.; Sayers, Thomas J.; Chertov, Oleg

    2014-01-01

    The precise molecular mechanisms enabling cancer cells to metastasize from the primary tumor to different tissue locations are still largely unknown. Secretion of some proteins by metastatic cells could facilitate metastasis formation. The comparison of secreted proteins from cancer cells with different metastatic capabilities in vivo might provide insight into proteins involved in the metastatic process. Comparison of the secreted proteins from the mouse breast cancer cell line 4T1 and its highly metastatic 4T1.2 clone revealed a prominent differentially secreted protein which was identified as SLPI (secretory leukocyte protease inhibitor). Western blotting indicated higher levels of the protein in both conditioned media and whole cell lysates of 4T1.2 cells. Additionally higher levels of SLPI were also observed in 4T1.2 breast tumors in vivo following immunohistochemical staining. A comparison of SLPI mRNA levels by gene profiling using microarrays and RT-PCR did not detect major differences in SLPI gene expression between the 4T1 and 4T1.2 cells indicating that SLPI secretion is regulated at the protein level. Our results demonstrate that secretion of SLPI is drastically increased in highly metastatic cells, suggesting a possible role for SLPI in enhancing the metastatic behavior of breast cancer cell line 4T1. PMID:25110884

  12. Targeted calretinin expression in granule cells of calretinin-null mice restores normal cerebellar functions

    Microsoft Academic Search

    Bertrand Bearzatto; Laurent Servais; Céline Roussel; David Gall; Fawzia Baba-Aďssa; Stéphane Schurmans; Alban de Kerchove d'Exaerde; Guy Cheron; Serge N. Schiffmann

    2005-01-01

    Ca 2 binding proteins such as calretinin, characterized by the presence of EF-hand motifs that bind Ca 2+ ions, are involved in the shaping of intraneuronal Ca 2+ fluxes. In the cerebellar cortex, information processing tightly relies on variations in intracellular Ca 2+ concentration in Purkinje and granule cells. Calretinin-deficient (Cr ?\\/? ) mice present motor discoordination, suggesting cellular and

  13. Secretory cell outgrowth, PAX2 and serous carcinogenesis in the Fallopian tube.

    PubMed

    Chen, Eleanor Y; Mehra, Karishma; Mehrad, Mitra; Ning, Gang; Miron, Alexander; Mutter, George L; Monte, Nicholas; Quade, Bradley J; McKeon, Frank D; Yassin, Yosuf; Xian, Wa; Crum, Christopher P

    2010-09-01

    The 'p53 signature' is a benign secretory cell outgrowth in the distal Fallopian tube that shares properties with ovarian serous cancer-including p53 mutations-and is a putative serous cancer precursor. We expanded the precursor definition to all secretory cell outgrowths (SCOUTs) of 30 or more cells and scored normal (N) and altered (A) expression of both p53 and PAX2, a gene down-regulated in ovarian and endometrial cancer. SCOUTs were identified by BCL2/p73 staining in tubes from women with serous carcinoma, inherited mutations in BRCA1 or BRCA2 and controls. SCOUTs were prevalent in both proximal and distal tube and significantly associated with serous carcinoma versus the others (p < 0.001); 89% were PAX2 (A) and 26% were PAX2 (A)/p53 (A) (p53 signatures). PAX2 (A)/p53 (N) SCOUTs were free of p53 mutations; however, 12 of 13 p53 signatures were PAX2 (A). A tubal carcinoma and contiguous SCOUT were p53 (A)/PAX2 (A) and shared the same p53 mutation. SCOUTs are discretely localized alterations commonly containing altered expression of multiple genes within histologically benign tubal epithelium. Geographic distribution in the tube varies by genotype and immunophenotype, from regionally unrestricted (PAX2) to greater likelihood specific area (fimbria) of shared prevalence (PAX2 and p53). This study reveals, for the first time, an entity (SCOUT) that is associated with serous cancer, expands the topography of altered PAX2 expression in the female genital tract mucosa and highlights another potential pathway disturbance involved in early serous carcinogenesis in the Fallopian tube. PMID:20597068

  14. Secretory cell outgrowth, PAX2 and serous carcinogenesis in the fallopian tube

    PubMed Central

    Chen, Eleanor Y.; Mehra, Karishma; Mehrad, Mitra; Ning, Gang; Miron, Alexander; Mutter, George L.; Monte, Nicholas; Quade, Bradley J.; McKeon, Frank D.; Yassin, Yosuf; Xian, Wa; Crum, Christopher P.

    2010-01-01

    The “p53 signature” is a benign secretory cell outgrowth in the distal fallopian tube that shares properties with ovarian serous cancer – including p53 mutations - and is a putative serous cancer precursor. We expanded the precursor definition to all secretory cell outgrowths (SCOUTs) of 30 or more cells and scored normal (N) and altered (A) expression of both p53 and PAX2, a gene down-regulated in ovarian and endometrial cancer. SCOUTs were identified by BCL2/p73 staining in tubes from women with serous carcinoma, inherited mutations in BRCA1 or BRCA2, and controls. SCOUTs were prevalent in both proximal and distal tube and significantly associated with serous carcinoma versus the others (p <0.001). Eighty-nine percent were PAX2 (A); 26% were PAX2 (A)/p53 (A) (p53 signatures). PAX2 (A)/p53 (N) SCOUTs were free of p53 mutations; however, 12 of 13 p53 signatures were PAX2 (A). A tubal carcinoma and contiguous SCOUT were p53 (A)/PAX2 (A) and shared the same p53 mutation. SCOUTs are discretely localized alterations commonly containing altered expression of multiple genes within histologically benign tubal epithelium. Geographic distribution in the tube varies by genotype and immunophenotype, from widespread (PAX2) to confinement to a specific area (fimbria) of shared prevalence (PAX2 and p53). This study reveals, for the first time, an entity (SCOUT) that is associated with serous cancer, expands the topography of altered PAX2 expression in the female genital tract mucosa and highlights another potential pathway disturbance involved in early serous carcinogenesis in the fallopian tube. PMID:20597068

  15. Regulation of output spike patterns by phasic inhibition in cerebellar granule cells.

    PubMed

    Nieus, Thierry R; Mapelli, Lisa; D'Angelo, Egidio

    2014-01-01

    The complex interplay of multiple molecular mechanisms taking part to synaptic integration is hard to disentangle experimentally. Therefore, we developed a biologically realistic computational model based on the rich set of data characterizing the cerebellar glomerulus microcircuit. A specific issue was to determine the relative role of phasic and tonic inhibition in dynamically regulating granule cell firing, which has not been clarified yet. The model comprised the excitatory mossy fiber-granule cell and the inhibitory Golgi cell-granule cell synapses and accounted for vesicular release processes, neurotransmitter diffusion and activation of different receptor subtypes. Phasic inhibition was based on stochastic GABA release and spillover causing activation of two major classes of postsynaptic receptors, ?1 and ?6, while tonic inhibition was based on steady regulation of a Cl(-) leakage. The glomerular microcircuit model was validated against experimental responses to mossy fiber bursts while metabotropic receptors were blocked. Simulations showed that phasic inhibition controlled the number of spikes during burst transmission but predicted that it specifically controlled time-related parameters (firing initiation and conclusion and first spike precision) when the relative phase of excitation and inhibition was changed. In all conditions, the overall impact of ?6 was larger than that of ?1 subunit-containing receptors. However, ?1 receptors controlled granule cell responses in a narrow ±10 ms band while ?6 receptors showed broader ±50 ms tuning. Tonic inhibition biased these effects without changing their nature substantially. These simulations imply that phasic inhibitory mechanisms can dynamically regulate output spike patterns, as well as calcium influx and NMDA currents, at the mossy fiber-granule cell relay of cerebellum without the intervention of tonic inhibition. PMID:25202237

  16. Regulation of output spike patterns by phasic inhibition in cerebellar granule cells

    PubMed Central

    Nieus, Thierry R.; Mapelli, Lisa; D'Angelo, Egidio

    2014-01-01

    The complex interplay of multiple molecular mechanisms taking part to synaptic integration is hard to disentangle experimentally. Therefore, we developed a biologically realistic computational model based on the rich set of data characterizing the cerebellar glomerulus microcircuit. A specific issue was to determine the relative role of phasic and tonic inhibition in dynamically regulating granule cell firing, which has not been clarified yet. The model comprised the excitatory mossy fiber—granule cell and the inhibitory Golgi cell—granule cell synapses and accounted for vesicular release processes, neurotransmitter diffusion and activation of different receptor subtypes. Phasic inhibition was based on stochastic GABA release and spillover causing activation of two major classes of postsynaptic receptors, ?1 and ?6, while tonic inhibition was based on steady regulation of a Cl? leakage. The glomerular microcircuit model was validated against experimental responses to mossy fiber bursts while metabotropic receptors were blocked. Simulations showed that phasic inhibition controlled the number of spikes during burst transmission but predicted that it specifically controlled time-related parameters (firing initiation and conclusion and first spike precision) when the relative phase of excitation and inhibition was changed. In all conditions, the overall impact of ?6 was larger than that of ?1 subunit-containing receptors. However, ?1 receptors controlled granule cell responses in a narrow ±10 ms band while ?6 receptors showed broader ±50 ms tuning. Tonic inhibition biased these effects without changing their nature substantially. These simulations imply that phasic inhibitory mechanisms can dynamically regulate output spike patterns, as well as calcium influx and NMDA currents, at the mossy fiber—granule cell relay of cerebellum without the intervention of tonic inhibition. PMID:25202237

  17. Maturation of postnatally generated olfactory bulb granule cells depends on functional ?-protocadherin expression.

    PubMed

    Ledderose, Julia; Dieter, Sandra; Schwarz, Martin K

    2013-01-01

    ?-protocadherins (?-pcdhs) are transmembrane receptor proteins ubiquitously expressed in the postnatal and adult mouse brain. ?-pcdhs are required for normal neuronal development as shown for spinal cord interneurons, retinal ganglion cells and cortical neurons. To test the role of ?-pcdhs during development of subventricular zone progenitor cells and their subsequent differentiation into olfactory granule cells we generated a conditional ?-pcdh(lox/lox) allele (?-pcdh(lox/lox)) allowing for functional ?-pcdh inactivation upon lentivirus-mediated Cre-recombinase expression selectively in subventricular zone progenitor cells. While ?-pcdh loss did not alter the proliferation of subventricular zone progenitors, ?-pcdh ko progenitors that reached the main olfactory bulb showed a significant reduction in dendritic arborization and failed to develop dendritic spines. Our results suggest that olfactory bulb granule cell maturation necessitates functional ?-pcdh expression. PMID:23515096

  18. Maturation of postnatally generated olfactory bulb granule cells depends on functional ?-protocadherin expression

    PubMed Central

    Ledderose, Julia; Dieter, Sandra; Schwarz, Martin K.

    2013-01-01

    ?-protocadherins (?-pcdhs) are transmembrane receptor proteins ubiquitously expressed in the postnatal and adult mouse brain. ?-pcdhs are required for normal neuronal development as shown for spinal cord interneurons, retinal ganglion cells and cortical neurons. To test the role of ?-pcdhs during development of subventricular zone progenitor cells and their subsequent differentiation into olfactory granule cells we generated a conditional ?-pcdhlox/lox allele (?-pcdhlox/lox) allowing for functional ?-pcdh inactivation upon lentivirus-mediated Cre-recombinase expression selectively in subventricular zone progenitor cells. While ?-pcdh loss did not alter the proliferation of subventricular zone progenitors, ?-pcdh ko progenitors that reached the main olfactory bulb showed a significant reduction in dendritic arborization and failed to develop dendritic spines. Our results suggest that olfactory bulb granule cell maturation necessitates functional ?-pcdh expression. PMID:23515096

  19. 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

  20. Secretory Leukocyte Protease Inhibitor (SLPI) Expression and Tumor Invasion in Oral Squamous Cell Carcinoma

    PubMed Central

    Wen, Jie; Nikitakis, Nikolaos G.; Chaisuparat, Risa; Greenwell-Wild, Teresa; Gliozzi, Maria; Jin, Wenwen; Adli, Azita; Moutsopoulos, Niki; Wu, Tanxia; Warburton, Gary; Wahl, Sharon M.

    2011-01-01

    Differential expression of secretory leukocyte protease inhibitor (SLPI) impacts on tumor progression. SLPI directly inhibits elastase and other serine proteases, and regulates matrix metalloproteinases, plasminogen activation, and plasmin downstream targets to influence invasion. We examined tissues from human oral squamous cell carcinoma (OSCC) for SLPI expression in parallel with proteases associated with tumor progression and evaluated their relationships using tumor cell lines. Significantly decreased SLPI was detected in OSCC compared to normal oral epithelium. Furthermore, an inverse correlation between SLPI and histological parameters associated with tumor progression, including stage of invasion, pattern of invasion, invasive cell grade, and composite histological tumor score was evident. Conversely, elevated plasmin and elastase were positively correlated with histological parameters of tumor invasion. In addition to its known inhibition of elastase, we identify SLPI as a novel inhibitor of plasminogen activation through its interaction with annexin A2 with concomitant reduced plasmin generation by macrophages and OSCC cell lines. In an in vitro assay measuring invasive activity, SLPI blocked protease-dependent tumor cell migration. Our data suggest that SLPI may possess antitumorigenic activity by virtue of its ability to interfere with multiple requisite proteolytic steps underlying tumor cell invasion and may provide insight into potential stratification of oral cancer according to risk of occult metastasis, guiding treatment strategies. PMID:21641406

  1. Ultrastructural and functional analysis of secretory goblet cells in the midgut of the lepidopteran Anticarsia gemmatalis.

    PubMed

    Gomes, F M; Carvalho, D B; Machado, E A; Miranda, K

    2013-05-01

    Defoliation caused by Anticarsia gemmatalis larvae affects the commercial production of the soybean. Although regulation of the digestion of soybean components has become part of the suggested strategy to overcome problems caused by Anticarsia larvae, few studies have focused on the morphological and cellular aspects of Anticarsia intestinal tissue. We have therefore further analyzed the morphology and ultrastructure of the midgut of 5th instar larvae of A. gemmatalis. Dissected midgut was subjected to chemical or cryo-fixation and then to several descriptive and analytical techniques associated with both light and electron microscopy in order to correlate anatomical and physiological aspects of this organ. Histological analysis revealed typical anatomy composed of a cell layer limited by a peritrophic membrane. The identified lepidoptera-specific goblet cells were shown to contain several mitochondria inside microvilli of the goblet cell cavity and a vacuolar H(+)-ATPase possibly coupled to a K(+)-pumping system. Columnar cells were present and exhibited microvilli dispersed along the apical region that also presented secretory characteristics. We additionally found evidence for the secretion of polyphosphate (PolyP) into the midgut, a result corroborating previous reports suggesting an excretion route from the goblet cell cavity toward the luminal space. Thus, our results suggest that the Anticarsia midgut not only possesses several typical lepidopteran features but also presents some unique aspects such as the presence of a tubular network and PolyP-containing apocrine secretions, plus an apparent route for the release of cellular debris by the goblet cells. PMID:23397424

  2. [The role of mitochondrial uniporter in calcium-homeostasis of the exorbital lacrimal gland secretory cells].

    PubMed

    Kotliarova, A B; Merlavs'ky?, V M; Dorosh, O M; Man'ko, V V

    2014-01-01

    The role of mitochondrial calcium-uniporter in calcium-homeostasis maintenance and correlations of calcium-uniporter with other calcium-transport systems of the rat exorbital lacrimal gland secretory cells were studied. The experiments were performed on intact and digitonin-permeabilized cells. The interdependence of calcium-uniporter and other calcium-transporting systems functioning was estimated on the basis of additivity of their inhibitors/agonists effects, which was accompanied with a decrease in the Ca2+ content in the gland cells. It was found that in conditions of simultaneously inhibition of sarco endoplasmic reticulum Ca2+-ATPase (SERCA) and mitochondrial calcium-uniporter Ca2+ passively released from different calcium stores, because the effects of these calcium-transport systems inhibitors (thapsigargin and ruthenium red, respectively) were additive. Similarly, the processes of inositol-1,4,5-trisphosphate receptors (IP3Rs) activation and calcium-uniporter inhibition were additive. In contrast, the effects of ryanodine and ruthenium red on the Ca2+ content in cells were significantly non-additive. In addition, ryanodine at concentrations 1-3 ?M reduced respiration rate of studied cells in dose-dependent manner, and this effect was persisted at cells preincubation with ruthenium red or tapsigargin. Thus, besides the activation of ryanodine receptors (RyRs) in endoplasmic reticulum, ryanodine inhibits Ca2+ influx to the mitochondrial matrix, that was insensitive to ruthenium red. PMID:25566673

  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.; Rhodes, Christopher J.

    2002-01-01

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

  4. Eosinophil Secretion of Granule-Derived Cytokines

    PubMed Central

    Spencer, Lisa A.; Bonjour, Kennedy; Melo, Rossana C. N.; Weller, Peter F.

    2014-01-01

    Eosinophils are tissue-dwelling leukocytes, present in the thymus, and gastrointestinal and genitourinary tracts of healthy individuals at baseline, and recruited, often in large numbers, to allergic inflammatory foci and sites of active tissue repair. The biological significance of eosinophils is vast and varied. In health, eosinophils support uterine and mammary gland development, and maintain bone marrow plasma cells and adipose tissue alternatively activated macrophages, while in response to tissue insult eosinophils function as inflammatory effector cells, and, in the wake of an inflammatory response, promote tissue regeneration, and wound healing. One common mechanism driving many of the diverse eosinophil functions is the regulated and differential secretion of a vast array of eosinophil-derived cytokines. Eosinophils are distinguished from most other leukocytes in that many, if not all, of the over three dozen eosinophil-derived cytokines are pre-synthesized and stored within intracellular granules, poised for very rapid, stimulus-induced secretion. Eosinophils engaged in cytokine secretion in situ utilize distinct pathways of cytokine release that include classical exocytosis, whereby granules themselves fuse with the plasma membrane and release their entire contents extracellularly; piecemeal degranulation, whereby granule-derived cytokines are selectively mobilized into vesicles that emerge from granules, traverse the cytoplasm and fuse with the plasma membrane to release discrete packets of cytokines; and eosinophil cytolysis, whereby intact granules are extruded from eosinophils, and deposited within tissues. In this latter scenario, extracellular granules can themselves function as stimulus-responsive secretory-competent organelles within the tissue. Here, we review the distinctive processes of differential secretion of eosinophil granule-derived cytokines. PMID:25386174

  5. Relationship between Membrane Permeability and Specificity of Human Secretory Phospholipase A2 Isoforms during Cell Death

    PubMed Central

    Nelson, Jennifer; Gibbons, Elizabeth; Pickett, Katalyn R.; Streeter, Michael; Warcup, Ashley O.; Yeung, Celestine H.-Y.; Judd, Allan M.; Bell, John D.

    2011-01-01

    Summary During apoptosis, a number of physical changes occur in the cell membrane including a gradual increase in permeability to vital stains such as propidium iodide. This study explored the possibility that one consequence of membrane changes concurrent with early modest permeability is vulnerability to degradation by secretory phospholipase A2 (sPLA2). The activity of this hydrolytic enzyme toward mammalian cells depends on the health of the cell; healthy cells are resistant, but they become susceptible early during programmed death. Populations of S49 lymphoma cells during programmed death were classified by flow cytometry based on permeability to propidium iodide and susceptibility to sPLA2. The apoptotic inducers thapsigargin and dexamethasone caused modest permeability to propidium iodide and increased staining by merocyanine 540, a dye sensitive to membrane perturbations. Various sPLA2 isozymes (human groups IIa, V, X, and snake venom) preferentially hydrolyzed the membranes of cells that displayed enhanced permeability. In contrast, cells exposed briefly to a calcium ionophore showed the increase in cell staining intensity by merocyanine 540 without accompanying uptake of propidium iodide. Under that condition, only the snake venom and human group × enzymes hydrolyzed cells that were dying. These results suggested that cells showing modest permeability to propidium iodide during the early phase of apoptosis are substrates for sPLA2 and that specificity among isoforms of the enzyme depends on the degree to which the membrane has been perturbed during the death process. This susceptibility to hydrolysis may be important as part of the signal to attract macrophages toward apoptotic cells. PMID:21510917

  6. Ca˛? signaling and fluid secretion by secretory cells of the airway epithelium.

    PubMed

    Lee, Robert J; Foskett, J Kevin

    2014-06-01

    Cytoplasmic Ca(2+) is a master regulator of airway physiology; it controls fluid, mucus, and antimicrobial peptide secretion, ciliary beating, and smooth muscle contraction. The focus of this review is on the role of cytoplasmic Ca(2+) in fluid secretion by airway exocrine secretory cells. Airway submucosal gland serous acinar cells are the primary fluid secreting cell type of the cartilaginous conducting airways, and this review summarizes the current state of knowledge of the molecular mechanisms of serous cell ion transport, with an emphasis on their regulation by intracellular Ca(2+). Many neurotransmitters that regulate secretion from serous acinar cells utilize Ca(2+) as a second messenger. Changes in intracellular Ca(2+) concentration regulate the activities of ion transporters and channels involved in transepithelial ion transport and fluid secretion, including Ca(2+)-activated K(+) channels and Cl(-) channels. We also review evidence of interactions of Ca(2+) signaling with other signaling pathways (cAMP, NO) that impinge upon different ion transport pathways, including the cAMP/PKA-activated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel. A better understanding of Ca(2+) signaling and its targets in airway fluid secretion may identify novel strategies to intervene in airway diseases, for example to enhance fluid secretion in CF airways. PMID:24703093

  7. Evidence for specific TRPM8 expression in human prostate secretory epithelial cells: functional androgen receptor requirement.

    PubMed

    Bidaux, G; Roudbaraki, M; Merle, C; Crépin, A; Delcourt, P; Slomianny, C; Thebault, S; Bonnal, J-L; Benahmed, M; Cabon, F; Mauroy, B; Prevarskaya, N

    2005-06-01

    TRPM8 (melastatine-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily of cation channels, has been shown to be a calcium-channel protein. TRPM8 mRNA has also been shown to be overexpressed in prostate cancer and is considered to play an important role in prostate physiology. This study was designed to determine the androgen-regulation mechanisms for TRPM8 mRNA expression and to identify the phenotype of TRPM8-expressing cells in the human prostate. Our findings show that trpm8 gene expression requires a functional androgen receptor. Furthermore, this article argues strongly in favour of the fact that the trpm8 gene is a primary androgen-responsive gene. Single-cell reverse transcriptase PCR and immunohistochemical experiments also showed that the trpm8 gene was mainly expressed in the apical secretory epithelial cells of the human prostate and trpm8 down-regulation occurred during the loss of the apical differentiated phenotype of the primary cultured human prostate epithelial cells. The androgen-regulated trpm8 expression mechanisms are important in understanding the progression of prostate cancer to androgen-independence. These findings may contribute to design a strategy to predict prostate cancer status from the TRPM8 mRNA level. Furthermore, as the TRPM8 channel is localized in human prostate cells, it will be interesting to understand its physiological function in the normal prostate and its potential role in prostate cancer development. PMID:15947109

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

    PubMed

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

    2015-02-01

    Neuronal dendritic spines have been speculated to function as independent computational units, yet evidence for active electrical computation in spines is scarce. Here we show that strictly local voltage-gated sodium channel (Nav) activation can occur during excitatory postsynaptic potentials in the spines of olfactory bulb granule cells, which we mimic and detect via combined two-photon uncaging of glutamate and calcium imaging in conjunction with whole-cell recordings. We find that local Nav activation boosts calcium entry into spines through high-voltage-activated calcium channels and accelerates postsynaptic somatic depolarization, without affecting NMDA receptor-mediated signaling. Hence, Nav-mediated boosting promotes rapid output from the reciprocal granule cell spine onto the lateral mitral cell dendrite and thus can speed up recurrent inhibition. This striking example of electrical compartmentalization both adds to the understanding of olfactory network processing and broadens the general view of spine function. PMID:25619656

  9. The Role of CED3Related Cysteine Proteases in Apoptosis of Cerebellar Granule Cells

    Microsoft Academic Search

    Basil A. Eldadah; Alexander G. Yakovlev; Alan I. Faden

    1997-01-01

    The CED-3-related cysteine proteases (CRCPs) have been im- plicated as mediators of apoptosis, primarily in hematogenous cell systems, but their role in neuronal apoptosis remains un- clear. The present study examined the role of two CRCP families—CPP32- and interleukin-1b converting enzyme (ICE)- like cysteine proteases—in apoptosis of cerebellar granule cells (CGCs) caused by withdrawal of serum and\\/or potassium (K 1).

  10. GABA Acts as a Ligand Chaperone in the Early Secretory Pathway to Promote Cell Surface Expression of GABAA Receptors

    PubMed Central

    Eshaq, Randa S.; Stahl, Letha D.; Stone, Randolph; Smith, Sheryl S.; Robinson, Lucy C.; Leidenheimer, Nancy J.

    2010-01-01

    GABA (?-aminobutyric acid) is the primary inhibitory neurotransmitter in brain. The fast inhibitory effect of GABA is mediated through the GABAA receptor, a postsynaptic ligand-gated chloride channel. We propose that GABA can act as a ligand chaperone in the early secretory pathway to facilitate GABAA receptor cell surface expression. Forty-two hrs of GABA treatment increased the surface expression of recombinant receptors expressed in HEK 293 cells, an effect accompanied by an increase in GABA-gated chloride currents. In time-course experiments, a 1 hr GABA exposure, followed by a 5 hr incubation in GABA-free medium, was sufficient to increase receptor surface expression. A shorter GABA exposure could be used in HEK 293 cells stably transfected with the GABA transporter GAT-1. In rGAT-1HEK 293 cells, the GABA effect was blocked by the GAT-1 inhibitor NO-711, indicating that GABA was acting intracellularly. The effect of GABA was prevented by brefeldin A (BFA), an inhibitor of early secretory pathway trafficking. Coexpression of GABAA receptors with the GABA synthetic enzyme glutamic acid decarboxylase 67 (GAD67) also resulted in an increase in receptor surface levels. GABA treatment failed to promote the surface expression of GABA binding site mutant receptors, which themselves were poorly expressed at the surface. Consistent with an intracellular action of GABA, we show that GABA does not act by stabilizing surface receptors. Furthermore, GABA treatment rescued the surface expression of a receptor construct that was retained within the secretory pathway. Lastly, the lipophilic competitive antagonist (+)bicuculline promoted receptor surface expression, including the rescue of an secretory pathway-retained receptor. Our results indicate that a neurotransmitter can act as a ligand chaperone in the early secretory pathway to regulate the surface expression of its receptor. This effect appears to rely on binding site occupancy, rather than agonist-induced structural changes, since chaperoning is observed with both an agonist and a competitive antagonist. PMID:20580636

  11. Identification of hepatocyte nuclear factor-3 binding sites in the Clara cell secretory protein gene.

    PubMed Central

    Bingle, C D; Gitlin, J D

    1993-01-01

    To determine the mechanisms of cell-specific gene expression in the developing pulmonary epithelium the Clara cell secretory protein (CCSP) gene promoter was analysed by DNAase I footprinting. A prominent site of protein-DNA interaction was detected from nucleotides -132 to -76 using nuclear extract from mouse lung and human H441 cells. Mobility shift analysis revealed that an oligonucleotide corresponding to this region interacted with multiple proteins from lung and H441 cell nuclear extracts. Analysis of the nucleotide sequence of this region identified two potential binding sites for hepatocyte nuclear factor 3 (HNF-3), and consistent with this finding binding to this CCSP oligonucleotide was specifically competed for by an oligonucleotide corresponding to the HNF-3-binding site from the mouse transthyretin gene. Mobility shift of the CCSP oligonucleotide was supershifted using antisera specific to HNF-3 alpha and HNF-3 beta, and HNF-3 alpha and HNF-3 beta translated in vitro were found to bind specifically to this same oligonucleotide. Co-transfection of HNF-3 alpha- and HNF-3 beta-expression plasmids increased cell-specific reporter gene activity in H441 cells transfected with a CCSP-CAT gene chimeric construct containing this -132 to -76 region. Taken together, these results suggest a role for HNF-3 in mediating cell-specific CCSP gene expression within the bronchiolar epithelium. These findings support the hypothesis that members of the HNF-3 'forkhead' family of transcription factors determine gene expression and cell fate in multiple cell lineages derived from the primitive gut endoderm. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8216221

  12. Hyperplastic effects of aerosolized sodium metabisulfite on rat airway mucus-secretory epithelial cells.

    PubMed

    Pon, D J; van Staden, C J; Boulet, L; Rodger, I W

    1994-09-01

    The ability of aerosolized sodium metabisulfite to induce hypertrophic and hyperplastic changes in rat airway secretory epithelial cells was investigated. A 10% solution of sodium metabisulfite was aerosolized into a Plexiglas exposure chamber, using an ultrasonic humidifier. The level of SO2 gas generated by this apparatus was measured to be 500 ppm. Measured levels of neutral and acidic mucous glycoproteins in extracts from tracheal and lung tissue were used as indices of hypertrophic (increases in mucus content per cell) and hyperplastic (increased numbers of cells containing mucus per gram of tissue) changes occurring in mucus-secreting cells of the airways. Exposing rats to sodium metabisulfite for 3 weeks resulted in profound increases in total neutral mucous glycoproteins found in tracheal and lung tissue (6.2-fold and 10.1-fold, respectively), compared with the H2O-treated counterparts. Total acidic mucous glycoproteins were significantly elevated in lung tissue only (13.5-fold). In addition, neutral and acidic mucous glycoproteins were elevated 20-fold and 9-fold, respectively, in bronchoalveolar lavage samples prepared from sodium metabisulfite exposed animals. These results indicate that aerosolized sodium metabisulfite may be a useful agent for developing small animal models of mucus hypersecretion. PMID:7842384

  13. Hippocampal granule cells are necessary for normal spatial learning but not for spatially-selective pyramidal cell discharge

    Microsoft Academic Search

    B. L. McNaughton; C. A. Barnes; J. Meltzer; R. J. Sutherland

    1989-01-01

    The effects of massive destruction of granule cells of the fascia dentata on the spatial and temporal firing characteristics of pyramidal cells in the CA1 and CA3 subfields of the hippocampus were examined in freely moving rats. Microinjections of the neurotoxin colchicine were made at a number of levels along the septo-temporal axis of the dentate gyri of both hemispheres,

  14. Distinct integrin-dependent signals define requirements for lytic granule convergence and polarization in natural killer cells.

    PubMed

    Hsu, Hsiang-Ting; Orange, Jordan S

    2014-10-01

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

  15. Regulators of Cerebellar Granule Cell Development Act Through Specific Signaling Pathways

    NSDL National Science Digital Library

    David Vaudry (European Institute for Peptide Research; Laboratoryof Cellular and Molecular Neuroendocrinology)

    2003-06-06

    The proper development of the central nervous system depends upon a finely tuned balance between cell proliferation and programmed cell death (PCD). Although PCD was initially believed to depend solely on the inability of certain neurons to obtain access to a limited supply of trophic factors, it has become apparent that the local production of death signals is also critical. In this Viewpoint, we discuss several pathways implicated in the survival of cerebellar granule cellsâ?? both pathways that protect from apoptosis and pathways that promote apoptosisâ??and describe how these disparate pathways converge on the final common mediators of PCD. Information on other important pathways implicated in granule cell survival may be found in the Connections Maps.

  16. GPR56-regulated granule cell adhesion is essential for rostral cerebellar development

    PubMed Central

    Koirala, Samir; Jin, Zhaohui; Piao, Xianhua; Corfas, Gabriel

    2009-01-01

    Mutations in GPR56, an orphan G-protein coupled receptor (GPCR), cause bilateral frontoparietal polymicrogyria (BFPP), a disorder characterized by mental retardation, seizures, motor developmental delay, and ataxia. BFPP patients have structural abnormalities of the cerebral cortex, cerebellum and pons. To shed light on the function of GPR56 and the anatomical and behavioral defects underlying BFPP, we analyzed the cerebellum of mice lacking this GPCR. Gpr56 ?/? mice display a severe malformation of the rostral cerebellum that develops perinatally. Defects involve fusion of adjacent lobules, disrupted layering of neurons and glia, and fragmentation of the pial basement membrane. At the age of defect onset, GPR56 expression is restricted specifically to developing granule cells in the rostral cerebellum, suggesting that GPR56 regulates properties of these cells. Indeed, granule cells from the rostral region of perinatal Gpr56 ?/? cerebella show loss of adhesion to extracellular matrix molecules of the pial basement membrane. RNAi-mediated knockdown of GPR56 recapitulates the loss of adhesion seen in knockouts and re-expression of GPR56 rescues the adhesion defect in knockout granule cells. Loss of GPR56 does not affect cell proliferation, migration or neurite outgrowth. These studies establish a novel role for GPR56 in the adhesion of developing neurons to basal lamina molecules, and suggest that this adhesion is critical for maintenance of the pia and proper cerebellar morphogenesis. PMID:19515912

  17. Clara Cell Secretory Protein in Tracheobronchial Aspirates and Umbilical Cord Serum of Extremely Premature Infants with Systemic Inflammation

    Microsoft Academic Search

    Wolfgang Thomas; Silvia Seidenspinner; Maria Chmielnicka-Kopaczyk; Alexander Marx; Johannes Wirbelauer; Marta Szymankiewicz; Christian P. Speer

    2010-01-01

    Background: A systemic fetal inflammatory response, reflected by chorioamnionitis with funisitis, is a risk factor for bronchopulmonary dysplasia. Clara cell secretory protein (CC10), a product of pulmonary Clara cells, has anti-inflammatory properties. Local down-regulation of CC10 has been associated with inflammatory lung disease. Increased serum levels of CC10 can indicate injury to alveolar-capillary integrity. Objective: We hypothesized that extremely premature

  18. Induction of FcεRII\\/CD23 on Human T Cells by Excretory and Secretory Antigen of Dirofilaria immitis

    Microsoft Academic Search

    Kunio A. Yamaoka; Setsuko Tsukidate; Megumu Higaki; Nobuyuki Miyasaka; Koichiro Fujita

    1991-01-01

    We investigated the capacity of excretory and secretory antigen (ES) derived from living filarial worms in the induction of CD23 expression on human peripheral blood T cells by using flow cytometry. ES (10 ?g\\/ml) significantly induced the expression of CD23 on human T cells. Moreover, increased CD23 expression was completely abolished by preincubation with specific antibody to ES. The results

  19. Binding and Selective Detection of the Secretory N-terminal Domain of the Alzheimer Amyloid Precursor Protein on Cell Surfaces

    Microsoft Academic Search

    Jens Hoffmann; Claus U. Pietrzik; Markus P. Kummer; Christiane Twiesselmann; Christoph Bauer; Volker Herzog

    1999-01-01

    SUMMARY The secretory N-terminal domain of the Alzheimer amyloid precursor protein (sAPP) evokes specific responses in cells on binding to their surfaces. Because APP is ex- pressed in a large variety of cell types, the localization of sAPP binding requires detection techniques that selectively recognize sAPP as a ligand. For this purpose, we prepared anti- bodies against recombinant sAPP695 (sAPPrec)

  20. Agglutinating secretory IgA preserves intestinal epithelial cell integrity during apical infection by Shigella flexneri.

    PubMed

    Mathias, Amandine; Longet, Stéphanie; Corthésy, Blaise

    2013-08-01

    Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-?B nuclear translocation, and inhibition of proinflammatory mediator secretion. Our results add to the understanding of the function of SIgA-mediated immune exclusion by identifying a mode of action whereby the formation of immune complexes translates into maintenance of the integrity of epithelial cells lining the mucosa. This novel mechanism of protection mediated by SIgA is important to extend the arsenal of effective strategies to fight against S. flexneri mucosal invasion. PMID:23753631

  1. Agglutinating Secretory IgA Preserves Intestinal Epithelial Cell Integrity during Apical Infection by Shigella flexneri

    PubMed Central

    Mathias, Amandine; Longet, Stéphanie

    2013-01-01

    Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-?B nuclear translocation, and inhibition of proinflammatory mediator secretion. Our results add to the understanding of the function of SIgA-mediated immune exclusion by identifying a mode of action whereby the formation of immune complexes translates into maintenance of the integrity of epithelial cells lining the mucosa. This novel mechanism of protection mediated by SIgA is important to extend the arsenal of effective strategies to fight against S. flexneri mucosal invasion. PMID:23753631

  2. Morphology and monoterpene biosynthetic capabilities of secretory cell clusters isolated from glandular trichomes of peppermint (Mentha piperita L.).

    PubMed

    McCaskill, D; Gershenzon, J; Croteau, R

    1992-07-01

    Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (Mr=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the vacuole. The molecular-weight exclusion limit for this permeability was shown to be less than approx. 1800, based on exclusion of fluorescein-conjugated dextran (Mr ? 1800). Intact secretory cell clusters very efficiently incorporated [(3)H]geranyl pyrophosphate into monoterpenes. The addition of exogenous cofactors and redox substrates affected the distribution of monoterpenes synthesized from [(3)H]geranyl pyrophosphate, demonstrating that the cell clusters were permeable to these compounds and that the levels of endogenous cofactors and redox substrates were depleted in the isolated cells. When provided with the appropriate cofactors, such as NADPH, NAD(+), ATP, ADP and coenzyme A, the isolated secretory cell clusters incorporated [(14)C]sucrose into monoterpenes, indicating that these structures are capable of the de-novo biosynthesis of monoterpenes from a primary carbon source, and that they maintain a high degree of metabolic competence in spite of their permeable nature. PMID:24178138

  3. Perfluoroalkylated compounds induce cell death and formation of reactive oxygen species in cultured cerebellar granule cells.

    PubMed

    Reistad, Trine; Fonnum, Frode; Mariussen, Espen

    2013-03-27

    The present communication investigates the effects of different perfluoroalkylated compounds (PFCs) on formation of reactive oxygen species (ROS) and cell death in cultured cerebellar granule cells. This allows direct comparison with similar effects found for other environmental contaminants like polychlorinated biphenyls and brominated flame-retardants. The increase in ROS formation and cell death was assayed using the fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA) and the trypan blue exclusion assay. The effects of the PFCs were structure dependent. Cell death was induced at relatively low concentrations by perfluorooctyl sulfonate (PFOS), perfluorooctane sulfonylamide (PFOSA) and the fluorotelomer alcohol 1H, 1H, 2H, 2H-perfluorodecanol (FTOH 8:2) with EC(50)-values of 62 ± 7.6, 13 ± 1.8 and 15 ± 4.2 ?M (mean ± SD) respectively. PFOS, perfluorooctanoic acid (PFOA) and PFOSA induced a concentration dependent increase in ROS formation with EC(50)-values of 27 ± 9.0, 25 ± 11 and 57 ± 19?M respectively. Reduced cell viability and ROS formation were observed at concentration level close to what is found in serum of occupationally exposed workers. The effect of PFCs on ROS formation and cell viability was compared with other halogenated compounds and future investigations should emphasize effects of mixtures and how physical chemical properties of the compounds influence their toxicity. PMID:23340305

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

    SciTech Connect

    Kendall, M.D.

    1988-03-01

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

  5. Inhibition of cerebellar granule cell turning by alcohol

    Microsoft Academic Search

    T. Kumada; Y. Komuro; Y. Li; T. Hu; Z. Wang; Y. Littner; H. Komuro

    2010-01-01

    Ectopic neurons are often found in the brains of fetal alcohol spectrum disorders (FASD) and fetal alcohol syndrome (FAS) patients, suggesting that alcohol exposure impairs neuronal cell migration. Although it has been reported that alcohol decreases the speed of neuronal cell migration, little is known about whether alcohol also affects the turning of neurons. Here we show that ethanol exposure

  6. Expression of S100 beta in sensory and secretory cells of the vertebrate inner ear

    NASA Technical Reports Server (NTRS)

    Fermin, C. D.; Martin, D. S.

    1995-01-01

    We evaluated anti-S100 beta expression in the chick (Gallus domesticus) inner ear and determined that: 1) the monomer anti-S100 beta is expressed differentially in the vestibular and auditory perikarya; 2) expression of S100 beta in the afferent nerve terminals is time-related to synapse and myelin formation; 3) the expression of the dimer anti-S100 alpha alpha beta beta and monomer anti-S100 beta overlaps in most inner ear cell types. Most S100 alpha alpha beta beta positive cells express S100 beta, but S100 beta positive cells do not always express S100 alpha alpha beta beta. 4) the expression of S100 beta is diffused over the perikaryal cytoplasm and nuclei of the acoustic ganglia but is concentrated over the nuclei of the vestibular perikarya. 6) S100 beta is expressed in secretory cells, and it is co-localized with GABA in sensory cells. 7) Color thresholding objective quantitation indicates that the amount of S100 beta was higher (mean 22, SD +/- 4) at E19 than at E9 (mean 34, SD +/- 3) in afferent axons. 8) Moreover, S100 beta was unchanged between E11-E19 in the perikaryal cytoplasm, but did change over the nuclei. At E9, 74%, and at E21, 5% of vestibular perikarya were positive. The data suggest that S100 beta may be physically associated with neuronal and ionic controlling cells of the vertebrate inner ear, where it could provide a dual ionic and neurotrophic modulatory function.

  7. Kinetic Evaluation of Cell Membrane Hydrolysis during Apoptosis by Human Isoforms of Secretory Phospholipase A2*

    PubMed Central

    Olson, Erin D.; Nelson, Jennifer; Griffith, Katalyn; Nguyen, Thaothanh; Streeter, Michael; Wilson-Ashworth, Heather A.; Gelb, Michael H.; Judd, Allan M.; Bell, John D.

    2010-01-01

    Some isoforms of secretory phospholipase A2 (sPLA2) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6–48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA2 were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform. PMID:20139082

  8. Multifaceted Roles of Cysteinyl Leukotrienes in Eliciting Eosinophil Granule Protein Secretion

    PubMed Central

    Baptista-dos-Reis, Renata; Muniz, Valdirene S.; Neves, Josiane S.

    2015-01-01

    Cysteinyl leukotrienes (cysLTs) are cell membrane-impermeant lipid mediators that play major roles in the pathogenesis of eosinophilic inflammation and are recognized to act via at least 2 receptors, namely, cysLT1 receptor (cysLT1R) and cysLT2 receptor (cysLT2R). Eosinophils, which are granulocytes classically associated with host defense against parasitic helminthes and allergic conditions, are distinguished from leukocytes by their dominant population of cytoplasmic crystalloid (also termed secretory, specific, or secondary) granules that contain robust stores of diverse preformed proteins. Human eosinophils are the main source of cysLTs and are recognized to express both cysLTs receptors (cysLTRs) on their surface, at the plasma membrane. More recently, we identified the expression of cysLTRs in eosinophil granule membranes and demonstrated that cysLTs, acting via their granule membrane-expressed receptors, elicit secretion from cell-free human eosinophil granules. Herein, we review the multifaceted roles of cysLTs in eliciting eosinophil granule protein secretion. We discuss the intracrine and autocrine/paracrine secretory responses evoked by cysLTs in eosinophils and in cell-free extracellular eosinophil crystalloid granules. We also discuss the importance of this finding in eosinophil immunobiology and speculate on its potential role(s) in eosinophilic diseases. PMID:25866815

  9. GABAA Receptors of Cerebellar Granule Cells in Culture: Interaction with Benzodiazepines.

    PubMed

    Cupello, Aroldo; Di Braccio, Mario; Gatta, Elena; Grossi, Giancarlo; Nikas, Periklis; Pellistri, Francesca; Robello, Mauro

    2013-10-12

    GABAA receptor mediated inhibition plays an important role in modulating the input/output dynamics of cerebellum. A characteristic of cerebellar GABAA receptors is the presence in cerebellar granule cells of subunits such as ?6 and ? which give insensitivity to classical benzodiazepines. In fact, cerebellar GABAA receptors have generally been considered a poor model for testing drugs which potentially are active at the benzodiazepine site. In this overview we show how rat cerebellar granule cells in culture may be a useful model for studying new benzodiazepine site agonists. This is based on the pharmacological separation of diazepam-sensitive ?1 ?2/3 ?2 receptors from those which are diazepam-insensitive and contain the ?6 subunit. This is achieved by utilizing furosemide/Zn(2+) which block ?6 containing and incomplete receptors. PMID:24122079

  10. Leaf volatiles and secretory cells of Alpinia zerumbet (Pers.) Burtt et Smith (Zingiberaceae).

    PubMed

    Victório, C P; Arruda, R do Carmo de O; Riehl, C A S; Lage, C L S

    2011-06-01

    Plant leaves are commonly used in folk medicine and food industry. Their volatile composition is an important determinant in such applications. However, to properly assess the quality of volatiles, proper analytic tools must be utilised. Accordingly, the static headspace technique was used to evaluate the main volatiles emitted from in vitro-grown Alpinia zerumbet plants cultured with indole-3-acetic acid, thidiazuron, benzyladenine or kinetin, under standard physical conditions, as compared to those of field-grown donor plants. Although the leaf aroma of the donor plants was found to be a complex mixture, mainly consisting of sabinene, ? and ?-terpinene, 1,8-cineole and caryophyllene, volatile analyses from most of the in vitro samples only revealed the presence of sabinene and caryophyllene. Many alkanes were found in the aromas after treating plantlets with cytokinins. Histochemical analysis of leaf sections was also carried out. Secretory cells found in the epidermis and mesophyll showed a strong positive reaction to lipophilic compounds using Oil red and Nile blue reagents. These findings demonstrated how in vitro conditions may alter the quality of volatiles in micropropagation systems, while leaf anatomy analysis revealed a large quantity of oil cells in the mesophyll as a constant feature responsible for the production of volatile compounds in both donor and in vitro-grown plants. PMID:21644174

  11. Elevated Plasma Clara Cell Secretory Protein Concentration is Associated with High-Grade Primary Graft Dysfunction

    PubMed Central

    Diamond, Joshua M.; Kawut, Steven M.; Lederer, David J.; Ahya, Vivek N.; Kohl, Benjamin; Sonett, Joshua; Palmer, Scott M.; Crespo, Maria; Wille, Keith; Lama, Vibha; Shah, Pali D.; Orens, Jonathan; Bhorade, Sangeeta; Weinacker, Ann; Demissie, Ejigayehu; Bellamy, Scarlett; Christie, Jason D.; Ware, Lorraine B.

    2011-01-01

    Primary graft dysfunction (PGD) is the leading cause of early post-transplant morbidity and mortality after lung transplantation. Clara cell secretory protein (CC16) is produced by the non-ciliated lung epithelium and may serve as a plasma marker of epithelial cell injury. We hypothesized that elevated levels of CC16 would be associated with increased odds of PGD. We performed a prospective cohort study of 104 lung transplant recipients. Median plasma CC16 levels were determined at three time points: pre-transplant and 6 and 24 hours post transplant. The primary outcome was the development of grade 3 PGD within the first 72 hours after transplantation. Multivariable logistic regression was performed to evaluate for confounding by donor and recipient demographics and surgical characteristics. Twenty-nine patients (28%) developed grade 3 PGD within the first 72 hours. The median CC16 level 6 hours after transplant was significantly higher in patients with PGD (13.8 ng/ml (IQR 7.9, 30.4 ng/ml)) than in patients without PGD (8.2 ng/ml (IQR 4.5, 19.1 ng/ml)), p = 0.02. Elevated CC16 levels were associated with increased odds of PGD after lung transplantation. Damage to airway epithelium or altered alveolar permeability as a result of lung ischemia and reperfusion may explain this association. PMID:21299834

  12. A Cell-Specific Transgenic Approach in Xenopus Reveals the Importance of a Functional p24 System for a Secretory Cell

    PubMed Central

    Bouw, Gerrit; Van Huizen, Rick; Jansen, Eric J.R.; Martens, Gerard J.M.

    2004-01-01

    The p24?, -?, -?, and -? proteins are major multimeric constituents of cycling endoplasmic reticulum-Golgi transport vesicles and are thought to be involved in protein transport through the early secretory pathway. In this study, we targeted transgene overexpression of p24?2 specifically to the Xenopus intermediate pituitary melanotrope cell that is involved in background adaptation of the animal and produces high levels of its major secretory cargo proopiomelanocortin (POMC). The transgene product effectively displaced the endogenous p24 proteins, resulting in a melanotrope cell p24 system that consisted predominantly of the transgene p24?2 protein. Despite the severely distorted p24 machinery, the subcellular structures as well as the level of POMC synthesis were normal in these cells. However, the number and pigment content of skin melanophores were reduced, impairing the ability of the transgenic animal to fully adapt to a black background. This physiological effect was likely caused by the affected profile of POMC-derived peptides observed in the transgenic melanotrope cells. Together, our results suggest that in the early secretory pathway an intact p24 system is essential for efficient secretory cargo transport or for supplying cargo carriers with the correct protein machinery to allow proper secretory protein processing. PMID:14699062

  13. Effects of adult-generated granule cells on coordinated network activity in the dentate gyrus

    PubMed Central

    Lacefield, Clay O.; Itskov, Vladimir; Reardon, Thomas; Hen, René; Gordon, Joshua A.

    2012-01-01

    Throughout the adult life of most mammals, new neurons are continuously generated in the dentate gyrus of the hippocampal formation. Recent work has documented specific cognitive deficits after elimination of adult hippocampal neurogenesis in rodents, suggesting that these neurons may contribute to information processing in hippocampal circuits. Young adult-born neurons exhibit enhanced excitability and have altered capacity for synaptic plasticity in hippocampal slice preparations in vitro. Still, little is known about the effect of adult-born granule cells on hippocampal activity in vivo. In order to assess the impact of these new neurons on neural circuits in the dentate, we recorded perforant-path evoked responses and spontaneous network activity from the dentate gyrus of urethane-anesthetized mice whose hippocampus had been focally X-irradiated to eliminate the population of young adult-born granule cells. After X-irradiation, perforant-path responses were reduced in magnitude. In contrast, there was a marked increase in the amplitude of spontaneous gamma-frequency bursts in the dentate gyrus and hilus, as well as increased synchronization of dentate neuron firing to these bursts. A similar increase in gamma burst amplitude was also found in animals in which adult neurogenesis was eliminated using the GFAP:TK pharmacogenetic ablation technique. These data suggest that young neurons may inhibit or destabilize recurrent network activity in the dentate and hilus. This unexpected result yields a new perspective on how a modest number of young adult–generated granule cells may modulate activity in the larger population of mature granule cells, rather than acting solely as independent encoding units. PMID:20882540

  14. Prenatal ethanol exposure reduces phosphoinositide hydrolysis stimulated by quisqualate in rat cerebellar granule cell cultures

    Microsoft Academic Search

    Philip G. Rhodes; Zhengwei Cai; Nannan Zhu

    1994-01-01

    Prenatal ethanol exposure-induced alteration in poly-phosphoinositide (PPI) hydrolysis stimulated by excitatory amino acids\\u000a (EAA) was studied in rat cerebellar granule cells previously labeled with [3H]myoinositol. The prenatal exposure to ethanol was achieved via maternal consumption of a Sustacal (chocolate flavored) liquid\\u000a diet containing either 5% ethanol (w\\/v, 35% of calories) or isocaloric sucrose (pair-fed) substituted for ethanol from gestation\\u000a d

  15. High Ratio of Synaptic Excitation to Synaptic Inhibition in Hilar Ectopic Granule Cells of Pilocarpine-Treated Rats

    PubMed Central

    Zhan, Ren-Zhi; Timofeeva, Olga

    2010-01-01

    After experimental status epilepticus, many dentate granule cells born into the postseizure environment migrate aberrantly into the dentate hilus. Hilar ectopic granule cells (HEGCs) have also been found in persons with epilepsy. These cells exhibit a high rate of spontaneous activity, which may enhance seizure propagation. Electron microscopic studies indicated that HEGCs receive more recurrent mossy fiber innervation than normotopic granule cells in the same animals but receive much less inhibitory innervation. This study used hippocampal slices prepared from rats that had experienced pilocarpine-induced status epilepticus to test the hypothesis that an imbalance of synaptic excitation and inhibition contributes to the hyperexcitability of HEGCs. Mossy fiber stimulation evoked a much smaller GABAA receptor–mediated inhibitory postsynaptic currents (IPSC) in HEGCs than in normotopic granule cells from either control rats or rats that had experienced status epilepticus. However, recurrent mossy fiber-evoked excitatory postsynaptic currents (EPSCs) of similar size were recorded from HEGCs and normotopic granule cells in status epilepticus–experienced rats. HEGCs exhibited the highest frequency of miniature excitatory postsynaptic currents (mEPSCs) and the lowest frequency of miniature inhibitory postsynaptic currents (mIPSCs) of any granule cell group. On average, both mEPSCs and mIPSCs were of higher amplitude, transferred more charge per event, and exhibited slower kinetics in HEGCs than in granule cells from control rats. Charge transfer per unit time in HEGCs was greater for mEPSCs and much less for mIPSCs than in the normotopic granule cell groups. A high ratio of excitatory to inhibitory synaptic function probably accounts, in part, for the hyperexcitability of HEGCs. PMID:20881195

  16. The role of cell density in the survival of cultured cerebellar granule neurons.

    PubMed

    Young, T H; Huang, J H; Hung, S H; Hsu, J P

    2000-12-15

    The dependence for survival of cerebellar granule neurons on the cell density was examined both experimentally and theoretically. The results of batch experiments revealed that the cell survival index (CSI) was inappreciable, if cell density was below a critical level. If cell density exceeded this critical value, CSI increased with the increase in cell density. In addition, CSI was significantly increased by using a conditioned medium from the dense cultures. This suggests that not only cell density promotes survival of neurons, but also an increased concentration of growth factors produced by neurons has a direct effect on the survival of the neurons. A quantitative model describing the distribution of the growth factor at different cell densities was proposed to investigate the role of cell density in the survival of the neurons. We showed the existence of a critical level for cell density, and good agreement in the improvement of CSI was found between the theoretical prediction and the experimental result. Finally, the average concentration of growth factor necessary for cell survival based on our model was in a reasonable range compared to the practice of the addition of neurotrophic factors to the medium of cultured cerebellar granule neurons. PMID:11033558

  17. Protein kinase D2 regulates chromogranin A secretion in human BON neuroendocrine tumour cells

    Microsoft Academic Search

    Götz von Wichert; Teresa Edenfeld; Julia von Blume; Holger Krisp; Denis Krndija; Heidrun Schmid; Franz Oswald; Ulrike Lother; Paul Walther; Guido Adler; Thomas Seufferlein

    2008-01-01

    Chromogranin A is a member of the granin family of acidic secretory glycoproteins that is found in secretory granules of many endocrine cells including neuroendocrine tumour cells. This hormone serves as a model system for autonomous hormone secretion by the so called functional neuroendocrine tumours of the gastrointestinal tract. The precise regulation of chromogranin secretion at the level of the

  18. Cyclic AMP-dependent protein kinase (cAPK) regulatory subunits are packaged and secreted by many exocrine and endocrine cells

    SciTech Connect

    Mednieks, M.I.; Hand, A.R.

    1986-05-01

    Regulatory (R) subunits of cAPK were identified by us as components of rat and human saliva by photoaffinity labeling with (/sup 32/P)-8-azido cyclic AMP. Photoaffinity labeling of purified rat parotid granule contents and immunogold labeling of thin sections with monoclonal antibodies showed the presence of R subunits in granules. The authors now report that cAPK R subunits are present in secretory granules and are apparently secreted by many exocrine and endocrine cell types. Labeling of thin sections of rat tissues with antibody to R subunits and protein A-gold shows gold particles over secretory granules of endocrine cells of the pituitary, pancreas and intestine. Zymogen granules of exocrine pancreatic acinar cells, the dense cores of secretory granules of seminal vesicle epithelial cells and secretory product in the seminal vesicle lumina were prominently labeled with gold. Photoaffinity labeling shows that pancreatic secretions and seminal vesicle contents have cAPK components. Phosphorylative modification of cellular proteins by cAMP controls hormonally stimulated protein secretion by many cell types. Although no catalytic activity was detected, identification of R subunits in granules and as secretory products indicates that they may have multiple roles in cellular mechanisms of action of cyclic AMP-mediated events in secretory cells.

  19. Secretory cells of the supraoptic nucleus have central as well as neurohypophysial projections.

    PubMed

    Inyushkin, A N; Orlans, H O; Dyball, R E J

    2009-10-01

    Conventional neuroanatomical methods may fail to demonstrate the presence of axons that are finer than 1 microm in diameter because such processes are near or below the limit of resolution of the light microscope. The presence of such axons can, however, be readily demonstrated by recording. The most easily interpreted type of recording for this purpose is the demonstration of antidromic activation of the cell body following stimulation of the region through which the axon passes. We have exploited this technique in the hypothalamus and have demonstrated the presence of double axonal projections or axons branching very near the cell bodies of the secretory cells of the neurohypophysial system in the rat supraoptic nucleus. We found that a small proportion of supraoptic magnocellular cells could be antidromically activated both from the neural stalk and from elsewhere in the hypothalamus, including the suprachiasmatic nucleus (8 cells of a total of 182) and the antero-ventral third ventricular region (AV3V; 4 of 182 cells) near the organum vasculosum of the lamina terminalis (OVLT). Collision of antidromic and orthodromic spikes showed that the cells were clearly antidromically (rather than synaptically, or orthodromically) activated from both sites. A stimulus applied to one of the axons prevented propagation of a spike evoked by a pulse delivered to the other axon until sufficient time had elapsed after the first stimulus for the resultant spike to have propagated from the first stimulus site along one cell process (towards the cell body or branch point), and from this point along the other axonal branch to the second stimulus site (there was also a short additional delay period during which the axon at the site of the second stimulus recovered from its absolute refractory period). If the interval between the stimuli was progressively reduced, there came a point where the second spike failed. Such a clear demonstration of dual projections in a system where the cells were previously thought to have only a single axon raises the possibility that many nerve cells in the CNS have previously unsuspected projections. PMID:19754684

  20. Cellular Barcodes for Efficiently Profiling Single-Cell Secretory Responses by Microengraving

    E-print Network

    Yamanaka, Yvonne Joy

    We present a method that uses fluorescent cellular barcodes to increase the number of unique samples that can be analyzed simultaneously by microengraving, a nanowell array-based technique for quantifying the secretory ...

  1. Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells1

    PubMed Central

    Turner, Glenn; Gershenzon, Jonathan; Nielson, Erik E.; Froehlich, John E.; Croteau, Rodney

    1999-01-01

    Circumstantial evidence based on ultrastructural correlation, specific labeling, and subcellular fractionation studies indicates that at least the early steps of monoterpene biosynthesis occur in plastids. (4S)-Limonene synthase, which is responsible for the first dedicated step of monoterpene biosynthesis in mint species, appears to be translated as a preprotein bearing a long plastidial transit peptide. Immunogold labeling using polyclonal antibodies raised to the native enzyme demonstrated the specific localization of limonene synthase to the leucoplasts of peppermint (Mentha × piperita) oil gland secretory cells during the period of essential oil production. Labeling was shown to be absent from all other plastid types examined, including the basal and stalk cell plastids of the secretory phase glandular trichomes. Furthermore, in vitro translation of the preprotein and import experiments with isolated pea chloroplasts were consistent in demonstrating import of the nascent protein to the plastid stroma and proteolytic processing to the mature enzyme at this site. These experiments confirm that the leucoplastidome of the oil gland secretory cells is the exclusive location of limonene synthase, and almost certainly the preceding steps of monoterpene biosynthesis, in peppermint leaves. However, succeeding steps of monoterpene metabolism in mint appear to occur outside the leucoplasts of oil gland cells. PMID:10398724

  2. Secretory expression of Lentinula edodes intracellular laccase by yeast high-cell-density system: sub-milligram production of difficult-to-express secretory protein.

    PubMed

    Kurose, Takeshi; Saito, Yuta; Kimata, Koichi; Nakagawa, Yuko; Yano, Akira; Ito, Keisuke; Kawarasaki, Yasuaki

    2014-06-01

    While a number of heterologous expression systems have been reported for extracellular laccases, there are few for the intracellular counterparts. The Lentinula edodes intracellular laccase Lcc4 is an industrially potential enzyme with its unique substrate specificity. The heterologous production of the intracellular laccase, however, had been difficult because of its expression-dependent toxicity. We previously demonstrated that recombinant yeast cells synthesized and, interestingly, secreted Lcc4 only when they were suspended to an inducing medium in a high cell-density (J. Biosci. Bioeng., 113, 154-159, 2012). The high cell-density system was versatile and applicable to other difficult-to-express secretory proteins. Nevertheless, the system's great dependence on aeration, which was a practical obstacle to scale-up production of the enzyme and some other proteins, left the secretion pathway and enzymatic properties of the Lcc4 uncharacterized. In this report, we demonstrate a successful production of Lcc4 by applying a jar-fermentor to the high cell-density system. The elevated yield (0.6 mg L(-1)) due to the sufficient aeration allowed us to prepare and purify the enzyme to homogeneity. The enzyme had been secreted as a hyper-glycosylated protein, resulting in smear band-formations in SDS-PAGE. The amino acid sequencing analysis suggested that the N-terminal 17 residues had been recognized as a secretion signal. The recombinant enzyme showed similar enzymatic properties to the naturally occurring Lcc4. The characteristics of the scale-upped expression system, which includes helpful information for the potential users, have also been described. PMID:24411669

  3. 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 ?M. 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. The mode of interaction of H+ at the single-channel level and implications of such interactions at cerebellar granule cell GABAA receptors are discussed. PMID:11208970

  4. Cell-Specific Expression of a Clara Cell Secretory Protein-Human Growth Hormone Gene in the Bronchiolar Epithelium of Transgenic Mice

    Microsoft Academic Search

    Brian P. Hackett; Jonathan D. Gitlin

    1992-01-01

    Clara cell secretory protein (CCSP) is an abundant 10-kDa protein synthesized and secreted by nonciliated epithelial cells lining the respiratory and terminal bronchioles of the lung. CCSP gene expression is an informative developmental marker within the bronchiolar epithelium recapitulating cellular differentiation in the distal respiratory epithelium during late fetal and early postnatal life. To define the mechanisms that establish and

  5. 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.

  6. A reinforcing circuit action of extrasynaptic GABAA receptor modulators on cerebellar granule cell inhibition.

    PubMed

    Santhakumar, Vijayalakshmi; Meera, Pratap; Karakossian, Movses H; Otis, Thomas S

    2013-01-01

    GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of ? subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (?6100Q) allele show that modulators and agonists selective for ?-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that ? subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR ? subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants. PMID:23977374

  7. A Reinforcing Circuit Action of Extrasynaptic GABAA Receptor Modulators on Cerebellar Granule Cell Inhibition

    PubMed Central

    Santhakumar, Vijayalakshmi; Otis, Thomas S.

    2013-01-01

    GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of ? subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (?6100Q) allele show that modulators and agonists selective for ?-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that ? subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR ? subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants. PMID:23977374

  8. Secretory phospholipase A2-IIa upregulates HER/HER2-elicited signaling in lung cancer cells.

    PubMed

    Dong, Zhongyun; Meller, Jaroslaw; Succop, Paul; Wang, Jiang; Wikenheiser-Brokamp, Kathryn; Starnes, Sandra; Lu, Shan

    2014-09-01

    Lung cancer is the leading cause of cancer death worldwide. There is an urgent need for early diagnostic tools and novel therapies in order to increase lung cancer survival. Secretory phospholipase A2 group IIa (sPLA2-IIa) is involved in inflammation, tumorigenesis and metastasis. We were the first to uncover that cancer cells secrete sPLA2?IIa. sPLA2?IIa is overexpressed in almost all specimens of human lung cancers examined and is significantly elevated in the plasma of lung cancer patients. High levels of plasma sPLA2-IIa are significantly associated with advanced stage and decreased overall cancer survival. In this study, we further showed that elevated HER/HER2?PI3K-Akt-NF-?B signaling contributes to sPLA2-IIa overexpression in lung cancer cells. sPLA2-IIa in turn phosphorylates and activates HER2 and HER3 in a time- and dose?dependent manner in lung cancer cells. The structure and sequence?based docking analysis revealed that sPLA2-IIa ? hairpin shares structural similarity with the corresponding EGF hairpin. sPLA2-IIa forms an extensive interface with EGFR and brings the two lobes of EGFR into an active conformation. sPLA2-IIa also enhances the NF-?B promoter activity. Anti-sPLA2-IIa antibody, but not the small molecule sPLA2-IIa inhibitor LY315920, significantly inhibits sPLA2?IIa-induced activation of NF-?B promoter. Our findings support the notion that sPLA2-IIa functions as a ligand for the EGFR family of receptors leading to an elevated HER/HER2-elicited signaling. Plasma sPLA2-IIa can potentially serve as lung cancer biomarker and sPLA2?IIa is a potential therapeutic target against lung cancer. PMID:24913497

  9. Combined Inhibition of p97 and the Proteasome Causes Lethal Disruption of the Secretory Apparatus in Multiple Myeloma Cells

    PubMed Central

    Auner, Holger W.; Moody, Anne Marie; Ward, Theresa H.; Kraus, Marianne; Milan, Enrico; May, Philippa; Chaidos, Aristeidis; Driessen, Christoph; Cenci, Simone; Dazzi, Francesco; Rahemtulla, Amin; Apperley, Jane F.; Karadimitris, Anastasios; Dillon, Niall

    2013-01-01

    Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma. PMID:24069311

  10. Combined inhibition of p97 and the proteasome causes lethal disruption of the secretory apparatus in multiple myeloma cells.

    PubMed

    Auner, Holger W; Moody, Anne Marie; Ward, Theresa H; Kraus, Marianne; Milan, Enrico; May, Philippa; Chaidos, Aristeidis; Driessen, Christoph; Cenci, Simone; Dazzi, Francesco; Rahemtulla, Amin; Apperley, Jane F; Karadimitris, Anastasios; Dillon, Niall

    2013-01-01

    Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma. PMID:24069311

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

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan); Akimoto, Yoshihiro [Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan); Nakamichi, Yoko; Nishiwaki, Chiyono [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan); Kawakami, Hayato [Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan); Nagamatsu, Shinya, E-mail: shinya@ks.kyorin-u.ac.jp [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)

    2009-12-04

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

  12. The composition of intracellular granules from the metal-accumulating cells of the common garden snail (Helix aspersa).

    PubMed Central

    Howard, B; Mitchell, P C; Ritchie, A; Simkiss, K; Taylor, M

    1981-01-01

    Certain cells in the hepatopancreas of the common garden snail (Helix aspersa) contain intracellular granules that are sites of metal-ion accumulation. These granules have been extracted and investigated by u.v. and i.r. spectroscopy, atomic-absorption spectroscopy, X-ray microanalysis, thermogravimetric analysis, enzymic assay and microanalysis. The deposits contain about 18% (w/w) water, 5% (w/w) organic matter and 76% (w/w) inorganic material of which the main components are Ca2+, Mg2+ and P2O7(4)-. The possible origin of these granules is discussed, as is their role in detoxifying heavy-metal ions. PMID:6272732

  13. Calnuc plays a role in dynamic distribution of Galphai but not Gbeta subunits and modulates ACTH secretion in AtT-20 neuroendocrine secretory cells.

    PubMed

    Lin, Ping; Fischer, Thierry; Lavoie, Christine; Huang, Haining; Farquhar, Marilyn Gist

    2009-01-01

    In AtT-20 cells ACTH secretion is regulated by both Ca2+ and G proteins. We previously demonstrated that calnuc, an EF-hand Ca2+ binding protein which regulates Alzheimer's beta-amyloid precursor protein (APP) biogenesis, binds both Ca2+ as well as Galpha subunits. Here we investigate calnuc's role in G protein-mediated regulation of ACTH secretion in AtT-20 neuroendocrine secretory cells stably overexpressing calnuc-GFP. Similar to endogenous calnuc, calnuc-GFP is mainly found in the Golgi, on the plasma membrane (PM), and associated with regulated secretion granules (RSG). By deconvolution immunofluorescence, calnuc-GFP partially colocalizes with Galphai1/2 and Galphai3 at the PM and on RSG. Cytosolic calnuc(DeltaSS)-CFP with the signal sequence deleted also partially colocalizes with RSG and partially cosediments with Galphai1/2 in fractions enriched in RSG. Overexpression of calnuc-GFP specifically increases the distribution of Galphai1/2 on the PM whereas the distribution of Gbeta subunits and synaptobrevin 2 (Vamp 2) is unchanged. Overexpression of calnuc-GFP or cytosolic calnuc(DeltaSS)-CFP enhances ACTH secretion two-fold triggered by mastoparan or GTPgammaS but does not significantly affect glycosaminoglycan (GAG) chain secretion along the constitutive pathway or basal secretion of ACTH. Calnuc's facilitating effects on ACTH secretion are decreased after introducing anti-Galphai1/2, Galphai3, Gbeta or calnuc IgG into permeabilized cells but not when Galpha12 or preimmune IgG is introduced. The results suggest that calnuc binds to Galpha subunits on the Golgi and on RSG and that overexpression of calnuc causes redistribution of Galphai subunits to the PM and RSG, indicating that calnuc plays a role in dynamic distribution of only Galpha but not Gbeta subunits. Thus calnuc may connect G protein signaling and calcium signaling during regulated secretion. PMID:19320978

  14. Syntaxin 11 marks a distinct intracellular compartment recruited to the immunological synapse of NK cells to colocalize with cytotoxic granules

    PubMed Central

    Dabrazhynetskaya, Alena; Ma, Jinxia; Guerreiro-Cacais, Andre Ortlieb; Arany, Zita; Rudd, Eva; Henter, Jan-Inge; Karre, Klas; Levitskaya, Jelena; Levitsky, Victor

    2012-01-01

    Abstract The syntaxin 11 (STX11) gene is mutated in a proportion of patients with familial haemophagocytic lymphohistiocytosis (FHL) and exocytosis of cytotoxic granules is impaired in STX11-deficient NK cells. However, the subcellular localization, regulation of expression and molecular function of STX11 in NK cells and other cytotoxic lymphocytes remain unknown. Here we demonstrate that STX11 expression is strictly controlled by several mechanisms in a cell-type-specific manner and that the enzymatic activity of the proteasome is required for STX11 expression in NK cells. In resting NKL cells, STX11 was localized in the cation-dependent mannose-6-phosphate receptor (CD-M6PR)-containing compartment, which was clearly distinct from cytotoxic granules or Rab27a-expressing vesicles. These subcellular structures appeared to fuse at the contact area with NK-sensitive target cells as demonstrated by partial colocalization of STX11 with perforin and Rab27a. Although STX11-deficent allo-specific cytotoxic T-lymphocytes efficiently lysed target cells and released cytotoxic granules, they exhibited a significantly lower extent of spontaneous association of perforin with Rab27a as compared with STX11-expressing T cells. Thus, our results suggest that STX11 promotes the fusion of Rab27a-expressing vesicles with cytotoxic granules and reveal an additional level of complexity in the spatial/temporal segregation of subcellular structures participating in the process of granule-mediated cytotoxicity. PMID:21342435

  15. [IP3-sensitive Ca(2+)-channels of endoplasmic reticulum in secretory cells of the rat exorbital lacrimal gland].

    PubMed

    Kotliarova, A B; Man'ko, V V

    2013-01-01

    The role of inositol-1,4,5-trisphosphate of (IP3)-sensitive Ca2+ channels in Ca2+ homeostasis maintenance under activation of M-cholinergic receptors and P2Y receptors in the secretory cells of the rat lacrimal gland was investigated. The study was carried out on intact and permeabilized secretory cells of exorbital lacrimal glands of rats. The cells were isolated using the modified Herzog, Sides, Miller method (1976) and permeabilized with digitonin (50 mg per 0.5 million cells). The functioning of the Ca(2+)-transport systems was estimated by changes of Ca2+ content in the studied cells, which was determined by the spectrophotometric method using arsenazo III. It was shown that IP3-sensitive Ca2+ channels (IP3Rs) of investigated cells are directly inhibited by 2-APB (10 microM/l). On the other hand, the channels are activated by IP3, cholinomimetic (carbacholine) and purine receptor agonist (ATP). When both M-cholinergic receptors and P2Y receptors were activated Ca2+ was released from the same IP3-sensitive store because the effects of ATP and carbacholine at high concentrations (1mM/l and 10 microM/l, respectively) on the Ca2+ content were non-additive. The presence of the store-operated Ca(2+)-channels in secretory cells of the lacrimal gland is confirmed by the observed increase of cellular Ca2+ content as a result of Ca2+ mobilization from the store by carbacholine or thapsigargin and following restoration of Ca2+ concentration in the extracellular solution. PMID:24479320

  16. The transmembrane domain of the prohormone convertase PC3: A key motif for targeting to the regulated secretory pathway

    PubMed Central

    Lou, Hong; Smith, Angela M.; Coates, Leigh C.; Cawley, Niamh X.; Peng Loh, Y.; Birch, Nigel P.

    2007-01-01

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617–638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC31–638 (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC31–616 (PC3-616) and PC3-?TM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-?TM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway. PMID:17240044

  17. Modulation of secretory leukoprotease inhibitor gene expression in human bronchial epithelial cells by phorbol ester.

    PubMed Central

    Maruyama, M; Hay, J G; Yoshimura, K; Chu, C S; Crystal, R G

    1994-01-01

    Secretory leukoprotease inhibitor (SLPI), a 12-kD nonglycosylated serine antiprotease, helps to protect the epithelial surface of the airways from the destructive capacity of neutrophil elastase. Based on the recognition that SLPI levels can increase in the presence of airway inflammation, we hypothesized that inflammatory stimuli should modulate the expression of the SLPI gene in airway epithelial cells. To evaluate this, the modulation of SLPI gene expression with various inflammatory stimuli was evaluated in the HS-24 human bronchial epithelial cell line. After preliminary studies showed that several inflammatory mediators enhanced SLPI messenger RNA (mRNA) levels, PMA was used as a model inflammatory stimulus. PMA significantly increased the level of 0.7-kb SLPI mRNA transcripts in HS-24 cells in a dose- and time-dependent fashion and increased the amount of SLPI protein in the culture supernatant. Nuclear run-on analyses showed that the SLPI gene transcription rate increased approximately twofold after PMA stimulation. Transfection studies using fusion genes composed of fragments of up to 1.2 kb of the 5' flanking sequence of the SLPI gene and a luciferase reporter gene demonstrated potent promoter activity in the 131-bp segment (-115 to +16 relative to the transcription start site), and all longer segments up to 1.2 kb, whereas smaller segments showed low promoter activity. An 18-bp element (-98 to -115), in a region with homology to PMA-responsive regions in the Moloney murine leukemia virus enhancer and the IL-8 gene, was shown to be of importance in the level of transcription of the SLPI gene. However, this element was not responsible for the upregulation of SLPI gene expression by PMA. Evaluation of HS-24 cells in the presence of actinomycin D demonstrated that SLPI mRNA transcripts were very stable and became more so in the presence of PMA. Thus, SLPI gene expression in airway epithelial cells can be upregulated by an inflammatory stimulus, and this modulation is regulated at both the transcriptional and posttranscriptional levels. These mechanisms of SLPI upregulation likely play a role in defending the epithelial surface in the local milieu of inflammatory lung diseases. Images PMID:7913712

  18. The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells

    PubMed Central

    Didonna, Alessandro; Sussman, Joshua; Benetti, Federico; Legname, Giuseppe

    2012-01-01

    Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrPC is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis. PMID:22561161

  19. The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells.

    PubMed

    Didonna, Alessandro; Sussman, Joshua; Benetti, Federico; Legname, Giuseppe

    2012-07-01

    Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrP (C) is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis. PMID:22561161

  20. Fate of afferents to the dentate gyrus following destruction of granule cells with colchicine.

    PubMed

    Goldschmidt, R B; Steward, O

    1992-01-01

    Granule cells of the dentate gyrus can be selectively destroyed by intrahippocampal injections of colchicine. The present study evaluates the consequences of this selective neuronal destruction on the afferent axon terminals which have been deprived of their normal targets. The area of the neuropil in the dentate gyrus (the molecular layer) was evaluated in sections stained using the Timm's method for heavy metals, which selectively marks the terminal fields of the different afferent systems. The molecular layer was examined electron microscopically to determine the fate of afferent terminals. Anterograde transport of HRP or [3H]proline was used to define the location and extent of afferent terminal fields of the entorhinal and commissural projections to the dentate gyrus in which the granule cells had been destroyed. There was a substantial reduction in the size of the dentate gyrus molecular layer after destruction of granule cells with colchicine. Electron microscopic analyses revealed that there were very few axon terminals or synapses remaining in the shrunken molecular layer. Tract tracing methods revealed that both the entorhinal and commissural pathways were still present in their normal terminal zones in the dentate gyrus, however, the density of the projections was greatly reduced. There was no evidence to suggest the formation of ectopic projections to unusual locations, such as the contralateral dentate gyrus. Pathways passing through the hippocampus appeared to survive the colchicine injections. These results suggest that target destruction in adult animals leads to the disappearance of the afferent axon terminals which normally innervate the cells which die. PMID:21551890

  1. Cdk5 Regulates Accurate Maturation of Newborn Granule Cells in the Adult Hippocampus

    PubMed Central

    Jessberger, Sebastian; Aigner, Stefan; Clemenson, Gregory D; Toni, Nicolas; Lie, D. Chichung; Karalay, Özlem; Overall, Rupert; Kempermann, Gerd; Gage, Fred H

    2008-01-01

    Newborn granule cells become functionally integrated into the synaptic circuitry of the adult dentate gyrus after a morphological and electrophysiological maturation process. The molecular mechanisms by which immature neurons and the neurites extending from them find their appropriate position and target area remain largely unknown. Here we show that single-cell–specific knockdown of cyclin-dependent kinase 5 (cdk5) activity in newborn cells using a retrovirus-based strategy leads to aberrant growth of dendritic processes, which is associated with an altered migration pattern of newborn cells. Even though spine formation and maturation are reduced in cdk5-deficient cells, aberrant dendrites form ectopic synapses onto hilar neurons. These observations identify cdk5 to be critically involved in the maturation and dendrite extension of newborn neurons in the course of adult neurogenesis. The data presented here also suggest a mechanistic dissociation between accurate dendritic targeting and subsequent synapse formation. PMID:18998770

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

    SciTech Connect

    Aziz, Gulzeb [Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo (Norway); Akselsen, Oyvind W.; Hansen, Trond V. [Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo (Norway); Paulsen, Ragnhild E., E-mail: r.e.paulsen@farmasi.uio.n [Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo (Norway)

    2010-09-15

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

  3. Distinct fusion properties of synaptotagmin-1 and synaptotagmin-7 bearing dense core granules

    PubMed Central

    Rao, Tejeshwar C.; Passmore, Daniel R.; Peleman, Andrew R.; Das, Madhurima; Chapman, Edwin R.; Anantharam, Arun

    2014-01-01

    Adrenal chromaffin cells release hormones and neuropeptides that are essential for physiological homeostasis. During this process, secretory granules fuse with the plasma membrane and deliver their cargo to the extracellular space. It was once believed that fusion was the final regulated step in exocytosis, resulting in uniform and total release of granule cargo. Recent evidence argues for nonuniform outcomes after fusion, in which cargo is released with variable kinetics and selectivity. The goal of this study was to identify factors that contribute to the different outcomes, with a focus on the Ca2+-sensing synaptotagmin (Syt) proteins. Two Syt isoforms are expressed in chromaffin cells: Syt-1 and Syt-7. We find that overexpressed and endogenous Syt isoforms are usually sorted to separate secretory granules and are differentially activated by depolarizing stimuli. In addition, overexpressed Syt-1 and Syt-7 impose distinct effects on fusion pore expansion and granule cargo release. Syt-7 pores usually fail to expand (or reseal), slowing the dispersal of lumenal cargo proteins and granule membrane proteins. On the other hand, Syt-1 diffuses from fusion sites and promotes the release of lumenal cargo proteins. These findings suggest one way in which chromaffin cells may regulate cargo release is via differential activation of synaptotagmin isoforms. PMID:24943843

  4. Role of calcium binding proteins in the control of cerebellar granule cell neuronal excitability: experimental and modeling studies

    Microsoft Academic Search

    D. Gall; C. Roussel; T. Nieus; G. Cheron; L. Servais; E. D’Angelo; S. N. Schiffmann

    2005-01-01

    Calcium binding proteins, such as calretinin, are abundantly expressed in distinctive patterns in the central nervous system but their physiological function remains poorly understood. Calretinin is expressed in cerebellar granule cells which provide the major excitatory input to Purkinje cells through parallel fibers. Calretinin deficient mice exhibit dramatic alterations in motor coordination and in Purkinje cell firing recorded in vivo

  5. The protozoan parasite Toxoplasma gondii targets proteins to dense granules and the vacuolar space using both conserved and unusual mechanisms.

    PubMed

    Karsten, V; Qi, H; Beckers, C J; Reddy, A; Dubremetz, J F; Webster, P; Joiner, K A

    1998-06-15

    All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli beta-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events. PMID:9628889

  6. Enhanced acoustic startle responding in rats with radiation-induced hippocampal granule cell hypoplasia

    SciTech Connect

    Mickley, G.A.; Ferguson, J.L.

    1989-01-01

    Irradiation of the neonatal rat hippocampus reduces the proliferation of granule cells in the dentate gyrus and results in locomotor hyperactivity, behavioral preservation, and deficits on some learned tasks. In order to address the role of changes in stimulus salience and behavioral inhibition in animals with this type of brain damage, irradiated and normal rats were compared in their startle reactions to an acoustic stimulus. Irradiated rats startled with a consistently higher amplitude than control and were more likely to exhibit startle responses. These animals with hippocampal damage also failed to habituate to the startle stimulus and, under certain circumstances, showed potentiated startle responses after many tone presentations.

  7. 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. PMID:25226298

  8. Simulating Spinal Border Cells and Cerebellar Granule Cells under Locomotion – A Case Study of Spinocerebellar Information Processing

    PubMed Central

    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. PMID:25226298

  9. Synaptic sodium spikes trigger long-lasting depolarizations and slow calcium entry in rat olfactory bulb granule cells.

    PubMed

    Egger, Veronica

    2008-04-01

    In the mammalian olfactory bulb, axonless granule cells mediate self- and lateral inhibitory interactions between mitral/tufted cells via reciprocal dendrodendritic synapses. Synaptic output from granule cells occurs on both fast and slow timescales, allowing for multiple granule cell functions during olfactory processing. We find that granule cell sodium action potentials evoked by synaptic activation of the sensory input via mitral/tufted cells are followed by a long-lasting depolarization that is not observed after current-evoked action potentials or large excitatory postsynaptic potentials in the same cell. Using two-photon imaging in acute rat brain slices, we demonstrate that this prolonged electrical response is paralleled by an unusual, long-lasting postsynaptic calcium signal. We find that this slow synaptic Ca(2+) signal requires sequential activation of NMDA receptors, a nonselective cation conductance I(CAN) and T-type voltage-dependent Ca(2+) channels. Remarkably, T-type Ca(2+) channels are of critical importance for the 'globalization' of Ca(2+) transients. In individual active spines, the local synaptic Ca(2+) signal summates at least linearly with the global spike-mediated Ca(2+) signal. We suggest that this robust slow synaptic Ca(2+) signal triggers dendritic transmitter release and thus contributes to slow synaptic output of the granule cell. Therefore, the synaptic sodium spike signal could represent a special adaptation of granule cells to the wide range of temporal requirements for their dendritic output. Our findings demonstrate with respect to neuronal communication in general that action potentials evoked by somatic current injection may lack some of the information content of 'true' synaptically evoked spikes. PMID:18412627

  10. The toxin helothermine affects potassium currents in newborn rat cerebellar granule cells.

    PubMed

    Nobile, M; Magnelli, V; Lagostena, L; Mochca-Morales, J; Possani, L D; Prestipino, G

    1994-04-01

    Helothermine, a recently isolated toxin from the venom of the Mexican beaded lizard Heloderma horridum horridum was tested on K+ currents of newborn rat cerebellar granule cells. In whole-cell voltage-clamp experiments, cerebellar granule neurons exhibited at least two different K+ current components: a first transient component which is similar to an IA-type current, is characterized by fast activating and inactivating kinetics and blocked by 4-aminopyridine; a second component which is characterized by noninactivating kinetics, is blocked by tetraetylammonium ions and resembles the classical delayed-rectifier current. When added to the standard external solution at concentrations ranging between 0.1 and 2 microM, helothermine reduced the pharmacologically isolated IA-type current component in a voltage- and dose-dependent way, with a half-maximal inhibitory concentration (IC50) of 0.52 microM. A comparison between control and helothermine-modified peak transient currents shows a slowdown of activation and inactivation kinetics. The delayed-rectifier component inhibition was concentration dependent (IC50 = 0.86 microM) but not voltage dependent. No frequency- or use-dependent block was observed on both K+ current types. Perfusing the cells with control solution resulted in quite a complete current recovery. We conclude that helothermine acts with different affinities on two types of K+ current present in central nervous system neurons. PMID:8071987

  11. Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions

    PubMed Central

    1975-01-01

    A fraction of secretion granules has been isolated from rabbit parotid by a procedure which was found to be especially effective in reducing contamination resulting from aggregation and/or cosedimentation of granules with other cell particulates. The fraction, representing 15 percent (on the average) of the total tissue amylase activity, was homogeneous as judged by electron microscopy and contaminated to exceedingly low levels by other cellular organelles as judged by marker enzymatic and chemical assays. Lysis of the granules was achieved by their gradual exposure to hypotonic NaHCO3, containing 0.5 mM EDTA. The content and the membranes separated by centrifugation of the granule lysate were characterized primarily by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis which indicated that the content was composed of a limited number of molecular weight classes of polypeptides of which three bands (having approximate mol wt 58,000, 33, 000, and 12,000) could be considered major components. The gel profile of the membrane subfraction was characterized by 20-30 Coomassie brilliant blue-staining bands of which a single species of mol wt 40,000 was the conspicuous major polypeptide. Two types of experiments employing gel electrophoretic analysis were carried out for identifying and assessing the extent of residual secretory protein adsorbed to purified granule membranes: (a) examination of staining and radioactivity profiles after mixing of radioactive secretion granule extract with nonradioactively labeled granule membranes and (b) comparison of gel profiles of secretion granule extract and granule membranes with those of unlysed secretion granules and secretory protein dischraged from lobules in vitro or collected by cannulation of parotid ducts, the last two samples being considered physiologic secretory standards. The results indicated that the membranes were contaminated to a substantial degree by residual, poorly extractable secretory protein even though assays of membrane fractions for a typical secretory enzyme activity (amylase) indicated quite through separation of membranes and content. Hence, detailed examination of membrane subfractions for residual content species by gel electrophoresis points to the general unity and sensitivity of this technique as a means for accurately detecting a defined set of polypeptides occurring as contaminants in cellular fractions or organelle subfractions. PMID:162790

  12. Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein

    SciTech Connect

    Plopper, C.G. [Department of Anatomy, Physiology and Cell Biology, California National Primate Center, School of Veterinary Medicine, 1 Shields Avenue, University of California, Davis, CA 95616 (United States)]. E-mail: cgplopper@ucdavis.edu; Mango, G.W. [Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 (United States); Hatch, G.E. [Pulmonary Toxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (United States); Wong, V.J. [Department of Anatomy, Physiology and Cell Biology, California National Primate Center, School of Veterinary Medicine, 1 Shields Avenue, University of California, Davis, CA 95616 (United States); Toskala, E. [Department of Anatomy, Physiology and Cell Biology, California National Primate Center, School of Veterinary Medicine, 1 Shields Avenue, University of California, Davis, CA 95616 (United States); Reynolds, S.D. [Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 (United States); Tarkington, B.K. [Department of Anatomy, Physiology and Cell Biology, California National Primate Center, School of Veterinary Medicine, 1 Shields Avenue, University of California, Davis, CA 95616 (United States); Stripp, B.R. [Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 (United States)

    2006-05-15

    Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O{sub 3}). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.

  13. Impact of simulated microgravity on the secretory and adhesive activity of cultured human vascular endothelial cells.

    NASA Astrophysics Data System (ADS)

    Rudimov, Evgeny; Buravkova, Ludmila; Pogodina, Margarita; Andrianova, Irina

    The layer of vascular endothelial cells (ECs) is a dynamic,disseminated organ that perform the function of an interface between the blood and vascular wall. The endothelial monolayer is able to quickly respond to changes in the microenvironment due to its synthesis of vasoactive substances, chemokines, adhesion molecules expression, etc. ECs are highly sensitive to gravitational changes and capable of short-term and long-term responses (Sangha et al., 2001; Buravkova et al., 2005; Infanger et al., 2006, 2007. However, the question remains how to reflect the impact of microgravity on endothelium under the inflammatory process. Therefore, the aim of this study was to investigate secretory and adhesive activity of human umbilical vein endothelial cells (HUVECs) during simulated microgravity and TNF-a activation. HUVECs were isolated according to Gimbrone et al. (1978) in modification A. Antonov (1981) and used for experiments at 2-4 passages. HUVECs were activated by low level of TNF-a (2 ng/ml). Microgravity was generated by Random Positioning Machine (RPM, Dutch Space, Leiden) placed into the thermostat at 37°C. After 24 hours of clinorotation we measured adhesion molecules expression on the cell surface (ICAM-1, VCAM-1, PECAM-1, E-selectin, CD144, endoglin (CD105)) and cell viability using a flow cytometry. To evaluate the level of target gene expression was used the real time RT-PCR. IL-6 and IL-8 concentration was measured in the conditioned medium of HUVECs by using the ELISA test. We found that simulated microgravity within 24 hours caused a decrease of ICAM-1, CD144, and E-selectin expression, at the same time not affect the cell viability, endoglin and PECAM-1 expression on the surface HUVEC. Furthermore, there were no changes of the level of IL-6 and IL-8 gene expression and their products in the culture medium. TNF-activated HUVECs showed an increase in gene expression of interleukins and molecules involved in the adhesion process, which also was confirmed by the higher level of cytokines in the medium and elevated share of CD144, ICAM-1 and VCAM-1-positive cells. Comparative analysis of the level TNF-induced secretion of IL-6 and IL-8, as well as the share of cells bearing ICAM-1 and VCAM-1, showed significant variability depending on the donors. Simultaneous exposure to simulated microgravity and proinflammatory activation did not potentiate and did not cancel the effect caused by TNF-a. In summary, our findings indicate that the simulated microgravity is not activating and additional pro-inflammatory stimulus to HUVEC in vitro model. This work was supported in part by Grant from RFBR ? 12-04-31763 and Grant ? NSh-371.2014.4

  14. A secretory cell type develops alongside multiciliated cells, ionocytes and goblet cells, and provides a protective, anti-infective function in the frog embryonic mucociliary epidermis

    PubMed Central

    Dubaissi, Eamon; Rousseau, Karine; Lea, Robert; Soto, Ximena; Nardeosingh, Siddarth; Schweickert, Axel; Amaya, Enrique; Thornton, David J.; Papalopulu, Nancy

    2014-01-01

    The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences. PMID:24598166

  15. A secretory cell type develops alongside multiciliated cells, ionocytes and goblet cells, and provides a protective, anti-infective function in the frog embryonic mucociliary epidermis.

    PubMed

    Dubaissi, Eamon; Rousseau, Karine; Lea, Robert; Soto, Ximena; Nardeosingh, Siddarth; Schweickert, Axel; Amaya, Enrique; Thornton, David J; Papalopulu, Nancy

    2014-04-01

    The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences. PMID:24598166

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

    SciTech Connect

    Rowe, Alexander M. [Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506 (United States); Brundage, Kathleen M. [Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506 (United States); Center for Immunopathology and Microbial Pathogenesis, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506 (United States); Barnett, John B. [Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506 (United States) and Center for Immunopathology and Microbial Pathogenesis, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506 (United States)]. E-mail: jbarnett@hsc.wvu.edu

    2007-06-01

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

  17. The chromogranins: Their roles in secretion from neuroendocrine cells and as markers for neuroendocrine neoplasia

    Microsoft Academic Search

    Steven A. Feldman; Lee E. Eiden

    2003-01-01

    Chromogranins are the major components of the secretory granules of most neuroendocrine cells. Within the secretory pathway,\\u000a chromogranins are involved in granulogenesis, and in sorting and processing of secretory proteincargo prior to, secretion.\\u000a Once secreted, they have hormonal, autocrine, and paracrine activities. The chromogranin family includes chromogranins A (CgA)\\u000a and B (CgB)and secretogranin II (Sgll, once called chromogranin C). The

  18. Platelet ?–granules: Basic biology and clinical correlates

    PubMed Central

    Blair, Price; Flaumenhaft, Robert

    2009-01-01

    Summary ?–Granules are essential to normal platelet activity. These unusual secretory granules derive their cargo from both regulated secretory and endocytotic pathways in megakaryocytes. Rare, inheritable defects of ?–granule formation in mice and man have enabled identification of proteins that mediate cargo trafficking and ?–granule formation. In platelets, ?–granules fuse with the plasma membrane upon activation, releasing their cargo and increasing platelet surface area. The mechanisms that control ?–granule membrane fusion have begun to be elucidated at the molecular level. SNAREs and SNARE accessory proteins that control ?–granule secretion have been identified. Proteomic studies demonstrate that hundreds of bioactive proteins are released from ?–granules. This breadth of proteins implies a versatile functionality. While initially known primarily for their participation in thrombosis and hemostasis, the role of ?–granules in inflammation, atherosclerosis, antimicrobial host defense, wound healing, angiogenesis, and malignancy has become increasingly appreciated as the function of platelets in the pathophysiology of these processes has been defined. This review will consider the formation, release, and physiologic roles of ?–granules with special emphasis on work performed over the last decade. PMID:19450911

  19. Cholinergic Afferent Stimulation Induces Axonal Function Plasticity in Adult Hippocampal Granule Cells

    PubMed Central

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

    2015-01-01

    Summary Acetylcholine critically influences hippocampal-dependent learning. Cholinergic fibers innervate hippocampal neuron axons, dendrites, and somata. The effects of acetylcholine on axonal information processing, though, remain unknown. By stimulating cholinergic fibers and making electrophysiological recordings from hippocampal dentate gyrus granule cells, we show that synaptically released acetylcholine preferentially lowered the action potential threshold, enhancing intrinsic excitability and synaptic potential-spike coupling. These effects persisted for at least 30 min after the stimulation paradigm and were due to muscarinic receptor activation. This caused sustained elevation of axonal intracellular Ca2+ via T-type Ca2+ channels, as indicated by two-photon imaging. The enhanced Ca2+ levels inhibited an axonal KV7/M current, decreasing the spike threshold. In support, immunohistochemistry revealed muscarinic M1 receptor, CaV3.2, and KV7.2/7.3 subunit localization in granule cell axons. Since alterations in axonal signaling affect neuronal firing patterns and neurotransmitter release, this is an unreported cellular mechanism by which acetylcholine might, at least partly, enhance cognitive processing. PMID:25578363

  20. Imaging activation of adult-generated granule cells in spatial memory.

    PubMed

    Kee, Nohjin; Teixeira, Cátia M; Wang, Afra H; Frankland, Paul W

    2007-01-01

    New neurons are continuously generated in the subgranular zone of the hippocampus throughout adulthood, and there is increasing interest as to whether these new neurons become functionally integrated into memory circuits. This protocol describes the immunohistochemical procedures to visualize the recruitment of new neurons into circuits supporting spatial memory in intact mice. To label adult-generated granule cells, mice are injected with the proliferation marker 5-bromo-2'-deoxyuridine (BrdU). At different delays after BrdU treatment, mice are trained to locate a hidden platform in the Morris water maze, and spatial memory can then be tested in a probe test with the platform removed from the pool. Ninety minutes after this probe test, mice are perfused and tissue is sectioned. Immunohistochemical procedures are used to quantify BrdU-labeled cells and expression of the immediate early gene, Fos. Because Fos expression is regulated by neuronal activity, the degree of overlap between BrdU-labeled and Fos-labeled neurons provides an indication of whether adult-generated granule neurons have been incorporated into spatial memory circuits. PMID:18079702

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

    PubMed

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

    2015-01-21

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

  2. Continuous exposure to low concentrations of methylmercury impairs cerebellar granule cell migration in organotypic slice culture.

    PubMed

    Mancini, Jayme D; Autio, Dawn M; Atchison, William D

    2009-03-01

    Chronic, low-level perinatal exposure to methylmercury (MeHg) is associated with neurological and motor deficits that appear to result from cerebellar dysfunction. Neuropathological studies suggest that these deficits are due to impaired cerebellar granule cell (CGC) migration. Although neuronal migration in vivo and in vitro has been shown to be impaired during acute and/or high level exposure to MeHg, the cellular effects of chronic exposure to submicromolar and micromolar levels of MeHg during development are not clear. The majority of CGC migration in rats occurs between postnatal days 8 and 14 (P8 and 14); migration peaks on P10 and 11. Organotypic cultures of parasagittal slices of cerebellum from P8 rats were exposed to low levels of MeHg (0.2-5.0microM) for 3 or 7 days, and CGC viability and migration were assessed. MeHg-induced cell death was time- and concentration-dependent. After 3 days of exposure CGC viability decreased in 3microM MeHg and declined to 42.7% in 5microM MeHg. Cultures treated with MeHg for 7 days showed decreased CGC viability in 1microM MeHg, which declined to 62.8% in 3microM MeHg. CGC migration was assessed by BrdU pulse-chase labeling. Migration into the internal granule cell layer (IGL) was impaired in cultures exposed to >or=1microM MeHg for 3 days or >or=0.5microM for 7 days. CGCs failed to initiate migration from the external germinal cell layer at the same level of exposure. For those cells which initiated migration, MeHg reduced the number that migrated into the IGL. This implied a slowing of migration once it had begun. These effects occurred with no overall change in cerebellar cortical structure, or loss of granule cell viability. Thus, chronic exposure to low micromolar concentrations of MeHg impairs development of the cerebellar cortex in a slice culture model. PMID:19152806

  3. Lytic Granule Loading of CD8+ T Cells Is Required for HIV-Infected Cell Elimination Associated with Immune Control

    PubMed Central

    Migueles, Stephen A.; Osborne, Christine M.; Royce, Cassandra; Compton, Alex A.; Joshi, Rohan P.; Weeks, Kristin A.; Rood, Julia E.; Berkley, Amy M.; Sacha, Jonah B.; Cogliano-Shutta, Nancy A.; Lloyd, Margaret; Roby, Gregg; Kwan, Richard; McLaughlin, Mary; Stallings, Sara; Rehm, Catherine; O’Shea, Marie A.; Mican, JoAnn; Packard, Beverly Z.; Komoriya, Akira; Palmer, Sarah; Wiegand, Ann P.; Maldarelli, Frank; Coffin, John M.; Mellors, John W.; Hallahan, Claire W.; Follman, Dean A.; Connors, Mark

    2009-01-01

    SUMMARY Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity. PMID:19062316

  4. Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy

    PubMed Central

    Brown, Alice C. N.; Oddos, Stephane; Dobbie, Ian M.; Alakoskela, Juha-Matti; Parton, Richard M.; Eissmann, Philipp; Neil, Mark A. A.; Dunsby, Christopher; French, Paul M. W.; Davis, Ilan; Davis, Daniel M.

    2011-01-01

    Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ?100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology. PMID:21931537

  5. Antibody-dependent cell-mediated antibacterial activity of intestinal lymphocytes with secretory IgA

    Microsoft Academic Search

    A. Tagliabue; L. Nencioni; L. Villa; D. F. Keren; G. H. Lowell; D. Boraschi

    1983-01-01

    Secretory antibodies of the IgA class (sIgA) are thought to have an important role in the defence against bacteria at mucosal surfaces-the level at which the infectious agents first come into contact with the host. However, the mechanism by which sIgA exert their antibacterial activity is still a matter of debate. After the recent discovery of receptors for the Fc

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

    PubMed

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

    2015-04-01

    In the developing brain, immature neurons migrate from their sites of origin to their final destination, where they reside for the rest of their lives. This active movement of immature neurons is essential for the formation of normal neuronal cytoarchitecture and proper differentiation. Deficits in migration result in the abnormal development of the brain, leading to a variety of neurological disorders. A myriad of extracellular guidance molecules and intracellular effector molecules is involved in controlling the migration of immature neurons in a cell type, cortical layer and birth-date-specific manner. To date, little is known about how extracellular guidance molecules transfer their information to the intracellular effector molecules, which regulate the migration of immature neurons. In this article, to fill the gap between extracellular guidance molecules and intracellular effector molecules, using the migration of cerebellar granule cells as a model system of neuronal cell migration, we explore the role of second messenger signaling (specifically Ca(2+) and cyclic nucleotide signaling) in the regulation of neuronal cell migration. We will, first, describe the cortical layer-specific changes in granule cell migration. Second, we will discuss the roles of Ca(2+) and cyclic nucleotide signaling in controlling granule cell migration. Third, we will present recent studies showing the roles of Ca(2+) and cyclic nucleotide signaling in the deficits in granule cell migration in mouse models of fetal alcohol spectrum disorders and fetal Minamata disease. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 369-387, 2015. PMID:25066767

  7. Degranulation of acinar cells in von Ebner's gland of the rat.

    PubMed

    Ueba, H; Uchihashi, K

    1991-04-01

    We investigated the effect of sympathetic agonists, parasympathetic muscarinic agonists and substance P on depletion of secretory granules in acinar cells of rat von Ebner's gland. Drugs were injected intraperitoneally at several different concentrations. Antagonists were given 15 minutes before injection of the agonist, and the extent of depletion of secretory granules in glandular acini was calculated using a computerized color image analyzer. The specific alpha 2-sympathetic agonist clonidine and the beta 1-sympathetic agonist dobutamine produced a depletion of secretory granules. When combined with injections of the alpha 2-sympathetic antagonist yohimbine and the beta 1-sympathetic antagonist acebutolol, depletion of secretory granules was blocked. The parasympathetic muscarinic agonist carbachol also produced a depletion of secretory granules. QNB blocked the depletion caused by carbachol, while atropine partially inhibited depletion. The specific M1-muscarinic agonist McN-A-343 caused some depletion, although there was no significant differences between it and the control. Complete depletion of the secretory granules was achieved by carbachol stimulation superimposed on substance P stimulation. We concluded that the activation of the sympathetic alpha 2- and beta 1-receptors, as well as the M2 (M2 beta)-muscarinic and substance P receptors, results in degranulation of acinar cells in von Ebner's gland of the rat. PMID:1725993

  8. 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 75-125?? 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 ?M (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

  9. Enhanced dense core granule function and adrenal hypersecretion in a mouse model of Rett syndrome.

    PubMed

    Ladas, Thomas; Chan, Shyue-An; Ogier, Michael; Smith, Corey; Katz, David M

    2009-08-01

    Rett syndrome (RTT) is a progressive developmental disorder resulting from loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2), a transcription regulatory protein. The RTT phenotype is complex and includes severe cardiorespiratory abnormalities, dysautonomia and behavioral symptoms of elevated stress. These findings have been attributed to an apparent hyperactivity of the sympathetic nervous system due to defects in brainstem development; however, the possibility that the peripheral sympathoadrenal axis itself is abnormal has not been explored. The present study demonstrates that the adrenal medulla and sympathetic ganglia of Mecp2 null mice exhibit markedly reduced catecholamine content compared with wild-type controls. Despite this, null animals exhibit significantly higher plasma epinephrine levels, suggesting enhanced secretory granule function in adrenal chromaffin cells. Indeed, we find that Mecp2 null chromaffin cells exhibit a cell autonomous hypersecretory phenotype characterized by significant increases in the speed and size of individual secretory granule fusion events in response to electrical stimulation. These findings appear to indicate accelerated formation and enhanced dilation of the secretory granule fusion pore, resulting in elevated catecholamine release. Our data therefore highlight abnormal catecholamine function in the sympathoadrenal axis as a potential source of autonomic dysfunction in RTT. These findings may help to explain the apparent 'overactivity' of the sympathetic nervous system reported in patients with RTT. PMID:19674087

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

    PubMed Central

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

    2013-01-01

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

  11. Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell.

    PubMed Central

    Kuiper, R P; Bouw, G; Janssen, K P; Rötter, J; van Herp, F; Martens, G J

    2001-01-01

    Members of the p24 family of putative cargo receptors (subdivided into p24-alpha, -beta, -gamma and -delta) are localized in the intermediate-and cis-Golgi compartments of the early secretory pathway, and are thought to play an important role in protein transport. In the present study, we wondered what effect increased biosynthetic cell activity with resulting high levels of protein transport would have on the subcellular localization of p24. We examined p24 localization in Xenopus intermediate pituitary melanotrope cells, which in black- and white-adapted animals are biosynthetically highly active and virtually inactive respectively. In addition, p24 localization was studied in Xenopus anterior pituitary cells whose activity is not changed during background adaptation. Using organelle fractionation, we found that in the inactive melanotropes and moderately active anterior pituitary cells of white-adapted animals, the p24-alpha, -beta, -gamma and -delta proteins are all located in the Golgi compartment. In the highly active melanotropes, but not in the anterior cells of black-adapted animals, the steady-state distribution of all four p24 members changed towards the intermediate compartment and subdomains of the endoplasmic reticulum (ER), most probably the ER exit sites. In the active melanotropes, the major cargo protein pro-opiomelanocortin was mostly localized to ER subdomains and partially co-localized with the p24 proteins. Furthermore, in the active cells, in vitro blocking of protein biosynthesis by cycloheximide or dispersion of the Golgi complex by brefeldin A led to a redistribution of the p24 proteins, indicating their involvement in ER-to-Golgi protein transport and extensive cycling in the early secretory pathway. We conclude that the subcellular localization of p24 proteins is dynamic and depends on the biosynthetic activity of the cell. PMID:11716771

  12. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    ERIC Educational Resources Information Center

    Hood-DeGrenier, Jennifer K.

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in…

  13. Early-life status epilepticus induces ectopic granule cells in adult mice dentate gyrus.

    PubMed

    Muramatsu, Rieko; Ikegaya, Yuji; Matsuki, Norio; Koyama, Ryuta

    2008-06-01

    A large number of aberrant hilar granule cells (GCs) are found in the patients and animal models of adult temporal lobe epilepsy (TLE), and these "ectopic" GCs have synchronous epileptiform bursting with other hippocampal neurons. In this study, we investigated whether early-life status epilepticus (SE) induces hilar ectopic GCs that remain in the adulthood because TLE patients frequently experience seizures in the early childhood when a large number of postnatally born GCs migrate in the hilus. To label newborn GCs, bromodeoxyuridine (BrdU) was injected daily for three consecutive days to C57BL/6J mice at different postnatal days starting at postnatal-0-day-old (P0) (Group1), P7 (Group2), or P35 (Group3). Mice in each group underwent pilocarpine-induced SE at P14. Six months later, to determine whether SE induces ectopic GCs, we plotted the distribution of postnatally born GCs which were immunohistochemically defined as BrdU- and the GC marker Prox1-colabeled cells. We also examined whether SE causes the granule cell layer (GCL) dispersion and/or the mossy fiber (MF) sprouting, other representative pathologies of TLE hippocampus. Only SE-experiencing mice in Group1 had significantly more neonatally born ectopic GCs compared with control mice. Neither control nor SE mice had dispersed GCL. All mice that underwent SE had sprouted MFs in CA3. We conclude that early-life SE disrupts a normal incorporation of GCs born pre-SE but not post-SE, inducing ectopic GCs in the adult hilus. Interestingly, the results also indicate that developmentally earlier born GCs are more responsive to early-life SE in terms of the emergence of ectopic GCs. PMID:18420198

  14. Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K+ channels.

    PubMed

    Malinowska, Danuta H; Sherry, Ann M; Tewari, Kirti P; Cuppoletti, John

    2004-03-01

    Our objective was to identify and localize a K+ channel involved in gastric HCl secretion at the parietal cell secretory membrane and to characterize and compare the functional properties of native and recombinant gastric K+ channels. RT-PCR showed that mRNA for Kir2.1 was abundant in rabbit gastric mucosa with lesser amounts of Kir4.1 and Kir7.1, relative to beta-actin. Kir2.1 mRNA was localized to parietal cells of rabbit gastric glands by in situ RT-PCR. Resting and stimulated gastric vesicles contained Kir2.1 by Western blot analysis at approximately 50 kDa as observed with in vitro translation. Immunoconfocal microscopy showed that Kir2.1 was present in parietal cells, where it colocalized with H+ -K+ -ATPase and ClC-2 Cl- channels. Function of native K+ channels in rabbit resting and stimulated gastric mucosal vesicles was studied by reconstitution into planar lipid bilayers. Native gastric K+ channels exhibited a linear current-voltage relationship and a single-channel slope conductance of approximately 11 pS in 400 mM K2SO4. Channel open probability (Po) in stimulated vesicles was high, and that of resting vesicles was low. Reduction of extracellular pH plus PKA treatment increased resting channel Po to approximately 0.5 as measured in stimulated vesicles. Full-length rabbit Kir2.1 was cloned. When stably expressed in Chinese hamster ovary (CHO) cells, it was activated by reduced extracellular pH and forskolin/IBMX with no effects observed in nontransfected CHO cells. Cation selectivity was K+ = Rb+ > Na+ = Cs+ = Li+ = NMDG+. These findings strongly suggest that the Kir2.1 K+ channel may be involved in regulated gastric acid secretion at the parietal cell secretory membrane. PMID:14602583

  15. Reconstituted human polyclonal plasma-derived secretory-like IgM and IgA maintain the barrier function of epithelial cells infected with an enteropathogen.

    PubMed

    Longet, Stéphanie; Vonarburg, Cédric; Lötscher, Marius; Miescher, Sylvia; Zuercher, Adrian; Corthésy, Blaise

    2014-08-01

    Intravenous administration of polyclonal and monoclonal antibodies has proven to be a clinically valid approach in the treatment, or at least relief, of many acute and chronic pathologies, such as infection, immunodeficiency, and a broad range of autoimmune conditions. Plasma-derived IgG or recombinant IgG are most frequently used for intravenous or subcutaneous administration, whereas a few IgM-based products are available as well. We have established recently that secretory-like IgA and IgM can be produced upon association of plasma-derived polymeric IgA and IgM with a recombinant secretory component. As a next step toward potential future mucosal administration, we sought to unravel the mechanisms by which these secretory Igs protect epithelial cells located at the interface between the environment and the inside of the body. By using polarized epithelial Caco-2 cell monolayers and Shigella flexneri as a model enteropathogen, we found that polyspecific plasma-derived SIgA and SIgM fulfill many protective functions, including dose-dependent recognition of the antigen via formation of aggregated immune complexes, reduction of bacterial infectivity, maintenance of epithelial cell integrity, and inhibition of proinflammatory cytokine/chemokine production by epithelial cells. In this in vitro model devoid of other cellular or molecular interfering partners, IgM and secretory IgM showed stronger bacterial neutralization than secretory IgA. Together, these data suggest that mucosally delivered antibody preparations may be most effective when combining both secretory-like IgA and IgM, which, together, play a crucial role in preserving several levels of epithelial cell integrity. PMID:24951593

  16. 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 naďve 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

  17. Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L.

    PubMed

    Olsson, Mikael E; Olofsson, Linda M; Lindahl, Ann-Louise; Lundgren, Anneli; Brodelius, Maria; Brodelius, Peter E

    2009-06-01

    A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Delta11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1alpha was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes. PMID:19664791

  18. Excessive activation of mTOR in postnatally generated granule cells is sufficient to cause epilepsy.

    PubMed

    Pun, Raymund Y K; Rolle, Isaiah J; Lasarge, Candi L; Hosford, Bethany E; Rosen, Jules M; Uhl, Juli D; Schmeltzer, Sarah N; Faulkner, Christian; Bronson, Stefanie L; Murphy, Brian L; Richards, David A; Holland, Katherine D; Danzer, Steve C

    2012-09-20

    The dentate gyrus is hypothesized to function as a "gate," limiting the flow of excitation through the hippocampus. During epileptogenesis, adult-generated granule cells (DGCs) form aberrant neuronal connections with neighboring DGCs, disrupting the dentate gate. Hyperactivation of the mTOR signaling pathway is implicated in driving this aberrant circuit formation. While the presence of abnormal DGCs in epilepsy has been known for decades, direct evidence linking abnormal DGCs to seizures has been lacking. Here, we isolate the effects of abnormal DGCs using a transgenic mouse model to selectively delete PTEN from postnatally generated DGCs. PTEN deletion led to hyperactivation of the mTOR pathway, producing abnormal DGCs morphologically similar to those in epilepsy. Strikingly, animals in which PTEN was deleted from ? 9% of the DGC population developed spontaneous seizures in about 4 weeks, confirming that abnormal DGCs, which are present in both animals and humans with epilepsy, are capable of causing the disease. PMID:22998871

  19. Formation of a large Vasa-positive germ granule and its inheritance by germ cells in the enigmatic Chaetognaths

    Microsoft Academic Search

    Daničle Carré; Chakib Djediat; Christian Sardet

    2002-01-01

    Chaetognaths (arrow worms) are abundant hermaphrodite marine organisms whose phylogenetic position amongst protostomes and deuterostomes is still debated. Ancient histological observations dating from a century ago described the presence in eggs of a large granule, presumed to be a germ plasm, and its probable inheritance in four primary germ cells (PGCs). Using videomicroscopy, electron microscopy and immunocytochemistry (labelling with anti-Vasa

  20. Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma

    Microsoft Academic Search

    Trudy G. Oliver; Tracy Ann Read; Jessica D. Kessler; Anriada Mehmeti; Jonathan F. Wells; Trang T. T. Huynh; Simon M. Lin; Robert J. Wechsler-Reya

    2005-01-01

    Medulloblastoma is the most common malignant brain tumor in children. It is thought to result from the transformation of granule cell precursors (GCPs) in the developing cerebellum, but little is known about the early stages of the disease. Here, we identify a pre-neoplastic stage of medulloblastoma in patched heterozygous mice, a model of the human disease. We show that pre-neoplastic

  1. Ethanol Inhibits L1-mediated Neurite Outgrowth in Postnatal Rat Cerebellar Granule Cells

    PubMed Central

    Bearer, Cynthia F.; Swick, Alan R.; O’Riordan, Mary Ann; Cheng, Guanghui

    2014-01-01

    The neuropathology of the effects of ethanol on the developing central nervous system are similar to those of patients with mutations in L1, a neural cell adhesion molecule. This observation suggests that inhibition of L1 plays a role in the pathogenesis of alcohol-related neurodevelopmental disorders. Here we examine the effects of ethanol on L1 homophilic binding and on L1-mediated neurite outgrowth. Ethanol had no effect on cell adhesion or aggregation in a myeloma cell line expressing full-length human L1. In contrast, the rate of L1-mediated neurite outgrowth of rat postnatal day 6 cerebellar granule cells grown on a substratum of Ng-CAM, the chick homologue of L1, was inhibited by 48.6% in the presence of ethanol with a half-maximal concentration of 4.7mm. The same effect was found with soluble L1-Fc, thus showing that the inhibitory effect is not dependent on cell adhesion. In contrast, neither laminin nor N-cadherin-mediated neurite outgrowth was inhibited by physiologic concentrations of ethanol. We conclude that one mechanism of ethanol’s toxicity to the developing central nervous system may be the inhibition of L1-mediated neurite outgrowth. PMID:10224086

  2. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

    SciTech Connect

    Coppé, Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua; Nelson, Peter; Desprez, Pierre-Yves; Campisi, Judith

    2008-10-24

    Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial-mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

  3. Optofluidic platform for real-time monitoring of live cell secretory activities using Fano resonance in gold nanoslits.

    PubMed

    Wu, Shu-Han; Lee, Kuang-Li; Chiou, Arthur; Cheng, Xuanhong; Wei, Pei-Kuen

    2013-10-25

    An optofluidic platform for real-time monitoring of live cell secretory activities is constructed via Fano resonance in a gold nanoslit array. Large-area and highly sensitive gold nanoslits with a period of 500 nm are fabricated on polycarbonate films using the thermal-annealed template-stripping method. The coupling between gap plasmon resonance in the slits and surface plasmon polariton Bloch waves forms a sharp Fano resonance with intensity sensitivity greater than 11 000% per refractive index unit. The nanoslit array is integrated with a cell-trapping microfluidic device to monitor dynamic secretion of matrix metalloproteinase 9 (MMP-9) from human acute monocytic leukemia cells in situ. Upon continuous lipopolysaccharide (LPS) stimulation, MMP-9 secretion is detected within 2 h due to ultrahigh surface sensitivity and close proximity of the sensor to the target cells. In addition to the advantage of detecting early cell responses, the sensor also allows interrogation of cell secretion dynamics. Furthermore, the average secretion per cell measured using our system well matches previous reports while it requires orders of magnitude less cells. The optofluidic platform may find applications in fundamental studies of cell functions and diagnostics based on secretion signals. PMID:23606668

  4. Adverse influence of coumestrol on secretory function of bovine luteal cells in the first trimester of pregnancy.

    PubMed

    M?ynarczuk, J; Wróbel, M H; Kotwica, J

    2013-07-01

    Coumestrol is one of a few biologically active substances present in leguminous plants, which are widely used as fodder for ruminants. Depending on the doses, coumestrol acts on the reproductive processes as an estrogen-like factor or antiestrogen to evoke a decrease in ovulation frequency, elongation of estrous cycle duration. The aim of the current investigations was to study the influence of coumestrol on secretory function of luteal cells obtained from first trimester of pregnant cows. Luteal cells (2.5 × 10(5) /mL) from 3rd to 5th, 6th to 8th, and 9th to 12th week of pregnancy were preincubated for 24 h and incubated with coumestrol (1 × 10(-6) M) for successive 48 h and the medium concentrations of progesterone (P4), oxytocin (OT), prostaglandin (PG) E2 and F2? were determined. Moreover, the expression of mRNA for neurophysin-I/oxytocin (NP-I/OT; precursor of OT) and peptidyl-glycine-?-amidating mono-oxygenase (PGA, an enzyme responsible for post-translational OT synthesis) was determined after 8 h of treatment. Coumestrol did not affect P4 secretion but increased the secretion of OT from the cells collected at all stages of gestation studied. Hence, the ratio of P4 to OT was markedly decreased. Simultaneously, coumestrol increased the expression of NP-I/OT mRNA during 9th to 12th weeks of pregnancy, and mRNA for PGA during 3rd to 5th and 9th to 12th weeks of gestation. Furthermore, coumestrol decreased PGE2 secretion from luteal cells in all studied stages of pregnancy, while it affected PGF2? metabolite (PGFM) concentration only from week 3 to 5 of pregnancy. Obtained results suggest that coumestrol impairs secretory function of the corpus luteum (CL) and this way it can affect the maintenance of pregnancy in the cow. PMID:21656645

  5. Neuroanatomical Clues to Altered Neuronal Activity in Epilepsy: From Ultrastructure to Signaling Pathways of Dentate Granule Cells

    PubMed Central

    Houser, Carolyn R.; Zhang, Nianhui; Peng, Zechun; Huang, Christine S.; Cetina, Yliana

    2014-01-01

    SUMMARY The dynamic aspects of epilepsy, in which seizures occur sporadically and are interspersed with periods of relatively normal brain function, present special challenges for neuroanatomical studies. While numerous morphological changes can be identified during the chronic period, the relationship of many of these changes to seizure generation and propagation remain unclear. Mossy fiber sprouting is an example of a fequently observed morphological change for which a functional role in epilepsy continues to be debated. This review will focus on neuroanatomically-identified changes that would support high levels of activity in reorganized mossy fibers and potentially associated granule cell activation. Early ultrastructural studies of reorganized mossy fiber terminals in human temporal lobe epilepsy tissue have identified morphological substrates for highly efficacious excitatory connections among granule cells. If similar connections in animal models contribute to seizure activity, activation of granule cells would be expected. Increaed labeling with two activity-related markers, Fos and phosphorylated extracellular signal-regulated kinase, has suggested increased activity of dentate granule cells at the time of a spontaneous seizures in a mouse model of epilepsy. However, neuroanatomical support for a direct link between activation of reorganized mossy fiber terminals and increased granule cell activity remains elusive. As novel activity-related markers are developed, it may yet be possible to demonstrate such functional links and allow mapping of seizure activity throughout the brain. Relating patterns of neuronal activity during seizures to the underlying morphological changes could provide important new insights into the basic mechanisms of epilepsy and seizure generation. PMID:22612811

  6. Cytoskeletal Dependence of Insulin Granule Movement Dynamics in INS-1 Beta-Cells in Response to Glucose

    PubMed Central

    Heaslip, Aoife T.; Nelson, Shane R.; Lombardo, Andrew T.; Beck Previs, Samantha; Armstrong, Jessica; Warshaw, David M.

    2014-01-01

    For pancreatic ?-cells to secrete insulin in response to elevated blood glucose, insulin granules retained within the subplasmalemmal space must be transported to sites of secretion on the plasma membrane. Using a combination of super-resolution STORM imaging and live cell TIRF microscopy we investigate how the organization and dynamics of the actin and microtubule cytoskeletons in INS-1 ?-cells contribute to this process. GFP-labeled insulin granules display 3 different modes of motion (stationary, diffusive-like, and directed). Diffusive-like motion dominates in basal, low glucose conditions. Upon glucose stimulation no gross rearrangement of the actin cytoskeleton is observed but there are increases in the 1) rate of microtubule polymerization; 2) rate of diffusive-like motion; and 3) proportion of granules undergoing microtubule-based directed motion. By pharmacologically perturbing the actin and microtubule cytoskeletons, we determine that microtubule-dependent granule transport occurs within the subplasmalemmal space and that the actin cytoskeleton limits this transport in basal conditions, when insulin secretion needs to be inhibited. PMID:25310693

  7. Interactions between Ca2+ mobilizing mechanisms in cultured rat cerebellar granule cells.

    PubMed Central

    Irving, A J; Collingridge, G L; Schofield, J G

    1992-01-01

    1. The interactions between IP3 receptor-mediated and Ca(2+)-induced Ca2+ release were investigated in cerebellar granule cell bodies, using the techniques of microfluorimetry and image analysis. 2. The IP3-sensitive Ca2+ release mechanism was activated using acetylcholine (ACh) and the selective metabotropic glutamate receptor agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD). Caffeine was used to activate, and ryanodine to inhibit, the Ca(2+)-induced Ca2+ release process. Thapsigargin was used to deplete intracellular Ca2+ stores. 3. Transient applications of caffeine (5-50 mM), ACPD (50-500 microM) and ACh (0.05-1 microM) mobilized intracellular Ca2+ ([Ca2+]i). Ca2+ mobilizing responses to 50 mM caffeine and 1 microM ACh increased with time in culture until day 4. However, beyond this period the responsiveness of cells to caffeine, but not to ACh, declined markedly. 4. Responses induced by ACPD and ACh were inhibited in the presence of caffeine at concentrations below those which mobilized Ca2+ (1-5 mM). This effect was not due to Ca2+ pool depletion, elevation of cAMP or inhibition of phosphodiesterases. 5. Prior challenge with ACh or ACPD inhibited Ca2+ mobilization induced by caffeine (50 mM). Transient exposure to caffeine inhibited subsequent responses to ACh through a mechanism which involved store depletion. 6. Thapsigargin (0.1-1 microM) inhibited, to a similar extent, Ca2+ mobilization induced by caffeine, ACPD and ACh. 7. Ryanodine (10 microM) antagonized Ca2+ mobilization induced by caffeine, ACh and ACPD. However, the ability of ryanodine to block inositol 1,4,5-trisphosphate-linked agonist responses varied considerably between cells. The sensitivity of ACh-induced responses to ryanodine correlated with the sensitivity of the cells to caffeine. 8. The possible explanations for the pronounced interactions between IP3 receptor-mediated and Ca(2+)-induced Ca2+ release processes in cerebellar granule cells are discussed. PMID:1338107

  8. 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

  9. Tocotrienols prevent hydrogen peroxide-induced axon and dendrite degeneration in cerebellar granule cells.

    PubMed

    Fukui, Koji; Ushiki, Keisuke; Takatsu, Hirokatsu; Koike, Tatsuro; Urano, Shiro

    2012-02-01

    It is well known that reactive oxygen species (ROS) attack several living tissues and increase the risk of development and progression of serious diseases. In neuronal level, ROS induce cell death in concentration-dependent fashion. However, little is known about the mechanisms of neuronal changes by ROS prior to induction of cell death. Here we found that treatment of cerebellar granule neurons (CGCs) with 0.5 ?M hydrogen peroxide induced axonal injury, but not cell death. The number of dendrites remarkably decreased in hydrogen peroxide-treated CGCs, and extensive beading was observed on survival dendrites. In addition, an abnormal band of the original collapsin response mediator protein (CRMP)-2 was detected by Western blotting in hydrogen peroxide-treated CGCs. Treatment with each tocotrienol isoform prevented axonal and dendrite degeneration and induction of the abnormal band of the original band of CRMP-2 in hydrogen peroxide-treated CGCs. These results indicate that treatment with tocotrienols may therefore be neuroprotective in the presence of hydrogen peroxide by preventing changes to the CRMP-2 that occur before neuron death. PMID:22149330

  10. Molecular and functional characterization of GAD67-expressing, newborn granule cells in mouse dentate gyrus

    PubMed Central

    Cabezas, Carolina; Irinopoulou, Theano; Cauli, Bruno; Poncer, Jean Christophe

    2013-01-01

    Dentate gyrus granule cells (GCs) have been suggested to synthesize both GABA and glutamate immediately after birth and under pathological conditions in the adult. Expression of the GABA synthesizing enzyme GAD67 by GCs during the first few weeks of postnatal development may then allow for transient GABA synthesis and synaptic release from these cells. Here, using the GAD67-EGFP transgenic strain G42, we explored the phenotype of GAD67-expressing GCs in the mouse dentate gyrus. We report a transient, GAD67-driven EGFP expression in differentiating GCs throughout ontogenesis. EGFP expression correlates with the expression of GAD and molecular markers of GABA release and uptake in 2–4 weeks post-mitotic GCs. These rather immature cells are able to fire action potentials (APs) and are synaptically integrated in the hippocampal network. Yet they show physiological properties that differentiate them from mature GCs. Finally, GAD67-expressing GCs express a specific complement of GABAA receptor subunits as well as distinctive features of synaptic and tonic GABA signaling. Our results reveal that GAD67 expression in dentate gyrus GCs is a transient marker of late differentiation that persists throughout life and the G42 strain may be used to visualize newborn GCs at a specific, well-defined differentiation stage. PMID:23565079

  11. Recovery of amorphous polyhydroxybutyrate granules from Cupriavidus necator cells grown on used cooking oil.

    PubMed

    Martino, Lucrezia; Cruz, Madalena V; Scoma, Alberto; Freitas, Filomena; Bertin, Lorenzo; Scandola, Mariastella; Reis, Maria A M

    2014-11-01

    Used cooking oil (UCO) was employed as the sole carbon source for the production of polyhydroxybutyrate (PHB) by cultivation in batch mode of Cupriavidus necator DSM 428. The produced biomass was used for extraction of the PHB granules with a solvent-free approach using sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and the enzyme Alcalase in an aqueous medium. The recovered PHB granules showed a degree of purity higher than 90% and no crystallization (i.e., granules were recovered in their 'native' amorphous state) as demonstrated by wide angle X-ray diffraction (WAXS). Granules were characterized according to their thermal properties and stability by differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Results show that UCO can be used as a renewable resource to produce amorphous PHB granules with excellent properties in a biocompatible manner. PMID:24751509

  12. KIF20A-Mediated RNA Granule Transport System Promotes the Invasiveness of Pancreatic Cancer Cells1

    PubMed Central

    Taniuchi, Keisuke; Furihata, Mutsuo; Saibara, Toshiji

    2014-01-01

    Pancreatic cancers are aggressive because they are highly invasive and highly metastatic; moreover, effective treatments for aggressive pancreatic cancers are lacking. Here, we report that the motor kinesin protein KIF20A promoted the motility and invasiveness of pancreatic cancer cells through transporting the RNA-binding protein IGF2BP3 and IGF2BP3-bound transcripts toward cell protrusions along microtubules. We previously reported that IGF2BP3 and its target transcripts are assembled into cytoplasmic stress granules of pancreatic cancer cells, and that IGF2BP3 promotes the motility and invasiveness of pancreatic cancer cells through regulation of localized translation of IGF2BP3-bound transcripts in cell protrusions. We show that knockdown of KIF20A inhibited accumulation of IGF2BP3-containing stress granules in cell protrusions and suppressed local protein expression from specific IGF2BP3-bound transcripts, ARF6 and ARHGEF4, in the protrusions. Our results provide insight into the link between regulation of KIF20A-mediated trafficking of IGF2BP3-containing stress granules and modulation of the motility and invasiveness in pancreatic cancers. PMID:25499221

  13. Protein sorting among two distinct export pathways occurs from the content of maturing exocrine storage granules

    Microsoft Academic Search

    Mark von Zastrow; J. David Castle

    1987-01-01

    We have developed a method for separating purified parotid secretory granules according to their degree of maturation, and we have used this method to examine the relationship between granule formation and stimulus-independent (constitutive) protein secre- tion. Constitutive export of pulse-labeled secretory proteins occurs almost entirely after their appearance in newly formed granules, and this secretion can be resolved kineticaUy into

  14. 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

    2014-01-01

    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. PMID:25313870

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

    PubMed Central

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

    2014-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.04390.001 PMID:25313870

  16. Cognitive Enhancing Treatment with a PPAR? Agonist Normalizes Dentate Granule Cell Presynaptic Function in Tg2576 APP Mice

    PubMed Central

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

    2014-01-01

    Hippocampal network hyperexcitability is considered an early indicator of Alzheimer's disease (AD) memory impairment. Some AD mouse models exhibit similar network phenotypes. In this study we focused on dentate gyrus (DG) granule cell spontaneous and evoked properties in 9-month-old Tg2576 mice that model AD amyloidosis and cognitive deficits. Using whole-cell patch-clamp recordings, we found that Tg2576 DG granule cells exhibited spontaneous EPSCs that were higher in frequency but not amplitude compared with wild-type mice, suggesting hyperactivity of DG granule cells via a presynaptic mechanism. Further support of a presynaptic mechanism was revealed by increased I–O relationships and probability of release in Tg2576 DG granule cells. Since we and others have shown that activation of the peroxisome proliferator-activated receptor gamma (PPAR?) axis improves hippocampal cognition in mouse models for AD as well as benefitting memory performance in some humans with early AD, we investigated how PPAR? agonism affected synaptic activity in Tg2576 DG. We found that PPAR? agonism normalized the I–O relationship of evoked EPSCs, frequency of spontaneous EPSCs, and probability of release that, in turn, correlated with selective expression of DG proteins essential for presynaptic SNARE function that are altered in patients with AD. These findings provide evidence that DG principal cells may contribute to early AD hippocampal network hyperexcitability via a presynaptic mechanism, and that hippocampal cognitive enhancement via PPAR? activation occurs through regulation of presynaptic vesicular proteins critical for proper glutamatergic neurotransmitter release, synaptic transmission, and short-term plasticity. PMID:24431460

  17. The expression pattern of a Cav3-Kv4 complex differentially regulates spike output in cerebellar granule cells.

    PubMed

    Heath, N Colin; Rizwan, Arsalan P; Engbers, Jordan D T; Anderson, Dustin; Zamponi, Gerald W; Turner, Ray W

    2014-06-25

    The cerebellum receives sensory information by mossy fiber input from a multitude of sources that require differential signal processing. A compartmentalization of function begins with the segregation of mossy fibers across 10 distinct lobules over the rostrocaudal axis, with tactile receptor afferents prevalent in anterior lobules and vestibular input in caudal lobules. However, it is unclear how these unique signals might be differentially processed at the circuit level across the cerebellum. As granule cells receive mossy fiber input, they represent a key stage at which postsynaptic mechanisms could influence signal processing. Granule cells express an A-type current mediated by Kv4 potassium channels that modify the latency and frequency of spike output. The current study examined the potential for a Cav3 calcium-Kv4 channel complex to regulate the response of granule cells to mossy fiber input in lobules 2 and 9 of the rat cerebellum. Similar A-type currents were recorded in both regions, but the Cav3 calcium current was expressed at a substantially higher density in lobule 9 cells, acting to increase A-type current availability through its influence on Kv4 voltage for inactivation. The difference in excitability imparted by Cav3-Kv4 interactions proves to allow lobule 2 granule cells to respond more effectively to tactile stimulus-like burst input and lobule 9 cells to slow shifts in input frequency characteristic of vestibular input. The expression pattern of Cav3 channels and its control of Kv4 availability thus provides a novel means of processing widely different forms of sensory input across cerebellar lobules. PMID:24966380

  18. Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line.

    PubMed Central

    Weymer, A; Huott, P; Liu, W; McRoberts, J A; Dharmsathaphorn, K

    1985-01-01

    Confluent T84 monolayers grown on permeable supports and mounted in a modified Ussing chamber secrete chloride (Cl-) in response to prostaglandin E1. The threshold stimulation was observed at 10(-9) M and a maximal effect at 10(-6) M. Unidirectional flux studies showed an increase in both serosal to mucosal and mucosal to serosal Cl- fluxes with 10(-6) M prostaglandin E1; the increase in serosal to mucosal Cl- flux exceeded the increase in mucosal to serosal flux, resulting in net Cl- secretion. Na+ transport was not affected in either direction and the changes in net Cl- flux correlated well with the changes in short circuit current. To identify the electrolyte transport pathways involved in the Cl- secretory process, the effect of prostaglandin E1 on ion fluxes was tested in the presence of putative inhibitors. Bumetanide was used as an inhibitor for the basolaterally localized Na+,K+,Cl- cotransport system whose existence and bumetanide sensitivity have been verified in earlier studies (Dharmsathaphorn et al. 1984. J. Clin. Invest. 75:462-471). Barium was used as an inhibitor for the K+ efflux pathway on the basolateral membrane whose existence and barium sensitivity were demonstrated in this study by preloading the monolayers with 86Rb+ (as a tracer for K+) and simultaneously measuring 86Rb+ efflux into both serosal and mucosal reservoirs. Both bumetanide and barium inhibited the net chloride secretion induced by prostaglandin E1 suggesting the involvement of the Na+,K+,Cl- cotransport and a K+ efflux pathways on the basolateral membrane in the Cl- secretory process. The activation of another Cl- transport pathway on the apical membrane by prostaglandin E1 was suggested by Cl- uptake studies. Our findings indicate that the prostaglandin E1-stimulated Cl- secretion, which is associated with an increase in cyclic AMP level, intimately involves (a) a bumetanide-sensitive Na+,K+,Cl- cotransport pathway that serves as a Cl- uptake step across the basolateral membrane, (b) the stimulation of a barium-sensitive K+ efflux mechanism on the basolateral membrane that most likely acts to recycle K+, and (c) the activation of a Cl- transport pathway on the apical membrane that serves as a Cl- exit pathway. PMID:2997290

  19. Posttraining ablation of adult-generated olfactory granule cells degrades odor-reward memories.

    PubMed

    Arruda-Carvalho, Maithe; Akers, Katherine G; Guskjolen, Axel; Sakaguchi, Masanori; Josselyn, Sheena A; Frankland, Paul W

    2014-11-19

    Proliferation of neural progenitor cells in the subventricular zone leads to the continuous generation of new olfactory granule cells (OGCs) throughout life. These cells synaptically integrate into olfactory bulb circuits after ?2 weeks and transiently exhibit heightened plasticity and responses to novel odors. Although these observations suggest that adult-generated OGCs play important roles in olfactory-related memories, global suppression of olfactory neurogenesis does not typically prevent the formation of odor-reward memories, perhaps because residual OGCs can compensate. Here, we used a transgenic strategy to selectively ablate large numbers of adult-generated OGCs either before or after learning in mice. Consistent with previous studies, pretraining ablation of adult-generated OGCs did not prevent the formation of an odor-reward memory, presumably because existing OGCs can support memory formation in their absence. However, ablation of a similar cohort of adult-generated OGCs after training impaired subsequent memory expression, indicating that if these cells are available at the time of training, they play an essential role in subsequent expression of odor-reward memories. Memory impairment was associated with the loss of adult-generated OGCs that were >10 d in age and did not depend on the developmental stage in which they were generated, suggesting that, once sufficiently mature, OGCs generated during juvenility and adulthood play similar roles in the expression of odor-reward memories. Finally, ablation of adult-generated OGCs 1 month after training did not produce amnesia, indicating that adult-generated OGCs play a time-limited role in the expression of odor-reward memories. PMID:25411506

  20. Heparan sulphate proteoglycans interact with neurocan and promote neurite outgrowth from cerebellar granule cells

    PubMed Central

    2004-01-01

    We found that neurocan, a major brain chondroitin sulphate proteoglycan, interacts with HSPGs (heparan sulphate proteoglycans) such as syndecan-3 and glypican-1. Binding of these HSPGs to neurocan was prevented by treatment of the HSPGs with heparitinases I and II, but not by treatment of neurocan with chondroitinase ABC. Scatchard plot analysis indicated that neurocan has two binding sites for these HSPGs with different affinities. It is known that neurocan in the rodent brain is proteolytically processed with aging into N- and C-terminal fragments. When a mixture of whole neurocan and N- and C-terminal fragments prepared from neonatal mouse brains or recombinant N- and C-terminal fragments was applied to a heparin column, the whole molecule and both the N- and C-terminal fragments bound to heparin. A centrifugation cell adhesion assay indicated that both the N- and C-terminal neurocan fragments could interact with these HSPGs expressed on the cell surface. To examine the biological significance of the HSPG–neurocan interaction, cerebellar granule cells expressing these HSPGs were cultured on the recombinant neurocan substrate. A significant increase in the rate of neurite outgrowth was observed on the wells coated with the C-terminal neurocan fragment, but not with the N-terminal one. Neurite outgrowth-promoting activity was inhibited by pretreatment of neurocan substrate with heparin or the addition of heparitinase I to culture medium. These results suggest that HSPGs such as syndecan-3 and glypican-1 serve as the cell-surface receptor of neurocan, and that the interaction of these HSPGs with neurocan through its C-terminal domain is involved in the promotion of neurite outgrowth. PMID:15198637

  1. Differentiation of Mucilage Secretory Cells of the Arabidopsis Seed Coat1

    E-print Network

    Western, Tamara L.

    genetics to identify genes controlling cell morphogenesis and complex poly- saccharide biosynthesis the structure and differentiation of Arabidopsis seed coat mucilage cells, including cell morphogenesis, morphogenesis, mucilage biosynthesis and secretion, and secondary cell wall synthesis. The angiosperm seed coat

  2. Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells.

    PubMed

    Boding, Lasse; Hansen, Ann K; Meroni, Germana; Johansen, Bo B; Braunstein, Thomas H; Bonefeld, Charlotte M; Kongsbak, Martin; Jensen, Benjamin A H; Woetmann, Anders; Thomsen, Allan R; Odum, Niels; von Essen, Marina R; Geisler, Carsten

    2014-10-01

    Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2A activity. Loss-of-function mutations in MID1 lead to the X-linked Opitz G/BBB syndrome characterized by defective midline development during embryogenesis. Here, we show that MID1 is strongly upregulated in murine cytotoxic lymphocytes (CTLs), and that it controls TCR signaling, centrosome trafficking, and exocytosis of lytic granules. In accordance, we find that the killing capacity of MID1(-/-) CTLs is impaired. Transfection of MID1 into MID1(-/-) CTLs completely rescued lytic granule exocytosis, and vice versa, knockdown of MID1 inhibited exocytosis of lytic granules in WT CTLs, cementing a central role for MID1 in the regulation of granule exocytosis. Thus, MID1 orchestrates multiple events in CTL responses, adding a novel level of regulation to CTL activation and cytotoxicity. PMID:25043946

  3. Electrical responses of three classes of granule cells of the olfactory bulb to synaptic inputs in different dendritic locations

    PubMed Central

    Simőes-de-Souza, Fábio M.; Antunes, Gabriela; Roque, Antonio C.

    2014-01-01

    This work consists of a computational study of the electrical responses of three classes of granule cells of the olfactory bulb to synaptic activation in different dendritic locations. The constructed models were based on morphologically detailed compartmental reconstructions of three granule cell classes of the olfactory bulb with active dendrites described by Bhalla and Bower (1993, pp. 1948–1965) and dendritic spine distributions described by Woolf et al. (1991, pp. 1837–1854). The computational studies with the model neurons showed that different quantities of spines have to be activated in each dendritic region to induce an action potential, which always was originated in the active terminal dendrites, independently of the location of the stimuli, and the morphology of the dendritic tree. These model predictions might have important computational implications in the context of olfactory bulb circuits. PMID:25360108

  4. Metabotropic glutamate receptors in the main olfactory bulb drive granule cell-mediated inhibition.

    PubMed

    Heinbockel, Thomas; Laaris, Nora; Ennis, Matthew

    2007-01-01

    Main olfactory bulb (MOB) granule cells (GCs) express high levels of the group I metabotropic glutamate receptor (mGluR), mGluR5. We investigated the role of mGluRs in regulating GC activity in rodent MOB slices using whole cell patch-clamp electrophysiology. The group I/II mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) or the selective group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) depolarized ( approximately 20 mV) and increased the firing rate of GCs. In the presence of ionotropic glutamate and GABA receptor antagonists, DHPG evoked a more modest depolarization ( approximately 8 mV). In voltage clamp, DHPG, but not group II [(2S,2'R,3)-2-(2',3'-dicarboxycyclopropyl)glycine, DCG-IV] or group III [L(+)-2-amino-4-phosphonobutyric acid, L-AP4] mGluR agonists, induced an inward current. The inward current reversed polarity near the potassium equilibrium potential, suggesting mediation by closure of potassium channels. The DHPG-evoked inward current was unaffected by the mGluR1 antagonist (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), was blocked by the group I/II mGluR antagonist (alphaS)-alpha-amino-alpha-[(1S,2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid (LY341495), and was absent in GCs from mGluR5 knockout mice. LY341495 also attenuated mitral cell-evoked voltage-sensitive dye signals in the external plexiform layer and mitral cell-evoked spikes in GCs. These results suggest that activation of mGluR5 increases GC excitability, an effect that should increase GC-mediated GABAergic inhibition of mitral cells. In support of this: DHPG increased the frequency of spontaneous GABAergic inhibitory postsynaptic currents in mitral cells and LY341495 attenuated the feedback GABAergic postsynaptic potential elicited by intracellular depolarization of mitral cells. Our results suggest that activation of mGluR5 participates in feedforward and/or feedback inhibition at mitral cell to GC dendrodendritic synapses, possibly to modulate lateral inhibition and contrast in the MOB. PMID:17093122

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

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

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

  6. Tumor necrosis factor alpha maintains denervation-induced homeostatic synaptic plasticity of mouse dentate granule cells

    PubMed Central

    Becker, Denise; Zahn, Nadine; Deller, Thomas; Vlachos, Andreas

    2013-01-01

    Neurons which lose part of their input respond with a compensatory increase in excitatory synaptic strength. This observation is of particular interest in the context of neurological diseases, which are accompanied by the loss of neurons and subsequent denervation of connected brain regions. However, while the cellular and molecular mechanisms of pharmacologically induced homeostatic synaptic plasticity have been identified to a certain degree, denervation-induced homeostatic synaptic plasticity remains not well understood. Here, we employed the entorhinal denervation in vitro model to study the role of tumor necrosis factor alpha (TNF?) on changes in excitatory synaptic strength of mouse dentate granule cells following partial deafferentation. Our experiments disclose that TNF? is required for the maintenance of a compensatory increase in excitatory synaptic strength at 3–4 days post lesion (dpl), but not for the induction of synaptic scaling at 1–2 dpl. Furthermore, laser capture microdissection combined with quantitative PCR demonstrates an increase in TNF?-mRNA levels in the denervated zone, which is consistent with our previous finding on a local, i.e., layer-specific increase in excitatory synaptic strength at 3–4 dpl. Immunostainings for the glial fibrillary acidic protein and TNF? suggest that astrocytes are a source of TNF? in our experimental setting. We conclude that TNF?-signaling is a major regulatory system that aims at maintaining the homeostatic synaptic response of denervated neurons. PMID:24385951

  7. Tumor necrosis factor alpha maintains denervation-induced homeostatic synaptic plasticity of mouse dentate granule cells.

    PubMed

    Becker, Denise; Zahn, Nadine; Deller, Thomas; Vlachos, Andreas

    2013-01-01

    Neurons which lose part of their input respond with a compensatory increase in excitatory synaptic strength. This observation is of particular interest in the context of neurological diseases, which are accompanied by the loss of neurons and subsequent denervation of connected brain regions. However, while the cellular and molecular mechanisms of pharmacologically induced homeostatic synaptic plasticity have been identified to a certain degree, denervation-induced homeostatic synaptic plasticity remains not well understood. Here, we employed the entorhinal denervation in vitro model to study the role of tumor necrosis factor alpha (TNF?) on changes in excitatory synaptic strength of mouse dentate granule cells following partial deafferentation. Our experiments disclose that TNF? is required for the maintenance of a compensatory increase in excitatory synaptic strength at 3-4 days post lesion (dpl), but not for the induction of synaptic scaling at 1-2 dpl. Furthermore, laser capture microdissection combined with quantitative PCR demonstrates an increase in TNF?-mRNA levels in the denervated zone, which is consistent with our previous finding on a local, i.e., layer-specific increase in excitatory synaptic strength at 3-4 dpl. Immunostainings for the glial fibrillary acidic protein and TNF? suggest that astrocytes are a source of TNF? in our experimental setting. We conclude that TNF?-signaling is a major regulatory system that aims at maintaining the homeostatic synaptic response of denervated neurons. PMID:24385951

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  9. Dendritic patch-clamp recordings from cerebellar granule cells demonstrate electrotonic compactness

    PubMed Central

    Delvendahl, Igor; Straub, Isabelle; Hallermann, Stefan

    2015-01-01

    Cerebellar granule cells (GCs), the smallest neurons in the brain, have on average four short dendrites that receive high-frequency mossy fiber inputs conveying sensory information. The short length of the dendrites suggests that GCs are electrotonically compact allowing unfiltered integration of dendritic inputs. The small average diameter of the dendrites (~0.7 µm), however, argues for dendritic filtering. Previous studies based on somatic recordings and modeling indicated that GCs are electrotonically extremely compact. Here, we performed patch-clamp recordings from GC dendrites in acute brain slices of mice to directly analyze the electrotonic properties of GCs. Strikingly, the input resistance did not differ significantly between dendrites and somata of GCs. Furthermore, spontaneous excitatory postsynaptic potentials (EPSP) were similar in amplitude at dendritic and somatic recording sites. From the dendritic and somatic input resistances we determined parameters characterizing the electrotonic compactness of GCs. These data directly demonstrate that cerebellar GCs are electrotonically compact and thus ideally suited for efficient high-frequency information transfer. PMID:25852483

  10. Pathological ?-synuclein impairs adult-born granule cell development and functional integration in the olfactory bulb

    PubMed Central

    Neuner, Johanna; Ovsepian, Saak V.; Dorostkar, Mario; Filser, Severin; Gupta, Aayush; Michalakis, Stylianos; Biel, Martin; Herms, Jochen

    2014-01-01

    Although the role of noxious ?-synuclein (?-SYN) in the degeneration of midbrain dopaminergic neurons and associated motor deficits of Parkinson’s disease is recognized, its impact on non-motor brain circuits and related symptoms remains elusive. Through combining in vivo two-photon imaging with time-coded labelling of neurons in the olfactory bulb of A30P ?-SYN transgenic mice, we show impaired growth and branching of dendrites of adult-born granule cells (GCs), with reduced gain and plasticity of dendritic spines. The spine impairments are especially pronounced during the critical phase of integration of new neurons into existing circuits. Functionally, retarded dendritic expansion translates into reduced electrical capacitance with enhanced intrinsic excitability and responsiveness of GCs to depolarizing inputs, while the spine loss correlates with decreased frequency of AMPA-mediated miniature EPSCs. Changes described here are expected to interfere with the functional integration and survival of new GCs into bulbar networks, contributing towards olfactory deficits and related behavioural impairments. PMID:24867427

  11. Pharmacological characterization of mGlu1 receptors in cerebellar granule cells reveals biased agonism.

    PubMed

    Hathaway, Hannah A; Pshenichkin, Sergey; Grajkowska, Ewa; Gelb, Tara; Emery, Andrew C; Wolfe, Barry B; Wroblewski, Jarda T

    2015-06-01

    The majority of existing research on the function of metabotropic glutamate (mGlu) receptor 1 focuses on G protein-mediated outcomes. However, similar to other G protein-coupled receptors (GPCR), it is becoming apparent that mGlu1 receptor signaling is multi-dimensional and does not always involve G protein activation. Previously, in transfected CHO cells, we showed that mGlu1 receptors activate a G protein-independent, ?-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response. Here we set out to investigate the physiological relevance of these findings in a native system using primary cultures of cerebellar granule cells. We tested the ability of a panel of compounds to stimulate two mGlu1 receptor-mediated outcomes: (1) protection from decreased cell viability after withdrawal of trophic support and (2) G protein-mediated phosphoinositide (PI) hydrolysis. We report that the commonly used mGlu1 receptor ligands quisqualate, DHPG, and ACPD are completely biased towards PI hydrolysis and do not induce mGlu1 receptor-stimulated neuroprotection. On the other hand, endogenous compounds including glutamate, aspartate, cysteic acid, cysteine sulfinic acid, and homocysteic acid stimulate both responses. These results show that some commonly used mGlu1 receptor ligands are biased agonists, stimulating only a fraction of mGlu1 receptor-mediated responses in neurons. This emphasizes the importance of utilizing multiple agonists and assays when studying GPCR function. PMID:25700650

  12. Ultrastructural changes in contents of membrane-coating granules after extrusion from epithelial cells of hamster cheek pouch

    Microsoft Academic Search

    A. F. Hayward

    1978-01-01

    Membrane-coating granules of the epithelium of the hamster cheek pouch displayed a lamellated internal structure consisting of alternating thick and thin electron dense bands separated by translucent bands of equal width. The repeat distance of the contents was 8.5nm±sd 0.7nm and the thickness of the major electron dense band was 3 nm±sd 0.4 nm. After exocytosis from the cells the

  13. Expression of NR2B in Cerebellar Granule Cells Specifically Facilitates Effect of Motor Training on Motor Learning

    Microsoft Academic Search

    Jianwei Jiao; Akira Nakajima; William G. M. Janssen; Vytautas P. Bindokas; Xiaoli Xiong; John H. Morrison; James R. Brorson; Ya-Ping Tang; Martin Giurfa

    2008-01-01

    It is believed that gene\\/environment interaction (GEI) plays a pivotal role in the development of motor skills, which are acquired via practicing or motor training. However, the underlying molecular\\/neuronal mechanisms are still unclear. Here, we reported that the expression of NR2B, a subunit of NMDA receptors, in cerebellar granule cells specifically enhanced the effect of voluntary motor training on motor

  14. Secretory vesicle formation in the secretory cavity of glandular trichomes of Cannabis sativa L. (Cannabaceae).

    PubMed

    Kim, Eun Soo; Mahlberg, Paul G

    2003-06-30

    The disc cell wall facing the secretory cavity in lipophilic glands of Cannabis was studied for origin and distribution of hyaline areas, secretory vesicles, fibrillar matrix and particulate material. Secretions evident as light areas in the disc cell cytoplasm pass through modified regions in the plasma membrane and appear as hyaline areas in the cell wall. Hyaline areas, surrounded with a filamentous outline, accumulate near the wall surface facing the secretory cavity where they fuse to form enlarged hyaline areas. Fibrillar matrix is related to and may originate from the dense outer layer of the plasma membrane. This matrix becomes distributed throughout the wall material and contributes in part to the composition of the surface feature of secretory vesicles. Thickening of the cell wall is associated with secretions from the disc cells that facilitates movement of hyaline areas, fibrillar matrix and other possible secretions through the wall to form secretory vesicles and intervesicular materials in the secretory cavity. The outer wall of disc cells in aggregate forms the basilar wall surface of the secretory cavity which facilitates the organization of secretory vesicles that fill the secretory cavity. PMID:12872998

  15. Elastic behavior of zymogen granule membranes in response to changes in pH and pCa.

    PubMed Central

    Miyamoto, S; Fujime, S

    1990-01-01

    In the process of secretion, the membrane of secretory granules is expected to change its elastic behavior. Elastic modulus of the membrane of zymogen granules, prepared from the rat pancreas acinar cell, was measured by an osmotic swelling method. The elastic modulus of the granule membrane at pCa 8 reduced from the maximal value of 230 dyn/cm at pH 6.0 to almost zero at pH 7.5. In a cytosol of an acinar cell, calcium ions play an important role as a second messenger in secretion. The elastic modulus of the granule membrane reduced in a sigmoidal fashion at pCa between 7.0 and 6.0. This range of pCa corresponds to a physiological rise of free Ca2+ concentrations in the cell cytosol when stimulated by external secretagogues. Reduction of the elastic modulus indicates that the state of the granule membrane switches to a more flexible one in which the granule is easy to appose to the cell plasma membrane and then swell as a final step of exocytosis. PMID:2306504

  16. Cytoarchitectural features of Ucides cordatus (Crustacea Decapoda) hepatopancreas: structure and elemental composition of electron-dense granules.

    PubMed

    Corręa, J D; Farina, M; Allodi, S

    2002-10-01

    Hepatopancreal tissue of the crab Ucides cordatus was investigated by light and electron microscopy. The observed epithelial cells were: E-cells (embryonic), located in the distal portion of the hepatopancreal tubules, R-cells (resorptive) F-cells (fibrillar) and B-cells (blister or secretory), found in its intermediate and proximal regions. Two types of electron-dense granules (EDGs) were found frequently in the cells of the proximal portion of the hepatopancreal tubule. Both types of EDGs presented alternating concentric electron-dense and electron-lucent layers. In order to better characterize these granules, energy dispersive X-ray analysis (EDXA) and glucose-6-phosphatase (G6Pase) cytochemistry were performed. One type of spherical granule was seen inside vacuoles surrounded by an association of myelin-like membranes as well as some small membrane-bound vesicles. This type of granule neither presented detectable Ca and P on EDXA spectra nor G6Pase cytochemical reaction products. The second type of granule had O, P and Ca characteristic peaks. G6Pase cytochemical products were observed inside these structures and showed that this mineralized type was surrounded by endoplasmic reticulum membranes. This result suggests that in U. cordatus the endoplasmic reticulum is associated with the genesis of mineralized EDGs. While amorphous mineral granules may be associated with a storage of Ca and P for the new carapace synthesis, EDGs covered by the non-mineralized spherical multi-layered membranes may be associated with late endosomes. No specific secretory pathway however was determined for the EDGs at the epithelial proximal portion. PMID:12270258

  17. Acute and long-term inhibition of agonist-stimulated phosphoinositide hydrolysis by pulse treatment of cerebellar granule cells with TPA

    Microsoft Academic Search

    Ragnhild E. Paulsen; Robert Raulli; Dennis R. Grayson; Jarda T. Wroblewski

    1994-01-01

    Acute pretreatment (30 min) of primary cultures of cerebellar granule cells with TPA (10 nM) resulted in a decrease in carbachol- and glutamate-stimulated phosphoinositide hydrolysis, but not in basal levels of PI\\u000a hydrolysis. To investigate the mechanism of TPA action, phospholipase C was assayed in membranes prepared from cerebellar\\u000a granule cells acutely treated with TPA. TPA had no effect on

  18. Effect of oral N-acetylcysteine (NAC) on volume and albumin content of respiratory tract fluid but not on epithelial secretory cell number in "smoking" rats.

    PubMed

    Robinson, N; Brattsand, R; Dahlbäck, M

    1990-03-01

    This study was designed to look at the effect of N-acetylcysteine (NAC) on epithelial secretory cells and the respiratory tract fluid volume and albumin content from the lower airways of "bronchitic" rats. Rats were exposed either to tobacco smoke (TS), TS and NAC, or NAC alone. TS caused a significant increase in epithelial secretory cell number which was not reduced by concomitant NAC administration; NAC alone had no effect on cell numbers. TS increased respiratory tract fluid volume and albumin content by a small but non-significant amount, whereas TS and NAC increased the volume and albumin content by a greater and significant amount; NAC alone was also shown to significantly increase both fluid volume and albumin content. PMID:2340888

  19. Dentate granule cell GABAA receptors in epileptic hippocampus: enhanced synaptic efficacy and altered pharmacology

    PubMed Central

    Cohen, Akiva S.; Lin, Dean D.; Quirk, Gerald L.; Coulter, Douglas A.

    2008-01-01

    The dentate gyrus (DG) normally functions as a filter, preventing propagation of synchronized activity into the seizure-prone hippocampus. This filter or ‘gatekeeper’ attribute of the DG is compromised in various pathological states, including temporal lobe epilepsy (TLE). This study examines the role that altered inhibition may play in the deterioration of this crucial DG function. Using the pilocarpine animal model of TLE, we demonstrate that inhibitory synaptic function is altered in principal cells of the DG. Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in dentate granule cells (DGCs) from epileptic animals were larger, more sensitive to blockade by zinc and less sensitive to augmentation by the benzodiazepine type site 1 modulator zolpidem. Furthermore, mIPSCs examined during a quiescent period following injury but preceding onset of epilepsy were significantly smaller than those present either in control or in TLE DGCs, and had already acquired sensitivity to blockade by zinc prior to the onset of spontaneous seizures. Rapid agonist application experiments demonstrated that prolonged (>35 ms) exposure to zinc is required to block GABAA receptors (GABAARs) in patches pulled from epileptic DGCs. Therefore, zinc must be tonically present to block DGC GABAARs and alter DG function. This would occur only during repetitive activation of mossy fibres. Thus, in the pilocarpine animal model of TLE, an early, de novo, expression of zinc-sensitive GABAARs is coupled with delayed, epilepsy-induced development of a zinc delivery system provided by aberrant sprouting of zinc-containing mossy fibre recurrent collaterals. The temporal and spatial juxtaposition of these pathophysiological alterations may compromise normal ‘gatekeeper’ function of the DG through dynamic zinc-induced failure of inhibition, predisposing the hippocampal circuit to generate seizures. PMID:12752378

  20. High cell-density expression system: yeast cells in a phalanx efficiently produce a certain range of "difficult-to-express" secretory recombinant proteins.

    PubMed

    Kawarasaki, Yasuaki; Kurose, Takeshi; Ito, Keisuke

    2015-01-01

    Yeast's extracellular expression provides a cost-efficient means of producing recombinant proteins of academic or commercial interests. However, depending on the protein to be expressed, the production occasionally results in a poor yield, which is frequently accompanied with a deteriorated growth of the host. Here we describe our simple approach, high cell-density expression, to circumvent the cellular toxicity and achieve in a production of a certain range of "difficult-to-express" secretory protein in preparative amount. The system features an ease of performing: (1) precultivate yeast cells to the stationary phase in non-inducing condition, (2) suspend the cells to a small aliquot of inducing medium to form a high cell-density suspension or "a phalanx," and then (3) give a sufficient aeration to the phalanx. Factors and pitfalls that affect the system's performance are also described. PMID:25447864

  1. The mast cell nature of granule cells in the digestive tract of the pike, Esox lucius : similarity to mammalian mucosal mast cells and globule leucocytes

    Microsoft Academic Search

    OLA B. REITE

    1996-01-01

    Observations were made on sections of intestinal tissue from the pike,Esox lucius, fixed in a solution containing 4% formaldehyde and 5% acetic acid in methanol. Four staining procedures, using May-Grünwald Giemsa combi-nation dye, hematoxylin and eosin, toluidine blue, and alcian blue in sequence with safranin, were applied. Numerous granule cells were found in the area of stratum compactum and in

  2. Enzymatic isolation and culture of cement secreting cells from cypris larvae of the barnacle Megabalanus rosa

    Microsoft Academic Search

    Keiju Okano; Katsuhiko Shimizu; Cyril Glenn Satuito; Nobuhiro Fusetani

    1998-01-01

    Isolation of viable secretory cells from cyprid cement glands of the barnacle Megabalanus rosa was achieved by trypsin digestion followed by mechanical treatment. Isolated cells, which were classified into two groups based on cell and granule size, were comparable to earlier descriptions of ?? and ß?cells in the cement glands of Balanus balanoides. \\

  3. Isolated secretion granules from parotid glands of chronically stimulated rats possess an alkaline internal pH and inward-directed H/sup +/ pump activity

    SciTech Connect

    Arvan, P.; Castle, J.D.

    1986-10-01

    Secretion granules have been isolated from the parotid glands of rats that have been chronically stimulated with the ..beta..-adrenergic agonist, isoproterenol. These granules are of interest because they package a quantitatively different set of secretory proteins in comparison with granules from the normal gland. Polypeptides enriched in proline, glycine, and glutamine, which are known to have pI's >10, replace ..cap alpha..-amylase (pI's = 6.8) as the principal content species. The internal pH of granules from the treated rats changes from 7.8 in a potassium sulfate medium to 6.9 in a choline chloride medium. The increased pH over that of normal parotid granules (approx.6.8) appears to protect the change in composition of the secretory contents. Whereas normal mature parotide granules have practically negligible levels of H/sup +/ pumping ATPase activity, the isolated granules from isoproterenol-treated rats undergo a time-dependent internal acidification that requires the presence of ATP and is abolished by an H/sup +/ ionophore. Additionally, an inside-positive granule transmembrane potential develops after ATP addition that depends upon ATP hydrolysis. Two independent methods have been used that exclude the possibility that contaminating organelles are the source of the H/sup +/-ATPase activity. Together these data provide clear evidence for the presence of an H/sup +/ pump in the membranes of parotid granules from chronically stimulated rats. However, despite the presence of H/sup +/-pump activity, fluorescence microscopy with the weak base, acridine orange, reveals that the intragranular pH in live cells is greater than that of the cytoplasm.

  4. A biophysical model of the chromaffin granule. Accurate description of the kinetics of ATP and Cl- dependent granule lysis.

    PubMed Central

    Creutz, C E; Pollard, H B

    1980-01-01

    A model is constructed to describe the behavior of isolated chromaffin granules (secretory vesicles of the adrenal medulla) when they are induced to release their contents by incubation with MgATP and Cl-. The model is based on the assumption that the release event is osmotic lysis due to the ATPase dependent influx of protons and osmotically active Cl- ions. The consequences of this influx of osmotically active particles are predicted from osmotic fragility curves determined by suspending granules in hypotonic media. Turbidity measurements on granule suspensions undergoing the ATP and Cl- dependent release reaction are used to fit the parameters of the model. The model then successfully described the time course, Cl- dependence, ATP dependence, and osmotic strength suppression of the release event as monitored either by measurement of turbidity changes or of epinephrine release. The degree of suppression of release predicted in hypertonic media is also in agreement with published data on hypertonic suppression of exocytosis from several cell types: chromaffin cells, blood platelets, and parathyroid cells. Therefore, the model may also provide an accurate description of some of the events occurring during exocytosis. PMID:6455169

  5. Anoctamin 1 in secretory epithelia.

    PubMed

    Jang, Yongwoo; Oh, Uhtaek

    2014-06-01

    Fluid and electrolyte releasing from secretory epithelia are elaborately regulated by orchestrated activity of ion channels. The activity of chloride channel at the apical membrane decides on the direction and the rate of secretory fluid and electrolyte. Chloride-dependent secretion is conventionally associated with intracellular increases in two second messengers, cAMP and Ca(2+), responding to luminal purinergic and basolateral adrenergic or cholinergic stimulation. While it is broadly regarded that cAMP-dependent Cl(-) secretion is regulated by cystic fibrosis transmembrane conductance regulator (CFTR), Ca(2+)-activated Cl(-) channel (CaCC) had been veiled for quite some time. Now, Anoctamin 1 (ANO1 or TMEM16A) confers Ca(2+)-activated Cl(-) currents. Ano 1 and its paralogs have been actively investigated for multiple functions underlying Ca(2+)-activated Cl(-) efflux and fluid secretion in a variety of secretory epithelial cells. In this review, we will discuss recent advances in the secretory function and signaling of ANO1 in the secretory epithelia, such as airways, intestines, and salivary glands. PMID:24636668

  6. Cytopathologic Features of Mammary Analogue Secretory Carcinoma

    PubMed Central

    Bishop, Justin A.; Yonescu, Raluca; Batista, Denise A. S.; Westra, William H.; Ali, Syed Z.

    2013-01-01

    BACKGROUND Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland neoplasm that is defined by ETV6-NTRK3 gene fusion. To the best of the authors’ knowledge, only rare case reports of the cytopathologic features of MASC have been published to date. METHODS A wide variety of archival salivary gland tumors were tested for ETV6 translocation by break-apart fluorescent in situ hybridization. Positive cases with preoperative fine-needle aspiration (FNA) specimens or intraoperative touch preparations were retrieved from the archives of The Johns Hopkins Hospital. All smears were reviewed and the cytologic characteristics were described. RESULTS Five cases of MASC with cytopathologic material (4 FNA specimens and 1 touch preparation) were identified. The cases occurred in 3 men and 2 women ranging in age from 21 years to 78 years (mean, 52 years). On the cytologic smears, the MASCs were variably cellular and exhibited 2 different architectural patterns: 1) intact tissue fragments with isomorphic cells arranged in a sheet-like or papillary configuration; and 2) dispersed and dissociated cells with a mostly “histiocyte-like” appearance with large cells containing abundant vacuolated cytoplasm. No matrix tissue or stromal spindled cells were present. The cells did not display acinic differentiation in the form of cytoplasmic zymogen granules. In each case, the preoperative FNA correctly identified a neoplasm, and the most frequent diagnostic considerations were acinic cell carcinoma, mucoepidermoid carcinoma, and pleomorphic adenoma. CONCLUSIONS MASC is a newly described salivary gland tumor that should be considered in the differential diagnosis of low-grade salivary gland neoplasms. Its cytologic features overlap considerably with those of other tumors, especially acinic cell carcinoma and mucoepidermoid carcinoma. PMID:23042752

  7. Activation of mitogen-activated protein kinase and p70S6 kinase is not correlated with cerebellar granule cell survival.

    PubMed Central

    Gunn-Moore, F J; Williams, A G; Toms, N J; Tavaré, J M

    1997-01-01

    We have investigated the role of mitogen-activated protein (MAP) kinase in the survival of cerebellar granule cells in primary culture. Brain-derived neurotrophic factor (BDNF) and insulin, but not epidermal growth factor (EGF), promoted the survival of P6 cerebellar granule neurons. BDNF promoted a sustained activation of MAP kinase, whereas that induced by EGF was only transient. Insulin promoted a small but transient activation of MAP kinase that was completely blocked by PD98059, an inhibitor of MAP kinase kinase activation. PD98059 had no effect on the insulin- or BDNF-induced survival of cerebellar granule cells. We also investigated the role of p70S6 kinase in survival. The activation of p70S6 kinase by EGF was transient, whereas BDNF and insulin promoted a sustained activation of p70S6 kinase. Rapamycin, which blocked p70S6 kinase activation, had no effect on the BDNF- or insulin-induced survival of cerebellar granule cells. We conclude that sustained activation of MAP kinase is not correlated with the survival response of cerebellar granule cells; indeed insulin-mediated survival is independent of MAP kinase. Survival of cerebellar granule cells is also independent of the activation of p70S6 kinase. PMID:9182692

  8. Attenuation of Pulmonary Neuroendocrine Differentiation in Mice Lacking Clara Cell Secretory Protein

    Microsoft Academic Search

    Cesar M Castro; Yongping Yang; Zhongjian Zhang; R Ilona Linnoila

    2000-01-01

    During development and injury, pulmonary neuroendocrine (NE) cells may transiently express Clara cell 10 kD protein (CC10), a major product of the nonciliated progenitor cells for normal and neoplastic airway epithelia suggesting a close relationship between the cells. To assess the role of CC10 during NE differentiation, we studied CC10-deficient mouse lungs by immunohistochemistry and digital imaging. The knockout model

  9. The secretory apparatus of Xerophyta viscosa (Velloziaceae): Epidermis anatomy and chemical composition of the secretory product

    Microsoft Academic Search

    Gonasageran Naidoo; Seventhilingam Kaliamoorthy; Yougasphree Naidoo

    2009-01-01

    In this study, secretory activity on the adaxial surface of the leaves of the desiccation tolerant plant, Xerophyta viscosa Baker was investigated, using light, scanning and transmission electron microscopy. Glandular activity was associated with sunken cavities which appear to be modifications of infolded epidermal cells. The secretory cavity consisted of a globose lumen surrounded by two layers of cells. The

  10. Interplay between sodium and calcium dynamics in granule cell presynaptic terminals.

    PubMed Central

    Regehr, W G

    1997-01-01

    Fluorescent indicators were used to detect stimulus-evoked changes in presynaptic levels of intracellular sodium (Na(i)) and calcium (Ca(i)) in granule cell parallel fibers in brain slices from rat cerebellum. Ca(i) increased during stimulation, and three exponentials were needed to approximate its return to prestimulus levels. Ca(i) decayed to approximately 10% of peak levels with tau approximately 100 ms, to approximately 1% of peak values with tau approximately 6 s, and then returned to prestimulus levels with tau approximately 1-2 min. After stimulation, Na(i) accumulated in two phases; one rapid, the other continuing for several hundred milliseconds. The return of Na(i) to prestimulus levels was well approximated by a double exponential decay with time constants of 6-17 s and 2-3 min. Manipulations that prevented calcium entry eliminated both the slow component of sodium entry and the rapid component of Na(i) decay. Reductions of extracellular sodium slowed the rapid phase of Ca(i) decay. These Ca(i) and Na(i) transients were well described by a model in which the plasma membrane of presynaptic boutons contained both a sodium/calcium exchanger and a calcium ATPase (Ca-ATPase). According to this model, immediately after stimulation the sodium/calcium exchanger removes calcium from the terminal more rapidly than does the Ca-ATPase. Eventually, the large concomitant sodium influx brings the exchanger into steady-state, leaving only the Ca-ATPase to remove calcium. This perturbs the equilibrium of the sodium/calcium exchanger, which opposes the Ca-ATPase, leading to a slow return of Ca(i) and Na(i) to resting levels. PMID:9370441

  11. GABAB receptor subtypes differentially modulate synaptic inhibition in the dentate gyrus to enhance granule cell output

    PubMed Central

    Foster, Joshua D; Kitchen, Ian; Bettler, Bernhard; Chen, Ying

    2013-01-01

    Background and Purpose Activation of GABAB receptors in the dentate gyrus (DG) enhances granule cell (GC) activity by reducing synaptic inhibition imposed by hilar interneurons. This disinhibitory action facilitates signal transfer from the perforant path to the hippocampus. However, as the two main molecular subtypes, GABAB(1a,2) and GABAB(1b,2) receptors, prefer axonal terminal and dendritic compartments, respectively, they may modulate the hilar pathways at different synaptic localizations. We examined their relative expression and functions in the DG. Experimental Approach The localization of GABAB subtypes was revealed immunohistochemically using subunit-selective antibodies in GABAB1a–/– and GABAB1b–/– mice. Effects of subtype activation by the GABAB receptor agonist, baclofen, were examined on the perforant path-stimulated GC population activities in brain slices. Key Results GABAB(1a,2) receptors were concentrated in the inner molecular layer, the neuropil of the hilus and hilar neurons at the border zone; while GABAB(1b,2) receptors dominated the outer molecular layer and hilar neurons in the deep layer, showing their differential localization on GC dendrite and in the hilus. Baclofen enhanced the GC population spike to a larger extent in the GABAB1b–/– mice, demonstrating exclusively disinhibitory roles of the GABAB(1a,2) receptors. Conversely, in the GABAB1a–/– mice baclofen not only enhanced but also inhibited the population spike during GABAA blockade, revealing both disinhibitory and inhibitory effects of GABAB(1b,2) receptors. Conclusions and Implications The GABAB(1a,2) and GABAB(1b,2) receptor subtypes differentially modulate GC outputs via selective axonal terminal and dendritic locations in the hilar pathways. The GABAB(1a,2) receptors exclusively mediate disinhibition, thereby playing a greater role in gating signal transfer for hippocampal spatial and pattern learning. PMID:23186302

  12. Acute and long-term effects of metformin on the function and insulin secretory responsiveness of clonal ?-cells.

    PubMed

    McKiney, Joan M; Irwin, Nigel; Flatt, Peter R; Bailey, Clifford J; McClenaghan, Neville H

    2010-12-01

    Functional effects of acute and prolonged (48 h) exposure to the biguanide drug metformin were examined in the clonal pancreatic ?-cell line, BRIN-BD11. Effects of metformin on prolonged exposure to excessive increased concentrations of glucose and palmitic acid were also assessed. In acute 20-min incubations, 12.5-50 ?m metformin did not alter basal (1.1 mm glucose) or glucose-stimulated (16.7 mm glucose) insulin secretion. However, higher concentrations of metformin (100-1000 ?m) increased (1.3-1.5-fold; p<0.001) insulin release at basal glucose concentrations, but had no effect on glucose-stimulated insulin secretion. There were no apparent acute effects of metformin on intracellular Ca˛(+) concentrations, but metformin enhanced (p<0.05 to p<0.01) the acute insulinotropic actions of GIP and GLP-1. Exposure for 48 h to 200 ?m metformin improved aspects of ?-cell insulin secretory function, whereas these benefits were lost at 1 mm metformin. Prolonged glucotoxic and lipotoxic conditions impaired ?-cell viability and insulin release in response to glucose and to a broad range of insulin secretagogues. Concomitant culture with 200 ?m metformin partially reversed many of the adverse effects of prolonged glucotoxic conditions. However, there were no beneficial effects of metformin under prolonged culture with elevated concentrations of palmitic acid. The results suggest that metformin exerts direct effects on ?-cell viability, function and survival that could contribute to the use of this agent in the treatment of type 2 diabetes. PMID:20868233

  13. Investigation into the Role of Phosphatidylserine in Modifying the Susceptibility of Human Lymphocytes to Secretory Phospholipase A2 using Cells Deficient in the Expression of Scramblase

    PubMed Central

    Nelson, Jennifer; Francom, Lyndee L.; Anderson, Lynn; Damm, Kelly; Baker, Ryan; Chen, Joseph; Franklin, Sarah; Hamaker, Amy; Izidoro, Izadora; Moss, Eric; Orton, Mikayla; Stevens, Evan; Yeung, Celestine; Judd, Allan M.; Bell, John D.

    2012-01-01

    Summary Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt’s lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. PMID:22266334

  14. Cholinergic Stimulation of Lacrimal Acinar Cells Promotes Redistribution of Membrane-associated Kinesin and the Secretory Protein, ?-hexosaminidase, and Increases Kinesin Motor Activity

    Microsoft Academic Search

    SARAH F. HAMM-ALVAREZ; SILVIA DA COSTA; TAO YANG; XINHUA WEI; PETER J. GIEROW; AUSTIN K. MIRCHEFF

    1997-01-01

    The role of the microtubule-based motor, kinesin, in membrane trafficking has been investigated in resting and stimulated acinar cells from rabbit lacrimal gland, a cholinergically controlled secretory tissue. Microtubule-dependent motors from extracts of control and carbachol-treated acini were isolated by microtubule-affinity purification and their activity was determined using a video-enhanced differential interference contrast microscopy assay for microtubule gliding. The observation

  15. Characterisation of glycoproteins in the secretory cells in the operculum of an Indian hill stream fish Garra lamta (Hamilton) (Cyprinidae, Cypriniformes)

    Microsoft Academic Search

    S. Mittal; Pinky; A. K. Mittal

    2002-01-01

    Glycoproteins (GPs) elaborated by the secretory cells in the opercular epidermis (OE) and the epithelium lining the inner\\u000a surface of the operculum (EISO), of an Indian hill stream fish Garra lamta have been analysed by means of a battery of histochemical methods. These included methods for the characterisation and simultaneous\\u000a visualisation of GPs with oxidizable vicinal diols, O-acyl sugars, O-sulphate

  16. CD28-stimulated ERK2 phosphorylation is required for polarization of the microtubule organizing center and granules in YTS NK cells

    Microsoft Academic Search

    Xi Chen; David S. J. Allan; Konrad Krzewski; Baoxue Ge; Hernan Kopcow; Jack L. Strominger

    2006-01-01

    Activation of natural killer (NK) cell cytotoxicity requires adhesion and formation of a conjugate with a susceptible target cell, followed by actin polymerization, and polarization of the microtubule organizing center (MTOC) and cytolytic granules to the NK cell immune synapse. Here, by using the YTS NK cell line as a model, CD28 is shown to be an activating receptor. It

  17. Analysis of constitutive and constitutive-like secretion in semi-intact pituitary cells.

    PubMed

    Dumermuth, E; Moore, H P

    1998-10-01

    To study biosynthetic transport through the constitutive and regulated secretory pathways, we have designed a semi-intact mammalian cell system that restores the transport of secretory proteins from the trans-Golgi/trans-Golgi network (TGN) to the cell surface. The mouse pituitary AtT-20 cell line is a suitable model to biochemically analyze molecular sorting in the secretory pathway. The prohormone proopiomelanocortin is sulfated on N-linked carbohydrate chains in the trans-Golgi prior to proteolytic processing in the secretory granule. Radiolabeling with [35S]sulfate therefore provides a convenient tool to selectively follow molecular events in the regulated secretory pathway without interference from earlier steps. Likewise, transport through the constitutive secretory pathway may be monitored using sulfate-labeled glycosaminoglycan chains. We show that export from the TGN is efficiently reconstituted in cells made semi-intact with streptolysin O, and is dependent on temperature, ATP and GTP hydrolysis, and cytosol. Packaging of proopiomelanocortin into immature secretory granules also activates the proteolytic processing machinery which eventually converts the prohormone to its bioactive mature product, adrenocorticotropic hormone. In addition, a large fraction of incompletely processed proopiomelanocortin is secreted as the processing intermediates from immature secretory granules. This process of constitutive-like secretion can be clearly distinguished from direct constitutive secretion from the trans-Golgi network by kinetic and compositional criteria. Furthermore, we have found that specific inhibitors of different protein phosphatases and kinases are potent blockers of constitutive and constitutive-like secretion. This experimental model should provide a valuable system to elucidate the molecular mechanism regulating post-Golgi traffic during secretory granule biogenesis. PMID:9790865

  18. Phosducin regulates secretory activity in TT line of thyroid parafollicular C cells.

    PubMed

    Piotrowska, U; Adler, G; Kozicki, I

    2015-02-01

    The endocrine activity of the thyroid gland is accomplished by its follicular and parafollicular cells. In these cells, numerous G proteins-dependent pathways are active and potentially could be regulated by a 33-kDa cytoplasmic protein phosducin, which interacts with the G? subunit and may compete with G? or G?? dimer effectors. Significant expression of phosducin has been shown in the retina, pineal gland, and some neurons. Here, we studied postoperative thyroid tissue samples collected from patients with nodular goiter and 2 thyroid-derived cell lines for the presence of phosducin. Using reverse transcription PCR with product sequencing and highly sensitive immunodetection we identified phosducin mRNA and protein in the thyroid gland and parafollicular C TT cells, but not in the follicular Nthy-ori 3-1 cell line. We also observed that siRNA-mediated silencing of phosducin gene expression decreased Ca(2+)-stimulated secretion of calcitonin and serotonin by TT cells. PMID:25153685

  19. Export of major cell surface proteins is blocked in yeast secretory mutants

    Microsoft Academic Search

    PETER NOVICK; RANDY SCHEKMAN

    1983-01-01

    The transport of newly synthesized proteins to the yeast cell surface has been analyzed by a modification of the technique developed by Kaplan et al. (Kaplan, G., C. Unkeless, and Z. A. Cohn, 1979, Proc. Natl. Acad. Sci. USA, 76:3824-3828). Cells metabolically labeled with 35SO42- are treated with trinitrobenzenesulfonic acid (TNBS) at 0°C under conditions where cell-surface proteins are tagged

  20. Long-term intermittent exposure to sulfuric acid aerosol, ozone, and their combination: alterations in tracheobronchial mucociliary clearance and epithelial secretory cells

    SciTech Connect

    Schlesinger, R.B.; Gorczynski, J.E.; Dennison, J.; Richards, L.; Kinney, P.L.; Bosland, M.C. (Department of Environmental Medicine, New York University Medical Center, NY (United States))

    1992-07-01

    Understanding the effects from long-term exposure to individual ambient air pollutants and mixtures of pollutants is necessary for adequate assessment of health risk. This study examined quantitative and temporal alterations in tracheobronchial mucociliary clearance function and bronchial epithelial secretory cells in rabbits exposed to sulfuric acid (125 micrograms/m3), ozone (0.1 ppm), and their combination for 2 h/d, 5 d/wk for up to 1 yr; some animals were allowed a 6-month post-exposure period. Clearance times were altered during exposure to sulfuric acid or to the mixture, and became progressively slower following the end of exposures to each of the pollutant atmospheres. There was no indication of any interaction in terms of clearance response between the acid and ozone in the group exposed to the mixture. Histological examination of intrapulmonary conducting airways was performed after 4, 8, or 12 months of exposure, and after the post-exposure period. Sulfuric acid resulted in an increase in the number of secretory cells in small airways by 12 months of exposure. Ozone and the mixture resulted in an increase in secretory cell number by 4 months, but the response became attenuated with continued exposure. There was evidence for synergistic interaction between ozone and acid at 4 months, and antagonistic interaction at subsequent times. No inflammation or other biologically significant histological effects were found in any of the animals.

  1. Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

    PubMed Central

    1992-01-01

    Mutant V.24.1, a member of the End4 complementation group of temperature-sensitive CHO cells, is defective in secretion at the restrictive temperature (Wang, R.-H., P. A. Colbaugh, C.-Y. Kao, E. A. Rutledge, and R. K. Draper. 1990. J. Biol. Chem. 265:20179-20187; Presley, J. F., R. K. Draper, and D. T. Brown. 1991. J. Virol. 65:1332- 1339). We have further investigated the secretory lesion and report three main findings. First, the block in secretion is not due to aberrant folding or oligomerization of secretory proteins in the endoplasmic reticulum because the hemagglutinin of influenza virus folded and oligomerized at the same rate in mutant and parental cells at the restrictive temperature. Second, secretory proteins accumulated in a compartment intermediate between the ER and the Golgi. Several lines of evidence support this conclusion, the most direct being the colocalization by immunofluorescence microscopy of influenza virus hemagglutinin with a 58-kD protein that is known to reside in an intermediate compartment. Third, at the resolution of fluorescence microscopy, the Golgi complex in the mutant cells vanished at the restrictive temperature. PMID:1577851

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

    PubMed Central

    Slesinger, P A; Lansman, J B

    1991-01-01

    1. Granule cells were dissociated from mouse cerebellum and grown in vitro. Currents through single Ca2+ channels were recorded from the cell body with the patch clamp technique. 2. Voltage steps to 0 mV produced brief channel openings with a mean open time of approximately 0.5 ms. The single-channel conductance measured from the amplitude of the single-channel current with 90 mM-Ba2+ in the patch electrode was 22 pS. 3. The probability of Ca2+ channel opening increased with test potentials more positive than -30 mV, with half-activation near 0 mV, and followed the Boltzmann relation for the activation of whole-cell Ca2+ current. 4. Voltage steps to potentials more positive than 0 mV produced more channel activity at the beginning than at the end of the voltage step. The average of the single-channel currents decayed to a non-zero level with a time course similar to that of the whole-cell Ca2+ current. 5. The amplitude as well as the decay of the mean current measured during a test pulse to 0 mV was reduced as the holding potential was made more positive than approximately -90 mV. The change in the open channel probability with holding potential followed the Boltzmann relation which described the inactivation of the whole-cell Ca2+ current. 6. Ca2+ channel activity persisted for over several minutes after excising the patch from the cell body when intracellular cyclic AMP was increased. After patch excision, the number of functional channels decreased to a level where only one channel at a time was active. Ca2+ channel openings appeared as either short bursts at the beginning of the voltage step or long bursts that lasted throughout the pulse. 7. Exposing the cell to the dihydropyridine agonist +(S)-202-791 markedly increased the fraction of sweeps with long openings and produced a non-decaying mean current that was approximately 5 times larger than control. In a fraction of the sweeps, however, long openings occurred more frequently at the beginning than at the end of the voltage step and these produced a decaying mean current. 8. Shifting the holding potential to more positive potentials in the presence of the dihydropyridine agonist preferentially reduced the number of brief openings while sparing the long openings. The amplitude of the mean current was similar to that obtained from the more negative holding potential and there was no change in the fraction of sweeps with long openings that occurred at the beginning of the voltage pulse.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1654414

  3. Analysis of immunosuppressive properties of iris and ciliary body cells and their secretory products.

    PubMed

    Streilein, J W; Bradley, D

    1991-09-01

    The anterior chamber of the eye is an immunosuppressive microenvironment as shown experimentally by immune privilege, anterior chamber-associated immune deviation, and inability to display local delayed-type hypersensitivity responses. It recently was reported that both the aqueous humor and the cells of the iris and ciliary body (I-CB) have immune inhibitory properties in vitro, suggesting that these components of the anterior segment might contribute to the unique properties of this microenvironment. To explore the cellular sources of immunosuppressive factors in the anterior chamber, cultures of I-CB cells were established from normal eyes of BALB/c mice. Supernatants were harvested from these cultures and assayed in vitro for their ability to inhibit T-lymphocyte activation. It was found that I-CB cell-derived supernatants profoundly suppressed alloantigen-driven T-cell proliferation (mixed lymphocyte response) and interleukin-2 production by a T-cell hybridoma that responds to stimulator cells bearing I-Ad. The inhibitory activity of I-CB supernatants did not appear to be related to prostaglandins; supernatants of I-CB cells cultured with indomethacin retained their suppressive properties, as did supernatants to which neutralizing antiprostaglandin E2 antibodies had been added. Moreover, suppression by I-CB supernatants was not relieved by antibodies specific for transforming growth factor-beta, even though this cytokine is known to be present in normal aqueous humor. Thus, the identity of the suppressive factor(s) in cultured I-CB cell supernatants remains elusive. Finally, by separating I-CB cell suspensions into bone marrow-derived (T-200-positive) and those not derived from bone marrow (parenchymal) subpopulations with a fluorescence-activated cell sorter, it was determined that the inhibitory activity of I-CB cell suspensions was produced by parenchymal, rather than hematogenous, cells. It is proposed and discussed that inhibitory factors and cytokines secreted by parenchymal I-CB cells contribute to the immunosuppressive qualities of the anterior chamber. PMID:1894470

  4. Activation of the ancestral polarity regulator protein kinase C zeta at the immunological synapse drives polarization of Th cell secretory machinery toward APCs.

    PubMed

    Bertrand, Florie; Esquerré, Michael; Petit, Anne-Elisabeth; Rodrigues, Magda; Duchez, Sophie; Delon, Jérôme; Valitutti, Salvatore

    2010-09-01

    A key feature in T lymphocyte biology is that Th cells rapidly polarize their secretory machinery toward cognate APCs. The molecular mechanisms of these dynamic Th cell responses and their impact on APC biology remain to be elucidated. In this study, we demonstrate that protein kinase Czeta (PKCzeta) is rapidly activated at the immunological synapse (IS) in human Th cells interacting with cognate dendritic cells (DCs) and that a functional PKCzeta is required for the polarization of Th cell secretory machinery toward DCs. We also show that PKCzeta-dependent Th cell polarization allows dedicated delivery of IFN-gamma and CD40L at the IS and is required for the activation of cognate DCs to IL-12 production. PKCzeta synaptic activation is a low-threshold phenomenon and, in Th cells interacting with multiple DCs, selectively occurs at the IS formed with the DCs offering the strongest stimulus leading to dedicated Th cell polarization. Our results identify the PKCzeta signaling pathway as a key component of the Th cell polarization machinery and provide a molecular basis for T cell-dedicated activation of cognate DCs. PMID:20679531

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

    PubMed Central

    Slesinger, P A; Lansman, J B

    1991-01-01

    1. Cells dissociated from mouse cerebellum were grown in vitro. Ca2+ channel currents were recorded from granule cells with the patch-clamp technique under conditions which suppressed currents through Na+ and K+ channels and minimized run-down of current through Ca2+ channels. 2. A strong depolarizing voltage step from a hyperpolarized holding potential produced inward Ca2+ channel current that decayed exponentially to a non-zero level. Inward current decayed to approximately 40% of its peak amplitude (range 20-90%). 3. The inward current increased in amplitude when Ca2+ was replaced with Ba2+ or after raising the concentration of extracellular Ba2+, but the rate of decay of current was unaffected. 4. The current-voltage (I-V) relation showed that peak or sustained current increased with voltage pulses more positive than approximately -30 mV, reached a maximum amplitude near +20 mV and became progressively smaller with larger depolarizations. 5. The tail currents produced after rapidly repolarizing the membrane potential to -70 mV from a positive test pulse decayed along a single exponential time course with a time constant of approximately 0.5 ms. The amplitude of tail current measured at a fixed repolarization potential increased as the pre-pulse was made more positive and reached a maximum with pre-pulses more positive than +40 mV. A plot of normalized amplitude of the tail current as a function of the pre-pulse potential was fitted with a Boltzmann relation with V1/2 = approximately + 8 mV and steepness k = 14 mV. 6. Shifting the holding potential to more positive potentials reduced the amplitude of the Ca2+ channel current elicited by the fixed voltage step and abolished the decay of the inward current. The peak current was normalized to the maximum peak current elicited from a very negative holding potential and plotted as a function of holding potential. The points were fitted with a Boltzmann relation for inactivation with V1/2 = approximately -57 mV and steepness k = 14 mV. 7. The onset of inactivation was studied in two-pulse experiments in which the duration of conditioning pre-pulse was varied. Increasing the duration of a pre-pulse to a fixed potential reduced the peak inward current evoked by the second test pulse. Plotting normalized current as a function of pre-pulse duration showed that inactivation developed along a double exponential time course. Both fast and slow time constants decreased as the pre-pulse potential was made more positive.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1663157

  6. Increase of GABAA receptor-mediated tonic inhibition in dentate granule cells after traumatic brain injury.

    PubMed

    Mtchedlishvili, Zakaria; Lepsveridze, Eka; Xu, Hong; Kharlamov, Elena A; Lu, Bo; Kelly, Kevin M

    2010-06-01

    Traumatic brain injury (TBI) can result in altered inhibitory neurotransmission, hippocampal dysfunction, and cognitive impairments. GABAergic spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) and tonic (extrasynaptic) whole cell currents were recorded in control rat hippocampal dentate granule cells (DGCs) and at 90days after controlled cortical impact (CCI). At 34 degrees C, in CCI DGCs, sIPSC frequency and amplitude were unchanged, whereas mIPSC frequency was decreased (3.10+/-0.84Hz, n=16, and 2.44+/-0.67Hz, n=7, p<0.05). At 23 degrees C, 300nM diazepam increased peak amplitude of mIPSCs in control and CCI DGCs, but the increase was 20% higher in control (26.81+/-2.2pA and 42.60+/-1.22pA, n=9, p=0.031) compared to CCI DGCs (33.46+/-2.98pA and 46.13+/-1.09pA, n=10, p=0.047). At 34 degrees C, diazepam did not prolong decay time constants (6.59+/-0.12ms and 6.62+/-0.98ms, n=9, p=0.12), the latter suggesting that CCI resulted in benzodiazepine-insensitive pharmacology in synaptic GABA(A) receptors (GABA(A)Rs). In CCI DGCs, peak amplitude of mIPSCs was inhibited by 100microM furosemide (51.30+/-0.80pA at baseline and 43.50+/-5.30pA after furosemide, n=5, p<0.001), a noncompetitive antagonist of GABA(A)Rs with an enhanced affinity to alpha4 subunit-containing receptors. Potentiation of tonic current by the GABA(A)R delta subunit-preferring competitive agonist THIP (1 and 3microM) was increased in CCI DGCs (47% and 198%) compared to control DGCs (13% and 162%), suggesting the presence of larger tonic current in CCI DGCs; THIP (1microM) had no effect on mIPSCs. Taken together, these results demonstrate alterations in synaptic and extrasynaptic GABA(A)Rs in DGCs following CCI. PMID:20304069

  7. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    PubMed Central

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor. PMID:18316814

  8. Secretory competence in a gateway endocrine cell conferred by the nuclear receptor ?FTZ-F1 enables stage-specific ecdysone responses throughout development in Drosophila.

    PubMed

    Cho, Kook-Ho; Daubnerová, Ivana; Park, Yoonseong; Zitnan, Dusan; Adams, Michael E

    2014-01-15

    Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor ?FTZ-F1. Selective RNA silencing of ?ftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in ?ftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted ?FTZ-F1 expression. Moreover, premature ?ftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a "gateway cell", whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed ?FTZ-F1 expression late in the molt as steroids decline. PMID:24247008

  9. Secretory Competence in a Gateway Endocrine Cell Conferred by the Nuclear Receptor ?FTZ-F1 Enables Stage-specific Ecdysone Responses Throughout Development in Drosophila

    PubMed Central

    Cho, Kook-Ho; Daubnerová, Ivana; Park, Yoonseong; Zitnan, Dusan; Adams, Michael E.

    2014-01-01

    Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETH). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor ?FTZ-F1. Selective RNA silencing of ?ftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in ?ftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted ?FTZ-F1 expression. Moreover, premature ?ftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a “gateway cell”, whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed ?FTZ-F1 expression late in the molt as steroids decline. PMID:24247008

  10. FTY720 suppresses interleukin-1?-induced secretory phospholipase A2 expression in renal mesangial cells by a transcriptional mechanism

    PubMed Central

    Xin, C; Ren, S; Eberhardt, W; Pfeilschifter, J; Huwiler, A

    2007-01-01

    Background and purpose: FTY720 is a potent immunomodulatory prodrug that is converted to its active phosphorylated form by a sphingosine kinase. Here we have studied whether FTY720 mimicked the action of sphingosine-1-phosphate (S1P) and exerted an anti-inflammatory potential in renal mesangial cells. Experimental approach: Prostaglandin E2 (PGE2) was quantified by an enzyme-linked immunosorbent-assay. Secretory phospholipase A2 (sPLA2) protein was detected by Western blot analyses. mRNA expression was determined by Northern blot analysis and sPLA2-promoter activity was measured by a luciferase-reporter-gene assay. Key results: Stimulation of cells for 24?h with interleukin-1? (IL-1?) is known to trigger increased PGE2 formation which coincides with an induction of the mRNA for group-IIA-sPLA2 and protein expression. FTY720 dose-dependently suppressed IL-1?-induced IIA-sPLA2 protein secretion and activity in the supernatant. This effect is due to a suppression of cytokine-induced sPLA2 mRNA expression which results from a reduced promoter activity. As a consequence of suppressed sPLA2 activity, PGE2 formation is also reduced by FTY720. Mechanistically, the FTY720-suppressed sPLA2 expression results from an activation of the TGF?/Smad signalling cascade since inhibition of the TGF? receptor type I by a specific kinase inhibitor reverses the FTY720-mediated decrease of sPLA2 protein expression and sPLA2 promoter activity. Conclusions and implications: In summary, our data show that FTY720 was able to mimic the anti-inflammatory activity of TGF? and blocked cytokine-triggered sPLA2 expression and subsequent PGE2 formation. Thus, FTY720 may exert additional in vivo effects besides the well reported immunomodulation and its anti-inflammatory potential should be considered. PMID:17325654

  11. Munc13-3 superprimes synaptic vesicles at granule cell-to-basket cell synapses in the mouse cerebellum.

    PubMed

    Ishiyama, Shimpei; Schmidt, Hartmut; Cooper, Benjamin H; Brose, Nils; Eilers, Jens

    2014-10-29

    Munc13-3 is a presynaptic protein implicated in vesicle priming that is strongly expressed in cerebellar granule cells (GCs). Mice deficient of Munc13-3 (Munc13-3(-/-)) show an increased paired-pulse ratio (PPR), which led to the hypothesis that Munc13-3 increases the release probability (pr) of vesicles. In the present study, we analyzed unitary synaptic connections between GCs and basket cells in acute cerebellar slices from wild-type and Munc13-3(-/-) mice. Unitary EPSCs recorded from Munc13-3(-/-) GCs showed normal kinetics and synaptic latency but a significantly increased PPR and fraction of synaptic failures. A quantal analysis revealed that neither the charge of single quanta nor the binominal parameter N were affected by loss of Munc13-3 but that pr was almost halved in Munc13-3(-/-). Neither presynaptic Ca(2+) influx was affected by deletion of Munc13-3 nor replenishment of the readily releasable vesicle pool. However, a high concentration of EGTA led to a reduction in EPSCs that was significantly stronger in Munc13-3(-/-). We conclude that Munc13-3 is responsible for an additional step of molecular and/or positional "superpriming" that substantially increases the efficacy of Ca(2+)-triggered release. PMID:25355221

  12. Insufficient ER-stress response causes selective mouse cerebellar granule cell degeneration resembling that seen in congenital disorders of glycosylation

    PubMed Central

    2013-01-01

    Background Congenital disorders of glycosylation (CDGs) are inherited diseases caused by glycosylation defects. Incorrectly glycosylated proteins induce protein misfolding and endoplasmic reticulum (ER) stress. The most common form of CDG, PMM2-CDG, is caused by deficiency in the cytosolic enzyme phosphomannomutase 2 (PMM2). Patients with PMM2-CDG exhibit a significantly reduced number of cerebellar Purkinje cells and granule cells. The molecular mechanism underlying the specific cerebellar neurodegeneration in PMM2-CDG, however, remains elusive. Results Herein, we report that cerebellar granule cells (CGCs) are more sensitive to tunicamycin (TM)-induced inhibition of total N-glycan synthesis than cortical neurons (CNs). When glycan synthesis was inhibited to a comparable degree, CGCs exhibited more cell death than CNs. Furthermore, downregulation of PMM2 caused more CGCs to die than CNs. Importantly, we found that upon PMM2 downregulation or TM treatment, ER-stress response proteins were elevated less significantly in CGCs than in CNs, with the GRP78/BiP level showing the most significant difference. We further demonstrate that overexpression of GRP78/BiP rescues the death of CGCs resulting from either TM-treatment or PMM2 downregulation. Conclusions Our results indicate that the selective susceptibility of cerebellar neurons to N-glycosylation defects is due to these neurons’ inefficient response to ER stress, providing important insight into the mechanisms of selective neurodegeneration observed in CDG patients. PMID:24305089

  13. [Effect of modeled microgravity on the secretory activity of cultivated human endothelium cells].

    PubMed

    Rudimov, E G; Pogodina, M V; Buravkova, L B

    2014-01-01

    Endothelial cells monolayers are capable to respond to microenvironmental changes immediately by synthesis of vasoactive substances, chemokines, and expression of adhesion molecules. A variety of microgravity models were used to demonstrate the high mechanical and gravity sensitivity of the endothelium. The work was aimed at studying the effects of modeled microgravity on soluble cytokines synthesis by normal and TNF-activated human umbilical vein endotheliocytes. It was found that intact endothelial cells may differ in the ability to secrete IL-6 and IL-8 and that proinflammatory activation increases secretion of these interleukins. Manifestation of this effect is inversely proportional to basal cytokines secretion. Microgravity generated in the Random Positioning Machine does not affect the paracrine effects of proinflammatory induction. This suggests that microgravity is not a proinflammatory stimulant for endothelial cells; at least, it does not increase cytokines secretion or impede the development of inflammatory reaction. PMID:25163336

  14. Delayed Expulsion of the Nematode Trichinella spiralis in Mice Lacking the Mucosal Mast Cell-specific Granule Chymase, Mouse Mast Cell Protease1

    Microsoft Academic Search

    Pamela A. Knight; Steven H. Wright; Catherine E. Lawrence; Yvonne Y. W. Paterson; Hugh R. P. Miller

    Expulsion of gastrointestinal nematodes is associated with pronounced mucosal mast cell (MMC) hyperplasia, differentiation, and activation, accompanied by the systemic release of MMC granule chymases (chymotrypsin-like serine proteases). The b -chymase mouse mast cell protease-1 (mMCP-1) is expressed predominantly by intraepithelial MMCs, and levels in the bloodstream and intestinal lumen are maximal at the time of worm expulsion in parasitized

  15. RNA Interference Analysis of Legionella in Drosophila Cells: Exploitation of Early Secretory Apparatus Dynamics

    Microsoft Academic Search

    Marion S Dorer; Donald Kirton; Joel S Bader; Ralph R Isberg

    2006-01-01

    Legionella pneumophila translocates multiple bacterial effector proteins into host cells to direct formation of a replication vacuole for the bacterium. The emerging consensus is that formation of this compartment involves recruitment of membrane material that traffics between the endoplasmic reticulum (ER) and Golgi. To investigate this model, a targeted approach was used to knock down expression of proteins involved in

  16. Secretory activity of Poecilia latipinna (Teleostei) pituitary in vitro: rostral pars distalis and proximal pars distalis.

    PubMed

    Batten, T F; Young, G; Ball, J N

    1983-07-01

    The ultrastructure of each adenohypophysial secretory cell type was examined in pituitaries of adult female mollies (Poecilia latipinna) after various periods in vitro, and with varied medium osmotic pressure (OP), Na+, and Ca2+ concentrations. Prolactin (PRL) cells were markedly activated by 18 hr, and after 7 or 14 days were almost totally degranulated, with massive arrays of Golgi and RER. Reduction in OP, but not Na+ or Ca2+, caused an additional activation of PRL cells after periods of 18 hr or longer. Corticotroph (ACTH) cells became noticeably activated by 4 hr, and were possibly affected by OP, but not Na+ or Ca2+. Growth hormone (GH) cells were activated by 6 hr, and after 18 hr were quite degranulated with extensive arrays of RER. OP had no effect on GH cells before 3 days, when reduced OP appeared to cause an additional activation, with the appearance of large irregular secretory granule (SG)-like inclusions. Na+ and Ca2+ again had no effect. Gonadotrophic (GtH) cells appeared to be little affected by in vitro incubation; however, the very active cells from vitellogenic fish underwent a reduction in dilated RER after prolonged culture. Thyrotrophic (TSH) cells gradually became activated in vitro, but the response again varied with the sexual condition of the fish. Neither GtH nor TSH cells were affected by OP, Na+, or Ca2+. The findings are discussed in relation to hypothalamic control, via releasing/inhibiting factors, of adenohypophysial cell activity. PMID:6309607

  17. Preventing effect of L-type calcium channel blockade on electrophysiological alterations in dentate gyrus granule cells induced by entorhinal amyloid pathology.

    PubMed

    Pourbadie, Hamid Gholami; Naderi, Nima; Mehranfard, Nasrin; Janahmadi, Mahyar; Khodagholi, Fariba; Motamedi, Fereshteh

    2015-01-01

    The entorhinal cortex (EC) is one of the earliest affected brain regions in Alzheimer's disease (AD). EC-amyloid pathology induces synaptic failure in the dentate gyrus (DG) with resultant behavioral impairment, but there is little known about its impact on neuronal properties in the DG. It is believed that calcium dyshomeostasis plays a pivotal role in the etiology of AD. Here, the effect of the EC amyloid pathogenesis on cellular properties of DG granule cells and also possible neuroprotective role of L-type calcium channel blockers (CCBs), nimodipine and isradipine, were investigated. The amyloid beta (A?) 1-42 was injected bilaterally into the EC of male rats and one week later, electrophysiological properties of DG granule cells were assessed. Voltage clamp recording revealed appearance of giant sIPSC in combination with a decrease in sEPSC frequency which was partially reversed by CCBs in granule cells from A? treated rats. EC amyloid pathogenesis induced a significant reduction of input resistance (Rin) accompanied by a profound decreased excitability in the DG granule cells. However, daily administration of CCBs, isradipine or nimodipine (i.c.v. for 6 days), almost preserved the normal excitability against A?. In conclusion, lower tendency to fire AP along with reduced Rin suggest that DG granule cells might undergo an alteration in the membrane ion channel activities which finally lead to the behavioral deficits observed in animal models and patients with early-stage Alzheimer's disease. PMID:25689857

  18. Preventing Effect of L-Type Calcium Channel Blockade on Electrophysiological Alterations in Dentate Gyrus Granule Cells Induced by Entorhinal Amyloid Pathology

    PubMed Central

    Pourbadie, Hamid Gholami; Naderi, Nima; Mehranfard, Nasrin; Janahmadi, Mahyar; Khodagholi, Fariba; Motamedi, Fereshteh

    2015-01-01

    The entorhinal cortex (EC) is one of the earliest affected brain regions in Alzheimer’s disease (AD). EC-amyloid pathology induces synaptic failure in the dentate gyrus (DG) with resultant behavioral impairment, but there is little known about its impact on neuronal properties in the DG. It is believed that calcium dyshomeostasis plays a pivotal role in the etiology of AD. Here, the effect of the EC amyloid pathogenesis on cellular properties of DG granule cells and also possible neuroprotective role of L-type calcium channel blockers (CCBs), nimodipine and isradipine, were investigated. The amyloid beta (A?) 1–42 was injected bilaterally into the EC of male rats and one week later, electrophysiological properties of DG granule cells were assessed. Voltage clamp recording revealed appearance of giant sIPSC in combination with a decrease in sEPSC frequency which was partially reversed by CCBs in granule cells from A? treated rats. EC amyloid pathogenesis induced a significant reduction of input resistance (Rin) accompanied by a profound decreased excitability in the DG granule cells. However, daily administration of CCBs, isradipine or nimodipine (i.c.v. for 6 days), almost preserved the normal excitability against A?. In conclusion, lower tendency to fire AP along with reduced Rin suggest that DG granule cells might undergo an alteration in the membrane ion channel activities which finally lead to the behavioral deficits observed in animal models and patients with early-stage Alzheimer’s disease. PMID:25689857

  19. Yizhijiannao Granule and a combination of its effective monomers, icariin and Panax notoginseng saponins, inhibit early PC12 cell apoptosis induced by beta-amyloid (25–35)?

    PubMed Central

    Zhang, Ting; Zhang, Zhanwei; Dong, Keli; Li, Guangcheng; Zhu, Hong

    2012-01-01

    One of our previous studies showed that Yizhijiannao Granule, a compound Chinese medicine, effectively improved the clinical symptoms of Alzheimer's disease. In the present study, we established a model of Alzheimer's disease using beta-amyloid (25–35) in PC12 cells, and treated the cells with Yizhijiannao Granule and its four monomers, i.e., icariin, catechin, Panax notoginseng saponins, and eleutheroside E. Flow cytometry showed that Yizhijiannao Granule-containing serum, icariin, Panax notoginseng saponins, and icariin + Panax notoginseng saponins were protective against beta-amyloid (25–35)-induced injury in PC12 cells. Icariin in combination with Panax notoginseng saponins significantly inhibited early apoptosis of PC12 cells with beta-amyloid (25–35)-induced injury compared to icariin or Panax notoginseng saponins alone. The effects of icariin + Panax notoginseng saponins were similar to the effects of Yizhijiannao Granule. The findings indicate that two of the effective monomers of Yizhijiannao Granule, icariin and Panax notoginseng saponins, can synergistically inhibit early apoptosis of PC12 cells induced by beta-amyloid (25–35). PMID:25624809

  20. Regulation of lipid synthesis genes and milk fat production in human mammary epithelial cells during secretory activation

    PubMed Central

    Mohammad, Mahmoud A.

    2013-01-01

    Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our goal was to study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretory activation. Gene expression from mRNA isolated from milk fat globule (MFG) and milk FA composition were measured from 6 h to 42 days postpartum in seven normal women. Over the first 96 h postpartum, daily milk fat output increased severalfold and mirrored expression of genes for all aspects of lipid metabolism and milk FA production, including lipolysis at the MEC membrane, FA uptake from blood, intracellular FA transport, de novo FA synthesis, FA and glycerol activation, FA elongation, FA desaturation, triglyceride synthesis, cholesterol synthesis, and lipid droplet formation. Expression of the gene for a key lipid synthesis regulator, sterol regulatory element-binding transcription factor 1 (SREBF1), increased 2.0-fold by 36 h and remained elevated over the study duration. Expression of genes for estrogen receptor 1, thyroid hormone-responsive protein, and insulin-induced 2 increased progressively to plateau by 96 h. In contrast, mRNA of peroxisome proliferator-activated receptor-? decreased severalfold. With onset of lactation, increased de novo synthesis of FA was the most prominent change in milk FA composition and mirrored the expression of FA synthesis genes. In conclusion, milk lipid synthesis and secretion in humans is a complex process requiring the orchestration of a wide variety of pathways of which SREBF1 may play a primary role. PMID:23880316

  1. Two distinct secretory vesicle-priming steps in adrenal chromaffin cells.

    PubMed

    Liu, Yuanyuan; Schirra, Claudia; Edelmann, Ludwig; Matti, Ulf; Rhee, JeongSeop; Hof, Detlef; Bruns, Dieter; Brose, Nils; Rieger, Heiko; Stevens, David R; Rettig, Jens

    2010-09-20

    Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process. PMID:20855507

  2. Developmental expression of N-methyl- d -aspartate (NMDA)-induced neurotoxicity, NMDA receptor function, and the NMDAR1 and glutamate-binding protein subunits in cerebellar granule cells in primary cultures

    Microsoft Academic Search

    Yue Xia; Ronald E. Ragan; E. E. Ching Seah; Mary L. Michaelis; Elias K. Michaelis

    1995-01-01

    Cerebellar granule cells maintained in vitro as primary cultures are a relatively homogeneous neuronal population that can be used to evaluate the developmental expression of neurotransmitter receptors and to assess their role in cell survival and degeneration. The toxicity induced by N-methyl-d-aspartate (NMDA) in granule cells maintained under partially depolarizing conditions and in the presence of physiologic extracellular concentrations of

  3. Ultrastructural distribution of glycinergic and GABAergic neurons and axon terminals in the rat dorsal cochlear nucleus, with emphasis on granule cell areas

    PubMed Central

    Alibardi, Lorenzo

    2003-01-01

    A knowledge of neurotransmitters in the neurons of the rat cochlear nuclear complex is of importance in understanding the function of auditory circuits. Using post-embedding ultrastructural immunogold labelling, the distribution of glycinergic and GABAergic neurons and axonal terminals has been studied in the molecular, fusiform and polymorphic layers of the rat dorsal cochlear nucleus (DCN). This technique is not limited by the penetration of antibodies into the nervous tissue as in pre-embedding methods, and allows a fine neurochemical mapping of the nervous tissue. Numerous glycinergic and GABAergic axon terminals contain pleomorphic and flat synaptic vesicles, and are present in all layers (1, 2, 3) of the dorsal cochlear nucleus. Glycine and GABA-negative large terminals (mossy fibres) are mainly seen in granule cell areas of layer 2 (fusiform layer). Mossy fibres contact the dendrites of GABA- and glycine-negative granule cells and of the few unipolar brush cells (excitatory neurons). The least common cells in the granule cell areas are GABAergic and glycinergic Golgi-stellate neurons. In unipolar brush cells, aggregations of vesicles seem to be the origin of their characteristic ringlet-bodies. Golgi-stellate cells send their inhibitory terminals to the dendrites of granule and unipolar brush cells, occasionally directly to mossy fibres. Small or (less frequently) large GABAergic terminals contact the soma or the main dendrite of unipolar brush cells. The circuit of a hypothetical functional unit of neurons in the DCN is proposed. The inputs from auditory tonotopic or non-auditory non-tonotopic mossy fibres eventually reach pyramidal cells through axons from the granule cells or unipolar brush cells. Pyramidal cells convey an excitatory signal from the DCN to higher mesencephalic nuclei for further elaboration of the acoustic signal. PMID:12892405

  4. Distinctive population of Gfap-expressing neural progenitors arising around the dentate notch migrate and form the granule cell layer in the developing hippocampus.

    PubMed

    Seki, Tatsunori; Sato, Toru; Toda, Keiko; Osumi, Noriko; Imura, Tetsuya; Shioda, Seiji

    2014-02-01

    In the adult hippocampus, granule cells continue to be generated from astrocyte-like progenitors expressing glial fibrillary acidic protein (GFAP) that differ from embryonic neocortical progenitors. However, during the embryonic period, dentate granule neurons and neocortical pyramidal neurons are derived from the ventricular zone (VZ) of the pallium. Our question is when do GFAP+ progenitors of granule neurons appear in the developing hippocampus during the embryonic period, and how do they form the granule cell layer. The present analysis using Gfap-GFP transgenic mice shows that the GFP+ distinct cell population first appears in the VZ of the medial pallium at the dorsal edge of the fimbria on embryonic day 13.5. During the perinatal period, they form a migratory stream from the VZ to the developing dentate gyrus, and establish the germinal zones in the migratory stream, and the marginal and hilar regions in the developing dentate gyrus. GFP+ cells in these regions were positive for Sox2 and Ki67, but negative for BLBP. GFP+ cells with Neurogenin2 expression were largely distributed in the VZ, whereas GFP+ cells with Tbr2 and NeuroD expressions were seen in the migratory stream and developing dentate gyrus. Prox1-expressing GFP+ cells were restricted to the developing dentate gyrus. These results suggest that distinctive Gfap-expressing progenitors arising around the dentate notch form germinal regions in the migratory stream and the developing dentate gyrus where they differentiate into granule neurons, indicating that distinct astrocyte-like neural progenitors continue to generate granule neurons, from the beginning of dentate development and throughout life. J. Comp. Neurol. 522:261-283, 2014. © 2013 Wiley Periodicals, Inc. PMID:23983092

  5. siRNA knock-down of mutant torsinA restores processing through secretory pathway in DYT1 dystonia cells

    PubMed Central

    Hewett, Jeffrey W.; Nery, Flávia C.; Niland, Brian; Ge, Pei; Tan, Pamela; Hadwiger, Philipp; Tannous, Bakhos A.; Sah, Dinah W.Y.; Breakefield, Xandra O.

    2008-01-01

    Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the C-terminal region (torsinA?E). TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinA?E when compared with control fibroblasts expressing only torsinA. In this study, small interfering RNA (siRNA) oligonucleotides were identified, which downregulate the levels of torsinA or torsinA?E mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared with the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinA?E message in DYT fibroblasts increased luciferase secretion when compared with cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinA?E acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinA?E to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia. PMID:18258738

  6. MIST1 Regulates the Pancreatic Acinar Cell Expression of Atp2c2, the gene encoding Secretory Pathway Calcium ATPase 2

    PubMed Central

    Garside, Victoria C.; Kowalik, Agnes S.; Johnson, Charis L.; DiRenzo, Daniel; Konieczny, Stephen F.; Pin, Christopher L.

    2012-01-01

    MIST1 is a transcription factor expressed in pancreatic acinar cells and other serous exocrine cells. Mice harboring a targeted deletion of the Mist1 gene (Mist1?/?) exhibit alterations in acinar regulated exocytosis and aberrant Ca2+ signaling that are normally controlled by acinar cell Ca2+-ATPases. Previous studies indicated that total sarcoendoplasmic reticulum Ca2+-ATPases (SERCA) and plasma membrane Ca2+-ATPases (PMCA) remained unaffected in Mist1?/? acinar cultures. Therefore, we have assessed the expression of Atp2c2, the gene that encodes the secretory pathway Ca2+-ATPase 2 (SPCA2). We revealed a dramatic decrease in pancreatic expression of Atp2a2 mRNA and SPCA2 protein in Mist1?/? mice. Surprisingly, this analysis indicated that the acinar-specific Atp2c2 mRNA is a novel transcript, consisting of only the 3? end of the gene and the protein and localizes to the endoplasmic reticulum. Expression of SPCA2 was also lost in Mist1?/? secretory cells of the salivary glands and seminal vesicles, suggesting that Atp2c2 transcription is regulated by MIST1. Indeed, inducible MIST1 expression in Mist1?/? pancreatic acinar cells restored normal Atp2c2 expression, supporting a role for MIST1 in regulating the Atp2c2 gene. Based on these results, we have identified a new Atp2c2 transcript, the loss of which may be linked to the Mist1?/? phenotype. PMID:20599950

  7. Low molecular weight GTP-binding proteins in human neutrophil granule membranes.

    PubMed

    Philips, M R; Abramson, S B; Kolasinski, S L; Haines, K A; Weissmann, G; Rosenfeld, M G

    1991-01-15

    Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments. PMID:1898732

  8. Endogenous proBDNF is a negative regulator of migration of cerebellar granule cells in neonatal mice.

    PubMed

    Xu, Zhi-Qiang; Sun, Ying; Li, Hong-Yun; Lim, Yoon; Zhong, Jin-Hua; Zhou, Xin-Fu

    2011-04-01

    The majority of newborn neurons migrate from their birthplace to final destination in the developing brain. Migration of cerebellar granule cells (CGCs) requires multiple factors. Mature brain-derived neurotrophic factor (BDNF) positively regulates the proliferation, migration, survival and differentiation of CGCs in rodents. However, the role of the BDNF precursor, proBDNF, in neuronal development remains unknown. In this study, we investigated the effect of proBDNF in vivo and in vitro on migration of CGCs. We demonstrate that proBDNF and its receptors p75 neurotrophin receptor (p75NTR) and sortilin are highly expressed in the cerebella as determined by immunohistochemistry and Western blot. ProBDNF is released from cultured cerebellar neurons, and this release is increased by high potassium stimulation. ProBDNF inhibits migration of CGCs in vitro, and the neutralizing antibodies to proBDNF enhance such migration as assayed by transwell culture. In addition, proBDNF incorporated into an agarose plug reduces granule cell migration from such plugs, whereas the neutralizing antibodies attract these cells towards the plug. The application of proBDNF into the lateral ventricle significantly inhibits migration of CGCs out of the proliferative zone into the internal granular cell layer, whereas the neutralizing antibodies enhance this migration. Furthermore, the effects of proBDNF on cell migration are lost in p75NTR(-/-) mice. Our data suggest that proBDNF negatively regulates migration of CGCs and this effect is mediated by p75NTR. We conclude that proBDNF has an opposing role in migration of CGCs to that of mature BDNF. PMID:21366730

  9. Cultured cerebellar granule cells, but not astrocytes, produce an ester of ganglioside GD1b, presumably GD1b monolactone, from exogenous GD1b.

    PubMed Central

    Bassi, R; Riboni, L; Tettamanti, G

    1994-01-01

    Granule cells and astrocytes from rat cerebellum were fed in culture with 2 microM ganglioside [Gal-3H]GD1b and then analysed for the presence of carboxyl esters of that ganglioside. Before extraction and purification of gangliosides, cells were treated with NaBH4 under conditions that would allow complete reductive cleavage of carboxyl ester linkages, [Gal-3H]GD1b monolactone and dilactone being used as reference esters of GD1b. These conditions, established by adding harvested cells (250 micrograms of protein) with 0.01-2 nmol of standard [Gal-3H]GD1b monolactone or dilactone and [Gal-3H]GD1b-1ol or -2ol formed respectively, consisted of an NaBH4/cell protein ratio of 2:1 (w/w). Cerebellar granule cells, but not astrocytes, were able to produce a radioactive compound which was identified as GD1b-1ol. The formation of this compound increased with pulse (up to 4 h) and chase (up to 3 h) time after a 2 h pulse and also occurred when ganglioside endocytosis was blocked. It can be concluded that cerebellar granule cells are able to convert ganglioside GD1b into a carboxyl ester form, presumably GD1b monolactone. The natural occurrence of the same GD1b carboxyl ester in cerebellar granule cells was also demonstrated. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:7945223

  10. Calcium-independent phospholipase A2 mediates store-operated calcium entry in rat cerebellar granule cells.

    PubMed

    Singaravelu, Karthika; Lohr, Christian; Deitmer, Joachim W

    2008-01-01

    Store-operated Ca(2+) entry (SOCE) has been extensively studied in non-neuronal cells, such as glial cells and smooth muscle cells, in which Ca(2+)-independent phospholipase A(2) (iPLA(2)) has been shown to play a key role in the regulation of SOCE channels. In the present study, we have investigated the role of iPLA(2) for store-operated Ca(2+) entry in rat cerebellar granule neurons in acute brain slices using confocal Ca(2+) imaging. Depletion of Ca(2+) stores by cyclopiazonic acid (CPA) induced a Ca(2+) influx, which could be inhibited by SOCE channel blockers 2-aminoethoxy-diphenylborate (2-APB) and 3,5-bistrifluoromethyl pyrazole derivative (BTP2), but not by the voltage-operated Ca(2+) channel blocker diltiazem and by the Na+ channel blocker tetrodotoxin. The inhibitors of iPLA(2), bromoenol lactone (BEL) and 1,1,1-trifluoro-2-heptadecanone, and the selective suppression of iPLA(2) expression by antisense oligodeoxynucleotides, inhibited CPA-induced Ca(2+) influx. Calmidazolium, which relieves the block of inhibitory calmodulin from iPLA(2), elicited a Ca(2+) influx similar to CPA-induced Ca(2+) entry. The product of iPLA(2), lysophosphatidylinositol, elicited a 2-APB- and BTP2-sensitive, but BEL-insensitive, Ca(2+) influx. Spontaneous Ca(2+) oscillations in granule cells in acute brain slices were reduced after inhibiting iPLA(2) activity or by blocking SOCE channels. The results suggest that depletion of Ca(2+) stores activates iPLA(2) to trigger Ca(2+) influx by the formation of lysophospholipids in these neurons. PMID:18784973

  11. Chymase in exocytosed rat mast cell granules effectively proteolyzes apolipoprotein AI-containing lipoproteins, so reducing the cholesterol efflux-inducing ability of serum and aortic intimal fluid.

    PubMed Central

    Lindstedt, L; Lee, M; Castro, G R; Fruchart, J C; Kovanen, P T

    1996-01-01

    Degranulated mast cells are present in human fatty streaks. Chymase in granules released from degranulated rat serosal mast cells, i.e., in granule remnants, proteolyzes human high density lipoprotein3 (HDL3), and so reduces its ability to induce cholesterol efflux from macrophage foam cells in vitro. In this study we found that remnant chymase, by proteolyzing human serum and human aortic intimal fluid, prevents these two physiologic fluids from effectively inducing cholesterol efflux from cultured macrophage foam cells. Inhibition was strongest when remnants were added to apolipoprotein AI (apoAI)-containing lipoproteins; the remnants had no effect on the weaker efflux produced by apoAI-deficient serum. Western blot analysis showed that granule remnants degrade apoAI in serum and in internal fluid. When released from remnants, chymase lost its ability to proteolyze HDL3 in the presence of serum. Thus, remnant chymase (but not isolated chymase) was able to resist the natural protease inhibitors present in serum and in intimal fluid. The results imply participation of exocytosed mast cell granules in foam cell formation in atherogenesis. PMID:8636396

  12. PTBP1 is required for glucose-stimulated cap-independent translation of insulin granule proteins and Coxsackieviruses in beta cells

    PubMed Central

    Knoch, Klaus-Peter; Nath-Sain, Suchita; Petzold, Antje; Schneider, Hendryk; Beck, Mike; Wegbrod, Carolin; Sönmez, Anke; Münster, Carla; Friedrich, Anne; Roivainen, Merja; Solimena, Michele

    2014-01-01

    Glucose and GLP-1 stimulate not only insulin secretion, but also the post-transcriptional induction of insulin granule biogenesis. This process involves the nucleocytoplasmic translocation of the RNA binding protein PTBP1. Binding of PTBP1 to the 3?-UTRs of mRNAs for insulin and other cargoes of beta cell granules increases their stability. Here we show that glucose enhances also the binding of PTBP1 to the 5?-UTRs of these transcripts, which display IRES activity, and their translation exclusively in a cap-independent fashion. Accordingly, glucose-induced biosynthesis of granule cargoes was unaffected by pharmacological, genetic or Coxsackievirus-mediated inhibition of cap-dependent translation. Infection with Coxsackieviruses, which also depend on PTBP1 for their own cap-independent translation, reduced instead granule stores and insulin release. These findings provide insight into the mechanism for glucose-induction of insulin granule production and on how Coxsackieviruses, which have been implicated in the pathogenesis of type 1 diabetes, can foster beta cell failure. PMID:25061557

  13. Mutant Human FUS Is Ubiquitously Mislocalized and Generates Persistent Stress Granules in Primary Cultured Transgenic Zebrafish Cells

    PubMed Central

    Acosta, Jamie Rae; Goldsbury, Claire; Winnick, Claire; Badrock, Andrew P.; Fraser, Stuart T.; Laird, Angela S.; Hall, Thomas E.; Don, Emily K.; Fifita, Jennifer A.; Blair, Ian P.; Nicholson, Garth A.; Cole, Nicholas J.

    2014-01-01

    FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS), a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C) human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG) inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons. PMID:24912067

  14. Transcription factor Foxq1 controls mucin gene expression and granule content in mouse stomach surface mucous cells

    PubMed Central

    Verzi, Michael P.; Khan, Abdul H.; Ito, Susumu; Shivdasani, Ramesh A.

    2010-01-01

    Background and Aims The gastric mucosa provides a stringent epithelial barrier and produces acid and enzymes that initiate digestion. In this regenerating tissue, progenitors differentiate continually into 4 principal specialized cell types, yet underlying mechanisms of differentiation are poorly understood. We identified stomach-restricted expression of the forkhead transcription factor FOXQ1. Methods We used a combination of genetic, histochemical, ultrastructural and molecular analysis to study gastric cell lineages with respect to FOXQ1. Results Within the developing and adult gastrointestinal tract, Foxq1 mRNA is restricted to the stomach and expressed predominantly in foveolar (pit) cells, the abundant mucin-producing cells that line the mucosal surface. Mice carrying Foxq1 coding mutations show virtual absence of mRNA and protein for the backbone of the major stomach mucin, MUC5AC. These observations correspond to a paucity of foveolar-cell secretory vesicles and notable loss of stomach but not intestinal mucus. Transcriptional profiling identified a surprisingly restricted set of genes with altered expression in Foxq1 mutant stomachs. MUC5AC is a highly tissue-restricted product that similarly depends on FOXQ1 in its other major site of expression, conjunctival goblet cells. Conclusions Taken together, these observations imply that promotion of gastric MUC5AC synthesis is a primary, cell-autonomous function of FOXQ1. This study is the first to implicate a transcription factor in terminal differentiation of foveolar cells and begins to define the requirements to assemble highly specialized organelles and cells in the gastric mucosa. PMID:18558092

  15. Radiation-induced alterations in synaptic neurotransmission of dentate granule cells depend on the dose and species of charged particles.

    PubMed

    Marty, V N; Vlkolinsky, R; Minassian, N; Cohen, T; Nelson, G A; Spigelman, I

    2014-12-01

    The evaluation of potential health risks associated with neuronal exposure to space radiation is critical for future long duration space travel. The purpose of this study was to evaluate and compare the effects of low-dose proton and high-energy charged particle (HZE) radiation on electrophysiological parameters of the granule cells in the dentate gyrus (DG) of the hippocampus and its associated functional consequences. We examined excitatory and inhibitory neurotransmission in DG granule cells (DGCs) in dorsal hippocampal slices from male C57BL/6 mice at 3 months after whole body irradiation with accelerated proton, silicon or iron particles. Multielectrode arrays were used to investigate evoked field synaptic potentials, an extracellular measurement of synaptic excitability in the perforant path to DG synaptic pathway. Whole-cell patch clamp recordings were used to measure miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) in DGCs. Exposure to proton radiation increased synaptic excitability and produced dose-dependent decreases in amplitude and charge transfer of mIPSCs, without affecting the expression of ?-aminobutyric acid type A receptor ?2, ?3 and ?2 subunits determined by Western blotting. Exposure to silicon radiation had no significant effects on synaptic excitability, mEPSCs or mIPSCs of DGCs. Exposure to iron radiation had no effect on synaptic excitability and mIPSCs, but significantly increased mEPSC frequency at 1 Gy, without changes in mEPSC kinetics, suggesting a presynaptic mechanism. Overall, the data suggest that proton and HZE exposure results in radiation dose- and species-dependent long-lasting alterations in synaptic neurotransmission, which could cause radiation-induced impairment of hippocampal-dependent cognitive functions. PMID:25402556

  16. Amyloid ?-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-? (A?) concentration. A single injection of A? (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. A?-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn2+ is involved in A? action. When A? was injected into the dentate gyrus, intracellular Zn2+ levels were increased only in the injected area in the dentate gyrus, suggesting that A? induces the influx of Zn2+ into cells in the injected area. When A? was added to hippocampal slices, A? 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 A?-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 A?-induced cognition deficits. PMID:25536033

  17. The RNA binding protein Musashi1 regulates apoptosis, gene expression and stress granule formation in urothelial carcinoma cells

    PubMed Central

    Nikpour, Parvaneh; Baygi, Modjtaba Emadi; Steinhoff, Christine; Hader, Christiane; Luca, Anna C; Mowla, Seyed J; Schulz, Wolfgang A

    2011-01-01

    Abstract The RNA-binding protein Musashi1 (MSI1) is a marker of progenitor cells in the nervous system functioning as a translational repressor. We detected MSI1 mRNA in several bladder carcinoma cell lines, but not in cultured normal uroepithelial cells, whereas the paralogous MSI2 gene was broadly expressed. Knockdown of MSI1 expression by siRNA induced apoptosis and a severe decline in cell numbers in 5637 bladder carcinoma cells. Microarray analysis of gene expression changes after MSI1 knockdown significantly up-regulated 735 genes, but down-regulated only 31. Up-regulated mRNAs contained a highly significantly greater number and density of Musashi binding sites. Therefore, a much larger set of mRNAs may be regulated by Musashi1, which may affect not only their translation, but also their turnover. The study confirmed p21CIP1 and Numb proteins as targets of Musashi1, suggesting additionally p27KIP1 in cell-cycle regulation and Jagged-1 in Notch signalling. A significant number of up-regulated genes encoded components of stress granules (SGs), an organelle involved in translational regulation and mRNA turnover, and impacting on apoptosis. Accordingly, heat shock induced SG formation was augmented by Musashi1 down-regulation. Our data show that ectopic MSI1 expression may contribute to tumorigenesis in selected bladder cancers through multiple mechanisms and reveal a previously unrecognized function of Musashi1 in the regulation of SG formation. PMID:20477901

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

    PubMed

    Temprana, Silvio G; Mongiat, Lucas A; Yang, Sung M; Trinchero, Mariela F; Alvarez, Diego D; Kropff, Emilio; Giacomini, Damiana; Beltramone, Natalia; Lanuza, Guillermo M; Schinder, Alejandro F

    2015-01-01

    Developing granule cells (GCs) of the adult dentate gyrus undergo a critical period of enhanced activity and synaptic plasticity before becoming mature. The impact of developing GCs on the activity of preexisting dentate circuits remains unknown. Here we combine optogenetics, acute slice electrophysiology, and in vivo chemogenetics to activate GCs at different stages of maturation to study the recruitment of local target networks. We show that immature (4-week-old) GCs can efficiently drive distal CA3 targets but poorly activate proximal interneurons responsible for feedback inhibition (FBI). As new GCs transition toward maturity, they reliably recruit GABAergic feedback loops that restrict spiking of neighbor GCs, a mechanism that would promote sparse coding. Such inhibitory loop impinges only weakly in new cohorts of young GCs. A computational model reveals that the delayed coupling of new GCs to FBI could be crucial to achieve a fine-grain representation of novel inputs in the dentate gyrus. PMID:25533485

  19. Langerin, a Novel C-Type Lectin Specific to Langerhans Cells, Is an Endocytic Receptor that Induces the Formation of Birbeck Granules

    Microsoft Academic Search

    Jenny Valladeau; Odile Ravel; Colette Dezutter-Dambuyant; Kevin Moore; Monique Kleijmeer; Ying Liu; Valérie Duvert-Frances; Claude Vincent; Daniel Schmitt; Jean Davoust; Christophe Caux; Serge Lebecque; Sem Saeland

    2000-01-01

    We have identified a type II Ca2+-dependent lectin displaying mannose-binding specificity, exclusively expressed by Langerhans cells (LC), and named Langerin. LC are uniquely characterized by Birbeck granules (BG), which are organelles consisting of superimposed and zippered membranes. Here, we have shown that Langerin is constitutively associated with BG and that antibody to Langerin is internalized into these structures. Remarkably, transfection

  20. 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. PMID:25429123

  1. Hyperpolarization-activated cation current Ih of dentate gyrus granule cells is upregulated in human and rat temporal lobe epilepsy

    PubMed Central

    Surges, Rainer; Kukley, Maria; Brewster, Amy; Rüschenschmidt, Christiane; Schramm, Johannes; Baram, Tallie Z.; Beck, Heinz; Dietrich, Dirk

    2012-01-01

    The hyperpolarization-activated cation current Ih is an important regulator of neuronal excitability and may contribute to the properties of the dentate gyrus granule (DGG) cells, which constitute the input site of the canonical hippocampal circuit. Here, we investigated changes in Ih in DGG cells in human temporal lobe epilepsy (TLE) and the rat pilocarpine model of TLE using the patch-clamp technique. Messenger-RNA (mRNA) expression of Ih-conducting HCN1, 2 and 4 isoforms was determined using semi-quantitative in-situ hybridization. Ih density was ~1.8-fold greater in DGG cells of TLE patients with Ammon’s horn sclerosis (AHS) as compared to patients without AHS. The magnitude of somatodendritic Ih was enhanced also in DGG cells in epileptic rats, most robustly during the latent phase after status epilepticus and prior to the occurrence of spontaneous epileptic seizures. During the chronic phase, Ih was increased ~1.7-fold. This increase of Ih was paralleled by an increase in HCN1 and HCN4 mRNA expression, whereas HCN2 expression was unchanged. Our data demonstrate an epilepsy-associated upregulation of Ih likely due to increased HCN1 and HCN4 expression, which indicate plasticity of Ih during epileptogenesis and which may contribute to a compensatory decrease in neuronal excitability of DGG cells. PMID:22405820

  2. Arsenite-Activated JNK Signaling Enhances CPEB4-Vinexin Interaction to Facilitate Stress Granule Assembly and Cell Survival

    PubMed Central

    Chang, Yu-Wei; Huang, Yi-Shuian

    2014-01-01

    Stress granules (SGs) are compartmentalized messenger ribonucleoprotein particles (mRNPs) where translationally repressed mRNAs are stored when cells encounter environmental stress. Cytoplasmic polyadenylation element-binding protein (CPEB)4 is a sequence-specific RNA-binding protein and translational regulator. In keeping with the results obtained from the study of other RNA-binding proteins, we found CPEB4 localized in SGs in various arsenite-treated cells. In this study, we identified that Vinexin, a CPEB4-interacting protein, is a novel component of SGs. Vinexin is a SH3-domain-containing adaptor protein and affects cell migration through its association with Vinculin to localize at focal adhesions (FAs). Unexpectedly, Vinexin is translocated from FAs to SGs under arsenite-induced stress. The recruitment of Vinexin to SGs depends on its interaction with CPEB4 and influences SG formation and cell survival. Arsenite-activated c-Jun N-terminal kinase (JNK) signaling enhances the association between CPEB4 and Vinexin, which consequently facilitates SG localization of Vinexin. Taken together, this study uncovers a novel interaction between a translational regulator and an adaptor protein to influence SG assembly and cell survival. PMID:25237887

  3. Cytochemistry of trichohyalin granules: a possible role for cornification of inner root sheath cell in the hair follicle.

    PubMed

    Sugiyama, S

    1989-04-01

    We studied ultracytochemically how trichohyalin granules (TGs) were involved in the cornification of the inner root sheath (IRS) cells in the hair follicle. After storing unfixed mouse skin samples in glycerol and then low ionic strength salt solution (G-LISS), the TGs reduced electron-dense amorphous material (EDM) to various degrees, with the appearance of filamentous material in routine staining. As the IRS cells were differentiating, these changes in the TGs became successively prominent; Some TGs consisted of the filamentous material and residual granular EDM, showing a "filamentogranular structure". In the cells in transition to cornified cells, or transitional cells, the EDM of the TGs disappeared and consisted of only filamentous material which was intertwined with keratin filaments (KFs). The internal substructure of the TGs induced by the G-LISS treatment stained well with ethanolic phosphotungstic acid, which reacts with basic proteins, and the KFs associated with the TGs also stained well. In skin samples not treated with G-LISS, the TGs of the transitional cells often exhibited fibrogranular structure after PTA staining to detect tissue basic proteins, suggesting a release of PTA stainable proteins(s) by the TGs in living transitional cells. The KFs of the cornified cells were more intensively stained and thicker (about 20 nm wide) than those of the differentiating IRS cells. These findings suggest that the TGs may contain at least two kinds of basic proteins; One is G-LISS-soluble, and the other is a very insoluble filamentous protein. These protein materials may be added to or incorporated into the KFs in the cornified cells with the disappearance of the TGs, since the KFs are markedly thick with PTA staining. PMID:2778195

  4. Acute and long-term effects of peroxisome proliferator-activated receptor-? activation on the function and insulin secretory responsiveness of clonal beta-cells.

    PubMed

    Irwin, N; McKinney, J M; Bailey, C J; McClenaghan, N H; Flatt, P R

    2011-04-01

    Thiazolidinediones (TZDs) are used as antidiabetic therapy. The purpose of the present study was to examine whether the TZD rosiglitazone has direct actions on pancreatic beta-cells that contribute to its overall effects. Effects of acute and prolonged (48 h) exposure to rosiglitazone, as a model glitazone compound, were assessed in clonal pancreatic BRIN-BD11 beta-cells maintained in standard, glucotoxic and lipotoxic cultures. In acute 20-min incubations, rosiglitazone (0.2-100 ?M) did not alter basal or glucose-stimulated insulin secretion. However, rosiglitazone (6.25 ?M) enhanced (p<0.001) the acute insulinotropic action of GLP-1. Prolonged exposure to 6.25 ?M rosiglitazone in standard media had no effect on cell viability or cellular insulin content, but slightly reduced the insulin secretory response to glucose and alanine (p<0.05). Prolonged (48 h) exposure to glucotoxic or lipotoxic conditions reduced beta-cell viability (p<0.05), cellular insulin content (p<0.001 and p<0.05, respectively), and insulin release in response to glucose and a range of secretagogues. The adverse effect of lipotoxicity on beta-cell viability was prevented by concomitant exposure to 6.25 ?M rosiglitazone. Culture with 6.25 ?M rosiglitazone further decreased acute insulin release under glucotoxic conditions. However, when insulin secretion was expressed as percentage cellular insulin content, rosiglitazone (6.25 ?M) significantly improved many of the adverse effects of gluco- and lipotoxic conditions on insulin secretory responsiveness. The results suggest that despite decrease in cellular insulin content TZDs exert direct beneficial effects on beta-cell viability and function during gluco- or lipotoxicity. PMID:21165811

  5. Secretion of urocortin I by human glioblastoma cell lines, possibly via the constitutive pathway.

    PubMed

    Ikeda, Keiichi; Fujioka, Kouki; Tachibana, Toshiaki; Kim, Seung U; Tojo, Katsuyoshi; Manome, Yoshinobu

    2015-01-01

    Corticotropin-releasing factor (CRF) and its family of peptides, i.e., urocortins (UCNs), play a critical role in systemic and peripheral stress-response systems and are widely expressed not only in normal tissues but also in various types of cancer cells. Given limited understanding of the mechanism of UCN I secretion, we investigated the UCN I secretory pathway in human neural stem cells (HNSCs) and in two glioblastoma cell lines, e.g., A172 and U-138 MG. Immunoreactivities for CRF receptors were detected in A172 glioblastoma cells, but not in HNSCs or U-138 glioblastoma cells, while UCN I immunoreactivity was detected in A172 and U-138 MG glioblastoma cell lines by both light field and electron microscopy. Interestingly, electron microscopy revealed UCN I immunoreactivtiy in vesicle-like structures in the plasma membrane of the glioblastoma cells. Tracking of a hybrid fluorescent protein containing a UCN I signal peptide expressed in A172 human glioblastoma cells revealed that fluorescence in secretory granules could be decreased by cycloheximide (100?g/ml), indicating that the forward transport of secretory granules containing fluorescent protein was not altered by the inhibition of protein synthesis by cycloheximide. Retrograde transport and the fusion of fluorescent granules in A172 human glioblastoma cells was induced by brefeldin A (10?g/ml), indicating that UCN I secretory granules may be transported via the constitutive pathway. Based on these results, it appears that UCN I is secreted from human glioblastoma cells by exocytosis through constitutive secretory granules, indicating that transcription of UCN I mRNA may be correlated to secretion of UCN I protein. PMID:25239507

  6. Selective disruption of Tcf7l2 in the pancreatic ? cell impairs secretory function and lowers ? cell mass

    PubMed Central

    Mitchell, Ryan K.; Mondragon, Angeles; Chen, Lingling; Mcginty, James A.; French, Paul M.; Ferrer, Jorge; Thorens, Bernard; Hodson, David J.; Rutter, Guy A.; Da Silva Xavier, Gabriela

    2015-01-01

    Type 2 diabetes (T2D) is characterized by ? cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired ? cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the ? cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in ? cells from the earliest expression of the Ins1 gene (?E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2fl/fl::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks, respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcfl2fl/fl::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 (?20%) and Glp1r (?40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca2+ increases, and connectivity between individual ? cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed ?30% decrease in ? cell mass in pancreata from Tcfl2fl/fl::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of ? cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed. PMID:25355422

  7. Cytological Features of Mammary Analogue Secretory Carcinoma of the Parotid Gland in a 15-Year-Old Girl: A Case Report with Review of the Literature

    PubMed Central

    Inaba, Takako; Fukumura, Yuki; Saito, Tsuyoshi; Yokoyama, Junkichi; Ohba, Shinichi; Arakawa, Atsushi; Yao, Takashi

    2015-01-01

    Mammary analogue secretory carcinoma (MASC) is a recently recognized tumor of salivary glands characterized by the ETV6-NTRK3 fusion gene. This tumor is very rare in children and adolescents. We report a case of MASC in a 15-year-old girl, the fifth youngest case so far reported. The patient complained of a left infra-auricular mass that gradually enlarged for a year. Fine-needle aspiration cytology/imprint cytology showed individual tumor cells that had faintly eosinophilic granular cytoplasm with secretion granules sometimes seen adjacent to the tumor cells. These cytological features overlapped between those of zymogen granule-poor acinic cell carcinoma (AciCC) and MASC. In addition to the case report, we present a review of the related literature with a focus on the cytological features of MASC. The differential diagnostic clues are also discussed.

  8. Cytological features of mammary analogue secretory carcinoma of the parotid gland in a 15-year-old girl: a case report with review of the literature.

    PubMed

    Inaba, Takako; Fukumura, Yuki; Saito, Tsuyoshi; Yokoyama, Junkichi; Ohba, Shinichi; Arakawa, Atsushi; Yao, Takashi

    2015-01-01

    Mammary analogue secretory carcinoma (MASC) is a recently recognized tumor of salivary glands characterized by the ETV6-NTRK3 fusion gene. This tumor is very rare in children and adolescents. We report a case of MASC in a 15-year-old girl, the fifth youngest case so far reported. The patient complained of a left infra-auricular mass that gradually enlarged for a year. Fine-needle aspiration cytology/imprint cytology showed individual tumor cells that had faintly eosinophilic granular cytoplasm with secretion granules sometimes seen adjacent to the tumor cells. These cytological features overlapped between those of zymogen granule-poor acinic cell carcinoma (AciCC) and MASC. In addition to the case report, we present a review of the related literature with a focus on the cytological features of MASC. The differential diagnostic clues are also discussed. PMID:25815230

  9. Topically Delivered Adipose Derived Stem Cells Show an Activated-Fibroblast Phenotype and Enhance Granulation Tissue Formation in Skin Wounds

    PubMed Central

    Hong, Seok Jong; Xu, Wei; Leung, Kai P.; Mustoe, Thomas A.; Galiano, Robert D.

    2013-01-01

    Multipotent mesenchymal stem cells (MSCs) are found in various tissues and can proliferate extensively in vitro. MSCs have been used in preclinical animal studies and clinical trials in many fields. Adipose derived stem cells (ASCs) have several advantages compared to other MSCs for use in cell-based treatments because they are easy to isolate with relative abundance. However, quantitative approaches for wound repair using ASCs have been limited because of lack of animal models which allow for quantification. Here, we addressed the effect of topically delivered ASCs in wound repair by quantitative analysis using the rabbit ear model. We characterized rabbit ASCs, and analyzed their multipotency in comparison to bone marrow derived-MSCs (BM-MSCs) and dermal fibroblasts (DFs) in vitro. Topically delivered ASCs increased granulation tissue formation in wounds when compared to saline controls, whereas BM-MSCs or DFs did not. These studies suggest that ASCs and BM-MSCs are not identical, though they have similar surface markers. We found that topically delivered ASCs are engrafted and proliferate in the wounds. We showed that transplanted ASCs exhibited activated fibroblast phenotype, increased endothelial cell recruitment, and enhanced macrophage recruitment in vivo. PMID:23383253

  10. Topically delivered adipose derived stem cells show an activated-fibroblast phenotype and enhance granulation tissue formation in skin wounds.

    PubMed

    Hong, Seok Jong; Jia, Sheng-Xian; Xie, Ping; Xu, Wei; Leung, Kai P; Mustoe, Thomas A; Galiano, Robert D

    2013-01-01

    Multipotent mesenchymal stem cells (MSCs) are found in various tissues and can proliferate extensively in vitro. MSCs have been used in preclinical animal studies and clinical trials in many fields. Adipose derived stem cells (ASCs) have several advantages compared to other MSCs for use in cell-based treatments because they are easy to isolate with relative abundance. However, quantitative approaches for wound repair using ASCs have been limited because of lack of animal models which allow for quantification. Here, we addressed the effect of topically delivered ASCs in wound repair by quantitative analysis using the rabbit ear model. We characterized rabbit ASCs, and analyzed their multipotency in comparison to bone marrow derived-MSCs (BM-MSCs) and dermal fibroblasts (DFs) in vitro. Topically delivered ASCs increased granulation tissue formation in wounds when compared to saline controls, whereas BM-MSCs or DFs did not. These studies suggest that ASCs and BM-MSCs are not identical, though they have similar surface markers. We found that topically delivered ASCs are engrafted and proliferate in the wounds. We showed that transplanted ASCs exhibited activated fibroblast phenotype, increased endothelial cell recruitment, and enhanced macrophage recruitment in vivo. PMID:23383253

  11. Vesicular distribution of Secretory Pathway Ca2+-ATPase isoform 1 and a role in manganese detoxification in liver-derived polarized cells

    PubMed Central

    Leitch, Sharon; Feng, Mingye; Muend, Sabina; Braiterman, Lelita T.; Hubbard, Ann L.

    2011-01-01

    Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn2+ leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn2+ detoxification by excretion into bile. Although many pathways of cellular Mn2+ uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn2+ detoxification by the Secretory Pathway Ca2+, Mn2+-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca2+-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn2+ as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn2+. Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn2+ toxicity. Taken together, our findings suggest a role for SPCA1 in Mn2+ detoxification in liver. PMID:20981470

  12. Human microRNA-24 modulates highly pathogenic avian-origin H5N1 influenza A virus infection in A549 cells by targeting secretory pathway furin.

    PubMed

    Loveday, Emma-Kate; Diederich, Sandra; Pasick, John; Jean, François

    2015-01-01

    A common critical cellular event that many human enveloped viruses share is the requirement for proteolytic cleavage of the viral glycoprotein by furin in the host secretory pathway. For example, the furin-dependent proteolytic activation of highly pathogenic (HP) influenza A (infA) H5 and H7 haemagglutinin precursor (HA0) subtypes is critical for yielding fusion-competent infectious virions. In this study, we hypothesized that viral hijacking of the furin pathway by HP infA viruses to permit cleavage of HA0 could represent a novel molecular mechanism controlling the dynamic production of fusion-competent infectious virus particles during the viral life cycle. We explored the biological role of a newly identified furin-directed human microRNA, miR-24, in this process as a potential post-transcriptional regulator of the furin-mediated activation of HA0 and production of fusion-competent virions in the host secretory pathway. We report that miR-24 and furin are differentially expressed in human A549 cells infected with HP avian-origin infA H5N1. Using miR-24 mimics, we demonstrated a robust decrease in both furin mRNA levels and intracellular furin activity in A549 cells. Importantly, pretreatment of A549 cells with miR-24 mimicked these results: a robust decrease of H5N1 infectious virions and a complete block of H5N1 virus spread that was not observed in A549 cells infected with low-pathogenicity swine-origin infA H1N1 virus. Our results suggest that viral-specific downregulation of furin-directed microRNAs such as miR-24 during the life cycle of HP infA viruses may represent a novel regulatory mechanism that governs furin-mediated proteolytic activation of HA0 glycoproteins and production of infectious virions. PMID:25234642

  13. Boric acid induces cytoplasmic stress granule formation, eIF2? phosphorylation, and ATF4 in prostate DU-145 cells.

    PubMed

    Henderson, Kimberly A; Kobylewski, Sarah E; Yamada, Kristin E; Eckhert, Curtis D

    2015-02-01

    Dietary boron intake is associated with reduced prostate and lung cancer risk and increased bone mass. Boron is absorbed and circulated as boric acid (BA) and at physiological concentrations is a reversible competitive inhibitor of cyclic ADP ribose, the endogenous agonist of the ryanodine receptor calcium (Ca(+2)) channel, and lowers endoplasmic reticulum (ER) [Ca(2+)]. Low ER [Ca(2+)] has been reported to induce ER stress and activate the eIF2?/ATF4 pathway. Here we report that treatment of DU-145 prostate cells with physiological levels of BA induces ER stress with the formation of stress granules and mild activation of eIF2?, GRP78/BiP, and ATF4. Mild activation of eIF2? and its downstream transcription factor, ATF4, enables cells to reconfigure gene expression to manage stress conditions and mild activation of ATF4 is also required for the differentiation of osteoblast cells. Our results using physiological levels of boric acid identify the eIF2?/ATF pathway as a plausible mode of action that underpins the reported health effects of dietary boron. PMID:25425213

  14. 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.

  15. Actin depolymerisation and crosslinking join forces with myosin II to contract actin coats on fused secretory vesicles.

    PubMed

    Miklavc, Pika; Ehinger, Konstantin; Sultan, Ayesha; Felder, Tatiana; Paul, Patrick; Gottschalk, Kay-Eberhard; Frick, Manfred

    2015-03-15

    In many secretory cells actin and myosin are specifically recruited to the surface of secretory granules following their fusion with the plasma membrane. Actomyosin-dependent compression of fused granules is essential to promote active extrusion of cargo. However, little is known about molecular mechanisms regulating actin coat formation and contraction. Here, we provide a detailed kinetic analysis of the molecules regulating actin coat contraction on fused lamellar bodies in primary alveolar type II cells. We demonstrate that ROCK1 and myosin light chain kinase 1 (MLCK1, also known as MYLK) translocate to fused lamellar bodies and activate myosin II on actin coats. However, myosin II activity is not sufficient for efficient actin coat contraction. In addition, cofilin-1 and ?-actinin translocate to actin coats. ROCK1-dependent regulated actin depolymerisation by cofilin-1 in cooperation with actin crosslinking by ?-actinin is essential for complete coat contraction. In summary, our data suggest a complementary role for regulated actin depolymerisation and crosslinking, and myosin II activity, to contract actin coats and drive secretion. PMID:25637593

  16. Actin depolymerisation and crosslinking join forces with myosin II to contract actin coats on fused secretory vesicles

    PubMed Central

    Miklavc, Pika; Ehinger, Konstantin; Sultan, Ayesha; Felder, Tatiana; Paul, Patrick; Gottschalk, Kay-Eberhard; Frick, Manfred

    2015-01-01

    ABSTRACT In many secretory cells actin and myosin are specifically recruited to the surface of secretory granules following their fusion with the plasma membrane. Actomyosin-dependent compression of fused granules is essential to promote active extrusion of cargo. However, little is known about molecular mechanisms regulating actin coat formation and contraction. Here, we provide a detailed kinetic analysis of the molecules regulating actin coat contraction on fused lamellar bodies in primary alveolar type II cells. We demonstrate that ROCK1 and myosin light chain kinase 1 (MLCK1, also known as MYLK) translocate to fused lamellar bodies and activate myosin II on actin coats. However, myosin II activity is not sufficient for efficient actin coat contraction. In addition, cofilin-1 and ?-actinin translocate to actin coats. ROCK1-dependent regulated actin depolymerisation by cofilin-1 in cooperation with actin crosslinking by ?-actinin is essential for complete coat contraction. In summary, our data suggest a complementary role for regulated actin depolymerisation and crosslinking, and myosin II activity, to contract actin coats and drive secretion. PMID:25637593

  17. A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu 5 receptor in cerebellar granule cells

    Microsoft Academic Search

    Fabrice Ango; Serenella Albani-Torregrossa; Cécile Joly; David Robbe; Jean-Marie Michel; Jean-Philippe Pin; Joël Bockaert; Laurent Fagni

    1999-01-01

    We describe a method to transfer cDNA into neuronal primary cultures with a commercialised cationic lipid, Transfast. Cultures were transfected at a rate of about 5% with green fluorescent protein (GFP) cDNA. Comparing Transfast to other transfection reagents, we found this compound to be the most efficient. GFP-transfected mouse cerebellar granule cells displayed normal whole-cell voltage-sensitive and unitary big K+

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

    Microsoft Academic Search

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

    2008-01-01

    Aims\\/hypothesis  We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage\\u000a and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked\\u000a whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli.\\u000a \\u000a \\u000a \\u000a Methods  The intracellular

  19. Adverse effects of 2,4-dichlorophenoxyacetic acid on rat cerebellar granule cell cultures were attenuated by amphetamine.

    PubMed

    Bongiovanni, B; Ferri, A; Brusco, A; Rassetto, M; Lopez, L M; Evangelista de Duffard, A M; Duffard, R

    2011-05-01

    2,4-Dichlorophenoxyacetic acid (2,4-D), a worldwide-used herbicide, has been shown to produce a wide range of adverse effects in the health--from embryotoxicity and teratogenicity to neurotoxicity--of animals and humans. In this study, neuronal morphology and biochemical events in rat cerebellar granule cell (CGC) cultures have been analyzed to define some of the possible mechanisms involved in 2,4-D-induced cell death. For that purpose, amphetamine (AMPH) that has been shown to accelerate the recovery of several functions in animals with brain injury has been used as a pharmacologycal tool and was also investigated as a possible protecting agent. Addition of 2,4-D to CGC cultures produced a drastic decrease in cell viability, in association with an increased incidence of necrosis and apoptosis, and an increased level of reactive oxygen species, a decrease in glutathione content, and an abnormal activity of some enzymes with respect to the control group. The adverse effects of 2,4-D were partly attenuated in presence of AMPH. Some deleterious effects on several ultrastructural features of the cells, as well as the enhanced incidence of apoptosis, were partially preserved in AMPH-protected cultures as compared with those which were exposed to 2,4-D alone. The collected evidences (1) confirms the previously observed, deleterious effects of 2.4D on the same or a similar model; (2) suggests that the 2,4-D-induced apoptosis could have been mediated by or associated to an oxidative imbalance in the affected cells, and (3) shows some evidence of a protective effect of AMPH on 2,4-D-induced cell death, which could have been exerted through a reduction in the oxidative stress. PMID:20383622

  20. Effects of glucagon and insulin on the paneth cells of the mouse duodenum

    Microsoft Academic Search

    A. Ahonen; A. Penttilä

    1975-01-01

    Summary  The effect of glucagon and insulin on the paneth cells (PC) of the duodenum of the mouse was investigated using light microscopy.\\u000a Both glucagon and insulin were able to increase significantly the number of the secretory granules of PC. This possibly means\\u000a that these hormones are capable of inhibiting the secretion of PC.

  1. 5-HT receptor-mediated modulation of granule cell inhibition after juvenile stress recovers after a second exposure to adult stress.

    PubMed

    Gruber, D; Gilling, K E; Albrecht, A; Bartsch, J C; Çal??kan, G; Richter-Levin, G; Stork, O; Heinemann, U; Behr, J

    2015-05-01

    Aversive experiences in early life are thought to dispose to psychopathologies such as mood or anxiety disorders. In a two-hit stress model, we assessed the effects of juvenile and/or adult stress on the 5-HT-mediated modulation of synaptic inhibition of ventral dentate gyrus granule cells. Combined but not single stress exposure led to a significant reduction in activity and increased anxiety-like behavior. Similarly, the 5-HT1A receptor-mediated inhibition of evoked inhibitory postsynaptic currents (IPSCs) of granule cells was only reduced in single stress exposed animals. This was also true for the number of granule cells responding with a 5-HT3 receptor-dependent burst of miniature IPSCs. 5-HT3 receptors are expressed on cholecystokinin (CCK)+ basket cells in the hippocampus. In fact, we observed a reduction of steady-state mRNA levels of CCK+ basket cell markers after single juvenile or adult stress and partial recovery after combined stress, thus matching the electrophysiological findings. Adaptive changes in 5-HT-mediated modulation of synaptic inhibition and CCK+ basket cells in the DG may help to maintain normal levels of anxiety after single juvenile or adult stress exposure, as indicated by the increased anxiety that accompanies the loss of this regulation upon combined stress. PMID:25748530

  2. Ultrastructural Study on Colocalization of Glucagon-Like Peptide (GLP)-1 with GLP-2 in Chicken Intestinal L-Cells

    PubMed Central

    NISHIMURA, Kei; HIRAMATSU, Kohzy; MONIR, Mohammad M.; TAKEMOTO, Chihiro; WATANABE, Takafumi

    2013-01-01

    ABSTRACT Colocalization of glucagon-like peptide (GLP)-1 with GLP-2 in L-cells was investigated in the chicken ileum by using double immunofluorescent and immunocytochemical techniques. Ultrastructural features of L-cells were also clarified in this study. L-cells showing immunoreactivity for both GLP-1 and GLP-2 were distributed in the whole ileum. They showed comma-like or flask-like shape and were located in epithelium of crypts and lower part of intestinal villi. L-cells showing GLP-1-immunoreactivity only were found in epithelium of lower and middle parts of intestinal villi. Transmission electron microscopy indicated that L-cells identified by colloidal gold-labeled immunocytochemistry were covered apically with microvilli, open-type and contained many secretory granules in their perikarya. These secretory granules without halo were round to oval in shape and showed moderate electron density. The longest and shortest diameters of secretory granules were 355 ± 62 nm (mean ± SD) and 287 ± 48 nm, respectively. Double labeling immunocytochemistry using two different sizes of particles (6 and 12 nm in diameter) of colloidal gold revealed that GLP-1 colocalized with GLP-2 in the same secretory granules. This study advances new morphological data about the endocrine system of the chicken small intestine. PMID:23759686

  3. 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 shows that localised iron increase may be an early event contributing to cell death. (4) The iron content in granules found in both the MPTP-injected and contra-lateral SNs is correlated with the degree of bulk SN cell loss (assessed by TH-immunohistochemistry) in individual models. This indicates a correlation between localised iron increase and cell loss, at least at the whole SN level. Our results are consistent with the observation that in Parkinson's disease (PD), neuronal cell death seems to be related to their neuromelanin content and support the proposal that iron-melanin interaction may play a role in oxidative neuronal cell death. Indeed, iron-saturated neuromelanin granules may act as centres of free radical production, contributing to localised cell death.

  4. Rapid upregulation and granule-independent transport of perforin to the immunological synapse define a novel mechanism of antigen-specific CD8+ T cell cytotoxic activity

    PubMed Central

    Makedonas, George; Banerjee, Pinaki P.; Pandey, Rahul; Hersperger, Adam R.; Sanborn, Keri B.; Hardy, Gareth A.D.; Orange, Jordan S.; Betts, Michael R.

    2009-01-01

    Cytotoxic T lymphocytes (CTL) are endowed with the ability to eliminate pathogens through perforin-mediated cytotoxic activity. The mechanism for perforin-mediated antigen-specific killing has been solely attributed to cytotoxic granule exocytosis from activated CD8+ T cells. Here we redefine this mechanism, demonstrating that virus-specific CD8+ T cells rapidly upregulate perforin in response to stimulation temporally with IFN-? and CD107a expression. Following antigen-specific activation, newly synthesized perforin rapidly appears at the immunological synapse, both in association with and independent of cytotoxic granules, where it functions to promote cytotoxicity. Our work redefines the current mechanism of CTL cytotoxicity and identifies a novel correlate of CD8+ T cell mediated immunity. PMID:19380804

  5. Effect of Aggregated ?-Amyloid (1-42) on Synaptic Plasticity of Hippocampal Dentate Gyrus Granule Cells in Vivo

    PubMed Central

    Babri, Shirin; Amani, Mohammad; Mohaddes, Gisou; Alihemmati, Alireza; Ebrahimi, Hadi

    2012-01-01

    Introduction Alzheimer’s disease (AD) is a common neurodegenerative disorder in elderly people with an impairment of cognitive decline and memory loss. ?-amyloid (A?) as a potent neurotoxic peptide has a pivotal role in the pathogenesis of AD. This disease begins with impairment in synaptic functions before developing into later neuro¬degeneration and neuronal loss. The aim of this study was to evaluate the synaptic plasticity and electrophysiological function of granule cells in hippocampal dentate gyrus (DG) after intracerebroventricular (i.c.v.) administration of aggregated A? (1-42) peptide in vivo. Methods Animals were divided to control and A? (1-42) groups. Long-term potentia¬tion (LTP) in perforant path-DG synapses was assessed in order to investigate the effect of aggregated A? (1-42) on synaptic plasticity. Field excitatory post-synaptic potential (fEPSP) slope and population spike (PS) amplitude were measured. Results Administration of A? (1-42) significantly decreased fEPSP slope and PS amplitude in A? (1-42) group comparing with the control group and had no effect on baseline activity of neurons. Conclusion The present study indicates that administration of aggregated form of A? (1-42) into the lateral ventricle effectively inhibits LTP in granular cells of the DG in hippocampus in vivo. PMID:23678459

  6. Glycolytic enzyme upregulation and numbness of mitochondrial activity characterize the early phase of apoptosis in cerebellar granule cells.

    PubMed

    Bobba, A; Amadoro, G; La Piana, G; Calissano, P; Atlante, A

    2015-01-01

    Alzheimer's disease (AD) and cancer proceed via one or more common molecular mechanisms: a metabolic shift from oxidative phosphorylation to glycolysis-corresponding to the activation of the Warburg effect-occurs in both diseases. The findings reported in this paper demonstrate that, in the early phase of apoptosis, glucose metabolism is enhanced, i.e. key proteins which internalize and metabolize glucose-glucose transporter, hexokinase and phosphofructokinase-are up-regulated, in concomitance with a parallel decrease in oxygen consumption by mitochondria and increase of L-lactate accumulation. Reversal of the glycolytic phenotype occurs in the presence of dichloroacetate, inhibitor of the pyruvate dehydrogenase kinase enzyme, which speeds up apoptosis of cerebellar granule cells, reawakening mitochondria and then modulating glycolytic enzymes. Loss of the adaptive advantage afforded by aerobic glycolysis, which occurs in the late phase of apoptosis, exacerbates the pathological processes underlying neurodegeneration, leading inevitably the cell to death. In conclusion, the data propose that both aerobic, i.e. Warburg effect, essentially due to the protective numbness of mitochondria, and anaerobic glycolysis, rather due to the mitochondrial impairment, characterize the entire time frame of apoptosis, from the early to the late phase, which mimics the development of AD. PMID:25351440

  7. Decreased tonic inhibition in cerebellar granule cells causes motor dysfunction in a mouse model of Angelman syndrome.

    PubMed

    Egawa, Kiyoshi; Kitagawa, Kyoko; Inoue, Koichi; Takayama, Masakazu; Takayama, Chitoshi; Saitoh, Shinji; Kishino, Tatsuya; Kitagawa, Masatoshi; Fukuda, Atsuo

    2012-12-01

    Angelman syndrome is a neurodevelopmental disorder caused by loss of function of the UBE3A gene encoding a ubiquitin E3 ligase. Motor dysfunction is a characteristic feature of Angelman syndrome, but neither the mechanisms of action nor effective therapeutic strategies have yet been elucidated. We report that tonic inhibition is specifically decreased in cerebellar granule cells of Ube3a-deficient mice, a model of Angelman syndrome. As a mechanism underlying this decrease in tonic inhibition, we show that Ube3a controls degradation of ?-aminobutyric acid (GABA) transporter 1 (GAT1) and that deficiency of Ube3a induces a surplus of GAT1 that results in a decrease in GABA concentrations in the extrasynaptic space. Administering low doses of 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP), a selective extrasynaptic GABA(A) receptor agonist, improves the abnormal firing properties of a population of Purkinje cells in cerebellar brain slices and reduces cerebellar ataxia in Ube3a-deficient mice in vivo. These results suggest that pharmacologically increasing tonic inhibition may be a useful strategy for alleviating motor dysfunction in Angelman syndrome. PMID:23220633

  8. Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from ?-Granules to the Cell Surface

    PubMed Central

    Heijnen, Harry F.G.; Oorschot, Viola; Sixma, Jan J.; Slot, Jan W.; James, David E.

    1997-01-01

    Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most ?-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the ?-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant ?-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of ?-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in ?-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (?15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets. PMID:9230074

  9. Single channel properties of P2X ATP receptors in outside-out patches from rat hippocampal granule cells

    PubMed Central

    Wong, Adrian YC; Burnstock, Geoffrey; Gibb, Alasdair J

    2000-01-01

    The single channel properties of P2X ATP receptors were investigated in outside-out patches from hippocampal granule cells in brain slices from 12-day-old rats. The results demonstrate that functional P2X ATP receptors are expressed in hippocampal granule cells and, combined with previously published information on the P2X subunits expressed in the hippocampus, suggest that the receptors may be heteromers of the P2X4 and P2X6 subunits or P2X1, P2X2, P2X4 and P2X6 subunits. Two distinct types of P2X channel openings were observed. A flickery P2X receptor channel was observed in three patches with a mean chord conductance of 32 ± 6 pS, a mean open time of 1.0 ± 0.3 ms and a mean burst length of 11 ± 5 ms at a membrane potential of ?60 mV. A large conductance P2X receptor was observed in 19 out of 98 patches with a mean conductance of 56 ± 1.8 pS, a linear current-voltage relationship between ?80 and +60 mV with a reversal potential around 0 mV, a mean open time of 2.6 ± 0.2 ms and a mean burst length of 8.8 ± 1.8 ms at ?60 mV. At an ATP concentration of 1 mm, these channels exhibited a low steady-state open probability (Popen, 0.07 ± 0.008; n = 15), little apparent desensitisation and were also activated by ?,?-methylene ATP (?,?-meATP, 40 ?m; Popen, 0.007 ± 0.0002; conductance, 57 ± 1.1 pS; n = 3). No decrease in the single channel conductance was observed on increasing the free extracellular calcium concentration from 0.3 to 0.85 mm. Channel closed time distributions were fitted with five exponential components with time constants (and relative areas) of 90 ?s (20%), 0.77 ms (32%), 10 ms (15%), 90 ms (18%) and 403 ms (15%) at 1 mm ATP. Of these, the first two components are suggested to represent gaps within single activations of the receptor based on the lack of agonist concentration dependence of these two shut time components between 1 ?m and 1 mm ATP. Suramin (40 ?m) significantly increased the single channel conductance (19 ± 7%; n = 5) and produced a small decrease in Popen (39 ± 9%; n = 5) by decreasing mean open time, burst length and total open time per burst. These actions of suramin are not consistent with simple competitive antagonism. PMID:10990539

  10. Souffle/Spastizin Controls Secretory Vesicle Maturation during Zebrafish Oogenesis

    PubMed Central

    Riedel, Dietmar; Schomburg, Christoph; Cerdŕ, Joan; Vollack, Nadine; Dosch, Roland

    2014-01-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research. PMID:24967841

  11. Functional ryanodine receptors in the membranes of neurohypophysial secretory granules

    PubMed Central

    McNally, James M.; Custer, Edward E.; Ortiz-Miranda, Sonia; Woodbury, Dixon J.; Kraner, Susan D.; Salzberg, Brian M.

    2014-01-01

    Highly localized Ca2+ release events have been characterized in several neuronal preparations. In mouse neurohypophysial terminals (NHTs), such events, called Ca2+ syntillas, appear to emanate from a ryanodine-sensitive intracellular Ca2+ pool. Traditional sources of intracellular Ca2+ appear to be lacking in NHTs. Thus, we have tested the hypothesis that large dense core vesicles (LDCVs), which contain a substantial amount of calcium, represent the source of these syntillas. Here, using fluorescence immunolabeling and immunogold-labeled electron micrographs of NHTs, we show that type 2 ryanodine receptors (RyRs) are localized specifically to LDCVs. Furthermore, a large conductance nonspecific cation channel, which was identified previously in the vesicle membrane and has biophysical properties similar to that of an RyR, is pharmacologically affected in a manner characteristic of an RyR: it is activated in the presence of the RyR agonist ryanodine (at low concentrations) and blocked by the RyR antagonist ruthenium red. Additionally, neuropeptide release experiments show that these same RyR agonists and antagonists modulate Ca2+-elicited neuropeptide release from permeabilized NHTs. Furthermore, amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Collectively, our findings suggest that RyR-dependent syntillas could represent mobilization of Ca2+ from vesicular stores. Such localized vesicular Ca2+ release events at the precise location of exocytosis could provide a Ca2+ amplification mechanism capable of modulating neuropeptide release physiologically. PMID:24863930

  12. A Novel Class of mRNA-containing Cytoplasmic Granules Are Produced in Response to UV-Irradiation

    PubMed Central

    Gaillard, Hélčne

    2008-01-01

    Nucleic acids are substrates for different types of damage, but little is known about the fate of damaged RNAs. We addressed the existence of an RNA-damage response in yeast. The decay kinetics of GAL1p-driven mRNAs revealed a dose-dependent mRNA stabilization upon UV-irradiation that was not observed after heat or saline shocks, or during nitrogen starvation. UV-induced mRNA stabilization did not depend on DNA repair, damage checkpoint or mRNA degradation machineries. Notably, fluorescent in situ hybridization revealed that after UV-irradiation, polyadenylated mRNA accumulated in cytoplasmic foci that increased in size with time. In situ colocalization showed that these foci are not processing-bodies, eIF4E-, eIF4G-, and Pab1-containing bodies, stress granules, autophagy vesicles, or part of the secretory or endocytic pathways. These results point to the existence of a specific eukaryotic RNA-damage response, which leads to new polyadenylated mRNA-containing granules (UV-induced mRNA granules; UVGs). We propose that potentially damaged mRNAs, which may be deleterious to the cell, are temporarily stored in UVG granules to safeguard cell viability. PMID:18768757

  13. Wide distribution of cysteine-rich secretory proteins in snake venoms: Isolation and cloning of novel snake venom cysteine-rich secretory proteins

    Microsoft Academic Search

    Yasuo Yamazaki; Fumiko Hyodo; Takashi Morita

    2003-01-01

    Cysteine-rich secretory proteins (CRISPs) are found in epididymis and granules of mammals, and they are thought to function in sperm maturation and in the immune system. Recently, we isolated and obtained clones for novel snake venom proteins that are classified as CRISP family proteins. To elucidate the distribution of snake venom CRISP family proteins, we evaluated a wide range of

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

    PubMed

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

    2009-03-16

    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) 7-35. Thirty-nine days after the last MPH administration (PND 76), 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 per 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 PND 130 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 PND 135, 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 per 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

  15. Receptors with low affinity for neurosteroids and GABA contribute to tonic inhibition of granule cells in epileptic animals

    PubMed Central

    Rajasekaran, Karthik; Joshi, Suchitra; Sun, Chengsan; Mtchedlishvilli, Zakaria; Kapur, Jaideep

    2010-01-01

    Neurosteroid sensitivity of GABAA receptor mediated inhibition of the hippocampal dentate granule cells (DGCs) is reduced in animal models of temporal lobe epilepsy. However, the properties and subunit composition of GABAA receptors mediating tonic inhibition in DGCS of epileptic animals have not been described. In the DGCs of epileptic animals, allopregnanolone and L-65708 sensitivity of holding current was diminished and ? subunit was retained in the endoplasmic reticulum and its surface expression was decreased the in the hippocampus. Ro15–4513 and lanthanum had distinct effects on holding current recorded from DGCs of control and epileptic animals. The pharmacological properties of GABAA receptors maintaining tonic inhibition in DGCs of epileptic animals were similar to those containing the ?4?x?2 subunits. Furthermore, surface expression of the ?4 subunit increased and a larger fraction of the subunit was co-immunoprecipitated with the ?2 subunit in hippocampi of epileptic animals. Together these studies revealed that functional ?4?x? and ?5?x?2 receptors were reduced in the hippocampi of epileptic animals, and that novel ?4bx?2 receptors contributed to the maintenance of tonic inhibition. The presence of ?4?x?2 receptors resulted in low GABA affinity and neurosteroid sensitivity of tonic currents in the DGCs of epileptic animals that could potentially increase seizure vulnerability. These receptors may represent a novel therapeutic target for anticonvulsant drugs without sedative actions. PMID:20682339

  16. Selective silencing of individual dendritic branches by an mGlu2-activated potassium conductance in dentate gyrus granule cells

    PubMed Central

    Brunner, János; Ster, Jeanne; Van-Weert, Susan; Andrási, Tibor; Neubrandt, Máté; Corti, Corrado; Corsi, Mauro; Ferraguti, Francesco; Gerber, Urs; Szabadics, János

    2013-01-01

    Group II metabotropic glutamate receptors (mGlu-IIs) modulate hippocampal information processing through several presynaptic actions. We describe a novel postsynaptic inhibitory mechanism mediated by the mGlu2 subtype that activates an inwardly-rectifying potassium conductance in the dendrites of dentate gyrus granule cells (GCs) of rats and mice. Data from glutamate uncaging experiments and simulations indicate that the mGlu2-activated potassium conductance uniformly reduces the peak amplitude of synaptic inputs arriving in the distal two-thirds of dendrites with only minor effects on proximal inputs. This unique shunting profile is consistent with a peak expression of the mGlu2-activated conductance at the transition between the proximal and middle third of the dendrites. Further simulations under various physiologically relevant conditions show that when a shunting conductance is activated in the proximal third of a single dendrite it effectively modulates input to this specific branch while leaving inputs in neighboring dendrites relatively unaffected. Thus, the restricted expression of the mGlu2-activated potassium conductance in the proximal third of GC dendrites represents an optimal localization for achieving the opposing biophysical requirements for uniform yet selective modulation of individual dendritic branches. PMID:23616537

  17. Correlation between calbindin expression in granule cells of the resected hippocampal dentate gyrus and verbal memory in temporal lobe epilepsy.

    PubMed

    Karádi, Kázmér; Janszky, József; Gyimesi, Csilla; Horváth, Zsolt; Lucza, Tivadar; Dóczi, Tamás; Kállai, János; Abrahám, Hajnalka

    2012-09-01

    Calbindin expression of granule cells of the dentate gyrus is decreased in temporal lobe epilepsy (TLE) regardless of its etiology. In this study, we examined the relation between reduction of calbindin immunoreactivity and the verbal and visuo-spatial memory function of patients with TLE of different etiologies. Significant linear correlation was shown between calbindin expression and short-term and long-term percent retention and retroactive interference in auditory verbal learning test (AVLT) of patients including those with hippocampal sclerosis. In addition, we found significant linear regression between calbindin expression and short-term and long-term percent retention of AVLT in patients whose epilepsy was caused by malformation of cortical development or tumor and when no hippocampal sclerosis and substantial neuronal loss were detected. Together with the role of calbindin in memory established in previous studies on calbindin knock-out mice, our results suggest that reduction of calbindin expression may contribute to memory impairments of patients with TLE, particularly, when neuronal loss is not significant. PMID:22796338

  18. Excitatory Actions of Noradrenaline and Metabotropic Glutamate Receptor Activation in Granule Cells of the Accessory Olfactory Bulb

    PubMed Central

    Smith, Richard S.; Weitz, Christopher J.; Araneda, Ricardo C.

    2009-01-01

    Modulation of dendrodendritic synapses by the noradrenergic system in the accessory olfactory bulb (AOB) plays a key role in the formation of memory in olfactory-mediated behaviors. We have recently shown that noradrenaline (NA) inhibits mitral cells by increasing ?-aminobutyric acid inhibitory input onto mitral cells in the AOB, suggesting an excitatory action of NA on granule cells (GCs). Here, we show that NA (10 ?M) elicits a long-lasting depolarization of GCs. This effect is mediated by activation of ?1-adrenergic receptors as the depolarization is mimicked by phenylephrine (PE, 30 ?M) and completely blocked by the ?1-adrenergic receptor antagonist prazosin (300 nM). In addition to this depolarization, application of NA induced the appearance of a slow afterdepolarization (sADP) following a stimulus-elicited train of action potentials. Similarly, the group I metabotropic glutamate receptor (mGluR1) agonist DHPG (10–30 ?M) also produced a depolarization of GCs and the appearance of a stimulus-induced sADP. The ionic and voltage dependence and sensitivity to blockers of the sADP suggest that it is mediated by the nonselective cationic conductance ICAN. Thus the excitatory action resulting from the activation of these receptors could be mediated by a common transduction target. Surprisingly, the excitatory effect of PE on GCs was completely blocked by the mGluR1 antagonist LY367385 (100 ?M). Conversely, the effect of DHPG was not antagonized by the ?1-adrenergic receptor antagonist prazosin (300 nM). These results suggest that most of the noradrenergic effect on GCs in the AOB is mediated by potentiation of a basal activity of mGluR1s. PMID:19474170

  19. ONTOGENY OF PROTEINS ASSOCIATED WITH NEURITE GROWTH AND SYNAPTOGENESIS IN CEREBELLAR GRANULE CELLS IN VITRO.

    EPA Science Inventory

    In vitro techniques may be useful in screening for effects of developmental neurotoxicants. Previously, we characterized changes in biochemical markers associated with neuronal development in a PC12 cell model of differentiation and growth. The current research extended these stu...

  20. Bcl-x L protects cerebellar granule neurons against the late phase, but not against the early phase of glutamate-induced cell death

    Microsoft Academic Search

    Gunnar P. H. Dietz; Birgit Dietz; Mathias Bähr

    2007-01-01

    Neuronal death can take on many different forms, from well-defined apoptosis to caspase-independent processes. While members of the Bcl-2 family of intracellular proteins are known to be involved in classic apoptotic cascades, their role in necrosis has been less well defined. Here, we applied a cell-permeable form of the anti-apoptotic Bcl-2 family member Bcl-xL on glutamate-treated rat primary cerebellar granule

  1. Differential interaction of splicing snRNPs with coiled bodies and interchromatin granules during mitosis and assembly of daughter cell nuclei

    Microsoft Academic Search

    Joao A. Ferreira; Maria Carmo-Fonseca; Angus I. Lamond

    1994-01-01

    In the interphase nucleus of mammalian cells the U1, U2, U4\\/U6, and U5 small nuclear ribo- nucleoproteins (snRNPs), which are subunits of spliceosomes, associate with specific subnuclear do- mains including interchromatin granules and coiled bodies. Here, we analyze the association of splicing snRNPs with these structures during mitosis and re- assembly of daughter nuclei. At the onset of mitosis snRNPs

  2. Brain-derived neurotrophic factor induces rapid morphological changes in dendritic spines of olfactory bulb granule cells in cultured slices through the modulation of glutamatergic signaling

    Microsoft Academic Search

    S Matsutani; N Yamamoto

    2004-01-01

    While the acute physiological effects of brain-derived neurotrophic factor (BDNF) have been well demonstrated, little is known regarding possible morphological effects that occur within a short period of time. The acute effects of BDNF on dendritic spine morphology were examined in granule cells in cultured main olfactory bulb slices. Organotypic slices prepared from 7-day-old rats were cultured for 1 day,

  3. Perturbations in Maturation of Secretory Proteins and their Association with Endoplasmic Reticulum Chaperones in a Cell Culture Model for Epithelial Ischemia

    Microsoft Academic Search

    Galina Kuznetsov; Kevin T. Bush; Ping L. Zhang; Sanjay K. Nigam

    1996-01-01

    The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on

  4. Osteopontin is a myosphere-derived secretory molecule that promotes angiogenic progenitor cell proliferation through the phosphoinositide 3-kinase/Akt pathway

    SciTech Connect

    Ogata, Takehiro [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Ueyama, Tomomi [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan)]. E-mail: tueyama@kuhp.kyoto-u.ac.jp; Nomura, Tetsuya [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Asada, Satoshi [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Tagawa, Masashi [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Nakamura, Tomoyuki [Department of Pharmacology, Kansai Medical University, Moriguchi, Osaka 570-8507 (Japan); Takahashi, Tomosaburo [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Matsubara, Hiroaki [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Oh, Hidemasa [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan)]. E-mail: hidemasa@kuhp.kyoto-u.ac.jp

    2007-07-27

    We have reported that skeletal myosphere-derived progenitor cells (MDPCs) can differentiate into vascular cells, and that MDPC transplantation into cardiomyopathic hearts improves cardiac function. However, the autocrine/paracrine molecules and underlying mechanisms responsible for MDPC growth have not yet been determined. To explore the molecules enhancing the proliferation of MDPCs, we performed serial analysis of gene expression and signal sequence trap methods using RNA isolated from MDPCs. We identified osteopontin (OPN), a secretory molecule, as one of most abundant molecules expressed in MDPCs. OPN provided a proliferative effect for MDPCs. MDPCs treated with OPN showed Akt activation, and inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway repressed the proliferative effect of OPN. Furthermore, OPN-pretreated MDPCs maintained their differentiation potential into endothelial and vascular smooth muscle cells. These findings indicate an important role of OPN as an autocrine/paracrine molecule in regulating the proliferative growth of muscle-derived angiogenic progenitor cells via the PI3K/Akt pathway.

  5. Evolution of apicomplexan secretory organelles

    PubMed Central

    Gubbels, Marc-Jan; Duraisingh, Manoj T.

    2013-01-01

    The alveolate superphylum includes many free-living and parasitic organisms, which are united by the presence of alveolar sacs lying proximal to the plasma membrane, providing cell structure. All species comprising the apicomplexan group of alveolates are parasites and have adapted to the unique requirements of the parasitic lifestyle. Here the evolution of apicomplexan secretory organelles that are involved in the critical process of egress from one cell and invasion of another is explored. The variations within the Apicomplexa and how these relate to species-specific biology will be discussed. In addition, recent studies have identified specific calcium-sensitive molecules that coordinate the various events and regulate the release of these secretory organelles within apicomplexan parasites. Some aspects of this machinery are conserved outside the Apicomplexa, and are beginning to elucidate the conserved nature of the machinery. Briefly, the relationship of this secretion machinery within the Apicomplexa will be discussed, compared with free-living and predatory alveolates, and how these might have evolved from a common ancestor. PMID:23068912

  6. Stimulation of Fibroblast Cell Growth, Matrix Production, and Granulation Tissue Formation by Connective Tissue Growth Factor

    Microsoft Academic Search

    Ken Frazier; Shawn Williams; Devashish Kothapalli; Helene Klapper; Gary R. Grotendorst

    1996-01-01

    Connective tissue growth factor (CTGF) is a 36- to 38-kDa peptide that is selectively induced by transforming growth factor-? (TGF-?) in fibroblastic cell types. We compared the biologic activities of CTGF with TGF-? on fibroblasts in culture and in animal models of fibroplasia. CTGF was active as a mitogen in monolayer cultures of normal rat kidney fibroblasts. CTGF did not

  7. Mosaic organization of the hippocampal neuroepithelium and the multiple germinal sources of dentate granule cells

    Microsoft Academic Search

    Joseph Altman; Shirley A. Bayer

    1990-01-01

    This study deals with the site of origin, migration, and settling of the principal cell constituents of the rat hippocampus during the embryonic period. The results indicate that the hippocampal neuroepithelium consists of three morphogenetically discrete components--the Ammonic neuroepithelium, the primary dentate neuroepithelium, and the fimbrial glioepithelium--and that these are discrete sources of the large neurons of Ammon's horn, the

  8. COMPARATIVE EFFECTS OF TWO POLYCHLORINATED BIPHENYL CONGENERS ON CALCIUM HOMEOSTASIS IN RAT CEREBELLAR GRANULE CELLS

    EPA Science Inventory

    Some polychlorinated biphenyls (PCBs) have been reported to alter locomotor activity and decrease brain dopamine function in laboratory animals. CBs with orth- and/or para-chlorine substitutions are reportedly most potent in decreasing cell dopamine content in vitro and were dete...

  9. Linkage of azurophil granule secretion in neutrophils to chloride ion transport and endosomal transcytosis.

    PubMed Central

    Fittschen, C; Henson, P M

    1994-01-01

    Neutrophils contain at least two types of secretory granules. The present work links the secretion of the (lysosomal type) azurophil granules, but not that of specific granules, to endosomal transport mechanisms. (a) Selective stimulation of azurophil granule secretion by the Na-ionophore Monensin, or nonselective stimulation by FMLP after cytochalasin B pretreatment elicited marked pinocytic activity in parallel with azurophil granule release, whereas FMLP alone, selective for specific granules, elicited little fluid pinocytosis. (b) Pinosomes thus formed fused with azurophil granules, suggesting that exocytosis of azurophil granules might occur via endosomal organelles. This hypothesis was tested by determining the effect on the endosomal pathway(s) of two treatments that selectively prevent the release of azurophil granule contents without interfering with specific granule secretion, namely replacement of Cl- with gluconate- or the addition of zinc. Replacement of Cl- was found to impair the pinocytosis process itself, whereas ZnSO4 appeared to prevent the fusion between endosomes and azurophil granules. These data support the concept that the (lysosomal type) azurophil granules, but not the specific granules, are secreted through the endosomal pathway. Images PMID:8282794

  10. Melatonin protects rat cerebellar granule cells against electromagnetic field-induced increases in Na(+) currents through intracellular Ca(2+) release.

    PubMed

    Liu, Dong-Dong; Ren, Zhen; Yang, Guang; Zhao, Qian-Ru; Mei, Yan-Ai

    2014-06-01

    Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low-frequency electromagnetic field (ELF-EMF)-induced Nav activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF-EMF for 60 min. significantly increased the Nav current (INa ) densities by 62.5%. MT (5 ?M) inhibited the ELF-EMF-induced INa increase. This inhibitory effect of MT is mimicked by an MT2 receptor agonist and was eliminated by an MT2 receptor antagonist. The Nav channel steady-state activation curve was significantly shifted towards hyperpolarization by ELF-EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF-EMF. ELF-EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK-7 did not reduce the ELF-EMF-induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF-EMF-induced Nav activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca(2+) level, but it significantly elevated the intracellular Ca(2+) level evoked by the high K(+) stimulation in cerebellar GC that were either exposed or not exposed to ELF-EMF. In the presence of ruthenium red, a ryanodine-sensitive receptor blocker, the MT-induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal INa that result from ELF-EMF exposure through Ca(2+) influx-induced Ca(2+) release. PMID:24548607

  11. The effect of paxilline on early alterations of electrophysiological properties of dentate gyrus granule cells in pilocarpine-treated rats.

    PubMed

    Mehranfard, Nasrin; Gholamipour-Badie, Hamid; Motamedi, Fereshteh; Janahmadi, Mahyar; Naderi, Nima

    2014-01-01

    The dentate gyrus of hippocampus has long been considered as a focal point for studies on mechanisms responsible for the development of temporal lobe epilepsy (TLE). Change in intrinsic properties of dentate gyrus granule cells (GCs) has been considered as an important factor responsible in temporal lobe seizures. In this study, we evaluated the intrinsic properties of GCs, during acute phase of seizure (24 h after i.p. injection of pilocarpine) compared to sham group using whole cell patch-clamp recordings. Our results showed a significant increase in the number of action potentials (APs) after applying depolarizing currents of 200 pA (p < 0.01) and 250pA (p < 0.05) compared to sham group. The evaluation of AP properties revealed a decrease in half-width of AP in GCs of seizure group (1.27 ± 0.03 ms) compared to sham group (1.60 ± 0.11). Moreover, addition of BAPTA to pipette solution prevented changes in AP half-width in seizure group (1.71 ± 0.11 ms) compared to sham group (1.91 ± 0.08 ms). In contrast, an increase in the amplitude of fast afterhyperpolarization was observed in GCs of seizure group (-11.68 ± 0.72 mV) compared to sham group (-8.28 ± 0.59 mV). Also, GCs of seizure group showed a significant increase in both firing rate and instantaneous firing frequency at depolarizing currents of 200 pA (P < 0.01) and 250 pA (P < 0.05) compared to sham group. The changes in electrophysiological properties of GCs were attenuated after bath application of paxilline suggesting possible involvement of large conductance Ca(2+)- activated K(+) channel (BK channel). Our results suggested the possible involvement of certain potassium channels in early changes of intrinsic properties of GCs which eventually facilitate TLE development. PMID:24711838

  12. The Effect of Paxilline on Early Alterations of Electrophysiological Properties of Dentate Gyrus Granule Cells in Pilocarpine-Treated Rats

    PubMed Central

    Mehranfard, Nasrin; Gholamipour-Badie, Hamid; Motamedi, Fereshteh; Janahmadi, Mahyar; Naderi, Nima

    2014-01-01

    The dentate gyrus of hippocampus has long been considered as a focal point for studies on mechanisms responsible for the development of temporal lobe epilepsy (TLE). Change in intrinsic properties of dentate gyrus granule cells (GCs) has been considered as an important factor responsible in temporal lobe seizures. In this study, we evaluated the intrinsic properties of GCs, during acute phase of seizure (24 h after i.p. injection of pilocarpine) compared to sham group using whole cell patch-clamp recordings. Our results showed a significant increase in the number of action potentials (APs) after applying depolarizing currents of 200 pA (p < 0.01) and 250pA (p < 0.05) compared to sham group. The evaluation of AP properties revealed a decrease in half-width of AP in GCs of seizure group (1.27 ± 0.03 ms) compared to sham group (1.60 ± 0.11). Moreover, addition of BAPTA to pipette solution prevented changes in AP half-width in seizure group (1.71 ± 0.11 ms) compared to sham group (1.91 ± 0.08 ms). In contrast, an increase in the amplitude of fast afterhyperpolarization was observed in GCs of seizure group (-11.68 ± 0.72 mV) compared to sham group (-8.28 ± 0.59 mV). Also, GCs of seizure group showed a significant increase in both firing rate and instantaneous firing frequency at depolarizing currents of 200 pA (P < 0.01) and 250 pA (P < 0.05) compared to sham group. The changes in electrophysiological properties of GCs were attenuated after bath application of paxilline suggesting possible involvement of large conductance Ca2+- activated K+ channel (BK channel). Our results suggested the possible involvement of certain potassium channels in early changes of intrinsic properties of GCs which eventually facilitate TLE development. PMID:24711838

  13. Mefenamic acid bi-directionally modulates the transient outward K{sup +} current in rat cerebellar granule cells

    SciTech Connect

    Zhang Man; Shi Wenjie; Fei Xiaowei; Liu Yarong; Zeng Ximin [Institute of Brain Science, School of Life Sciences and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200433 (China); Mei Yanai [Institute of Brain Science, School of Life Sciences and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200433 (China)], E-mail: yamei@fudan.edu.cn

    2008-02-01

    The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on ion channels has been widely studied in several cell models, but less is known about their modulatory mechanisms. In this report, the effect of mefenamic acid on voltage-activated transient outward K{sup +} current (I{sub A}) in cultured rat cerebellar granule cells was investigated. At a concentration of 5 {mu}M to 100 {mu}M, mefenamic acid reversibly inhibited I{sub A} in a dose-dependent manner. However, mefenamic acid at a concentration of 1 {mu}M significantly increased the amplitude of I{sub A} to 113 {+-} 1.5% of the control. At more than 10 {mu}M, mefenamic acid inhibited the amplitude of I{sub A} without any effect on activation or inactivation. In addition, a higher concentration of mefenamic acid induced a significant acceleration of recovery from inactivation with an increase of the peak amplitude elicited by the second test pulse. Intracellular application of mefenamic acid could significantly increase the amplitude of I{sub A}, but had no effect on the inhibition induced by extracellular mefenamic acid, implying that mefenamic acid may exert its effect from both inside and outside the ion channel. Furthermore, the activation of current induced by intracellular application of mefenamic acid was mimicked by other cyclooxygenase inhibitors and arachidonic acid. Our data demonstrate that mefenamic acid is able to bi-directionally modulate I{sub A} channels in neurons at different concentrations and by different methods of application, and two different mechanisms may be involved.

  14. Glucose-6-phosphate tips the balance in modulating apoptosis in cerebellar granule cells.

    PubMed

    Bobba, A; Amadoro, G; La Piana, G; Petragallo, V A; Calissano, P; Atlante, A

    2015-02-27

    A metabolic shift from oxidative phosphorylation to glycolysis (i.e. the Warburg effect) occurs in Alzheimer's disease accompanied by an increase of both activity and level of HK-I. The findings reported here demonstrate that in the early phase of apoptosis VDAC1 activity, but not its protein level, progressively decreases, in concomitance with the physical interaction of HK-I with VDAC1. In the late phase of apoptosis, glucose-6-phosphate accumulation in the cell causes the dissociation of the two proteins, the re-opening of the channel and the recovery of VDAC1 function, resulting in a reawakening of the mitochondrial function, thus inevitably leading to cell death. PMID:25647035

  15. Mast cell chymase is present in uterine cervical carcinoma and it detaches viable and growing cervical squamous carcinoma cells from substratum in vitro

    Microsoft Academic Search

    Nicolae-Costin Diaconu; Jaana Rummukainen; Anita Naukkarinen; Mikko Mättö; Rauno J. Harvima; Jukka Pelkonen; Ilkka T. Harvima

    Increased numbers of mast cells is a typical feature of a variety of human cancers. The major mediators in the secretory granules\\u000a of the MCTC type of mast cells, serine proteinases tryptase and chymase, may be involved in squamous cell carcinoma (SCC) lesions by\\u000a inducing matrix remodeling and epithelial cell detachment. The objective of this study was to analyze immunohistochemically

  16. HEp-2 Cell-Adherent Escherichia coli and Intestinal Secretory Immune Response to Human Immunodeficiency Virus (HIV) in Outpatients with HIV-Associated Diarrhea

    PubMed Central

    Mathewson, John J.; Salameh, Bassam M.; DuPont, Herbert L.; Jiang, Zhi D.; Nelson, Andrew C.; Arduino, Roberto; Smith, Melinda A.; Masozera, Nicholas

    1998-01-01

    HEp-2 cell-adherent Escherichia coli and the human immunodeficiency virus (HIV) itself have recently been incriminated as causes of chronic HIV-associated diarrhea. This study sought to determine the prevalence of these two agents among HIV-infected patients with diarrhea in an outpatient setting in the United States and to compare their prevalence to that of other commonly recognized enteropathogens known to be present in this population. HEp-2 cell-adherent E. coli was found in 20 of 83 (24.1%) patients with diarrhea. A diffuse pattern of adherence was the most common, found in 14 of 20 (70%) patients, followed by a localized adherence pattern (6 of 20; 30%). An intestinal secretory immune response against the p24 antigen of HIV was found in 9 of 34 (27.5%) patients with HIV-associated diarrhea. The following pathogens or products were also detected in lower frequencies: Cryptosporidium spp. (10.8%), Clostridium difficile toxin (8.8%), microsporidia (6%), Isospora belli (3.6%), Blastocystis hominis (2.4%), Giardia spp. (1.2%), Salmonella spp. (1.2%), and Mycobacterium spp. (1.2%). The role of HEp-2 cell-adherent E. coli and HIV enteric infections in patients with HIV-associated diarrhea deserves further study. PMID:9455887

  17. Influence of 4-hydroxylated polychlorinated biphenyls on the secretory function of bovine ovarian cells: Role of the steroidogenic factor-1 receptor.

    PubMed

    Mlynarczuk, Jaroslaw; Kotwica, Jan

    2015-04-01

    The hydroxy-derivatives (OHPCBs) of polychlorinated biphenyls (PCBs) can accumulate in the tissues of the reproductive tract in animals and humans and may still have estrogen-like properties. Moreover, the "orphan" nuclear receptor Steroidogenic Factor-1 (SF-1) can be the target of PCBs. The aim of the present study was to determine the effect 4OH4CB and 4OH3CB on the secretion of estradiol (E2), progesterone (P4), and oxytocin (OT) from granulosa cells of follicles <1cm and >1cm in diameter and from luteal cells collected at four stages of the estrous cycle of cows. Furthermore, the possibility that 4OHPCBs have an effect on OT synthesis and secretion via the SF receptor was studied using receptor blocker (F0160). Used OHPCBs increased the secretion of P4 from the granulosa cells of follicles of both sizes and increased the secretion of OT from follicles with a diameter of >1cm. These increases were inhibited by an SF-1 receptor blocker. In luteal cells, 4OH3CB increased the secretion of P4 and OT from luteal cells at all phases of the estrous cycle, while 4OH4CB increased OT secretion during the first half of the estrous cycle. Concomitant with the increase in OT secretion from the cells, an increase in the expression of OT precursor mRNA (NP-I/OT) was observed. This effect was inhibited by SF-1 receptor blocker. These results indicate that 4OHPCBs impair the secretory function of ovarian steroidogenic cells by disrupting steroidogenesis and increasing OT secretion, and the receptor SF-1 appears to be essentially involved in these processes. PMID:25726440

  18. Beta-cell insulin secretory response to oral hypoglycemic agents is blunted in humans in vivo during moderate hypoglycemia.

    PubMed

    Aldhahi, Waleed; Armstrong, Jen; Bouche, Clara; Carr, Richard D; Moses, Alan; Goldfine, Allison B

    2004-09-01

    Oral hypoglycemic agents bind to the ATP-sensitive potassium channel and lower glucose levels effectively in individuals with diabetes. Although the principle mechanism of action can also promote hypoglycemia, clinically profound hypoglycemia is rare. Decreased stimulation of insulin secretion by these agents at mild hypoglycemia could provide protection from more profound hypoglycemia. Sulfonylureas and meglitinides bind to both shared and unique sites on the ATP-sensitive potassium receptor/channel complex but have different pharmacokinetic profiles. To evaluate the differential ability of both sulfonylureas and meglitinides to stimulate insulin release at modest hypoglycemia, we evaluated dextrose infusion rates necessary to maintain plasma glucose after oral administration of repaglinide (1 mg) or glipizide (5 mg) at euglycemia and again at modest hypoglycemia. Healthy subjects with no family history of diabetes underwent four clamp studies, two performed while maintaining isoglycemia (glucose levels at the fasted value) and two while maintaining modest hypoglycemia of 2.78 mmol/liter (50 mg/dl) induced by low-dose insulin infusion (3.6 pmol/kg.min). There was a marked decrease in the dextrose infusion rate with administration of either repaglinide or glipizide at hypoglycemia compared with drug administration at euglycemia (P secretory response to the agents during hypoglycemia and suggests that at modest hypoglycemia, low glucose or other metabolite(s) or altered counterregulatory hormone levels are sufficient to inhibit insulin release in response to potent insulin secretagogues. These findings may help to explain the relatively low incidence of severe hypoglycemia with clinical administration of these drugs. PMID:15356061

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

    PubMed Central

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

    2007-01-01

    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 Ptf1aCre/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. PMID:17360405

  20. The intracellular polyglucose storage granules of Spirochaeta aurantia

    Microsoft Academic Search

    Andrew M. Kropinski; William C. Ghiorse; E. Peter Greenberg

    1988-01-01

    Extracts of Spirochaeta aurantia contained granules approximately 36 nm in diameter. These granules were purified by isopycnic centrifugation on CsCl gradients and shown on the basis of chemical and spectroscopic evidence to be glycogen. Electron microscopic cytochemical methods revealed glycogen-like granules in S. aurantia cells.

  1. Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii.

    PubMed

    Rome, Michael E; Beck, Josh R; Turetzky, Jay M; Webster, Paul; Bradley, Peter J

    2008-11-01

    Toxoplasma gondii is an obligate intracellular parasite that resides in the cytoplasm of its host in a unique membrane-bound vacuole known as the parasitophorous vacuole (PV). The membrane surrounding the parasite is remodeled by the dense granules, secretory organelles that release an array of proteins into the vacuole and to the PV membrane (PVM). Only a small portion of the protein constituents of the dense granules have been identified, and little is known regarding their roles in infection or how they are trafficked within the infected host cell. In this report, we identify a novel secreted dense granule protein, GRA14, and show that it is targeted to membranous structures within the vacuole known as the intravacuolar network and to the vacuolar membrane surrounding the parasite. We disrupted GRA14 and exploited the knockout strain to show that GRA14 can be transferred between vacuoles in a coinfection experiment with wild-type parasites. We also show that GRA14 has an unexpected topology in the PVM with its C terminus facing the host cytoplasm and its N terminus facing the vacuolar lumen. These findings have important implications both for the trafficking of GRA proteins to their ultimate destinations and for expectations of functional domains of GRA proteins at the host-parasite interface. PMID:18765740

  2. Ameloblast transcriptome changes from secretory to maturation stages

    PubMed Central

    Simmer, James P.; Richardson, Amelia S.; Wang, Shih-Kai; Reid, Bryan M.; Bai, Yongsheng; Hu, Yuanyuan; Hu, Jan C.-C.

    2014-01-01

    The purpose of this study was to identify the major molecular components in the secretory and maturation stages of amelogenesis through transcriptome analyses. Ameloblasts (40 sections per age group) were laser micro-dissected from Day 5 (secretory stage) and Days 11–12 (maturation stage) first molars. PolyA+ RNA was isolated from the lysed cells, converted to cDNA, and amplified to generate a cDNA library. DNA sequences were obtained using next generation sequencing and analyzed to identify genes whose expression had increased or decreased at least 1.5-fold in maturation stage relative to secretory stage ameloblasts. Among the 9198 genes that surpassed the quality threshold, 373 showed higher expression in secretory stage, while 614 genes increased in maturation stage ameloblasts. The results were crosschecked against a previously published transcriptome generated from tissues overlying secretory and maturation stage mouse incisor enamel and 34 increasing and 26 decreasing expressers common to the two studies were identified. Expression of F2r, which encodes protease activated receptor 1 (PAR1) that showed 10-fold higher expression during the secretory stage in our transcriptome analysis, was characterized in mouse incisors by immunohistochemistry. PAR1 was detected in secretory, but not maturation stage ameloblasts. We conclude that transcriptome analyses are a good starting point for identifying genes/proteins that are critical for proper dental enamel formation and that PAR1 is specifically expressed by secretory stage ameloblasts. PMID:25158176

  3. Hookworm Excretory/Secretory Products Induce Interleukin-4 (IL-4)+ IL-10+ CD4+ T Cell Responses and Suppress Pathology in a Mouse Model of Colitis

    PubMed Central

    Ferreira, Ivana; Smyth, Danielle; Gaze, Soraya; Aziz, Ammar; Giacomin, Paul; Ruyssers, Nathalie; Artis, David; Laha, Thewarach; Navarro, Severine; McSorley, Henry J.

    2013-01-01

    Evidence from human studies and mouse models shows that infection with parasitic helminths has a suppressive effect on the pathogenesis of some inflammatory diseases. Recently, we and others have shown that some of the suppressive effects of hookworms reside in their excretory/secretory (ES) products. Here, we demonstrate that ES products of the hookworm Ancylostoma caninum (AcES) suppress intestinal pathology in a model of chemically induced colitis. This suppression was associated with potent induction of a type 2 cytokine response characterized by coexpression of interleukin-4 (IL-4) and IL-10 by CD4+ T cells, downregulation of proinflammatory cytokine expression in the draining lymph nodes and the colon, and recruitment of alternatively activated (M2) macrophages and eosinophils to the site of ES administration. Protease digestion and heat denaturation of AcES resulted in impaired induction of CD4+ IL-4+ IL-10+ cell responses and diminished ability to suppress colitis, indicating that protein component(s) are responsible for some of the immunosuppressive effects of AcES. Identification of the specific parasite-derived molecules responsible for reducing pathology during chemically induced colitis could lead to the development of novel therapeutics for the treatment of human inflammatory bowel disease. PMID:23545299

  4. Activation of ?1 and ?2 Noradrenergic Receptors Exert Opposing Effects on Excitability of Main Olfactory Bulb Granule Cells

    PubMed Central

    Nai, Qiang; Dong, Hong-Wei; Linster, Christiane; Ennis, Matthew

    2010-01-01

    The mammalian main olfactory bulb (MOB) receives a dense noradrenergic innervation from the pontine nucleus locus coeruleus that is important for neonatal odor preference learning and odor processing in mature animals. Modulation of GABAergic granule cells (GCs) is thought to play a key role in the net functional impact of norepinephrine (NE) release in the MOB, yet there are few direct studies of the influence of NE on these cells. In the present study we investigated noradrenergic modulation of GC excitability using electrophysiological approaches in rat MOB slices. A moderate concentration of NE (10 µM) and the ?1 receptor agonist phenylephrine (10 µM) depolarized and increased spontaneous or current injection-evoked spiking in GCs. By contrast, low NE concentrations (0.1–1.0 µM) or the ?2 receptor agonist clonidine (10 µM) hyperpolarized and decreased the discharge of GCs. The effects of NE (10 µM) were blocked by antagonism of ?1 and ?2 receptors. Inhibitory effects of low NE concentrations were blocked or converted to excitatory responses by ?2 receptor blockade, whereas excitatory effects of the moderate NE concentration were converted to inhibitory responses after ?1 receptor blockade. NE (10 µM) and phenylephrine elicited inward currents that reversed near the potassium equilibrium potential. The effects of NE and phenylephrine were associated with increased membrane input resistance. Clonidine elicited an outward current associated with decreased membrane input resistance that reversed near the potassium equilibrium potential. These results indicate that ?1 and ?2 receptor activation exert opposing effects on GC excitability. Low concentrations of NE acting via ?2 receptors suppress GC excitability, while higher concentrations of NE acting at ?1 receptors increase GC excitability. These findings are consistent with recent findings that ?1 and ?2 receptor activation increase and decrease, respectively, GABAergic inhibition of mitral cells. The differential affinities of ?1 and ?2 noradrenergic receptor subtypes may allow for differential modulation of GABA release and olfactory processing as a function of the level of NE release, which in turn, is regulated by behavioral state. PMID:20466037

  5. Modulation of calcium entry and glutamate release in cultured cerebellar granule cells by palytoxin.

    PubMed

    Vale, Carmen; Alfonso, Amparo; Suńol, Cristina; Vieytes, Mercedes R; Botana, Luis M

    2006-06-01

    A channel open on the membrane can be formed by palytoxin (PTX). Ten nanomolar PTX caused an irreversible increase in the cytosolic calcium concentration ([Ca(2+)](c)), which was abolished in the absence of external calcium. The increase was eliminated by saxitoxin (STX) and nifedipine (NIF). Calcium rise is secondary to the membrane depolarization. PTX effect on calcium was dependent on extracellular Na(+). Li(+) decreased the PTX-evoked rise in [Ca(2+)](c); replacement of Na(+) by N-methyl-D-glucamine (NMDG) abolished PTX-induced calcium increase. [Ca(2+)](c) increase by PTX was strongly reduced after inhibition of the reverse operation of the Na(+)/Ca(2+) exchanger, in the presence of antagonists of excitatory amino acid (EAA) receptors, and by inhibition of neurotransmitter release. PTX did not modify calcium extrusion by the plasma membrane Ca(2+)-ATPase (PMCA), because blockade of the calcium pump increased rather than decreased the PTX-induced calcium influx. Extracellular levels of glutamate and aspartate were measured by HPLC and exocytotic neurotransmitter release by determination of synaptic vesicle exocytosis using total internal reflection fluorescence microscopy (TIRFM). PTX caused a concentration-dependent increase in EAA release to the culture medium. Ten nanomolar PTX decreased cell viability by 30% within 5 min. PTX-induced calcium influx involves three pathways: Na(+)-dependent activation of voltage-dependent sodium channels (VDSC) and voltage-dependent calcium channels (VDCC), reverse operation of the Na(+)/Ca(2+) exchanger, and indirect activation of EAA receptors through glutamate release. The neuronal injury produced by the toxin could be partially mediated by the PTX-induced overactivation of EAA receptors, VDSC, VDCC and the glutamate efflux into the extracellular space. PMID:16547972

  6. A marked paucity of granule cells in the developing cerebellum of the Npc1(-/-) mouse is corrected by a single injection of hydroxypropyl-?-cyclodextrin.

    PubMed

    Nusca, S; Canterini, S; Palladino, G; Bruno, F; Mangia, F; Erickson, R P; Fiorenza, M T

    2014-10-01

    In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1(-/-) mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%-25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent. Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8-PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1(-/-) mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1(-/-) mice. A single injection of hydroxypropyl-?-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size. To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum. PMID:24969023

  7. A marked paucity of granule cells in the developing cerebellum of the Npc1?/? mouse is corrected by a single injection of hydroxypropyl-?-cyclodextrin

    PubMed Central

    Nusca, S.; Canterini, S.; Palladino, G.; Bruno, F.; Mangia, F.; Erickson, R.P.; Fiorenza, M.T.

    2014-01-01

    In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1?/? mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%–25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent. Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8–PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1?/? mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1?/? mice. A single injection of hydroxypropyl-?-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size. To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum. PMID:24969023

  8. Measurement of the intracellular ph in human stomach cells: a novel approach to evaluate the gastric acid secretory potential of coffee beverages.

    PubMed

    Weiss, Carola; Rubach, Malte; Lang, Roman; Seebach, Elisabeth; Blumberg, Simone; Frank, Oliver; Hofmann, Thomas; Somoza, Veronika

    2010-02-10

    As the consumption of coffee beverages sometimes is reported to cause gastric irritation, for which an increased stomach acid secretion is one of the promoting factors, different processing technologies such as steam-treatment have been developed to reduce putative stomach irritating compounds. There is evidence-based data neither on the effect of detailed processing variations nor on individual coffee components affecting the proton secretory activity (PSA). This work aimed at developing a screening model suitable for investigating the effects of commercial coffee beverages and components thereof on human parietal cells. Human gastric cancer cells (HGT-1) were treated with reconstituted freeze-dried coffee beverages prepared from customary coffee products such as regular coffee (RC, n = 4), mild bean coffee (MBC, n = 5), stomach friendly coffee (SFC, n = 4), and SFC decaffeinated (SFCD, n = 3). PSA was analyzed by flow cytometry using the pH-sensitive dye SNARF-AM. Treatment of the cells with MBC did not result in a PSA different from RC treatment (p cells treated with SFC (p

  9. Large-scale identification of endogenous secretory peptides using electron transfer dissociation mass spectrometry.

    PubMed

    Sasaki, Kazuki; Osaki, Tsukasa; Minamino, Naoto

    2013-03-01

    Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was a