Sample records for cell secretory granules

  1. Characterization of mast cell secretory granules and their cell biology.

    PubMed

    Azouz, Nurit Pereg; Hammel, Ilan; Sagi-Eisenberg, Ronit

    2014-10-01

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

  2. Menin immunoreactivity in secretory granules of human pancreatic islet cells.

    PubMed

    Debelenko, Larisa V; Agarwal, Sunita; Du, Qiang; Yan, Wusheng; Erickson, Heidi S; Abu-Asab, Mones; Raffeld, Mark A; Libutti, Steven K; Marx, Stephen J; Emmert-Buck, Michael R

    2014-01-01

    The protein product of the Multiple Endocrine Neoplasia Type I (MEN1) gene is thought to be involved in predominantly nuclear functions; however, immunohistochemical (IHC) analysis data on cellular localization are conflicting. To further investigate menin expression, we analyzed human pancreas (an MEN1 target organ) using IHC analyses and 6 antibodies raised against full-length menin or its peptides. In 10 normal pancreas specimens, 2 independently raised antibodies showed unexpected cytoplasmic immunoreactivity in peripheral cells in each islet examined (over 100 total across all 10 patients). The staining exhibited a distinct punctate pattern and subsequent immunoelectron microscopy indicated the target antigen was in secretory granules. Exocrine pancreas and pancreatic stroma were not immunoreactive. In MEN1 patients, unaffected islets stained similar to those in normal samples but with a more peripheral location of positive cells, whereas hyperplastic islets and tumorlets showed increased and diffuse cytoplasmic staining, respectively. Endocrine tumors from MEN1 patients were negative for menin, consistent with a 2-hit loss of a tumor suppressor gene. Secretory granule localization of menin in a subset of islet cells suggests a function of the protein unique to a target organ of familial endocrine neoplasia, although the IHC data must be interpreted with some caution because of the possibility of antibody cross-reaction. The identity, cellular trafficking, and role of this putative secretory granule-form of menin warrant additional investigation. PMID:25153502

  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. Rab3D Is Critical for Secretory Granule Maturation in PC12 Cells

    PubMed Central

    Kögel, Tanja; Rudolf, Rüdiger; Hodneland, Erlend; Copier, John; Regazzi, Romano; Tooze, Sharon A.; Gerdes, Hans-Hermann

    2013-01-01

    Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs. PMID:23526941

  5. The glycosylation pattern of secretory granules in binucleate trophoblast cells is highly conserved in ruminants.

    PubMed

    Klisch, K; Wooding, F B P; Jones, C J P

    2010-01-01

    The binucleate trophoblast cells (BNCs) in the ruminant placenta are a unique feature of this taxon. These cells produce several secretory proteins and transfer these across the fetomaternal barrier into the dam. We used lectin histochemistry with a panel of 24 lectins to characterise the glycosylation pattern of BNC secretory granules in a variety of ruminants. Seven species out of three ruminant families were thus investigated: greater malayan chevrotain (Tragulidae); fallow deer, red deer, chinese water deer (Cervidae); and domestic goat, springbok, impala (Bovidae). BNC granules in all species studied strongly expressed tri-/tetraantennary complex N-glycans and bisecting N-acetylglucosamine [GlcNAc] as shown by binding of leuco- and erythroagglutins of Phaseolus vulgaris respectively. The presence of terminal N-acetylgalactosamine [GalNAc]) in BNC granules is shown by intense staining with lectins from Dolichos biflorus, Vicia villosa and Wisteria floribunda. Terminal galactose or GalNAc was also present, bound by Glycine max agglutinin. Treatment of slides with neuraminidase strongly intensified staining of Erythrina cristagalli lectin (ECA) to terminal lactosamine in all species studied; this was otherwise absent except in goat. Sambucus nigra-1 lectin bound to BNC granules in all species except in Impala, indicating the presence of abundant alpha2,6 linked sialic acid. These results indicate that these unusual highly branched glycans, with bisecting GlcNAc and terminal GalNAc are a general feature of BNC granules in Ruminants, including the most basal Tragulid branch. It therefore appears that the specific glycosylation pattern of BNC granules evolved early in ruminant phylogenesis, together with the appearance of BNC. The conserved glycan structure in BNC secretory granules indicates that this pattern of glycosylation is likely to be of considerable functional importance for the secretory glycoproteins of ruminant BNC. PMID:19959226

  6. Protein Mobility within Secretory Granules

    PubMed Central

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

    2014-01-01

    We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (?64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10?10 cm2/s (?1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit. PMID:24988337

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

    PubMed Central

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

    2010-01-01

    Abstract Morphometric evidence derived from studies of mast cells, pancreatic acinar cells and other cell types supports a model in which the post-Golgi processes that generate mature secretory granules can be resolved into three steps: (1) fusion of small, Golgi-derived progranules to produce immature secretory granules which have a highly constrained volume; (2) transformation of such immature granules into mature secretory granules, a process often associated with a reduction in the maturing granule’s volume, as well as changes in the appearance of its content and (3) fusion of secretory granules of the smallest size, termed ‘unit granules’, forming granules whose volumes are multiples of the unit granule’s volume. Mutations which perturb this process can cause significant pathology. For example, Chediak–Higashi syndrome / lysosomal trafficking regulator (CHS)/(Lyst) mutations result in giant secretory granules in a number of cell types in human beings with the Chediak–Higashi syndrome and in ‘beige’ (Lystbg/Lystbg) mice. Analysis of the secretory granules of mast cells and pancreatic acinar cells in Lyst-deficient beige mice suggests that beige mouse secretory granules retain the ability to fuse randomly with other secretory granules no matter what the size of the fusion partners. By contrast, in normal mice, the pattern of granule–granule fusion occurs exclusively by the addition of unit granules, either to each other or to larger granules. The normal pattern of fusion is termed unit addition and the fusion evident in cells with CHS/Lyst mutations is called random addition. The proposed model of secretory granule formation has several implications. For example, in neurosecretory cells, the secretion of small amounts of cargo in granules constrained to a very narrow size increases the precision of the information conveyed by secretion. By contrast, in pancreatic acinar cells and mast cells, large granules composed of multiple unit granules 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

  8. 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.

  9. 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

  10. Localization of DNA and RNA in Eosinophil Secretory Granules

    Microsoft Academic Search

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

    2010-01-01

    Background: Although the accepted paradigm is that the proteins stored in eosinophil crystalloid granules are translated from messenger RNA transcribed in the cell nucleus, recent ultrastructural evidence suggests that protein synthesis may also take place within eosinophilic granules. Methods: We used 2 different methods to detect the presence of DNA and RNA in eosinophil secretory granules. Using bromodeoxyuridine, a thymidine

  11. Zymophagy: Selective Autophagy of Secretory Granules

    PubMed Central

    Vaccaro, Maria I.

    2012-01-01

    Timing is everything. That's especially true when it comes to the activation of enzymes created by the pancreas to break down food. Pancreatic enzymes are packed in secretory granules as precursor molecules called zymogens. In physiological conditions, those zymogens are activated only when they reach the gut, where they get to work releasing and distributing nutrients that we need to survive. If this process fails and the enzymes are prematurely activated within the pancreatic cell, before they are released from the gland, they break down the pancreas itself causing acute pancreatitis. This is a painful disease that ranges from a mild and autolimited process to a severe and lethal condition. Recently, we demonstrated that the pancreatic acinar cell is able to switch on a refined mechanism that could explain the autolimited form of the disease. This is a novel selective form of autophagy named zymophagy, a cellular process to specifically detect and degrade secretory granules containing activated enzymes before they can digest the organ. In this work, we revise the molecules and mechanisms that mediate zymophagy, a selective autophagy of secretory granules. PMID:22550490

  12. Localization of inositol trisphosphate receptor subtype 3 to insulin and somatostatin secretory granules and regulation of expression in islets and insulinoma cells.

    PubMed Central

    Blondel, O; Moody, M M; Depaoli, A M; Sharp, A H; Ross, C A; Swift, H; Bell, G I

    1994-01-01

    Calcium ions play a central role in stimulus-secretion coupling in pancreatic beta cells, and an elevation of cytosolic Ca2+ levels is necessary for insulin secretion. Inositol 1,4,5-trisphosphate mobilizes intracellular Ca2+ stores in the beta cell by binding to specific receptors that are ligand-activated Ca2+ channels. The inositol trisphosphate receptors comprise a family of structurally related proteins with distinct but overlapping tissue distributions. Previous studies indicated that the predominant inositol trisphosphate receptor subtype expressed in rat pancreatic islets was the protein designated IP3R-3. We have confirmed the expression of IP3R-3 in pancreatic islets by immunohistocytochemistry and localized this protein to the secretory granules of insulin-secreting beta cells and somatostatin-secreting delta cells by immunogold electron microscopy. Secretory granules contain high levels of Ca2+, and the presence of IP3R-3 in the granule provides a mechanism for mobilizing granule Ca2+ stores in response to glucose and/or hormones. The release of Ca2+ from granule stores would increase the Ca2+ concentration in the surrounding cytoplasm and promote rapid exocytosis of granules, especially those granules in close proximity to the plasma membrane. The levels of IP3R-3 were increased in pancreatic islets of diabetic rats and rats that had been refed after a period of fasting. They were also increased in rat insulinoma RINm5F cells cultured in 25 mM glucose compared with cells cultured in 5 mM glucose. The localization of IP3R-3 to secretory granules of insulin-secreting beta cells and somatostatin-secreting delta cells suggests that granule Ca2+ stores actively participate in the secretory process and that their release is regulated by inositol 1,4,5-trisphosphate. The regulation of IP3R-3 levels by glucose, diabetes, and refeeding may allow the beta cell to adjust the insulin secretory response to changing physiological conditions. Images PMID:7914371

  13. Secretory lysosomes

    Microsoft Academic Search

    Emma J. Blott; Gillian M. Griffiths

    2002-01-01

    Regulated secretion of stored secretory products is important in many cell types. In contrast to professional secretory cells, which store their secretory products in specialized secretory granules, some secretory cells store their secretory proteins in a dual-function organelle, called a secretory lysosome. Functionally, secretory lysosomes are unusual in that they serve both as a degradative and as a secretory compartment.

  14. The AP1 adaptor complex binds to immature secretory granules from PC12 cells, and is regulated by ADP-ribosylation factor

    Microsoft Academic Search

    A. S. Dittie; Nasser Hajibagheri; Sharon A. Tooze

    1996-01-01

    Immature secretory granules (ISGs) in endo- crine and neuroendocrine cells have been shown by morphological techniques to be partially clathrin coated (Orci, L., M. Ravazzola, M. Amherdt, D. Lon- vard, A. Perrelet. 1985a. Proc. Natl. Acad. Sci. USA. 82: 5385-5389; Tooze, J., and S.A. Tooze. 1986. J. Cell Biol. 103:839-850). The function, and composition, of this clathrin coat has remained

  15. 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

  16. Developing Dictyostelium Cells Contain the Lysosomal Enzyme ct-Mannosidase in a Secretory Granule

    Microsoft Academic Search

    James M. Lenhard; Alan Siegel; Stephen J. Free

    1989-01-01

    The prespore vesicle (PSV) is an organelle which secretes spore coat proteins and gal\\/galNAc polysaccharides from prespore cells of Dictyostelium. By combining the techniques of protein A-gold immu- nocytochemistry and ricin-gold affinity cytochemistry we have demonstrated colocalization of the lysosomal enzyme t~-mannosidase with gal\\/galNAc polysaccha- rides in prespore vesicles and the spore coat. To deter- mine the origin of prespore

  17. FRAP Analysis of Secretory Granule Lipids and Proteins in the Sea Urchin Egg

    E-print Network

    Wessel, Gary M.

    5 FRAP Analysis of Secretory Granule Lipids and Proteins in the Sea Urchin Egg Julian L. Wong of the egg plasma membrane prior to insemination such that the vesicle­plasma membrane complex may after photobleaching; FRAP; secretory vesicles. 1 Introduction Eggs undergo major cell surface changes

  18. AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

    PubMed Central

    Burgess, Jason; Jauregui, Miluska; Tan, Julie; Rollins, Janet; Lallet, Sylvie; Leventis, Peter A.; Boulianne, Gabrielle L.; Chang, Henry C.; Le Borgne, Roland; Krämer, Helmut; Brill, Julie A.

    2011-01-01

    ?Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing “glue granules” that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1– and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules. PMID:21490149

  19. Automated Kymograph Analysis for Profiling Axonal Transport of Secretory Granules

    E-print Network

    Radke, Rich

    to significant levels of noise and clutter, and can be used to capture and quantify trends in transport patterns the impact of oxidative stress on granule transport in which the fully auto- mated analysis correctly is to regulate transport of secretory granules containing Brain-derived Neurotrophic Factor (BDNF), and that loss

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

    PubMed Central

    Rohrbach, A

    2000-01-01

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

  1. Differential dynamics of Rab3A and Rab27A on secretory granules

    PubMed Central

    Handley, Mark T W; Haynes, Lee P; Burgoyne, Robert D

    2007-01-01

    SUMMARY We have assessed the dynamics of the association of Rab3A and Rab27A with secretory granules at various stages of their life in PC12 cells. Endogenous Rab3A colocalised with the secretory granule marker secretogranin II (SGII) and expressed EGFP-Rab3A and ECFP-Rab27A colocalised with one another. The extent of colocalisation between EGFP-Rab3A or EGFP-Rab27 and SGII increased after longer times post-transfection suggesting that these Rab proteins are preferentially recruited to newly synthesised granules. Following the release of immature secretory granules from the trans-Golgi network, Rab3A and Rab27A became associated with the immature granules after a lag period of around 20 minutes. Rab dynamics on granules were analysed in fluorescence recovery after photobleaching (FRAP) experiments. The recovery profile of EGFP-Rab27A was comparable to that of ppANF-EGFP, while the recovery profile of EGFP-Rab3A was significantly faster, indicating that Rab3A but not Rab27A may be rapidly exchanged between granules and cytosol. Inhibition of heat-shock protein 90 with 10?M geldanamycin did not affect the exchange process or regulated exocytosis. Rab dynamics during stimulation with 300?M ATP were analysed in live cells. Loss of granular ppANF-EGFP fluorescence was seen at the cell periphery after stimulation but only limited changes in EGFP-Rab3A and EGFP-Rab27A fluorescence was observed, indicating that the Rab proteins do not immediately dissociate or disperse on stimulation. The data suggest potentially distinct roles for Rab3A and Rab27A and we suggest that the finding that young secretory granules have a higher capacity for binding Rab3A and Rab27A may be functionally important for preferential exocytosis from these granules. PMID:17311845

  2. Fibroblast growth factor receptor like-1 (FGFRL1) interacts with SHP-1 phosphatase at insulin secretory granules and induces beta-cell ERK1/2 protein activation.

    PubMed

    Silva, Pamuditha N; Altamentova, Svetlana M; Kilkenny, Dawn M; Rocheleau, Jonathan V

    2013-06-14

    FGFRL1 is a newly identified member of the fibroblast growth factor receptor (FGFR) family expressed in adult pancreas. Unlike canonical FGFRs that initiate signaling via tyrosine kinase domains, the short intracellular sequence of FGFRL1 consists of a putative Src homology domain-2 (SH2)-binding motif adjacent to a histidine-rich C terminus. As a consequence of nonexistent kinase domains, FGFRL1 has been postulated to act as a decoy receptor to inhibit canonical FGFR ligand-induced signaling. In pancreatic islet beta-cells, canonical FGFR1 signaling affects metabolism and insulin processing. This study determined beta-cell expression of FGFRL1 as well as consequent effects on FGFR1 signaling and biological responses. We confirmed FGFRL1 expression at the plasma membrane and within distinct intracellular granules of both primary beta-cells and ?TC3 cells. Fluorescent protein-tagged FGFRL1 (RL1) induced a significant ligand-independent increase in MAPK signaling. Removal of the histidine-rich domain (RL1-?His) or entire intracellular sequence (RL1-?C) resulted in greater retention at the plasma membrane and significantly reduced ligand-independent ERK1/2 responses. The SHP-1 phosphatase was identified as an RL1-binding substrate. Point mutation of the SH2-binding motif reduced the ability of FGFRL1 to bind SHP-1 and activate ERK1/2 but did not affect receptor localization to insulin secretory granules. Finally, overexpression of RL1 increased cellular insulin content and matrix adhesion. Overall, these data suggest that FGFRL1 does not function as a decoy receptor in beta-cells, but rather it enhances ERK1/2 signaling through association of SHP-1 with the receptor's intracellular SH2-binding motif. PMID:23640895

  3. Expression of Secretogranin III in Chicken Endocrine Cells: Its Relevance to the Secretory Granule Properties of Peptide Prohormone Processing and Bioactive Amine Content.

    PubMed

    Gomi, Hiroshi; Morikawa, Satomi; Shinmura, Naoki; Moki, Hiroaki; Yasui, Tadashi; Tsukise, Azuma; Torii, Seiji; Watanabe, Tsuyoshi; Maeda, Yoshinori; Hosaka, Masahiro

    2015-05-01

    The expression of secretogranin III (SgIII) in chicken endocrine cells has not been investigated. There is limited data available for the immunohistochemical localization of SgIII in the brain, pituitary, and pancreatic islets of humans and rodents. In the present study, we used immunoblotting to reveal the similarities between the expression patterns of SgIII in the common endocrine glands of chickens and rats. The protein-protein interactions between SgIII and chromogranin A (CgA) mediate the sorting of CgA/prohormone core aggregates to the secretory granule membrane. We examined these interactions using co-immunoprecipitation in chicken endocrine tissues. Using immunohistochemistry, we also examined the expression of SgIII in a wide range of chicken endocrine glands and gastrointestinal endocrine cells (GECs). SgIII was expressed in the pituitary, pineal, adrenal (medullary parts), parathyroid, and ultimobranchial glands, but not in the thyroid gland. It was also expressed in GECs of the stomach (proventriculus and gizzard), small and large intestines, and pancreatic islet cells. These SgIII-expressing cells co-expressed serotonin, somatostatin, gastric inhibitory polypeptide, glucagon-like peptide-1, glucagon, or insulin. These results suggest that SgIII is expressed in the endocrine cells that secrete peptide hormones, which mature via the intragranular enzymatic processing of prohormones and physiologically active amines in chickens. PMID:25673289

  4. Cell type-specific gene expression in the neuroendocrine system. A neuroendocrine-specific regulatory element in the promoter of chromogranin A, a ubiquitous secretory granule core protein.

    PubMed Central

    Wu, H; Rozansky, D J; Webster, N J; O'Connor, D T

    1994-01-01

    The acidic secretory protein chromogranin A universally occurs in amine and peptide hormone and neurotransmitter storage granules throughout the neuroendocrine system. What factors govern the activity of the chromogranin A gene, to yield such a widespread yet neuroendocrine-selective pattern of expression? To address this question, we isolated the mouse chromogranin A gene promoter. The promoter conferred cell type-specific expression in several neuroendocrine cell types (adrenal medullary chromaffin cells, anterior pituitary corticotropes, and anterior pituitary somatolactotropes) but not in control (fibroblast or kidney) cells. In neuroendocrine cells, analysis of promoter deletions established both positive and negative transcriptional regulatory domains. A distal positive domain (-4.8/-2.2 kbp) was discovered, as well as negative (-258/-181 bp) and positive (-147/-61 bp) domains in the proximate promoter. The proximate promoter contained a minimal neuroendocrine-specific element between -77 and -61 bp. Sequence alignment of the mouse promoter with corresponding regions in rat and bovine clones indicated that the mouse sequence shares over 85% homology with rat and 52% with bovine promoters. DNaseI footprinting and electrophoretic gel mobility shift assays demonstrated the presence of nuclear factors in neuroendocrine cells that recognized the proximate promoter. We conclude that the chromogranin A promoter contains both positive and negative domains governing its cell type-specific pattern of transcription, and that a small proximate region of the promoter, containing novel as well as previously described elements, interacts specifically with neuroendocrine nuclear proteins, and is thereby sufficient to ensure widespread neuroendocrine expression of the gene. Images PMID:8040254

  5. 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.

  6. Compound exocytosis of secretory granules containing salivary chromogranin A in granular duct cells in rat submandibular gland: the last study in collaboration with the late Professor Noboru Yanaihara at Yanaihara Institute.

    PubMed

    Kanno, Tomio

    2004-12-15

    Chromogranin A (CgA) is a member of a family of chromogranins, which are co-stored and co-released with adrenaline and noradrenalin (NAd) in the adrenal medulla in response to stimulation of the splanchnic nerve. Double immunohistochemical staining is carried out by use of antibodies against CgA and 1,4,5-trisphosphate receptor type 2 (IP3R2) on the same sections prepared from the isolated and perfused submandibulllar gland of rat. In the control sections prepared from resting state, an intense IP3R2 immunoreactivity (IR) appeared preferentially at the apical pole of subpopulation of the granulated duct cells, in which CgA-like IR distributed throughout the cytoplasm. Electron-micrograph showed that the granular cells in the resting state stored numerous membrane-bound granules in the apical cytoplasm. Stimulation with 1 microM NAd caused rapid immediate increase in secretory responses. Sections prepared from the gland at the peak of secretory responses exhibited that, in the granular duct cells, the apically converge IP3R2 IR became diffuse and indistinguishable, and that the apical half of the cells was occupied, indicating that mobilization of Ca2+ from the IP3-sensitive pool may preferentially be involved in the secretory responses to alpha-adrenergic agonist. PMID:15518886

  7. ?-Synuclein binds the KATP channel at insulin-secretory granules and inhibits insulin secretion

    PubMed Central

    Geng, Xuehui; Lou, Haiyan; Wang, Jian; Li, Lehong; Swanson, Alexandra L.; Sun, Ming; Beers-Stolz, Donna; Watkins, Simon; Perez, Ruth G.

    2011-01-01

    ?-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic ?-cells, ?-synuclein might therefore play a similar role by interacting with organelles involved in insulin secretion. We tested for ?-synuclein localizing to insulin-secretory granules and characterized its role in glucose-stimulated insulin secretion. Immunohistochemistry and fluorescent sulfonylureas were used to test for ?-synuclein localization to insulin granules in ?-cells, immunoprecipitation with Western blot analysis for interaction between ?-synuclein and KATP channels, and ELISA assays for the effect of altering ?-synuclein expression up or down on insulin secretion in INS1 cells or mouse islets, respectively. Differences in cellular phenotype between ?-synuclein knockout and wild-type ?-cells were found by using confocal microscopy to image the fluorescent insulin biosensor Ins-C-emGFP and by using transmission electron microscopy. The results show that anti-?-synuclein antibodies labeled secretory organelles within ?-cells. Anti-?-synuclein antibodies colocalized with KATP channel, anti-insulin, and anti-C-peptide antibodies. ?-Synuclein coimmunoprecipitated in complexes with KATP channels. Expression of ?-synuclein downregulated insulin secretion at 2.8 mM glucose with little effect following 16.7 mM glucose stimulation. ?-Synuclein knockout islets upregulated insulin secretion at 2.8 and 8.4 mM but not 16.7 mM glucose, consistent with the depleted insulin granule density at the ?-cell surface membranes observed in these islets. These findings demonstrate that ?-synuclein interacts with KATP channels and insulin-secretory granules and functionally acts as a brake on secretion that glucose stimulation can override. ?-Synuclein might play similar roles in diabetes as it does in other degenerative diseases, including Alzheimer's and Parkinson's diseases. PMID:20858756

  8. Exocrine granule specific packaging signals are present in the polypeptide moiety of the pancreatic granule membrane protein GP2 and in amylase: implications for protein targeting to secretory granules.

    PubMed

    Colomer, V; Lal, K; Hoops, T C; Rindler, M J

    1994-08-15

    The mechanisms for segregation of secretory and membrane proteins incorporated into storage granules from those transported constitutively have been thought to be conserved in diverse cell types, including exocrine and endocrine cells. However, GP2, the major protein of pancreatic zymogen granule membranes, in its native glycosyl phosphatidylinositol (GPI)-linked form, is incorporated into secretory granules when expressed in exocrine pancreatic AR42J cells, but not in the endocrine cells such as pituitary AtT20. To determine whether the protein moiety of GP2 contains the cell-type specific information for packaging into granules, a secretory form of GP2 (GP2-GPI-), with the GPI attachment site deleted, was generated and introduced into AR42J and AtT20 cells. Like native GP2, GP2-GPI- localized to the zymogen-like granules of AR42J cells and underwent regulated secretion. In AtT20 cells expressing GP2-GPI-, however, the protein was secreted by the constitutive pathway. Thus, a granule packaging signal is present in the luminal portion of GP2 that is functional only in the exocrine cells. However, this cell-type dependent sorting process is not limited to GP2 or membrane proteins. Amylase, a major content protein of pancreatic acinar and serous salivary gland granules, was also secreted exclusively by the constitutive pathway when expressed in AtT20 cells. The cell-type specific targeting of GP2 to granules correlated with its behavior in an in vitro aggregation assay where it co-aggregated more effectively with content proteins from pancreatic zymogen granules than with those from pituitary granules.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7520866

  9. Granule maturation in mast cells: histamine in control.

    PubMed

    Hallgren, Jenny; Gurish, Michael F

    2014-01-01

    Mast cells are derived from committed progenitors that originate in the BM. They mature into histochemically distinguishable, metachromatic mast cells containing numerous cytoplasmic secretory granules. Accumulating evidence demonstrates that mast cell granule maturation is very tightly regulated by many factors including different granule components such as proteoglycans. In this issue of the European Journal of Immunology, Nakazawa et al. [Eur. J. Immunol. 2014. 44: 204-214] highlight a role for mast cell derived histamine as another factor critical for mast cell maturation. Using histidine decarboxylase (HDC) deficient mice that are unable to make histamine, they show poorly formed secretory granules and decreased secretory granule protease expression in peritoneal mast cells. Co-culturing BM-derived mast cells with fibroblasts normally drives granule maturation, but HDC-deficient BM-derived mast cells fail to do so. Exogenously provided histamine partly restores granule differentiation as evidenced by increased tryptase and chymase activity, and this is histamine receptor type H4 -dependent. However, H4 -deficient mice have intact granule formation in peritoneal mast cells, suggesting that when HDC is functional, the intrinsic histamine production is sufficient for most granule maturation processes and H4 is dispensable. This study highlights the role of histamine in the regulation of mast cell maturation, although the cytosolic target remains unknown. PMID:24319003

  10. A quantitative ultrastructural study of microtubule content and secretory granule accumulation in parathyroid glands of phosphate- and colchicine-treated rats.

    PubMed Central

    Reaven, E P; Reaven, G M

    1975-01-01

    Microtubule involvement in secretory events of the parathyroid gland was investigated in rats treated with colchicine and/or phosphorus, agents which have been shown to modify parathyroid secretion. Quantitative ultrastructural techniques were used in an effort to assess the cytoplasmic microtubule and secretory granule content of chief cells 3 h after treatment, when hypocalcemia was well established. After cochicine administration, the chief cells appeared to have lost all assembled microtubules and accumulated greater than normal amounts of cytoplasmic secretory granules. On the other hand, phosphorus treatment was associated with increased microtubule content although the cytoplasmic content of secretory granules remained unchanged. When colchicine and phosphorus were given concomitantly, microtubules were again absent, but the secretory granule content of the cells was markedly increased. These data provide direct evidence that colchicine disrupts assembled microtubules in chief cells of rat parathyroids; the consequence of this effect appears to be a blockage of hormone release which is reflected in the accumulation of secretory granules in the cell. The fact that microtubules also show a significant increase in content when hormone release from chief cells is presumed to increase, suggests that microtubules may participate in the physiological control of parathyroid hormone secretion. Images PMID:1141440

  11. A functional cyclic AMP response element plays a crucial role in neuroendocrine cell type-specific expression of the secretory granule protein chromogranin A.

    PubMed Central

    Wu, H; Mahata, S K; Mahata, M; Webster, N J; Parmer, R J; O'Connor, D T

    1995-01-01

    Chromogranin A, a soluble acidic protein, is a ubiquitous component of secretory vesicles throughout the neuroendocrine system. We reported previously the cloning and initial characterization of the mouse chromogranin A gene promoter, which showed that the promoter contains both positive and negative domains and that a proximal promoter spanning nucleotides -147 to +42 bp relative to the transcriptional start site is sufficient for neuroendocrine cell type-specific expression. The current study was undertaken to identify the particular elements within this proximal promoter that control tissue-specific expression. We found that deletion or point mutations in the potential cAMP response element (CRE) site at -68 bp virtually abolished promoter activity specifically in neuroendocrine (PC12 chromaffin or AtT20 corticotrope) cells, with little effect on activity in control (NIH3T3 fibroblast) cells; thus, the CRE box is necessary for neuroendocrine cell type-specific activity of the chromogranin A promoter. Furthermore, the effect of the CRE site is enhanced in the context of intact (wild-type) promoter sequences between -147 and -100 bp. DNase I footprint analysis showed that these regions (including the CRE box) bind nuclear proteins present in both neuroendocrine (AtT20) and control (NIH3T3) cells. In AtT20 cells, electrophoretic mobility shift assays and factor-specific antibody supershifts showed that an oligonucleotide containing the chromogranin A CRE site formed a single, homogeneous protein-DNA complex containing the CRE-binding protein CREB. However, in control NIH3T3 cells we found evidence for an additional immunologically unrelated protein in this complex. A single copy of this oligonucleotide was able to confer neuroendocrine-specific expression to a heterologous (thymidine kinase) promoter, albeit with less fold selectivity than the full proximal chromogranin A promoter. Hence, the CRE site was partially sufficient to explain the neuroendocrine cell type specificity of the promoter. The functional activity of the CRE site was confirmed through studies of the endogenous chromogranin A gene. Northern mRNA analysis showed that expression of the endogenous chromogranin A gene was stimulated seven- to eightfold by cAMP in PC12 cells, whereas no induction occurred in the NIH3T3 cells. Similar cAMP induction was obtained with the transfected chromogranin A promoter in PC12 cells, and abolition of the CRE site (by deletion or point mutation) eliminated the induction. Thus, the CRE site in the chromogranin A proximal promoter is functional and plays a crucial, indeed indispensable, role in neuroendocrine-specific expression of the gene. These results also provide insight into transcriptional mechanisms governing acquisition of the neuroendocrine secretory phenotype. Images PMID:7615829

  12. 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

  13. Morphological and morphometric study of atrial specific granules and other secretory components in dogs experimentally infected with Trypanosoma cruzi.

    PubMed Central

    Caliari, M. V.; Lana, M.; Leite, V. H.; Tafuri, W. L.

    1995-01-01

    Changes in blood volume can induce morphometric and morphological alterations in the secretory complex of the myoendocrine cells due to the stretching of atrial walls. These alterations were studied by electron microscopy, using dogs infected intraperitonially with Trypanosoma cruzi and necropsied during the acute phase of the infection when congestive heart failure was present. Several changes were observed in the myoendocrine cells of the heart: hypertrophy and hyperplasia of rough endoplasmic reticulum and Golgi complex, increase in telenuclear secretory complex, increase in fusion of type B atrial specific granules (ASG), decrease of the total number of ASG, enlargement of the maximum diameter of type A ASG and a relative increase in the number of type B ASG. These alterations suggest a larger secretory activity of the atrial myoendocrine cells with a larger secretion of atrial natriuretic peptide (ANP). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7547444

  14. IP3 Receptor Type 2 Deficiency Is Associated with a Secretory Defect in the Pancreatic Acinar Cell and an Accumulation of Zymogen Granules

    PubMed Central

    Orabi, Abrahim I.; Luo, Yuhuan; Ahmad, Mahwish U.; Shah, Ahsan U.; Mannan, Zahir; Wang, Dong; Sarwar, Sheharyar; Muili, Kamaldeen A.; Shugrue, Christine; Kolodecik, Thomas R.; Singh, Vijay P.; Lowe, Mark E.; Thrower, Edwin; Chen, Ju; Husain, Sohail Z.

    2012-01-01

    Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is often multi-factorial. It is of great importance to understand the interplay between factors that predispose patients to develop the disease. One such factor is an excessive elevation in pancreatic acinar cell Ca2+. These aberrant Ca2+ elevations are triggered by release of Ca2+ from apical Ca2+ pools that are gated by the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3. In this study, we examined the role of IP3R type 2 (IP3R2) using mice deficient in this Ca2+ release channel (IP3R2?/?). Using live acinar cell Ca2+ imaging we found that loss of IP3R2 reduced the amplitude of the apical Ca2+ signal and caused a delay in its initiation. This was associated with a reduction in carbachol-stimulated amylase release and an accumulation of zymogen granules (ZGs). Specifically, there was a 2-fold increase in the number of ZGs (P<0.05) and an expansion of the ZG pool area within the cell. There was also a 1.6- and 2.6-fold increase in cellular amylase and trypsinogen, respectively. However, the mice did not have evidence of pancreatic injury at baseline, other than an elevated serum amylase level. Further, pancreatitis outcomes using a mild caerulein hyperstimulation model were similar between IP3R2?/? and wild type mice. In summary, IP3R2 modulates apical acinar cell Ca2+ signals and pancreatic enzyme secretion. IP3R-deficient acinar cells accumulate ZGs, but the mice do not succumb to pancreatic damage or worse pancreatitis outcomes. PMID:23185258

  15. Carboxypeptidase B-like converting enzyme activity in secretory granules of rat pituitary.

    PubMed Central

    Hook, V Y; Loh, Y P

    1984-01-01

    Recent amino acid sequence data suggest that trypsin-like and carboxypeptidase B-like activities are required for the processing of pituitary prohormones--e.g., pro-opiocortin (pro-adrenocorticotropin/lipotropin) and provasopressin in secretory granules. In this study the existence of a carboxypeptidase B activity in purified secretory granules from anterior, intermediate, and neural lobes of rat pituitary has been examined. A carboxypeptidase B activity that cleaved the COOH-terminal -Lys-Lys-Arg residues from the adrenocorticotropin fragment ACTH-(1-17) (a potential hormone product liberated from pro-opiocortin by a trypsin-like enzyme) was detected in anterior and intermediate lobe granules. A similar carboxypeptidase B activity was also present in purified secretory granules from rat pituitary neural lobes that cleaved the -Lys-Arg residues from [Arg8]vasopressin-Gly-Lys-Arg, a potential product cleaved from provasopressin. Secretory granule carboxypeptidase(s) from the three lobes of the pituitary was shown to cleave 125I-[Met]enkephalin-Arg6 to form 125I-[Met]enkephalin as well. 125I-[Met]Enkephalin was used as a model substrate for the quantitative assay of pituitary carboxypeptidase activity. The carboxypeptidase B in secretory granules from all three lobes was shown to be active at pH 5.5, but not at pH 7.4. Inhibition by the zinc metallocarboxypeptidase inhibitors guanidinopropylsuccinic acid, aminomercaptosuccinic acid, benzylsuccinic acid, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and the potato carboxypeptidase B inhibitor, and inhibition by the metal chelators EDTA and 1,10-phenanthroline demonstrate metal ion dependence of the pituitary granule carboxypeptidase activities. However, Co2+ stimulated the secretory granule carboxypeptidase B activities. Thiol protease inhibitors such as Cu2+ and p-chloromercuriphenylsulfonic acid also inhibited the activity. Thus, the secretory granule carboxypeptidase B-like activities in all three lobes of the pituitary appear to be similar thiol-metallopeptidases that differ from other carboxypeptidase activities previously described and may play an exclusive role in hormone biosynthesis in the pituitary. PMID:6326144

  16. Mannose 6Phosphate Receptors Are Sorted from Immature Secretory Granules via Adaptor Protein AP1, Clathrin, and Syntaxin 6-positive Vesicles

    Microsoft Academic Search

    Judith Klumperman; Regina Kuliawat; Janice M. Griffith; Hans J. Geuze; Peter Arvan

    1998-01-01

    The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cy- tosolic adaptor proteins. By quantitative immunoelec- tron microscopy of rat endocrine pancreatic b cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6-phosphate receptors (MPRs)

  17. Conformational study of the proline rich peptide from bovine neurohypophysis secretory granules

    NASA Astrophysics Data System (ADS)

    Alieva, Irada; Velieva, Lala; Aliev, Dshavanchir; Gojayev, Niftali; Demukhamedova, Svetlana

    2004-01-01

    The spatial organization and conformational properties of the Proline Rich Peptide (PRP) from bovine neurohypophysis secretory granules have been established by the methods of molecular mechanics and molecular dynamics simulations in water solution. Conformational studies showed the peptide with limited conformational flexibility. Two ?-type III turns are observed in PRP spatial organization.

  18. Serous cutaneous glands in anurans: Fourier transform analysis of the repeating secretory granule substructure

    NASA Astrophysics Data System (ADS)

    Nosi, D.; Delfino, G.; Quercioli, F.

    2013-03-01

    A combined transmission electron microscopy (TEM) and Fourier transform analysis has been performed on the secretory granules storing active peptides/proteins in serous cutaneous glands of n = 12 anuran species. Previous TEM investigation showed that the granules are provided with remarkable repeating substructures based on discrete subunits, arranged into a consistent framework. Furthermore, TEM findings revealed that this recurrent arrangement is acquired during a prolonged post-Golgian (or maturational) processing that affects the secretory product. Maturation leads to a variety of patterns depending on the degree of subunit clustering. This variety of recurrent patterns has been plotted into a range of frequency spectra. Through this quantitative approach, we found that the varying granule substructure can be reduced to a single mechanism of peptide/protein aggregation.

  19. LYSOSOME FUNCTION IN THE REGULATION OF THE SECRETORY PROCESS IN CELLS OF THE ANTERIOR PITUITARY GLAND

    PubMed Central

    Smith, Robert E.; Farquhar, Marilyn G.

    1966-01-01

    The nature and content of lytic bodies and the localization of acid phosphatase (AcPase) activity were investigated in mammotrophic hormone-producing cells (MT) from rat anterior pituitary glands. MT were examined from lactating rats in which secretion of MTH1 was high and from postlactating rats in which MTH secretion was suppressed by removing the suckling young. MT from lactating animals contained abundant stacks of rough-surfaced ER, a large Golgi complex with many forming secretory granules, and a few lytic bodies, primarily multivesicular bodies and dense bodies. MT from postlactating animals, sacrificed at selected intervals up to 96 hr after separation from their suckling young, showed (a) progressive involution of the protein synthetic apparatus with sequestration of ER and ribosomes in autophagic vacuoles, and (b) incorporation of secretory granules into multivesicular and dense bodies. The content of mature granules typically was incorporated into dense bodies whereas that of immature granules found its way preferentially into multivesicular bodies. The secretory granules and cytoplasmic constituents segregated within lytic bodies were progressively degraded over a period of 24 to 72 hr to yield a common residual body, the vacuolated dense body. In MT from lactating animals, AcPase reaction product was found in lytic bodies, and in several other sites not usually considered to be lysosomal in nature, i.e., inner Golgi cisterna and associated vesicles, and around most of the immature, and some of the mature secretory granules. In MT from postlactating animals, AcPase was concentrated in lytic bodies; reaction product and incorporated secretory granules were frequently recognizable within the same multivesicular or dense body which could therefore be identified as "autolysosomes" connected with the digestion of endogenous materials. Several possible explanations for the occurrence of AcPase in nonlysosomal sites are discussed. From the findings it is concluded that, in secretory cells, lysosomes function in the regulation of the secretory process by providing a mechanism which takes care of overproduction of secretory products. PMID:19866704

  20. 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.

  1. The GM2-1 Ganglioside Islet Autoantigen in Insulin-Dependent Diabetes Mellitus Is Expressed in Secretory Granules and Is Not  Cell Specific

    Microsoft Academic Search

    FRANCESCO DOTTA; MARCELLO PREVITI; MARGUERITE NEERMAN-ARBEZ; SABRINA DIONISI; DOMENICO CUCINOTTA; LUISA LENTI; UMBERTO DI MARIO; PHILIPPE A. HALBAN

    1998-01-01

    The pancreatic islet monosialo-ganglioside (GM2-1), an autoanti- gen in insulin-dependent diabetes mellitus (IDDM) recently shown to be the target of autoantibodies associated with diabetes development in relatives of IDDM patients, is islet specific within the pancreas, and its expression is metabolically regulatable. In the present study we sought to establish 1) whether GM2-1 is b-cell specific, and 2) its intracellular

  2. Type II phosphatidylinositol 4-kinase regulates trafficking of secretory granule proteins in Drosophila

    PubMed Central

    Burgess, Jason; Del Bel, Lauren M.; Ma, Cheng-I J.; Barylko, Barbara; Polevoy, Gordon; Rollins, Janet; Albanesi, Joseph P.; Krämer, Helmut; Brill, Julie A.

    2012-01-01

    Type II phosphatidylinositol 4-kinase (PI4KII) produces the lipid phosphatidylinositol 4-phosphate (PI4P), a key regulator of membrane trafficking. Here, we generated genetic models of the sole Drosophila melanogaster PI4KII gene. A specific requirement for PI4KII emerged in larval salivary glands. In PI4KII mutants, mucin-containing glue granules failed to reach normal size, with glue protein aberrantly accumulating in enlarged Rab7-positive late endosomes. Presence of PI4KII at the Golgi and on dynamic tubular endosomes indicated two distinct foci for its function. First, consistent with the established role of PI4P in the Golgi, PI4KII is required for sorting of glue granule cargo and the granule-associated SNARE Snap24. Second, PI4KII also has an unforeseen function in late endosomes, where it is required for normal retromer dynamics and for formation of tubular endosomes that are likely to be involved in retrieving Snap24 and Lysosomal enzyme receptor protein (Lerp) from late endosomes to the trans-Golgi network. Our genetic analysis of PI4KII in flies thus reveals a novel role for PI4KII in regulating the fidelity of granule protein trafficking in secretory tissues. PMID:22791894

  3. Origin of the bursal secretory dendritic cell

    Microsoft Academic Search

    N. Nagy; A. Magyar; M. Tóth; I. Oláh

    2004-01-01

    The origin of vimentin-positive secretory dendritic cells of the bursa of Fabricius was studied by chick–quail chimera, parabiosis and immunohistochemistry using species-specific monoclonal antibodies. Quail bursal primordia of different ages were transferred to coelomic cavity of 3-day-old chicken embryos and further incubated for 18 days. In transplanted quail bursas the secretory dendritic cells of chicken and quail origin were detected

  4. Platelet granule exocytosis: a comparison with chromaffin cells.

    PubMed

    Fitch-Tewfik, Jennifer L; Flaumenhaft, Robert

    2013-01-01

    The rapid secretion of bioactive amines from chromaffin cells constitutes an important component of the fight or flight response of mammals to stress. Platelets respond to stresses within the vasculature by rapidly secreting cargo at sites of injury, inflammation, or infection. Although chromaffin cells derive from the neural crest and platelets from bone marrow megakaryocytes, both have evolved a heterogeneous assemblage of granule types and a mechanism for efficient release. This article will provide an overview of granule formation and exocytosis in platelets with an emphasis on areas in which the study of chromaffin cells has influenced that of platelets and on similarities between the two secretory systems. Commonalities include the use of transporters to concentrate bioactive amines and other cargos into granules, the role of cytoskeletal remodeling in granule exocytosis, and the use of granules to provide membrane for cytoplasmic projections. The SNAREs and SNARE accessory proteins used by each cell type will also be considered. Finally, we will discuss the newly appreciated role of dynamin family proteins in regulated fusion pore formation. This evaluation of the comparative cell biology of regulated exocytosis in platelets and chromaffin cells demonstrates a convergence of mechanisms between two disparate cell types both tasked with responding rapidly to physiological stimuli. PMID:23805129

  5. Clara Cell Secretory Protein Deficiency Alters Clara Cell Secretory Apparatus and the Protein Composition of Airway Lining Fluid

    Microsoft Academic Search

    Barry R. Stripp; Susan D. Reynolds; Inger-Margrethe Boe; Johan Lund; John H. T. Power; John T. Coppens; Virginia Wong; Paul R. Reynolds; Charles G. Plopper

    Clara cells represent the predominant secretory cell within distal conducting airways of mammals and exhibit functional alterations with chronic lung disease. We previously demon- strated that Clara cell secretory protein (CCSP) deficiency re- sults in enhanced susceptibility to environmental agents. The present study was undertaken to define changes in Clara cell secretory function associated with CCSP deficiency in knock- out

  6. Biogenesis of the Secretory Granule: Chromogranin a Coiled-Coil Structure Results in Unusual Physical Properties And Suggests a Mechanism for Granule Core Condensation

    SciTech Connect

    Mosley, C.A.; Taupenot, L.; Biswas, N.; Taulane, J.P.; Olson, N.H.; Vaingankar, S.M.; Wen, G.; Schork, N.J.; Ziegler, M.G.; Mahata, S.K.; O'Connor, D.T.

    2009-06-03

    The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein 'packing' in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a binding complex by the CHGA core. Inhibition of CHGA expression, by siRNA, disrupted regulated secretory protein traffic by approximately 65%, while targeted ablation of the CHGA gene in the mouse reduced chromaffin granule cotransmitter concentrations by approximately 40-80%. These results suggest new roles for secretory protein tertiary structure in hormone and transmitter storage, with implications for secretory cargo condensation (or dense core 'packing' structure) within the regulated pathway.

  7. SPECIFIC GRANULES IN ATRIAL MUSCLE CELLS

    Microsoft Academic Search

    J. D. JAMIESON; G. E. PALADE

    2006-01-01

    Large populations (up to 600\\/cell) of spherical, electron-opaque granules ~ 0.3 to 0.4\\/~ in diameter are characteristically found in muscle fibers of mammalian atria. They are absent in muscle fibers of the ventricles. The granules arc concentrated in the sarcoplasmic core and occur in lesser numbers in the sarcoplasmic layers between myofibrils and under the plasma membrane. Their intimate association

  8. Proinsulin Intermolecular Interactions during Secretory Trafficking in Pancreatic ? Cells*

    PubMed Central

    Haataja, Leena; Snapp, Erik; Wright, Jordan; Liu, Ming; Hardy, Alexandre B.; Wheeler, Michael B.; Markwardt, Michele L.; Rizzo, Mark; Arvan, Peter

    2013-01-01

    Classically, exit from the endoplasmic reticulum (ER) is rate-limiting for secretory protein trafficking because protein folding/assembly occurs there. In this study, we have exploited “hPro-CpepSfGFP,” a human proinsulin bearing “superfolder” green fluorescent C-peptide expressed in pancreatic ? cells where it is processed to human insulin and CpepSfGFP. Remarkably, steady-state accumulation of hPro-CpepSfGFP and endogenous proinsulin is in the Golgi region, as if final stages of protein folding/assembly were occurring there. The Golgi regional distribution of proinsulin is dynamic, influenced by fasting/refeeding, and increased with ? cell zinc deficiency. However, coexpression of ER-entrapped mutant proinsulin-C(A7)Y shifts the steady-state distribution of wild-type proinsulin to the ER. Endogenous proinsulin coprecipitates with hPro-CpepSfGFP and even more so with hProC(A7)Y-CpepSfGFP. Using Cerulean and Venus-tagged proinsulins, we find that both WT-WT and WT-mutant proinsulin pairs exhibit FRET. The data demonstrate that wild-type proinsulin dimerizes within the ER but accumulates at a poorly recognized slow step within the Golgi region, reflecting either slow kinetics of proinsulin hexamerization, steps in formation of nascent secretory granules, or other unknown molecular events. However, in the presence of ongoing misfolding of a subpopulation of proinsulin in ? cells, the rate-limiting step in transport of the remaining proinsulin shifts to the ER. PMID:23223446

  9. 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.

  10. Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis

    PubMed Central

    Gandasi, Nikhil R.; Vestö, Kim; Helou, Maria; Yin, Peng; Saras, Jan; Barg, Sebastian

    2015-01-01

    Fluorescent proteins (FPs) have proven to be valuable tools for high-resolution imaging studies of vesicle transport processes, including exo- and endocytosis. Since the pH of the vesicle lumen changes between acidic and neutral during these events, pH-sensitive FPs with near neutral pKa, such as pHluorin, are particularly useful. FPs with pKa>6 are readily available in the green spectrum, while red-emitting pH-sensitive FPs are rare and often not well characterized as reporters of exo- or endocytosis. Here we tested a panel of ten orange/red and two green FPs in fusions with neuropeptide Y (NPY) for use as secreted vesicle marker and reporter of dense core granule exocytosis and release. We report relative brightness, bleaching rate, targeting accuracy, sensitivity to vesicle pH, and their performance in detecting exocytosis in live cells. Tandem dimer (td)-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP. Single exocytosis events were readily observed, which allowed measurements of fusion pore lifetime and the dynamics of the exocytosis protein syntaxin at the release site during membrane fusion and cargo release. PMID:26091288

  11. 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

  12. Functional and structural characterization of a dense core secretory granule sorting domain from the PC1/3 protease

    PubMed Central

    Dikeakos, Jimmy D.; Di Lello, Paola; Lacombe, Marie-Josée; Ghirlando, Rodolfo; Legault, Pascale; Reudelhuber, Timothy L.; Omichinski, James G.

    2009-01-01

    Several peptide hormones are initially synthesized as inactive precursors. It is only on entry of these prohormones and their processing proteases into dense core secretory granules (DCSGs) that the precursors are cleaved to generate their active forms. Prohormone convertase (PC)1/3 is a processing protease that is targeted to DCSGs. The signal for targeting PC1/3 to DCSGs resides in its carboxy-terminal tail (PC1/3617–753), where 3 regions (PC1/3617–625, PC1/3665–682, and PC1/3711–753) are known to aid in sorting and membrane association. In this article, we have determined a high-resolution structure of the extreme carboxy-terminal sorting domain, PC1/3711–753 in micelles by NMR spectroscopy. PC1/3711–753 contains 2 alpha helices located between residues 722–728 and 738–750. Functional assays demonstrate that the second helix (PC1/3738–750) is necessary and sufficient to target a constitutively secreted protein to granules, and that L745 anchors a hydrophobic patch that is critical for sorting. Also, we demonstrate that calcium binding by the second helix of PC1/3711–753 promotes aggregation of the domain via the hydrophobic patch centered on L745. These results provide a structure-function analysis of a DCSG-sorting domain, and reveal the importance of a hydrophobic patch and calcium binding in controlling the sorting of proteins containing alpha helices to DCSGs. PMID:19376969

  13. An aspartyl cathepsin, CTH3, is essential for proprotein processing during secretory granule maturation in Tetrahymena thermophila

    PubMed Central

    Kumar, Santosh; Briguglio, Joseph S.; Turkewitz, Aaron P.

    2014-01-01

    In Tetrahymena thermophila, peptides secreted via dense-core granules, called mucocysts, are generated by proprotein processing. We used expression profiling to identify candidate processing enzymes, which localized as cyan fluorescent protein fusions to mucocysts. Of note, the aspartyl cathepsin Cth3p plays a key role in mucocyst-based secretion, since knockdown of this gene blocked proteolytic maturation of the entire set of mucocyst proproteins and dramatically reduced mucocyst accumulation. The activity of Cth3p was eliminated by mutation of two predicted active-site mutations, and overexpression of the wild-type gene, but not the catalytic-site mutant, partially rescued a Mendelian mutant defective in mucocyst proprotein processing. Our results provide the first direct evidence for the role of proprotein processing in this system. Of interest, both localization and the CTH3 disruption phenotype suggest that the enzyme provides non–mucocyst-related functions. Phylogenetic analysis of the T. thermophila cathepsins, combined with prior work on the role of sortilin receptors in mucocyst biogenesis, suggests that repurposing of lysosomal enzymes was an important step in the evolution of secretory granules in ciliates. PMID:24943840

  14. 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

  15. Decidualization of human endometrial stromal cells in vitro: effects of progestin and relaxin on the ultrastructure and production of decidual secretory proteins.

    PubMed

    Lane, B; Oxberry, W; Mazella, J; Tseng, L

    1994-02-01

    Decidual cells arise by proliferation and differentiation of endometrial stromal cells of the uterus after appropriate stimulation by ovarian hormones. Previously we have shown that progestin and relaxin stimulate the secretion of several decidual-cell-specific secretory proteins in a long-term primary cell culture system. We now report the effects of progestin and relaxin on the morphology of stromal cells in association with the production rate of two major decidual secretory proteins, prolactin and insulin-like growth factor binding protein-1 (IGFBP-1). Stromal cells were cultured in RPMI 1640 and 2% fetal bovine serum for 22 days under control conditions (no hormone), with relaxin or medroxyprogesterone acetate (MPA), or MPA plus relaxin. Cells treated with MPA alone or MPA plus porcine relaxin grew to a high density with many areas of heaping while control cells and cells grown in medium containing relaxin alone formed discontinuous layers. The cytoplasm was distinguished by aggregates of rough endoplasmic reticulum and secretory granules. Surfaces of cells treated with MPA plus relaxin had clusters of short blunt processes containing secretory granules. The processes were rarely seen in cells exposed to MPA alone and completely absent in control cells or cells exposed to relaxin alone. Intercellular space was greatly widened in cells treated with MPA alone or MPA plus relaxin. There were many extended gap junctions in MPA- and relaxin-treated cells in contrast to controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7517949

  16. Tumor Necrosis Factor a Stimulates Human Clara Cell Secretory Protein Production by Human Airway Epithelial Cells

    Microsoft Academic Search

    X. L. Yao; S. J. Levine; M. J. Cowan; C. Logun; J. H. Shelhamer

    Clara cell secretory protein (CCSP), or CC10, is an inhibitor of secretory phospholipase A 2 which may be produced by phagocytic cells and by a variety of other cells in the airway. Tumor necrosis factor- a (TNF- a ) is capable of activating phospholipases and inducing the expression of a variety of genes in the airway epi- thelium which may

  17. 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

  18. 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

  19. Structural Requirements for Sorting Pro-Vasopressin to the Regulated Secretory Pathway in a Neuronal Cell Line

    PubMed Central

    Cool, David R.; Jackson, Steven B.; Waddell, Karen S.

    2009-01-01

    Vasopressin is a peptide hormone normally secreted via the regulated secretory pathway in neuro-endocrine cells. In an effort to determine which region of vasopressin contains sufficient information for sorting, we created five constructs with the cDNA for vasopressin or regions of vasopressin in frame with the gene for green fluorescent protein (GFP). Fluorescence microscopy of Neuro-2a cells expressing the constructs revealed full-length vasopressin-GFP (VP-GFP), neurophysin-GFP (NP-GFP) and arginine-vasopressin/neurophysin-GFP (AN-GFP), were localized to punctate granules in the neurites and accumulated at the tips of neurites, characteristic of regulated secretory granules. These fusion proteins were secreted in a regulated manner as determined by pulse-chase labeling experiments. Two other chimeric proteins, signalpeptide-GFP and AVP-GFP were localized to a perinuclear region, characteristic of the endoplasmic reticulum. Pulse/chase [35S]labeling followed by immunoprecipitation using anti-GFP antibody indicated that these two fusion proteins were constitutively secreted. We conclude that the neurophysin region of pro-vasopressin contains information that is both sufficient and necessary for sorting GFP into the regulated secretory pathway. PMID:19830265

  20. Pancreatic beta-cell granule peptides form heteromolecular complexes which inhibit islet amyloid polypeptide fibril formation.

    PubMed Central

    Jaikaran, Emma T A S; Nilsson, Melanie R; Clark, Anne

    2004-01-01

    Islet amyloid polypeptide (IAPP), or 'amylin', is co-stored with insulin in secretory granules of pancreatic islet beta-cells. In Type 2 diabetes, IAPP converts into a beta-sheet conformation and oligomerizes to form amyloid fibrils and islet deposits. Granule components, including insulin, inhibit spontaneous IAPP fibril formation in vitro. To determine the mechanism of this inhibition, molecular interactions of insulin with human IAPP (hIAPP), rat IAPP (rIAPP) and other peptides were examined using surface plasmon resonance (BIAcore), CD and transmission electron microscopy (EM). hIAPP and rIAPP complexed with insulin, and this reaction was concentration-dependent. rIAPP and insulin, but not pro-insulin, bound to hIAPP. Insulin with a truncated B-chain, to prevent dimerization, also bound hIAPP. In the presence of insulin, hIAPP did not spontaneously develop beta-sheet secondary structure or form fibrils. Insulin interacted with pre-formed IAPP fibrils in a regular repeating pattern, as demonstrated by immunoEM, suggesting that the binding sites for insulin remain exposed in hIAPP fibrils. Since rIAPP and hIAPP form complexes with insulin (and each other), this could explain the lack of amyloid fibrils in transgenic mice expressing hIAPP. It is likely that IAPP fibrillogenesis is inhibited in secretory granules (where the hIAPP concentration is in the millimolar range) by heteromolecular complex formation with insulin. Alterations in the proportions of insulin and IAPP in granules could disrupt the stability of the peptide. The increase in the proportion of unprocessed pro-insulin produced in Type 2 diabetes could be a major factor in destabilization of hIAPP and induction of fibril formation. PMID:14565847

  1. 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

  2. Cytolytic T cell granules. Isolation, structural, biochemical, and functional characterization

    PubMed Central

    1984-01-01

    The cytoplasmic, dense granules of cloned T cell lines were isolated and analyzed for their functional and biochemical properties. Isolated granules of approximately 90% homogeneity, in the presence of Ca, effect strong tumoricidal and hemolytic activity. Tumor cell lysis is complete in less than 30 min, with less than 2 micrograms granule protein corresponding to a killer/target ratio of 3-6:1 by assuming 50% yield for granule isolation. The granules contain a set of unique proteins, responsible for cytolytic activity and designated K1 to K6, in the molecular weight range of 14,000 to 75,000, as defined by sodium dodecyl sulfate (SDS) polyacrylamide slab gel analysis under reducing and nonreducing conditions. Cytolysis mediated by isolated granules is accompanied by the assembly of tubular complexes of 160 A (poly P1) and of approximately 70 A width (poly P2) that are inserted into membranes and form ultrastructural membrane lesions. As shown by immunofluorescence and by Percoll gradient fractionation, cytolytic granules are detected in cells of cytolytic T cell lineage and not in the T cell lymphomas E14 and S194. Poly perforin 1 assembled by CTLL-2 upon stimulation with concanavalin A (Con A) and phorbol myristate acetate (PMA) was isolated by detergent extraction and gel filtration. Poly P1 is composed of disulfide-linked subunits that, after reduction, co-migrate with certain granule proteins. The results are compatible with the hypothesis that the dense granules of cytolytic T cells contain cytolytic proteins that polymerize to disulfide-linked tubular poly perforins in a Ca-dependent reaction and may cause cytolysis by membrane insertion and transmembrane channel formation. PMID:6332169

  3. Video rate bioluminescence imaging of secretory proteins in living cells: localization, secretory frequency, and quantification.

    PubMed

    Suzuki, Takahiro; Kondo, Chihiro; Kanamori, Takao; Inouye, Satoshi

    2011-08-15

    We have developed a method of video rate bioluminescence imaging to investigate protein secretion from a single mammalian cell and analyzed the localization, secretory frequency, and quantification of secreted protein. By detecting the luminescence signals from the Gaussia luciferase (GLase) reaction using a high-speed electron-multiplying charge-coupled device (EM-CCD) camera, video rate imaging was performed with a time resolution within 500 ms/image over 30 min in living cells. As a model study, we applied the method to visualize the glucose-stimulated insulin secretion from clustered pancreatic MIN6 ? cells using the fused protein of GLase with preproinsulin. High-quality video images clearly showed that the glucose-stimulated insulin secretion from the clustered MIN6 ? cells oscillated within a period of a few minutes over 10 min. In addition, the glibenclamide-induced insulin secretion from the clustered MIN6 ? cells was visualized, suggesting that bioluminescence video rate imaging is a useful method for validating drug action in living cells. PMID:21477579

  4. 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...

  5. 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

  6. Discovery of the ‘porosome’ the universal secretory machinery in cells

    PubMed Central

    Anderson, L L

    2006-01-01

    The release of neurotransmitters at the nerve terminal for neurotransmission, release of insulin from ?-cells of the endocrine pancreas for regulating blood glucose levels, the release of growth hormone from GH cells of the pituitary gland to regulate body growth, or the expulsion of zymogen from exocrine pancreas to digest food, are only a few examples of key physiological processes made possible by cell secretion. It comes as no surprise that defects in cell secretion are the cause for numerous diseases, and have been under intense investigation for over half century. Only in the last decade, the molecular machinery and mechanism of cell secretion has become clear. Cell secretion involves the docking and transient fusion of membrane-bound secretory vesicles at the base of plasma membrane structures called porosomes, and the regulated expulsion of intravesicular contents to the outside, by vesicle swelling. The discovery of the porosome in live cells, its morphology and dynamics at nanometer resolution and in real time, its isolation, its composition, and its structural and functional reconstitution in lipid membrane, are complete. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and the regulated expulsion of intravesicular contents during cell secretion, are also resolved. In this minireview, the monumental discovery of the porosome, a new cellular structure at the cell plasma membrane, is briefly discussed. PMID:16563225

  7. A Spatial Model of Insulin-Granule Dynamics in Pancreatic ?-Cells.

    PubMed

    Dehghany, Jaber; Hoboth, Peter; Ivanova, Anna; Mziaut, Hassan; Müller, Andreas; Kalaidzidis, Yannis; Solimena, Michele; Meyer-Hermann, Michael

    2015-08-01

    Insulin secretion from pancreatic ?-cells in response to sudden glucose stimulation is biphasic. Prolonged secretion in vivo requires synthesis, delivery to the plasma membrane (PM) and exocytosis of insulin secretory granules (SGs). Here, we provide the first agent-based space-resolved model for SG dynamics in pancreatic ?-cells. Using recent experimental data, we consider a single ?-cell with identical SGs moving on a phenomenologically represented cytoskeleton network. A single exocytotic machinery mediates SG exocytosis on the PM. This novel model reproduces the measured spatial organization of SGs and insulin secretion patterns under different stimulation protocols. It proposes that the insulin potentiation effect and the rising second-phase secretion are mainly due to the increasing number of docking sites on the PM. Furthermore, it shows that 6 min after glucose stimulation, the 'newcomer' SGs are recruited from a region within less than 600 nm from the PM. PMID:25809669

  8. Ultrastructure and Secretion of the Secretory Cells of Two Species of Fagonia L. (Zygophyllaceae)

    Microsoft Academic Search

    ABRAHAM FAHN; CARMELA SHIMONY

    1998-01-01

    The ultrastructure and secretion of the secretory cells of the glandular trichomes ofFagonia mollisandF. glutinosawere studied. The most important finding of this study is that two species of the same genus produce the lipophilic component of the secretory material in completely different ways and at different sites within the cell. In the early stages of development of secretory cells ofF.

  9. Dual protein trafficking to secretory and non-secretory cell compartments: clear or double vision?

    PubMed

    Porter, Brad W; Yuen, Christen Y L; Christopher, David A

    2015-05-01

    Approximately 18% of Arabidopsis thaliana proteins encode a signal peptide for translocation to the endoplasmic reticulum (ER), the gateway of the eukaryotic secretory pathway. However, it was recently discovered that some ER proteins can undergo both co-translational import into the ER/secretory pathway and trafficking to compartments outside of the secretory pathway. This phenomenon is observed among members of the protein disulfide isomerase (PDI) family, which are traditionally regarded as ER enzymes involved in protein folding. Although classical PDIs possess an N-terminal signal peptide and a C-terminal ER retention signal, some also dual localize to secretory and non-secretory compartments, including mammalian PDI ERp57, Chlamydomonas reinhardtii PDI RB60, and A. thaliana AtPDI2. ERp57 is present in both the ER and nucleus where it influences gene transcription. RB60 localizes to the ER and chloroplast where it modulates the redox state of polyadenylate-binding protein RB47. AtPDI2, which interacts with transcription factor MEE8, localizes to the ER-secretory pathway and the nucleus. A model proposing secretory trafficking of AtPDI2 and nuclear co-translocation of an AtPDI2-MEE8 complex illustrates the diversity of dual targeting mechanisms, the multifunctional roles of some PDIs, and the potential co-translocation of other proteins to multiple subcellular compartments. PMID:25804820

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

    E-print Network

    van Oudenaarden, Alexander

    ­8 weeks7 . Paneth cells serve as niche cells, providing Wnt, Notch and EGF signals to stem cells8L 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

  11. Cholesterol accumulation increases insulin granule size and impairs membrane trafficking

    PubMed Central

    Bogan, Jonathan S.; Xu, Yingke; Hao, Mingming

    2012-01-01

    The formation of mature secretory granules is essential for proper storage and regulated release of hormones and neuropeptides. In pancreatic ?-cells, cholesterol accumulation causes defects in insulin secretion and may participate in the pathogenesis of type 2 diabetes. Using a novel cholesterol analog, we show for the first time that insulin granules are the major sites of intracellular cholesterol accumulation in live ?-cells. This is distinct from other, non-secretory cell types, in which cholesterol is concentrated in the recycling endosomes and the trans-Golgi network. Excess cholesterol was delivered specifically to insulin granules, which caused granule enlargement and retention of syntaxin 6 and VAMP4 in granule membranes, with concurrent depletion of these proteins from the trans-Golgi network. Clathrin also accumulated in the granules of cholesterol-overloaded cells, consistent with a possible defect in the last stage of granule maturation, during which clathrin-coated vesicles bud from the immature granules. Excess cholesterol also reduced the docking and fusion of insulin granules at the plasma membrane. Together, the data support a model in which cholesterol accumulation in insulin secretory granules impairs the ability of these vesicles to respond to stimuli, and thus reduces insulin secretion. PMID:22889194

  12. Cerebellar Granule Cells in Culture: Monosynaptic Connections with Purkinje Cells and Ionic Currents

    Microsoft Academic Search

    Tomoo Hirano; Yoshihiro Kubo; Michael M. Wu

    1986-01-01

    Electrophysiological properties of cerebellar granule cells and synapses between granule and Purkinje cells were studied in dissociated cultures. Electrophysiological properties of neurons and synapses in the mammalian central nervous system are best studied in dissociated cell cultures because of good target cell visibility, control over the contents of the extracellular solution, and the feasibility of whole-cell patch electrode recording, which

  13. Integration of quanta in cerebellar granule cells during sensory processing

    Microsoft Academic Search

    Paul Chadderton; Troy W. Margrie; Michael Häusser

    2004-01-01

    To understand the computations performed by the input layers of cortical structures, it is essential to determine the relationship between sensory-evoked synaptic input and the resulting pattern of output spikes. In the cerebellum, granule cells constitute the input layer, translating mossy fibre signals into parallel fibre input to Purkinje cells. Until now, their small size and dense packing have precluded

  14. 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

  15. EBV specific secretory IgA in saliva of NPC patients. Presence of secretory piece in epithelial malignant cells.

    PubMed

    Desgranges, C; Li, J Y; De-Thé

    1977-12-15

    Saliva samples from 59 patients with nasopharyngeal carcinoma (NPC) and from 20 normal individuals were studied to determine the nature and origin of the EBV-specific IgA in NPC saliva. About 50% of NPC saliva samples contained secretory IgA specific for EBV. The corresponding tumor IgA(alpha) was found in plasma cells surrounding the epithelial tumor cells and the secretory piece at the surface of epithelial cells. A slightly higher proportion of NPC saliva samples containing IgA was found in patients from Tunis than in samples from Hong Kong. Attention is drawn to the clinical value of the salivary IgA in diagnosis and monitoring of treatment of NPC. PMID:201574

  16. 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.

  17. Clara cell secretory protein is reduced in equine recurrent airway obstruction.

    PubMed

    Katavolos, P; Ackerley, C A; Viel, L; Clark, M E; Wen, X; Bienzle, D

    2009-07-01

    Horses are prone to recurrent airway obstruction (RAO), an inflammatory lung disease induced by repeated exposure to environmental mold, dust, and bacterial components. Active disease manifests with mucus hyperproduction, neutrophilic inflammation, bronchoconstriction, and coughing. Chronically affected animals have lung remodeling characterized by smooth muscle hyperplasia, collagen deposition, lymphoid hyperplasia, and impaired aerobic performance. Clara cell secretory protein (CCSP) counters inflammation in the lung, hence we hypothesized that CCSP depletion is a key feature of RAO in horses. Recombinant equine CCSP and specific antiserum were produced, and percutaneous lung biopsies were obtained from 3 healthy horses and from 3 RAO-affected horses before and after induction of RAO. CCSP relative gene expression in tissue, as well as protein concentration in lung lavage fluid, was determined. Immunocytochemical analysis, using both light and immunogold ultrastructural methods, demonstrated reduced CCSP staining in lung tissue of animals with RAO. Immunogold label in Clara cell granules was less in animals with chronic RAO than in normal animals, and absent in animals that had active disease. Median lung lavage CCSP concentration was 132 and 129 ng/ml in healthy horses, and 62 and 24 ng/ml in RAO horses before and after challenge, respectively. CCSP lung gene expression was significantly higher in healthy animals than in animals with chronic RAO. Together, these preliminary findings suggest that reduced production of CCSP and subcellular changes in Clara cells are features of chronic environmentally induced lung inflammation in horses. PMID:19276063

  18. The Enzymology and Intracellular Organization of Peptide Precursor Processing: The Secretory Vesicle Hypothesis

    Microsoft Academic Search

    Harold Gainer; James T. Russell; Y. Peng Loh

    1985-01-01

    The ‘secretory vesicle hypothesis of precursor processing’ states that the initial endopeptidase cleavages which excise the nascent, biologically active peptides from their protein precursors occur primarily in secretory vesicles (or granules). Hence, all the processing steps subsequent to these cleavages must also occur within these organelles. Two types of evidence are presented in support of this view: (1) cell biological

  19. 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.

  20. Secretion marker proteins and cell-wall polysaccharides move through different secretory pathways

    Microsoft Academic Search

    Maria Rosaria Leucci; Gian-Pietro Di Sansebastiano; Massimiliano Gigante; Giuseppe Dalessandro; Gabriella Piro

    2007-01-01

    The building up of the cell wall is tightly dependent on the functionality of the secretory pathway. Syntaxins as well as\\u000a other SNARE proteins play important roles during vesicle secretion and fusion. We have compared the secretion of newly synthesised\\u000a cell-wall polysaccharides to that of secretory marker proteins such as secreted green-fluorescent protein (secGFP) and secreted\\u000a rat preputial ?-glucuronidase (secRGUS)

  1. Histamine synthesis is required for granule maturation in murine mast cells.

    PubMed

    Nakazawa, Shunsuke; Sakanaka, Mariko; Furuta, Kazuyuki; Natsuhara, Mayuko; Takano, Hirotsugu; Tsuchiya, Soken; Okuno, Yasushi; Ohtsu, Hiroshi; Nishibori, Masahiro; Thurmond, Robin L; Hirasawa, Noriyasu; Nakayama, Kazuhisa; Ichikawa, Atsushi; Sugimoto, Yukihiko; Tanaka, Satoshi

    2014-01-01

    Mast cells are the major sources of histamine, which is released in response to immunological stimulations. The synthesis of histamine is catalyzed by histidine decarboxylase (HDC). Previous studies have shown that Hdc(-/-) mast cells exhibit aberrant granule morphology with severely decreased granule content. Here, we investigated whether the histamine synthesized in mast cells regulates the granule maturation of murine mast cells. Several genes, including those encoding granule proteases and enzymes involved in heparin biosynthesis, were downregulated in Hdc(-/-) peritoneal mast cells. Impaired granule maturation was also found in Hdc(-/-) BM-derived cultured mast cells when they were cocultured with fibroblasts in the presence of c-kit ligand. Exogenous application of histamine and several H4 receptor agonists restored the granule maturation of Hdc(-/-) cultured mast cells. However, the maturation of granules was largely normal in Hrh4(-/-) peritoneal mast cells. Depletion of cellular histamine with tetrabenazine, an inhibitor of vesicular monoamine transporter-2, did not affect granule maturation. In vivo experiments with mast cell deficient Kit(W) /Kit(W-v) mice indicated that the expression of the Hdc gene in mast cells is required for granule maturation. These results suggest that histamine promotes granule maturation in mast cells and acts as an proinflammatory mediator. PMID:24002822

  2. Granule Cell Activation of Complex-Spiking Neurons in Dorsal Cochlear Nucleus

    Microsoft Academic Search

    Kevin A. Davis; Eric D. Young

    1997-01-01

    Dorsal cochlear nucleus (DCN) principal cells receive, in addi- tion to their well known auditory inputs, various nonauditory inputs via a cerebellar-like granule cell circuit located in the superficial layers of the DCN. Activation of this circuit (granule cell axons make excitatory synapses on the principal cells but also contact inhibitory interneurons that project to the principal cells) produces strong

  3. Passive sorting in maturing granules of AtT-20 cells: the entry and exit of salivary amylase and proline-rich protein.

    PubMed

    Castle, A M; Huang, A Y; Castle, J D

    1997-07-14

    Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093- 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711- 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry. PMID:9214380

  4. Inhibition of human mast cell chymase by secretory leukocyte proteinase inhibitor: enhancement of the interaction by heparin.

    PubMed

    Walter, M; Plotnick, M; Schechter, N M

    1996-03-01

    The inhibition of human chymase, a chymotrypsin-like proteinase stored in mast cell granules, by secretory leukocyte proteinase inhibitor (SLPI) is investigated in this study. SLPI is a serine proteinase inhibitor present in human mucus secretions and tissues. It binds heparin, a highly sulfated glycosaminoglycan also found in mast cell secretary granules, and the interaction increases its effectiveness as an inhibitor of neutrophil elastase. Analysis of the chymase-SL interaction by equilibrium and kinetic methods indicates that the inhibition of chymase results from the reversible formation of a stable 1:1 enzyme-inhibitor complex. The dissociation equilibrium constant (determined in reactions containing 0.18 M or 1.0M NaCl (pH 8.0, 25 degrees C) was 5 X 10(-8) and 2 x 10(-8) M, respectively. Addition of heparin to the low-salt reaction decreased the Ki approximately 10-fold to a value of 3 x 10(-9) M, making SLPI a more effective inhibitor of human chymase. The decrease was due primarily to an approximately 10-fold increase in the association rate constant (kass) from 2 X 10(4) to 3 X 10(5) M-1 s-1. The magnitudes of the rate and dissociation equilibrium constants indicate that SLPI has the potential to be a good chymase inhibitor in vivo, especially if chymase and heparin are released from mast cell granules simultaneously. The enhanced interaction in the presence of heparin supports the importance of this glycosaminoglycan to the inhibitory function of SLPI. PMID:8615699

  5. Origin of granules in granular cell tumor. Intracellular myelin formation with autodigestion.

    PubMed

    Mittal, K R; True, L D

    1988-03-01

    Eight cases of granular cell tumor were stained with Luxol fast blue to look for the presence of myelin. Luxol fast blue-positive granules were present in all cases. One case was studied ultrastructurally. It showed evidence of origin of granules as infoldings of cell membrane by a process similar to myelin formation around axons. These infoldings subsequently appear to be phagocytosed by lysosomes, resulting in the typical granules of the granular cell tumor. PMID:2830865

  6. Morphological and functional analysis of PTA granules in human NK cells

    Microsoft Academic Search

    S. A. Kolb; Peter Groscurth

    1997-01-01

    Human natural killer (NK) cells contain unique granules with parallel tubular arrays (PTA granules) of approximately 30 nm\\u000a diameter that can be seen only by electron microscopy. In order to clarify the role of PTA granules in NK cell-mediated cytolysis\\u000a we examined these structures with regard to frequency and expression of lytic proteins (perforin, granzymes). NK cells (CD3?, CD16+, CD56+)

  7. Stochastic Differential Equation Model for Cerebellar Granule Cell Excitability

    PubMed Central

    Yli-Harja, Olli

    2008-01-01

    Neurons in the brain express intrinsic dynamic behavior which is known to be stochastic in nature. A crucial question in building models of neuronal excitability is how to be able to mimic the dynamic behavior of the biological counterpart accurately and how to perform simulations in the fastest possible way. The well-established Hodgkin-Huxley formalism has formed to a large extent the basis for building biophysically and anatomically detailed models of neurons. However, the deterministic Hodgkin-Huxley formalism does not take into account the stochastic behavior of voltage-dependent ion channels. Ion channel stochasticity is shown to be important in adjusting the transmembrane voltage dynamics at or close to the threshold of action potential firing, at the very least in small neurons. In order to achieve a better understanding of the dynamic behavior of a neuron, a new modeling and simulation approach based on stochastic differential equations and Brownian motion is developed. The basis of the work is a deterministic one-compartmental multi-conductance model of the cerebellar granule cell. This model includes six different types of voltage-dependent conductances described by Hodgkin-Huxley formalism and simple calcium dynamics. A new model for the granule cell is developed by incorporating stochasticity inherently present in the ion channel function into the gating variables of conductances. With the new stochastic model, the irregular electrophysiological activity of an in vitro granule cell is reproduced accurately, with the same parameter values for which the membrane potential of the original deterministic model exhibits regular behavior. The irregular electrophysiological activity includes experimentally observed random subthreshold oscillations, occasional spontaneous spikes, and clusters of action potentials. As a conclusion, the new stochastic differential equation model of the cerebellar granule cell excitability is found to expand the range of dynamics in comparison to the original deterministic model. Inclusion of stochastic elements in the operation of voltage-dependent conductances should thus be emphasized more in modeling the dynamic behavior of small neurons. Furthermore, the presented approach is valuable in providing faster computation times compared to the Markov chain type of modeling approaches and more sophisticated theoretical analysis tools compared to previously presented stochastic modeling approaches. PMID:18463700

  8. Differentiation of apical and basal dendrites in pyramidal cells and granule cells in dissociated hippocampal cultures.

    PubMed

    Wu, You Kure; Fujishima, Kazuto; Kengaku, Mineko

    2015-01-01

    Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons. PMID:25705877

  9. The secretory ependymal cells of the subcommissural organ: Which role in hydrocephalus?

    Microsoft Academic Search

    Annie Meiniel

    2007-01-01

    Ependyma in the central nervous system gives rise to several specialized cell types, including the secretory ependymal cells located in the subcommissural organ. These elongated cells show large cisternae in their cytoplasm, which are filled with material secreted into the cerebrospinal fluid and toward the leptomeningeal spaces. A specific secretion of the subcommissural organ was named SCO-spondin, regarding its marked

  10. Glial activation modulates glutamate neurotoxicity in cerebellar granule cell cultures.

    PubMed

    Pérez-Capote, Kamil; Serratosa, Joan; Solà, Carme

    2004-02-01

    We studied the influence of glial cells on the neuronal response to glutamate toxicity in cerebellar granule cell cultures. We compared the effect of glutamate on neuronal viability in neuronal vs. neuronal-glial cultures and determined this effect after pretreating the cultures with the lipopolysaccharide (LPS) of Escherichia coli, agent widely used to induce glial activation. Morphological changes in glial cells and nitric oxide (NO) production were evaluated as indicators of glial activation. We observed that glutamate neurotoxicity in neuronal-glial cultures was attenuated in a certain range of glutamate concentration when compared to neuronal cultures, but it was enhanced at higher glutamate concentrations. This enhanced neurotoxicity was associated with morphological changes in astrocytes and microglial cells in the absence of NO production. LPS treatment induced morphological changes in glial cells in neuronal-glial cultures as well as NO production. These effects occurred in the absence of significant neuronal death. However, when LPS-pretreated cultures were treated with glutamate, the sensitivity of neuronal-glial cultures to glutamate neurotoxicity was increased. This was accompanied by additional morphological changes in glial cells in the absence of a further increase in NO production. These results suggest that quiescent glial cells protect neuronal cells from glutamate neurotoxicity, but reactive glial cells increase glutamate neurotoxicity. Therefore, glial cells play a key role in the neuronal response to a negative stimulus, suggesting that this response can be modified through an action on glial cells. PMID:14730699

  11. Anion channel blockers cause apparent inhibition of exocytosis by reacting with agonist or secretory product, not with cell.

    PubMed Central

    Vostal, J G; Reid, D M; Jones, C E; Shulman, N R

    1989-01-01

    Agents that act as anion channel blockers (ACBs) and do not permeate cells appear to inhibit exocytosis in platelets, parathyroid cells, and neutrophils. Based in large part on these observations, anion influx through plasma membrane channels has been considered a factor controlling cellular secretion, but there have been no direct anion influx measurements in cells or granules to support this concept. We have found that ACBs inhibit only thrombin-induced platelet secretion, not secretion induced by ADP, collagen, or A23187. ACBs inhibit thrombin esterolytic activity, binding of thrombin to platelets, and thrombin-stimulated platelet production of malondialdehyde in proportion to the degree of inhibition of thrombin-induced platelet secretion. Thus inhibition of platelet secretion by ACBs is due to inactivation of the stimulatory agonist, thrombin, and not to interference with cellular secretion per se. We have also found that previously reported inhibition of secretion of parathyroid cells and neutrophils by ACBs can be explained by the ability of ACBs to interfere with detection of the cellular secretory products that were measured to assess exocytosis. Our measurements of parathyroid hormone and beta-glucuronidase in the presence of ACBs were reduced to the same degree as the reported reduction in apparent cellular secretion produced by these agents. We conclude that plasma membrane anion channels of the type that can be blocked by ACBs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, suramin, and probenecid do not participate in cellular secretory processes. Whether other types of anion channels exist that are not affected by these ACBs and whether there are mechanisms of anion flux during secretion not dependent on channels remain open questions. PMID:2474820

  12. MUM ENHANCERS are important for seed coat mucilage production and mucilage secretory cell differentiation in Arabidopsis thaliana

    Microsoft Academic Search

    Andrej A. Arsovski; Maria M. Villota; Owen Rowland; Rajagopal Subramaniam

    2009-01-01

    Pollination triggers not only embryo development but also the differentiation of the ovule integuments to form a specialized seed coat. The mucilage secretory cells of the Arabidopsis thaliana seed coat undergo a complex differentiation process in which cell growth is followed by the synthesis and secretion of pectinaceous mucilage. A number of genes have been identified affecting mucilage secretory cell

  13. A histochemical study of the secretory gland cells of Cercaria shikokuensis and their role during development from cercaria to metacercaria

    Microsoft Academic Search

    Masakazu Harada; Setsuo Suguri

    2001-01-01

    The roles of secretory glands during the developmental process from an immature cercaria to a metacercaria in Cercaria shikokuensis were studied. Four types of secretory cells were identified in this species. On maturation of the cercaria in redia, the products of ventral gland cells and mucoid gland cells formed a thick surface coat on the mature cercaria, and the products

  14. Cytoskeleton disruption causes apoptotic degeneration of dentate granule cells in hippocampal slice cultures

    Microsoft Academic Search

    Jeong-Ah Kim; Kayo Mitsukawa; Maki K. Yamada; Nobuyoshi Nishiyama; Norio Matsuki; Yuji Ikegaya

    2002-01-01

    Colchicine, a potent microtubule-depolymerizing agent, is well known to selectively kill dentate granule cells in the hippocampal formation in vivo. Using organotypic cultures of rat entorhino-hippocampal slices, we confirmed that in vitro exposure to 1 ?M and 10 ?M of colchicine reproduced a specific degeneration of the granule cells after 24 h. Similar results were obtained with other types of

  15. 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

  16. Integration of newly born dentate granule cells into adult brains: hypotheses based on normal and epileptic rodents

    Microsoft Academic Search

    Lee A. Shapiro; Charles E. Ribak

    2005-01-01

    The granule cells of the dentate gyrus are a population of neurons continously generated throughout life. In the rat, the morphological development of newly born granule cells generated in the adult share many similarities with granule cells generated during development. These include a specific migration pattern, orientation and progression of neurite outgrowth. It appears as though varied dendritic morphology occurs

  17. Multiple mechanisms for oxygen-induced regulation of the Clara cell secretory protein gene

    Microsoft Academic Search

    Patricia L. Ramsay; Ziqiang Luo; Angela Major; Moon S. Park; Milton Finegold; Steven E. Welty; Inseok Kwak; Gretchen Darlington; Francesco J. DeMayo

    2003-01-01

    The Clara cell secretory protein (CCSP) imparts a protective effect to the lung during oxidant injury. However, exposure to supplemental oxygen, a common therapeutic modality for lung disease, represses the expression of CCSP in the adult mouse lung. We investigated the mechanisms of hyperoxia-induced repression of the mouse CCSP promoter. Deletion experiments in vivo and in vitro indicated that the

  18. Altered Pulmonary Response to Hyperoxia in Clara Cell Secretory Protein Deficient Mice

    Microsoft Academic Search

    Carl J. Johnston; Gregory W. Mango; Jacob N. Finkelstein; Barry R. Stripp

    1997-01-01

    Clara cell secretory protein (CCSP) is an abundant component of the extracellular lining fluid of airways. Even though the in vivo function of CCSP is unknown, in vitro studies support a potential role of CCSP in the control of inflammatory responses. CCSP-deficient mice (CCSP 2 \\/ 2 ) were generated to investigate the in vivo function of this protein (13).

  19. Clara cell secretory protein increases phagocytic and decreases oxidative activity of neutrophils

    Microsoft Academic Search

    P. Katavolos; C. A. Ackerley; M. E. Clark; D. Bienzle

    2011-01-01

    Horses suffer from recurrent airway obstruction, an asthma-like condition induced by repeat inhalation of environmental substances present in barn air. Clara cell secretory protein (CCSP) is much reduced during active inflammation when neutrophils predominate in the airways, and in chronic asthmatics. We sought to investigate morphologic and functional interactions of CCSP with neutrophils. Bronchoalveolar and blood neutrophils from healthy control

  20. 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

  1. Acute Exposure to Methylmercury Opens the Mitochondrial Permeability Transition Pore in Rat Cerebellar Granule Cells

    Microsoft Academic Search

    Tobi L. Limke; William D. Atchison

    2002-01-01

    Cerebellar granule cells are preferentially targeted during methylmercury (MeHg) poisoning. Following acute MeHg exposure, granule cells in culture undergo an increase in intracellular Ca2+ concentration ([Ca2+]i) that apparently contributes to cell death. This effect consists of several temporally and kinetically distinct phases. The initial elevation arises from release of Ca2+i stores; the second phase results from entry of Ca2+e. In

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

    SciTech Connect

    Gianotti, A.J.; Clark, D.T.; Dash, J. (Portland State Univ., OR (USA))

    1991-04-01

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

  3. Alcohol Enhances GABAergic Transmission to Cerebellar Granule Cells via an Increase in Golgi Cell Excitability

    Microsoft Academic Search

    Mario Carta; Manuel Mameli; C. Fernando

    2004-01-01

    Alcohol intoxication alters coordination and motor skills, and this is responsible for a significant number of traffic accident-related deaths around the world. Although the precise mechanism of action of ethanol (EtOH) is presently unknown, studies suggest that it acts, in part, by interfering with normal cerebellar functioning. An important component of cerebellar circuits is the granule cell. The excit- ability

  4. 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

  5. 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

  6. Cell death and immunityThe ABCs of granule-mediated cytotoxicity: new weapons in the arsenal

    Microsoft Academic Search

    Judy Lieberman

    2003-01-01

    Granule exocytosis is the main pathway for the immune elimination of virus-infected cells and tumour cells by cytotoxic T lymphocytes and natural killer cells. After target-cell recognition, release of the cytotoxic granule contents into the immunological synapse formed between the killer cell and its target induces apoptosis. The granules contain two membrane-perturbing proteins, perforin and granulysin, and a family of

  7. Early Postnatal Surge of Serum Clara Cell Secretory Protein in Newborn Infants

    Microsoft Academic Search

    A. S. Loughran-Fowlds; J. W. Lin; J. Oei; J. Michalowski; R. Henry; K. Lui

    2012-01-01

    Background: Clara cell secretory protein (CCSP) is an anti-inflammatory mediator, but its role in neonatal lung adaptation and diseases is uncertain. Objective: To characterize postnatal changes in serum CCSP in relation to gestation, respiratory disease (RDS) and bronchopulmonary dysplasia (BPD) in comparison with other anti-inflammatory cytokines (IL-4, -10 and -13). Methods: Blood was collected from 76 infants (26 of 23–29

  8. Exocyst Sec5 Regulates Exocytosis of Newcomer Insulin Granules Underlying Biphasic Insulin Secretion

    PubMed Central

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

    2013-01-01

    The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet ? cells in part by tethering insulin secretory granules to Ca2+ channels to assist excitosome assembly. Here, we assessed ? cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet ? cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in ? cells are more sensitive than predocked granules to Sec5 regulation. PMID:23844030

  9. The transcription factor Cux1 in cerebellar granule cell development and medulloblastoma pathogenesis.

    PubMed

    Topka, Sabine; Glassmann, Alexander; Weisheit, Gunnar; Schüller, Ulrich; Schilling, Karl

    2014-12-01

    Cux1, also known as Cutl1, CDP or Cut is a homeodomain transcription factor implicated in the regulation of normal and oncogenic development in diverse peripheral tissues and organs. We studied the expression and functional role of Cux1 in cerebellar granule cells and medulloblastoma. Cux1 is robustly expressed in proliferating granule cell precursors and in postmitotic, migrating granule cells. Expression is lost as postmigratory granule cells mature. Moreover, Cux1 is also strongly expressed in a well-established mouse model of medulloblastoma. In contrast, expression of CUX1 in human medulloblastoma tissue samples is lower than in normal fetal cerebellum. In these tumors, CUX1 expression tightly correlates with a set of genes which, when mapped on a global protein-protein interaction dataset, yields a tight network that constitutes a cell cycle control signature and may be related to p53 and the DNA damage response pathway. Antisense-mediated reduction of CUX1 levels in two human medulloblastoma cell lines led to a decrease in proliferation and altered motility. The developmental expression of Cux1 in the cerebellum and its action in cell lines support a role in granule cell and medulloblastoma proliferation. Its expression in human medulloblastoma shifts that perspective, suggesting that CUX1 is part of a network involved in cell cycle control and maintenance of DNA integrity. The constituents of this network may be rational targets to therapeutically approach medulloblastomas. PMID:25096634

  10. [Histochemical characteristics of the secretory cells of gastric glands compared].

    PubMed

    Shubich, M G; Mogil'naia, G M; Dudetski?, V I; Bogatyr', L Ia

    1978-02-01

    The work is dedicated to complex histological studies of the secreting cells in the gastric fundal glands, in their comparative aspect. In the representatives of Amphibia, Reptilians and birds, histochemical differentiation of oxyntopeptic cells was demonstrated to be independent on the peculiarities of the animal nutrition. In mammals, histochemical characteristic of the carbohydrate component in the glandular secreting cells depends on the type of nutrition. PMID:646638

  11. Mesenchymal cell activation is the rate-limiting step of granulation tissue induction.

    PubMed Central

    McClain, S. A.; Simon, M.; Jones, E.; Nandi, A.; Gailit, J. O.; Tonnesen, M. G.; Newman, D.; Clark, R. A.

    1996-01-01

    During wound repair a 3-day lag occurs between injury and granulation tissue development. When full-thickness, 8-mm-round, excisional wounds were made in the paravertebral skin of outbred Yorkshire pigs and harvested at various times, no granulation tissue was observed before day 4. Day 4 wounds were 3% filled with granulation tissue, day 5 wounds 48% filled, and day 7 wounds 88% filled. The prerequisites for granulation tissue induction are not known but hypothetically include fibrin matrix maturation or cell activation. To examine whether matrix maturation was necessary, wounds were allowed to heal for 5 or 7 days and then aggressively curetted, resulting in the formation of fresh fibrin clots in the newly formed wound spaces. In contrast to original wounds, no lag phase was observed; wounds curetted on day 5 were 23% filled with granulation tissue 1 day later and 99% filled 3 days later, whereas wounds curetted on day 7 were 47% filled 1 day later and completely filled within 2 days. Thus, granulation tissue formation resumed promptly and independently of fibrin clot matrix maturation. This observation suggested that mesenchymal cell activation might be the rate-limiting step in granulation tissue formation. To address this hypothesis more directly, cultured porcine or human fibroblasts, grown to 80% confluence in Dulbecco's minimal essential medium plus 10% fetal calf serum, were added to new wounds. These wounds were sealed with a freshly made exogenous fibrin clot. In some wounds, platelet releasate was added to the fibrin clot. Granulation tissue did not form in day 3 wounds, which had received either fibrin alone, fibrin and platelet releasate, or fibrin and fibroblasts. In contrast, granulation tissue was observed in wounds receiving fibrin, human fibroblasts, and platelet releasate. By day 4, wounds receiving cultured human fibroblasts, fibrin, and platelet releasate were 14% filled with granulation tissue compared with less than 4% granulation tissue in control wounds. Thus, fibroblast activation is a limiting step of granulation tissue formation, and continued cell stimulation is required for accelerated development. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8863674

  12. Effect of disturbance of nervous integration on relations between auricular granules and other components of the myocytes

    Microsoft Academic Search

    V. G. Tsyplenkova

    1973-01-01

    UDC 611.127-018.63 +616.125-018.63-02 : 616.833.191.5-089.85-092.9 The morphology of the specific auricular granules and their connection with the cell organelles of the cardiomyocytes were studied by electron microscopy in normal rats and after division of the right vagus nerve; The results suggest that the muscle cells of the mamalian auricles have a secretory function. The secretory activity of the cardiomyocytes is

  13. An ultrastructural study of the morphology and lectin-binding properties of human mast cell granules.

    PubMed

    Jones, C J; Kirkpatrick, C J; Stoddart, R W

    1988-08-01

    The morphological characteristics and lectin-binding properties of mast cell granules from four human neurofibromata are described. Ultrastructural examination of the granules revealed that some contained dense cores, others had membranous configurations and some forms were intermediate between the two. A round electron-lucent area was present in some granules. After treatment with biotinylated lectins (10 micrograms ml-1) followed by an avidin-peroxidase revealing system (5 micrograms ml-1 in 0.125 M Tris-buffered saline with 0.347 M NaCl, pH 7.6), mast cell granules strongly bound Concanavalin A, garden pea, lentil, wheatgerm, erythro- and leuco-kidney bean lectins. This indicated the presence of abundant N-linked complex-type saccharide sequences. Soybean and peanut lectins showed only weak binding, while the presence of sparse alpha-L-fucosyl terminals was indicated by the weak binding of winged pea lectin. The staining intensity of wheatgerm lectin was considerably reduced when incubated in the presence of its specific competing sugar tri-N-acetylchitotriose. Despite a wide variety of morphological differences between granules, all showed similar staining patterns and all granules within a single cell shared the same binding characteristics. PMID:3198420

  14. Natural secretory products of human neural and microvessel endothelial cells

    Microsoft Academic Search

    Yuhai Zhao; Jian-Guo Cui; Walter J. Lukiw

    2006-01-01

    Neurons, glia, and endothelial cells of the cerebral microvasculature co-exist in intimate proximity in nervous tissues, and\\u000a their homeostatic interactions in health, as well as coordinated response to injury, have led to the concept that they form\\u000a the basic elements of a functional neurovascular unit. During the course of normal cellular metabolism, growth, and development,\\u000a each of these brain cell

  15. Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

    Microsoft Academic Search

    Manus W. Ward; A. Cristina Rego; Bruno G. Frenguelli; David G. Nicholls

    2000-01-01

    The relationship between changes in mitochondrial membrane potential (Dcm) and the failure of cytoplasmic Ca 21 homeostasis, delayed Ca 21deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetrameth- ylrhodamine methyl ester (TMRM 1) or rhodamine-123, we de- vised and validated a mathematical simulation with well

  16. Expression of tryptophan 2,3-dioxygenase in mature granule cells of the adult mouse dentate gyrus.

    PubMed

    Ohira, Koji; Hagihara, Hideo; Toyama, Keiko; Takao, Keizo; Kanai, Masaaki; Funakoshi, Hiroshi; Nakamura, Toshikazu; Miyakawa, Tsuyoshi

    2010-01-01

    New granule cells are continuously generated in the dentate gyrus of the adult hippocampus. During granule cell maturation, the mechanisms that differentiate new cells not only describe the degree of cell differentiation, but also crucially regulate the progression of cell differentiation. Here, we describe a gene, tryptophan 2,3-dioxygenase (TDO), whose expression distinguishes stem cells from more differentiated cells among the granule cells of the adult mouse dentate gyrus. The use of markers for proliferation, neural progenitors, and immature and mature granule cells indicated that TDO was expressed in mature cells and in some immature cells. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, in which dentate gyrus granule cells fail to mature normally, TDO immunoreactivity was substantially downregulated in the dentate gyrus granule cells. Moreover, a 5-bromo-2'-deoxyuridine labeling experiment revealed that new neurons began to express TDO between 2 and 4 wk after the neurons were generated, when the axons and dendrites of the granule cells developed and synaptogenesis occurred. These findings indicate that TDO might be required at a late-stage of granule cell development, such as during axonal and dendritic growth, synaptogenesis and its maturation. PMID:20815922

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

    PubMed Central

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

    2011-01-01

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

  18. Structure of the secretory cells of male and female adult guinea pigs Harderian gland.

    PubMed

    Hussein, Ola A; Elgamal, Dalia A; Elgayar, Sanaa A M

    2015-06-01

    The main objective of this study was to investigate the structure of the Harderian gland (HG) in male and female guinea pigs. A total number of sixteen animals of 4 months age were divided according to sex into two groups; eight animals each. Unfixed glands were weighed and their length and width were measured. Specimens from fixed glands were processed and examined using light, transmission electron microscopy and immunohistochemistry for the detection of the presence of chromogranin A (CgA). The gland consisted of a well-developed duct system which included both intra and extra parenchymal ducts and secretory end pieces lined by many types of cells of variable morphological features and modes of secretion. However, the holocrine mode of secretion was rare as mitotic figures were occasionally present. The interstitial cells included fibroblasts and immune cells (mast cells, lymphocyte, plasma cells and macrophages). The secretion produced by the gland included lipid, protein, neutral mucin and CgA which may be a newly identified constituent of biologically potent proteins stored in the cells of the guinea pig HG. Neutral mucin and CgA may function in photoprotection. The gland revealed sexual dimorphism in mast cells and blood capillaries number and chromogranin secretory activity. PMID:25960413

  19. The sory of cell secretion: events leading to the dicovery of the ‘porosome’- the universal secretory machinery in cells

    PubMed Central

    Jefingija, S

    2006-01-01

    Cell secretio has come age, and a century old quest has been elegantly solved. We have come a long way since earlier observations of what appeared to be ‘fibrillar regions’ at teh cell plasama membrance, and electrophysological studies suggesting the presence of ‘fusion pores’ at the cell plasma membrane where secretion occurs. Finally, the fusion pore or ‘porosome’ has been discovered, and its morpholgy and dynamics determined at nm resolution and in real time in live secretory cells. The porosome has been isolated, its omposition determined at nm resolution and in real time in live secretory cells. The porosme has been isolated, its composition determmined and it has been jkboth structurally and functionally reconsituted n artificial lipid membrance. The discoversy of the porosome as the univeral secretory machinery in cell and the discovery of the molecular mechaninsm of vesicular content expulsion during cell secretin have fially enabled a clear understanding of this important cellular process. This review outlines the fascinating and exciting joumey leding to the dicovery of the porosme, ultimately solving one of the most difficut, significant, and fundamental cellular process- cell seretion. PMID:16796798

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

    PubMed Central

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

    2012-01-01

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

  1. [Effect of plant hormones on the components of secretory pathway in human normal and tumor cells].

    PubMed

    Vil'danova, M S; Savitskaia, M A; Onishchenko, G E; Smirnova, E A

    2014-01-01

    Plant hormones play a key role in plant growth and differentiation. Many hormones are known as potential antitumor agents, yet others appear to affect the secretory activity and are produced by mammalian cells as pro-inflammatory cytokines. The goal of this research was to study the effect of abscisic and gibberellic acids on the secretory system of human cultured epidermoid carcinoma cells A431 and keratinocytes HaCat. Immunocytochemical and morphometric analysis demonstrated that subtoxic concentration of plant hormones induced the broadening of the ER network and increased the size of Golgi complex. Electron microscopy studies confirmed the hypertrophic changes of the Golgi apparatus, specifically, the swelling of cisternae in the trans-compartment of dictyosomes after exposure to abscisic acid, and swelling of cis- and trans-compartment of dictyosomes after exposure to abscisic acid, and swelling of cis- and trans-compartments of dictyosomes after exposure to gibberellic acid. Using of Click-iT technique allowed to detect the elevation of the total protein synthesis only in A431 cells exposed to abscisic acid. Cumulative data suggests that, under these conditions, the hypertrophy of Golgi apparatus may reflect the enhanced secretory activity of cells. In other experiments, the hypertrophy of Golgi is not related to increased protein synthesis and therefore may suggest the stress-related changes of ER and Golgi apparatus. Our results demonstrate that morphologically similar reaction of cellular organelles, such as hypertrophy of Golgi apparatus, is the result of different functional activities, and that molecular mechanisms underlying the changes induced in cells need further investigations. PMID:25696996

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

    PubMed

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

    2014-12-01

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

  3. The a6 Subunit of the GABA, Receptor Is Concentrated in Both Inhibitory and Excitatory Synapses on Cerebellar Granule Cells

    Microsoft Academic Search

    Zoltan Nusser; Werner Sieghatt; F. Anne Stephenson; Peter Somogyil

    Although three distinct subunits seem to be sufficient to form a functional pentameric GABA, receptor channel, cerebellar granule cells express mRNA for nine subunits. They receive GABAergic input from a relatively homogeneous population of Golgi cells. It is not known whether all subunits are distributed similarly on the surface of granule cells or whether some of them have differential subcellular

  4. A General Role for Rab27a in Secretory CellsD?V?

    PubMed Central

    Tolmachova, Tanya; Anders, Ross; Stinchcombe, Jane; Bossi, Giovanna; Griffiths, Gillian M.; Huxley, Clare; Seabra, Miguel C.

    2004-01-01

    Vesicular transport is a complex multistep process regulated by distinct Rab GTPases. Here, we show for the first time that an EGFP-Rab fusion protein is fully functional in a mammalian organism. We constructed a PAC-based transgenic mouse, which expresses EGFP-Rab27a under the control of endogenous Rab27a promoter. The EGFP-Rab27a transgene was fully functional and rescued the two major defects of the ashen Rab27a knockout mouse. We achieved cell-specific expression of EGFP-Rab27a, which faithfully followed the pattern of expression of endogenous Rab27a. We found that Rab27a is expressed in an exceptionally broad range of specialized secretory cells, including exocrine (particularly in mucin- and zymogen-secreting cells), endocrine, ovarian, and hematopoietic cells, most of which undergo regulated exocytosis. We suggest that Rab27a acts in concert with Rab3 proteins in most regulated secretory events. The present strategy represents one way in which the complex pattern of expression and function of proteins involved in specialized cell types may be unraveled. PMID:14617806

  5. Changes in mobility of chromaffin granules in actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin.

    PubMed Central

    Miyamoto, S; Funatsu, T; Ishiwata, S; Fujime, S

    1993-01-01

    As a final stage of cell signal transduction, secretory cells release hormones by exocytosis. Before secretory granules contact with the cell membrane for fusion, an actin-network barrier must dissociate as a prelude. To elucidate dynamical behaviors of secretory granules in actin networks, in vitro assembly and disassembly processes of actin networks were examined by means of dynamic light-scattering spectroscopy. We studied actin polymerization in the presence of chromaffin granules isolated from bovine adrenal medullas and found that the entanglement of actin filaments rapidly formed cages that confined granules in them. We also studied the effect of gelsolin, one of actin-severing proteins, on the network of actin filaments preformed in the presence of chromaffin granules. It turned out that the cages that confined granules rapidly disappeared when gelsolin was added in the presence of free Ca2+ ions. A semiquantitative analysis of dynamic light-scattering spectra permitted us to estimate the changes in the mobility (or the translational diffusion coefficient) of chromaffin granules in the actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin. Based on the present results and some pieces of evidence in the literature, a model is proposed for biophysical situations before, during, and after an exocytotic event. PMID:8388266

  6. Benzodiazepine receptor increases after repeated seizures: evidence for localization to dentate granule cells.

    PubMed Central

    Valdes, F; Dasheiff, R M; Birmingham, F; Crutcher, K A; McNamara, J O

    1982-01-01

    Repeated seizures, whether induced by kindling or electroshock, result in increased numbers of benzodiazepine receptors in hippocampal formation membranes. We sought to determine the cellular constituents containing the receptor increases. Binding studies of microdissected samples localized the receptor increases to fascia dentata. [3H]Flunitrazepam autoradiographic studies showed increases of silver grain density over the granule cell and molecular layers of fascia dentata but not in other regions of hippocampal formation. Destruction of granule cells by colchicine or neonatal x-irradiation was associated with marked decrease of benzodiazepine receptor binding. Together, these results provide evidence for localization of the receptor increases to the somata and dendritic tree of the granule cells. We suggest that this cellular localization may provide a clue to the network of altered neural circuitry underlying amygdala kindling. Images PMID:6275387

  7. Benzodiazepine receptor increases after repeated seizures: evidence for localization to dentate granule cells.

    PubMed

    Valdes, F; Dasheiff, R M; Birmingham, F; Crutcher, K A; McNamara, J O

    1982-01-01

    Repeated seizures, whether induced by kindling or electroshock, result in increased numbers of benzodiazepine receptors in hippocampal formation membranes. We sought to determine the cellular constituents containing the receptor increases. Binding studies of microdissected samples localized the receptor increases to fascia dentata. [3H]Flunitrazepam autoradiographic studies showed increases of silver grain density over the granule cell and molecular layers of fascia dentata but not in other regions of hippocampal formation. Destruction of granule cells by colchicine or neonatal x-irradiation was associated with marked decrease of benzodiazepine receptor binding. Together, these results provide evidence for localization of the receptor increases to the somata and dendritic tree of the granule cells. We suggest that this cellular localization may provide a clue to the network of altered neural circuitry underlying amygdala kindling. PMID:6275387

  8. Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells

    Microsoft Academic Search

    Samantha L. Budd; Roger F. Castilho; David G. Nicholls

    1997-01-01

    Mitochondrial depolarisation has been reported to enhance the generation of superoxide anion (O??2) in a number of cell preparations while an inhibition has been observed with isolated mitochondria. Cerebellar granule cells equilibrated with >1 ?M hydroethidine (dihydroethidium) which is oxidised to the fluorescent ethidium cation by O??2 showed a large increase in fluorescence on protonophore addition. However, controls showed the

  9. Calcium Influx through NMDA Receptors Directly Evokes GABA Release in Olfactory Bulb Granule Cells

    E-print Network

    Strowbridge, Ben

    Calcium Influx through NMDA Receptors Directly Evokes GABA Release in Olfactory Bulb Granule Cells, Case Western Reserve University, Cleveland, Ohio 44106-4975 Recurrent inhibition in olfactory bulb on the activation of NMDA receptors. Using whole-cell recordings from rat olfactory bulb slices, we now show

  10. Glutathione Levels Modulate Domoic Acid Induced Apoptosis in Mouse Cerebellar Granule Cells

    Microsoft Academic Search

    Gennaro Giordano; Collin C. White; Isaac Mohar; Terrance J. Kavanagh; Lucio G. Costa

    2007-01-01

    Exposure of mouse cerebellar granule neurons (CGNs) to domoic acid induced cell death, either by apoptosis or by necrosis, depending on its concentration. Necrotic damage predominated in response to domoic acid above 0.1mM. In contrast, cell injury with apoptotic features (assessed by Hoechst staining and DNA laddering assay) was evident after exposure to lower concen- trations of domoic acid (£

  11. Non-secretory multiple myeloma presenting as primary plasma cell leukaemia.

    PubMed Central

    Sureda, A.; Pais, J. R.; Pascual, J.; Pérez Vaquero, M. A.; Hernando, J. C.

    1992-01-01

    A case of non-secretory multiple myeloma presenting as primary plasma cell leukaemia in a 65 year old woman is presented. Bone pain was the initial clinical manifestation. Laboratory analysis showed 20% of circulating immature plasma cells. Despite the presence of osteolytic lesions, no M-component could be demonstrated in serum protein electrophoresis, and serum and urine immunoelectrophoresis. Bone marrow aspirate demonstrated an 83% infiltration of plasma cells showing various degrees of immaturity. Immunofluorescence with monoclonal antisera demonstrated intracytoplasmic kappa light chains in a high percentage of plasma cells. Immature plasma cells without cellular capacity to synthesize and excrete complete immunoglobulins could be more aggressive, leading to an initial leukaemic process. Previous work regarding possible pathogenetic mechanisms, clinical and laboratory features, and response to treatment of this extremely rare association are reviewed. PMID:1437933

  12. The secretory ependymal cells of the subcommissural organ: which role in hydrocephalus?

    PubMed

    Meiniel, Annie

    2007-01-01

    Ependyma in the central nervous system gives rise to several specialized cell types, including the secretory ependymal cells located in the subcommissural organ. These elongated cells show large cisternae in their cytoplasm, which are filled with material secreted into the cerebrospinal fluid and toward the leptomeningeal spaces. A specific secretion of the subcommissural organ was named SCO-spondin, regarding its marked homology with developmental proteins of the thrombospondin superfamily (presence of thrombospondin type 1 repeats). The ependymal cells of the subcommissural organ and SCO-spondin secretion are suspected to play a crucial role in cerebrospinal fluid flow and/or homeostasis. There is a close correlation between absence of the subcommissural organ and hydrocephalus in rat and mouse strains exhibiting congenital hydrocephalus, and in a number of mice transgenic for developmental genes. The ependymal cells of the subcommissural organ are under research as a key factor in several developmental processes of the central nervous system. PMID:17150405

  13. Regulatory volume increase after secretory volume decrease in colonic epithelial cells under muscarinic stimulation.

    PubMed

    Manabe, Ken-ichi; Shimizu, Takahiro; Morishima, Shigeru; Okada, Yasunobu

    2004-09-01

    To address the question of whether colonic secretory cells change their volume in response to carbachol (CCh) stimulation and, if so, the mechanisms involved therein, we used two-photon laser scanning microscopy to measure the volume of individual epithelial cells in the fundus region of crypts isolated from the guinea-pig distal colon. We also measured the volume of human colonic epithelial T84 cells using an electronic sizing technique. Both types of colonocytes responded to stimulation by CCh with shrinkage and then underwent a regulatory volume increase (RVI), even during continued stimulation by CCh. The secretory volume decrease (SVD) induced by CCh was antagonized by atropine, BAPTA loading and niflumic acid, a blocker of Ca(2+)-activated Cl(-) channels. An increase in the intracellular free [Ca(2+)] was observed with fura-2 during these volume responses to CCh. Removal of all Na(+) or K(+) or of most of the Cl(-) from the extracellular solution abolished the RVI, but not the preceding SVD. The RVI, but not the preceding SVD, was abolished by bumetanide, a blocker of the Na(+)-K(+)-2Cl(-) cotransporter. We conclude that guinea-pig crypt colonocytes and human T84 cells exhibit a cytosolic Ca(2+)-dependent SVD and undergo a subsequent RVI that is dependent on the operation of Na(+)-K(+)-2Cl(-) cotransporters. PMID:15243741

  14. The origin and role of autophagy in the formation of cytoplasmic granules in canine lingual granular cell tumors.

    PubMed

    Suzuki, S; Uchida, K; Harada, T; Nibe, K; Yamashita, M; Ono, K; Nakayama, H

    2015-05-01

    Granular cell tumors (GCTs) are histologically characterized by polygonal neoplastic cells with abundant eosinophilic cytoplasmic granules. In humans, these cells are considered to be derived from Schwann cells, and the cytoplasmic granules are assumed to be autophagosomes or autophagolysosomes. However, the origin and nature of the cytoplasmic granules in canine GCTs have not been well characterized. The present study examined 9 canine lingual GCTs using immunohistochemistry, transmission electron microscopy (TEM), and cell culture and xenotransplantation experiments. In some cases, the tumor cells expressed S100, CD133, and desmin. The cytoplasmic granules were positive for LC3, p62, NBR1, and ubiquitin. TEM revealed autophagosome-like structures in the cytoplasm of the granule-containing cells. The cultured GCT cells were round to spindle shaped and expressed S100, nestin, Melan-A, CD133, LC3, p62, NBR1, and ubiquitin, suggesting that they were of neural crest origin, redifferentiated into melanocytes, and exhibited upregulated autophagy. The xenotransplanted tumors consisted of spindle to polygonal cells. Only a few cells contained cytoplasmic granules, and some had melanin pigments in their cytoplasm. The xenotransplanted cells expressed S100, nestin, Melan-A, and CD133. P62 and ubiquitin were detected, regardless of the presence or absence of cytoplasmic granules, while LC3 and NBR1 were detected only in the neoplastic cells containing cytoplasmic granules. These findings suggest that some xenotransplanted cells redifferentiated into melanocytes and that autophagy was upregulated in the cytoplasmic granule-containing cells. In conclusion, canine lingual GCTs originate from the neural crest and develop cytoplasmic granules via autophagy. In addition, the microenvironment of GCT cells affects their morphology. PMID:25161210

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

    PubMed Central

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

    2010-01-01

    Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons. PMID:20941361

  16. 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

  17. Bone marrow cells expressing clara cell secretory protein increase epithelial repair after ablation of pulmonary clara cells.

    PubMed

    Bustos, Martha L; Mura, Marco; Marcus, Paula; Hwang, David; Ludkovski, Olga; Wong, Amy P; Waddell, Thomas K

    2013-06-01

    We have previously reported a subpopulation of bone marrow cells (BMC) that express Clara cell secretory protein (CCSP), generally felt to be specific to lung Clara cells. Ablation of lung Clara cells has been reported using a transgenic mouse that expresses thymidine kinase under control of the CCSP promoter. Treatment with ganciclovir results in permanent elimination of CCSP(+) cells, failure of airway regeneration, and death. To determine if transtracheal delivery of wild-type bone marrow CCSP(+) cells is beneficial after ablation of lung CCSP(+) cells, transgenic mice were treated with ganciclovir followed by transtracheal administration of CCSP(+) or CCSP(-) BMC. Compared with mice administered CCSP(-) cells, mice treated with CCSP(+) cells had more donor cells lining the airway epithelium, where they expressed epithelial markers including CCSP. Although donor CCSP(+) cells did not substantially repopulate the airway, their administration resulted in increased host ciliated cells, better preservation of airway epithelium, reduction of inflammatory cells, and an increase in animal survival time. Administration of CCSP(+) BMC is beneficial after permanent ablation of lung Clara cells by increasing bronchial epithelial repair. Therefore, CCSP(+) BMC could be important for treatment of lung diseases where airways re-epithelialization is compromised. PMID:23609017

  18. Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota.

    PubMed

    Lindner, Cornelia; Thomsen, Irene; Wahl, Benjamin; Ugur, Milas; Sethi, Maya K; Friedrichsen, Michaela; Smoczek, Anna; Ott, Stephan; Baumann, Ulrich; Suerbaum, Sebastian; Schreiber, Stefan; Bleich, André; Gaboriau-Routhiau, Valérie; Cerf-Bensussan, Nadine; Hazanov, Helena; Mehr, Ramit; Boysen, Preben; Rosenstiel, Philip; Pabst, Oliver

    2015-08-01

    Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis. However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how maternal antibodies are optimized throughout life to protect the newborn. PMID:26147688

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

    PubMed Central

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

    2014-01-01

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

  20. Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness.

    PubMed

    Wang, Xiao-Yang; Keefe, Kathleen M; Jensen-Taubman, Sandra M; Yang, Danlei; Yan, Kai; Linnoila, R Ilona

    2012-01-01

    Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ?25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+) cells. Moreover, CCSP(+) cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-)expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP(+) cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models. PMID:22916196

  1. Status epilepticus increases mature granule cells in the molecular layer of the dentate gyrus in rats?

    PubMed Central

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

    2013-01-01

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

  2. The lack of extracellular Na+ exacerbates Ca2+-dependent damage of cultured cerebellar granule cells.

    PubMed

    Isaev, N K; Stelmashook, E V; Alexandrova, O P; Andreeva, N A; Polyakova, I A; Victorov, I V; Zorov, D B

    1998-08-28

    Rhodamine 123 staining, light and electron microscopy were used to evaluate the ultrastructural and functional state of cultured cerebellar granule cells after short treatment with the solution where NaCl was substituted by sucrose (sucrose balance salt medium, SBSM). Cell exposure to SBSM for 20 min resulted in the fact that mitochondria in the neurons lost their ability to sequester rhodamine 123. This effect could be prevented by: (i) non-competitive N-methyl-D-aspartate (NMDA) receptor channel blocker, 10(-5) M MK-801; (ii) a competitive specific antagonist of NMDA glutamate receptors, 0.25 x 10(-3) M D,L-2-amino-7-phosphonoheptanoate (APH); (iii) 10(-3) M cobalt chloride; (iv) removal of Ca2+ from the medium. Low Na+ in the Ca2+-containing medium caused considerable mitochondrial swelling in granule cells. However, the same treatment in the absence of calcium ions in the medium abolished the deleterious effect of SBSM on the neuronal mitochondrial structure and functions. It is suggested that (i) the exposure of cultured cerebellar granule cells to SBSM leads to a release of endogenous glutamate from cells; (ii) Ca2+ ions potentially de-energizing neuronal mitochondria enter the neuron preferentially through the NMDA channels rather than through the Na+/Ca2+ exchanger; (iii) mitochondrial swelling in granule cells is highly Ca2+-dependent; (iv) cellular overload with sodium ions can activate mitochondrial Na+/Ca2+ exchanger and thus prevent permeability transition pore opening in mitochondria. PMID:9738475

  3. Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells.

    PubMed

    Shomorony, A; Pfeifer, C R; Aronova, M A; Zhang, G; Cai, T; Xu, H; Notkins, A L; Leapman, R D

    2015-08-01

    A combination of two-dimensional (2D) and three-dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in the endocrine ? and ? cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of a ? cell. PMID:26139222

  4. Mining the granule proteome: a potential source of endocrine biomarkers.

    PubMed

    Albrethsen, Jakob; Goetze, Jens P; Johnsen, Anders H

    2015-01-01

    Proteomics of secretory granules is an emerging strategy for identifying secreted proteins, including potentially novel candidate biomarkers and peptide hormones. In addition, proteomics can provide information about the abundance, localization and structure (post-translational modification) of granule proteins and peptides. Analytical strategies within this research line include so-called 'subtractive proteomics', 'peptidomics' and granule purification by the use of multiple gradient centrifugations. Here we review the literature, and describe the challenges and opportunities in proteomics of secretory granules. PMID:25731211

  5. Evidence for evoked release of adenosine and glutamate from cultured cerebellar granule cells

    SciTech Connect

    Schousboe, A.; Frandsen, A.; Drejer, J. (Univ. of Copenhagen (Denmark))

    1989-09-01

    Evoked release of ({sup 3}H)-D-aspartate which labels the neurotransmitter glutamate pool in cultured cerebellar granule cells was compared with evoked release of adenosine from similar cultures. It was found that both adenosine and (3H)-D-aspartate could be released from the neurons in a calcium dependent manner after depolarization of the cells with either 10-100 microM glutamate or 50 mM KCl. Cultures of cerebellar granule cells treated with 50 microM kainate to eliminate GABAergic neurons behaved in the same way. This together with the observation that cultured astrocytes did not exhibit a calcium dependent, potassium stimulated adenosine release strongly suggest that cerebellar granule cells release adenosine in a neurotransmitter-like fashion together with glutamate which is the classical neurotransmitter of these neurons. Studies of the metabolism of adenosine showed that in the granule cells adenosine is rapidly metabolized to ATP, ADP, and AMP, but in spite of this, adenosine was found to be released preferential to ATP.

  6. Normal function and lack of fibronectin accumulation in kidneys of Clara cell secretory protein\\/uteroglobin deficient mice

    Microsoft Academic Search

    Susan D. Reynolds; Gregory W. Mango; Robert Gelein; Inger-Margrethe Bøe; Johan Lund; Barry R. Stripp

    1999-01-01

    Clara cell secretory protein (CCSP), also known as uteroglobin (Ug), is a 16-kDa homodimeric protein of unknown function. Within rodent species, CCSP is expressed predominantly by nonciliated Clara cells that line conducting airways of the lung. To investigate in vivo functions for CCSP, we established mice homozygous for a null allele of the CCSP gene (CCSP?\\/?). We previously showed no

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

    Microsoft Academic Search

    C. G.. Plopper; G. W. Mango; G. E. Hatch; V. J. Wong; E. Toskala; S. D. Reynolds; B. K. Tarkington; B. R. Stripp

    2006-01-01

    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 (O3). 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

  8. Postnatal lung development of rhesus monkey airways: cellular expression of Clara cell secretory protein.

    PubMed

    Coppens, John T; Plopper, Charles G; Murphy, Shannon R; Van Winkle, Laura S

    2009-12-01

    Clara cell secretory protein (CCSP) is a protective lung protein that is believed to have antioxidant, immunomodulatory, and anticarcinogenic properties. Evidence suggests that CCSP is involved in mitigating many lung disease states during development including asthma. This study's rationale is to define the distribution and abundance of CCSP in the airway epithelium of the rhesus monkey during postnatal lung development using carefully controlled site-specific morphometric approaches in defined airway regions. Immunoreactive CCSP was found in nonciliated cells and mucous cells, including glands, throughout the airway epithelium at all ages, with proximal and mid-level airways having the highest labeling. Overall airway CCSP levels were low at 1 week and 1 month, doubled between 1 and 3 months, and changed little from 3 months to 3 years. Thus, the critical developmental window for CCSP expression to reach adult levels in the rhesus conducting airways occurs between 1 and 3 months of age. PMID:19877270

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

    SciTech Connect

    Huang, C.M.; Liu, G.; Huang, R.H. (Univ. of Missouri, Kansas City (United States))

    1991-03-11

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

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

    PubMed Central

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

    2013-01-01

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

  11. Long-Term Potentiation in Cultures of Single Hippocampal Granule Cells: A Presynaptic Form of Plasticity

    Microsoft Academic Search

    Gang Tong; Robert C Malenka; Roger A Nicoll

    1996-01-01

    We have explored the mechanisms of mossy fiber long-term potentiation (LTP) at autapses in single-cell cultures of guinea pig hippocampal dentate granule cells. L?AP4-sensitive, but not insensitive, cells responded to a brief tetanus with a sustained potentiation in the synaptic responses. The induction of this LTP appeared identical to that observed in hippocampal mossy fiber synapses in situ, in that

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

    PubMed

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

    2014-08-01

    Colchicine has been discovered to inhibit many inflammatory processes such as gout, familial Mediterranean fever, pericarditis and Behcet disease. Other than these beneficial anti-inflammatory effects, colchicine blocks microtubule-assisted axonal transport, which results in the selective loss of dentate granule cells of the hippocampus. The mechanism of the colchicine-induced dentate granule cell death and depletion of mossy fiber terminals still remains unclear. In the present study, we hypothesized that colchicine-induced dentate granule cell death may be caused by accumulation of labile intracellular zinc. 10 ?g kg(-1) of colchicine was injected into the adult rat hippocampus and then brain sections were evaluated at 1 day or 1 week later. Neuronal cell death was evaluated by H&E staining or Fluoro-Jade B. Zinc accumulation and vesicular zinc were detected by N-(6-methoxy-8-quinolyl)-para-toluene sulfonamide (TSQ) staining. To test whether an extracellular zinc chelator can prevent this process, CaEDTA was injected into the hippocampus over a 5 min period with colchicine. To test whether other microtubule toxins also produce similar effects as colchicine, vincristine was injected into the hippocampus. The present study found that colchicine injection induced intracellular zinc accumulation in the dentate granule cells and depleted vesicular zinc from mossy fiber terminals. Injection of a zinc chelator, CaEDTA, did not block the zinc accumulation and neuronal death. Vincristine also produced intracellular zinc accumulation and neuronal death. These results suggest that colchicine-induced dentate granule cell death is caused by blocking axonal zinc flow and accumulation of intracellular labile zinc. PMID:24874779

  13. Lack of Evidence for the Direct Activation of Endothelial Cells by Adult Female and Microfilarial Excretory-Secretory Products

    PubMed Central

    Weinkopff, Tiffany; Lammie, Patrick

    2011-01-01

    Lymphangiectasia (dilation of the lymphatic vessel (LV)) is pathognomonic for lymphatic filariasis. In both infected humans and animal models of infection, lymphangiectasia is not restricted to the site of the worm nest, but is found along the infected vessel. These observations argue that soluble products secreted by the worm could be mediating this effect by activating the lymphatic endothelial cells (LEC) lining the vessel. We tested the ability of filarial Excretory-Secretory products to activate LECs, but were unable to detect a direct effect of the Excretory-Secretory products on the activation of LEC as assessed by a variety of approaches including cellular proliferation, cell surface molecule expression and cytokine and growth factor production (although other mediators used as positive controls did induce these effects). Collectively, these results do not support the hypothesis that Excretory-Secretory products directly activate LECs. PMID:21829611

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

    PubMed

    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

  15. The biology of cytotoxic cell granule exocytosis pathway: granzymes have evolved to induce cell death and inflammation.

    PubMed

    Pardo, Julián; Aguilo, Juan Ignacio; Anel, Alberto; Martin, Praxedis; Joeckel, Lars; Borner, Christoph; Wallich, Reiner; Müllbacher, Arno; Froelich, Christopher J; Simon, Markus M

    2009-04-01

    The granule exocytosis pathway of cytotoxic lymphocytes (Tc and NK cells) is critical for control of tumor development and viral infections. Granule-associated perforin and granzymes are key components in Tc cell-mediated function(s). On the basis of studies that showed granzymes A, B, C, K and M, to induce apoptosis in vitro, all granzymes were thought to also induce cell death in vivo. This review summarizes our present understanding of the biological processes elicited by purified granzyme A and granzyme as well as the processes induced by the more physiologically relevant cytotoxic cells secreting these proteases. The combined evidence supports the concept that the granule secretion pathway is not mono-specific but rather poly-functional including induction of pro-inflammatory cytokines, besides their widely appreciated apoptotic properties. PMID:19249384

  16. Secretory and radioligand binding studies on muscarinic receptors in bovine and feline chromaffin cells.

    PubMed

    Ballesta, J J; Borges, R; García, A G; Hidalgo, M J

    1989-11-01

    1. Muscarinic agonists enhanced catecholamine release from perfused cat adrenal glands with the following relative order of potencies: methacholine greater than oxotremorine greater than McN-A-343 greater than pilocarpine greater than bethanechol greater than muscarine. Because a continuous online electrochemical detection system was used to monitor catecholamine release, this sequence could be obtained at concentrations much lower (1-10 microM) and during much shorter stimulation times (3-30 s) than in previous reports. 2. All muscarinic agonists used secreted adrenaline preferentially over noradrenaline. Methacholine evoked a sustained, non-desensitizing response in the cat adrenal, which declined to basal levels of secretion immediately after Ca2+ removal: upon Ca2+ restoration secretion was restored to the previous plateau. 3. In addition to evoking a direct secretory response, low concentrations of methacholine, pilocarpine, bethanechol or muscarine clearly potentiated cat adrenal secretory responses evoked by pulses of nicotine (2 microM for 30 s) or high K+ (17.7 mM for 30 s). 4. [3H]Quinuclydinyl benzylate (QNB) specifically bound to cat adrenomedullary membranes with a saturating monophasic curve, suggesting a single binding site with a KD of 23 pM and a Bmax of 67 fmol (mg protein)-1. Preferential displacement by atropine over pirenzepine suggests that the binding site is associated to a M2-type muscarinoceptor. 5. Methacholine (3-300 microM) did not enhance the spontaneous catecholamine release from perfused bovine intact adrenal glands or superfused chromaffin cells. Neither did the drug affect secretion evoked by dimethylphenylpiperazinium (10 microM for 3 s) or K+ (35 mM for 3 s) from isolated superfused bovine adrenal chromaffin cells. 6. [3H]QNB bound to purified bovine adrenomedullary plasma membranes with a KD of 29 pM and a Bmax of 89 fmol (mg protein)-1. Displacement by pirenzepine suggests the presence of two binding sites (Hill coefficient = 0.64) with Ki1 of 39 nM and Ki2 of 2734 nM. 7. Because the ionophore A23187 enhanced K(+)-evoked secretion in both, bovine and cat adrenals, it seems that a similar cytosolic Ca2+ rise induced by muscarinic stimulation might constitute the underlying mechanism both to cause a secretory response per se as well as the potentiation of catecholamine release evoked by nicotinic or high K+ stimulation. However, it is unclear why the bovine behaves differently from the feline chromaffin cell as far as the muscarine-evoked effects are concerned.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2516125

  17. 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).

  18. Topiramate attenuates voltage-gated sodium currents in rat cerebellar granule cells

    Microsoft Academic Search

    Cristina Zona; Maria Teresa Ciotti; Massimo Avoli

    1997-01-01

    Whole-cell, voltage-clamp recordings were made from rat cerebellar granule cells in culture under experimental conditions designed to study voltage-gated Na+ currents that were elicited by depolarizing commands from a holding potential of ?60 mV up to +20 mV. These tetrodotoxin-sensitive inward currents were reduced in a dose-related manner by bath application of the structurally novel, anticonvulsant drug topiramate (10–1000 ?M;

  19. Neuroprotective activity of honokiol and magnolol in cerebellar granule cell damage

    Microsoft Academic Search

    Yi-Ruu Lin; Hwei-Hsien Chen; Chien-Hsin Ko; Ming-Huan Chan

    2006-01-01

    The aim of the present study was to investigate the neuroprotective effects of honokiol and magnolol, two major bioactive constituents of the bark of Magnolia officinalis, against neuron toxicity induced by glucose deprivation, excitatory amino acids and hydrogen peroxide (H2O2) in cultured rat cerebellar granule cells. Cell membrane damage was measured with a lactate dehydrogenase (LDH) release assay and 3-(4,5-dimethyl-2

  20. Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells

    Microsoft Academic Search

    CRAIG D. LOGSDON; JOACHIM MOESSNER; JOHN A. WILLIAMS; IRA D. GOLDFINE

    1985-01-01

    Previous studies have suggested a role for glucocorticoids in the differentiation of the acinar pancreas. We have now used the rat tumor cell line AR42J, derived from the acinar pancreas, to directly study this effect of glucocorticoids in vitro. The steroid hormones dexamethasone, corticosterone, aldosterone, and progesterone, but not estrogen, increased both the amylase content and the number of secretory

  1. VEGF stimulation of endothelial cell PAF synthesis is mediated by group V 14 kDa secretory phospholipase A2

    E-print Network

    Dennis, Edward A.

    VEGF stimulation of endothelial cell PAF synthesis is mediated by group V 14 kDa secretory Jolla, California, CA, 92093-0601, U.S.A. 1 Vascular endothelial growth factor (VEGF) is a potent assessed their contribution to VEGF-induced PAF synthesis in bovine aortic EC (BAEC) and human umbilical

  2. Regulation of Clara cell secretory protein gene expression by the CCAAT-binding factor NF-Y

    Microsoft Academic Search

    Adriana Acosta; Teresa Zariñán; Héctor Macías; Ana María Pasapera; Marco Allán Pérez-Solis; Aleida Olivares; Alfredo Ulloa-Aguirre; Rubén Gutiérrez-Sagal

    2007-01-01

    Analysis of the transcriptional regulation of the Clara cell secretory protein (CCSP) gene has resulted in the characterization of several trans-acting factors that regulate the activity of this gene. However, little is known about negative regulatory elements involved in CCSP gene transcription. Using transient transfections of luciferase reporter constructs driven by various fragments of the Neotomodon CCSP (nCCSP) promoter, we

  3. The secretory leukocyte protease inhibitor gene is a target of epidermal growth factor receptor action in endometrial epithelial cells

    Microsoft Academic Search

    M C Velarde; S I Parisek; R R Eason; F A Simmen; R C M Simmen

    2005-01-01

    The over-expression of epidermal growth factor receptor (EGFR) and its ligands, epidermal growth factor (EGF) and transforming growth factor-, is a common feature of epithelial carcinomas and correlates with neoplastic pro- gression. Secretory leukocyte protease inhibitor (SLPI), a member of the Kazal superfamily of serine anti-proteases, induces proliferation and promotes malignancy of epi- thelial cells and is expressed at high

  4. A histochemical study of the secretory gland cells of Cercaria shikokuensis and their role during development from cercaria to metacercaria.

    PubMed

    Harada, M; Suguri, S

    2001-07-01

    The roles of secretory glands during the developmental process from an immature cercaria to a metacercaria in Cercaria shikokuensis were studied. Four types of secretory cells were identified in this species. On maturation of the cercaria in redia, the products of ventral gland cells and mucoid gland cells formed a thick surface coat on the mature cercaria, and the products of cephalic gland cells also formed a thin cover on the surface coat. In the process leading to the formation of a metacercaria, the surface coat constituted the outer layer of the cyst, mucoid gland cells secreted mucous substances inside the wall, and then cystogenous gland cells discharged their products to the inner wall. The cyst wall was composed of four layers, and it was thought that the outermost surface layer helped the cyst wall to adhere to the matrix and the intermediate layers helped to put together outer and inner walls. PMID:11438439

  5. Optical monitoring of progressive synchronization in dentate granule cells during population burst activities.

    PubMed

    Murayama, Masanori; Miyazaki, Kenichi; Kudo, Yoshihisa; Miyakawa, Hiroyoshi; Inoue, Masashi

    2005-06-01

    Monitoring multiple neurons is essential for understanding neuronal network activities. While calcium imaging from a population of cells is an effective method to study the network dynamics of a neural structure, it has been difficult to image from densely packed structures, such as the granule cell layer of the dentate gyrus, due to overlap of the cells. We have developed a novel method to label multiple granule cells with a Ca(2+) indicator in rat hippocampal slices using Oregon Green 488 BAPTA-1 (OGB-1) AM. Synchronized burst activities (0.3-1.4 Hz), which were induced by applying 50 microm 4-aminopyridine, were monitored extracellularly with a glass electrode placed at the granule cell layer in the dentate gyrus. During the burst activities, spontaneously occurring action potential-induced Ca(2+) transients from multiple (4-12) granule cells were monitored with a cooled CCD camera with single-cell resolution. Temporal structures of firing patterns from the multiple neurons were determined from Ca(2+) transients. In each single-burst-event recorded from the extracellular electrode, each neuron fired synchronously within a 200 ms time window. The latency and its variance from the onset time of the single-burst-events to one of the Ca(2+) transients decreased over time (< 7.5 min). These results indicate that the synchrony of the action potentials within a single-burst-event was enhanced as the burst activities proceeded. This progressive synchronization may be a key feature in making self-organizing neuronal networks. PMID:16026472

  6. 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

  7. [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

  8. JC virus granule cell neuronopathy: A cause of infectious cerebellar degeneration.

    PubMed

    Henry, Carole; Jouan, Fanny; De Broucker, Thomas

    2015-07-15

    JC virus (JCV) infection of glial cells can lead to progressive multifocal leukoencephalopathy (PML) in immunocompromised patients. A newly described phenotype of the infection is infection of neurons. This distinct clinical and radiological syndrome is named JCV granule cell neuronopathy, characterized by exclusive or predominant cerebellar atrophy. We report the clinical and radiological longitudinal findings of 5 HIV-infected patients referred to us between September 2004 and November 2011 who exhibited JCV granule cell neuronopathy (4 probable cases and 1 possible). The association of immunocompromised status, progressive cerebellar syndrome, MRI abnormalities with cortical cerebellar atrophy and cerebrospinal fluid positive for JCV on PCR allowed for a highly probable diagnosis. The reversal of the immunocompromised status is the only way to stop the disease evolution. Motor functioning can remain impaired, but the illness itself, unlike progressive multifocal leukoencephalopathy, does not seem to threaten life. PMID:26003226

  9. 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.

  10. Pigment granule migration in isolated cells of the teleost retinal pigment epithelium.

    PubMed

    Bruenner, U; Burnside, B

    1986-11-01

    In the teleost eye, the melanin granules of the retinal pigment epithelium (RPE) move in response to changes in light conditions. In the dark, pigment granules aggregate toward the cell base, and in the light, they disperse into long apical projections. Isolated RPE cells from the green sunfish (Lepomis cyanellus) were used to investigate the mechanism and regulation of pigment movement. Changing light conditions did not elicit pigment migration in isolated cells. However, pigment aggregation was induced by 3',5' cyclic-adenosine monophosphate (cAMP), dibutyryl cAMP (dbcAMP), and forskolin (an adenylate cyclase activator). The effectiveness of forskolin suggests that an endogenous adenylate cyclase participates in regulating aggregation. Pigment dispersal was induced by the catecholamines epinephrine, phenylephrine, clonidine, dopamine, and apomorphine. Together the authors' studies suggest: that RPE cells contain the necessary motile machinery to support pigment granule transport in the absence of retina, but not the ability to respond to light; that elevating cAMP induces pigment aggregation; and that catecholamines induce dispersion by binding to receptors on the RPE cell. The authors' observations are consistent with previous suggestions that light regulation of RPE pigment migration is mediated by the retina. PMID:3021648

  11. Odorant-induced activation of extracellular signal-regulated kinase/mitogen-activated protein kinase in the olfactory bulb promotes survival of newly formed granule cells.

    PubMed

    Miwa, Naofumi; Storm, Daniel R

    2005-06-01

    Extracellular signal-regulated kinase 1/2 (Erk1/2)/mitogen-activated protein (MAP) kinase (MAPK) plays a significant role in neuronal survival, including odorant-induced, activity-dependent survival of olfactory sensory neurons in the main olfactory epithelium. Here, we examined the role of MAPK for the survival of neurons in the olfactory bulb. To study odorant-induced activation of MAPK in the olfactory bulb, mice were exposed to odorants in vivo, and MAPK was assayed. Exposure of mice to some odorants in vivo activated MAPK in granule cells 10 min after exposure. Activation of MAPK was particularly evident in the nucleus and dendrites of granule cells. Because MAPK activation can augment neuronal survival, odorant enhancement of granule cell survival was monitored by bromodeoxyuridine (BrdU) incorporation. Long-term exposure to odorants increased the survival of newly formed granule cells as well as the number of granule cells that were both BrdU+ and phospho-Erk+. Inhibition of MAPK by administration of SL327 in vivo blocked the odorant-induced increase in newly formed granule cells, suggesting that activation of MAPK promotes the survival of granule cells in the olfactory bulb. Studies using cultured granule cells confirmed that activation of MAPK in granule cells protects them against strong apoptotic signals. These data suggest that stimulation of MAPK in olfactory bulb granule cells by some odorants may contribute to the survival of newly formed granule cells caused by odorant exposure. PMID:15930390

  12. Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans

    PubMed Central

    Ueki, Shigeharu; Melo, Rossana C. N.; Ghiran, Ionita; Spencer, Lisa A.; Dvorak, Ann M.; Weller, Peter F.

    2013-01-01

    Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane–enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets. PMID:23303825

  13. 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.

  14. Inositol 1,4,5-triphosphate-stimulated calcium release from permeabilized cerebellar granule cells.

    PubMed Central

    Whitham, E. M.; Challiss, R. A.; Nahorski, S. R.

    1991-01-01

    1. Muscarinic cholinoceptor stimulation of phosphoinositide hydrolysis in rat cultured cerebellar granule cells results in a rapid, transient accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which has been implicated in the release of non-mitochondrial intracellular Ca2+ stores. In the present study, the release of Ca2+ from intracellular stores and the Ins(1,4,5)P3 receptor responsible for this process have been investigated. 2. Monolayers of saponin-permeabilized granule cells accumulate 45Ca2+ in an ATP-dependent manner and the sequestered 45Ca2+ can be concentration-dependently released by Ins(1,4,5)P3 by a stereospecific and heparin-sensitive mechanism. The EC50 for Ins(1,4,5)P3-stimulated 45Ca2+ release was 80 +/- 3 nM. 3. Radioligand binding studies performed on a crude granule cell membrane fraction indicated the presence of an apparently homogeneous population of stereo-specific Ins(1,4,5)P3 receptors (KD 54.7 +/- 2.0 nM; Bmax 1.37 +/- 0.29 pmol mg-1 protein). 4. This study provides evidence for Ins(1,4,5)P3-sensitive intracellular Ca2+ stores in primary cultures of cerebellar granule cells and suggest that these cells provide an excellent model neuronal system in which to study the relative functional roles of Ca2+ release from intracellular stores and Ca(2+)-entry in neuronal Ca2+ homeostasis. PMID:1786511

  15. 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

  16. The Possible Roles of the Dentate Granule Cell's Leptin and Other Ciliary Receptors in Alzheimer's Neuropathology.

    PubMed

    Whitfield, James F; Chiarini, Anna; Prà, Ilaria Dal; Armato, Ubaldo; Chakravarthy, Balu

    2015-01-01

    Dentate-gyral granule cells in the hippocampus plus dentate gyrus memory-recording/retrieving machine, unlike most other neurons in the brain, are continuously being generated in the adult brain with the important task of separating overlapping patterns of data streaming in from the outside world via the entorhinal cortex. This "adult neurogenesis" is driven by tools in the mature granule cell's cilium. Here we report our discovery of leptin's LepRb receptor in this cilium. In addition, we discuss how ciliary LepRb signaling might be involved with ciliary p75NTR and SSTR3 receptors in adult neurogenesis and memory formation as well as attenuation of Alzheimer's neuropathology by reducing the production of its toxic amyloid-?-derived drivers. PMID:26184316

  17. Protective role for club cell secretory protein-16 (CC16) in the development of COPD.

    PubMed

    Laucho-Contreras, Maria E; Polverino, Francesca; Gupta, Kushagra; Taylor, Katherine L; Kelly, Emer; Pinto-Plata, Victor; Divo, Miguel; Ashfaq, Naveed; Petersen, Hans; Stripp, Barry; Pilon, Aprile L; Tesfaigzi, Yohannes; Celli, Bartolome R; Owen, Caroline A

    2015-06-01

    Club cell secretory protein-16 (CC16) is the major secreted product of airway club cells, but its role in the pathogenesis of chronic obstructive pulmonary disease (COPD) is unclear. We measured CC16 airway expression in humans with and without COPD and CC16 function in a cigarette smoke (CS)-induced COPD murine model. Airway CC16 expression was measured in COPD patients, smokers without COPD and non-smokers. We exposed wildtype (WT) and CC16(-/-)mice to CS or air for up to 6?months, and measured airway CC16 expression, pulmonary inflammation, alveolar septal cell apoptosis, airspace enlargement, airway mucin 5AC (MUC5AC) expression, small airway remodelling and pulmonary function. Smokers and COPD patients had reduced airway CC16 immunostaining that decreased with increasing COPD severity. Exposing mice to CS reduced airway CC16 expression. CC16(-/-) mice had greater CS-induced emphysema, airway remodelling, pulmonary inflammation, alveolar cell apoptosis, airway MUC5AC expression, and more compliant lungs than WT mice. These changes were associated with increased nuclear factor-?B (NF-?B) activation in CC16(-/-) lungs. CS-induced acute pulmonary changes were reversed by adenoviral-mediated over-expression of CC16. CC16 protects lungs from CS-induced injury by reducing lung NF-?B activation. CS-induced airway CC16 deficiency increases CS-induced pulmonary inflammation and injury and likely contributes to the pathogenesis of COPD. PMID:25700379

  18. Club cell secretory protein improves survival in a murine obliterative bronchiolitis model.

    PubMed

    Wendt, Christine; Tram, Kevin; Price, Andrew; England, Kristen; Stiehm, Andrew; Panoskaltsis-Mortari, Angela

    2013-11-01

    Club cell secretory protein (CCSP) is an indirect phospholipase A2 inhibitor with some immunosuppressive and antiproliferative properties that is expressed in bronchiolar Club cells. In our murine bone marrow transplant (BMT) model of obliterative bronchiolitis (OB), CCSP is diminished; however, its role is unknown. To determine the role of CCSP, B6 wild-type (WT) or CCSP-deficient (CCSP(-/-)) mice were lethally conditioned and given allogeneic bone marrow with a sublethal dose of allogeneic splenic T cells to induce OB. We found that CCSP(-/-) mice demonstrated a higher mortality following BMT-induced OB compared with WT mice. Mice were analyzed 60 days post-BMT for protein expression, pulmonary function, and histology. CCSP levels were reduced in WT mice with BMT-induced OB, and lower levels correlated to decreased lung compliance. CCSP(-/-) had a higher degree of injury and fibrosis as measured by hydroxy proline, along with an increased lung resistance and the inflammatory markers, leukotriene B4 and CXCL1. Replacement with recombinant intravenous CCSP partially reversed the weight loss and improved survival in the CCSP(-/-) mice. In addition, CCSP replacement improved histology and decreased inflammatory cells and markers. These findings indicate that CCSP has a regulatory role in OB and may have potential as a preventive therapy. PMID:23997179

  19. Spatial and structural interrelationships between secretory cells of the subcommissural organ and blood vessels. An immunocytochemical study.

    PubMed

    Rodríguez, E M; Oksche, A; Hein, S; Rodríguez, S; Yulis, R

    1984-01-01

    In 76 specimens (amphibians, reptilians, mammals) belonging to 25 different vertebrate species, the region of the subcommissural organ (SCO) was investigated with the use of a primary antiserum raised against an extract of bovine Reissner's fiber + the immunoperoxidase procedure according to Sternberger et al. (1970). In the SCO of a toad (Bufo arenarum) and several species of reptiles (lacertilians, ophidians, crocodilians), the ependymal cells were the only type of secretory cell displaying vascular contacts, whereas in mammals ependymal and hypendymal cells established intimate spatial contacts with blood vessels. In Bufo arenarum, but especially in the reptilian species examined, the ependymo-vascular relationship was exerted by a population of ependymal cells having a rather constant location within the SCO and projecting to capillaries that showed a remarkably constant pattern of anatomical distribution. In the SCO of mammals the modality and degree of the structural relationships between secretory cells and blood vessels varied greatly from species to species. In the SCO of the armadillo and dog the secretory tissue was organized as a thick, highly vascularized layer with most of the cells oriented toward the capillaries. A rather opposite situation was found in the SCO of New- and Old-World monkeys, where vascular contacts were restricted to a few ependymal cells. PMID:6435877

  20. 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

  1. Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells

    Microsoft Academic Search

    Liang Peng; Arne Schousboe; Leif Hertz

    1991-01-01

    Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited

  2. Role of Ionic Fluxes in the Apoptotic Cell Death of Cultured Cerebellar Granule Neurons

    Microsoft Academic Search

    A. Franco-Cea; A. Valencia; S. Sánchez-Armass; G. Domínguez; J. Morán

    2004-01-01

    Cultured cerebellar granule neurons (CGC) increase survival in a medium containing 25 mM KCl (K25), and they die apoptotically when cultures are treated with staurosporine (St) or are transferred to a 5-mM KCl containing medium (K5). Apoptotic CGC show nuclear condensation and caspase-3 activation. Cell death induced by these conditions was partially prevented when cultures were maintained under alkaline conditions,

  3. Ontogenic changes in secretory component expression by villous and crypt cells of rat small intestine.

    PubMed Central

    Buts, J P; Delacroix, D L

    1985-01-01

    In order to determine whether the rat small intestine exhibits quantitative changes in the synthesis of the secretory component (SC) during growth, epithelial villus and crypt cells were isolated from jejunal segments at intervals after birth up to adulthood. SC concentration was measured in each cell fraction by immunoradiometric assay and compared to sucrase activity, an enzyme marker of the differentiated villus enterocyte. The following results were observed. (i) Adult rats showed a characteristic decreasing concentration gradient of SC from the crypts (mean concentration in crypt cells: 636 +/- 173 ng/mg protein) to the villus tip (mean concentration in villus cells: 152 +/- 17 ng/mg protein). This gradient was the reverse of that found for sucrase activity. (ii) In young sucklings (10 days old), SC was virtually absent in both villus and crypt cells, but its concentration progressively increased in weanling rats and reached adult levels by day 40 postpartum. (iii) The crypt to villus cell gradient of SC, absent in sucklings up to day 20, developed during the fourth postnatal week. (iv) Treatment of 10-day-old suckling pups with pharmacological doses of either corticosterone or L-thyroxine for 3 consecutive days failed to induce the precocious synthesis of SC by jejunal enterocytes, but produced significant (P less than 0.01) decreases in concentration. Under the same conditions, sucrase activity was markedly enhanced. In conclusion, major changes in the ability of the immature crypt cell to produce the specific receptor for transepithelial transport of polymeric immunoglobulins occur during the fourth week of rat life. The initiation of this ontogenic process is not triggered by the dietary and hormonal changes known to control the maturation of other functions linked to the differenciated villus cell, such as sucrase activity. PMID:3972432

  4. 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

  5. Oncospheral Penetration Glands and Secretory Blebs Are the Sources of Taenia ovis Vaccine Antigens? †

    PubMed Central

    Jabbar, Abdul; Crawford, Simon; Gauci, Charles G.; Walduck, Anna K.; Anderson, Garry A.; Lightowlers, Marshall W.

    2010-01-01

    Taenia ovis is a cestode parasite infecting primarily sheep as intermediate hosts and dogs as definitive hosts. The first highly effective, recombinant vaccine against a parasitic organism was developed against T. ovis infection in sheep. Three separate host-protective antigens (To16, To18, and To45W) have been cloned from the oncosphere of the parasite. We localize these antigens in the oncosphere by using quantitative immunogold labeling and transmission electron microscopy. The three antigens were uniquely associated with penetration gland cells. The cytoplasm and secretory granules of both penetration gland type 1 and type 2 cells exhibited statistically significant levels of staining for each of the three antigens. The intensity of labeling of the penetration gland type 1 cell was approximately three to five times greater (P < 0.01) compared to the level of staining intensity seen in the penetration gland type 2 cell. In activated oncospheres, secretory blebs were found to contain granules with a structure similar to those observed in the penetration gland cells. The granules within the secretory blebs were shown to stain specifically for the presence of each of the three host-protective antigens. The absence of surface location of the T. ovis antigens suggests that the parasite may not be susceptible to vaccine-induced antibody- and complement-mediated attack until some postoncospheral development has occurred after infection of the intermediate host. PMID:20643854

  6. High-frequency stimulation induces gradual immediate early gene expression in maturing adult-generated hippocampal granule cells.

    PubMed

    Jungenitz, Tassilo; Radic, Tijana; Jedlicka, Peter; Schwarzacher, Stephan W

    2014-07-01

    Increasing evidence shows that adult neurogenesis of hippocampal granule cells is advantageous for learning and memory. We examined at which stage of structural maturation and age new granule cells can be activated by strong synaptic stimulation. High-frequency stimulation of the perforant pathway in urethane-anesthetized rats elicited expression of the immediate early genes c-fos, Arc, zif268 and pCREB133 in almost 100% of mature, calbindin-positive granule cells. In contrast, it failed to induce immediate early gene expression in immature doublecortin-positive granule cells. Furthermore, doublecortin-positive neurons did not react with c-fos or Arc expression to mild theta-burst stimulation or novel environment exposure. Endogenous expression of pCREB133 was increasingly present in young cells with more elaborated dendrites, revealing a close correlation to structural maturation. Labeling with bromodeoxyuridine revealed cell age dependence of stimulation-induced c-fos, Arc and zif268 expression, with only a few cells reacting at 21 days, but with up to 75% of cells activated at 35-77 days of cell age. Our results indicate an increasing synaptic integration of maturing granule cells, starting at 21 days of cell age, but suggest a lack of ability to respond to activation with synaptic potentiation on the transcriptional level as long as immature cells express doublecortin. PMID:23425888

  7. 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

  8. Secretory Leukocyte Protease Inhibitor, SLPI, Antagonizes Paclitaxel in Ovarian Cancer Cells

    PubMed Central

    Rasool, Nabila; LaRochelle, William; Zhong, Haihong; Ara, Gulshan; Cohen, Joshua; Kohn, Elise C.

    2009-01-01

    Purpose Ovarian cancer (OvCa) recurrence with development of paclitaxel resistance is an obstacle to long term survival. We demonstrated that secretory leukocyte protease inhibitor (SLPI) is a survival factor for OvCa. We hypothesize SLPI may antagonize paclitaxel injury. Experimental design Differential SLPI induction in response to paclitaxel, and response to stable forced expression of SLPI was demonstrated in A2780-1A9 cells and their paclitaxel-resistant sublines, PTX10 and PTX22 and confirmed with HEY-A8 cells. SLPI-mediated survival was reduced by the MEK inhibitor, U0126 and a humanized neutralizing monoclonal anti-SLPI antibody, CR012. OVCAR3 xenographs tested the role of CR012 in vivo. Results SLPI expression was lower in A2780-1A9 OvCa cells than PTX10 and PTX22 and SLPI was induced by paclitaxel exposure. Stable SLPI expression yielded a proliferation advantage (p=0.01); expression of and response to SLPI in OVCAR3 cells was abrogated by exposure to CR012. SLPI reduced paclitaxel susceptibility of 1A9 and HEY-A8 cells (p?0.05) and SLPI expression did not increase resistance of PTX10 and ?22 cells. Both paclitaxel and SLPI overexpression induced ERK activation. Inhibition of MEK with U0126 increased paclitaxel injury and overcame SLPI-mediated cell protection. It did not reinstate PTX10 sensitivity to paclitaxel, which was associated with AKT activation. Significant inhibition of OVCAR3 xenograft growth was observed with CR012 and paclitaxel, over single agents (p?0.001). Conclusions A two-pronged approach confirmed SLPI overcomes paclitaxel in part through activation of ERK1/2. These results credential SLPI as a molecular target for OvCa and suggest CR012 as a tool for proof of concept. PMID:20068074

  9. P2X7 Receptors Trigger ATP Exocytosis and Modify Secretory Vesicle Dynamics in Neuroblastoma Cells*

    PubMed Central

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R.

    2011-01-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca2+-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca2+ concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca2+ and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation. PMID:21292765

  10. Granule cells born in the adult rat hippocampus can regulate the expression of GABAergic markers.

    PubMed

    Lara, Erika; Beltrán, Jesús Q; Segovia, José; Gutiérrez, Rafael

    2012-09-01

    The granule cells (GCs) of the dentate gyrus transiently express markers of the GABAergic phenotype early during development. However, GCs are generated throughout life, posing the question of whether the newborn neurons in the adult rodent recapitulate the development of the neurotransmitter phenotype of GCs generated during embryonic and early postnatal development. In this work we asked whether newborn GCs transiently express a GABAergic phenotype during their development in the adult rat. Using retroviral infection, we labeled dividing cells in the dorsal hippocampus with GFP, identified them as granule cells, and determined their expression of GABAergic markers at different developmental stages. We found that GFP-positive cells express Prox-1 and calbindin, identifying them as GCs. GABA or GAD(67) was expressed in 13% of GFP-positive cells at 7 dpi, in 16% at 10 dpi and in 20% at 15 dpi. At 30 dpi, however, no GFP-positive cell somata containing GABAergic markers were detected, but their mossy fiber boutons did contain GAD(67). Interestingly, developing GCs detected with doublecortin and PSA-NCAM in non-injected adult rats, did not express GABAergic markers, suggesting that retroviral injection/infection stimulates their transient expression. However, in non-injected rats, a number of mossy fiber boutons of newborn granule cells detected with PSA-NCAM did express GAD(67). Our findings reveal that developing GCs born in the adult are able to transiently up-regulate the expression of GABAergic markers to be detected in their soma in response to insults, while they constitutively express GAD(67) in their mossy fibers. PMID:22750325

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

    PubMed Central

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

    2013-01-01

    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. PMID:22266469

  12. Possible mechanisms inducing granule cell dispersion in humans with temporal lobe epilepsy.

    PubMed

    Lurton, D; Sundstrom, L; Brana, C; Bloch, B; Rougier, A

    1997-01-01

    The stratum granulosum (SG) of the fascia dentata from 17 human epileptic hippocampi was assessed in terms of width, volumetric cell density (VCD) and percentage of cell loss to study the granule cell dispersion (GCD) phenomenon described by Houser. GCD was considered when three conditions were observed, the SG was wider than 120 microns, granule cell (GC) somata did not remain in close apposition to one another the normal clear boundary between the molecular layer and the SG was not maintained. GCD involved a partial zone of the SG in six cases and the whole SG in two cases. Dynorphin mRNA in-situ hybridization was performed in two cases and allowed us to affirm that dispersed cells are actually GC. A close correlation linked GCD, GC loss and VCD decrease in diffuse CA4, laminated CA4, CA3, CA2 and CA1. The discussion is focused on the possible causes of dispersion. Some arguments did not suggest for a migration arrest during development. Nevertheless, in one case, a cluster of horizontal cells in the inner part of the molecular layer could evoke the persistence of normally transient cells during ontogenesis. A neo-migration due to permissive phenomenon induced by gliogenesis, mossy fibers sprouting in the supra-granular layer and over-expression of growth factors is suggested from experimental data. Nevertheless a straining due to the tissue shrinkage observed in severe hippocampal sclerosis (HS) could also be involved in the origin of GCD. PMID:9095397

  13. 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

  14. 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.

  15. Neurotrophins Protect Cultured Cerebellar Granule Neurons against the Early Phase of Cell Death by a Two Component Mechanism

    Microsoft Academic Search

    Michael J. Courtney; Karl E. O. Åkerman; Eleanor T. Coffey

    1997-01-01

    Cerebellar granule neurons cultured with serum develop a ma- ture neuronal phenotype, including stimulus-coupled release of glutamate, and depend on elevated potassium for survival. We find that cells cultured with serum undergo two phases of cell death. By 6 d in vitro, 30 -50% of the cells present are dead; after this time the remaining cells die. Elevated potassium prevents

  16. 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

  17. Granzymes, cytotoxic granules and cell death: the early work of Dr. Jurg Tschopp

    PubMed Central

    Trapani, J A

    2012-01-01

    Within the powerful legacy left by Jurg Tschopp, we should not forget his early work that helped to elucidate the molecular pathways responsible for the clearance of virus-infected and transformed cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Jurg's skilful biochemical approach formed a firm platform upon which the work of so many other biochemists, cell biologists and immunologists would come to rely. Jurg coined the shorthand term ‘granzyme' to denote the individual members of a family of serine proteases sequestered in and secreted from the cytotoxic granules of CTL/NK cells. He was also one of the first to describe the lytic properties of purified perforin and to postulate the synergy of perforin and granzymes, which we now know to underpin target cell apoptosis. Jurg was a major protagonist in the debate that raged throughout the 1980's and early 1990's on the physiological relevance of the ‘granule exocytosis' pathway. Ultimately, resolving this issue led Jurg and his colleagues to even greater and impactful discoveries in the broader field of apoptosis research. Jurg Tschopp ranks with other pioneers, particularly Gideon Berke, Chris Bleackley, Pierre Golstein, Pierre Henkart and Eckhard Podack for making seminal discoveries on our understanding of how the immune system eliminates dangerous cells. PMID:22095283

  18. 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 ...

  19. 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

  20. Neuroendocrine secretory protein 7B2: structure, expression and functions.

    PubMed Central

    Mbikay, M; Seidah, N G; Chrétien, M

    2001-01-01

    7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2-7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin ('ACTH') hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2 has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation. PMID:11439082

  1. The secretory dynamics of the CHH-producing cell group in the eyestalk of the crayfish, Astacus leptodactylus , in the course of the day\\/night cycle

    Microsoft Academic Search

    Janine L. Gorgels-Kallen; Christina E. M. Voorter

    1985-01-01

    The secretory dynamics of the Crustacean Hyperglycemic Hormone (CHH)-producing cells in the eyestalk of the crayfish Astacus leptodactylus were studied during the daily cycle (12 h light\\/12 h dark). The different secretory stages of individual cells were determined by means of immunocytochemistry combined with morphometric analysis at the light-microscopic level. The data obtained were correlated with the 24-h rhythmicity of

  2. Clara cell secretory protein (CC16) as a peripheral blood biomarker of lung injury in ventilated preterm neonates

    Microsoft Academic Search

    Kosmas Sarafidis; Theodora Stathopoulou; Elisavet Diamanti; Vasiliki Soubasi; Charalambos Agakidis; Aikaterini Balaska; Vasiliki Drossou

    2008-01-01

    The aim of this study was to assess the serum concentrations of Clara cell secretory protein (CC16) in association with acute\\u000a and chronic lung injury in mechanically ventilated preterm neonates. Thirty-five preterm neonates (gestational age [GA] ?31 weeks)\\u000a with acute respiratory failure were enrolled. Of these, 23 neonates requiring ventilatory support within 2 h after birth comprised\\u000a the mechanically ventilated group

  3. IL8 Released Constitutively by Primary Bronchial Epithelial Cells in Culture Forms an Inactive Complex with Secretory Component1

    Microsoft Academic Search

    Lindsay J. Marshall; Beatrice Perks; Thomas Ferkol; Janis K. Shute

    The bronchial epithelium is a source of both and chemokines and, uniquely, of secretory component (SC), the extracellular ligand-binding domain of the polymeric IgA receptor. Ig superfamily relatives of SC, such as IgG and 2-macroglobulin, bind IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying its activity as a neutrophil chemoattractant. Primary bronchial epithelial cells were cultured under

  4. cDNA sequence, 5 ?-flanking region, and promoter activity of the Neotomodon alstoni alstoni Clara cell secretory protein gene

    Microsoft Academic Search

    Héctor Mac??as; Ana Mar??a Pasapera; Marco Allán Pérez-Solis; Alfredo Ulloa-Aguirre; Rubén Gutiérrez-Sagal

    2004-01-01

    To better understand the phylogenetic divergence and the species-specific characteristics of the Clara cell secretory protein (CCSP), we cloned the cDNA encoding the neotomodon CCSP (nCCSP) and analyzed its tissue-specific expression. The full-length cDNA is 451bp long and predicts an amino acid sequence of 93 residues. Northern blot analysis from different neotomodon tissues demonstrated that the mRNA of CCSP appears

  5. Expression of Clara cell secretory protein in the lungs of rats exposed to silicon carbide whisker in vivo

    Microsoft Academic Search

    Yasuo Morimoto; Li Ding; Takako Oyabu; Masami Hirohashi; Heungnam Kim; Akira Ogami; Hiroshi Yamato; Izumi Akiyama; Hajime Hori; Toshiaki Higashi; Isamu Tanaka

    2003-01-01

    Intratracheal instillation studies have shown that exposure to silicon carbide whiskers (SiCW), an asbestos substitute, produces pulmonary fibrotic changes, suggesting that SiCW might have fibrogenic potential.It has been theorized that Clara cell secretory protein (CCSP) plays a critical role in regulating the acute inflammatory response in the lung. The present study was conducted to investigate the time course of the

  6. Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

    PubMed Central

    Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

    2014-01-01

    In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4?h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the ?-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of ?-cells. PMID:24535122

  7. Effect of mycobacterial secretory proteins on the cellular integrity and cytokine profile of type II alveolar epithelial cells

    PubMed Central

    Adlakha, Nidhi; Vir, Pooja; Verma, Indu

    2012-01-01

    Background: Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis (M. tb). In lungs, alveolar macrophages and type II alveolar epithelial cells serve as a replicative niche for this pathogen. Secretory proteins released by actively replicating tubercle bacilli are known to interact with host cells at the initial stages of infection. To understand the role of these cells in TB pathogenesis, it is important to identify the mycobacterial components involved in interaction with alveolar epithelial cells. Materials and Methods: We fractionated the whole secretory proteome of M. tb H37Rv into 10 narrow molecular mass fractions (A1-A10; <20 kDa to >90 kDa) that were studied for their binding potential with A549; type II alveolar epithelial cell line. We also studied the consequences of this interaction in terms of change in epithelial cell viability by MTT assay and cytokine release by ELISA. Results: Our results show that several mycobacterial proteins bind and confer cytolysis in epithelial cells. Amongst all the fractions, proteins ranging from 35-45 kDa (A5) exhibited highest binding to A549 cells with a consequence of cytolysis of these cells. This fraction (A5) also led to release of various cytokines important in anti-mycobacterial immunity. Conclusion: Fraction A5 (35-45 kDa) of mycobacterial secretory proteome play an important role in mediating M. tb interaction with type II alveolar epithelial cells with the consequences detrimental for the TB pathogenesis. Further studies are being carried out to identify the candidate proteins from this region. PMID:23243342

  8. 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

  9. Sputum and BAL Clara cell secretory protein and surfactant protein D levels in asthma.

    PubMed

    Emmanouil, P; Loukides, S; Kostikas, K; Papatheodorou, G; Papaporfyriou, A; Hillas, G; Vamvakaris, I; Triggidou, R; Katafigiotis, P; Kokkini, A; Papiris, S; Koulouris, N; Bakakos, P

    2015-06-01

    Clara cell secretory protein (CC16) is associated with Th2 modulation. Surfactant protein D (SPD) plays an important role in surfactant homeostasis and eosinophil chemotaxis. We measured CC16 and SPD in sputum supernatants of 84 asthmatic patients and 12 healthy controls. In 22 asthmatics, we additionally measured CC16 and SPD levels in BAL and assessed smooth muscle area (SMA), reticular basement membrane (RBM) thickness, and epithelial detachment (ED) in bronchial biopsies. Induced sputum CC16 and SPD were significantly higher in patients with severe asthma (SRA) compared to mild-moderate and healthy controls. BAL CC16 and SPD levels were also higher in SRA compared to mild-moderate asthma. CC16 BAL levels correlated with ED, while SPD BAL levels correlated with SMA and RBM. Severity represented a significant covariate for these associations. CC16 and SPD levels are upregulated in SRA and correlate with remodeling indices, suggesting a possible role of these biomarkers in the remodeling process. PMID:25728058

  10. Clara cell secretory protein increases phagocytic and decreases oxidative activity of neutrophils.

    PubMed

    Katavolos, P; Ackerley, C A; Clark, M E; Bienzle, D

    2011-01-01

    Horses suffer from recurrent airway obstruction, an asthma-like condition induced by repeat inhalation of environmental substances present in barn air. Clara cell secretory protein (CCSP) is much reduced during active inflammation when neutrophils predominate in the airways, and in chronic asthmatics. We sought to investigate morphologic and functional interactions of CCSP with neutrophils. Bronchoalveolar and blood neutrophils from healthy control animals, and from animals with recurrent airway obstruction in remission and exacerbation, were evaluated by immuno-cytochemistry and immuno-electron microscopy for presence of CCSP. Blood neutrophil oxidative burst and phagocytic activities were determined in the presence of different concentrations of recombinant equine CCSP. Bronchoalveolar lavage neutrophils from horses with exacerbated lung inflammation, but not from control horses, and not blood neutrophils from either group of animal, contained abundant immunoreactive CCSP. On immuno-electron microscopy, CCSP localized to the cytoplasm and nucleus. Incubation of blood neutrophils with CCSP significantly reduced oxidative burst activity (P<0.0001) and increased phagocytosis (P<0.001) of neutrophils. These findings indicate that CCSP enters neutrophils in horses with active neutrophilic lung inflammation and alters the function of neutrophils in blood. Presence in the nucleus suggests a potential transcriptional role of CCSP in neutrophils. PMID:20728946

  11. Loss of PPAR? expression in mammary secretory epithelial cells creates a pro-breast tumorigenic environment.

    PubMed

    Apostoli, Anthony J; Skelhorne-Gross, Graham E A; Rubino, Rachel E; Peterson, Nichole T; Di Lena, Michael A; Schneider, Mark M; SenGupta, Sandip K; Nicol, Christopher J B

    2014-03-01

    Breast cancer is the leading cause of new cancer diagnoses among women. Using peroxisome proliferator-activated receptor (PPAR)?((+/-)) mice, we showed normal expression of PPAR? was critical to stop 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast tumorigenesis. PPAR? is expressed in many breast cell types including mammary secretory epithelial (MSE) cells. MSEs proliferate as required during pregnancy, and undergo apoptosis or reversible transdifferentiation during involution once lactation is complete. Thus, MSE-specific loss of PPAR? was hypothesized to enhance DMBA-mediated breast tumorigenesis. To test this, MSE cell-specific PPAR? knockout (PPAR?-MSE KO) and control (PPAR?-WT) mice were generated, mated and allowed to nurse for three days. One week after involution, dams were treated with DMBA to initiate breast tumors, and randomized on week 7 to continue receiving a normal chow diet (DMBA Only: PPAR?-WT, n = 15; PPAR?-MSE KO, n = 25) or one supplemented with a PPAR? activating drug (DMBA + ROSI: PPAR?-WT, n = 17; PPAR?-MSE KO, n = 24), and monitored for changes in breast tumor outcomes. PPAR?-MSE KOs had significantly lower overall survival and decreased mammary tumor latency as compared to PPAR?-WT controls. PPAR? activation significantly reduced DMBA-mediated malignant mammary tumor volumes irrespective of genotype. MSE-specific PPAR? loss resulted in decreased mammary gland expression of PTEN and Bax, increased superoxide anion production, and elevated serum eotaxin and RANTES, creating a protumorigenic environment. Moreover, PPAR? activation in MSEs delayed mammary tumor growth in part by down-regulating Cox-1, Cox-2 and cyclin D1. Collectively, these studies highlight a protective role of MSE-specific PPAR? during breast tumorigenesis, and support a novel chemotherapeutic role of PPAR? activation in breast cancer. PMID:23934545

  12. Organization of spinocerebellar projection map in three types of agranular cerebellum: Purkinje cells vs. granule cells as organizer element

    SciTech Connect

    Arsenio Nunes, M.L.; Sotelo, C.; Wehrle, R.

    1988-07-01

    The organization of the spinocerebellar projection was analysed by the anterograde axonal WGA-HRP (horseradish peroxidase-wheat germ agglutinin conjugate) tracing method in three different types of agranular cerebellar cortex either induced experimentally by X-irradiation or occurring spontaneously in weaver (wv/wv) and staggerer (sg/sg) mutant mice. The results of this study show that in the X-irradiated rat and weaver mouse, in both of which the granule cells are directly affected and die early in development, the spinal axons reproduce, with few differences, the normal spinocerebellar pattern. Conversely, in staggerer mouse, in which the Purkinje cells are intrinsically affected and granule neurons do not seem to be primarily perturbed by the staggerer gene action, the spinocerebellar organization is severely modified. These findings appear somewhat paradoxical because if granule cells, the synaptic targets of mossy spinocerebellar fibers, were necessary for the organization of spinocerebellar projection, the staggerer cerebellum would exhibit a much more normal projectional map than the weaver and the X-irradiated cerebella. It is, therefore, obvious that granule cells, and even specific synaptogenesis, are not essential for the establishment of the normal spinocerebellar topography. On the other hand, the fact that the Purkinje cells are primarily affected in the unique agranular cortex in which the spinocerebellar organization is severely modified suggests that these neurons could be the main element in the organization of the spinocerebellar projection map. This hypothesis is discussed in correlation with already-reported findings on the zonation of the cerebellar cortex by biochemically different clusters of Purkinje cells.

  13. 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.

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

    PubMed Central

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

    2013-01-01

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

  15. Differential roles of cyclin-dependent kinase 5 in tangential and radial migration of cerebellar granule cells.

    PubMed

    Umeshima, Hiroki; Kengaku, Mineko

    2013-01-01

    The cerebellar granule cell is a unique neuron which undergoes tangential migration along axonal tracts and radial migration along glial fibers sequentially during postnatal development. Little is known about molecular bases of the differential kinetics of tangential and radial migration. Here we developed a time-lapse imaging assay for tangential migration of cerebellar granule cells, and investigated comparative contributions of cyclin-dependent kinase 5 (CDK5), a key regulator of neuronal migration, in tangential and radial migration of granule cells in vivo and in organotypic cultures. Overexpression of a dominant-negative form of CDK5 severely disrupted cell morphology and somal movement during radial migration, while it only moderately affected tangential migration. Dominant-negative inhibition of CDK5 induced formation of ectopic radial processes in granule cells in vivo which aberrantly elongated into the white matter in the cerebellum. Live imaging of granule cell migration in cerebellar slices revealed that CDK5 regulates not only nuclear migration but also centrosome movement during radial migration. These findings suggest a mode-specific function of CDK5 in neuronal migration. PMID:22995860

  16. Effects of the actin-stabilizing drug, jasplakinolide, on pigment granule motility in isolated retinal pigment epithelial (RPE) cells of green sunfish, Lepomis cyanellus.

    PubMed

    King-Smith, C; Basciano, P A; Pham, N B

    2001-02-01

    The retinal pigment epithelium (RPE) of teleosts contains pigment granules that migrate in response to changes in light condition. Dissociated, cultured RPE cells in vitro can be triggered to aggregate or disperse pigment granules by the application of cAMP or dopamine, respectively. Previous research using the actin-disrupting drug, cytochalasin D, suggested that pigment granule motility is actin dependent. To further examine the role of actin in pigment granule motility, we tested the effects of the actin-stabilizing drug, jasplakinolide, on pigment granule motility. Pigment granules in previously dispersed RPE cells remained dispersed after jasplakinolide exposure (0.1-1 microM), but the drug halted movement of most pigment granules and stimulated rapid bi-directional movements in a small subset of granules. Jasplakinolide also blocked net pigment granule aggregation and interfered with the maintenance of full aggregation. Although jasplakinolide did not block pigment granule dispersion, it did alter the motility of dispersing granules compared to control cells; rather than the normal saltatory, primarily centrifugal movements, granules of jasplakinolide-treated cells demonstrated slow, creeping centrifugal movements and more rapid bi-directional movements. Jasplakinolide also altered cell morphology; the length and thickness of apical projections increased, and enlarged, paddle-like structures, which contained F-actin appeared at the tips of projections. Actin antibody labeling of jasplakinolide-treated cells revealed a more reticulated network of actin compared to antibody-labeled control cells. These results indicate that jasplakinolide-induced disruption of the actin network compromises normal pigment granule dispersion and aggregation in isolated RPE cells, thus providing further evidence that these movements are actin dependent. PMID:11277489

  17. MMP-13 Regulates Growth of Wound Granulation Tissue and Modulates Gene Expression Signatures Involved in Inflammation, Proteolysis, and Cell Viability

    PubMed Central

    Toriseva, Mervi; Laato, Matti; Carpén, Olli; Ruohonen, Suvi T.; Savontaus, Eriika; Inada, Masaki; Krane, Stephen M.; Kähäri, Veli-Matti

    2012-01-01

    Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13?/?) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13?/? mice. Granulation tissue in Mmp13?/? mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13?/? mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13?/? mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13?/? granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13?/? mice compared to WT mice. Mmp13?/? mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis. PMID:22880047

  18. Kindled seizure-induced reduction of muscarinic cholinergic receptors in rat hippocampal formation: evidence for localization to dentate granule cells.

    PubMed

    Savage, D D; Dasheiff, R M; McNamara, J O

    1983-11-20

    The binding of [3H] quinuclidinyl benzilate ( [3H] QNB) to muscarinic cholinergic receptors in dentate gyrus of rat hippocampal formation was analyzed by membrane binding assay and in vitro autoradiography. The destruction of dentate granule cells, either by neonatal irradiation or colchicine injection, resulted in nearly complete elimination of [3H] QNB binding sites in the molecular and granule cell layers. By contrast, neither perforant path transection nor destruction of the septal-hippocampal cholinergic afferents caused a decline of [3H] QNB binding sites. Amygdala kindled seizures resulted in a 30% reduction of [3H] QNB binding sites which was distributed uniformly across the entire molecular and granule cell layers. Thus, most, if not all, of the muscarinic cholinergic receptors present in dentate gyrus appear to reside on the somata and dendritic trees of the dentate granule cells. We propose that this kindled seizure-induced decline of muscarinic receptors represents an endogenous compensatory mechanism designed to stabilize granule cell excitability. PMID:6643742

  19. 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

  20. Temporal and spacial relationships between PSA-NCAM-expressing, newly generated granule cells, and radial glia-like cells in the adult dentate gyrus.

    PubMed

    Seki, T; Arai, Y

    1999-08-01

    The granule cell layer of the adult dentate gyrus possesses two characteristics of an immature nervous system. The first is that granule cells continue to be generated in the innermost region of the granule cell layer, and newly generated and developing granule cells in the adult express highly polysialylated neural cell adhesion molecule (PSA-NCAM). PSA-NCAM-expressing apical dendrites have dynamically unstable processes such as irregular shafts and many stick-like or fan-shaped fine processes. The second is that radial glia-like cells expressing glial fibrillary acidic protein (GFAP) remain in a similar region of the granular layer. The numbers of PSA-NCAM-expressing granule cells and GFAP-expressing radial glia-like cells show a parallel age-dependent decrease during aging. Moreover, by using confocal laser scanning microscopy and immunoelectron microscopy, we demonstrated that PSA-NCAM-expressing dendrites and GFAP-expressing radial processes are partly in contact with each other, and occasionally the radial glial processes envelop the PSA-NCAM-positive dendritic processes. The temporal and spatial relationship between the two immature elements suggests that the processes of the radial glia-like cells are closely associated with the dendritic growth of the newly generated granule cells in the adult dentate gyrus and that these two immature features of neurons and glia in the dentate gyrus diminish with age. PMID:10404415

  1. 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.

  2. Function suggests nano-structure: electrophysiology supports that granule membranes play dice.

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2012-10-01

    Cellular communication depends on membrane fusion mechanisms. SNARE proteins play a fundamental role in all intracellular fusion reactions associated with the life cycle of secretory vesicles, such as vesicle-vesicle and vesicle plasma membrane fusion at the porosome base in the cell plasma membrane. We present growth and elimination (G&E), a birth and death model for the investigation of granule growth, its evoked and spontaneous secretion and their information content. Using a statistical mechanics approach in which SNARE components are viewed as interacting particles, the G&E model provides a simple 'nano-machine' of SNARE self-aggregation behind granule growth and secretion. Results from experimental work, mathematical calculations and statistical modelling suggest that for vesicle growth a minimal aggregation of three SNAREs is required, while for the evoked secretion one SNARE is enough. Furthermore, the required number of SNARE aggregates (which varies between cell types and is nearly proportional to the square root of the mean granule diameter) affects and is statistically identifiable from the size distributions of spontaneous and evoked secreted granules. The new statistical mechanics approach to granule fusion is bound to have a significant changing effect on the investigation of the pathophysiology of secretory mechanisms and methodologies for the investigation of secretion. PMID:22628211

  3. Enhanced Tonic GABA Current in Normotopic and Hilar Ectopic Dentate Granule Cells After Pilocarpine-Induced Status Epilepticus

    PubMed Central

    Zhan, Ren-Zhi; Nadler, J. Victor

    2009-01-01

    In temporal lobe epilepsy, loss of inhibitory neurons and circuit changes in the dentate gyrus promote hyperexcitability. This hyperexcitability is compensated to the point that dentate granule cells exhibit normal or even subnormal excitability under some conditions. This study explored the possibility that compensation involves enhanced tonic GABA inhibition. Whole cell patch-clamp recordings were made from normotopic granule cells in hippocampal slices from control rats and from both normotopic and hilar ectopic granule cells in slices from rats subjected to pilocarpine-induced status epilepticus. After status epilepticus, tonic GABA current was an order of magnitude greater than control in normotopic granule cells and was significantly greater in hilar ectopic than in normotopic granule cells. These differences could be observed whether or not the extracellular GABA concentration was increased by adding GABA to the superfusion medium or blocking plasma membrane transport. The enhanced tonic GABA current had both action potential–dependent and action potential–independent components. Pharmacological studies suggested that the small tonic GABA current of granule cells in control rats was mediated largely by high-affinity ?4?x? GABAA receptors but that the much larger current recorded after status epilepticus was mediated largely by the lower-affinity ?5?x?2 GABAA receptors. A large ?5?x?2-mediated tonic current could be recorded from controls only when the extracellular GABA concentration was increased. Status epilepticus seemed not to impair the control of extracellular GABA concentration by plasma membrane transport substantially. Upregulated tonic GABA inhibition may account for the unexpectedly modest excitability of the dentate gyrus in epileptic brain. PMID:19474175

  4. 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

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

    PubMed Central

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

    2008-01-01

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

  6. Expression and role of a2 vacuolar-ATPase (a2V) in trafficking of human neutrophil granules and exocytosis.

    PubMed

    Gilman-Sachs, Alice; Tikoo, Anjali; Akman-Anderson, Leyla; Jaiswal, Mukesh; Ntrivalas, Evangelos; Beaman, Kenneth

    2015-06-01

    Neutrophils kill microorganisms by inducing exocytosis of granules with antibacterial properties. Four isoforms of the "a" subunit of V-ATPase-a1V, a2V, a3V, and a4V-have been identified. a2V is expressed in white blood cells, that is, on the surface of monocytes or activated lymphocytes. Neutrophil associated-a2V was found on membranes of primary (azurophilic) granules and less often on secondary (specific) granules, tertiary (gelatinase granules), and secretory vesicles. However, it was not found on the surface of resting neutrophils. Following stimulation of neutrophils, primary granules containing a2V as well as CD63 translocated to the surface of the cell because of exocytosis. a2V was also found on the cell surface when the neutrophils were incubated in ammonium chloride buffer (pH 7.4) a weak base. The intracellular pH (cytosol) became alkaline within 5 min after stimulation, and the pH increased from 7.2 to 7.8; this pH change correlated with intragranular acidification of the neutrophil granules. Upon translocation and exocytosis, a2V on the membrane of primary granules remained on the cell surface, but myeloperoxidase was secreted. V-ATPase may have a role in the fusion of the granule membrane with the cell surface membrane before exocytosis. These findings suggest that the granule-associated a2V isoform has a role in maintaining a pH gradient within the cell between the cytosol and granules in neutrophils and also in fusion between the surface and the granules before exocytosis. Because a2V is not found on the surface of resting neutrophils, surface a2V may be useful as a biomarker for activated neutrophils. PMID:25877929

  7. LOCALIZATION OF ACID PHOSPHATASE IN LIPOFUSCIN GRANULES AND POSSIBLE AUTOPHAGIC VACUOLES IN INTERSTITIAL CELLS OF THE GUINEA PIG TESTIS

    Microsoft Academic Search

    ARTHUR L. FRANK; A. KENT CHRISTENSEN

    1968-01-01

    The intracellular localization of acid phosphatase in guinea pig testicular interstitial cells was investigated by incubating nonfrozen thick sections of glutaraldehyde-perfused testis in a modified Comori medium and preparing the tissue for electron microscopy. Lipofuscin pigment granules in these cells contain dense pigment, granular matrix, and often a lipid droplet. Reaction product is seen in the matrix of the pigment

  8. Neurodegeneration in cerebellar granule cells of p/q type voltage gated calcium channel mutant leaner mice 

    E-print Network

    Bawa, Bhupinder

    2009-05-15

    the extent of neuronal cell death, basal intracellular calcium and mitochondrial (dys)function in cerebellar granule cells (CGC) of adult leaner mice; 2) to analyze the role of the leaner calcium channel mutation on postnatal development of CGCs; and 3...

  9. Munc18c mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose stimulated insulin secretion in human pancreatic beta-cells

    PubMed Central

    Zhu, Dan; Xie, Li; Karimian, Negar; Liang, Tao; Kang, Youhou; Huang, Ya-Chi; Gaisano, Herbert Y.

    2015-01-01

    Objective Pancreatic beta-cells express three Munc18 isoforms. Much is known about the roles of Munc18a (pre-docked secretory granules-SGs) and Munc18b (newcomer SGs and SG–SG fusion) in insulin exocytosis. Although shown to influence glucose-stimulated insulin secretion (GSIS) in rodents the precise role of Munc18c in insulin SG exocytosis has not been elucidated. We here examined the role of Munc18c in human pancreatic beta-cells. Methods Munc18c-shRNA/RFP lenti-virus (versus control virus) was used to knock down the expression level of Munc18c in human islets or single beta-cells. Insulin secretion and granule exocytosis were measured by performing islets perifusion, single-cell patch clamp capacitance measurements and total internal reflection fluorescence microscopy (TIRFM). Results Munc18c is most abundant in the cytosol of human beta-cells. Endogenous function of Munc18c was assessed by knocking down (KD) its islet expression by 70% employing lenti-shRNA virus. Munc18c-KD caused reduction in cognate syntaxin-4 islet expression but not in other exocytotic proteins, resulting in the reduction in GSIS in first- (by 42%) and second phases (by 35%). Single cell analyses of RFP-tagged Munc18c-KD beta-cells by patch clamp capacitance measurements showed inhibition in both readily-releasable pool (by 52%) and mobilization from the reserve pool (by 57%). TIRFM to assess single SG behavior showed that Munc18c-KD inhibition of first phase GSIS was attributed to reduction in exocytosis of pre-docked and newcomer SGs, and second phase inhibition attributed entirely to reduction in newcomer SG fusion (SGs that undergo minimal residence or docking time at the plasma membrane before fusion). Conclusion Munc18c is involved in the distinct molecular machineries that affect exocytosis of both predocked and newcomer SG pools that underlie Munc18c's role in first and second phases of GSIS, respectively. PMID:25973389

  10. 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

  11. 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.

  12. Secretory expression of Par-4 SAC-HA2TAT following adeno-associated virus-mediated gene transfer induces apoptosis in HepG2 cells.

    PubMed

    Qin, Tian-Jie; Ma, Wei; Liu, Shan-Xi; Yang, Guang-Xiao; Wang, Quan-Ying; Zhao, Xin-Han

    2010-01-01

    Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that induces apoptosis in cancer cells, but not in normal cells. The cancer-specific pro-apoptotic action of Par-4 is encoded in its centrally located SAC domain. In this study, to further enhance the anti-cancer effect of Par-4 in order to overcome the limitations of peptide therapy, a recombinant adeno-associated virus was constructed using the following strategies: the secretory expression of therapeutic peptide, a HA2TAT-mediated cytosolic delivery technique, and an adeno-associated virus gene transfer system. To test the hypothesis that Par-4 has an additive bystander effect as an anti-cancer therapy, we designed a secretory protein by adding a secretory signal peptide NT4(Si) to the Par-4 SAC-HA2TAT peptide gene sequence [NT4(Si)-Par-4 SAC-HA2TAT]. The results indicated that, compared to the normal NIH3T3 cell line, AAV-NT4(Si)-Par-4 SAC-HA2TAT significantly suppressed cell growth and induced rapid cell death in HepG2 cells in a time-dependent manner through successful gene transfer and secretory expression of therapeutic peptide at 48 h post-transfection. In addition, the secretory properties of Par-4 may greatly increase its effectiveness in cancer therapy when delivered in vivo. PMID:21472309

  13. Appearance of eosinophilic granule cells as pathological changes in the uteri of two aged virgin Donryu rats.

    PubMed

    Yoshida, M; Sasahara, K; Ando-Lu, J; Takahashi, M; Maekawa, A

    1997-01-01

    The morphological features of cells containing eosinophilic granules (EG cells) present as pathological changes in the uteri of 2 aged virgin Donryu rats were described and compared with granulated metrial gland (GMG) cells of a pregnant rat. Microscopically, EG cells distributed diffusely from the mesometrial triangle to the subendometrium with partly focal accumulation in Case 1 and infiltrated diffusely from the endometrium to the serosa without any clumping in Case 2. They were large cells with eosinophilic and hyaline intracytoplasmic granules, positive for the periodic acid-Schiff reaction. Both EG and GMG cells reacted positively to concanavalin A, wheat germ agglutinin, and phytohemagglutinin on lectin histochemical investigation, although common results were not always obtained for immunohistochemistry. These results show that EG cells are morphologically similar to GMG cells, suggesting a possibility that EG cells are GMG cells. EG cells were without neoplastic properties or pronounced cell-proliferative activity. In Case 1, histological changes of the ovaries and vagina were indicative of a pseudopregnant status. In Case 2, the rat had been maintained under abnormal hormonal conditions, and no evidence of any pseudopregnant-like status was found. These results suggest, thus, that the appearance of EG cells is evidence of a reactive pathological condition in the uteri of aged virgin rats, although the precise pathogenesis and biological significance of EG cells remain to be determined. PMID:9210264

  14. CTLs respond with activation and granule secretion when serving as targets for T-cell recognition.

    PubMed

    Milstein, Oren; Hagin, David; Lask, Assaf; Reich-Zeliger, Shlomit; Shezen, Elias; Ophir, Eran; Eidelstein, Yaki; Afik, Ran; Antebi, Yaron E; Dustin, Michael L; Reisner, Yair

    2011-01-20

    Cytotoxic T lymphocytes (CTLs) suppress T cell responses directed against their antigens regardless of their own T cell receptor (TCR) specificity. This makes the use of CTLs promising for tolerance induction in autoimmunity and transplantation. It has been established that binding of the CTL CD8 molecule to the major histocompatibility complex (MHC) class I ?3 domain of the recognizing T cell must be permitted for death of the latter cell to ensue. However, the signaling events triggered in the CTL by this molecular interaction in the absence of TCR recognition have never been clarified. Here we use single-cell imaging to study the events occurring in CTLs serving as targets for recognition by specific T cells. We demonstrate that CTLs actively respond to recognition by polarizing their cytotoxic granules to the contact area, releasing their lethal cargo, and vigorously proliferating. Using CTLs from perforin knockout (KO) mice and lymphocyte specific kinase (Lck) knockdown with specific small interfering RNA (siRNA), we show that the killing of the recognizing CD8 T cell is perforin dependent and is initiated by Lck signaling in the CTL. Collectively, these data suggest a novel mechanism in which the entire cascade generally triggered by TCR engagement is "hijacked" in CTLs serving as targets for T cell recognition without TCR ligation. PMID:21045195

  15. Serum levels of club (Clara) cell secretory protein predict cancer mortality in adults

    PubMed Central

    Guerra, Stefano; Vasquez, Monica M.; Spangenberg, Amber; Halonen, Marilyn; Martinez, Fernando D.

    2014-01-01

    Background Club (formerly Clara) cell secretory protein (CC16) is produced mainly by bronchiolar club cells and has been shown to have protective effects against airway inflammation and oxidative stress from cigarette smoking and related carcinogens. The goal of this study was to determine whether serum CC16 levels predict all-cause and cancer-specific mortality in adults. Methods We used data from the population-based TESAOD study, a prospective cohort study of respiratory health initiated in Tucson, AZ in 1972. At baseline, participants completed standardized respiratory questionnaires and lung function tests. Serum CC16 was measured in cryopreserved serum samples. A review of vital status of participants as of January 1st, 2011 was completed through contact with next of kin, collection of death certificates, and linkage with the National Death Index. Findings A total of 1086 participants who were 21 to 70 years old at enrollment were included. Of these, 653 (60%) died by 2011 and cause of death was ascertained for 649 (99%). In Cox proportional hazards models adjusted for sex, age, education, body mass index categories, smoking and pack-years, and baseline levels of lung function, serum CC16 levels at the baseline survey were inversely associated with mortality risk over the study follow-up. Mortality risk increased by 16% for each standard deviation (SD) decrease in CC16 (Hazard Ratio (HR), 95% CI: 1.16, 1.06 – 1.26; p = 0.0007). When data on cause-specific mortality were analyzed, each SD decrease in serum CC16 was associated with >40% increased risk of dying of cancer (adjusted HR=1.41, 1.19 – 1.67; p < 0.0001). Among smokers, the corresponding adjusted HRs for mortality by lung cancer were 1.52 (1.14 – 2.03; p = 0.004). Interpretation Serum CC16 levels predict mortality risk in the general adult population. The excess risk associated with lower CC16 is largely explained by cancer, particularly lung cancer. PMID:24461757

  16. Expressed Cell-penetrating Peptides Can Induce a Bystander Effect, but Passage Through the Secretory Pathway Reduces Protein Transduction Activity

    PubMed Central

    Shen, Ying; Yu, William; Hay, John G; Sauthoff, Harald

    2011-01-01

    Despite advances in vector technology, inefficient gene transfer still limits clinical efficacy of cancer gene therapy. Cell-penetrating peptides (CPPs), such as the basic domain of the transactivator of transcription (Tat) protein of HIV-1, are internalized by intact cells and have been used to deliver purified recombinant proteins. A combination of gene therapy with protein transduction technology could induce a strong bystander effect and represent a platform to deliver proteins to target cells. However, whether expressed CPP can facilitate intercellular trafficking, i.e., a bystander effect, is controversial. Our data suggest that expressed fusion proteins that contain the basic domain of Tat do not induce a detectable bystander effect. However, Tat-fusion proteins that also contain a secretory signal peptide (SP) can induce a bystander effect in vitro, although the in vivo effect is small. Surprisingly, despite the presence of a SP, the bystander effect does not seem to be related to secretion of the fusion protein. In fact, Tat-fusion proteins are secreted very inefficiently, and protein transduction seems largely mediated by fusion proteins that are released by cell lysis. Modification of Tat can improve secretion efficacy and prevent cleavage by the endoprotease furin, but passage through the secretory pathway is associated with reduced transduction activity of Tat-fusion proteins. PMID:21179011

  17. Expressed cell-penetrating peptides can induce a bystander effect, but passage through the secretory pathway reduces protein transduction activity.

    PubMed

    Shen, Ying; Yu, William; Hay, John G; Sauthoff, Harald

    2011-05-01

    Despite advances in vector technology, inefficient gene transfer still limits clinical efficacy of cancer gene therapy. Cell-penetrating peptides (CPPs), such as the basic domain of the transactivator of transcription (Tat) protein of HIV-1, are internalized by intact cells and have been used to deliver purified recombinant proteins. A combination of gene therapy with protein transduction technology could induce a strong bystander effect and represent a platform to deliver proteins to target cells. However, whether expressed CPP can facilitate intercellular trafficking, i.e., a bystander effect, is controversial. Our data suggest that expressed fusion proteins that contain the basic domain of Tat do not induce a detectable bystander effect. However, Tat-fusion proteins that also contain a secretory signal peptide (SP) can induce a bystander effect in vitro, although the in vivo effect is small. Surprisingly, despite the presence of a SP, the bystander effect does not seem to be related to secretion of the fusion protein. In fact, Tat-fusion proteins are secreted very inefficiently, and protein transduction seems largely mediated by fusion proteins that are released by cell lysis. Modification of Tat can improve secretion efficacy and prevent cleavage by the endoprotease furin, but passage through the secretory pathway is associated with reduced transduction activity of Tat-fusion proteins. PMID:21179011

  18. Role of platelet activating factor in the intestinal epithelial secretory and Chinese hamster ovary cell cytoskeletal responses to cholera toxin.

    PubMed Central

    Guerrant, R L; Fang, G D; Thielman, N M; Fonteles, M C

    1994-01-01

    With the recent heightened concern about cholera around the world come new questions about the mechanism by which cholera toxin causes diarrhea. Peterson and Ochoa have suggested that prostaglandin synthesis is key to both the intestinal epithelial secretory and the CHO cell responses to cholera toxin [Peterson, J. W. and Ochoa, G. (1989) Science 245, 857-859]. Because platelet activating factor (PAF) can be a potent stimulus for prostaglandin synthesis, we examined its role in the intestinal and tissue culture effects of cholera toxin. We report that the specific PAF receptor antagonists BN 52021 and SR 27417 inhibit the effects of cholera toxin on intestinal secretion in rabbit ileal loops in vivo and on the cytoskeleton of Chinese hamster ovary cells in vitro. We also show that PAF itself can cause net fluid secretion in the rabbit model and that PAF potentiates the effects of cholera toxin on intestinal secretion. Finally, we demonstrate that cholera toxin stimulates significant PAF production (2.6-fold) in isolated T-84 intestinal epithelial cells. We conclude that cholera toxin stimulates PAF production and that PAF is involved in both the secretory and cytoskeletal responses to cholera toxin. These findings further support the involvement of additional mediators of cholera toxin effects other than mucosal cell cyclic AMP and help explain the effects of cholera toxin on prostaglandin synthesis. PMID:7937824

  19. 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

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

    PubMed

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

    2013-01-01

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

  1. Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli.

    PubMed

    Orecná, M; Hafko, R; Bacová, Z; Podskocová, J; Chorvát, D; Strbák, V

    2008-01-01

    Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium. PMID:18052676

  2. The Rice  Amylase Glycoprotein Is Targeted from the Golgi Apparatus through the Secretory Pathway to the Plastids

    Microsoft Academic Search

    A. Kitajima; S. Asatsuma; H. Okada; Y. Hamada; K. Kaneko; Y. Nanjo; Y. Kawagoe; K. Toyooka; K. Matsuoka; M. Takeuchi; A. Nakano; T. Mitsui

    2009-01-01

    The well-characterized secretory glycoprotein, rice (Oryza sativa) a-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa)

  3. Whole cell K and Cl currents in dissociated eccrine secretory coil cells during stimulation

    Microsoft Academic Search

    Kenzo Sato; Minoru Ohtsuyama; Fusako Sato

    1993-01-01

    Using the whole-cell voltage clamp (to determine the membrane current) and current clamp (to determine membrane potential) methods in conjunction with the nystatin-perforation technique, we studied the effect of methacholine (MCh) and other secretagogues on whole cell K and Cl currents in dissociated rhesus palm eccrine sweat clear cells. Application of MCh by local superfusion induced a net outward current

  4. Selective vulnerability of dentate granule cells prior to amyloid deposition in PDAPP mice: Digital morphometric analyses

    PubMed Central

    Wu, Chi-Cheng; Chawla, Faisal; Games, Dora; Rydel, Russell E.; Freedman, Stephen; Schenk, Dale; Young, Warren G.; Morrison, John H.; Bloom, Floyd E.

    2004-01-01

    Increasing evidence from mouse models of Alzheimer's disease shows that overexpression of a mutant form of the amyloid precursor protein (APP) and its product, ?-amyloid peptide, initiate pathological changes before amyloid deposition. To evaluate the cytological basis for one of these early changes, namely reduced volume of the dentate gyrus (DG), we have used high-throughput diOlistic cell loading and 3D neuronal reconstruction to investigate potential dendritic pathology of granule cells (GCs) in 90-day-old PDAPP mice. Labeled GCs from fixed hippocampal slices were selected randomly and imaged digitally by using confocal laser-scanning microscopy. The dendritic complexity of GCs was quantified according to subordinate morphological parameters, including soma position within the granule cell layer (superficial versus deep) and topographic location within the DG (dorsal versus ventral blade) along the anterior-posterior hippocampal axis. Initial analysis, which included all sampled GC types, revealed a 12% reduction of total dendritic length in PDAPP mice compared with littermate controls. Further analysis, performed with refined subgroups, found that superficially located GCs in the dorsal blade were profoundly altered, exhibiting a 23% loss in total dendritic length, whereas neurons in the ventral blade were unaffected. Superficial GCs were particularly vulnerable (a 32% reduction) in the posterior region of the DG. Furthermore, the dendritic reductions of this select group were uniformly localized within middle-to-outer portions of the dentate molecular layer. We conclude that substantial dendritic pathology is evident in 90-day-old PDAPP mice for a spatially defined subset of GCs well before amyloid accumulation occurs. PMID:15118092

  5. Dipeptidyl peptidase-like protein 6 is required for normal electrophysiological properties of cerebellar granule cells.

    PubMed

    Nadin, Brian M; Pfaffinger, Paul J

    2010-06-23

    In cerebellar granule (CG) cells and many other neurons, A-type potassium currents play an important role in regulating neuronal excitability, firing patterns, and activity-dependent plasticity. Protein biochemistry has identified dipeptidyl peptidase-like protein 6 (DPP6) as an auxiliary subunit of Kv4-based A-type channels and thus a potentially important regulator of neuronal excitability. In this study, we used an RNA interference (RNAi) strategy to examine the role DPP6 plays in forming and shaping the electrophysiological properties of CG cells. DPP6 RNAi delivered by lentiviral vectors effectively disrupts DPP6 protein expression in CG cells. In response to the loss of DPP6, I(SA) peak conductance amplitude is reduced by >85% in parallel with a dramatic reduction in the level of I(SA) channel protein complex found in CG cells. The I(SA) channels remaining in CG cells after suppression of DPP6 show alterations in gating similar to Kv4 channels expressed in heterologous systems without DPP6. In addition to these effects on A-type current, we find that loss of DPP6 has additional effects on input resistance and Na(+) channel conductance that combine with the effects on I(SA) to produce a global change in excitability. Overall, DPP6 expression seems to be critical for the expression of a high-frequency electrophysiological phenotype in CG cells by increasing leak conductance, A-type current levels and kinetics, and Na(+) current amplitude. PMID:20573902

  6. Mutation in the Neural Cell Adhesion Molecule Interferes with the Differentiation of Anterior Pituitary Secretory Cells

    Microsoft Academic Search

    Olena Gubkina; Harold Cremer; Geneviève Rougon

    2001-01-01

    The neural cell adhesion molecule (NCAM) and its polysialylated isoform (PSA-NCAM) have been shown to influence the proliferation, differentiation and survival of different cell types. Here, we report the pattern of expression of NCAM and PSA-NCAM in the anterior lobe (AL) of the pituitary gland of the adult mouse. We demonstrate that the majority of cells express NCAM, while PSA-NCAM

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

    ERIC Educational Resources Information Center

    Spanswick, Simon C.; Sutherland, Robert J.

    2010-01-01

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

  8. Pituitary adenylate cyclase-activating polypeptide protects rat cerebellar granule neurons against ethanol-induced apoptotic cell death

    Microsoft Academic Search

    David Vaudry; Cécile Rousselle; Magali Basille; Anthony Falluel-Morel; Tommy F. Pamantung; Marc Fontaine; Alain Fournier; Hubert Vaudry; Bruno J. Gonzalez

    2002-01-01

    Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Here, we report that pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibited

  9. Differential Modulation of Sonic-Hedgehog-Induced Cerebellar Granule Cell Precursor Proliferation by the IGF Signaling Network

    Microsoft Academic Search

    Carla Fernandez; Valérie M. Tatard; Nicolas Bertrand; Nadia Dahmane

    2010-01-01

    The molecular mechanisms regulating organ growth and size remain unclear. Sonic hedgehog (SHH) signaling is a major player in the regulation of cerebellar development: SHH is secreted by Purkinje neurons and acts on the proliferation of granule cell precursors (GCPs) in the external germinal layer. These then become postmitotic and form the internal granular layer but do so in the

  10. T-cadherin (Cdh13) in association with pancreatic ?-cell granules contributes to second phase insulin secretion

    PubMed Central

    Tyrberg, Björn; Miles, Philip; Azizian, Krist T.; Denzel, Martin S.; Nieves, Maria L.; Monosov, Edward Z.; Levine, Fred; Ranscht, Barbara

    2011-01-01

    Glucose homeostasis depends on adequate control of insulin secretion. We report the association of the cell-adhesion and adiponectin (APN)-binding glycoprotein T-cadherin (Cdh13) with insulin granules in mouse and human ?-cells. Immunohistochemistry and electron microscopy of islets in situ and targeting of RFP-tagged T-cadherin to GFP-labeled insulin granules in isolated ?-cells demonstrate this unusual location. Analyses of T-cadherin-deficient (Tcad-KO) mice show normal islet architecture and insulin content. However, T-cadherin is required for sufficient insulin release in vitro and in vivo. Primary islets from Tcad-KO mice were defective in glucose-induced but not KCl-mediated insulin secretion. In vivo, second phase insulin release in T-cad-KO mice during a hyperglycemic clamp was impaired while acute first phase release was unaffected. Tcad-KO mice showed progressive glucose intolerance by 5 mo of age without concomitant changes in peripheral insulin sensitivity. Our analyses detected no association of APN with T-cadherin on ?-cell granules although colocalization was observed on the pancreatic vasculature. These data identify T-cadherin as a novel component of insulin granules and suggest that T-cadherin contributes to the regulation of insulin secretion independently of direct interactions with APN. PMID:21975561

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

    Microsoft Academic Search

    Danièle 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

  12. MID2 can substitute for MID1 and control exocytosis of lytic granules in cytotoxic T cells.

    PubMed

    Boding, Lasse; Hansen, Ann K; Meroni, Germana; Levring, Trine B; Woetmann, Anders; Ødum, Niels; Bonefeld, Charlotte M; Geisler, Carsten

    2015-08-01

    We have recently shown that the E3 ubiquitin ligase midline 1 (MID1) is upregulated in murine cytotoxic lymphocytes (CTL), where it controls exocytosis of lytic granules and the killing capacity. Accordingly, CTL from MID1 knock-out (MID1(-/-) ) mice have a 25-30% reduction in exocytosis of lytic granules and cytotoxicity compared to CTL from wild-type (WT) mice. We wondered why the MID1 gene knock-out did not affect exocytosis and cytotoxicity more severely and speculated whether MID2, a close homologue of MID1, might partially compensate for the loss of MID1 in MID1(-/-) CTL. Here, we showed that MID2, like MID1, is upregulated in activated murine T cells. Furthermore, MID1(-/-) CTL upregulated MID2 two-twenty-fold stronger than CTL from WT mice, suggesting that MID2 might compensate for MID1. In agreement, transfection of MID2 into MID1(-/-) CTL completely rescued exocytosis of lytic granules in MID1(-/-) CTL, and vice versa, knock-down of MID2 inhibited exocytosis of lytic granules in both WT and MID1(-/-) CTL, demonstrating that both MID1 and MID2 play a central role in the regulation of granule exocytosis and that functional redundancy exists between MID1 and MID2 in CTL. PMID:25924778

  13. Clonal analysis reveals granule cell behaviors and compartmentalization that determine the folded morphology of the cerebellum.

    PubMed

    Legué, Emilie; Riedel, Elyn; Joyner, Alexandra L

    2015-05-01

    The mammalian cerebellum consists of folds of different sizes and shapes that house distinct neural circuits. A crucial factor underlying foliation is the generation of granule cells (gcs), the most numerous neuron type in the brain. We used clonal analysis to uncover global as well as folium size-specific cellular behaviors that underlie cerebellar morphogenesis. Unlike most neural precursors, gc precursors divide symmetrically, accounting for their massive expansion. We found that oriented cell divisions underlie an overall anteroposteriorly polarized growth of the cerebellum and gc clone geometry. Clone geometry is further refined by mediolateral oriented migration and passive dispersion of differentiating gcs. Most strikingly, the base of each fissure acts as a boundary for gc precursor dispersion, which we propose allows each folium to be regulated as a developmental unit. Indeed, the geometry and size of clones in long and short folia are distinct. Moreover, in engrailed 1/2 mutants with shorter folia, clone cell number and geometry are most similar to clones in short folia of wild-type mice. Thus, the cerebellum has a modular mode of development that allows the plane of cell division and number of divisions to be differentially regulated to ensure that the appropriate number of cells are partitioned into each folium. PMID:25834018

  14. 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…

  15. Electron probe X-ray microanalysis of cellular ions in the eccrine secretory coil cells during methacholine stimulation.

    PubMed

    Saga, K; Sato, K

    1989-01-01

    Intracellular concentrations of Na, K, Cl ([Na], [K] and [Cl], respectively) and other elements were determined in isolated monkey eccrine sweat secretory coil cells using quantitative electron probe X-ray microanalysis of freeze dried cryosections. The validity of the methodology was partially supported by qualitative agreement of the X-ray microanalysis data with those obtained by micro-titration with a helium glow spectrophotometer. [Na], [K] and [Cl] of the cytoplasm were the same as those in the nucleus in both clear and dark cells. [Na], [K], and [Cl] of the clear cells were also the same as those of the dark cells at rest and after stimulation with methacholine (MCh), suggesting that these two cell types behave like a functional syncytium. MCh stimulation induced a pharmacologically specific, dose-dependent decrease in [K] and [Cl] (as much as 65%), and a 3.7-fold increase in [Na]. In myoepithelial cells, a similar change in [Na] and [K] was noted after MCh stimulation although the decrease in [Cl] was only 20%. The MCh-induced change in [Na], [K] and [Cl] was almost completely inhibited by removal of Ca2+ from the medium. 10(-4) M bumetanide inhibited the MCh-induced increase in [Na], reduced the decrease in [K] by about 50%, but slightly augmented the MCh-induced decrease in [Cl]. 10(-4) M ouabain increased [Na] and decreased [K] as did MCh; however, unlike MCh, ouabain increased [Cl] by 56% after 30 min of incubation. Thus the data may be best interpreted to indicate that Ca-dependent K efflux and (perhaps also Ca-dependent) Cl efflux are the predominant initial ionic movement in muscarinic cholinergic stimulation of the eccrine sweat secretory coils and that the ouabain-sensitive Na pump plays an important role in maintenance of intracellular ions and sweat secretion. PMID:2646452

  16. Role of ionic fluxes in the apoptotic cell death of cultured cerebellar granule neurons.

    PubMed

    Franco-Cea, A; Valencia, A; Sánchez-Armass, S; Domínguez, G; Morán, J

    2004-01-01

    Cultured cerebellar granule neurons (CGC) increase survival in a medium containing 25 mM KCl (K25), and they die apoptotically when cultures are treated with staurosporine (St) or are transferred to a 5-mM KCl containing medium (K5). Apoptotic CGC show nuclear condensation and caspase-3 activation. Cell death induced by these conditions was partially prevented when cultures were maintained under alkaline conditions, which also induced a marked reduction of the caspase-3 activation. The acidification of the medium further increased cell death induced by both stimuli. Cultures transferred to K5 suffered an immediate intracellular alkalinization that remained constant during the time K5 was present. In contrast, St did not modify cytosolic pH at any of the evaluated times. On the other hand, DIDS, furosemide, and bumetanide prevented CGC death induced by K5 and St. Other drugs such as amiloride, EIPA, tamoxifen, NEM, or NPPB did not modify cell death induced by these conditions. Both DIDS and bumetanide markedly inhibited the processing and activation of caspase-3, and DIDS prevented the nuclear condensation induced by K5 and St. These findings suggest that pH is a condition that could contribute to the modulation of cell death induced by some stimuli and that other ions, such as potassium, could have a role in the initial phase of apoptotic death of CGC. PMID:14992282

  17. Phosphorylation of the myosin IIA tailpiece regulates single myosin IIA molecule association with lytic granules to promote NK-cell cytotoxicity

    PubMed Central

    Sanborn, Keri B.; Mace, Emily M.; Rak, Gregory D.; Difeo, Analisa; Martignetti, John A.; Pecci, Alessandro; Bussel, James B.; Favier, Rémi

    2011-01-01

    Natural killer (NK) cells are innate immune lymphocytes that provide critical defense against virally infected and transformed cells. NK-cell cytotoxicity requires the formation of an F-actin rich immunologic synapse (IS), as well as the polarization of perforin-containing lytic granules to the IS and secretion of their contents at the IS. It was reported previously that NK-cell cytotoxicity requires nonmuscle myosin IIA function and that granule-associated myosin IIA mediates the interaction of granules with F-actin at the IS. In the present study, we evaluate the nature of the association of myosin IIA with lytic granules. Using NK cells from patients with mutations in myosin IIA, we found that the nonhelical tailpiece is required for NK-cell cytotoxicity and for the phosphorylation of granule-associated myosin IIA. Ultra-resolution imaging techniques demonstrated that single myosin IIA molecules associate with NK-cell lytic granules via the nonhelical tailpiece. Phosphorylation of myosin IIA at residue serine 1943 (S1943) in the tailpiece is needed for this linkage. This defines a novel mechanism for myosin II function, in which myosin IIA can act as a single-molecule actin motor, claiming granules as cargo through tail-dependent phosphorylation for the execution of a pre-final step in human NK-cell cytotoxicity. PMID:22123909

  18. Phosphorylation of the myosin IIA tailpiece regulates single myosin IIA molecule association with lytic granules to promote NK-cell cytotoxicity.

    PubMed

    Sanborn, Keri B; Mace, Emily M; Rak, Gregory D; Difeo, Analisa; Martignetti, John A; Pecci, Alessandro; Bussel, James B; Favier, Rémi; Orange, Jordan S

    2011-11-24

    Natural killer (NK) cells are innate immune lymphocytes that provide critical defense against virally infected and transformed cells. NK-cell cytotoxicity requires the formation of an F-actin rich immunologic synapse (IS), as well as the polarization of perforin-containing lytic granules to the IS and secretion of their contents at the IS. It was reported previously that NK-cell cytotoxicity requires nonmuscle myosin IIA function and that granule-associated myosin IIA mediates the interaction of granules with F-actin at the IS. In the present study, we evaluate the nature of the association of myosin IIA with lytic granules. Using NK cells from patients with mutations in myosin IIA, we found that the nonhelical tailpiece is required for NK-cell cytotoxicity and for the phosphorylation of granule-associated myosin IIA. Ultra-resolution imaging techniques demonstrated that single myosin IIA molecules associate with NK-cell lytic granules via the nonhelical tailpiece. Phosphorylation of myosin IIA at residue serine 1943 (S1943) in the tailpiece is needed for this linkage. This defines a novel mechanism for myosin II function, in which myosin IIA can act as a single-molecule actin motor, claiming granules as cargo through tail-dependent phosphorylation for the execution of a pre-final step in human NK-cell cytotoxicity. PMID:22123909

  19. Defects in the cerebella of conditional Neurod1 null mice correlate with effective Tg(Atoh1-cre) recombination and granule cell requirements for Neurod1 for differentiation

    Microsoft Academic Search

    Ning Pan; Israt Jahan; Jacqueline E. Lee; Bernd Fritzsch

    2009-01-01

    Neurod1 is a crucial basic helix-loop-helix gene for most cerebellar granule cells and mediates the differentiation of these cells\\u000a downstream of Atoh1-mediated proliferation of the precursors. In Neurod1 null mice, granule cells die throughout the posterior two thirds of the cerebellar cortex during development. However, Neurod1 is also necessary for pancreatic ?-cell development, and therefore Neurod1 null mice are diabetic,

  20. Endocrine pancreatic cells from Xenopus laevis: light and electron microscopic studies.

    PubMed

    Lozano, M T; Hernández, M P; Agulleiro, B

    1999-05-01

    Insulin, glucagon, pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), somatostatin (SST)-28 (1-12), salmon (s) SST-25, and SST-14 immunoreactivities were demonstrated in the pancreatic endocrine cells of Xenopus laevis using light and electron microscopic immunocytochemistry. Insulin-, SST-28 (1-12)/SST-14-, and PYY-immunoreactive (ir) cells were found throughout the pancreas either isolated in small clusters of a single cell type or, except in the case of PYY-ir cells, forming islets consisting of various cell types. Anti-sSST-25 serum detected the invariant SST-14 form. Cells that were only immunoreactive to glucagon were isolated or clustered in the duodenal lobe, while in the splenic lobe cells immunoreactive to both glucagon and PP were observed in isolation, clustered, or in the periphery of the islets. There were no cells that were immunoreactive only to PP or to NPY. Ultrastructurally, the endocrine cells were characterized by their secretory granules, which were immunogold labeled with the corresponding antisera. Insulin cells had large round secretory granules with a round, irregular, or crystalline-like dense core. Glucagon-ir cells had round secretory granules with a dense core and a clear halo. Glucagon/PP- and PYY-ir cells showed round, ovoid, or pear-shaped secretory granules, which were larger and less electron dense in the latter cell type. The secretory granules of SST-ir cells were ovoid or bacillary with a medium electron-dense content. A sixth cell type with very small secretory granules could only be characterized by conventional electron microscopy, since it did not immunoreact with any of the antisera applied in this study. PMID:10208768

  1. 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.

  2. From differentiation to proliferation: The secretory amyloid precursor protein as a local mediator of growth in thyroid?epithelial?cells

    PubMed Central

    Pietrzik, Claus Ulrich; Hoffmann, Jens; Stöber, Kai; Chen, Chun-Yan; Bauer, Christoph; Otero, Deborah A. C.; Roch, Jean-Marc; Herzog, Volker

    1998-01-01

    In various species, thyrotropin (TSH) is known to stimulate both differentiation and proliferation of thyroid follicle cells. This cell type has also been shown to express members of the Alzheimer amyloid precursor (APP) protein family and to release the secretory N-terminal domain of APP (sAPP) in a TSH-dependent fashion. In this study on binding to the cell surfaces, exogenously added recombinant sAPP stimulated phosphorylation mediated by mitogen-activated protein kinase and effectively evoked proliferation in the rat thyroid epithelial cell line FRTL-5. To see whether this proliverative effect of sAPP is of physiological relevance, we used antisense techniques to selectively inhibit the expression of APP and the proteolytic release of sAPP by cells grown in the presence of TSH. The antisense-induced inhibition was detected by immunoblot, immunoprecipitation, and immunocytochemical analyses. After the reduced APP expression and sAPP secretion, we observed a strong suppression of the TSH-induced cell proliferation down to 35%. Recombinant sAPP but not TSH was able to overcome this antisense effect and to completely restore cell proliferation, indicating that sAPP acts downstream of TSH, in that it is released from thyroid epithelial cells during TSH-induced differentiation. We propose that sAPP operates as an autocrine growth factor mediating the proliferative effect of TSH on neighboring thyroid epithelial cells. PMID:9465092

  3. Adult-born granule cells mature through two functionally distinct states

    PubMed Central

    Brunner, János; Neubrandt, Máté; Van-Weert, Susan; Andrási, Tibor; Kleine Borgmann, Felix B; Jessberger, Sebastian; Szabadics, János

    2014-01-01

    Adult-born granule cells (ABGCs) are involved in certain forms of hippocampus-dependent learning and memory. It has been proposed that young but functionally integrated ABGCs (4-weeks-old) specifically contribute to pattern separation functions of the dentate gyrus due to their heightened excitability, whereas old ABGCs (>8 weeks old) lose these capabilities. Measuring multiple cellular and integrative characteristics of 3- 10-week-old individual ABGCs, we show that ABGCs consist of two functionally distinguishable populations showing highly distinct input integration properties (one group being highly sensitive to narrow input intensity ranges while the other group linearly reports input strength) that are largely independent of the cellular age and maturation stage, suggesting that ‘classmate’ cells (born during the same period) can contribute to the network with fundamentally different functions. Thus, ABGCs provide two temporally overlapping but functionally distinct neuronal cell populations, adding a novel level of complexity to our understanding of how life-long neurogenesis contributes to adult brain function. DOI: http://dx.doi.org/10.7554/eLife.03104.001 PMID:25061223

  4. Polygalae radix extract protects cultured rat granule cells against damage induced by NMDA.

    PubMed

    Lee, Hyun Joo; Ban, Ju Yeon; Koh, Sang Bum; Seong, Nak Sul; Song, Kyung Sik; Bae, Ki Whan; Seong, Yeon Hee

    2004-01-01

    Polygalae Radix (PR) from Polygala tenuifolia (Polygalaceae) is traditionally used in China and Korea, as this herb has a sedative, anti-inflammatory and antibacterial agent. To extend our understanding of the pharmacological actions of PR in the CNS on the basis of its CNS inhibitory effect, the present study examined whether PR has the neuroprotective action against N-methyl-D-aspartate (NMDA)-induced cell death in primarily cultured rat cerebellar granule neurons. PR, over a concentration range of 0.05 to 5 microg/ml, inhibited NMDA (1 mM)-induced neuronal cell death, which was measured by a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. PR (0.5 microg/ml) inhibited glutamate release into medium induced by NMDA (1 mM), which was measured by HPLC. Pre-treatment of PR (0.5 microg/ml) inhibited NMDA (1 mM)-induced elevation of intracellular Ca2+ concentration ([Ca2+]i), which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that PR prevents NMDA-induced neuronal cell damage in vitro. PMID:15481649

  5. Raising cytosolic Cl? in cerebellar granule cells affects their excitability and vestibulo-ocular learning

    PubMed Central

    Seja, Patricia; Schonewille, Martijn; Spitzmaul, Guillermo; Badura, Aleksandra; Klein, Ilse; Rudhard, York; Wisden, William; Hübner, Christian A; De Zeeuw, Chris I; Jentsch, Thomas J

    2012-01-01

    Cerebellar cortical throughput involved in motor control comprises granule cells (GCs) and Purkinje cells (PCs), both of which receive inhibitory GABAergic input from interneurons. The GABAergic input to PCs is essential for learning and consolidation of the vestibulo-ocular reflex, but the role of GC excitability remains unclear. We now disrupted the Kcc2 K-Cl cotransporter specifically in either cell type to manipulate their excitability and inhibition by GABAA-receptor Cl? channels. Although Kcc2 may have a morphogenic role in synapse development, Kcc2 disruption neither changed synapse density nor spine morphology. In both GCs and PCs, disruption of Kcc2, but not Kcc3, increased [Cl?]i roughly two-fold. The reduced Cl? gradient nearly abolished GABA-induced hyperpolarization in PCs, but in GCs it merely affected excitability by membrane depolarization. Ablation of Kcc2 from GCs impaired consolidation of long-term phase learning of the vestibulo-ocular reflex, whereas baseline performance, short-term gain-decrease learning and gain consolidation remained intact. These functions, however, were affected by disruption of Kcc2 in PCs. GC excitability plays a previously unknown, but specific role in consolidation of phase learning. PMID:22252133

  6. Endogenous intracellular calcium buffering and the activation\\/inactivation of HVA calcium currents in rat dentate gyrus granule cells

    Microsoft Academic Search

    GEORG KOHR; ISTVAN MODY

    1991-01-01

    A B ST R ACT Granule cells acutely dissociated from the dentate gyrus of adult rat brains displayed a single class of high-threshold, voltage-activated (HVA) Ca 2+ channels. The kinetics of whole-cell Ca ~+ currents recorded with pipette solutions containing an intracellular ATP regenerating system but devoid of exogenous Ca ~+ buffers, were fit best by Hodgkin-Huxley kinetics (m2h), and

  7. Effects of Calretinin on Ca 2+ Signals in Cerebellar Granule Cells: Implications of Cooperative Ca 2+ Binding

    Microsoft Academic Search

    Elena È. Saftenku

    Calretinin is thought to be the main endogenous calcium buffer in cerebellar granule cells (GrCs). However, little is known\\u000a about the impact of cooperative Ca2+ binding to calretinin on highly localized and more global (regional) Ca2+ signals in these cells. Using numerical simulations, we show that an essential property of calretinin is a delayed equilibration\\u000a with Ca2+. Therefore, the amount

  8. Neurodegeneration in cerebellar granule cells of p/q type voltage gated calcium channel mutant leaner mice

    E-print Network

    Bawa, Bhupinder

    2009-05-15

    paradigm, to have organizing potential on surrounding neural tissue by virtue of its morphogenetic properties and expression of many regulatory genes like Fgf8, Wnt1, Gbx2 and Otx2 (Sotelo, 2004). During the embryonic phase of murine cerebellar... from precursors to mature neurons have suggested that Purkinje cell- derived Sonic hedgehog morphogen plays a role in proliferation of granule cell precursors (Wallace, 1999; Wechsler-Reya and Scott, 1999). Postnatal cerebellar development processes...

  9. 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

  10. Transcriptome profiling of human hippocampus dentate gyrus granule cells in mental illness

    PubMed Central

    Kohen, R; Dobra, A; Tracy, J H; Haugen, E

    2014-01-01

    This study is, to the best of our knowledge, the first application of whole transcriptome sequencing (RNA-seq) to cells isolated from postmortem human brain by laser capture microdissection. We investigated the transcriptome of dentate gyrus (DG) granule cells in postmortem human hippocampus in 79 subjects with mental illness (schizophrenia, bipolar disorder, major depression) and nonpsychiatric controls. We show that the choice of normalization approach for analysis of RNA-seq data had a strong effect on results; under our experimental conditions a nonstandard normalization method gave superior results. We found evidence of disrupted signaling by miR-182 in mental illness. This was confirmed using a novel method of leveraging microRNA genetic variant information to indicate active targeting. In healthy subjects and those with bipolar disorder, carriers of a high- vs those with a low-expressing genotype of miR-182 had different levels of miR-182 target gene expression, indicating an active role of miR-182 in shaping the DG transcriptome for those subject groups. By contrast, comparing the transcriptome between carriers of different genotypes among subjects with major depression and schizophrenia suggested a loss of DG miR-182 signaling in these conditions. PMID:24594777

  11. Retromer vesicles interact with RNA granules in haploid male germ cells.

    PubMed

    Da Ros, Matteo; Hirvonen, Noora; Olotu, Opeyemi; Toppari, Jorma; Kotaja, Noora

    2015-02-01

    Spermatozoa are produced during spermatogenesis as a result of mitotic proliferation, meiosis and cellular differentiation. Postmeiotic spermatids are exceptional cells given their haploid genome and remarkable sperm-specific structural transformations to compact and reshape the nucleus and to construct the flagellum and acrosome. These processes require delicate coordination and active communication between distinct cellular compartments. In this study, we elucidated the interplay between the haploid RNA regulation and the vesicular transport system. We identified a novel interaction between VPS26A/VPS35-containing retromer vesicles and the chromatoid body (CB), which is a large ribonucleoprotein (RNP) granule unique to haploid male germ cells. VPS26A/VPS35-positive vesicles were shown to be involved in the endosomal pathway, as well as in acrosomal formation that is dependent on the Golgi complex-derived vesicular trafficking. While the exact role of the retromer vesicles in the CB function remains unclear, our results suggest a direct functional link between vesicle transport and CB-mediated RNA regulation. PMID:25486514

  12. Protracted Postnatal Development of Sparse, Specific Dentate Granule Cell Activation in the Mouse Hippocampus

    PubMed Central

    Yu, Esther P.; Dengler, Christopher G.; Frausto, Shanti F.; Putt, Mary E.; Yue, Cuiyong; Takano, Hajime; Coulter, Douglas A.

    2013-01-01

    The dentate gyrus (DG) is a critical entry point regulating function of the hippocampus. Integral to this role are the sparse, selective activation characteristics of the principal cells of the DG, dentate granule cells (DGCs). This sparse activation is important both in cognitive processing, and in regulation of pathological activity in disease states. Using a novel, combined dynamic imaging approach capable of resolving sequentially both synaptic potentials and action potential firing in large populations of DGCs, we characterized the postnatal development of firing properties of DG neurons in response to afferent activation in mouse hippocampal-entorhinal cortical slices. During postnatal development, there was a protracted, progressive sparsification of responses, accompanied by increased temporal precision of activation. Both of these phenomena were primarily mediated by changes in local circuit inhibition, and not by alterations in afferent innervation of DGCs, since GABAA antagonists normalized developmental differences. There was significant theta and gamma frequency-dependent synaptic recruitment of DGC activation in adult, but not developing, animals. Finally, we found that the decision to fire or not fire by individual DGCs was robust and repeatable at all stages of development. The protracted postnatal development of sparse, selective firing properties, increased temporal precision and frequency dependence of activation, and the fidelity with which the decision to fire is made are all fundamental circuit determinants of DGC excitation, critical in both normal and pathological function of the DG. PMID:23407953

  13. Electrophysiological characterization of granule cells in the dentate gyrus immediately after birth.

    PubMed

    Pedroni, Andrea; Minh, Do Duc; Mallamaci, Antonello; Cherubini, Enrico

    2014-01-01

    Granule cells (GCs) in the dentate gyrus are generated mainly postnatally. Between embryonic day 10 and 14, neural precursors migrate from the primary dentate matrix to the dentate gyrus where they differentiate into neurons. Neurogenesis reaches a peak at the end of the first postnatal week and it is completed at the end of the first postnatal month. This process continues at a reduced rate throughout life. Interestingly, immediately after birth, GCs exhibit a clear GABAergic phenotype. Only later they integrate the classical glutamatergic trisynaptic hippocampal circuit. Here, whole cell patch clamp recordings, in current clamp mode, were performed from immature GCs, intracellularly loaded with biocytin (in hippocampal slices from P0 to P3 old rats) in order to compare their morphological characteristics with their electrophysiological properties. The vast majority of GCs were very immature with small somata, few dendritic branches terminating with small varicosities and growth cones. In spite of their immaturity their axons reached often the cornu ammonis 3 area. Immature GCs generated, upon membrane depolarization, either rudimentary sodium spikes or more clear overshooting action potentials that fired repetitively. They exhibited also low threshold calcium spikes. In addition, most spiking neurons showed spontaneous synchronized network activity, reminiscent of giant depolarizing potentials (GDPs) generated in the hippocampus by the synergistic action of glutamate and GABA, both depolarizing and excitatory. This early synchronized activity, absent during adult neurogenesis, may play a crucial role in the refinement of local neuronal circuits within the developing dentate gyrus. PMID:24592213

  14. Ultrastructural and cytochemical studies of resorptive and digestive functions of secretory ameloblasts in kitten tooth germs.

    PubMed

    Sasaki, T

    1983-04-01

    Ultrastructural and cytochemical studies of kitten secretory ameloblasts were made in order to clarify their functions in the resorption and digestion of extracellular organic materials. The secretory ameloblast had triangular Tomes' processes whose profile was divided into type 1 and type 2 faces. Type 1 face was associated with tubular structures, coated pits, coated vesicles, and irregularly shaped vesicles presumably representing phagosomes. Freeze-fracture replicas clearly showed the presence of large, particle-rich depressions and small depressions on the cell membrane P face in the type 1 face of the Tomes' process. Exocytosis of secretory granules was seldom observed. In both thin sections and replicas, the type 2 face possessed cell membrane microinvaginations. From the supranuclear region to a zone near the Tomes' process, many dense bodies, multivesicular bodies, and vacuoles were present; and many of them showed intense acid phosphatase reactions. Reaction products of acid phosphatase were demonstrated in the Golgi apparatus, GERL, and the lateral cell membrane. These results suggest that kitten secretory ameloblasts resorb and digest extracellular organic materials. PMID:6845973

  15. Immunohistochemical localization of Clara cell secretory proteins (CC10-CC26) and Annexin-1 protein in rat major salivary glands

    PubMed Central

    Cecchini, Maria Paola; Merigo, Flavia; Cristofoletti, Mirko; Osculati, Francesco; Sbarbati, Andrea

    2009-01-01

    The oral cavity is continuously bathed by saliva secreted by the major and minor salivary glands. Saliva is the first biological medium to confront external materials that are taken into the body as part of food or drink or inhaled volatile substances, and it contributes to the first line of oral defence. In humans, it has been shown that sputum and a variety of biological fluids contain Clara cell secretory proteins (CC10–CC26). Various studies of the respiratory apparatus have suggested their protective effect against inflammatory response and oxidative stress. Recently, CC10 deficiency has been related to the protein Annexin-1 (ANXA1), which has immunomodulatory and anti-inflammatory properties. Considering the defensive role of both Clara cell secretory proteins and ANXA1 in the respiratory apparatus, and the importance of salivary gland secretion in the first line of oral defence, we decided to evaluate the expression of CC10, CC26 and ANXA1 proteins in rat major salivary glands using immunohistochemistry. CC10 expression was found only in the ductal component of the sublingual gland. Parotid and submandibular glands consistently lacked CC10 immunoreactivity. In the parotid gland, both acinar and ductal cells were always CC26-negative, whereas in the submandibular gland, immunostaining was localized in the ductal component and in the periodic acid Schiff (PAS)-positive area. In the sublingual gland, ductal cells were always positive. Acinar cells were not immunostained at all. ANXA1 was expressed in ductal cells in all three major glands. In parotid and sublingual glands, acinar cells were negative. In submandibular glands, immunostaining was present in the mucous PAS-positive portion, whereas serous acinar cells were consistently negative. The existence of some CC10-CC26–ANXA1-positive cells in rat salivary glandular tissue is an interesting preliminary finding which could support the hypothesis, suggested for airway tissue, that these proteins have a defensive and protective role. Protein expression heterogeneity in the different portions of the glands could be an important clue in further investigations of their role. PMID:19438769

  16. Sodium, calcium and late potassium currents are reduced in cerebellar granule cells cultured in the presence of a protein complex conferring resistance to excitatory amino acids.

    PubMed

    Zona, C; Ragozzino, D; Ciotti, M T; Mercanti, D; Avoli, M; Brancati, A; Calissano, P

    1993-11-01

    Whole-cell, patch-clamp recordings were used to study voltage-gated currents generated by cerebellar granule cells that were cultured in medium containing either 10% fetal calf serum (hereafter termed S + granules) or neurite outgrowth and adhesion complex (NOAC, hereafter called NOAC granules). NOAC is a protein complex found in rabbit serum that renders granules resistant to the excitotoxic action of excitatory amino acids. During depolarizing commands both S+ and NOAC granules generated Na+ and Ca2+ inward currents and an early and a late K+ outward currents. However, Na+ and Ca2+ inward currents and late outward K+ currents recorded in NOAC granules were smaller than those seen in S+ granules. Furthermore, although of similar amplitude, early K+ currents displayed different kinetics in the two types of neurons. Thus, these data demonstrate that the electrophysiological properties of cerebellar granules, and probably of other neuronal populations, depend upon serum components and raise the possibility that an analogous modulation might be operative in vivo, and play a role in development, synaptic plasticity or neuropathological processes. PMID:8287194

  17. Isolation of a sesquiterpene synthase expressing in specialized epithelial cells surrounding the secretory cavities in rough lemon (Citrus jambhiri).

    PubMed

    Uji, Yuya; Ozawa, Rika; Shishido, Hodaka; Taniguchi, Shiduku; Takabayashi, Junji; Akimitsu, Kazuya; Gomi, Kenji

    2015-05-15

    Volatile terpenoids such as monoterpenes and sesquiterpenes play multiple roles in plant responses and are synthesized by terpene synthases (TPSs). We have previously isolated a partial TPS gene, RlemTPS4, that responds to microbial attack in rough lemon. In this study, we isolated a full length RlemTPS4 cDNA from rough lemon. RlemTPS4 localized in the cytosol. The recombinant RlemTPS4 protein was obtained using a prokaryotic expression system and GC-MS analysis of the terpenes produced by the RlemTPS4 enzymatic reaction determined that RlemTPS4 produces some sesquiterpenes such as ?-elemene. The RlemTPS4 gene was specifically expressed in specialized epithelial cells surrounding the oil secretory cavities in rough lemon leaf tissue. PMID:25899729

  18. Secretory Low Molecular Weight Phospholipase A2 Plays Important Roles in Cell Elongation and Shoot Gravitropism in Arabidopsis

    PubMed Central

    Lee, Hyoung Yool; Bahn, Sung Chul; Kang, Yoon-Mi; Lee, Kyu Hee; Kim, Hae Jin; Noh, Eun Kyeung; Palta, Jiwan P.; Shin, Jeong Sheop; Ryu, Stephen B.

    2003-01-01

    To elucidate the cellular functions of phospholipase A2 in plants, an Arabidopsis cDNA encoding a secretory low molecular weight phospholipase A2 (AtsPLA2?) was isolated. Phenotype analyses of transgenic plants showed that overexpression of AtsPLA2? promotes cell elongation, resulting in prolonged leaf petioles and inflorescence stems, whereas RNA interference–mediated silencing of AtsPLA2? expression retards cell elongation, resulting in shortened leaf petioles and stems. AtsPLA2? is expressed in the cortical, vascular, and endodermal cells of the actively growing tissues of inflorescence stems and hypocotyls. AtsPLA2? then is secreted into the extracellular spaces, where signaling for cell wall acidification is thought to occur. AtsPLA2?-overexpressing or -silenced transgenic plants showed altered gravitropism in inflorescence stems and hypocotyls. AtsPLA2? expression is induced rapidly by auxin treatment and in the curving regions of inflorescence stems undergoing the gravitropic response. These results suggest that AtsPLA2? regulates the process of cell elongation and plays important roles in shoot gravitropism by mediating auxin-induced cell elongation. PMID:12953106

  19. Primary cutaneous apocrine carcinoma arising within a congenital nevus: Keratins and filaggrin expression suggesting differentiation into the secretory cells of apocrine glands

    PubMed Central

    SENBA, YUKO; KUROKAWA, ICHIRO; TOKIME, KAZUYA; HABE, KOJI; ISODA, KEN-ICHI; YAMANAKA, KEI-ICHI; TSUBURA, AIRO; MIZUTANI, HITOSHI

    2010-01-01

    Primary cutaneous apocrine carcinoma (PCAC) is a rare neoplasm of skin appendages. To determine the differentiation of apocrine carcinoma, we studied the expression of epithelial keratins and filaggrin immunohistochemically using 10 anti-keratin antibodies againt keratin (K) 1, 7, 8, 10, 14, 15, 16, 17, 18, 19 and the anti-filaggrin antibody. PCAC demonstrated strong positivity for K7, K8, K18 and K19. These keratins are distributed in secretory cells of normal apocrine glands. The tumor cells were negative for K14 and K17. The two keratins exist in myoepithelial cells in normal apocrine glands. Results suggest that PCAC shows differentiation into secretory cells of apocrine glands, although it does not differentiate into myoepithelial cells. K14 is also known as undifferentiated keratin, whereas K17 is considered to be a hyperproliferative keratin. Absence of the expression of K14 and K17 may reflect an indolent clinical course of PCAC. PMID:22966317

  20. Secretory expression of p53(N15)-Ant following lentivirus-mediated gene transfer induces cell death in human cancer cells.

    PubMed

    Li, Yueping; Qiu, Shudong; Song, Liping; Yan, Qingfeng; Yang, Guangxiao

    2008-02-01

    p53 (N15)-Ant 32-peptide has been considered a novel and effective peptide for cancer therapy. To further enhance its anticancer effect and overcome the limitation of peptide therapy, a recombinant lentivirus was constructed in this study with the following strategies: the secretory expression of therapeutic peptide and lentivirus gene transfer system. The results demonstrated that LV-NT4(Si)-p53 (N15)-Ant could significantly suppress cell growth and induce rapid cell death in MCF-7 (overexpressed wild-type p53), HepG2 (wide-type p53), OVCAR-3 (mutant type p53) and H1299 (null p53) cells in time-dependent manners through successful gene transfer and secretory expression of therapeutic peptide at 48 h post-infection. Transmission electron microscopy and flow cytometric analysis revealed that LV-NT4(Si)-p53 (N15)-Ant could induce two different kinds of cell death (necrosis and apoptosis) by two different mechanisms, since p53 (N15)-Ant peptide has the potential of blocking interaction of mdm-2 with other protein target, and on the other hand, it could form S-shape helix-loop-helix structures, which is able to rapidly disrupt cancer cell membranes. Based on these finding, LV-NT4 (Si)-p53 (N15)-Ant may be a novel recombinant virus because it induces cell death by two different pathways. PMID:18181042

  1. Proposed antagonists at GABAB receptors that inhibit adenylyl cyclase in cerebellar granule cell cultures of rat.

    PubMed

    Holopainen, I; Rau, C; Wojcik, W J

    1992-10-01

    The effects of various proposed GABAB receptor antagonists on baclofen-mediated inhibition of adenylyl cyclase were studied in cultured cerebellar granule cells from rat. (+/-)-Baclofen maximally inhibited adenylyl cyclase by approximately 60% of the basal enzyme activity with an EC50 value of 10 microM. 3-Aminopropane sulfonic acid (3-APS) and 5-aminovaleric acid (5-AVA) produced similar responses to that seen with (+/-)-baclofen. Saclofen reversed the action of (+/-)-baclofen, 50 microM, with a half maximal inhibitory concentration (IC50) of about 1.0 mM. The most effective antagonist in blocking the action of (+/-)-baclofen was 3-aminopropyl-diethoxy-methyl-phosphonic acid (CGP 35,348). In the presence of (+/-)-baclofen, 50 microM, the IC50 for CGP 35,348 was 290 microM and its inhibitory constant (KA) was 180 microM. The agonist-like actions of 3-APS and 5-AVA were antagonized by CGP 35,348 suggesting that 3-APS and 5-AVA may act as weak agonists at the GABAB receptor that inhibits adenylyl cyclase. All antagonists tested, except the new compound CGP 35,348, have very low potencies at GABAB receptors that inhibit adenylyl cyclase, though these compounds have been quite effective at other GABAB receptor-mediated events. Thus, the GABAB receptor which inhibits adenylyl cyclase differs pharmacologically from other reported GABAB receptor/effector systems and supports the existence of multiple receptor subtypes. PMID:1330652

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

    PubMed

    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

  3. Rapid induction of granule cell elimination in the olfactory bulb by noxious stimulation in mice.

    PubMed

    Komano-Inoue, Sayaka; Murata, Koshi; Mori, Kensaku; Yamaguchi, Masahiro

    2015-06-26

    Elimination of granule cells (GCs) in the olfactory bulb (OB) is not a continuous event but is rather promoted during short time windows associated with the animal's behavior. We previously showed that apoptotic GC elimination is enhanced during food eating and subsequent rest or sleep, and that top-down inputs from the olfactory cortex (OC) to the OB during the postprandial period are the crucial signal promoting GC elimination. However, whether enhanced GC elimination occurs during behaviors other than postprandial behavior is not clear. Here, we investigated whether exposure to noxious stimulation promotes apoptotic GC elimination in mice. Mice were delivered a brief electrical foot shock, during and immediately after which they showed startle and fear responses. Surprisingly, the number of apoptotic GCs increased 2-fold within 10min after the start of foot shock delivery. This enhancement of GC apoptosis was significantly suppressed by injection of the GABAA receptor agonist muscimol in the OC, despite these muscimol-injected mice showing similar behavioral responses by foot shock as control mice. These results indicate that GC elimination is promoted in foot shock-delivered mice within a short time period of startle and fear responses. They also indicate that OC activity plays a central role in the enhanced GC elimination during this period, as is also the case in GC elimination during the postprandial period. PMID:25943284

  4. 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

  5. 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

  6. Differences between GABA Receptor Binding to Membranes from Cerebellum during Postnatal Development and from Cultured Cerebellar Granule Cells

    Microsoft Academic Search

    Eddi Meier; Arne Schousboe

    1982-01-01

    GABA receptor binding sites were determined in membranes from cerebella of 7-, 15- and 60-day-old rats and cerebellar granule cells derived from 7-day-old rats and cultured for 8 days using [3H]GABA as the ligand and nonradioactive GABA to measure non-specific binding. Membranes from cerebellum at all postnatal stages exhibited at least two binding sites for GABA with binding constants around

  7. Patterns of endogenous gangliosides and metabolic processing of exogenous gangliosides in cerebellar granule cells during differentiation in culture

    Microsoft Academic Search

    Laura Riboni; Alessandro Prinetti; Marina Pitto; Guido Tettamanti

    1990-01-01

    The qualitative and quantitative pattern of endogenous gangliosides and the routes of metabolic processing of exogenous GM1,3H labeled in the sphingosine moiety (Sph-3H GM1) were studied in cerebellar granule cells during differentiation in vitro. During the first 7–8 days in culture the ganglioside content markedly increased, and the qualitative pattern showed, in percentage terms, a drastic decrease of GD3 and

  8. Coexpression of CD1a, langerin and Birbeck's granules in Langerhans cell histiocytoses (LCH) in children: ultrastructural and immunocytochemical studies.

    PubMed

    Dziegiel, Piotr; Dolil?ska-Krajewska, Barbara; Duma?ska, Malgorzata; Wec?awek, Jadwiga; Jele?, Micha?; Podhorska-Oko?ów, Marzena; Jagoda, Ewa; Fic, Magdalena; Zabel, Maciej

    2007-01-01

    Langerhans cell histiocytoses (LCH) represent rare diseases of unclear etiology and pathogenesis. Most of the cases include children, 1 to 15 years of age, and various organs are involved (bones, skin, liver, lymph nodes, bone marrow and other). The diagnosis of LCH used to be established by biopsy of the inflamed tissue and demonstration of expression of markers specific for Langerhans cells: CD1a and langerin. The diagnosis can be ultimately confirmed by demonstration of Birbeck's granules in the electron microscopy. The present study was aimed at immunocytochemical demonstration, in the examined LCH material (skin, bones, lymph nodes), of the specific antigen expression and at comparing it with the presence of Birbeck's granules. In the examined 11 cases co-expression of CD1a with langerin and with the presence of Birbeck's granules was noted. Also in all examined biopsies the expression of S-100 protein on inflammatory cells was found. The results corroborate the usefulness of immunocytochemical studies on CD 1 a and langerin expression in diagnosis of LCH. PMID:17378241

  9. The Gs-Linked Receptor GPR3 Inhibits the Proliferation of Cerebellar Granule Cells during Postnatal Development

    PubMed Central

    Tanaka, Shigeru; Shaikh, Imran Mohammed; Chiocca, E. Antonio; Saeki, Yoshinaga

    2009-01-01

    Background During postnatal murine and rodent cerebellar development, cerebellar granule precursors (CGP) gradually stop proliferating as they differentiate after migration to the internal granule layer (IGL). Molecular events that govern this program remain to be fully elucidated. GPR3 belongs to a family of Gs-linked receptors that activate cyclic AMP and are abundantly expressed in the adult brain. Methodology/Principal Findings To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation. In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression. In wild-type mice, GPR3 expression increased postnatally and its expression was concentrated in the internal granular layer (IGL). In GPR3 ?/? mice, the IGL was widened with increased proliferation of CGPs, as measured by bromodeoxyuridine incorporation. Cell cycle kinetics of GPR3-transfected medulloblastoma cells revealed a G0/G1 block, consistent with cell cycle exit. Conclusions/Significance These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum. PMID:19526062

  10. GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors

    Microsoft Academic Search

    Bo Belhage; Eddi Meier; Arne Schousboe

    1986-01-01

    The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [3H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 µM) or THIP plus the antagonist bicuculline methobromide (150 µM of

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

    PubMed

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

    2015-02-01

    Inhibiting the mammalian target of rapamycin (mTOR) signaling pathway with rapamycin blocks granule cell axon (mossy fiber) sprouting after epileptogenic injuries, including pilocarpine-induced status epilepticus. However, it remains unclear whether axons from other types of neurons sprout into the inner molecular layer and synapse with granule cell dendrites despite rapamycin treatment. If so, other aberrant positive-feedback networks might develop. To test this possibility stereological electron microscopy was used to estimate the numbers of excitatory synapses in the inner molecular layer per hippocampus in pilocarpine-treated control mice, in mice 5 days after pilocarpine-induced status epilepticus, and after status epilepticus and daily treatment beginning 24 hours later with rapamycin or vehicle for 2 months. The optical fractionator method was used to estimate numbers of granule cells in Nissl-stained sections so that numbers of excitatory synapses in the inner molecular layer per granule cell could be calculated. Control mice had an average of 2,280 asymmetric synapses in the inner molecular layer per granule cell, which was reduced to 63% of controls 5 days after status epilepticus, recovered to 93% of controls in vehicle-treated mice 2 months after status epilepticus, but remained at only 63% of controls in rapamycin-treated mice. These findings reveal that rapamycin prevented excitatory axons from synapsing with proximal dendrites of granule cells and raise questions about the recurrent excitation hypothesis of temporal lobe epilepsy. PMID:25234294

  12. Secretory Phospholipase A2 Elicits Proinflammatory Changes and Upregulates the Surface Expression of Fas Ligand in Monocytic Cells Potential Relevance for Atherogenesis

    Microsoft Academic Search

    Marita Hernández; Lucía Fuentes; Francisco Javier Fernández; Mariano Sánchez Crespo Avilés; María Luisa Nieto

    Type IIA secretory phospholipase A2 (sPLA2) is an acute-phase reactant that plays a role in atherogenesis and is expressed in atherosclerotic arterial walls displaying inflammatory features. This generates a relevant question addressing the biological effects of this enzyme on monocytic cells, in view of the role of these cells in the inflammatory process associated with atherosclerosis. sPLA2 produced a mild

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

    PubMed Central

    1986-01-01

    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 greater than 10, replace alpha-amylase (pI's = 6.8) as the principal content species. The internal pH of granules from the treated rats ranges 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 (approximately 6.8) appears to reflect the change in composition of the secretory content. Whereas normal mature parotid granules have practically negligible levels of H+ pumping ATPase activity (Arvan, P., G. Rudnick, and J. D. Castle, 1985, J. Biol. Chem., 260, 14945-14952) the isolated granules from isoproterenol-treated rats undergo a time-dependent internal acidification (approximately 0.2 pH unit) that requires the presence of ATP and is abolished by an H+ 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+-ATPase activity. Together these data provide clear evidence for the presence of an H+ pump in the membranes of parotid granules from chronically stimulated rats. However, despite the presence of H+-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. PMID:3021780

  14. Mitochondrial oxidative stress mediates high-phosphate-induced secretory defects and apoptosis in insulin-secreting cells.

    PubMed

    Nguyen, Tuyet Thi; Quan, Xianglan; Hwang, Kyu-Hee; Xu, Shanhua; Das, Ranjan; Choi, Seong-Kyung; Wiederkehr, Andreas; Wollheim, Claes B; Cha, Seung-Kuy; Park, Kyu-Sang

    2015-06-01

    Inorganic phosphate (Pi) plays an important role in cell signaling and energy metabolism. In insulin-releasing cells, Pi transport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated Pi concentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of Pi on secretory function and apoptosis in INS-1E clonal ?-cells and rat pancreatic islets. Elevated extracellular Pi (1?5 mM) increased the mitochondrial membrane potential (??m), superoxide generation, caspase activation, and cell death. Depolarization of the ??m abolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters, prevented ??m hyperpolarization, superoxide generation, and cytotoxicity caused by Pi. High Pi also promoted the opening of the mitochondrial permeability transition (PT) pore, leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants mitoTEMPO or MnTBAP prevented Pi-triggered PT pore opening and cytotoxicity. Elevated extracellular Pi diminished ATP synthesis, cytosolic Ca(2+) oscillations, and insulin content and secretion in INS-1E cells as well as in dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high-Pi-induced phosphorylation of ER stress proteins. We propose that elevated extracellular Pi causes mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content and defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states. PMID:25852001

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

    PubMed

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

    2015-02-01

    The hippocampus organizes sequential memory composed of non-spatial information (such as objects and odors) and spatial information (places). The dentate gyrus (DG) in the hippocampus receives two types of information from the lateral and medial entorhinal cortices. Non-spatial and spatial information is delivered respectively to distal and medial dendrites (MDs) of granule cells (GCs) within the molecular layer in the DG. To investigate the role of the association of those two inputs, we measured the response characteristics of distal and MDs of a GC in a rat hippocampal slice and developed a multi-compartment GC model with dynamic synapses; this model reproduces the response characteristics of the dendrites. Upon applying random inputs or input sequences generated by a Markov process to the computational model, it was found that a high-frequency random pulse input to distal dendrites (DDs) and, separately, regular burst inputs to MDs were effective for inducing GC activation. Furthermore, when the random and theta burst inputs were simultaneously applied to the respective dendrites, the pattern discrimination for theta burst input to MDs that caused slight GC activation was enhanced in the presence of random input to DDs. These results suggest that the temporal pattern discrimination of spatial information is originally involved in a synaptic characteristic in GCs and is enhanced by non-spatial information input to DDs. Consequently, the co-activation of two separate inputs may play a crucial role in the information processing on dendrites of GCs by usefully combing each temporal sequence. PMID:26052358

  16. Postsynaptic gephyrin clustering controls the development of adult-born granule cells in the olfactory bulb.

    PubMed

    Deprez, Francine; Pallotto, Marta; Vogt, Fabia; Grabiec, Marta; Virtanen, Mari A; Tyagarajan, Shiva K; Panzanelli, Patrizia; Fritschy, Jean-Marc

    2015-09-01

    In adult rodent olfactory bulb, GABAergic signaling regulates migration, differentiation, and synaptic integration of newborn granule cells (GCs), migrating from the subventricular zone. Here we show that these effects depend on the formation of a postsynaptic scaffold organized by gephyrin-the main scaffolding protein of GABAergic synapses, which anchors receptors and signaling molecules to the postsynaptic density-and are regulated by the phosphorylation status of gephyrin. Using lentiviral vectors to selectively transfect adult-born GCs, we observed that overexpression of the phospho-deficient gephyrin mutant eGFP-gephyrin(S270A), which facilitates the formation of supernumerary GABAergic synapses in vitro, favors dendritic branching and the formation of transient GABAergic synapses on spines, identified by the presence of ?2-GABAA Rs. In contrast, overexpression of the dominant-negative eGFP-gephyrin(L2B) (a chimera that is enzymatically active but clustering defective), curtailed dendritic growth, spine formation, and long-term survival of GCs, pointing to the essential role of gephyrin cluster formation for its function. We could exclude any gephyrin overexpression artifacts, as GCs infected with eGFP-gephyrin were comparable to those infected with eGFP alone. The opposite effects induced by the two gephyrin mutant constructs indicate that the gephyrin scaffold at GABAergic synapses orchestrates signaling cascades acting on the cytoskeleton to regulate neuronal growth and synapse formation. Specifically, gephyrin phosphorylation emerges as a novel mechanism regulating morphological differentiation and long-term survival of adult-born olfactory bulb neurons. J. Comp. Neurol. 523:1998-2016, 2015 © 2015 Wiley Periodicals, Inc. PMID:25772192

  17. Complementary Postsynaptic Activity Patterns Elicited in Olfactory Bulb by Stimulation of Mitral/Tufted and Centrifugal Fiber Inputs to Granule Cells

    PubMed Central

    Laaris, Nora; Puche, Adam; Ennis, Matthew

    2009-01-01

    Main olfactory bulb (MOB) granule cells receive spatially segregated glutamatergic synaptic inputs from the dendrites of mitral/tufted cells as well as from the axons of centrifugal fibers (CFFs) originating in olfactory cortical areas. Dendrodendritic synapses from mitral/tufted cells occur on granule cell distal dendrites in the external plexiform layer (EPL), whereas CFFs preferentially target the somata/proximal dendrites of granule cells in the granule cell layer (GCL). In the present study, tract tracing, and recordings of field potentials and voltage-sensitive dye optical signals were used to map activity patterns elicited by activation of these two inputs to granule cells in mouse olfactory bulb slices. Stimulation of the lateral olfactory tract (LOT) produced a negative field potential in the EPL and a positivity in the GCL. CFF stimulation produced field potentials of opposite polarity in the EPL and GCL to those elicited by LOT. LOT-evoked optical signals appeared in the EPL and spread subsequently to deeper layers, whereas CFF-evoked responses appeared in the GCL and then spread superficially. Evoked responses were reduced by N-methyl-d-aspartate (NMDA) receptor antagonists and completely suppressed by AMPA receptor antagonists. Reduction of extracellular Mg2+ enhanced the strength and spatiotemporal extent of the evoked responses. These and additional findings indicate that LOT- and CFF-evoked field potentials and optical signals reflect postsynaptic activity in granule cells, with moderate NMDA and dominant AMPA receptor components. Taken together, these results demonstrate that LOT and CFF stimulation in MOB slices selectively activate glutamatergic inputs to the distal dendrites versus somata/proximal dendrites of granule cells. PMID:17035366

  18. Adverse Effects of 2,4-Dichlorophenoxyacetic Acid on Rat Cerebellar Granule Cell Cultures Were Attenuated by Amphetamine

    Microsoft Academic Search

    B. Bongiovanni; A. Ferri; A. Brusco; M. Rassetto; L. M. Lopez; A. M. Evangelista de Duffard; R. Duffard

    2011-01-01

    2,4-Dichlorophenoxyacetic acid (2,4-D), a worldwide-used herbicide, has been shown to produce a wide range of adverse effects\\u000a in the health—from embryotoxicity and teratogenicity to neurotoxicity—of animals and humans. In this study, neuronal morphology\\u000a and biochemical events in rat cerebellar granule cell (CGC) cultures have been analyzed to define some of the possible mechanisms\\u000a involved in 2,4-D-induced cell death. For that

  19. The effect of intracellular Ca 2+ on GABA-activated currents in cerebellar granule cells in culture

    Microsoft Academic Search

    M. Martina; G. Kili?; E. Cherubini

    1994-01-01

    The patch clamp technique was used to study the effects of intracellular free calcium ([Ca2+]\\u000ai\\u000a) on GABAA-evoked whole-cell and single channel currents of cultured cerebellar granule cells. Changes in [Ca2+]\\u000ai\\u000a were obtained by adding to the extracellular solution the calcium ionophore A23187 (2 m). The relationship between [Ca2+]\\u000ai\\u000a and [Ca2+]\\u000aO\\u000a in the presence or absence

  20. Heparin-binding secretory transforming gene (hst) facilitates rat lactotrope cell tumorigenesis and induces prolactin gene transcription.

    PubMed Central

    Shimon, I; Hüttner, A; Said, J; Spirina, O M; Melmed, S

    1996-01-01

    We have shown previously that human prolactinomas express transforming sequences of the heparin-binding secretory transforming gene (hst) which encodes fibroblast growth factor-4 (FGF-4). To elucidate the role of hst in pituitary tumorigenesis we treated primary rat pituitary and pituitary tumor cell cultures with recombinant FGF-4 and also stably transfected pituitary cell lines with full-length human hst cDNA. Transfectants were screened for hst mRNA expression and FGF-4 production. FGF-4 (0.1-50 ng/ml) caused a dose-dependent 2.5-fold increase of prolactin (PRL) secretion (P < 0.001) in GH4 cells and up to 60% (P < 0.05) in primary cultures, while decreasing growth hormone release (P < 0.001). GH4 hst transfectants displayed markedly enhanced basal PRL secretion (threefold, P < 0.001) and also proliferated faster (P < 0.001). FGF-4 treatment of wild-type GH4 cells, transiently transfected with an expression construct (rPRL.luc) containing a luciferase reporter driven by the rPRL promoter, resulted in a dose-dependent increase of up to 3.3-fold in PRL transcriptional activity. Tumors derived from in vivo subcutaneous injection of GH4 hst-transfected cells strongly expressing FGF-4 grew more aggressively as assessed by histologic invasiveness and proliferating cell nuclear antigen staining (P < 0.01). The results indicate that hst overexpression mediates lactotrope tumor growth and potently stimulates PRL synthesis. Thus, hst may directly facilitate prolactinoma development via paracrine or autocrine action of its secreted protein, FGF-4. PMID:8550832

  1. Posttranslational isomerization of a neuropeptide in crustacean neurosecretory cells studied by ultrastructural immunocytochemistry.

    PubMed

    Gallois, Dominique; Brisorgueil, Marie-Jeanne; Conrath, Marie; Mailly, Philippe; Soyez, Daniel

    2003-08-01

    Isomerization of the third amino acid residue (a phenylalanine) of crustacean hyperglycemic hormone (CHH) has been previously reported to occur as a late step of hormone precursor maturation in a few neurosecretory cells in the X-organ-sinus gland complex of the crayfish Orconectes limosus. In the present report, using conformation-specific antisera combined with immunogold labeling, we have studied, at the ultrastructural level, the distribution of L- and D-CHH immunoreactivity in CHH-secreting cells of the crayfish Astacus leptodactylus. Two CHH-secreting cell populations were observed, the first one (L-cells), the most numerous, exhibited only labeling for L-CHH. In the second one (D-cells), four secretory granule populations were distinguished according to their labeling: unlabeled, either L- or D- exclusively or both L- and D-granules. Labeling quantification by image analysis in D-cells showed a marked increase in D-labeling from the cell body to the axon terminal. However some L- and mixed granules remain in axon terminals. Our results demonstrate that Phe3 isomerization of CHH occurs within the secretory granules of specialized neurosecretory cells and progresses as the granules migrate along the axonal tract. The observation that not all the CHH synthesized is isomerized, and the great variability in the proportion of L- and D-immunoreactivity in granules in every cell region may suggest an heterogeneous distribution of the putative enzyme involved in Phe3 isomerization, a peptide isomerase, within the secretory pathway. PMID:14533741

  2. Extracellular Vesicles from Parasitic Helminths Contain Specific Excretory/Secretory Proteins and Are Internalized in Intestinal Host Cells

    PubMed Central

    Marcilla, Antonio; Trelis, María; Cortés, Alba; Sotillo, Javier; Cantalapiedra, Fernando; Minguez, María Teresa; Valero, María Luz; Sánchez del Pino, Manuel Mateo; Muñoz-Antoli, Carla; Toledo, Rafael; Bernal, Dolores

    2012-01-01

    The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30–100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents. PMID:23029346

  3. Multicolor RGB Marking Allows Morphometric and Functional Analysis of Hippocampal Granule Neurons at the Single-Cell Level.

    PubMed

    Gomez-Nicola, Diego; Riecken, Kristoffer; Perry, V Hugh; Fehse, Boris

    2015-06-01

    Identifying and tracking cells in the brain is a challenging task, requiring advanced molecular biology techniques and precise anatomical information. We have applied the principle of RGB marking to the long-term marking and tracking of progenitor and mature cells in the adult brain. Briefly, multicolor RGB marking is based on the simultaneous, lentiviral vector-mediated expression of three genes encoding fluorescent proteins in the three basic colors (red, green, and blue). Here, we show the application of RGB marking to the stable multicolor marking of adult granule cells in the hippocampus. This technique provides each individual cell with a characteristic hue, which facilitates their anatomical identification and spatial tracking. Multicolor RBG marking of mature neurons can provide an effective approach to dissect the function of individual cells, as it allows combination with functional techniques, facilitating the understanding of complex neuronal populations such as that of the dentate gyrus. PMID:25894663

  4. 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

  5. Vitamin A deficiency and inflammation: the pivotal role of secretory cells in the development of atrophic, hyperplastic and metaplastic change in the tracheal epithelium in vivo

    Microsoft Academic Search

    Xin-min Zhang; Elizabeth M. McDowell

    1992-01-01

    Summary  We showed previously that the proliferation of hamster airway secretory cells decreases during vitamin A deficiency (VAD)\\u000a but later increases when submucosal inflammation develops (Virchows Arch [B] 59:231–242, 1990). This observation has important\\u000a biological implications since two morphological extremes (atrophy and quiescence versus hyperplasia and hyperproliferation)\\u000a are reported in the literature for VAD tracheal epithelium in vivo. In the present

  6. Actin-dependent, retrograde motility of surface-attached beads and aggregating pigment granules in dissociated teleost retinal pigment epithelial cells.

    PubMed

    Basciano, Paul A; King-Smith, Christina

    2002-06-01

    Teleost retinal pigment epithelial (RPE) cells contain pigment granules within apical projections which undergo actin-dependent, bi-directional motility. Dissociated RPE cells in culture attach to the substrate and extend apical projections in a radial array from the central cell body. Pigment granules within projections can be triggered to aggregate or disperse by the presence or absence of 1 mM cAMP. Aminated, fluorescent latex beads attached to the dorsal surface of apical projections and moved in the retrograde direction, towards the cell body. Bead rates on RPE cells with aggregating or fully aggregated pigment granules were 2.2 +/- 0.5 and 2.6 +/- 0.2 microm/min (mean +/- SEM), respectively, similar to rates of aggregating (retrograde) pigment granule movement (2.0 +/- 0.4 microm/min). Bead rates were slightly slower on cells with fully dispersed or dispersing pigment granules (1.5 +/- 0.1 and 1.5 +/- 0.4 microm/min). Movements of surface-attached beads and aggregating pigment granules were closely correlated in the distal portions of apical projections, but were more independent of each other in proximal regions of the projections. The actin disrupting drug, cytochalasin D (CD), reversibly halted retrograde bead movements, suggesting that motility of surface-attached particles is actin-dependent. In contrast, the microtubule depolymerizing drug, nocodazole, had no effect on retrograde bead motility. The similar characteristics and actin-dependence of retrograde bead movements and aggregating pigment granules suggest a correlation between these two processes. PMID:12028582

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

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Nakamichi, Yoko; Nishiwaki, Chiyono [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); Sakurai, Takashi [Photon Medical Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192 Shizuoka (Japan)] [Photon Medical Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192 Shizuoka (Japan); Nagamatsu, Shinya, E-mail: shinya@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)

    2009-07-31

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

  8. 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

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

    PubMed Central

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

    2009-01-01

    Polychlorinated biphenyls (PCBs) are persistent organic pollutants that bioaccumulate in the body, however, they can be metabolized to more water-soluble products. Although they are more readily excreted than the parent compounds, some of the metabolites are still hydrophobic and may be more available to target tissues, such as the brain. They can also cross the placenta and reach a developing foetus. Much less is known about the toxicity of PCB metabolites than about the parent compounds. In the present study, we have investigated the effects of eight hydroxylated (OH) PCB congeners (2?-OH PCB 3, 4-OH PCB 14, 4-OH PCB 34, 4?-OH PCB 35, 4-OH PCB 36, 4?-OH PCB 36, 4-OH PCB 39, and 4?-OH PCB 68) on reactive oxygen species (ROS) formation and cell viability in rat cerebellar granule cells. We found that, similar to their parent compounds, OH-PCBs are potent ROS inducers with potency 4-OH PCB 14 < 4-OH PCB 36 < 4-OH PCB 34 < 4?-OH PCB 36 < 4?-OH PCB 68 < 4-OH PCB 39 < 4?-OH PCB 35. 4-OH PCB 36 was the most potent cell death inducer, and caused apoptotic or necrotic morphology depending on concentration. Inhibition of ERK1/2 kinase with U0126 reduced both cell death and ROS formation, suggesting that ERK1/2 activation is involved in OH-PCB toxicity. The results indicate that the hydroxylation of PCBs may not constitute a detoxification reaction. Since OH-PCBs like their parent compounds are retained in the body and may be more widely distributed to sensitive tissues, it is important that not only the levels of the parent compounds but also the levels of their metabolites are taken into account during risk assessment of PCBs and related compounds. PMID:19631230

  10. The Role of Secretory Immunoglobulin A in the Natural Sensing of Commensal Bacteria by Mouse Peyer's Patch Dendritic Cells*

    PubMed Central

    Rol, Nicolas; Favre, Laurent; Benyacoub, Jalil; Corthésy, Blaise

    2012-01-01

    The mammalian gastrointestinal (GI) tract harbors a diverse population of commensal species collectively known as the microbiota, which interact continuously with the host. From very early in life, secretory IgA (SIgA) is found in association with intestinal bacteria. It is considered that this helps to ensure self-limiting growth of the microbiota and hence participates in symbiosis. However, the importance of this association in contributing to the mechanisms ensuring natural host-microorganism communication is in need of further investigation. In the present work, we examined the possible role of SIgA in the transport of commensal bacteria across the GI epithelium. Using an intestinal loop mouse model and fluorescently labeled bacteria, we found that entry of commensal bacteria in Peyer's patches (PP) via the M cell pathway was mediated by their association with SIgA. Preassociation of bacteria with nonspecific SIgA increased their dynamics of entry and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c+CD11b+CD8? dendritic cell subset located in the subepithelial dome region of PPs, confirming that the host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria. PMID:23027876

  11. 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

  12. The Secretory System of Arabidopsis

    PubMed Central

    Bassham, Diane C.; Brandizzi, Federica; Otegui, Marisa S.; Sanderfoot, Anton A.

    2008-01-01

    Over the past few years, a vast amount of research has illuminated the workings of the secretory system of eukaryotic cells. The bulk of this work has been focused on the yeast Saccharomyces cerevisiae, or on mammalian cells. At a superficial level, plants are typical eukaryotes with respect to the operation of the secretory system; however, important differences emerge in the function and appearance of endomembrane organelles. In particular, the plant secretory system has specialized in several ways to support the synthesis of many components of the complex cell wall, and specialized kinds of vacuole have taken on a protein storage role—a role that is intended to support the growing seedling, but has been co-opted to support human life in the seeds of many crop plants. In the past, most research on the plant secretory system has been guided by results in mammalian or fungal systems but recently plants have begun to stand on their own as models for understanding complex trafficking events within the eukaryotic endomembrane system. PMID:22303241

  13. 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

  14. 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

  15. Regulation of Clara cell secretory protein gene transcription by thyroid transcription factor-1

    Microsoft Academic Search

    Liqian Zhang; Jeffrey A. Whitsett; Barry R. Stripp

    1997-01-01

    CCSP is a 16 kDa protein expressed selectively in the Clara cells of the lung. Cis-acting elements conferring Clara cell-specific expression of rat CCSP gene were contained within the sequences ?320 to +57 of the rCCSP promoter. DNA-TTF-1 protein interactions were identified within the sequences ?302 to ?278 and ?90 to ?66 from the transcriptional start site of the rat

  16. The effects of dentate granule cell destruction on behavioural activity and Fos protein expression induced by systemic methamphetamine in rats.

    PubMed

    Tani, K; Iyo, M; Matsumoto, H; Kawai, M; Suzuki, K; Iwata, Y; Won, T; Tsukamoto, T; Sekine, Y; Sakanoue, M; Hashimoto, K; Ohashi, Y; Takei, N; Mori, N

    2001-12-01

    1. We destroyed dentate granule cells unilaterally or bilaterally by means of intrahippocampal injection of colchicine in rats. Subsequently, we observed behavioural changes following the intraperitoneal injection of 2 mg kg(-1) methamphetamine or saline, in addition to quantitatively assessing Fos protein expression in several brain regions, including the medial prefrontal cortex, cingulate cortex, piriform cortex, dorsal striatum, and nucleus accumbens. 2. Bilaterally lesioned animals, when administered saline, showed a marked increase in locomotor activity compared with those of non-lesioned animals. With respect to the methamphetamine response, bilateral destruction resulted in a marked enhancement of locomotor activity, while the unilateral destruction led to a marked increase in rotation predominantly contralateral to the lesioned side, with no identifiable change in locomotor activity. 3. Bilaterally lesioned animals, when administered saline and having undergone an immunohistological examination, showed a marked increase in Fos expression in both sides of the nucleus accumbens. Bilaterally lesioned animals administered methamphetamine showed a marked increase in Fos expression in the right and left sides of all regions tested. Unilaterally lesioned animals administered methamphetamine showed a significant and bilateral enhancement in Fos expression in the medial prefrontal and cingulate cortices, and a marked and unilateral (ipsilateral to the lesioned side) enhancement of Fos protein in the piriform cortex, dorsal striatum, and nucleus accumbens. 4. The present findings suggest that dentate granule cells regulate methamphetamine-associated behavioural changes through the function of widespread areas of the brain, mostly the nucleus accumbens. PMID:11724746

  17. Macrophage secretory products selectively stimulate dermatan sulfate proteoglycan production in cultured arterial smooth muscle cells

    SciTech Connect

    Edwards, I.J.; Wagner, W.D.; Owens, R.T. (Wake Forest Univ., Winston-Salem, NC (USA))

    1990-03-01

    Arterial dermatan sulfate proteoglycan has been shown to increase with atherosclerosis progression, but factors responsible for this increase are unknown. To test the hypothesis that smooth muscle cell proteoglycan synthesis may be modified by macrophage products, pigeon arterial smooth muscle cells were exposed to the media of either cholesteryl ester-loaded pigeon peritoneal macrophages or a macrophage cell line P388D1. Proteoglycans radiolabeled with (35S)sulfate and (3H)serine were isolated from culture media and smooth muscle cells and purified following precipitation with 1-hexadecylpyridinium chloride and chromatography. Increasing concentrations of macrophage-conditioned media were associated with a dose-response increase in (35S)sulfate incorporation into secreted proteoglycans, but there was no change in cell-associated proteoglycans. Incorporation of (3H)serine into total proteoglycan core proteins was not significantly different (5.2 X 10(5) dpm and 5.5 X 10(5) disintegrations per minute (dpm) in control and conditioned media-treated cultures, respectively), but selective effects were observed on individual proteoglycan types. Twofold increases in dermatan sulfate proteoglycan and limited degradation of chondroitin sulfate proteoglycan were apparent based on core proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoinhibition studies indicated that interleukin-1 was involved in the modulation of proteoglycan synthesis by macrophage-conditioned media. These data provide support for the role of macrophages in alteration of the matrix proteoglycans synthesized by smooth muscle cells and provide a mechanism to account for the reported increased dermatan sulfate/chondroitin sulfate ratios in the developing atherosclerotic lesion.

  18. Isolation and Characterization of Mutants Defective in Seed Coat Mucilage Secretory Cell

    E-print Network

    Haughn, George

    cell wall. Differentiation involves a regulated series of cytological events including growth in embryo protection and the regulation of germination. One specialization is known as myxospermy-Wyssling, 1976; Esau, 1977), where they foster root tip and pollen tube growth, respectively. Mucilages

  19. Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

    PubMed Central

    Barlowe, Charles K.; Miller, Elizabeth A.

    2013-01-01

    The secretory pathway is responsible for the synthesis, folding, and delivery of a diverse array of cellular proteins. Secretory protein synthesis begins in the endoplasmic reticulum (ER), which is charged with the tasks of correctly integrating nascent proteins and ensuring correct post-translational modification and folding. Once ready for forward traffic, proteins are captured into ER-derived transport vesicles that form through the action of the COPII coat. COPII-coated vesicles are delivered to the early Golgi via distinct tethering and fusion machineries. Escaped ER residents and other cycling transport machinery components are returned to the ER via COPI-coated vesicles, which undergo similar tethering and fusion reactions. Ultimately, organelle structure, function, and cell homeostasis are maintained by modulating protein and lipid flux through the early secretory pathway. In the last decade, structural and mechanistic studies have added greatly to the strong foundation of yeast genetics on which this field was built. Here we discuss the key players that mediate secretory protein biogenesis and trafficking, highlighting recent advances that have deepened our understanding of the complexity of this conserved and essential process. PMID:23396477

  20. Substrate specificity of proinsulin conversion in the constitutive pathway of transfected FAO (hepatoma) cells

    Microsoft Academic Search

    F. Vollenweider; J.-C. Irminger; P. A. Halban

    1993-01-01

    Summary  Proinsulin is usually targetted to the regulated secretory pathway of beta cells, and converted to insulin in beta granules. Under certain pathological situations, a significant amount of proinsulin becomes diverted to the constitutive pathway. To study the kinetics of proinsulin conversion in the constitutive pathway, FAO (hepatoma) cells, which secrete proteins uniquely via this pathway and not the regulated pathway,

  1. The secretory nature of the lesion of carrot cell variant ts11, rescuable by endochitinase

    Microsoft Academic Search

    Barbara Baldan; Flavia Guzzo; Francesco Filippini; Marine Gasparian; Fiorella LoSchiavo; Alessandro Vitale; Sacco C. de Vries; Paola Mariani; Mario Terzi

    1997-01-01

    .   The carrot cell variant ts11 is unable to form somatic embryos at the non-permissive temperature of 32?°C, but the block\\u000a can be overcome by the addition of a 32-kDa acidic endochitinase to the medium. In this work we conducted a cyto-histological\\u000a analysis of the blocked embryo forms. The morphology of the endomembrane system is altered; in particular, the ER

  2. 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

  3. Matrix stiffness-modulated proliferation and secretory function of the airway smooth muscle cells.

    PubMed

    Shkumatov, Artem; Thompson, Michael; Choi, Kyoung M; Sicard, Delphine; Baek, Kwanghyun; Kim, Dong Hyun; Tschumperlin, Daniel J; Prakash, Y S; Kong, Hyunjoon

    2015-06-01

    Multiple pulmonary conditions are characterized by an abnormal misbalance between various tissue components, for example, an increase in the fibrous connective tissue and loss/increase in extracellular matrix proteins (ECM). Such tissue remodeling may adversely impact physiological function of airway smooth muscle cells (ASMCs) responsible for contraction of airways and release of a variety of bioactive molecules. However, few efforts have been made to understand the potentially significant impact of tissue remodeling on ASMCs. Therefore, this study reports how ASMCs respond to a change in mechanical stiffness of a matrix, to which ASMCs adhere because mechanical stiffness of the remodeled airways is often different from the physiological stiffness. Accordingly, using atomic force microscopy (AFM) measurements, we found that the elastic modulus of the mouse bronchus has an arithmetic mean of 23.1 ± 14 kPa (SD) (median 18.6 kPa). By culturing ASMCs on collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we found that gels designed to be softer than average airway tissue significantly increased cellular secretion of vascular endothelial growth factor (VEGF). Conversely, gels stiffer than average airways stimulated cell proliferation, while reducing VEGF secretion and agonist-induced calcium responses of ASMCs. These dependencies of cellular activities on elastic modulus of the gel were correlated with changes in the expression of integrin-?1 and integrin-linked kinase (ILK). Overall, the results of this study demonstrate that changes in matrix mechanics alter cell proliferation, calcium signaling, and proangiogenic functions in ASMCs. PMID:25724668

  4. COBRA-LIKE2, a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE family, plays a role in cellulose deposition in arabidopsis seed coat mucilage secretory cells.

    PubMed

    Ben-Tov, Daniela; Abraham, Yael; Stav, Shira; Thompson, Kevin; Loraine, Ann; Elbaum, Rivka; de Souza, Amancio; Pauly, Markus; Kieber, Joseph J; Harpaz-Saad, Smadar

    2015-03-01

    Differentiation of the maternally derived seed coat epidermal cells into mucilage secretory cells is a common adaptation in angiosperms. Recent studies identified cellulose as an important component of seed mucilage in various species. Cellulose is deposited as a set of rays that radiate from the seed upon mucilage extrusion, serving to anchor the pectic component of seed mucilage to the seed surface. Using transcriptome data encompassing the course of seed development, we identified COBRA-LIKE2 (COBL2), a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE gene family in Arabidopsis (Arabidopsis thaliana), as coexpressed with other genes involved in cellulose deposition in mucilage secretory cells. Disruption of the COBL2 gene results in substantial reduction in the rays of cellulose present in seed mucilage, along with an increased solubility of the pectic component of the mucilage. Light birefringence demonstrates a substantial decrease in crystalline cellulose deposition into the cellulosic rays of the cobl2 mutants. Moreover, crystalline cellulose deposition into the radial cell walls and the columella appears substantially compromised, as demonstrated by scanning electron microscopy and in situ quantification of light birefringence. Overall, the cobl2 mutants display about 40% reduction in whole-seed crystalline cellulose content compared with the wild type. These data establish that COBL2 plays a role in the deposition of crystalline cellulose into various secondary cell wall structures during seed coat epidermal cell differentiation. PMID:25583925

  5. Theta-gamma-modulated synaptic currents in hippocampal granule cells in vivo define a mechanism for network oscillations.

    PubMed

    Pernía-Andrade, Alejandro Javier; Jonas, Peter

    2014-01-01

    Theta-gamma network oscillations are thought to represent key reference signals for information processing in neuronal ensembles, but the underlying synaptic mechanisms remain unclear. To address this question, we performed whole-cell (WC) patch-clamp recordings from mature hippocampal granule cells (GCs) in vivo in the dentate gyrus of anesthetized and awake rats. GCs in vivo fired action potentials at low frequency, consistent with sparse coding in the dentate gyrus. GCs were exposed to barrages of fast AMPAR-mediated excitatory postsynaptic currents (EPSCs), primarily relayed from the entorhinal cortex, and inhibitory postsynaptic currents (IPSCs), presumably generated by local interneurons. EPSCs exhibited coherence with the field potential predominantly in the theta frequency band, whereas IPSCs showed coherence primarily in the gamma range. Action potentials in GCs were phase locked to network oscillations. Thus, theta-gamma-modulated synaptic currents may provide a framework for sparse temporal coding of information in the dentate gyrus. PMID:24333053

  6. Secretory Pathway of Trypanosomatid Parasites

    PubMed Central

    McConville, Malcolm J.; Mullin, Kylie A.; Ilgoutz, Steven C.; Teasdale, Rohan D.

    2002-01-01

    The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Trypanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles in the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory transport and protein glycosylation that may be exploited in developing new antiparasite drugs. PMID:11875130

  7. 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

  8. Effects of 5-aza-2'-deoxycytidine and trichostatin A on high glucose- and interleukin-1?-induced secretory mediators from human retinal endothelial cells and retinal pigment epithelial cells

    PubMed Central

    Xie, Manyun; Tian, Jingyi; Luo, Yan; Wei, Liqing; Lin, Shaofen

    2014-01-01

    Purpose We aimed to elucidate the effects of two epigenetic inhibitors, 5-aza-2’-deoxycytidine (5-aza-dC) and trichostatin A (TSA), on several key secretory mediators of diabetic retinopathy (DR) in human retinal endothelial cells (HRECs) and human retinal pigment epithelial (HRPE) cells treated with high glucose or interleukin-1? (IL-1?). Methods HRECs and HRPE cells were incubated in 30 mM D-glucose or 10 ng/ml IL-1? with or without the presence of various concentrations of 5-aza-dC or TSA. The production of pigment epithelium derived factor (PEDF), vascular endothelial cell growth factor (VEGF), intercellular cell adhesion molecule-1 (ICAM-1), IL-1?, and matrix metalloproteinase 2 (MMP2) was evaluated at the mRNA and protein levels using real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Results In the 30 mM D-glucose and the 10 ng/ml IL-1? condition, the expression of VEGF, ICAM-1, IL-1?, and MMP2 was induced in the HRECs and the HRPE cells. PEDF was downregulated in the HRPE cells but upregulated in the HRECs. However, the PEDF-to-VEGF ratio, which is thought to be critical in DR, was downregulated in both cell types. 5-aza-dC dose-dependently alleviated VEGF, ICAM-1, IL-1?, and MMP2 and reversed PEDF or the PEDF/VEGF ratio in both cell types. TSA had similar effects as 5-aza-dC on the target mediators. However, ICAM-1 production was aggravated in the HRECs while remaining unchanged in the HRPE cells after TSA was administered. Conclusions Our results demonstrated that 5-aza-dC and TSA enhance the protective PEDF and the PEDF/VEGF ratio and ameliorate the adverse effects of diabetic stimuli in vitro, suggesting that these two drugs may be of potential therapeutic value in DR. PMID:25352747

  9. The zymogen granule protein 2 (GP2) binds to scavenger receptor expressed on endothelial cells I (SREC-I)

    PubMed Central

    Hölzl, Markus A.; Hofer, Johannes; Kovarik, Johannes J.; Roggenbuck, Dirk; Reinhold, Dirk; Goihl, Alexander; Gärtner, Miriam; Steinberger, Peter; Zlabinger, Gerhard J.

    2011-01-01

    The pancreatic zymogen granule membrane protein (GP2) is expressed by pancreatic acinar cells and M cells of the ileum. GP2 is the closest related homologue of the urine resident Tamm–Horsfall protein (THP). Recently, it was shown that THP is a ligand of various scavenger receptors (SRs). Therefore, we were interested, if GP2 has similar properties. cDNA of different SRs was stably transfected into a murine thymoma cell line. GP2 was recombinantly expressed, purified and biotinylated. Binding or uptake of GP2 by transfected cells or monocyte-derived dendritic cells (moDCs) was analyzed by flow-cytometry. GP2 is a binding partner of the scavenger receptor expressed on endothelial cells I (SREC-I) but not of SR-AI and SR-BI. The dissociation constant (Kd) of GP2 binding to SREC-I is 41.3 nM. SREC transfected cells are able to internalize GP2. moDCs express SREC-I and also bind and internalize GP2. Inhibition of SREC-I on moDCs with anti-SREC-I antibodies does not result in a decreased GP2 binding. Interaction of GP2 with SREC-I and uptake might have profound effects in antigen clearance and mediation of the immune response. In addition to SREC-I other presently unknown receptors for GP2 on DCs might be involved in this process. PMID:21190681

  10. Activation of PAC1 Receptors in Rat Cerebellar Granule Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through N-Type Calcium Channels

    PubMed Central

    Basille-Dugay, Magali; Vaudry, Hubert; Fournier, Alain; Gonzalez, Bruno; Vaudry, David

    2013-01-01

    High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca2+]i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca2+ response. Preincubation of granule cells with the Ca2+ ATPase inhibitor, thapsigargin, or the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca2+]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca2+ channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca2+]i increase. Preincubation of granule neurons with the N-type Ca2+ channel blocker, ?-conotoxin GVIA, decreased the PACAP-induced [Ca2+]i response, whereas the L-type Ca2+ channel blocker, nifedipine, and the P- and Q-type Ca2+ channel blocker, ?-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca2+]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca2+ channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes. PMID:23675369

  11. Ultrastructural enzyme-cytochemical study of the intrinsic glandular cells in the corpus cardiacum of Locusta migratoria : relation to the secretory and endocytotic pathways, and to the lysosomal system

    Microsoft Academic Search

    W. F. Jansen; J. H. B. Diederen; M. Dorland; J. Langermans; B. P. M. Meesen; K. Mink; H. G. B. Vullings

    1989-01-01

    Ultrastructural aspects of the secretory and the endocytotic pathways and the lysosomal system of corpus cardiacum glandular cells (CCG cells) of migratory locusts were studied using morphological, marker enzyme, immunocytochemical and tracer techniques. It is concluded that (1) the distribution of marker enzymes of trans Golgi cisternae and trans Golgi network (TGN) in locust CCG cells corresponds to that in

  12. 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

  13. Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen

    PubMed Central

    Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

    2011-01-01

    Background In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. Results To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. Conclusions This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. PMID:21383842

  14. 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

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

    PubMed Central

    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.

    2012-01-01

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

  16. Restricted diffusion of calretinin in cerebellar granule cell dendrites implies Ca2+-dependent interactions via its EF-hand 5 domain

    PubMed Central

    Arendt, Oliver; Schwaller, Beat; Brown, Edward B; Eilers, Jens; Schmidt, Hartmut

    2013-01-01

    Ca2+-binding proteins (CaBPs) are important regulators of neuronal Ca2+ signalling, acting either as buffers that shape Ca2+ transients and Ca2+ diffusion and/or as Ca2+ sensors. The diffusional mobility represents a crucial functional parameter of CaBPs, describing their range-of-action and possible interactions with binding partners. Calretinin (CR) is a CaBP widely expressed in the nervous system with strong expression in cerebellar granule cells. It is involved in regulating excitability and synaptic transmission of granule cells, and its absence leads to impaired motor control. We quantified the diffusional mobility of dye-labelled CR in mouse granule cells using two-photon fluorescence recovery after photobleaching. We found that movement of macromolecules in granule cell dendrites was not well described by free Brownian diffusion and that CR diffused unexpectedly slow compared to fluorescein dextrans of comparable size. During bursts of action potentials, which were associated with dendritic Ca2+ transients, the mobility of CR was further reduced. Diffusion was significantly accelerated by a peptide embracing EF-hand 5 of CR. Our results suggest long-lasting, Ca2+-dependent interactions of CR with large and/or immobile binding partners. These interactions render CR a poorly mobile Ca2+ buffer and point towards a Ca2+ sensor function of CR. PMID:23732647

  17. 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

  18. Distinct Role of Rab27a in Granule Movement at the Plasma Membrane and in the Cytosol of NK Cells

    PubMed Central

    Long, Eric O.

    2010-01-01

    Protocols were developed to automate image analysis and to track the movement of thousands of vesicular compartments in live cells. Algorithms were used to discriminate among different types of movement (e.g. random, caged, and directed). We applied these tools to investigate the steady-state distribution and movement of lytic granules (LG) in live natural killer (NK) cells by high-speed 3-dimensional (3D) spinning disc confocal and 2-dimensional total internal reflection fluorescence microscopy. Both mouse NK cells and a human NK cell line deficient in the small GTPase Rab27a were examined. The unbiased analysis of large datasets led to the following observations and conclusions. The majority of LG in the cytosol and at the plasma membrane of unstimulated NK cells are mobile. The use of inhibitors indicated that movement in the cytosol required microtubules but not actin, whereas movement at the plasma membrane required both. Rab27a deficiency resulted in fewer LG, and in a reduced fraction of mobile LG, at the plasma membrane. In contrast, loss of Rab27a increased the fraction of mobile LG and the extent of their movement in the cytosol. Therefore, in addition to its documented role in LG delivery to the plasma membrane, Rab27a may restrict LG movement in the cytosol. PMID:20877725

  19. 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

  20. Twin screw granulation: steps in granule growth.

    PubMed

    Dhenge, Ranjit M; Cartwright, James J; Hounslow, Michael J; Salman, Agba D

    2012-11-15

    The present work focuses on the study of the progression of granules in different compartments along the length of screws in a twin screw granulator (TSG). The effects of varying powder feed rate; liquid to solid ratio and viscosity of granulation liquid on properties of granules was studied. The bigger granules produced at the start of the process were found to change in terms of size, shape and strength along the screw length at all the conditions investigated. The granules became more spherical and their strength increased along the screw length. Tracer granules were also introduced in order to understand the role of kneading and conveying elements in the TSG. The kneading elements promoted consolidation and breakage while the conveying elements led to coalescence, breakage and some consolidation. The results presented here help to provide a qualitative and quantitative understanding of the twin screw granulation process. PMID:22960611

  1. The neuroendocrine protein VGF is sorted into dense-core granules and is secreted apically by polarized rat thyroid epithelial cells.

    PubMed

    Gentile, Flaviana; Calì, Gaetano; Zurzolo, Chiara; Corteggio, Annunziata; Rosa, Patrizia; Calegari, Federico; Levi, Andrea; Possenti, Roberta; Puri, Claudia; Tacchetti, Carlo; Nitsch, Lucio

    2004-04-15

    We have expressed the neuroendocrine VGF protein in FRT rat thyroid cells to study the molecular mechanisms of its sorting to the regulated and polarized pathways of secretion. By immunoelectron microscopy, we have demonstrated that VGF localizes in dense-core granules. Rapid secretion of VGF is induced by PMA stimulation. Moreover, human chromogranin B, a protein of the regulated pathway, co-localizes in the same granules with VGF. In confluent, FRT monolayers on filters protein secretion occur from the apical cell domain. VGF deletion mutants have been generated. By confocal microscopy, we have found that in transient transfection, all mutant proteins are sorted into granules and co-localize with the full-length VGF. They all retain the apical polarity of secretion. We also found that intracellular VGF and its deletion mutants are largely in an aggregated form. We conclude that FRT thyroid cells correctly decode the sorting information of VGF. The signals present on the protein to enter the granules and to be secreted apically cannot be separated from each other and are not in just one discrete portion of the protein. We propose that selective aggregation might represent the signal for sorting VGF to the regulated, apical route. PMID:15051509

  2. TLQP-21, a neuroendocrine VGF-derived peptide, prevents cerebellar granule cells death induced by serum and potassium deprivation.

    PubMed

    Severini, Cinzia; Ciotti, Maria Teresa; Biondini, Laura; Quaresima, Stefania; Rinaldi, Anna Maria; Levi, Andrea; Frank, Claudio; Possenti, Roberta

    2008-01-01

    Different VGF peptides derived from Vgf, originally identified as a nerve growth factor responsive gene, have been detected in neurons within the central and peripheral nervous system and in various endocrine cells. In the current study, we have evaluated the ability of TLQP-21, a VGF-derived peptide, to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death. We demonstrated that TLQP-21 increased survival of CGCs by decreasing the degree of apoptosis as assessed by cell viability and DNA fragmentation. Moreover, TLQP-21 significantly activated extracellular signal-regulated kinase 1/2, serine/threonine protein kinase, and c-jun N-terminal kinase phosphorylation, while decreased the extent of protein kinase C phosphorylation, as demonstrated by western blot analysis. In addition, TLQP-21 induced significant increase in intracellular calcium (as measured by fura-2AM) in about 60% of the recorded neurons. Taken together, the present results demonstrate that TLQP-21 promotes the survival of CGCs via pathways involving, within few minutes, modulation of kinases associated with CGCs survival, and by increasing intracellular calcium which can contribute to the neuroprotective effect of the peptide. PMID:18173805

  3. 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. PMID:25815230

  4. Characterisation of secretory calcium-binding phosphoprotein-proline-glutamine-rich 1: a novel basal lamina component expressed at cell-tooth interfaces.

    PubMed

    Moffatt, Pierre; Wazen, Rima M; Dos Santos Neves, Juliana; Nanci, Antonio

    2014-12-01

    Functional genomic screening of the rat enamel organ (EO) has led to the identification of a number of secreted proteins expressed during the maturation stage of amelogenesis, including amelotin (AMTN) and odontogenic ameloblast-associated (ODAM). In this study, we characterise the gene, protein and pattern of expression of a related protein called secretory calcium-binding phosphoprotein-proline-glutamine-rich 1 (SCPPPQ1). The Scpppq1 gene resides within the secretory calcium-binding phosphoprotein (Scpp) cluster. SCPPPQ1 is a highly conserved, 75-residue, secreted protein rich in proline, leucine, glutamine and phenylalanine. In silico data mining has revealed no correlation to any known sequences. Northern blotting of various rat tissues suggests that the expression of Scpppq1 is restricted to tooth and associated tissues. Immunohistochemical analyses show that the protein is expressed during the late maturation stage of amelogenesis and in the junctional epithelium where it localises to an atypical basal lamina at the cell-tooth interface. This discrete localisation suggests that SCPPPQ1, together with AMTN and ODAM, participates in structuring the basal lamina and in mediating attachment of epithelia cells to mineralised tooth surfaces. PMID:25193156

  5. A Highly Ca2+-sensitive Pool of Granules Is Regulated by Glucose and Protein Kinases in Insulin-secreting INS-1 Cells

    PubMed Central

    Yang, Yan; Gillis, Kevin D.

    2004-01-01

    We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca2+ to directly measure the Ca2+ sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca2+ sensitivity of release in that a small proportion of granules makes up a highly Ca2+-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca2+. A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca2+ are not located close to Ca2+ channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca2+-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca2+, suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca2+ sensor that is poised to respond to modest, global elevations of [Ca2+]i. PMID:15572344

  6. Disruption of cell cycle kinetics and cyclin-dependent kinase system by ethanol in cultured cerebellar granule progenitors.

    PubMed

    Li, Z; Lin, H; Zhu, Y; Wang, M; Luo, J

    2001-12-14

    An in vitro model of neuronal precursors, primary culture of cerebellar granule progenitors (CGPs), was used to investigate the mechanisms underlying ethanol-induced cell cycle damage. The CGP cultures were generated from 3-day-old rats. Ethanol significantly inhibited the proliferation of the CGPs in culture. Analysis of cell cycle kinetics by a cumulative 5-bromo-2'-deoxyuridine (BrdU) labeling technique demonstrated that ethanol exposure increased the duration of the cell cycle and decreased the growth fraction (the cycling population). The duration of the S-phase and total cell cycle was significantly prolonged by ethanol exposure by 220% and 135%, respectively, while the growth fraction was decreased from 44% in the control groups to 22% in the ethanol-exposed cultures. Cyclin-dependent kinase 2 (Cdk2) is a key protein that regulates both the passage from G1 into S, and the S phase progression. The results from in vitro phosphorylation assay and Western blot demonstrated that ethanol dramatically down-regulated both the activity and the expression of Cdk2. In addition, ethanol significantly decreased the expression of Cyclin A and Cyclin D(2). Further studies using in situ TUNEL assay and DNA fragmentation ELISA showed that ethanol caused a delayed apoptosis, i.e. the ethanol-induced apoptosis was evident only after chronic exposure. On the other hand, ethanol did not affect the necrotic index. In conclusion, ethanol decreases the cycling pool of CGPs by inducing cell cycle delay and promoting apoptosis. Ethanol-mediated disturbance of the cyclin-dependent kinase system may be an important mechanism to account for cell cycle arrest in neuronal precursor cells. PMID:11744106

  7. Metastatic intestinal carcinomas simulating primary ovarian clear cell carcinoma and secretory endometrioid carcinoma: a clinicopathologic and immunohistochemical study of five cases.

    PubMed

    Young, R H; Hart, W R

    1998-07-01

    Five cases of ovarian metastases of intestinal adenocarcinomas that suggested the diagnosis of clear cell adenocarcinoma or the secretory variant of endometrioid carcinoma of the ovary are reported. Patient age ranged from 27 to 71 years at the time of diagnosis of the ovarian neoplasms. In four, the ovarian and intestinal tumors were discovered synchronously, and, in the fifth, the ovarian metastasis occurred 1 year after the intestinal primary was diagnosed. The ovarian tumors were unilateral in three patients and bilateral in two. They were up to 18 cm (mean, 12 cm) in maximum dimension and were characterized on microscopic evaluation by glands and cysts lined by cells whose most striking feature was abundant clear cytoplasm. In two cases, striking subnuclear or supranuclear vacuoles were present. An important clue to the diagnosis of metastatic intestinal adenocarcinoma was the presence in all cases of "dirty necrosis." The metastatic nature of the ovarian tumors was supported by the immunohistochemical findings. All tumors stained were strongly positive for carcinoembryonic antigen and cytokeratin 20 and failed to stain for CA125, whereas staining for HAM56 and cytokeratin 7 was absent or only focally positive in one case each. Three intestinal primary tumors involved the small bowel. Microscopic evaluation of the intestinal tumors in three cases and metastases in a fourth, in which the intestinal primary was not resected, showed the features of the uncommon clear cell variant of intestinal adenocarcinoma; the fifth was predominantly a conventional intestinal adenocarcinoma with only a focal clear cell component. Although intestinal adenocarcinomas metastatic in the ovary typically simulate endometrioid adenocarcinoma of the usual type or mucinous adenocarcinoma, they may mimic either primary clear cell adenocarcinoma or the secretory variant of endometrioid adenocarcinoma, particularly when the primary tumor is, even focally, the clear cell variant of intestinal adenocarcinoma. PMID:9669343

  8. 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

  9. ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons

    PubMed Central

    Lenzi, Jessica; De Santis, Riccardo; de Turris, Valeria; Morlando, Mariangela; Laneve, Pietro; Calvo, Andrea; Caliendo, Virginia; Chiò, Adriano; Rosa, Alessandro; Bozzoni, Irene

    2015-01-01

    ABSTRACT Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUSR514S and FUSR521C patient fibroblasts, whereas in the case of the severe FUSP525L mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis. PMID:26035390

  10. A differential display protocol to identify differentially expressed mRNAs in potassium-deprived cerebellar granule cells.

    PubMed

    Roschier, M; Kuusisto, E; Salminen, A

    2000-04-01

    Differential display (DD) has become a popular technique for the identification of differentially expressed genes. Here we present a DD protocol for studying mRNA expression changes during neuronal apoptosis. Neuronal apoptosis is typically dependent on macromolecular synthesis, thus suggesting that regulation of gene expression is involved in control of the activation of the cell-death machinery. In order to identify some of the genes involved, we employed the widely used cell culture model in which apoptosis is induced in rat cerebellar granule cells (CGCs) using potassium deprivation. Although DD has been applied productively in the study of various biological phenomena, the method has its drawbacks. In particular, the cloning and verification of cDNA fragments is frequently described as problematic or laborious, and often produces many "false positives". Here we report the successful use of DD including an efficient protocol for cDNA clone screening and verification. This protocol avoids some of the problems presented by heterogeneous DD bands, which may be a major cause of false-positive results. To identify the desired clones, we apply single-stranded conformational polymorphism (SSCP) and slot blot techniques. PMID:10775830

  11. ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons.

    PubMed

    Lenzi, Jessica; De Santis, Riccardo; de Turris, Valeria; Morlando, Mariangela; Laneve, Pietro; Calvo, Andrea; Caliendo, Virginia; Chiò, Adriano; Rosa, Alessandro; Bozzoni, Irene

    2015-07-01

    Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUS(R514S) and FUS(R521C) patient fibroblasts, whereas in the case of the severe FUS(P525L) mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis. PMID:26035390

  12. Fluorescent Labeling of Newborn Dentate Granule Cells in GAD67GFP Transgenic Mice: A Genetic Tool for the Study of Adult Neurogenesis

    Microsoft Academic Search

    Shengli Zhao; Yang Zhou; Jimmy Gross; Pei Miao; Li Qiu; Dongqing Wang; Qian Chen; Guoping Feng; Alain Chédotal

    2010-01-01

    Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice) that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP+ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express

  13. Mast Cell Tryptase Activates Peripheral Blood Eosinophils to Release Granule-Associated Enzymes

    Microsoft Academic Search

    Harissios Vliagoftis; Paige Lacy; Betty Luy; Darryl Adamko; Morley Hollenberg; Dean Befus; Redwan Moqbel

    2004-01-01

    Background: Mast cells and eosinophils are important effector cells in asthma. Understanding their interactions is essential for studying asthma pathophysiology. Inflammatory mediators released from mast cells, such as arachidonic acid metabolites, TNF and IL-5, are important in eosinophil biology. However, little is known about the effects of mast cell-specific mediators, such as tryptase, on eosinophils. Our objective was to investigate

  14. 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

  15. 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

  16. Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

    PubMed

    Rahimpour, Azam; Vaziri, Behrouz; Moazzami, Reza; Nematollahi, Leila; Barkhordari, Farzaneh; Kokabee, Leila; Adeli, Ahmad; Mahboudi, Fereidoun

    2013-08-01

    Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells. PMID:23676904

  17. The effect of cyclic phosphatidic acid on the proliferation and differentiation of mouse cerebellar granule precursor cells during cerebellar development.

    PubMed

    Konakazawa, Misa; Gotoh, Mari; Murakami-Murofushi, Kimiko; Hamano, Ayana; Miyamoto, Yasunori

    2015-07-21

    The proliferation and differentiation of cerebellar granule cell precursors (GCPs) are highly regulated spatiotemporally during development. We focused on cyclic phosphatidic acid (cPA) as a lipid mediator with a cyclic phosphate group as a regulatory factor of GCPs. While its structure is similar to that of lysophosphatidic acid (LPA), its function is very unique. cPA is known to be present in the cerebellum at high levels, but its function has not been fully elucidated. In this study, we examined the role of cPA on the proliferation and differentiation of GCPs. A cell cycle analysis of GCPs revealed that cPA reduced the number of phospho-histone H3 (Phh3)-positive cells and bromodeoxy uridine (BrdU)-incorporated cells and increased an index of the cell cycle exit. We next analyzed the effect of cPA on GCP differentiation using Tuj1 as a neuronal marker of final differentiation. The results show that cPA increased the number of Tuj1-positive cells. Further analysis of the proliferation of GCPs showed that cPA suppressed Sonic hedgehog (Shh)-dependent proliferation, but did not suppress insulin-like growth factor-1 (IGF-1)-dependent proliferation. P2Y5 (LPA6), an LPA receptor, is highly expressed in GCPs. The knockdown of P2Y5 suppressed the inhibitory effect of cPA on the proliferation of GCPs, suggesting that P2Y5 is a candidate receptor for cPA. Thus, cPA suppresses the Shh-dependent proliferation of GCPs and promotes the differentiation of GCPs through P2Y5. These results demonstrate that cPA plays a critical role in the development of GCPs. PMID:25896936

  18. 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

  19. 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

  20. 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

  1. Comparison of several parameters related to the secretory activity of the subcommissural organ in European green frogs

    Microsoft Academic Search

    J. H. B. Diederen; H. G. B. Vullings

    1980-01-01

    In European green frogs the secretory activity of the subcommissural organ (SCO) was investigated and quantified measuring three parameters considered to be closely related to the cellular processes of synthesis and release of secretory material by the cells of the SCO: (1) the amount of stained secretory material in the SCO; (2) the amount of secretory material in the SCO

  2. 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.

  3. Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis

    PubMed Central

    1986-01-01

    We describe an in vitro system with all components derived from the yeast Saccharomyces cerevisiae that can translocate a yeast secretory protein across microsomal membranes. In vitro transcribed prepro-alpha- factor mRNA served to program a membrane-depleted yeast translation system. Translocation and core glycosylation of prepro-alpha-factor were observed when yeast microsomal membranes were added during or after translation. A membrane potential is not required for translocation. However, ATP is required for translocation and nonhydrolyzable analogues of ATP cannot serve as a substitute. These findings suggest that ATP hydrolysis may supply the energy required for translocation of proteins across the endoplasmic reticulum. PMID:3517001

  4. Intrinsic rescaling of granule cells restores pattern separation ability of a dentate gyrus network model during epileptic hyperexcitability.

    PubMed

    Yim, Man Yi; Hanuschkin, Alexander; Wolfart, Jakob

    2015-03-01

    The dentate gyrus (DG) is thought to enable efficient hippocampal memory acquisition via pattern separation. With patterns defined as spatiotemporally distributed action potential sequences, the principal DG output neurons (granule cells, GCs), presumably sparsen and separate similar input patterns from the perforant path (PP). In electrophysiological experiments, we have demonstrated that during temporal lobe epilepsy (TLE), GCs downscale their excitability by transcriptional upregulation of "leak" channels. Here we studied whether this cell type-specific intrinsic plasticity is in a position to homeostatically adjust DG network function. We modified an established conductance-based computer model of the DG network such that it realizes a spatiotemporal pattern separation task, and quantified its performance with and without the experimentally constrained leaky GC phenotype. Two proposed TLE seizure mechanisms were implemented in various degrees and combinations: recurrent GC excitation via mossy fiber sprouting and increased PP input. While increasing PP strength degraded pattern separation only gradually, already the slight elevation of sprouting drastically (non-linearly) impaired pattern separation. In most tested hyperexcitable networks, leaky GCs ameliorated pattern separation. However, in some sprouting situations with all-or-none seizure behavior, pattern separation was disabled with and without leaky GCs. In the mild sprouting (and PP increase) region of non-linear impairment, leaky GCs were particularly effective in restoring pattern separation performance. These results are compatible with the hypothesis that the experimentally observed intrinsic rescaling of GCs serves to maintain the physiological function of the DG network. PMID:25269417

  5. 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

  6. 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

  7. The effects of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 on the secretory capacity of human blood eosinophils.

    PubMed Central

    Tai, P C; Spry, C J

    1990-01-01

    The effects of recombinant human GM-CSF and interleukin-3 (IL-3) on human blood eosinophil survival, activation, and secretion were studied. Purified normal density eosinophils from patients with the idiopathic hypereosinophilic syndrome (HES) survived in culture for 7 days (50% viable) in the presence of 50 nM GM-CSF or 50 nM IL-3. Neutrophils did not survive after 4 days. No eosinophils survived in the absence of GM-CSF or IL-3. In two out of five patients studied, the cultured eosinophils became elongated with numerous processes. In all five patients the cells became adherent, but there were no morphological signs of degranulation. Both GM-CSF and IL-3 activated eosinophils, transforming the storage form of eosinophil cationic protein (ECP) into the secreted form. The proportion of activated cells increased from less than 20% to over 50% after 4 days in culture. However, GM-CSF and IL-3 did not induce secretion on their own. On the other hand, when GM-CSF/IL-3-activated eosinophils were exposed to known secretory stimuli, there was a six-fold increase in the amount of ECP released when the cells were stimulated with sepharose coated with C3b, and a two-fold increase when they were stimulated with sepharose-activated whole autologous serum. Eosinophils from patients taking steroids were unable to secrete their granule contents, even though they became activated by GM-CSF and IL-3. A novel finding was that sepharose-activated whole serum was an extremely potent secretory signal for ECP, releasing up to 50% of the total ECP content. These studies showed that GM-CSF and IL-3 prime eosinophil effector function by initiating granule solubilization which is the first step in the secretory event, without affecting the subsequent extracellular release of granule proteins. Images Fig. 2 Fig. 3 PMID:2197048

  8. Exacerbation of lung radiation injury by viral infection: the role of Clara cells and Clara cell secretory protein.

    PubMed

    Manning, Casey M; Johnston, Carl J; Hernady, Eric; Miller, Jen-nie H; Reed, Christina K; Lawrence, B Paige; Williams, Jacqueline P; Finkelstein, Jacob N

    2013-06-01

    Viral infections have been associated with exacerbation of disease in human cases of idiopathic pulmonary fibrosis. Since pulmonary fibrosis is a common outcome after irradiation to the lung, we hypothesized that viral infection after radiation exposure would exacerbate radiation-induced lung injury. Epithelial injury, a frequent outcome after infection, has been hypothesized to contribute to the pathogenesis of pulmonary fibrosis and bronchiolar epithelial Clara cells participate in epithelial repair. Therefore, it was further hypothesized that altered responses after irradiation involve the bronchiolar epithelial Clara cells. C57BL/6J or CCSP(-/-) mice were irradiated with 0 (sham), 5, 10 or 15 Gy to the whole thorax. At ten weeks post-irradiation, animals were mock infected or infected with influenza A virus and body weight and survival were monitored. Pulmonary function was assessed by whole-body plethysmography. The Clara cell markers, CCSP and Cyp2f2, were measured in the lung by qRT-PCR, and protein expression was visualized in the lung by immunofluorescence. Following pulmonary function tests, mice were sacrificed and tissues were collected for pathological analysis. In 15 Gy irradiated animals infected with influenza A virus, accelerated respiratory rates, reduced pulmonary function, and exacerbated lung pathology occurred earlier post-irradiation than previously observed after irradiation alone, suggesting infection accelerates the development of radiation injury. After irradiation alone, CCSP and Cyp2f2 mRNA levels were reduced, correlating with reductions in the number of Clara cells lining the airways. When combined with infection, these markers further declined and an apparent delay in recovery of mRNA expression was observed, suggesting that radiation injury leads to a chronic reduction in the number of Clara cells that may potentiate the epithelial injury observed after influenza A virus infection. This novel finding may have considerable therapeutic implications with respect to both thoracic tumor patients and recipients of bone marrow transplants. PMID:23621375

  9. Aquaporin 1 regulates GTP-induced rapid gating of water in secretory vesicles

    Microsoft Academic Search

    Sang-Joon Cho; A. K. M. Abdus Sattar; Eun-Hwan Jeong; Mylan Satchi; Jin Ah Cho; Sudhansu Dash; Mary Sue Mayes; Marvin H. Stromer; Bhanu P. Jena

    2002-01-01

    The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remains largely unknown. Zymogen granules (ZGs), the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a Gi3 protein. Evidence is presented here that the water channel aquaporin-1 (AQP1) is present in the ZG membrane and participates in rapid

  10. Uptake and release of beta-alanine in cerebellar granule cells in primary culture: regulation of release by glutamatergic and GABAergic receptors.

    PubMed

    Saransaari, P; Oja, S S

    1993-03-01

    The uptake and release of beta-[3H]alanine were studied in cultured glutamatergic cerebellar granule cells of the rat. The uptake of beta-alanine was saturable and sodium-dependent, comprising one high-affinity transport component. It was inhibited by hypotaurine, taurine, GABA and homotaurine but not by glycine or glutamate. The release was enhanced by homoexchange, veratridine and high K+ concentrations (50 mM). The K(+)-stimulated release was at least partially Ca(2+)-dependent. The release was shown to be subject to regulation by GABAA receptors and glutamate receptors of the kainate type. The results signify that beta-alanine may have a functional role in cerebellar granule cells. PMID:8098513

  11. Quality control in the secretory assembly line.

    PubMed Central

    Helenius, A

    2001-01-01

    As a rule, only proteins that have reached a native, folded and assembled structure are transported to their target organelles and compartments within the cell. In the secretory pathway of eukaryotic cells, this type of sorting is particularly important. A variety of molecular mechanisms are involved that distinguish between folded and unfolded proteins, modulate their intracellular transport, and induce degradation if they fail to fold. This phenomenon, called quality control, occurs at several levels and involves different types of folding sensors. The quality control system provides a stringent and versatile molecular sorting system that guaranties fidelity of protein expression in the secretory pathway. PMID:11260794

  12. Neurotensinergic Excitation of Dentate Gyrus Granule Cells via G?q-Coupled Inhibition of TASK-3 Channels.

    PubMed

    Zhang, Haopeng; Dong, Hailong; Cilz, Nicholas I; Kurada, Lalitha; Hu, Binqi; Wada, Etsuko; Bayliss, Douglas A; Porter, James E; Lei, Saobo

    2014-11-18

    Neurotensin (NT) is a 13-amino acid peptide and serves as a neuromodulator in the brain. Whereas NT has been implicated in learning and memory, the underlying cellular and molecular mechanisms are ill-defined. Because the dentate gyrus receives profound innervation of fibers containing NT and expresses high density of NT receptors, we examined the effects of NT on the excitability of dentate gyrus granule cells (GCs). Our results showed that NT concentration dependently increased action potential (AP) firing frequency of the GCs by the activation of NTS1 receptors resulting in the depolarization of the GCs. NT-induced enhancement of AP firing frequency was not caused indirectly by releasing glutamate, GABA, acetylcholine, or dopamine, but due to the inhibition of TASK-3 K(+) channels. NT-mediated excitation of the GCs was G protein dependent, but independent of phospholipase C, intracellular Ca(2+) release, and protein kinase C. Immunoprecipitation experiment demonstrates that the activation of NTS1 receptors induced the association of G?q/11 and TASK-3 channels suggesting a direct coupling of G?q/11 to TASK-3 channels. Endogenously released NT facilitated the excitability of the GCs contributing to the induction of long-term potentiation at the perforant path-GC synapses. Our results provide a cellular mechanism that helps to explain the roles of NT in learning and memory. PMID:25405940

  13. 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

  14. Inhibitory effect of fangchinoline on excitatory amino acids-induced neurotoxicity in cultured rat cerebellar granule cells.

    PubMed

    Kim, S D; Oh, S K; Kim, H S; Seong, Y H

    2001-04-01

    Glutamate receptors-mediated excitotoxicity is believed to play a role in the pathophysiology of neurodegenerative diseases. The present study was performed to evaluate the inhibitory effect of fangchinoline, a bis-benzylisoquinoline alkaloid, which has a characteristic as a Ca2+ channel blocker, on excitatory amino acids (EAAs)-induced neurotoxicity in cultured rat cerebellar granule neuron. Fangchinoline (1 and 5 microM) inhibited glutamate (1 mM), N-methyl-D-aspartate (NMDA; 1 mM) and kainate (100 microM)-induced neuronal cell death which was measured by trypan blue exclusion test. Fangchinoline (1 and 5 microM) inhibited glutamate release into medium induced by NMDA (1 mM) and kainate (100 microM), which was measured by HPLC. And fangchinoline (5 microM) inhibited glutamate (1 mM)-induced elevation of intracellular calcium concentration. These results suggest that inhibition of Ca2+ influx by fangchinoline may contribute to the beneficial effects on neurodegenerative effect of glutamate in pathophysiological conditions. PMID:11339637

  15. Photo-hydrogen production rate of a PVA-boric acid gel granule containing immobilized photosynthetic bacteria cells

    Microsoft Academic Search

    Xin Tian; Qiang Liao; Wei Liu; Yong Zhong Wang; Xun Zhu; Jun Li; Hong Wang

    2009-01-01

    The polyvinyl alcohol (PVA)-boric acid gel granule facilitates the light penetration and mass transport as it has the features of the transparency and adequate porous structure. In this work, a hydrogen production bioreactor with the indigenous photosynthetic bacteria (PSB) Rhodopseudomonas palustris CQK 01 immobilized in a PVA-boric acid gel granule is developed to enhance the rate of photo-hydrogen production. Particular

  16. Respiratory Modulation of Spontaneous Subthreshold Synaptic Activity in Olfactory Bulb Granule Cells Recorded in Awake, Head-Fixed Mice.

    PubMed

    Youngstrom, Isaac A; Strowbridge, Ben W

    2015-06-10

    Although the firing patterns of principal neurons in the olfactory bulb are known to be modulated strongly by respiration even under basal conditions, less is known about whether inhibitory local circuit activity in the olfactory bulb (OB) is modulated phasically. The diverse phase preferences of principal neurons in the OB and olfactory cortex that innervate granule cells (GCs) may interfere and prevent robust respiratory coupling, as suggested by recent findings. Using whole-cell recording, we examined the spontaneous, subthreshold membrane potential of GCs in the OBs of awake head-fixed mice. We found that, during periods of basal respiration, the synaptic input to GCs was strongly phase modulated, leading to a phase preference in the average, cycle-normalized membrane potential. Subthreshold phase tuning was heterogeneous in both mitral and tufted cells (MTCs) and GCs but relatively constant within each GC during periods of increased respiratory frequency. The timing of individual EPSPs in GC recordings also was phase modulated with the phase preference imparted by large-amplitude EPSPs, with fast kinetics often matching the phase tuning of the average membrane potential. These results suggest that activity in a subset of excitatory afferents to GCs, presumably including cortical feedback projections and other sources of large-amplitude unitary EPSPs, function to provide a timing signal linked to respiration. The phase preference we find in the membrane potential may provide a mechanism to dynamically modulate recurrent and lateral dendrodendritic inhibition of MTCs and to selective engage a subpopulation of interneurons based on the alignment of their phase tuning relative to sensory-driven MTC discharges. PMID:26063910

  17. Brain derived neurotrophic factor induces a rapid upregulation of synaptophysin and tau proteins via the neurotrophin receptor TrkB in rat cerebellar granule cells

    Microsoft Academic Search

    Eleanor T Coffey; Karl E. O Åkerman; Michael J Courtney

    1997-01-01

    We have examined the effects of neurotrophins brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) on the expression of the maturation-specific proteins synaptophysin and tau, and the growth-associated protein (GAP)-43 in cerebellar granule cells. We find that BDNF but not NGF rapidly (within 2 h) upregulates levels of synaptophysin, tau and c-Fos correlating with expression of the neurotrophin

  18. Cellular/Molecular Dopamine Inhibits Mitral/Tufted3Granule Cell Synapses in

    E-print Network

    Delaney, Kerry R.

    potentials. Key words: reciprocal synapse; mitral cell; lateral dendrite; feedback inhibition; D2; Rana pipiens Introduction The olfactory bulb (OB) is responsible for the initial representa- tion of odor

  19. The Harderian gland, its secretory duct and porphyrin content in the mongolian gerbil (Meriones unguiculatus).

    PubMed Central

    Johnston, H S; McGadey, J; Thompson, G G; Moore, M R; Payne, A P

    1983-01-01

    The Harderian gland, its secretory duct and porphyrin content were examined in the mongolian gerbil (Meriones unguiculatus). The gland consisted of tubules lined by a single layer of epithelial cells and a myoepithelial network. The tubule cells were often binucleate and possessed lipid vacuoles in the apical half of the cell, a corona of granular endoplasmic reticulum surrounding the nucleus, and cytoplasmic 'slashes'. The latter are probably derived from dense membranous couplets and may be precursors of the lipid vacuoles. Holocrine and merocrine secretion was observed. Interstitial cells included plasma cells, mast cells and (predominantly) melanocytes which render the gland black. The gland was surrounded by a collagen capsule and an outer layer of highly attenuated (possibly endothelioid) cells. Within the gland, the secretory duct was lined by a single layer of normal tubule cells. Outside the gland, the duct enlarged to form an ampulla, from which clefts led off to deep crypts. The ampulla and clefts were lined by cells with small dense apical granules and stubby microvilli; some possessed lipid vacuoles. The crypts were lined by serous cells with active Golgi regions. At the duct opening, ampullary cells became squamous and goblet cells occurred. Geometric crystalloid deposits (with a layered structure of 7.6 nm periodicity) occurred at cleft-crypt junctions. Islets of extra-glandular ductal tissue were occasionally found within the gland. Porphyrins were detectable both by chemical assay and fluorescence microscopy. There was a trend for female glands to have a higher content than males. Solid intraluminal accretions of porphyrin and/or lipid were present. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 12 Fig. 13 Fig. 14 Fig. 15 Fig. 16 PMID:6654750

  20. Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco BY-2 cells.

    PubMed

    Laporte, Céline; Vetter, Guillaume; Loudes, Anne-Marie; Robinson, David G; Hillmer, Stefan; Stussi-Garaud, Christiane; Ritzenthaler, Christophe

    2003-09-01

    Grapevine fanleaf virus (GFLV) is one of a large class of plant viruses whose cell-to-cell transport involves the passage of virions through tubules composed of virus-encoded movement protein (MP). The tubules are embedded within modified plasmodesmata, but the mechanism of targeting of MP to these sites is unknown. To study intracellular GFLV MP trafficking, a green fluorescent protein-MP fusion (GFP:MP) was expressed in transgenic tobacco BY-2 suspension cells under the control of an inducible promoter. We show that GFP:MP is targeted preferentially to calreticulin-labeled foci within the youngest cross walls, where it assembles into tubules. During cell division, GFP:MP colocalizes in the cell plate with KNOLLE, a cytokinesis-specific syntaxin, and both proteins are linked physically, as shown by coimmunoprecipitation of the two proteins from the same microsomal fraction. In addition, treatment with various drugs has revealed that a functional secretory pathway, but not the cytoskeleton, is required for tubule formation. However, correct GFP:MP targeting to calreticulin-labeled foci seems to be cytoskeleton dependent. Finally, biochemical analyses have revealed that at least a fraction of the MP behaves as an intrinsic membrane protein. These findings support a model in which GFP:MP would be transported to specific sites via Golgi-derived vesicles along two different pathways: a microtubule-dependent pathway in normal cells and a microfilament-dependent default pathway when microtubules are depolymerized. PMID:12953111

  1. Immunohistochemical characterization of intracellular J-chain and binding site for secretory component (SC) in human immunoglobulin (Ig)-producing cells.

    PubMed

    Brandtzaeg, P

    1983-09-01

    J-chain staining of IgA- and IgM-producing immunocytes was significantly enhanced when tissue sections were pretreated with acid urea, apparently because molecular unfolding exposed concealed J-chains. This indicated substantial completion of the Ig polymers at the cytoplasmic level, which was verified by diffuse binding of SC in vitro to the cytoplasm of most J-chain-positive IgA and IgM cells. This process involved specific non-covalent forces which showed the same interrelation as that noted for isolated dimeric IgA and 19S IgM--the latter as well as IgM cells exhibiting stronger binding of SC than the IgA counterparts. Conversely, J-chain staining of IgD and IgG immunocytes was not enhanced by acid urea and these cells did not generally express affinity for SC; rare exceptions could apparently be ascribed to artifacts or dual isotype production including IgA or IgM polymers. Parallel demonstration of J-chain and SC binding seems to be the best available method for studies of polymer-producing immunocyte populations and offers the advantage of in situ evaluation of cell distribution in relation to morphology. The reliability of this approach was attested to by the fact that IgA immunocytes in all secretory tissues investigated (salivary, mammary and lacrimal glands; nasal and intestinal mucosae) expressed J-chain (87-97%) and SC affinity (84-87%) in comparable proportions, indicating that almost 90% of the cells were engaged mainly in dimer production. The observation that most IgD and 50-70% of the IgG immunocytes in secretory tissues expressed J-chain, has implications for the differentiation of B-cell clones homing to such sites. Conversely, IgG cells in extra-glandular tissues showed strikingly reduced J-chain production and such sites contained IgA immunocytes with heterogeneous expression of J-chain and SC affinity. Thus, in the extra-follicular area of palatine tonsils 70-80% of the IgA cells seemed to be pure monomer producers and the remainders apparently generated a mixed product. Most immunocytes in extra-glandular tissues may therefore belong to mature clones with completely or partially repressed J-chain synthesis. PMID:6417474

  2. Hormone-induced secretory and nuclear translocation of calmodulin: Oscillations of calmodulin concentration with the nucleus as an integrator

    PubMed Central

    Craske, Madeleine; Takeo, Teruko; Gerasimenko, Oleg; Vaillant, Camille; Török, Katalin; Petersen, Ole H.; Tepikin, Alexei V.

    1999-01-01

    Many important enzyme activities are regulated by Ca2+-dependent interactions with calmodulin (CaM). Some of the most important targets for CaM action are in the nucleus, and Ca2+-dependent CaM translocation into this organelle has been reported. Hormone-evoked cytosolic Ca2+ signals occur physiologically as oscillations, but, so far, oscillations in CaM concentration have not been described. We loaded fluorescent-labeled CaM into pancreatic acinar cells and monitored the fluorescence in various regions by confocal microscopy. Sustained high concentrations of the hormone cholecystokinin or the neurotransmitter acetylcholine evoked a transient movement of cytosolic CaM from the basal nonnuclear area into the secretory granule region and, thereafter, a more substantial and prolonged translocation of CaM into the nucleoplasm. About 50% of the CaM that bound Ca2+ translocated. At a lower hormone concentration, evoking Ca2+ oscillations, regular spikes of increased CaM concentration were seen in the secretory granule region with mirror image spikes of decreased CaM concentration in the basal nonnuclear region. The nucleus was able to integrate the Ca2+ spike-evoked pulses of CaM translocation into a sustained elevation of the nucleoplasmic concentration of this protein. PMID:10200278

  3. Uptake, storage and secretion of 5-hydroxytryptamine and its amino acid precursor by dispersed rat pancreas acinar cells.

    PubMed Central

    Stern, L; Tenenhouse, A; Yu, E W

    1983-01-01

    Rat pancreas acinar cells contain 5-hydroxytryptamine (5-HT; 10.86 +/- 2.52 ng/i.u. amylase), all of which can be accounted for by the 5-HT recovered from the zymogen granule fraction of these cells (10.70 +/- 3.06 ng/i.u. amylase). When incubated with [14C]5-HT dispersed acinar cells take up the amine and concentrate it in zymogen granules. These cells will also take up [14C]5-HTP (5-hydroxytryptophan), decarboxylate it and store the [14C]5-HT so produced in zymogen granules. 5-HTP itself is not taken up by the granules. 5-HT is incorporated into zymogen granules early in their formation; no amine is accumulated by mature zymogen granules and the amine within the mature granule is not exchangeable with extragranular amine. When dispersed acinar cells pre-labelled with [14C]5-HT and [3H]leucine are stimulated with caerulein, there is a synchronous increase in amylase activity and secretion of [14C]5-HT and [3H]protein; the ratios of [3H]protein/[14C]5-HT in zymogen granules and in the secretory products are identical. Pancreas acinar cells take up L-DOPA, decarboxylate it and store the dopamine produced in zymogen granules but the dopamine is not retained by the granules (t1/2 approximately equal to 90 min) and dopamine secretion from cells exposed to caerulein could not be demonstrated. It is concluded that 5-HT is a normal component of rat pancreas acinar cell zymogen granule. The granular amine has a turnover rate similar to that of granular protein and is released when the cells are stimulated to secrete protein. All the 5-HT released from the cell originates in zymogen granules. Images Fig. 6 PMID:6193271

  4. Dense granule-containing cells in the wall of the branchio-cardiac veins of a fresh water crayfish (Astacus leptodactylus).

    PubMed

    Kusakabe, T; Ishii, K; Ishii, K

    1991-01-01

    The anatomical structure of oxygen-sensitive receptors in branchio-cardiac veins of a crayfish (Astacus leptodactylus) was studied with fluorescence and electron microscopy. Fluorescent cells were observed in the wall of a restricted part of the branchio-cardiac veins which was electrophysiologically shown to be a chemoreceptor region. Electron microscopy disclosed that they were dense granule-containing cells, and form, together with supporting cells and nerve terminals, complex structures that show gross similarities with arterial chemoreceptor structures in vertebrates. PMID:1897743

  5. [Morphogenesis and reaction to hypoxia of atrial myoendocrine cells in chick embryos (Gallus gallus)].

    PubMed

    Maksimov, V F; Korostyshevskaia, I M

    2012-01-01

    The ultrastructural and stereomorphometrical study of the right atrium of chick embryos at the 14th day of incubation has shown the cardiomyocytes to divide by mitosis and to be at different stages of differentiation. In the cytoplasm of some muscle cells we detected secretory granules that by their sizes and morphology can be classified into the forming, mature, and dissolved forms. By the 18th day of incubation most cardiomyocytes are already differentiated, and the number of secretory granules in them rises. Under conditions of hypoxia, after 3 days, in myoendocrine cells there are noted signs of accelerated release of the peptides synthesized earlier and accumulated in granules, while after one week - of enhancement of their synthesis. It can be concluded that in chick embryos, beginning from at least the 14th incubation day, the system of natriuretic heart peptides takes part in regulation of hemodynamics and of water-salt balance and responds to hypoxia. PMID:23136760

  6. 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.

  7. 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

  8. Non-catalyzed cathodic oxygen reduction at graphite granules in microbial fuel cells

    Microsoft Academic Search

    Stefano Freguia; Korneel Rabaey; Zhiguo Yuan

    2007-01-01

    Oxygen is the most sustainable electron acceptor currently available for microbial fuel cell (MFC) cathodes. However, its high overpotential for reduction to water limits the current that can be produced. Several materials and catalysts have previously been investigated in order to facilitate oxygen reduction at the cathode surface. This study shows that significant stable currents can be delivered by using

  9. 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...

  10. Structure and development of the secretory cavities of Myrtus communis leaves

    Microsoft Academic Search

    D. Kalachanis; G. K. Psaras

    2005-01-01

    The structure and development of Myrtus communis L. secretory cavities has been studied in young and expanded leaves, using light and scanning electron microscope. Secretory cavities are continuously formed during leaf development, but in mature leaves the rhythm of their appearance shows steep decrease. Each secretory cavity is developed from a single epidermal cell, which undergoes a periclinal division followed

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

    PubMed

    Pfeiffer, Daniel; Wahl, Andreas; Jendrossek, Dieter

    2011-11-01

    A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ?phaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R.?eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1. PMID:22023320

  12. 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.

  13. Mast cell apoptosis induced by siramesine, a sigma-2 receptor agonist.

    PubMed

    Spirkoski, Jane; Melo, Fabio R; Grujic, Mirjana; Calounova, Gabriela; Lundequist, Anders; Wernersson, Sara; Pejler, Gunnar

    2012-12-15

    Mast cells (MCs) are well known for their detrimental effects in the context of allergic disorders. Strategies that limit MC function can therefore have a therapeutic value. Previous studies have shown that siramesine, a sigma-2 receptor agonist originally developed as an anti-depressant, can induce cell death in transformed cells through a mechanism involving lysosomal destabilization. Since MCs are remarkably rich in lysosome-like secretory granules we reasoned that MCs might be sensitive to siramesine. Here we show that murine and human MCs are highly sensitive to siramesine. Cell death was accompanied by secretory granule permeabilization, as shown by reduced acridine orange staining and leakage of granule proteases into the cytosol. Wild type siramesine-treated MCs underwent cell death with typical signs of apoptosis but MCs lacking serglycin, a proteoglycan crucial for promoting the storage of proteases within MC secretory granules, died predominantly by necrosis. A dissection of the underlying mechanism suggested that the necrotic phenotype of serglycin(-/-) cells was linked to defective Poly(ADP-ribose) polymerase-1 degradation. In vivo, siramesine treatment of mice caused a depletion of the MC populations of the peritoneum and skin. The present study shows for the first time that MCs are highly sensitive to apoptosis induced by siramesine and introduces the possibility of using siramesine as a therapeutic agent for treatment of MC-dependent disease. PMID:23058984

  14. GRANULES ASSOCIATED WITH THE CHLOROPLAST LAMELLAE OF PORPHYRIDIUM CRUENTUM

    Microsoft Academic Search

    E. GANTT; S. F. CONTI

    2009-01-01

    Small granules with a diameter of approximately 350 A are attached to the chloroplast lamellae of the red alga Porphyridium cruentum. To some extent, their size depends on the culture conditions and the age of the cell. It was possible to preserve the granules only with aldehyde prefixation. It can be seen that fixed or negatively stained granules are comprised

  15. Gametes Alter the Oviductal Secretory Proteome

    Microsoft Academic Search

    A. Stephen Georgiou; Edita Sostaric; Chi H. Wong; Ambrosius P. L. Snijders; Phillip C. Wright; Harry D. Moore; Alireza Fazeli

    2005-01-01

    The mammalian oviduct provides an optimal environment for the maturation of gametes, fertilization, and early em- bryonic development. Secretory cells lining the lumen of the mammalian oviduct synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. We hypothesized that the presence of gametes in the oviduct alters the oviduc- tal

  16. Activation of alpha1 and alpha2 noradrenergic receptors exert opposing effects on excitability of main olfactory bulb granule cells.

    PubMed

    Nai, Q; Dong, H W; Linster, C; Ennis, M

    2010-08-25

    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 microM) and the alpha1 receptor agonist phenylephrine (10 microM) depolarized and increased spontaneous or current injection-evoked spiking in GCs. By contrast, low NE concentrations (0.1-1.0 microM) or the alpha2 receptor agonist clonidine (Clon, 10 microM) hyperpolarized and decreased the discharge of GCs. The effects of NE (10 microM) were blocked by antagonism of alpha1 and alpha2 receptors. Inhibitory effects of low NE concentrations were blocked or converted to excitatory responses by alpha2 receptor blockade, whereas excitatory effects of the moderate NE concentration were converted to inhibitory responses after alpha1 receptor blockade. NE (10 microM) 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 alpha1 and alpha2 receptor activation exert opposing effects on GC excitability. Low concentrations of NE acting via alpha2 receptors suppress GC excitability, while higher concentrations of NE acting at alpha1 receptors increase GC excitability. These findings are consistent with recent findings that alpha1 and alpha2 receptor activation increase and decrease, respectively, GABAergic inhibition of mitral cells. The differential affinities of alpha1 and alpha2 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

  17. Continuous electricity generation by a graphite granule baffled air-cathode microbial fuel cell.

    PubMed

    Feng, Yujie; Lee, He; Wang, Xin; Liu, Yaolan; He, Weihua

    2010-01-01

    A baffled air-cathode microbial fuel cell (BAFMFC) was designed and operated under continuous flow. With glucose fed as substrate, an average voltage of 652 mV was obtained under the external resistance of 1000 Omega (30 degrees C). The maximum power density was 15.2 W/m(3) with the chemical oxygen demand (COD) removal rate of 88.0%. The overall resistance was 13.7 Omega while ohmic internal resistance was 10.8 Omega. Average COD removal rate was 69.7-88.0%, when COD loading varied from 4.11 kg COD/(m(3)NACd) to 16.0 kg COD/(m(3)NACd). The liquid from corn stover steam explosion process (COD=7160+/-50mg/L) was treated by BAFMFC, and the maximum power density was 10.7 W/m(3) with the average COD removal rate was 89.1%. The present study indicated BAFMFC can be comparable to the traditional anaerobic baffled reactor in COD removal rate for high-concentration wastewater and have an advantage in energy harvest from wastewater. PMID:19748267

  18. Refining the value of secretory phospholipase A2 as a predictor of acute chest syndrome in sickle cell disease: results of a feasibility study (PROACTIVE).

    PubMed

    Styles, Lori; Wager, Carrie G; Labotka, Richard J; Smith-Whitley, Kim; Thompson, Alexis A; Lane, Peter A; McMahon, Lillian E C; Miller, Robin; Roseff, Susan D; Iyer, Rathi V; Hsu, Lewis L; Castro, Oswaldo L; Ataga, Kenneth I; Onyekwere, Onyinye; Okam, Maureen; Bellevue, Rita; Miller, Scott T

    2012-06-01

    Acute chest syndrome (ACS) is defined as fever, respiratory symptoms and a new pulmonary infiltrate in an individual with sickle cell disease (SCD). Nearly half of ACS episodes occur in SCD patients already hospitalized, potentially permitting pre-emptive therapy in high-risk patients. Simple transfusion of red blood cells may abort ACS if given to patients hospitalized for pain who develop fever and elevated levels of secretory phospholipase A2 (sPLA2). In a feasibility study (PROACTIVE; ClinicalTrials.gov NCT00951808), patients hospitalized for pain who developed fever and elevated sPLA2 were eligible for randomization to transfusion or observation; all others were enrolled in an observational arm. Of 237 enrolled, only 10 were randomized; one of the four to receive transfusion had delayed treatment. Of 233 subjects receiving standard care, 22 developed ACS. A threshold level of sPLA2 ? 48 ng/ml gave optimal sensitivity (73%), specificity (71%) and accuracy (71%), but a positive predictive value of only 24%. The predictive value of sPLA2 was improved in adults and patients with chest or back pain, lower haemoglobin concentration and higher white blood cell counts, and in those receiving less than two-thirds maintenance fluids. The hurdles identified in PROACTIVE should facilitate design of a larger, definitive, phase 3 randomized controlled trial. PMID:22463614

  19. Refining the Value of Secretory Phospholipase A2 as a Predictor of Acute Chest Syndrome in Sickle Cell Disease: Results of a Feasibility Study (PROACTIVE)

    PubMed Central

    Styles, Lori; Wager, Carrie G.; Labotka, Richard J.; Smith-Whitley, Kim; Thompson, Alexis A.; Lane, Peter A.; McMahon, Lillian E.C; Miller, Robin; Roseff, Susan; Iyer, Rathi; Hsu, Lewis L.; Castro, Oswaldo; Ataga, Kenneth; Onyekwere, Onyinye; Okam, Maureen; Bellevue, Rita; Miller, Scott T.

    2012-01-01

    Acute chest syndrome (ACS) is defined as fever, respiratory symptoms and a new pulmonary infiltrate in an individual with sickle cell disease (SCD). Nearly half of ACS episodes occur in SCD patients already hospitalized, potentially permitting pre-emptive therapy in high-risk patients. Simple transfusion of red blood cells may abort ACS if given to patients hospitalized for pain who develop fever and elevated levels of secretory phospholipase A2 (sPLA2). In a feasibility study (PROACTIVE; ClinicalTrials.gov NCT00951808), patients hospitalized for pain who developed fever and elevated sPLA2 were eligible for randomization to transfusion or observation; all others were enrolled in an observational arm. Of 237 enrolled, only 10 were randomized; one of the four to receive transfusion had delayed treatment. Of 233 subjects receiving standard care, 22 developed ACS. A threshold level of sPLA2 ? 48 ng/ml gave optimal sensitivity (73%), specificity (71%) and accuracy (71%), but a positive predictive value of only 24%. The predictive value of sPLA2 was improved in adults and patients with chest or back pain, lower haemoglobin concentration and higher white blood cell counts; and those receiving less than two-thirds maintenance fluids. The hurdles identified in PROACTIVE should facilitate design of a larger, definitive, phase 3 randomized controlled trial. PMID:22463614

  20. Loss of cell surface microvilli on rat basophilic leukaemia cells precedes secretion and can be mimicked using the calmodulin antagonist trifluoperazine

    Microsoft Academic Search

    A. J. Edgar; G. R. Davies; M. A. Anwar; J. P. Bennett

    1997-01-01

    Objective: We studied changes in cell surface morphology following treatment with secretagogue or trifluoperazine in a mast cell model.¶Materials and Methods: Rat basophilic leukaemia (RBL) cells were treated with antigen and or the calmodulin antagonist, 0-50 7M trifluoperazine (TFP). The release of a secretory granule enzyme, #-hexosaminidase, into the external medium was used as a measure of secretion. Quantitation of

  1. Ursodeoxycholic acid attenuates colonic epithelial secretory function

    PubMed Central

    Kelly, Orlaith B; Mroz, Magdalena S; Ward, Joseph B J; Colliva, Carolina; Scharl, Michael; Pellicciari, Roberto; Gilmer, John F; Fallon, Padraic G; Hofmann, Alan F; Roda, Aldo; Murray, Frank E; Keely, Stephen J

    2013-01-01

    Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl? secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl? secretory responses to the Ca2+ and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n= 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na+/K+-ATPase activity and basolateral K+ channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg?1) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50–200 ?m) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6?-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea. PMID:23507881

  2. Transcription factors COUP-TFI and COUP-TFII are required for the production of granule cells in the mouse olfactory bulb.

    PubMed

    Zhou, Xing; Liu, Fang; Tian, Miao; Xu, Zhejun; Liang, Qifei; Wang, Chunyang; Li, Jiwen; Liu, Zhidong; Tang, Ke; He, Miao; Yang, Zhengang

    2015-05-01

    Neural stem cells (NSCs) persist in the adult mammalian subventricular zone (SVZ) of the lateral ventricle. Primary NSCs generate rapidly dividing intermediate progenitor cells, which in turn generate neuroblasts that migrate along the rostral migratory stream (RMS) to the olfactory bulb (OB). Here, we have examined the role of the COUP-TFI and COUP-TFII orphan nuclear receptor transcription factors in mouse OB interneuron development. We observed that COUP-TFI is expressed in a gradient of low rostral to high caudal within the postnatal SVZ neural stem/progenitor cells. COUP-TFI is also expressed in a large number of migrating neuroblasts in the SVZ and RMS, and in mature interneurons in the OB. By contrast, very few COUP-TFII-expressing (+) cells exist in the SVZ-RMS-OB pathway. Conditional inactivation of COUP-TFI resulted in downregulation of tyrosine hydroxylase expression in the OB periglomerular cells and upregulation of COUP-TFII expression in the SVZ, RMS and OB deep granule cell layer. In COUP-TFI/COUP-TFII double conditional mutant SVZ, cell proliferation was increased through the upregulation of the proneural gene Ascl1. Furthermore, COUP-TFI/II-deficient neuroblasts had impaired migration, resulting in ectopic accumulation of calretinin (CR)+ and NeuN+ cells, and an increase in apoptotic cell death in the SVZ. Finally, we found that most Pax6+ and a subset of CR+ granular cells were lost in the OB. Taken together, these results suggest that COUP-TFI/II coordinately regulate the proliferation, migration and survival of a subpopulation of Pax6+ and CR+ granule cells in the OB. PMID:25922524

  3. Antagonism by riluzole of entry of calcium evoked by NMDA and veratridine in rat cultured granule cells: evidence for a dual mechanism of action.

    PubMed Central

    Hubert, J P; Delumeau, J C; Glowinski, J; Prémont, J; Doble, A

    1994-01-01

    1. Intracellular calcium levels were measured in cultured cerebellar granule cells of the rat by use of the fluorescent dye, indo-1/AM. 2. Intracellular calcium levels were increased by depolarizing stimuli such as N-methyl-D-aspartate (NMDA) (100 microM), glutamic acid (20 microM), and veratridine (10 microM). This increase was essentially due to entry of external calcium. 3. Riluzole (10 microM) blocked responses to all the depolarizing agents. 4. Riluzole could still block the increase in intracellular calcium evoked by NMDA or glutamic acid when sodium channels were blocked by tetrodotoxin, suggesting that this effect is not mediated by a direct action of riluzole on the voltage-dependent sodium channel. 5. Pretreatment of the cells with pertussis toxin (0.1 micrograms ml-1) did not modify the increases in intracellular calcium evoked by NMDA, glutamic acid or veratridine. 6. In pertussis toxin-treated cells, riluzole could no longer block responses to excitatory amino acids, but still blocked responses to veratridine. 7. It is concluded that riluzole has a dual action on cerebellar granule cells, both blocking voltage-dependent sodium channels and interfering with NMDA receptor-mediated responses via a pertussis toxin-sensitive mechanism. Furthermore, these two processes have been shown to be independent. PMID:7812619

  4. Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry*

    PubMed Central

    Sasaki, Kazuki; Osaki, Tsukasa; Minamino, Naoto

    2013-01-01

    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 also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics. PMID:23250050

  5. 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 also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics. PMID:23250050

  6. PIKfyve negatively regulates exocytosis in neurosecretory cells.

    PubMed

    Osborne, Shona L; Wen, Peter J; Boucheron, Christine; Nguyen, Hao N; Hayakawa, Masahiko; Kaizawa, Hiroyuki; Parker, Peter J; Vitale, Nicolas; Meunier, Frederic A

    2008-02-01

    Regulated secretion depends upon a highly coordinated series of protein-protein and protein-lipid interactions. Two phosphoinositides, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate, are important for the ATP-dependent priming of the secretory apparatus prior to Ca(2+)-dependent exocytosis. Mechanisms that control phosphoinositide levels are likely to play an important role in priming fine tuning. Here we have investigated the involvement of PIKfyve, a phosphoinositide 5-kinase that can phosphorylate phosphatidylinositol 3-phosphate to produce phosphatidylinositol 3,5-bisphosphate on large dense core vesicle exocytosis from neuroendocrine cells. PIKfyve localizes to a subpopulation of secretory granules in chromaffin and PC12 cells. Nicotine stimulation promoted recruitment of PIKfyve-EGFP onto secretory vesicles in PC12 cells. YM-201636, a selective inhibitor of PIKfyve activity, and PIKfyve knockdown by small interfering RNA potentiated secretory granule exocytosis. Overexpression of PIKfyve or its yeast orthologue Fab1p inhibited regulated secretion in PC12 cells, whereas a catalytically inactive PIKfyve mutant had no effect. These results demonstrate a novel inhibitory role for PIKfyve catalytic activity in regulated secretion and provide further evidence for a fine tuning of exocytosis by 3-phosphorylated phosphoinositides. PMID:18039667

  7. Matrix-binding vascular endothelial growth factor (VEGF) isoforms guide granule cell migration in the cerebellum via VEGF receptor Flk1.

    PubMed

    Ruiz de Almodovar, Carmen; Coulon, Cathy; Salin, Paul Antoine; Knevels, Ellen; Chounlamountri, Naura; Poesen, Koen; Hermans, Karlien; Lambrechts, Diether; Van Geyte, Katie; Dhondt, Joke; Dresselaers, Tom; Renaud, Julie; Aragones, Julian; Zacchigna, Serena; Geudens, Ilse; Gall, David; Stroobants, Stijn; Mutin, Mireille; Dassonville, Karel; Storkebaum, Erik; Jordan, Bénédicte F; Eriksson, Ulf; Moons, Lieve; D'Hooge, Rudi; Haigh, Jody J; Belin, Marie-Françoise; Schiffmann, Serge; Van Hecke, Paul; Gallez, Bernard; Vinckier, Stefan; Chédotal, Alain; Honnorat, Jérôme; Thomasset, Nicole; Carmeliet, Peter; Meissirel, Claire

    2010-11-10

    Vascular endothelial growth factor (VEGF) regulates angiogenesis, but also has important, yet poorly characterized roles in neuronal wiring. Using several genetic and in vitro approaches, we discovered a novel role for VEGF in the control of cerebellar granule cell (GC) migration from the external granule cell layer (EGL) toward the Purkinje cell layer (PCL). GCs express the VEGF receptor Flk1, and are chemoattracted by VEGF, whose levels are higher in the PCL than EGL. Lowering VEGF levels in mice in vivo or ectopic VEGF expression in the EGL ex vivo perturbs GC migration. Using GC-specific Flk1 knock-out mice, we provide for the first time in vivo evidence for a direct chemoattractive effect of VEGF on neurons via Flk1 signaling. Finally, using knock-in mice expressing single VEGF isoforms, we show that pericellular deposition of matrix-bound VEGF isoforms around PC dendrites is necessary for proper GC migration in vivo. These findings identify a previously unknown role for VEGF in neuronal migration. PMID:21068311

  8. Perinatal exposure to low-dose methylmercury induces dysfunction of motor coordination with decreases in synaptophysin expression in the cerebellar granule cells of rats.

    PubMed

    Fujimura, Masatake; Cheng, Jinping; Zhao, Wenchang

    2012-06-29

    Methylmercury (MeHg) is an environmental pollutant that is toxic to the developing central nervous system (CNS) in children, even at low exposure levels. Perinatal exposure to MeHg is known to induce neurological symptoms with neuropathological changes in the CNS. However, the relationship between the neurological symptoms and neuropathological changes induced in offspring as a result of exposure to low-dose MeHg is not well defined. In the present study, neurobehavioral analyses revealed that exposure to a low level of MeHg (5 ppm in drinking water) during developmental caused a significant deficit in the motor coordination of rats in the rotating rod test. In contrast, general neuropathological findings, including neuronal cell death and the subsequent nerve inflammation, were not observed in the region of the cerebellum responsible for regulating motor coordination. Surprisingly, the expression of synaptophysin (SPP), a marker protein for synaptic formation, significantly decreased in cerebellar granule cells. These results showed that perinatal exposure to low-dose MeHg causes neurobehavioral impairment without general neuropathological changes in rats. We demonstrated for the first time that exposure to low-dose MeHg during development induces the dysfunction of motor coordination due to changes of synaptic homeostasis in cerebellar granule cells. PMID:22587888

  9. Phospholipase C treatment of certain human target cells reduces their susceptibility to NK lysis without affecting binding or sensitivity to lytic granules.

    PubMed

    Une, C; Grönberg, A; Axberg, I; Jondal, M; Orn, A

    1991-03-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) is an enzyme that has the capacity to release glycosyl-phosphatidyl inositol (G-PI)-anchored proteins from the cells surface. Pretreatment of the human T-cell leukemia cell line Molt-4 with PI-PLC resulted in a decrease in the susceptibility to lysis by natural killer (NK) cells. Treatment of the erythroleukemia cell line K562 with PI-PLC had no effect on its NK susceptibility. PI-PLC-treated and untreated Molt-4 bound equally well to lymphocytes in target-binding studies with effector cell preparations enriched for NK cells. Susceptibility to cytolytic granules isolated from rat LGL tumor cells remained the same after treatment of Molt-4 or K562 with PI-PLC. Combined treatment of Molt-4 with PI-PLC and rlFN-alpha or rlFN-gamma resulted in additive reductions of the NK susceptibility, suggesting that PI-PLC and interferons act on different mechanisms to protect cells from NK lysis. When expression of a number of antigens on Molt-4 and K562 was analyzed in flow cytometry, only the expression of CD58 was reduced after PI-PLC treatment. The susceptibility of Con A blasts to MLR derived cytotoxic T-cells was not altered by treatment with phospholipase. These data suggest that PI-PLC treatment reduces the capacity of some target cells to activate NK cells upon contract. The mechanism behind this phenomenon is presently unclear. PMID:1703925

  10. Protection of NMDA-induced neuronal cell damage by methanol extract of zizyphi spinosi semen in cultured rat cerebellar granule cells.

    PubMed

    Park, Jeong Hee; Lee, Hyun Joo; Koh, Sang Bum; Ban, Ju Yeon; Seong, Yeon Hee

    2004-11-01

    Zizypus is one of the herbs widely used in Korea and China due to the CNS calming effect. The present study aims to investigate the effect of the methanol extract of Zizyphi Spinosi Semen (ZSS), the seeds of Zizyphus jujuba Mill var. spinosa, on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in cultured rat cerebellar granule neuron. ZSS, over a concentration range of 0.05-5 microg/ml, inhibited NMDA (1 mM)-induced neuronal cell death, which was measured by a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. ZSS (0.5 microg/ml) inhibited glutamate release into medium induced by NMDA (1mM), which was measured by HPLC. Pretreatment of ZSS (0.5 microg/ml) inhibited NMDA (1mM)-induced elevation of cytosolic calcium concentration ([Ca(2+)](c)), which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that ZSS prevents NMDA-induced neuronal cell damage in vitro. PMID:15374605

  11. Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules

    SciTech Connect

    Darchen, F.; Hammel, F.; Monteils, M.P.; Scherman, D. (Centre National de la Recherche Scientifique, Paris (France)); Zahraoui, A.; Tavitian, A. (Institut National de la Sante et de la Recherche Medicale, Paris (France))

    1990-08-01

    The Rab3A protein belongs to a large family of small GTP-binding proteins that are present in eukaryotic cells and that share amino acid identities with the Ras proteins (products of the ras protooncogenes). Rab3A, which is specifically located in nervous and endocrine tissues, is suspected to play a key role in secretion. Its localization was investigated in bovine adrenal gland by using a polyclonal antibody. Rab3A was detected in adrenal medulla but not in adrenal cortex. In cultured adrenal medulla cells, Rab3A was specifically expressed in the catecholamine-secreting chromaffin cells. Subcellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage organelles) and with a second compartment likely to be the plasma membrane. The Rab3A localization on chromaffin granule membranes was confirmed by immunoadsorption with an antibody against dopamine {beta}-hydroxylase. Rab3A was not extracted from this membrane by NaCl or KBr but was partially extracted by urea and totally solubilized by Triton X-100, suggesting either an interaction with an intrinsic protein or a membrane association through fatty acid acylation. This study suggests that Rab3A, which may also be located on other secretory vesicles containing noncharacterized small GTP-binding proteins, is involved in their biogenesis or in the regulated secretion process.

  12. Inhibitory effect of fangchinoline on excitatory amino acids-induced neurotoxicity in cultured rat cerebellar granule cells

    Microsoft Academic Search

    Sun Don Kim; Sei Kwan Oh; Hack Seang Kim; Yeon Hee Seong

    2001-01-01

    Glutamate receptors-mediated excitotoxicity is believed to play a role in the pathophysiology of neurodegenerative diseases.\\u000a The present study was performed to evaluate the inhibitory effect of fangchinoline, abis-benzylisoquinoline alkaloid, which has a characteristic as a Ca2+ channel blocker, on excitatory amino acids (EAAs)-induced neurotoxicity in cultured rat cerebellar granule neuron. Fangchinoline\\u000a (1 and 5 ?M) inhibited glutamate (1 mM), N-methyl-D-aspartate

  13. Variability of solar granulation

    NASA Astrophysics Data System (ADS)

    Muller, R.

    The temporal variation of the mean size of granules discovered by Macris et al. (1983) and Muller and Roudier (1984) is confirmed with high-quality, homogenous photographs, showing that granules are smaller at the sunspot maximum. The amplitude of the variation is found to be about 5 percent. It is shown that the number of granules is highly dependent on image quality. It is suggested that the variation in granule size probably results from the interaction between the convection zone and the magnetic field at the global scale of the sun.

  14. Effects of methanolic extracts from edible plants on endogenous secretory receptor for advanced glycation end products induced by the high glucose incubation in human endothelial cells

    PubMed Central

    Okada, Yoshinori; Okada, Mizue

    2015-01-01

    Background: In diabetic populations, endogenous secretory receptor for advanced glycation end products (esRAGE) levels may be related to the degree of diabetic complications or to the protection from diabetic complications. Objective: We investigated the impact of 29 methanolic extracts from edible plants on esRAGE production in human umbilical vein endothelial cells (HUVECs) cultured in high (4.5 g/L) glucose. Materials and Methods: Edible plants were minced, and extracts were obtained with methanol overnight. The methanolic extracts from 29 edible plants were evaporated in a vacuum. For screening study purposes, HUVECs were seeded in culture dishes (1.5 × 105 cells). Then, HUVECs were incubated with 1 g/L or 4.5 g/L of glucose in SFM CS-C medium treated with methanolic extracts from edible plants (MEEP) for 96 h. Determination of esRAGE production in the cell culture-derived supernatants was performed by colorimetric ELISA. The 8-hydroxydeoxyguanosine (8-OHdG) level was determined by using the 8-OHdG Check ELISA kit. Peroxynitrite-dependent oxidation of 2’, 7’-dichlorodihydrofluorescein to 2’, 7’-dichlorofluorescein was estimated based on the method described by Crow. Because MEEP were methanolic extracts, we measured their total phenolic content (TPC). TPC was measured with a modified version of the Folin–Ciocalteu method. Results: The results showed eight extracts increased esRAGE production. The extract from white radish sprouts showed the highest esRAGE production activity, and then eggplant, carrot peel, young sweet corn, Jew's marrow, broad bean, Japanese radish and cauliflower. In order to understand the mechanism of esRAGE production, the eight extracts were examined for DNA damage, peroxynitrite scavenging activity, and TPC in correlation with their esRAGE production. The results showed esRAGE production correlates with the peroxynitrite level and TPC. Conclusion: This study supports the utilization of these eight extracts in folk medicine for improved treatment of diabetic complications. PMID:25883519

  15. Endoplasmic Reticulum Export Sites and Golgi Bodies Behave as Single Mobile Secretory Units in Plant Cells W

    E-print Network

    Snapp, Erik Lee

    , United Kingdom b Cell Biology and Metabolism Branch, National Institute of Child Health and Human on the ER membrane. Here, we have visualized two early components of the COPII machinery, the small GTPase Sar1p and its GTP exchanging factor Sec12p in live tobacco (Nicotiana tabacum) leaf epidermal cells

  16. EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase C?, Ca++, and Erk, leading to granule polarization

    PubMed Central

    Pérez-Quintero, Luis-Alberto; Roncagalli, Romain; Guo, Huaijian; Latour, Sylvain; Davidson, Dominique

    2014-01-01

    Ewing’s sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)–associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase C?, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation. PMID:24687958

  17. EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase C?, Ca++, and Erk, leading to granule polarization.

    PubMed

    Pérez-Quintero, Luis-Alberto; Roncagalli, Romain; Guo, Huaijian; Latour, Sylvain; Davidson, Dominique; Veillette, André

    2014-04-01

    Ewing's sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase C?, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation. PMID:24687958

  18. Mast cells that reside at different locations in the jejunum of mice infected with Trichinella spiralis exhibit sequential changes in their granule ultrastructure and chymase phenotype

    PubMed Central

    1996-01-01

    Whether or not a nontransformed, mature mouse mast cell (MC) or its committed progenitor can change its granule protease phenotype during inflammatory responses, has not been determined. To address this issue, the granule morphology and protease content of the MC in the jejunum of BALB/c mice exposed to Trichinella spiralis were assessed during the course of the infection. Within 1 wk after helminth infection of the mice, increased numbers of MC appeared in the crypts at the base of the villi, and by wk 2 the number of MC throughout the villi increased by approximately 25-fold. Shortly after the peak of the mastocytosis, the intraepithelial population of MC disappeared, followed by a progressive loss of lamina propria MC. The presence of stellate-shaped granules containing crystalline structures in intraepithelial MC at the height of infection and the retention of such granules with fragmented crystals in lamina propria MC during resolution of the mastocytosis suggest that MC migrate during the various phases of the inflammation. As assessed by immunohistochemical analyses of serial sections, predominant chymase phenotypes were observed at the height of the infection in the muscle that expressed mouse MC protease (mMCP) 5 without mMCP-1 or mMCP-2 and in the epithelium that expressed mMCP-1 and mMCP-2 without mMCP-5. Accompanying these two MC populations were transitional forms in the submucosa that expressed mMCP-2 and mMCP-5 without mMCP-1 and in the lamina propria that expressed mMCP-2 alone. These data suggest that jejunal MC sequentially express mMCP-2, cease expressing mMCP-5, and finally express mMCP-1 as the cells progressively appear in the submucosa, lamina propria, and epithelium, respectively. In the recovery phase of the disease, MC sequentially cease expressing mMCP-1, express mMCP-5, and finally cease expressing mMCP-2 as they present at the tips of the villi, the base of the villi, and the submucosa, respectively. That MC can reversibly alter their protease phenotypes suggests that a static nomenclature with fixed functional implications is inadequate to describe MC populations during an inflammatory process within a particular tissue. PMID:8858180

  19. Endogenous zinc depresses GABAergic transmission via T-type Ca2+ channels and broadens the time window for integration of glutamatergic inputs in dentate granule cells

    PubMed Central

    Grauert, Antonia; Engel, Dominique; Ruiz, Arnaud J

    2014-01-01

    Abstract?Zinc actions on synaptic transmission span the modulation of neurotransmitter receptors, transporters, activation of intracellular cascades and alterations in gene expression. Whether and how zinc affects inhibitory synaptic signalling in the dentate gyrus remains largely unexplored. We found that mono- and di-synaptic GABAergic inputs onto dentate granule cells were reversibly depressed by exogenous zinc application and enhanced by zinc chelation. Blocking T-type Ca2+ channels prevented the effect of zinc chelation. When recording from dentate fast-spiking interneurones, zinc chelation facilitated T-type Ca2+ currents, increased action potential half-width and decreased spike threshold. It also increased the offset of the input–output relation in a manner consistent with enhanced excitability. In granule cells, chelation of zinc reduced the time window for the integration of glutamatergic inputs originating from perforant path synapses, resulting in reduced spike transfer. Thus, zinc-mediated modulation of dentate interneurone excitability and GABA release regulates information flow to local targets and hippocampal networks. PMID:24081159

  20. A novel type of cytoplasmic granule in bovine neutrophils

    PubMed Central

    1983-01-01

    We obtained cell preparations containing greater than 95% neutrophils from freshly drawn bovine blood. The cells were suspended in sucrose and disrupted in a Dounce homogenizer, and the postnuclear supernate was fractionated by zonal differential sedimentation and by isopycnic equilibration. The subcellular fractions were characterized biochemically by testing for marker enzymes and other constituents known to occur in azurophil and specific granules of other species, and by electrophoretic analysis of extracts of the particulate material. In addition, each fraction was examined by random-sampling electron microscopy. We found that bovine neutrophils contain in addition to azurophil and specific granules a third type of granule, not known to occur in neutrophils of other species. These novel granules are larger, denser, and considerably more numerous than the two other types. Except for lactoferrin, they lack the characteristic constituents of azurophil granules (peroxidase, acid hydrolases, and neutral proteinases) and of specific granules (vitamin B12-binding protein). Instead, they contain a group of highly cationic proteins not found in the other granules, and they are the exclusive stores of powerful oxygen-independent bactericidal agents. We studied the fate of the large granules in bovine neutrophils exposed to opsonized particles, the ionophore A 23187, or phorbol myristate acetate. The appearance in the cell-free media of antibacterial activity and of the characteristic highly cationic proteins as revealed by electrophoresis was monitored and compared with the release of azurophil and specific granule markers. In addition, changes of the relative size of the large granule compartment induced by phagocytosis were assessed by morphometry. The results show that exocytosis of the large granules occurs following both phagocytosis and exposure to soluble stimuli. Like the specific granules, the large granules appear to be discharged by true secretion under conditions where the azurophil granules are fully retained. PMID:6406517

  1. Potential of yeast secretory vesicles in biodelivery systems.

    PubMed

    Kutralam-Muniasamy, Gurusamy; Flores-Cotera, Luis B; Perez-Guevara, Fermin

    2015-06-01

    Membranous vesicular organelles (MVOs), such as secretory vesicles and exosomes, perform a variety of biological functions ranging from secretion to cellular communication in eukaryotic cells. Exosomes, particularly those of mammalian cells, have been widely studied as potential carriers in human therapeutic applications. However, no study has yet demonstrated the use of yeast secretory vesicles for such applications. Therefore, we explore here the current state of knowledge on yeast secretory vesicles and their potential use in therapeutic delivery systems. We focus on the characteristics shared by exosomes and yeast secretory vesicles to provide insights into the use of the latter as delivery vehicles. From this perspective, we speculate on the potential application of post-Golgi vesicles (PGVs) in the biomedical field. PMID:25843637

  2. Cloning of the Zygosaccharomyces bailii GAS 1 homologue and effect of cell wall engineering on protein secretory phenotype

    Microsoft Academic Search

    Simone Passolunghi; Luca Riboldi; Laura Dato; Danilo Porro; Paola Branduardi

    2010-01-01

    Background  \\u000a Zygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which\\u000a related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell\\u000a factory. Gas1p is a ?-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining

  3. GATA6 Is Required for Proliferation, Migration, Secretory Cell Maturation, and Gene Expression in the Mature Mouse Colon

    PubMed Central

    Beuling, Eva; Aronson, Boaz E.; Tran, Luc M. D.; Stapleton, Kelly A.; ter Horst, Ellis N.; Vissers, Laurens A. T. M.; Verzi, Michael P.

    2012-01-01

    Controlled renewal of the epithelium with precise cell distribution and gene expression patterns is essential for colonic function. GATA6 is expressed in the colonic epithelium, but its function in the colon is currently unknown. To define GATA6 function in the colon, we conditionally deleted Gata6 throughout the epithelium of small and large intestines of adult mice. In the colon, Gata6 deletion resulted in shorter, wider crypts, a decrease in proliferation, and a delayed crypt-to-surface epithelial migration rate. Staining techniques and electron microscopy indicated deficient maturation of goblet cells, and coimmunofluorescence demonstrated alterations in specific hormones produced by the endocrine L cells and serotonin-producing cells. Specific colonocyte genes were significantly downregulated. In LS174T, the colonic adenocarcinoma cell line, Gata6 knockdown resulted in a significant downregulation of a similar subset of goblet cell and colonocyte genes, and GATA6 was found to occupy active loci in enhancers and promoters of some of these genes, suggesting that they are direct targets of GATA6. These data demonstrate that GATA6 is necessary for proliferation, migration, lineage maturation, and gene expression in the mature colonic epithelium. PMID:22733991

  4. Besides Purkinje cells and granule neurons: an appraisal of the cell biology of the interneurons of the cerebellar cortex

    Microsoft Academic Search

    Karl Schilling; John Oberdick; Ferdinando Rossi; Stephan L. Baader

    2008-01-01

    Ever since the groundbreaking work of Ramon y Cajal, the cerebellar cortex has been recognized as one of the most regularly\\u000a structured and wired parts of the brain formed by a rather limited set of distinct cells. Its rather protracted course of\\u000a development, which persists well into postnatal life, the availability of multiple natural mutants, and, more recently, the\\u000a availability

  5. Gangliosides Normalize Distorted Single-Cell Intracellular Free Ca2+ Dynamics After Toxic Doses of Glutamate in Cerebellar Granule Cells

    Microsoft Academic Search

    Gabriel A. de Erausquin; Hari Manev; Alessandro Guidotti; Erminio Costa; Gary Brooker

    1990-01-01

    Glutamate-induced delayed neurotoxicity after abusive and paroxismal activation of its receptors has been proposed to depend upon a sustained increased in intracellular free Ca2+ ([Ca2+]_i). To elucidate the temporal and causal relationship between glutamate-induced changes in [Ca2+]_i and neuronal death, we simultaneously studied the dynamics of [Ca2+]_i changes in single neurons with the acetoxymethyl ester of fura-2 and the cell

  6. Gene Expression Profiles Underlying Selective T-Cell-Mediated Immunity Activity of a Chinese Medicine Granule on Mice Infected with Influenza Virus H1N1

    PubMed Central

    Lu, Na-na; Liu, Qi; Gu, Li-gang; Ge, Shi-jie; Wu, Jun; Ze-ji, Qiu; Qiu, Ze-ji; Zhang, Hong-chun; Chao, En-xiang; Yu, Zhuo-nan

    2014-01-01

    A Chinese medicine granule, Shu-Feng-Xuan-Fei (SFXF), is critical for viral clearance in early phase of influenza virus infection. In this study, 72 ICR mice were randomly divided into six groups: normal control group, virus control group, Oseltamivir group, low-dose SFXF, medium-dose SFXF, and high-dose SFXF. Mice were anesthetized and inoculated with 4LD50 of influenza virus A (H1N1) except normal control group. Oseltamivir group received 11.375 mg·kg?1·d?1 Oseltamivir Phosphate. SFXF 3.76, 1.88 and 0.94 g·kg?1·d?1 were administrated to mice in all SFXF groups. Each group was in equal dose of 0.2ml daily for 4 consecutive days. Mice were sacrificed and then total RNA was extracted in lung tissue. Some genes involved in T-cell-mediated immunity were selected by DNA microarray. These candidate genes were verified by Real-Time PCR and western immunoblotting. Compared with virus control group, in Toll-like receptor signaling pathway, 12 virus-altered genes were significantly reduced following medium-dose SFXF treatment. Eighteen antigen processing presentation-associated genes were upregulated by medium-dose SFXF. In the process of T cell receptor signaling pathway, 19 genes were downregulated by medium-dose SFXF treatment. On exploration into effector T cells activation and cytokines, all of altered genes in virus control group were reversed by medium-dose SFXF. Real-time PCR and western immunoblotting showed that the regulation of medium-dose SFXF in IL-4, IFN-?, TNF-?, IL-1?, TLR7, MyD88, p38, and JNK was superior to Oseltamivir and high-dose SFXF group. Therefore, SFXF granules could reduce influenza infected cells and activation of T cells. PMID:24527057

  7. Vesicular Trafficking and Signaling for Cytokine and Chemokine Secretion in Mast Cells

    PubMed Central

    Blank, Ulrich; Madera-Salcedo, Iris Karina; Danelli, Luca; Claver, Julien; Tiwari, Neeraj; Sánchez-Miranda, Elizabeth; Vázquez-Victorio, Genaro; Ramírez-Valadez, Karla Alina; Macias-Silva, Marina; González-Espinosa, Claudia

    2014-01-01

    Upon activation mast cells (MCs) secrete numerous inflammatory compounds stored in their cytoplasmic secretory granules by a process called anaphylactic degranulation, which is responsible for type I hypersensitivity responses. Prestored mediators include histamine and MC proteases but also some cytokines and growth factors making them available within minutes for a maximal biological effect. Degranulation is followed by the de novo synthesis of lipid mediators such as prostaglandins and leukotrienes as well as a vast array of cytokines, chemokines, and growth factors, which are responsible for late phase inflammatory responses. While lipid mediators diffuse freely out of the cell through lipid bilayers, both anaphylactic degranulation and secretion of cytokines, chemokines, and growth factors depends on highly regulated vesicular trafficking steps that occur along the secretory pathway starting with the translocation of proteins to the endoplasmic reticulum. Vesicular trafficking in MCs also intersects with endocytic routes, notably to form specialized cytoplasmic granules called secretory lysosomes. Some of the mediators like histamine reach granules via specific vesicular monoamine transporters directly from the cytoplasm. In this review, we try to summarize the available data on granule biogenesis and signaling events that coordinate the complex steps that lead to the release of the inflammatory mediators from the various vesicular carriers in MCs. PMID:25295038

  8. Gramicidin-perforated Patch Recording Revealed the Oscillatory Nature of Secretory ClMovements in Salivary Acinar Cells

    Microsoft Academic Search

    Makoto Sugita; Chikara Hirono; Yoshiki Shiba

    2004-01-01

    Elevations of cytoplasmic free calcium concentrations ((Ca 2 ? ) i ) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Clthrough Ca 2 ? -activated Clchannels, while Clenters the cytoplasm against its electrochemical gradient via a loop diuretic- sensitive Na ? -K ? -2Clcotransporter (NKCC) and\\/or parallel

  9. A Novel Secretory Factor, Neurogenesin-1, Provides Neurogenic Environmental Cues for Neural Stem Cells in the Adult Hippocampus

    Microsoft Academic Search

    Takatoshi Ueki; Masamitsu Tanaka; Kanna Yamashita; Sumiko Mikawa; ZheFu Qiu; Nicholas J. Maragakis; Robert F. Hevner; Naoyuki Miura; Haruhiko Sugimura; Kohji Sato

    2003-01-01

    Neurogenesis occurs in restricted regions in the adult mammalian brain, among which the neurogenesis in the hippocampal dentate gyrus plays the crucial role in learning and memory. To date, little is known about neurogenic cues, which result in the neuronal fate adoption of neural stem cells residing in neurogenic regions, especially neurogenic cues in adult hippocampal neurogenesis. In the present

  10. High Frequency Burst Firing of Granule Cells Ensures Transmission at the Parallel Fiber to Purkinje Cell Synapse at the Cost of Temporal Coding

    PubMed Central

    van Beugen, Boeke J.; Gao, Zhenyu; Boele, Henk-Jan; Hoebeek, Freek; De Zeeuw, Chris I.

    2013-01-01

    Cerebellar granule cells (GrCs) convey information from mossy fibers (MFs) to Purkinje cells (PCs) via their parallel fibers (PFs). MF to GrC signaling allows transmission of frequencies up to 1 kHz and GrCs themselves can also fire bursts of action potentials with instantaneous frequencies up to 1 kHz. So far, in the scientific literature no evidence has been shown that these high-frequency bursts also exist in awake, behaving animals. More so, it remains to be shown whether such high-frequency bursts can transmit temporally coded information from MFs to PCs and/or whether these patterns of activity contribute to the spatiotemporal filtering properties of the GrC layer. Here, we show that, upon sensory stimulation in both un-anesthetized rabbits and mice, GrCs can show bursts that consist of tens of spikes at instantaneous frequencies over 800 Hz. In vitro recordings from individual GrC-PC pairs following high-frequency stimulation revealed an overall low initial release probability of ~0.17. Nevertheless, high-frequency burst activity induced a short-lived facilitation to ensure signaling within the first few spikes, which was rapidly followed by a reduction in transmitter release. The facilitation rate among individual GrC-PC pairs was heterogeneously distributed and could be classified as either “reluctant” or “responsive” according to their release characteristics. Despite the variety of efficacy at individual connections, grouped activity in GrCs resulted in a linear relationship between PC response and PF burst duration at frequencies up to 300 Hz allowing rate coding to persist at the network level. Together, these findings support the hypothesis that the cerebellar granular layer acts as a spatiotemporal filter between MF input and PC output (D’Angelo and De Zeeuw, 2009). PMID:23734102

  11. Matrix elasticity regulates the secretory profile of human bone marrow-derived multipotent mesenchymal stromal cells (MSCs)

    Microsoft Academic Search

    F. Philipp Seib; Marina Prewitz; Carsten Werner; Martin Bornhäuser

    2009-01-01

    The therapeutic efficacy of multipotent mesenchymal stromal cells (MSCs) is attributed to particular MSC-derived cytokines and growth factors. As MSCs are applied locally to target organs or home there after systemic administration, they experience diverse microenvironments that are biochemically and biophysically distinct. Here we use well-defined in vitro conditions to study the impact of substrate elasticity on MSC-derived trophic factors.

  12. Mitotic events in cerebellar granule progenitor cells that expand cerebellar surface area are critical for normal cerebellar cortical lamination in mice.

    PubMed

    Chang, Joshua C; Leung, Mark; Gokozan, Hamza Numan; Gygli, Patrick Edwin; Catacutan, Fay Patsy; Czeisler, Catherine; Otero, José Javier

    2015-03-01

    Late embryonic and postnatal cerebellar folial surface area expansion promotes cerebellar cortical cytoarchitectural lamination. We developed a streamlined sampling scheme to generate unbiased estimates of murine cerebellar surface area and volume using stereologic principles. We demonstrate that, during the proliferative phase of the external granular layer (EGL) and folial surface area expansion, EGL thickness does not change and thus is a topological proxy for progenitor self-renewal. The topological constraints indicate that, during proliferative phases, migration out of the EGL is balanced by self-renewal. Progenitor self-renewal must, therefore, include mitotic events yielding 2 cells in the same layer to increase surface area (? events) and mitotic events yielding 2 cells, with 1 cell in a superficial layer and 1 cell in a deeper layer (? events). As the cerebellum grows, therefore, ? events lie upstream of ? events. Using a mathematical model constrained by the measurements of volume and surface area, we could quantify intermitotic times for ? events on a per-cell basis in postnatal mouse cerebellum. Furthermore, we found that loss of CCNA2, which decreases EGL proliferation and secondarily induces cerebellar cortical dyslamination, shows preserved ?-type events. Thus, CCNA2-null cerebellar granule progenitor cells are capable of self-renewal of the EGL stem cell niche; this is concordant with prior findings of extensive apoptosis in CCNA2-null mice. Similar methodologies may provide another layer of depth to the interpretation of results from stereologic studies. PMID:25668568

  13. Cloning of the Zygosaccharomyces bailii GAS1 homologue and effect of cell wall engineering on protein secretory phenotype

    PubMed Central

    2010-01-01

    Background Zygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a ?-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion. Results The GAS1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS1 null mutant was able to restore the parental phenotype. The respective Z. bailii ?gas1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS1 gene with deletion cassettes having flanking regions of ~400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii ?gas1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1? secretion disruption had no relevant effect. Conclusions The data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as already widely reported in literature, our data confirmed that an "always working" solution to the problems related to recombinant protein production can be hardly, if never, found; instead, manipulations have to be finely tuned for each specific product and/or combination of host cell and product. PMID:20102600

  14. Secretory autoantibodies in primary biliary cirrhosis (PBC).

    PubMed

    Palmer, J M; Doshi, M; Kirby, J A; Yeaman, S J; Bassendine, M F; Jones, D E

    2000-12-01

    It is unclear how breakdown in immune tolerance to the ubiquitous self-antigen pyruvate dehydrogenase complex (PDC), seen in the autoimmune liver disease PBC, gives rise to tissue damage with such a limited distribution (restricted to the liver and salivary and lachrymal glands). One property shared by these tissues is the ability to export secretory IgA by the process of transcytosis. The aim of this study was to address whether active transcytosis of anti-PDC IgA occurs across epithelial surfaces in PBC, a finding that might implicate mucosal specific immune mechanisms in the pathogenesis of this disease. Parotid saliva was collected from PBC patients (n = 44), normal controls (n = 28) and PBC patients post-liver transplantation (n = 11). IgA and secretory component-positive antibodies specific for human PDC were quantified by ELISA and immunoblotting. PBC patients (but not control subjects) had anti-PDC IgA in their saliva. The strong correlation seen between titres detected using anti-IgA and anti-secretory component antibodies suggests that this is predominantly secretory IgA reaching the saliva by the active process of epithelial transcytosis. Titres of anti-PDC IgA remain high in PBC patients saliva post-liver transplant. Findings from studies of IgA in viral infection models raise the possibility that anti-PDC IgA could, whilst undergoing transcytosis, bind to newly translated PDC components in the cytoplasm of the epithelial cells transporting them out of the cell and inducing metabolic damage. This model would, if correct, help to explain the mechanism and tropism of tissue damage in PBC and the aberrant pattern of expression of PDC on the apical surface of biliary and salivary epithelial cells reported in this disease. PMID:11122250

  15. An essential role of syntaxin 3 protein for granule exocytosis and secretion of IL-1?, IL-1?, IL-12b, and CCL4 from differentiated HL-60 cells.

    PubMed

    Naegelen, Isabelle; Plançon, Sébastien; Nicot, Nathalie; Kaoma, Tony; Muller, Arnaud; Vallar, Laurent; Tschirhart, Eric J; Bréchard, Sabrina

    2015-03-01

    Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the rapid secretion of cytokines orchestrates the action of other immune cells at the infection site and thus, can contribute to the development and chronicity of inflammatory diseases, we aimed to determine the intracellular SNARE machinery responsible for the regulation of cytokine secretion and degranulation. From a constructed gene-expression network, we first selected relevant cytokines for functional validation by the CBA approach. We established a cytokine-secretion profile for human neutrophils and dHL-60 cells, underlining their similar ability to secrete a broad variety of cytokines within proinflammatory conditions mimicked by LPS stimulation. Secondly, after screening of SNARE genes by microarray experiments, we selected STX3 for further functional studies. With the use of a siRNA strategy, we show that STX3 is clearly required for the maximal release of IL-1?, IL-1?, IL-12b, and CCL4 without alteration of other cytokine secretion in dHL-60 cells. In addition, we demonstrate that STX3 is involved in MMP-9 exocytosis from gelatinase granules, where STX3 is partly localized. Our results suggest that the secretion of IL-1?, IL-1?, IL-12b, and CCL4 occurs during gelatinase degranulation, a process controlled by STX3. In summary, these findings provide first evidence that STX3 has an essential role in trafficking pathways of cytokines in neutrophil granulocytes. PMID:25548252

  16. Linking Albinism and Immunity: The Secrets of Secretory Lysosomes

    NSDL National Science Digital Library

    Jane Stinchcombe (Sir William Dunn School of Pathology; )

    2004-07-02

    Lysosomes are membrane-bound organelles that are found in all mammalian cells and contain hydrolases and lipases required for protein and membrane degradation. In many cells of the immune system, lysosomes also contain secretory proteins that can be released by regulated exocytosis in response to an external stimulus, providing different cell types with a wide range of effector functions. Melanosomes also use a lysosome-related organelle to secrete melanin for pigmentation. Links between albinism and immunity in patients have uncovered a number of key proteins required for lysosomal secretion and have revealed a versatile secretory mechanism that can be fine-tuned by distinct interactions in different cell types.

  17. Growth and Localization of Polyhydroxybutyrate Granules in Ralstonia eutropha

    PubMed Central

    Beeby, Morgan; Cho, Mimi

    2012-01-01

    The bacterium Ralstonia eutropha forms cytoplasmic granules of polyhydroxybutyrate that are a source of biodegradable thermoplastic. While much is known about the biochemistry of polyhydroxybutyrate production, the cell biology of granule formation and growth remains unclear. Previous studies have suggested that granules form either in the inner membrane, on a central scaffold, or in the cytoplasm. Here we used electron cryotomography to monitor granule genesis and development in 3 dimensions (3-D) in a near-native, “frozen-hydrated” state in intact Ralstonia eutropha cells. Neither nascent granules within the cell membrane nor scaffolds were seen. Instead, granules of all sizes resided toward the center of the cytoplasm along the length of the cell and exhibited a discontinuous surface layer more consistent with a partial protein coating than either a lipid mono- or bilayer. Putatively fusing granules were also seen, suggesting that small granules are continually generated and then grow and merge. Together, these observations support a model of biogenesis wherein granules form in the cytoplasm coated not by phospholipid but by protein. Previous thin-section electron microscopy (EM), fluorescence microscopy, and atomic force microscopy (AFM) results to the contrary may reflect both differences in nucleoid condensation and specimen preparation-induced artifacts. PMID:22178974

  18. Growth and localization of polyhydroxybutyrate granules in Ralstonia eutropha.

    PubMed

    Beeby, Morgan; Cho, Mimi; Stubbe, JoAnne; Jensen, Grant J

    2012-03-01

    The bacterium Ralstonia eutropha forms cytoplasmic granules of polyhydroxybutyrate that are a source of biodegradable thermoplastic. While much is known about the biochemistry of polyhydroxybutyrate production, the cell biology of granule formation and growth remains unclear. Previous studies have suggested that granules form either in the inner membrane, on a central scaffold, or in the cytoplasm. Here we used electron cryotomography to monitor granule genesis and development in 3 dimensions (3-D) in a near-native, "frozen-hydrated" state in intact Ralstonia eutropha cells. Neither nascent granules within the cell membrane nor scaffolds were seen. Instead, granules of all sizes resided toward the center of the cytoplasm along the length of the cell and exhibited a discontinuous surface layer more consistent with a partial protein coating than either a lipid mono- or bilayer. Putatively fusing granules were also seen, suggesting that small granules are continually generated and then grow and merge. Together, these observations support a model of biogenesis wherein granules form in the cytoplasm coated not by phospholipid but by protein. Previous thin-section electron microscopy (EM), fluorescence microscopy, and atomic force microscopy (AFM) results to the contrary may reflect both differences in nucleoid condensation and specimen preparation-induced artifacts. PMID:22178974

  19. Genetic Targeting of a Small Fluorescent Zinc Indicator to Cell Surface for Monitoring Zinc Secretion

    PubMed Central

    Li, Daliang; Liu, Lin; Li, Wen-Hong

    2015-01-01

    Numerous mammalian cells contain Zn2+ in their secretory granules. During secretion, Zn2+ is coreleased with granular cargos into extracellular medium so Zn2+ serves as a convenient surrogate marker for tracking the dynamics of secretion. Fluorescent Zn2+ sensors that can be selectively targeted to cells of interest would be invaluable tools for imaging Zn2+ release in multicellular systems including tissues and live animals. Exploiting the HaloTag labeling technology and using an optimized linker, we have engineered a fluorescent Zn2+ indicator that displayed a 15-fold fluorescence enhancement upon Zn2+ binding while reacting efficiently with a HaloTag enzyme in a cellular environment. Two-color imaging of ZIMIR-HaloTag and a red-emitting calcium indicator in pancreatic islet beta cells demonstrated that photoactivation of a channelrhodopsin was able to induce exocytosis of Zn2+/insulin granules and revealed heterogeneity in secretory activity along the cell membrane that was uncoupled from cellular Ca2+ activity. This integrated photonic approach for imaging and controlling the release of large dense core granules provides exquisite cellular selectivity and should facilitate future studies of stimulus-secretion coupling and paracrine signaling in secretory cells. PMID:25572404

  20. Developmental exposure to ethanol increases the neuronal vulnerability to oxygen-glucose deprivation in cerebellar granule cell cultures.

    PubMed

    Le Duc, Diana; Spataru, Ana; Ceanga, Mihai; Zagrean, Leon; Schöneberg, Torsten; Toescu, Emil C; Zagrean, Ana-Maria

    2015-07-21

    Prenatal alcohol exposure is associated with microencephaly, cognitive and behavioral deficits, and growth retardation. Some of the mechanisms of ethanol-induced injury, such as high level oxidative stress and overexpression of pro-apoptotic genes, can increase the sensitivity of fetal neurons towards hypoxic/ischemic stress associated with normal labor. Thus, alcohol-induced sequelae may be the cumulative result of direct ethanol toxicity and increased neuronal vulnerability towards metabolic stressors, including hypoxia. We examined the effects of ethanol exposure on the fetal cerebellar granular neurons? susceptibility to hypoxic/hypoglycemic damage. A chronic ethanol exposure covered the entire prenatal period and 5 days postpartum through breastfeeding, a time interval partially extending into the third-trimester equivalent in humans. After a binge-like alcohol exposure at postnatal day 5, glutamatergic cerebellar granule neurons were cultured and grown for 7 days in vitro, then exposed to a 3-h oxygen-glucose deprivation to mimic a hypoxic/ischemic condition. Cellular viability was monitored by dynamic recording of propidium iodide fluorescence over 20h reoxygenation. We explored differentially expressed genes on microarray data from a mouse embryonic ethanol-exposure model and validated these by real-time PCR on the present model. In the ethanol-treated cerebellar granule neurons we find an increased expression of genes related to apoptosis (Mapk8 and Bax), but also of genes previously described as neuroprotective (Dhcr24 and Bdnf), which might suggest an actively maintained viability. Our data suggest that neurons exposed to ethanol during development are more vulnerable to in vitro hypoxia/hypoglycemia and have higher intrinsic death susceptibility than unexposed neurons. PMID:25881894

  1. Stem cell engineered bone with calcium-phosphate coated porous titanium scaffold or silicon hydroxyapatite granules for revision total joint arthroplasty.

    PubMed

    García-Gareta, Elena; Hua, Jia; Rayan, Faizal; Blunn, Gordon W

    2014-06-01

    Aseptic loosening in total joint replacements (TJRs) is mainly caused by osteolysis which leads to a reduction of the bone stock necessary for implant fixation in revision TJRs. Our aim was to develop bone tissue-engineered constructs based on scaffolds of clinical relevance in revision TJRs to reconstitute the bone stock at revision operations by using a perfusion bioreactor system (PBRS). The hypothesis was that a PBRS will enhance mesenchymal stem cells (MSCs) proliferation and osteogenic differentiation and will provide an even distribution of MSCs throughout the scaffolds when compared to static cultures. A PBRS was designed and implemented. Scaffolds, silicon substituted hydroxyapatite granules and calcium-phosphate coated porous TiAl6V4 cylinders, were seeded with MSCs and cultured either in static conditions or in the PBRS at 0.75 mL/min. Statistically significant increased cell proliferation and alkaline phosphatase activity was found in samples cultured in the PBRS. Histology revealed a more even cell distribution in the perfused constructs. SEM showed that cells arranged in sheets. Long cytoplasmic processes attached the cells to the scaffolds. We conclude that a novel tissue engineering approach to address the issue of poor bone stock at revision operations is feasible by using a PBRS. PMID:24519756

  2. Dopamine D1 and D5 Receptors Modulate Spike Timing-Dependent Plasticity at Medial Perforant Path to Dentate Granule Cell Synapses

    PubMed Central

    Yang, Kechun

    2014-01-01

    Although evidence suggests that DA modulates hippocampal function, the mechanisms underlying that dopaminergic modulation are largely unknown. Using perforated-patch electrophysiological techniques to maintain the intracellular milieu, we investigated how the activation of D1-type DA receptors regulates spike timing-dependent plasticity (STDP) of the medial perforant path (mPP) synapse onto dentate granule cells. When D1-type receptors were inhibited, a relatively mild STDP protocol induced LTP only within a very narrow timing window between presynaptic stimulation and postsynaptic response. The stimulus protocol produced timing-dependent LTP (tLTP) only when the presynaptic stimulation was followed 30 ms later by depolarization-induced postsynaptic action potentials. That is, the time between presynaptic stimulation and postsynaptic response was 30 ms (?t = +30 ms). When D1-type receptors were activated, however, the same mild STDP protocol induced tLTP over a much broader timing window: tLTP was induced when ?30 ms ? ?t ? +30 ms. The result indicated that D1-type receptor activation enabled synaptic potentiation even when postsynaptic activity preceded presynaptic stimulation within this ?t range. Results with null mice lacking the Kv4.2 potassium channel and with the potassium channel inhibitor, 4-aminopyridine, suggested that D1-type receptors enhanced tLTP induction by suppressing the transient IA-type K+ current. Results obtained with antagonists and DA receptor knock-out mice indicated that endogenous activity of both D1 and D5 receptors modulated plasticity in the mPP. The DA D5 receptors appeared particularly important in regulating plasticity of the mPP onto the dentate granule cells. PMID:25429131

  3. Dual control of Shuanghuang Shengbai granule on upstream and downstream signal modulators of CyclinD-CDK4/6 signaling pathway of cell cycle in Lewis-bearing mice with cyclophosphamide-induced myelosuppression

    PubMed Central

    Gu, Xian; Xu, Zhen-ye; Zhu, Ling-yu; Wang, Li-fang; Li, Kai; Pei, Qiang

    2013-01-01

    Background This study investigated the dual control mechanism of the Shuanghuang Shengbai granule in modulating the cell cycle in Lewis-bearing mice with cyclophosphamide induced myelosuppression. Methods Thirty Lewis-bearing mice were randomly grouped into an untreated group, control group, and treated group. Both treated and untreated groups were intraperitoneally injected with cyclophosphamide to produce a myelosuppression model. Mice in the treated group were fed with the Shuanghuang Shengbai granule (40 g/day) for 6 consecutive days. Standard blood tests and the count of bone marrow nuclear cells were performed, and the cell reproductive cycles of bone marrow and tumors were measured in these mice. In addition, the western blot approach was used to measure the upstream activating signals of CyclinD-CDK4/6 such as c-Myc and CDC25A, the upstream suppression signals such as p16INK4a, and the expression of downstream activated signals such as Rb, pRB, and E2F. All of the tested results were validated by reverse transcription quantitative real-time polymerase chain reaction. Results The results showed that the Shuanghuang Shengbai granule could elevate the count of leukocyte and bone marrow nuclear cells of Lewis-bearing mice with cyclophosphamide induced myelosuppression. It could also stimulate bone marrow cells to move from G0/G1 phases to S phase, accelerating the progress of the cell reproductive cycle and increasing the cell proliferation index. Simultaneously, the Shuanghuang Shengbai granule could also suppress cancer cells moving from G0/G1 phase to S phase, reducing the proliferation index. The tumor weight of Lewis-bearing mice in the treated group was much less than those of the control group. Expression levels of c-Myc, CDC25A, Rb, pRb, and E2F of bone marrow in Shuanghuang Shengbai granule-treated mice was higher compared to the control group, whereas they were lower in the cancer cells. Conclusion The experimental results demonstrate that the Shuanghuang Shengbai granule has dual control on the cell reproductive cycles in cancer cells and bone marrow nuclear cells in Lewis-bearing mice. PMID:23569384

  4. Secretory pathway of cellulase: a mini-review

    PubMed Central

    2013-01-01

    Cellulase plays an important role in modern industry and holds great potential in biofuel production. Many different types of organisms produce cellulase, which go through secretory pathways to reach the extracellular space, where enzymatic reactions take place. Secretory pathways in various cells have been the focus of many research fields; however, there are few studies on secretory pathways of cellulases in the literature. It is therefore necessary and important to review the current knowledge on the secretory pathways of cellulases. In this mini-review, we address the subcellular locations of cellulases in different organisms, discuss the secretory pathways of cellulases in different organisms, and examine the secretory mechanisms of cellulases. These sections start with a description of general secreted proteins, advance to the situation of cellulases, and end with the knowledge of cellulases, as documented in UniProt Knowledgebase (UniProtKB). Finally, gaps in existing knowledge are highlighted, which may shed light on future studies for biofuel engineering. PMID:24295495

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

    Microsoft Academic Search

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

    2002-01-01

    In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron

  6. Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine

    PubMed Central

    1980-01-01

    The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen. PMID:7391135

  7. Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events

    Microsoft Academic Search

    J. K. Angleson; A. J. Cochilla; G. Kilic; I. Nussinovitch; W. J. Betz

    1999-01-01

    Using FM1-43 fluorescence, we have optically detected single exocytic and endocytic events in rat pituitary lactotrophs. About fifty discrete fluorescent spots abruptly appear around the entire surface of a cell bathed in FM1-43 and high-potassium saline. The spots, which also immunostain for prolactin, reflect the labeling of dense cores as well as membranes of exocytosed secretory granules. Stained cores are

  8. The prolactin cell of the white-crowned sparrow, Zonotrichia leucophrys pugetensis

    Microsoft Academic Search

    Shin-ichi Mikami; Donald S. Farner; Robert A. Lewis

    1973-01-01

    The anterior pituitaries from a series of female White-crowned Sparrows,Zonotrichia leucophrys pugetensis, in the periods of oviposition, incubation, and brooding under natural conditions, have been investigated by electron microscopy. The prolactin cells occur in cephalic lobe and are characterized by large (ca. 300–600 mµ), polymorphic electron-dense secretory granules and an extremely well developed, lamellated endoplasmic reticulum. During incubation and brooding

  9. Circadian Proteins CLOCK and BMAL1 in the Chromatoid Body, a RNA Processing Granule of Male Germ Cells

    E-print Network

    Peruquetti, Rita L; de Mateo, Sara; Sassone-Corsi, Paolo; Tora, Laszlo

    2012-01-01

    cells seem to play important roles during the gametogenesis process [cells, is characterized by a number of unique and remarkable features. This processprocess that involves genetic and epigenetic regulation, sophisticated hormonal control, and extensive structural changes in male germ cells.

  10. Formation of volutin granules in Corynebacterium glutamicum.

    PubMed

    Pallerla, Srinivas Reddy; Knebel, Sandra; Polen, Tino; Klauth, Peter; Hollender, Juliane; Wendisch, Volker F; Schoberth, Siegfried M

    2005-02-01

    Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted. PMID:15668011

  11. Developmental changes in gangliosides in cultured cerebellar granule neurons

    Microsoft Academic Search

    Wipawan Thangnipon; Robert Balázs

    1992-01-01

    The content and composition of gangliosides in cultures enriched in granule neurones and in astrocytes from rat cerebellum (P6–8) showed marked differences; astrocytes contained less than 10% of the amount of granule neurones and the profile was dominated by simple gangliosides with lactosyl ceramide backbone, while gangliosides of the ‘b’ series, which constitute about 40% in nerve cells, were virtually

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

    PubMed Central

    2012-01-01

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

  13. Fasciola hepatica excretory-secretory products induce CD4+T cell anergy via selective up-regulation of PD-L2 expression on macrophages in a Dectin-1 dependent way.

    PubMed

    Guasconi, Lorena; Chiapello, Laura S; Masih, Diana T

    2015-07-01

    Fasciola hepatica excretory-secretory products (FhESP) induce immunomodulatory effects on macrophages. Previously, we demonstrated that these effects are dependent on Dectin-1. Therefore, the aim of this study was to determine how this affects the CD4 T-cells immune response. We observed that FhESP induce an increased expression of PD-L2 in macrophages via Dectin-1. Furthermore, in co-cultures with CD4 T-cell we observed a suppressive effect on proliferative response, down-modulation of IFN-? and up-modulation of IL-10 via Dectin-1 on macrophages. These results suggest that FhESP induce T-cell anergy via selective up-regulation of PD-L2 expression on macrophages in a Dectin-1 dependent way. PMID:25758714

  14. Protection of NMDA-induced neuronal cell damage by methanol extract of Zizyphi Spinosi Semen in cultured rat cerebellar granule cells

    Microsoft Academic Search

    Jeong Hee Park; Hyun Joo Lee; Sang Bum Koh; Ju Yeon Ban; Yeon Hee Seong

    2004-01-01

    Zizypus is one of the herbs widely used in Korea and China due to the CNS calming effect. The present study aims to investigate the effect of the methanol extract of Zizyphi Spinosi Semen (ZSS), the seeds of Zizyphus jujuba Mill var. spinosa, on N-methyl-d-aspartate (NMDA)-induced neurotoxicity in cultured rat cerebellar granule neuron. ZSS, over a concentration range of 0.05–5?g\\/ml,

  15. Decreased cytogenesis in the granule cell layer of the hippocampus and impaired place learning after irradiation of the young mouse brain evaluated using the IntelliCage platform.

    PubMed

    Barlind, Anna; Karlsson, Niklas; Björk-Eriksson, Thomas; Isgaard, Jörgen; Blomgren, Klas

    2010-04-01

    Radiation therapy is used to treat malignant tumors in the brain and central nervous system involvement of leukemia and lymphomas in children. However, ionizing radiation causes a number of adverse long-term side effects in the brain, including cognitive impairment. Hippocampal neurogenesis is important for place learning and has been shown to be decreased by irradiation (IR) in rats and mice. In the present study, 10-day-old male mice received 6-Gy IR to the brain on postnatal day 10. We used BrdU labeling of the granule cell layer (GCL) of the hippocampus to evaluate cell proliferation and survival. An unbiased, automated platform for monitoring of behavior in a group housing environment (IntelliCage) was used to evaluate place learning 2 months after IR. We show that cranial IR impaired place learning and reduced BrdU labeling by 50% in the GCL. Cranial IR also reduced whole body weight gain 5%. We conclude that this experimental paradigm provides a novel and time-saving model to detect differences in place learning in mice subjected to IR. This method of detecting behavioral differences can be used for further studies of adverse effects of IR on hippocampal neurogenesis and possible new strategies to ameliorate the negative effects of IR on cognition. PMID:19943037

  16. [Effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expression of membrane transport proteins in K562/A02 cell xenografts].

    PubMed

    Li, Dong-Yun; Zheng, Zhi; Hou, Li; Jiang, Miao; Dong, Qing; Tian, Shao-Dan; Ma, Wei; Chen, Ju; Wang, Jing; Chen, Xin-Yi

    2010-02-01

    This study was purposed to investigate the effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expressions of P-gp, MRP, LRP in K562/A02 cell xenografts. Tumor xenograft model were established by injecting the multidrug resistant cell line K562/A02 in the axillary flank of BALB/c-nu-nu mice. CZBG-intragastric administration and doxorubicin-intraperitoneal injection in combination were given to the BALB/c-nu nude mice. The tumor xenografts were made into slice after the dissection, and the expression of P-gp, MRP, LRP in K562/A02 tumor xenografts in mice were investigated by immunohistochemical technique. The integral optical density (IOD) of P-gp, MRP, LRP in K562/A02 tumor xenografts were measured by Image Pro Plus 6.0. The results showed that as compared with the doxorubicin alone, the combination of the doxorubicin and CZBG with high, middle and low dosage could decrease IOD of P-gp, MRP in K562/A02 tumor xenografts with statistical significance (p < 0.05). The LRP expression in K562/A02 tumor xenografts was not observed in five groups. It is concluded that the combination of CZBG with doxorubicin decreases the expressions of P-gp, MRP in K562/A02 tumor xenografts of mice. PMID:20137116

  17. Video-microscopy for analysis of molecular dynamics in cells.

    PubMed

    Ozawa, K; Tamura, A; Ikeda, K; Kawai, E; Kondo, T; Fukano, Y; Nomura, S; Ishihara, Y; Masujima, T

    1997-06-01

    Real-time analysis of molecular dynamics in living cells was studied by developed video-microscopes. Two new detective methods were reported, one is for analysis of ciliary movement and the other is the qualitative analysis of exocytosis of insulin-containing granules with a video-enhanced light/fluorescent microscope. For analysis of ciliary movement, glass beads were migrated in the flow. The migration speed parallel to the flow produced by ciliary beating was used as an index of the beating activity. When tracheal epithelium isolated from mouse was incubated with ambroxol, and expectorant known to activate ciliary beat frequency, the floating speeds of glass beads were changed with 1 min of incubation. The results suggest that the present method is useful not only for screening of expectorants but also for the study of molecular mechanisms underlying ciliary beat of tracheal epithelium. Visualization of the moment of the release of contents from insulin-containing granules was achieved using video-enhanced fluorescent microscopy in MIN6 cells of mouse insulinoma cell line. A fluorescent amino acridine dye, quinacrine, was found to be incorporated into low-pH secretory granules, including insulin, in the cells. The granules which incorporated quinacrine emitted a slightly blue-green fluorescence. Upon stimulation with glucose, release of the quinacrine fluorescence from granules were observed. The present method would be useful for quantitative analysis of secretion of insulin from MIN6 cells as well as pancreatic beta-cells. PMID:9226579

  18. Mammary analogue secretory carcinoma (MASC) of salivary gland in four Mexican patients.

    PubMed

    Serrano-Arévalo, Mónica L; Mosqueda-Taylor, Adalberto; Domínguez-Malagón, Hugo; Michal, Michal

    2015-01-01

    The Clinco-pathological, immunohistochemical and molecular findings of four cases of Mammary Analogue Secretory Carcinoma (MASC) of salivary glands found in Mexico are described. The cases were extracted from 253 salivary gland tumors from a single institution in Mexico City. The 85 Candidates for initial selection were: low grade mucoepidermoid carcinoma (MEC) (N=70 ), Acinic cell cancinoma (AciCC) (N=14), papillary cystadenocarcinoma (N=1), and adenocarcinoma NOS (N=0). Tumors with some histological features consistent with MASC (N= 17, 6.7%) were studied by immunohistochemistry for mammaglobin, STAT5, and S-100 protein and four cases were positive (1.5%), thus the diagnosis of MASC was established, and these were submitted for molecular studies for ETV6-NTRK3. Fusion gene was demonstrated in three cases, two had been erroneously diagnosed as poorly granulated AciCC, and one as low grade MEC with microcystic pattern. Female gender predominated (3:1); one occurred in the parotid, two in minor salivary glands and one in the submaxillary gland; infiltrating borders, atypical mitosis and lymph node metastases were seen in the parotideal tumor. Two patients with major salivary gland tumors are alive and well at 10 and 20 months respectively, the two patients with minor salivary gland tumors are lost. It can be concluded that is important to think in MASC in poorly granulated AciCC and low grade MEC with microcystic pattern. Immunohistochemisty studies confirm the diagnosis, preferentially supported by molecular studies. MASC may follow aggressive behavior or transform into a high grade neoplasm. PMID:25481229

  19. Investigations of pigment granule transport systems in Gonodactylus oerstedii (Crustacea: Hoplocarida: Stomatopoda)

    Microsoft Academic Search

    T. W. Cronin

    1994-01-01

    Compound eyes of the stomatopod, Gonodactylus oerstedii, exhibit pupillary reflection responses which arise from migration of retinular cell pigment granules. In the light, reflectance from the eye increases as pigment granules accumulate around light-sensitive rhabdoms and scatter incoming light back out of the eye (pupillary closure). At dark onset, reflectance diminishes as pigment granules disperse centrifugally, enhancing photon capture by

  20. Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic beta-cells.

    PubMed

    Olofsson, Charlotta S; Salehi, Albert; Holm, Cecilia; Rorsman, Patrik

    2004-06-15

    We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic beta-cells by a combination of electrophysiological recordings, intracellular Ca(2+) ([Ca(2+)](i)) microfluorimetry and insulin release measurements. Addition of palmitate (1 mm, bound to fatty acid-free albumin) to intact islets exposed to 15 mm glucose increased the [Ca(2+)](i) by approximately 30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca(2+)](i) and insulin release in the presence of tolbutamide and in islets depolarized by high K(+) in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K(+) channels (K(ATP) channels). Palmitate (0.5 mm) augmented exocytosis (measured as an increase in cell capacitance) in single beta-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca(2+) currents rose by approximately 25% following addition of 0.5 mm palmitate, an effect that was abolished in the presence of 10 microm isradipine indicating that the free fatty acid specifically acts on L-type Ca(2+) channels. The actions of palmitate on exocytosis and Ca(2+) currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K(ATP) channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca(2+) currents and an increased size of the RRP. PMID:15090611

  1. Autofluorescence of Plant Secreting Cells as a Biosensor and Bioindicator Reaction

    Microsoft Academic Search

    Victoria V. Roshchina

    2003-01-01

    Autofluorescence of intact secretory cell of plants is considered as a possible biosensor for the cellular biomonitoring. The fluorescence of secretory cells was due to the chemical composition of their secretions. This phenomenon could be recommended: 1. for the diagnostics of secretory cells among non-secretory ones; 2. for the express-analysis of the content of secretory cells at norm and under

  2. Nonreutilizaton of adrenal chromaffin granule membranes following secretion

    SciTech Connect

    Nobiletti, J.B.

    1985-01-01

    The intracellular postexocytotic fate of the adrenal chromaffin granule membrane (reutilization vs. nonreutilization) was addressed through two experimental approaches. First, (/sup 3/H) leucine pulse-chase labeling experiments were conducted in two systems - the isolated retrograde perfused cat adrenal gland and cultured bovine adrenal chromaffin cells to compare chromaffin granule soluble dopamine-B-hydroxylase (DBH) turnover (marker for granule soluble content turnover) to that of membrane-bound DBH (marker for granule membrane turnover). Experiments in cat adrenal glands showed that at all chase periods the granule distribution of radiolabeled DBH was in agreement with the DBH activity distribution (73% membrane-bound/27% soluble) - a result consistent with parallel turnover of soluble and membrane-bound DBH. Experiments in cultured bovine cells showed that labeled soluble and membrane-bound DBH had parallel turnover patterns and at all chase period, the distribution of radiolabeled DBH between the soluble contents and membranes was similar to the DBH activity distribution (50% soluble/50% membrane-bound). The above experiments showed that the soluble contents and membranes turnover in parallel and are consistent with nonreutilization of chromaffin granule membranes following exocytosis. Isolated retrograde perfused bovine adrenal glands were subjected to repetitive acetylcholine stimulation to induce exocytosis and then the dense and less-dense chromaffin granule fractions were isolated. Since both approaches gave results consistent with membrane nonreutilization, the authors conclude that once a chromaffin granule is involved in exocytosis, its membrane is not reutilized for the further synthesis, storage, and secretion of catecholamines.

  3. Germ-granule components prevent somatic development in the C. elegans germline

    PubMed Central

    Knutson, Andrew Kek?pa'a; Egelhofer, Thea A.; Campbell, Anne C.; Strome, Susan

    2014-01-01

    Summary Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans [1]. In Drosophila, germ granules have been implicated in germline determination [2]. C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination [3], but their components are still needed for fertility [4–6]. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g. in germlines lacking MEX-3 and GLD-1 or upon neuronal induction by CHE-1 [7, 8]. However, it has not been established whether loss of P granules is the cause or effect of cell-fate transformation. To test cause-effect, we severely compromised P granules by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization (GLH-1 and GLH-4) [9], and investigated if that causes germ cells to lose totipotency and initiate somatic reprogramming. We found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate. PMID:24746798

  4. Endogenous Bax Translocation in SH-SY5Y Human Neuroblastoma Cells and Cerebellar Granule Neurons Undergoing Apoptosis

    Microsoft Academic Search

    Kim M. McGinnis; Margaret E. Gnegy; Kevin K. W. Wang

    2008-01-01

    Abstract: Changes at the mitochondria are an early, re- quired step in apoptosis,in various cell types. We used western,blot analysis to demonstrate,that the proapop- totic protein Bax translocated,from,the cytosolic to the mitochondrial,fraction in SH-SY5Y human,neuroblas- toma,cells undergoing,staurosporine- or EGTA-mediated apoptosis. Levels of mitochondrial,Bax increased,15 min after staurosporine treatment. In EGTA-treated cells, in- creased levels of mitochondrial Bax were seen at

  5. Top-down inputs from the olfactory cortex in the postprandial period promote elimination of granule cells in the olfactory bulb.

    PubMed

    Komano-Inoue, Sayaka; Manabe, Hiroyuki; Ota, Mizuho; Kusumoto-Yoshida, Ikue; Yokoyama, Takeshi K; Mori, Kensaku; Yamaguchi, Masahiro

    2014-09-01

    Elimination of granule cells (GCs) in the olfactory bulb (OB) is not a continual event but is promoted during a short time window in the postprandial period, typically with postprandial sleep. However, the neuronal mechanisms for the enhanced GC elimination during the postprandial period are not understood. Here, we addressed the question of whether top-down inputs of centrifugal axons from the olfactory cortex (OC) during the postprandial period are involved in the enhanced GC elimination in the OB. Electrical stimulation of centrifugal axons from the OC of anesthetized mice increased GC apoptosis. Furthermore, pharmacological suppression of top-down inputs from the OC to the OB during the postprandial period of freely behaving mice by ?-aminobutyric acid (GABA)A receptor agonist injection in the OC significantly decreased GC apoptosis. Remarkable apoptotic GC elimination in the sensory-deprived OB was also suppressed by pharmacological blockade of top-down inputs. These results indicate that top-down inputs from the OC to the OB during the postprandial period are the crucial signal promoting GC elimination, and suggest that the life and death decision of GCs in the OB is determined by the interplay between bottom-up sensory inputs from the external world and top-down inputs from the OC. PMID:25041475

  6. Mice deficient in carbonic anhydrase type 8 exhibit motor dysfunctions and abnormal calcium dynamics in the somatic region of cerebellar granule cells.

    PubMed

    Lamont, Matthew G; Weber, John T

    2015-06-01

    The waddles (wdl) mouse is characterized by a namesake "side-to-side" waddling gait due to a homozygous mutation of the Car8 gene. This mutation results in non-functional copies of the protein carbonic anhydrase type 8. Rota-rod testing was conducted to characterize the wdl mutations' effect on motor output. Results indicated that younger homozygotes outperformed their older cohorts, an effect not seen in previous studies. Heterozygotes, which were thought to be free of motor impairment, displayed motor learning deficiencies when compared with wild type performance. Acute cerebellar slices were then utilized for fluorescent calcium imaging experiments, which revealed significant alterations in cerebellar granule cell somatic calcium signaling when exposed to glutamate. The contribution of GABAergic signaling to these alterations was also verified using bath application of bicuculline. Changes in somatic calcium signals were found to be applicable to an in vivo scenario by comparing group responses to electrical stimulation of afferent mossy fiber projections. Finally, intracellular calcium store function was also found to be altered by the wdl mutation when slices were treated with thapsigargin. These findings, taken together with previous work on the wdl mouse, indicate a widespread disruption in cerebellar circuitry hampering proper neuronal communication. PMID:25721739

  7. NrCAM, cerebellar granule cell receptor for the neuronal adhesion molecule F3, displays an actin-dependent mobility in growth cones.

    PubMed

    Faivre-Sarrailh, C; Falk, J; Pollerberg, E; Schachner, M; Rougon, G

    1999-09-01

    The neuronal adhesion glycoprotein F3 is a multifunctional molecule of the immunoglobulin superfamily that displays heterophilic binding activities. In the present study, NrCAM was identified as the functional receptor mediating the inhibitory effect of F3 on axonal elongation from cerebellar granule cells. F3Fc-conjugated microspheres binding to neuronal growth cones resulted from heterophilic interaction with NrCAM but not with L1. Time-lapse video-microscopy indicated that F3Fc beads bind at the leading edge and move retrogradely to reach the base of the growth cone within a lapse of 30-60 seconds. Such velocity (5.7 microm/minute) is consistent with a coupling between F3 receptors and the retrograde flow of actin filaments. When actin filaments were disrupted by cytochalasin B, the F3Fc beads remained immobile at the leading edge. The retrograde mobility appeared to be dependent on NrCAM clustering since it was induced upon binding with cross-linked but not dimeric F3Fc chimera. These data indicate that F3 may control growth cone motility by modulating the linkage of its receptor, NrCAM, to the cytoskeleton. They provide further insights into the mechanisms by which GPI-anchored adhesion molecules may exert an inhibitory effect on axonal elongation. PMID:10462518

  8. Kaempferol blocks oxidative stress in cerebellar granule cells and reveals a key role for reactive oxygen species production at the plasma membrane in the commitment to apoptosis.

    PubMed

    Samhan-Arias, Alejandro Khalil; Martín-Romero, Francisco Javier; Gutiérrez-Merino, Carlos

    2004-07-01

    Micromolar concentrations of the flavonoid kaempferol were found to efficiently block cerebellar granule cell (CGC) death through low K+-induced apoptosis, as demonstrated by prevention of the activation of caspase-3, internucleosomal DNA fragmentation, and chromatin condensation, without a significant rise in intracellular free Ca2+ concentration. Half of the maximum protection against CGC apoptosis was attained with 8 +/- 2 microM kaempferol. Reactive oxygen species (ROS) were monitored with 2',7'-dichlorodihydrofluorescein diacetate. Quantitative analysis of intracellularly and extracellularly oriented ROS production up to 3 h from the onset of low K+-induced CGC apoptosis was carried out with acquired digital fluorescence microscopy images of CGC in culture plates using a CCD camera, and also with fluorescence measurements of resuspended CGCs. In both cases, nearly 90% of ROS production by CGCs during the early stages (up to 3 h) after induction of low-K+ apoptosis occurs at the plasma membrane. Kaempferol, at concentrations that blocked CGC apoptosis, has been found to be a particularly potent blocker of extracellularly oriented ROS production by CGCs, and to inhibit the ascorbate-dependent NADH oxidase and superoxide anion production activities of the neuronal plasma membrane redox chain. PMID:15183194

  9. Cell Signaling and Neurotoxicity: 3H-Arachidonic acid release (Phospholipase A2) in cerebellar granule neurons

    EPA Science Inventory

    Cell signaling is a complex process which controls basic cellular activities and coordinates actions to maintain normal cellular homeostasis. Alterations in signaling processes have been associated with neurological diseases such as Alzheimer's and cerebellar ataxia, as well as, ...