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Ca increase in secretory granules of stimulated mast cells  

Microsoft Academic Search

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray microanalysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous

S. Raison; I. Gillot; C. Choine; P. Pistone; S. Pagnotta; J.-P. Laugier; G. Nicaise



The Use of Permeabilized Cells to Investigate Secretory Granule Biogenesis  

Microsoft Academic Search

To investigate the mechanism of secretory granule biogenesis in endocrine cells, our laboratory used rat anterior pituitary GH3 cells which secrete growth hormone and prolactin. Here we describe a simple and rapid procedure for generating permeabilized cells to dissect molecular mechanisms involved in nascent secretory vesicle budding from the trans-Golgi network (TGN). Using this system, we demonstrate that vesicle budding

Wai Lam W. Ling; Anirban Siddhanta; Dennis Shields



Phospholipid Storage in the Secretory Granule of the Mast Cell,  

National Technical Information Service (NTIS)

A spontaneous membrane assembly process has been postulated to account for the rapid perigranular membrane enlargement which occurs during mast cell secretory granule activation. This process requires the presence of a phospholipid store in the quiescent ...

S. P. Chock E. A. Schumauder-Chock



Ultrastructural Similarity between Bat and Human Mast Cell Secretory Granules  

Microsoft Academic Search

Mast cells in the tongue of the bat (Artibeus lituratus) show a well-developed Golgi area and abundant mitochondria in the granule-free perinuclear cytoplasm. Rough endoplasmic reticulum profiles, free ribosomes, mitochondria, bundles of filaments and a great number of secretory granules are found throughout the remaining cytoplasm. The granules, of various shapes and sizes, are simple containing an electron-dense, homogeneous matrix,

Sônia Maria Oliani; Ithamar Vugman; Maria Célia Jamur



Maturation of Secretory Granules  

Microsoft Academic Search

\\u000a Exocrine, endocrine, and neuroendocrine cells store hormones and neuropeptides in secretory granules (SGs), which undergo\\u000a regulated exocytosis in response to an appropriate stimulus. These cargo proteins are sorted at the trans-Golgi network into forming immature secretory granules (ISGs). ISGs undergo maturation while they are transported to and\\u000a within the F-actin-rich cortex. This process includes homotypic fusion of ISGs, acidification of

Tanja Kögel; Hans-Hermann Gerdes


RNA Is Closely Associated with Human Mast Cell Secretory Granules, Suggesting a Role(s) for Granules in Synthetic Processes  

Microsoft Academic Search

The distribution of ribosomes in mature human mast cells, a major granulated secretory cell, does not resemble that in other secretory cells, such as pancreatic acinar cells and plasma cells. By routine ultrastructural analysis, ribosomes in human mast cells are often close to, attached to, or even appear to be within secretory granules. To document better these relationships, we used

Ann M. Dvorak; Ellen S. Morgan; Lawrence M. Lichtenstein; Peter F. Weller; Robert P. Schleimer



Polyamines Are Present in Mast Cell Secretory Granules and Are Important for Granule Homeostasis  

Microsoft Academic Search

BackgroundMast cell secretory granules accommodate a large number of components, many of which interact with highly sulfated serglycin proteoglycan (PG) present within the granules. Polyamines (putrescine, spermidine and spermine) are absolutely required for the survival of the vast majority of living cells. Given the reported ability of polyamines to interact with PGs, we investigated the possibility that polyamines may be

Gianni García-Faroldi; Carlos E. Rodríguez; José L. Urdiales; José M. Pérez-Pomares; José C. Dávila; Gunnar Pejler; Francisca Sánchez-Jiménez; Ignacio Fajardo; David Holowka



The Formation of the Secretory Granules in the Liver-cells of the Slug, Anadenus altivagus  

Microsoft Academic Search

SUMMARY The process of formation of secretory granules has been studied in the liver-cells of the slug, Anadenus altivagus, in fixed preparations. The following conclusions have been drawn. 1. The secretory granules, which are non-lipid in nature, are formed by direct transformation of the lipid granules. The lipid granules assume a duplex structure during this transformation. 2. The lipid granules,




Comparison of Secretory Protein and Membrane Composition of Secretory Granules Isolated from Normal and Neoplastic Pancreatic Acinar Cells of Rats  

Microsoft Academic Search

The diversity of cytodifferentiation in a transplantable rat pancreatic acinar carcinoma provides a biological model system for the study of regulatory molecular events that differ from those in normal acinar cells. Secretory (zymogen) granule proteins and granule membranes of neoplastic and normal pancreatic acinar cells were compared to determine the differences in gene expression between apparently well-differentiated secretory granule-containing neoplastic

Linnea J. Hansen; M. Kumudavalli Reddy; Janardan K. Reddy



Mitochondrion-secretory granules complexes in pancreatic islet B-cells.  


Mitochondrion-Secretory Granule Complexes (MSGC) are present in rodent pancreatic islet B-cells, characteristically showing fusion of mitochondria and secretory granules, absence of granule membrane and external mitochondrial membrane at place of contact, often invagination of internal mitochondrial membrane at place of contact forming an electron lucent space directly continuous with the "halo" of the secretory granules, and occurrence of potassium pyroantimonate precipitates in either or both of mitochondria and secretory granules. The MSGC are believed to possess functional significance, possibly playing a role in translocation of ions between mitochondria and secretory granules in the B-cells. PMID:382910

Boquist, L



Mast cells possess distinct secretory granule subsets whose exocytosis is regulated by different SNARE isoforms  

Microsoft Academic Search

Mast cells degranulate and release the contents of intracellular secretory granules in response to the cross-linking of Fc?RI by multivalent antigens. These granules contain a variety of biologically active inflammatory mediators; however, it is not clear whether these granules are homogenous or whether there is heterogeneity within the secretory granule population in mast cells. By using genetically altered mice lacking

Niti Puri; Paul A. Roche



Calcium dynamics in the secretory granules of neuroendocrine cells.  


Cellular Ca(2+)signaling results from a complex interplay among a variety of Ca(2+) fluxes going across the plasma membrane and across the membranes of several organelles, together with the buffering effect of large numbers of Ca(2+)-binding sites distributed along the cell architecture. Endoplasmic and sarcoplasmic reticulum, mitochondria and even nucleus have all been involved in cellular Ca(2+) signaling, and the mechanisms for Ca(2+) uptake and release from these organelles are well known. In neuroendocrine cells, the secretory granules also constitute a very important Ca(2+)-storing organelle, and the possible role of the stored Ca(2+) as a trigger for secretion has attracted considerable attention. However, this possibility is frequently overlooked, and the main reason for that is that there is still considerable uncertainty on the main questions related with granular Ca(2+) dynamics, e.g., the free granular [Ca(2+)], the physical state of the stored Ca(2+) or the mechanisms for Ca(2+) accumulation and release from the granules. This review will give a critical overview of the present state of knowledge and the main conflicting points on secretory granule Ca(2+) homeostasis in neuroendocrine cells. PMID:22209697

Alvarez, Javier



Two-dimensional electrophoretic analysis of secretory-granule, granule-membrane, and plasma-membrane proteins of rat parotid cells  

Microsoft Academic Search

Secretory granules and plasma membranes were isolated from rat parotid cells and characterized enzymatically and by electron microscopy. The proteins of the secretory granule membranes, the secretory granules and the plasma membranes were characterized by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining. The granule membrane contains 166 polypeptides of which only 26 are also present in the granule

Margaret A. Cascieri; Ethel W. Somberg



Formation of secretory granules by chromogranins.  


This review article covers the molecular mechanisms of secretory granule formation by chromogranin transfection. Recently, a few investigators have reported that the transfection of chromogranin A and B produces the structures of secretory granules. We used the GFP-chromogranin A transfection method to nonendocrine cells, COS-7 cells, which are not equipped with secretory granules. Despite the absence of endogenous secretory granules in nontransfected COS-7 cells, COS-7 cells transfected with chromogranin A contained granule-like structures in electron micrographs. The granules were composed of an outer limiting membrane with core structures that were interpreted as secretory granules. Human chromogranin A (CgA) labeled with 5-nm gold particles was present in several dense-core granules in our previous electron microscopy study. This review depicts the role of chromogranin A in the formation of secretory granules. It emphasizes the application of recently developed new technologies and the genesis of secretory granules. PMID:20033364

Inomoto, Chie; Osamura, Robert Yoshiyuki



Final Steps in Exocytosis Observed in a Cell with Giant Secretory Granules  

Microsoft Academic Search

Secretion by single mast cells was studied in normal and beige mice, a mutant with grossly enlarged secretory vesicles or granules. During degranulation, the membrane capacitance increased in steps, as single secretory vesicles fused with the cell membrane. The average step size was 10 times larger in beige than in normal mice, in agreement with the different granule sizes measured

L. J. Breckenridge; W. Almers



Immunocytochemical localization of secretory component in Paneth cell secretory granules-rat Paneth cells participate in acquired immunity  

Microsoft Academic Search

Summary  With the marker of Paneth cells-lysozyme, secretory component (SC) immunoreactivity was demonstrated exclusively in Paneth\\u000a cells of rat small intestine. The other types of epithelial cells (columnar, goblet, endocrine) were negative. On electron\\u000a microscopic level, many SC-positive colloidal gold particles were found in rough endoplasmic reticulum, Golgi complexes, basal\\u000a membrane and secretory granules of Paneth cells. These results suggest that

Qing-Juan Tang; Kai-Zhong Tao; Yun-Liu; Xue-Jun Sun; Mei-Yu Geng; Chun-Lei Jiang



Formation of secretory granules by chromogranins  

Microsoft Academic Search

This review article covers the molecular mechanisms of secretory granule formation by chromogranin transfection. Recently,\\u000a a few investigators have reported that the transfection of chromogranin A and B produces the structures of secretory granules.\\u000a We used the GFP-chromogranin A transfection method to nonendocrine cells, COS-7 cells, which are not equipped with secretory\\u000a granules. Despite the absence of endogenous secretory granules

Chie Inomoto; Robert Yoshiyuki Osamura



Secretory granule biogenesis: rafting to the SNARE  

Microsoft Academic Search

Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and

Sharon A. Tooze; Gerard J. M. Martens; Wieland B. Huttner



Reduction with dithiothreitol causes serglycin-specific defects in secretory granule integrity of bone marrow derived mast cells  

Microsoft Academic Search

Mast cell granule maturation and storage of granule components has previously been shown to be critically dependent on serglycin (SG), a proteoglycan abundantly stored in mast cell secretory granules. The N-terminal portion of serglycin contains a conserved disulfide motif that is similar to motifs found in secretory granule compounds of neuroendocrine cells. Interference with such motifs of neuroendocrine cells with

Tiago Braga; Maria Ringvall; Heidi Tveit; Magnus Ĺbrink; Gunnar Pejler



Biogenesis of secretory granules in the trans-Golgi network of neuroendocrine and endocrine cells  

Microsoft Academic Search

Secretory granule formation requires selection of soluble and membrane proteins into nascent secretory granules, and exclusion of proteins not required for the function of secretory granules. Both selection and exclusion presumably can occur in the compartment where assembly of the secretory granule begins, the trans most cisternae of the Golgi complex. Current research focused on the initial stages of secretory

Sharon A Tooze



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

SciTech Connect

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.

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:; 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)



Accumulation of Major Histocompatibility Complex Class II Molecules in Mast Cell Secretory Granules and Their Release upon Degranulation  

Microsoft Academic Search

To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by

Graca Raposo; Danielle Tenza; Salahedine Mecheri; Roger Peronet; Christian Bonnerot; Catherine Desaymard



Antibacterial Peptides Are Present in Chromaffin Cell Secretory Granules  

Microsoft Academic Search

1. Antibacterial activity has recently been associated with the soluble matrix of bovine chromaffin granules. Furthermore, this activity was detected in the contents secreted from cultured chromaffin cells following stimulation.

Marie-Hélčne Metz-Boutigue; Yannick Goumon; Karine Lugardon; Jean-Marc Strub; Dominique Aunis



Transport, docking and exocytosis of single secretory granules in live chromaffin cells  

NASA Astrophysics Data System (ADS)

Neurons maintain a limited pool of synaptic vesicles which are docked at active zones and are awaiting exocytosis. By contrast, endocrine cells releasing large, dense-core secretory granules have no active zones, and there is disagreement about the size and even the existence of the docked pool. It is not known how, and how rapidly, secretory vesicles are replaced at exocytic sites in either neurons or endocrine cells. By using electron microscopy, we have now been able to identify a pool of docked granules in chromaffin cells that is selectively depleted when cells secrete. With evanescent-wave fluorescence microscopy, we observed single granules undergoing exocytosis and leaving behind patches of bare plasmalemma. Fresh granules travelled to the plasmalemma at a top speed of 114 nm s-1, taking an average of 6 min to arrive. On arrival, their motility diminished 4-fold, probably as a result of docking. Some granules detached and returned to the cytosol. We conclude that a large pool of docked granules turns over slowly, that granules move actively to their docking sites, that docking is reversible, and that the `rapidly releasable pool' measured electrophysiologically represents a small subset of docked granules.

Steyer, J. A.; Horstmann, H.; Almers, W.



Coexistence of renin and cathepsin B in epithelioid cell secretory granules  

Microsoft Academic Search

Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin- and cathepsin B-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin B is suggested to be involved in the activation of renin prior to secretion.

R. Taugner; C. P. Bührle; R. Nobiling; H. Kirschke



Relationship between the golgi apparatus, gerl, and secretory granules in acinar cells of the rat exorbital lacrimal gland  

Microsoft Academic Search

The method of secretory granule formation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemi- cal techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A.




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


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

Tandler, Bernard



Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells  

PubMed Central

In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C- peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus- dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.



Unique features of secretory granules observed in the pituitary growth hormone-secreting (GH) cells of the musk shrew ( Suncus murinus L.)  

Microsoft Academic Search

Summary Unique rod-shaped secretory granules were observed among oval or spherical secretory granules in GH cells of the anterior pituitary gland of musk shrew using the protein A-gold procedure combined with electron microscopy. The rod-shaped and spherical secretory granules were both immunoreactive by the immuno-gold method using antiserum to sheep GH. The rod-shaped secretory granules, which seem to be formed

Toshiko Ishibashi; Masataka Shiino



An histochemical approach to characterization of anionic constituents in mast cell secretory granules  

Microsoft Academic Search

We used cationized colloidal gold in order to investigate the distribution of anionic sites in different secretory granules of rat and mouse mast cells. The localization of the anionic sites was performed by post-embedding labeling of thin sections of rat peritoneal cells or mouse skin tissue, fixed in Karnovsky's fixative and OsO4 and embedded in Araldite or LR white, respectively.

Ehud Skutelsky; Tanya Shoichetman; Han Hammel



The carbohydrates of secretory granules and the glycocalyx in developing mucoid cells  

Microsoft Academic Search

Complex carbohydrates in secretory granules and at the apical cell surface of mouse gastric mucoid cells were studied during embryogenesis and in the early postnatal period by various cytochemical methods; the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) and tannic acid-uranyl acetate (TA-UA) procedures made neutral mucosubstances (NMS) visible, whereas the hexose residues of glycoconjugates were identified using WGA-, RCA II- and ConA-ferritin.

Nada Pipan; Majda Pšeni?nik



?-SNAP Functions in Insulin Exocytosis from Mature, but Not Immature Secretory Granules in Pancreatic ? Cells  

Microsoft Academic Search

To explore ?-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic ? cells, we studied the effects of overexpression of adenovirus-mediated wild-type ?-SNAP and C-terminally deleted ?-SNAP mutant (1–285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing ?-SNAP and mutant ?-SNAP were pulse-chased. Exocytosis from immature and

Yoko Nakamichi; Shinya Nagamatsu



Translocation of Arf1 to the Secretory Granules in Rat Parotid Acinar Cells  

Microsoft Academic Search

We investigated the interaction of ADP-ribosylation factor (Arf) with the secretory granules in rat parotid acinar cells. The 20.5-kDa small-molecular-mass GTP-binding protein in the cytosolic fraction of rat parotid acinar cells was identified as ADP-ribosylation factor1 by using a pan-Arf monoclonal antibody and isotype-specific polyclonal antibodies for Arf proteins 1, 3, 5, and 6. Incubation of the cytosolic fraction with

Yoko Dohke; Miki Hara-Yokoyama; Junko Fujita-Yoshigaki; Richard A. Kahn; Yasunori Kanaho; Sadamitsu Hashimoto; Hiroshi Sugiya; Shunsuke Furuyama



Identification of Aminopeptidase Activity in the Secretory Granules of Mouse Mast Cells  

Microsoft Academic Search

Sonicates of mouse bone marrow-derived mast cells (BMMC) differentiated in vitro and of mouse serosal mast cells differentiated in vivo contained small but approximately equal amounts of aminopeptidase activity, as determined by cleavage of leucine-beta-naphthylamide and resolution of the reaction products by reverse-phase high-performance liquid chromatography. Aminopeptidase activity was exocytosed from antigen-activated, IgE-sensitized BMMC in proportion to the secretory granule

William E. Serafin; Ursula A. Guidry; Elahe T. Dayton; Mika M. Kamada; Richard L. Stevens; K. Frank Austen



Synthesis of Prostaglandins and Eicosanoids by the Mast Cell Secretory Granule,  

National Technical Information Service (NTIS)

The identification of a non-bilayer phospholipid storage in the secretory granule and the linking of the eicosanoid production with the release of histamine have prompted us to examine whether the secretory granule may also serve as both the source as wel...

E. A. Schmauder-Chock S. P. Chock



Lytic granules, secretory lysosomes and disease.  


Lytic granules harbour many of the dangerous apoptosis-inducing molecules of the immune system, including perforin, granzymes and Fas ligand. Safe transport, storage and release of these lytic components is vital. As a secretory lysosome, the lytic granule is able to accomplish these roles, as well as conferring the lysosomal functions of cytotoxic T lymphocytes and natural killer cells. Secretory lysosomes are common to many other haemopoietic cells and also melanocytes. Many of the proteins used in lysosomal secretion are found in both melanocytes and hemopoietic cells, and are dysfunctional in genetic diseases with defects in these proteins. The genetically heterogeneous Hermansky-Pudlak syndrome represents an excellent model for revealing proteins involved in secretory lysosome functioning. However, studies of this disease reveal differences between the various different types of secretory lysosomes, including lytic granules. PMID:14499259

Clark, Richard; Griffiths, Gillian M



Pro-hormone secretogranin II regulates dense core secretory granule biogenesis in catecholaminergic cells.  


Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H(+)-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network. PMID:20061385

Courel, Maďté; Soler-Jover, Alex; Rodriguez-Flores, Juan L; Mahata, Sushil K; Elias, Salah; Montero-Hadjadje, Maďté; Anouar, Youssef; Giuly, Richard J; O'Connor, Daniel T; Taupenot, Laurent



Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic cells  

Microsoft Academic Search

Insulin secretion is controlled by the cell's metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and

Hervřr L. Olsen; Marianne Hřy; Wei Zhang; Alejandro M. Bertorello; Krister Bokvist; Kirsten Capito; Alexander M. Efanov; Björn Meister; Peter Thams; Shao-Nian Yang; Patrik Rorsman; Per-Olof Berggren; Jesper Gromada



Proteolytic Processing of Pro-opiomelanocortin Occurs in Acidifying Secretory Granules of AtT-20 Cells  

Microsoft Academic Search

Using antibodies specific for pro-opiomelanocortin (POMC), amidated joining peptide (JP), and the prohormone convertase PC1, we showed immunocytochemically that PC1 in a corticotrophic tumor cell line, AtT-20, was co-localized either with POMC or with amidated JP in secretory granules, and also confirmed that POMC was cleaved mainly in secretory granules. Analysis using DAMP (3- [2,4-dinitroanilino]-3'-amino-N-methyldipropylamine) as the pH probe suggested

Shigeyasu Tanaka; Takao Yora; Kazuhisa Nakayama; Kinji Inoue; Kazumasa Kurosumi



Biogenesis of Dense-Core Secretory Granules  

Microsoft Academic Search

\\u000a Dense core granules (DCGs) are vesicular organelles derived from outbound traffic through the eukaryotic secretory pathway.\\u000a As DCGs are formed, the secretory pathway can also give rise to other types of vesicles, such as those bound for endosomes,\\u000a lysosomes, and the cell surface. DCGs differ from these other vesicular carriers in both content and function, storing highly\\u000a concentrated cores’ of

Grant R. Bowman; Andrew T. Cowan; Aaron P. Turkewitz


Differential effect of flavonoids on inhibition of secretion and accumulation of secretory granules in rat basophilic leukemia cells  

Microsoft Academic Search

Rat basophilic leukemia (RBL) cells resemble mucosal mast cells (MMC) and develop few secretory granules under normal culture conditions. RBL cells have been used for the study of secretion and for the possible involvement of MMC in food allergies and irritable bowel syndrome (IBS). The flavonoid quercetin is one of very few molecules that inhibit RBL cell proliferation and constitutive

Michael Alexandrakis; Leena Singh; William Boucher; Richard Letourneau; Panagiotis Theofilopoulos; Theoharis C. Theoharides



Proteolytic processing of pro-opiomelanocortin occurs in acidifying secretory granules of AtT-20 cells.  


Using antibodies specific for pro-opiomelanocortin (POMC), amidated joining peptide (JP), and the prohormone convertase PC1, we showed immunocytochemically that PC1 in a corticotrophic tumor cell line, AtT-20, was co-localized either with POMC or with amidated JP in secretory granules, and also confirmed that POMC was cleaved mainly in secretory granules. Analysis using DAMP (3- [2,4-dinitroanilino]-3'-amino-N-methyldipropylamine) as the pH probe suggested a correlation between POMC processing and acidic pH in the secretory granules. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, completely inhibited POMC processing and caused constitutive secretion of the unprocessed precursor. By contrast, chloroquine, a weak base that is known to neutralize acidic organelles, was unable to inhibit POMC processing. Electron microscopic analysis revealed that, in AtT-20 cells treated with bafilomycin A1, the trans-Golgi cisternae were dilated and few secretory granules were present in the cytoplasm. These observations suggest that acidic pH provides a favorable environment for proteolytic processing of POMC by PC1 but is not required, and that integrity of the trans-Golgi network and sorting of POMC into secretory granules are important for POMC processing. PMID:9071324

Tanaka, S; Yora, T; Nakayama, K; Inoue, K; Kurosumi, K



Stereological study of endoplasmic reticulum, golgi complex and secretory granules in the B-cells of normal and alloxan-treated mice  

Microsoft Academic Search

Summary  Different B-cell organelles (lamellar and vesicular endoplasmic reticulum, Golgi complex, whole secretory granules and secretory\\u000a granule cores) were studied stereologically in pancreatic islets from control mice and mice killed 10 or 60 min following\\u000a alloxan injection. Ten min following alloxan a significant decrease was observed in the volume, surface and numerical densities\\u000a of whole secretory granules and their cores, and

Lennart Boquist; Ronny Lorentzon



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

Microsoft Academic Search

The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na+ absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown.

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



Dense-Core Secretory Granule Biogenesis  

NSDL National Science Digital Library

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.

Taeyoon Kim (National Institutes of Health Section on Cellular Neurobiology, National Institute of Child Health and Human Development); Marjorie C. Gondré-Lewis (National Institutes of Health Section on Cellular Neurobiology, National Institute of Child Health and Human Development); Irina Arnaoutova (National Institutes of Health Section on Cellular Neurobiology, National Institute of Child Health and Human Development); Y. Peng Loh (National Institutes of Health Section on Cellular Neurobiology, National Institute of Child Health and Human Development)



Binding of microtubules to pituitary secretory granules and secretory granule membranes  

Microsoft Academic Search

Microtubules assembled in vitro were bound to purified porcine pituitary secre- tory granules and to isolated granule membranes. The interaction between micro- tubules and whole secretory granules was demonstrated by alteration in the sedimentation properties of the microtubules. Incubation of secretory granules with mierotubules resulted in pelleting of microtubules which increased as a function of the number of granules added.




Immuno-electron-microscopic localization of enkephalin in the secretory granules of C cells in the chicken ultimobranchial glands  

Microsoft Academic Search

In the chicken, enkephalin-immunoreactive cells and nerve fibers are distributed in the ultimobranchial glands, which consist of C-cell groups and cyst structures. Ultrastructural features of the enkephalin cells and nerve fibers were examined by immuno-electron microscopy using both the streptavidin-biotin-peroxidase method and the protein A-colloidal gold method. Immunoreactivity for enkephalin was located on the secretory granules of C cells. In

Yoko Kameda; Chiho Hirota; Masakatsu Murakami



Secretory granules of pituitary adenomas: quantitative study of hormonal antigenicity  

Microsoft Academic Search

Ultrastructurally, the antigenicity of major pituitary hormones in secretory granules was quantitatively investigated in\\u000a five growth hormone (GH)-secreting adenomas, five prolactin (PRL)-secreting adenomas and eight clinically non-functioning\\u000a (CN-F) adenomas. Sparsely granulated cells with a few or several small secretory granules (60–100 nm) exhibiting little or\\u000a only weak antigenicity of various biochemically unrelated hormones were commonly observed in CN-F adenomas and

Hiroshi Kamitani; Hideaki Masuzawa; Itaru Kanazawa; Toshiro Kubo



Localization of DNA and RNA in Eosinophil Secretory Granules  

Microsoft Academic Search

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

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



Yeast aspartic protease 3 is sorted to secretory granules and activated to process proopiomelanocortin in PC12 cells.  


The subcellular localization and functionality of transfected yeast aspartic protease 3 (YAP3p) in a mammalian cell line were investigated. The complementary DNAs encoding the prohormone-processing enzyme (YAP3p) and a prohormone, bovine POMC, were cotransfected into PC12 (rat pheochromocytoma) cells. Immunocytochemical analysis of the cells using a YAP3p antibody showed a perinuclear punctate distribution of YAP3p in the cell body as well as immunostaining in the tips of the neurites. This pattern of immunostaining indicates localization of YAP3p in secretory granules. Analysis of the processing of POMC showed that in cells transfected with the POMC complementary DNA alone, only POMC was found, indicating a lack of processing of the prohormone. However, in cells coexpressing YAP3p, the POMC was completely processed to yield ACTH-(1-39) and ACTH-(1-14), consistent with the specificity of YAP3p found in vitro. Pulse-chase studies showed that POMC was processed after 20 min of chase, suggesting that processing occurred in the late Golgi network and continued in the secretory granules. Western blot analysis determined that YAP3p was secreted from the cells in a regulated manner. This study provides the first demonstration that a yeast prohormone-processing enzyme (YAP3p) of the aspartic protease class can be sorted correctly to secretory granules and activated to process a prohormone (POMC) in a highly efficient manner in mammalian cells. PMID:8940369

Cool, D R; Louie, D Y; Loh, Y P



Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane  

Microsoft Academic Search

Secretory granules of sheep thyroid parafol- licular cells contain serotonin, a serotonin-binding pro- tein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete seroto- nin when activated by thyrotropin (TSH) or elevated (Ca2÷)~. TSH also induced a rise in (Ca2÷)~. We studied the effect of these secretogogues on the pH difference (ApH) across the membranes of the

Jonathan Barasch; Michael D. Gershon; Eladio A. Nunez; Hadassah Tamir; Qais AI-Awqati



Ultrastructural localization of chromogranin: a potential marker for the electron microscopical recognition of endocrine cell secretory granules  

Microsoft Academic Search

Summary  Using a monoclonal antibody (LK2H10) directed against human chromogranin, we have been able to localize this soluble glycoprotein to the matrix of secretory granules from a wide variety of endocrine cells. In the gut, enterochromaffin, enteroglucagon, glucose-dependent insulinotropic peptide, gastrin, and neurotensin-containing cells exhibit chromogranin immunoreactivity. In our system, chromogranin-immunoreactive material was restricted to the halo of human pancreatic glucagon-containing

Ian M. Varndell; Ricardo V. Lloyd; Barry S. Wilson; Julia M. Polak



Secretory granule biogenesis and chromogranin A: master gene, on\\/off switch or assembly factor?  

Microsoft Academic Search

Secretory granules are found in specialized cell types, including endocrine cells, suggesting that a coordinated programme of gene expression is involved in their biogenesis. Indeed, it has been proposed that chromogranin A (CgA) acts as an on\\/off switch for secretory granule biogenesis. However, this proposed function is difficult to reconcile with the large body of evidence suggesting that secretory granules

Robert Day; Sven-Ulrik Gorr



Spiperone: evidence for uptake into secretory granules.  

PubMed Central

Spiperone, a dopamine antagonist widely used as a specific ligand for dopamine and serotonin receptors, is actively accumulated into the F4C1 strain of rat pituitary tumor cells. The accumulation of 10 nM [3H]spiperone was linear for 3 min and reached a steady state after 10 min. Spiperone accumulation was reduced 50% by preincubation with 5 microM reserpine, an inhibitor of biogenic amine transport into secretory granules, and was also blocked by monensin and ammonium chloride, both of which increase the pH of intracellular storage organelles. Uptake was not affected by replacing sodium in the buffer with lithium at equimolar concentrations. Spiperone at 1 microM inhibited by over 50% serotonin transport into membrane vesicles isolated from platelet dense granules; this concentration inhibited the Na+-dependent plasma membrane transport system less than 10%. The data indicate spiperone specifically interacts with the secretory granule amine transport system and suggest that this transport system is found in the F4C1 pituitary cell strain as well as in platelets and neurons. The data also suggest that experiments utilizing spiperone to measure dopamine and serotonin receptors be interpreted with caution.

Dannies, P S; Rudnick, M S; Fishkes, H; Rudnick, G



Muscle actin filaments bind pituitary secretory granules in vitro  

Microsoft Academic Search

Hog anterior pituitary secretory granules sediment at 3,000 g. When rat or rabbit skeletal muscle actin filaments are present with the granules, the sedimentation decreases markedly. Depolymerized actin or viscous solutions of Ficoll and collagen have no effect on granule sedimentation. With this assay, actin filaments bind secretory granules (consisting of the proteinaceous core plus limiting mem- brane), secretory granule




Golgi Apparatus and Origin of the Secretory Granules in Adenohypophyseal Cells in the Cells in the Rat. Autoradiographic Studies with the Electron Microscope after Injection of Tritated Leucine.  

National Technical Information Service (NTIS)

Upon injection of tritiated leucine, the autoradiographic response first appears in the ergastoplasm. Then the first labelled secretory granules are detected in the Golgi apparatus (30 min.). Finally, the labelled secretory granules are dispersed in the c...

J. Racadot L. Olivier E. Porcile B. Droz



A synthetic mimic of the secretory granule for drug delivery  

Microsoft Academic Search

Secretory cells contain submicroscopic granules composed of a polyanionic polymer network that is collapsed owing to the presence of hydronium ions and weak base cations. The network is encapsulated within a lipid membrane, and functions as a vehicle for the osmotically inert storage of a variety of granule-bound endogenous mediator species, such as histamine, serotonin and proteases. These species are

Patrick F. Kiser; Glynn Wilson; David Needham



[Identification and classification of lysozyme-expressing cells in the mouse small intestinal crypt and their correlation with the morphology of secretory granules and labeling density of immunogold].  


The four principal epithelial cell lineages (absorptive enterocytes, goblet cells, enteroendocrine cells and Paneth cells) of the adult mouse small intestine derive from multipotent stem cells. Furthermore, the intermediate cells and granule goblet cells are located near the base of crypts of mouse intestine; the former has the characteristics of goblet and Paneth cells and the latter is transformed from the intermediate cells. However, the grounds and the definition for classifing these three cell types (Paneth, intermediate and granule goblet cells) are vague, making it difficult to discuss the structure and a function of those cells. The purpose of this study was to investigate the identification and classification of lysozyme-expressing cells in the mouse small intestinal crypt and their correlation with the morphology of secretory granules and labeling density of immunogold using quantitative immunoelectron microscopy analysis. The results were follows. (1) Paneth cells, intermediate cells and granule goblet cells showed lysozyme immunoreactivity in the electron-dense core of biphasic secretory granules, and therefore lysozyme-exprssing cells were identified in the mouse small intestinal crypt. The sizes of secretory granules were divided into ten groups (every 10%) according to area ratio (core/granule (%)). (2) This distribution of three type cells was classified statistically into "Paneth cell phase": 61% < or = (core/granule (%)), "intermediate cell phase": (core/granule (%)) 21 < or = 60%, "granule goblet cell phase": (core/granule (%)) < or = 20%. (3) Labeling density for lysozyme was commensurate with the size of the central dense core. The Paneth cells had the highest labeling density among the cells. When the transformation from intermediate to granule goblet cell occurred, it happened at the same time that the core of secretory granules gradually shrinks, and the labeling density for lysozyme disappears. (4) The labeling density of immunogold for lysozyme in the small intestine varied at different sites. The labeling density in the Paneth and intermediate cells of the ileal crypt was lower than those of the duodeal and jejunal crypts. (5) In the lysozyme-expressing cells in small intestinal crypt of 2- and 24-month old mouse, the ultrastructure and labeling density did not change. PMID:19803390

Satot, Toru; Kawamoto, Eiichi; Yamada, Jinzo



Enhanced expression of transforming growth factor-ß1 in inflammatory cells and secretory granules in Paneth cells in the small intestine of mice infected with Toxocara canis  

Microsoft Academic Search

The small intestine is the initial organ which Toxocara canis larvae invade. Information on intestinal pathological changes associated with transforming growth factor-?1 (TGF-?1) and secretory granules (SG) in Paneth cells (PCs) caused by T. canis is unclear. Mice orally inoculated with 250 T. canis infective eggs were evaluated by pathological and immunohistochemical assessments with a 294-day investigation. Pathologically, the inflammatory

Chia-Kwung Fan; Chien-Ching Hung; Yun-Ho Lin; Ming-Hsien Li; Kua-Eyre Su



Secretory Granules of an Anterior Pituitary Cell Line, AtT-20, Contain Only Mature Forms of Corticotropin and beta -lipotropin  

Microsoft Academic Search

The pituitary cell line, AtT-20, synthesizes the precursor to corticotropin (adrenocorticotropic hormone; ACTH) and beta -endorphin and correctly glycosylates and cleaves it to make the mature forms of the hormones before they are secreted. This cell line was used to study the intracellular transport, packaging, and secretion of these hormones. Secretory granules from the cells were isolated by homogenization and

Barry Gumbiner; Regis B. Kelly



Inositol 1,4,5-Trisphosphate Receptor Subtype 3 in Pancreatic Islet Cell Secretory Granules Revisited  

Microsoft Academic Search

It has been reported that the inositol 1,4,5-trisphosphate receptor subtype 3 is expressed in islet cells and is localized to both insulin and somatostatin granules [Blondel, O., Moody, M. M., Depaoli, A. M., Sharp, A. H., Ross, C. A., Swift, H. & Bell, G. I. (1994) Proc. Natl. Acad. Sci. USA 91, 7777-7781]. This subcellular localization was based on electron

Mariella Ravazzola; Philippe A. Halban; Lelio Orci



Age-dependent labeling and imaging of insulin secretory granules.  


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

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



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


Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation. PMID:16630574

Fujita-Yoshigaki, Junko; Katsumata, Osamu; Matsuki, Miwako; Yoshigaki, Tomoyoshi; Furuyama, Shunsuke; Sugiya, Hiroshi



Chromogranin A, an “On\\/Off” Switch Controlling Dense-Core Secretory Granule Biogenesis  

Microsoft Academic Search

We present evidence that regulation of dense-core secretory granule biogenesis and hormone secretion in endocrine cells is dependent on chromogranin A (CGA). Downregulation of CGA expression in a neuroendocrine cell line, PC12, by antisense RNAs led to profound loss of dense-core secretory granules, impairment of regulated secretion of a transfected prohormone, and reduction of secretory granule proteins. Transfection of bovine

Taeyoon Kim; Jung-Hwa Tao-Cheng; Lee E. Eiden; Y. Peng Loh



Mouse Mast Cell Secretory Granules Can Function as Intracellular Ionic Oscillators  

Microsoft Academic Search

Fluorescent Ca2+ probes and digital photo-sectioning techniques were used to directly study the dynamics of Ca2+ in isolated mast cell granules of normal (CB\\/J) and beige (Bgj\\/Bgj) mice. The resting intraluminal free Ca2+ concentration ([Ca2+]L) is 25±4.2?M (mean±SD, n=68). Exposure to 3?M inositol 1,4,5-trisphosphate (InsP3) induced periodic oscillations of luminal Ca2+ ([Ca2+]L) of ?10?M amplitude and a period around 8–10s.

Ivan Quesada; Wei-Chun Chin; Jordan Steed; Patricia Campos-Bedolla; Pedro Verdugo




Microsoft Academic Search

A method has been devised for the isolation of a secretory granule fraction from isolated rat islets of Langerhans . The islets were homogenized in buffered sucrose, and the homoge- nate was separated into nuclear, mitochondrial, secretory granule, and microsomal fractions by differential centrifugation . The secretory granule fraction was purified by differential centrifugation in discontinuous sucrose density gradients .

S. L. Howell; C. J. FINK; P. E. LACY



Eosinophil granules function extracellularly as receptor-mediated secretory organelles  

PubMed Central

Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-?, and G protein–coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.

Neves, Josiane S.; Perez, Sandra A. C.; Spencer, Lisa A.; Melo, Rossana C. N.; Reynolds, Lauren; Ghiran, Ionita; Mahmudi-Azer, Salahaddin; Odemuyiwa, Solomon O.; Dvorak, Ann M.; Moqbel, Redwan; Weller, Peter F.



Signaling from the secretory granule to the nucleus.  


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

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



Secretory lysosomes  

Microsoft Academic Search

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.

Emma J. Blott; Gillian M. Griffiths



A synthetic mimic of the secretory granule for drug delivery.  


Secretory cells contain submicroscopic granules composed of a polyanionic polymer network that is collapsed owing to the presence of hydronium ions and weak base cations. The network is encapsulated within a lipid membrane, and functions as a vehicle for the osmotically inert storage of a variety of granule-bound endogenous mediator species, such as histamine, serotonin and proteases. These species are excreted from the granule and thence from the cell in response to external biochemical signals. Hydrogels that swell and shrink in response to external stimuli might serve as synthetic analogues of secretory granules. Here we describe the systematic engineering of multi-component, environmentally responsive hydrogel microspheres, coated with a lipid bilayer to mimic more closely the natural secretory granule. These microspheres exhibit pH- and ion-dependent volume phase transitions and ion-sensitive exchange of bound cations when the encapsulating lipid membrane is porated. We stimulated poration electrically in individual microgel particles immobilized and manipulated with a micropipette. This system could find use for the triggered release of encapsulated drugs in the body. PMID:9697768

Kiser, P F; Wilson, G; Needham, D



A subclass of proteins and sulfated macromolecules secreted by AtT-20 (mouse pituitary tumor) cells is sorted with adrenocorticotropin into dense secretory granules  

Microsoft Academic Search

The AtT-20 cell, a mouse pituitary tumor line that secretes adrenocorticotropin and\\/3-endorphin, sorts the proteins it exernalizes into two exocytotic pathways. Cells that are labeled with (35S)methionine or (3SS)sulfate can be shown to transport three acidic polypep- tides (65,000, 60,000, and 37,000 mol wt) and at least two sulfated macromolecules into storage secretory granules. When the cells are stimulated by




Role of secretory granules in inositol 1,4,5-trisphosphate-dependent Ca 2+ signaling: From phytoplankton to mammals  

Microsoft Academic Search

The majority of secretory cell calcium is stored in secretory granules that serve as the major IP3-dependent intracellular Ca2+ store. Even in unicellular phytoplankton secretory granules are responsible for the IP3-induced Ca2+ release that triggers exocytosis. The number of secretory granules in the cell is directly related not only to the magnitude of IP3-induced Ca2+ release, which accounts for the

Seung Hyun Yoo



Is swelling of the secretory granule matrix the force that dilates the exocytotic fusion pore?  

PubMed Central

The swelling of the secretory granule matrix which follows fusion has been proposed as the driving force for the rapid expansion of the fusion pore necessary for exocytosis. To test this hypothesis, we have combined simultaneous measurements of secretory granule swelling using videomicroscopy with patch clamp measurements of the time course of the exocytotic fusion pore in mast cells from the beige mouse. We show that isotonic acidic histamine solutions are able to inhibit swelling of the secretory granule matrix both in purified secretory granules lysed by electroporation and in intact cells stimulated to exocytose by guanine nucleotides. In contrast to the inhibitory effects on granule swelling, the rate of expansion of the exocytotic fusion pore is unaffected. Therefore, as the rate of granule swelling was more than 20 times slower under these conditions, swelling of the secretory granule matrix due to water entry through the fusion pore cannot be the force responsible for the characteristic rapid expansion of the exocytotic fusion pore. We suggest that tension in the secretory granule membrane, which has recently been demonstrated in fused secretory granules, might be the force that drives the irreversible expansion of the fusion pore. Images FIGURE 1 FIGURE 3 p43-b

Monck, J R; Oberhauser, A F; Alvarez de Toledo, G; Fernandez, J M



Immunocytochemical localization of heat-shock protein 60-related protein in Beta-cell secretory granules and its altered distribution in non-obese diabetic mice  

Microsoft Academic Search

Summary  Immuno-electron microscopy technique was employed to investigate the cellular distribution of 60 kDa heat-shock protein (HSP60) in pancreatic Beta cells of control and non-obese diabetic mice. In thin sections prepared from control mice, antibody to mammalian HSP60 cross-reacted with protein(s) located to mitochondria and secretory granules. In particular, prominent binding of the antibody was seen to the insulin core of

K. Brudzynski; V. Martinez; R. S. Gupta



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

Microsoft Academic Search

The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the rans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horseradish peroxidase and by the endogenous marker

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



Kalirin/Trio Rho Guanine Nucleotide Exchange Factors Regulate a Novel Step in Secretory Granule Maturation  

PubMed Central

The molecular mechanisms involved in the maturation of secretory granules, organelles that store hormones and neuropeptides, are poorly understood. As granule content proteins are processed, the composition of granule membranes changes, yielding constitutive-like secretion of immature content proteins and producing secretagogue-responsive mature granules. Constitutive-like secretion was not previously recognized as a process subject to regulation. We show that Kalirin and Trio, homologous Rho guanine nucleotide exchange factors (GEFs), which interact with a secretory granule resident protein, modulate cargo secretion from immature granules. Some of the Kalirin and Trio isoforms expressed in neuroendocrine cells colocalize with immature granules. Overexpression of their N-terminal GEF domain (GEF1) enhances secretion from immature granules, depleting cells of secretory cargo in the absence of secretagogue. This response requires GEF1 activity and is mimicked by Kalirin/Trio substrates Rac1 and RhoG. Accordingly, selective pharmacological inhibition of endogenous GEF1 activity decreases secretagogue-independent release of hormone precursors, accumulating product peptide in mature secretory granules. Kalirin/Trio modulation of cargo secretion from immature granules provides secretory cells with an extra layer of control over the sets of peptides released. Control of this step enhances the range of physiological responses that can be elicited, whereas lack of control could have pathological consequences.

Ferraro, Francesco; Ma, Xin-Ming; Sobota, Jacqueline A.; Eipper, Betty A.



Strain-Specific and Tissue-Specific Expression of Mouse Mast Cell Secretory Granule Proteases  

Microsoft Academic Search

As assessed by RNA blot analyses with gene-specific probes, we report that the perivascular connective tissue mast cells (CTMCs) in the ear and skin of BALB\\/cJ mice contain abundant levels of the mouse mast cell protease (mMCP) 7 transcript, in addition to those protease transcripts present in their serosal mast cells (SMCs). High levels of the mMCP-7 transcript also were

Richard L. Stevens; Daniel S. Friend; H. Patrick McNeil; Vera Schiller; Namit Ghildyal; K. Frank Austen



The Structure of Mast Cell Secretory Granules in the Blind Mole Rat ( Spalax ehrenbergi)  

Microsoft Academic Search

Eosinophils, basophils, and mast cells produce and secrete active substances whose role is to attack invading parasites and protect the host. In this study we use morphometric methods to study mast cells in the blind mole rat (Spalax ehrenbergi). The subterranean and solitary way of life of this species has led to the evolutionary development of special anatomical, morphological, behavioral,

D Amihai; S Trachtenberg; J Terkel; I Hammel



Coexistence of renin and angiotensin II in epitheloid cell secretory granules of rat kidney  

Microsoft Academic Search

The distribution of renin and angiotensin II (ANG II) in juxtaglomerular epitheloid cells of control and adrenalectomized rats was studied, using specific antisera and the protein A-gold technique in Lowicryl- and glycol methacrylate-embedded tissue.

R. Taugner; E. Mannek; R. Nobiling; C. P. Bührle; E. Hackenthal; D. Ganten; T. Inagami; H. Schröder



Rab27b Is Expressed in a Wide Range of Exocytic Cells and Involved in the Delivery of Secretory Granules Near the Plasma Membrane  

PubMed Central

Rab proteins regulate multiple, complex processes of membrane traffic. Among these proteins, Rab27a has been shown to function specifically in regulated exocytic pathways. However, the roles of Rab27b, another Rab27 subfamily member, have not been well characterized. We disrupted the Rab27b gene in mice. The targeting vector was designed to insert LacZ downstream of the initiation codon of the Rab27b gene so that the authentic promoter should drive this reporter gene. A comprehensive analysis of Rab27b expression using this mouse strain indicated that it is widely expressed not only in canonical secretory cells, but also in neurons and cells involved in surface protection and mechanical extension. To evaluate the function in pituitary endocrine cells where the isoform Rab27a is coexpressed, we generated Rab27a/Rab27b double knockout mice by crossing Rab27b knockout mice with Rab27a-mutated ashen mice. The polarized distribution of secretory granules close to the plasma membrane was markedly impaired in the pituitary of double knockout mice, indicating that the Rab27 subfamily is involved in the delivery of granules near the exocytic site. In conjunction with a phenotype having a pituitary devoid of the Rab27 effector granuphilin, we discuss the relationship between the residence and the releasable pool of granules.

Gomi, Hiroshi; Mori, Kenichi; Itohara, Shigeyoshi



Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules  

PubMed Central

Peptidylglycine alpha-amidating monooxygenase (PAM: EC is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and mid-region lyase act in sequence to perform the peptide alpha-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry. Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM proteins were endoproteolytically processed. When the linker region was present between the monooxygenase and lyase domains, monofunctional soluble enzymes were generated from bifunctional PAM proteins; without the linker region, bifunctional enzymes were generated. Soluble forms of PAM expressed in AtT-20 cells and soluble proteins generated through selective endoproteolysis of membrane-associated PAM were secreted in an active form into the medium; secretion of the transfected proteins and endogenous hormone were stimulated in parallel by secretagogues. PAM proteins were localized by immunocytochemistry in the perinuclear region near the Golgi apparatus and in secretory granules, with the greatest intensity of staining in the perinuclear region in cell lines expressing integral membrane forms of PAM. Monofunctional and bifunctional PAM proteins that were soluble or membrane-associated were all packaged into regulated secretory granules in AtT-20 cells.



Observing secretory granules with a multiangle evanescent wave microscope.  

PubMed Central

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.

Rohrbach, A



Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage  

Microsoft Academic Search

Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins—including amylase, proline-rich proteins, and parotid secretory protein (PSP)—to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols

S.-U. Gorr; S. G. Venkatesh; D. S. Darling



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

PubMed Central

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

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



Role of secretory granules in inositol 1,4,5-trisphosphate-dependent Ca(2+) signaling: from phytoplankton to mammals.  


The majority of secretory cell calcium is stored in secretory granules that serve as the major IP(3)-dependent intracellular Ca(2+) store. Even in unicellular phytoplankton secretory granules are responsible for the IP(3)-induced Ca(2+) release that triggers exocytosis. The number of secretory granules in the cell is directly related not only to the magnitude of IP(3)-induced Ca(2+) release, which accounts for the majority of the IP(3)-induced cytoplasmic Ca(2+) release in neuroendocrine cells, but also to the IP(3) sensitivity of the cytoplasmic IP(3) receptor (IP(3)R)/Ca(2+) channels. Moreover, secretory granules contain the highest IP(3)R concentrations and the largest amounts of IP(3)Rs in any subcellular organelles in neuroendocrine cells. Secretory granules from phytoplankton to mammals contain large amounts of polyanionic molecules, chromogranins being the major molecules in mammals, in addition to acidic intragranular pH and high Ca(2+) concentrations. The polyanionic molecules undergo pH- and Ca(2+)-dependent conformational changes that serve as a molecular basis for condensation-decondensation phase transitions of the intragranular matrix. Likewise, chromogranins undergo pH- and Ca(2+)-dependent conformational changes with increased exposure of the structure and increased interactions with Ca(2+) and other granule components at acidic pH. The unique physico-chemical properties of polyanionic molecules appear to be at the center of biogenesis, and physiological functions of secretory granules in living organisms from primitive to advanced species. PMID:21176957

Yoo, Seung Hyun



Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common  

Microsoft Academic Search

In response to an external stimulus, neu- ronal cells release neurotransmitters from small synap- tic vesicles and endocrine cells release secretory pro- teins from large dense core granules. Despite these differences, endocrine cells express three proteins known to be components of synaptic vesicle mem- branes. To determine if all three proteins, p38, p65, and SV2, are present in endocrine dense

A. W. Lowe; L. Madeddu; R. B. Kelly



Association with Nitric Oxide Synthase on Insulin Secretory Granules Regulates Glucokinase Protein Levels  

PubMed Central

Glucokinase (GCK) association with insulin-secretory granules is controlled by interaction with nitric oxide synthase (NOS) and is reversed by GCK S-nitrosylation. Nonetheless, the function of GCK sequestration on secretory granules is unknown. Here we report that the S-nitrosylation blocking V367M mutation prevents GCK accumulation on secretory granules by inhibiting association with NOS. Expression of this mutant is reduced compared with a second S-nitrosylation blocking GCK mutant (C371S) that accumulates to secretory granules and is expressed at levels greater than wild type. Even so, the rate of degradation for wild type and mutant GCK proteins were not significantly different from one another, and neither mutation disrupted the ability of GCK to be ubiquitinated. Furthermore, gene silencing of NOS reduced endogenous GCK content but did not affect ?-actin content. Treatment of GCK(C371S) expressing cells with short interfering RNA specific for NOS also blocked accumulation of this protein to secretory granules and reduced expression levels to that of GCK(V367M). Conversely, cotransfection of catalytically inactive NOS increased GCK-mCherry levels. Expression of GCK(C371S) in ?TC3 cells enhanced glucose metabolism compared with untransfected cells and cells expressing wild type GCK, even though this mutant has slightly reduced enzymatic activity in vitro. Finally, molecular dynamics simulations revealed that V367M induces conformational changes in GCK that are similar to S-nitrosylated GCK, thereby suggesting a mechanism for V367M-inhibition of NOS association. Our findings suggest that sequestration of GCK on secretory granules regulates cellular GCK protein content, and thus cellular GCK activity, by acting as a storage pool for GCK proteins.

Markwardt, Michele L.; Nkobena, Andongfac; Ding, Shi-Ying



Involvement of AQP6 in the Mercury-sensitive osmotic lysis of rat parotid secretory granules.  


In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73-80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg˛?, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg˛? (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5-2.0 ?M Hg˛?, concentrations that activate AQP6. The Hg lysis was completely blocked by ?-mercaptoethanol which disrupts Hg˛?-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO(3) (-) > Br? > I? > Cl? and was facilitated by acidic pH. The anion selectivity for NO(3) (-) and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg˛?-sensitive anion channel in rat parotid secretory granules. PMID:23183829

Matsuki-Fukushima, Miwako; Fujita-Yoshigaki, Junko; Murakami, Masataka; Katsumata-Kato, Osamu; Yokoyama, Megumi; Sugiya, Hiroshi



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


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

Bendayan, Moise; Londono, Irene; Paransky, Eugene



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


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

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



Lumenal protein multimerization in the distal secretory pathway\\/secretory granules  

Microsoft Academic Search

Differences in protein solubility appear to play an important role in lumenal protein trafficking through Golgi\\/post-Golgi compartments. Recent advances indicate that multimeric protein assembly is one of the factors regulating the efficiency of protein storage within secretory granules, by mechanisms that, with slight modification, might be considered to represent the culmination of a process of Golgi cisternal maturation.

Peter Arvan; Bao-yan Zhang; Lijun Feng; Ming Liu; Regina Kuliawat



Molecular Cloning of a cDNA that Encodes the Peptide Core of a Mouse Mast Cell Secretory Granule Proteoglycan and Comparison with the Analogous Rat and Human cDNA  

Microsoft Academic Search

A cDNA that encodes a mouse secretory granule proteoglycan peptide core was isolated from a cDNA library prepared from nontransformed mouse bone marrow-derived mast cells (BMMC) using as a probe a 280-base-pair fragment of a rat cDNA that encodes the proteoglycan peptide core of rat basophilic leukemia (RBL)-1 cells. Based on the consensus nucleotide sequence and deduced amino acid sequence

Shalom Avraham; Richard L. Stevens; Christopher F. Nicodemus; Michael C. Gartner; K. Frank Austen; John H. Weis



ATP-Independent Luminal Oscillations and Release of Ca 2+ and H + from Mast Cell Secretory Granules: Implications for Signal Transduction  

Microsoft Academic Search

InsP3 is an important link in the intracellular information network. Previous observations show that activation of InsP3-receptor channels on the granular membrane can turn secretory granules into Ca2+ oscillators that deliver periodic trains of Ca2+ release to the cytosol (T. Nguyen, W. C. Chin, and P. Verdugo, 1998, Nature, 395:908–912; I. Quesada, W. C. Chin, J. Steed, P. Campos-Bedolla, and

Ivan Quesada; Wei-Chun Chin; Pedro Verdugo



Storage and release of secretory granules in human prolactinomas: modification by bromocriptine.  


Electron microscopic morphometric evaluation of the effects of bromocriptine on human prolactinomas during and after short-term parenteral (7 days) and long-term peroral (4-6 weeks) treatment showed that a reduction in size of prolactinoma cells occurs within a few days (half-time to maximum shrinkage, 2.2 days). The number of secretory granules discharged into the intercellular space increased after short-term treatment by a factor of three but the total volume of stored secretory granules did not change significantly. The total volume of cellular lysosomes, both primary and secondary, decreased significantly to about one-half of the pretreatment value. The amount of stored lipoids, end-products of lysosomal activity, decreased in specimens treated for 7 days, but returned to pretreatment levels in specimens treated for 4-6 weeks, suggesting that lysosomal material is discharged from the cells together with the secretory material. PMID:3625099

Landolt, A M; Osterwalder, V; Landolt, T A



A phogrin-aequorin chimaera to image free Ca2+ in the vicinity of secretory granules.  

PubMed Central

Microdomains of high Ca2+ concentration ([Ca2+]) may be critical to the control of intracellular processes such as secretion and metabolism without compromising other cell functions. To explore changes in [Ca2+] in the outer mantle (< 30 nm deep) that surrounds the surface of dense-core secretory granules, we have designed a recombinant chimaera between the granule protein phogrin and aequorin. When expressed in populations of insulin-secreting MIN6 or phaeochromocytoma PC12 cells, the chimaera was targeted to secretory granules as expected. The recombinant protein reported a similar [Ca2+] at the granule surface to that in the bulk cytosol, measured with untargeted aequorin. This was the case both at rest (-Ca2+- = 80-120 nM) and after stimulation with agents that provoke Ca2+ entry or Ca2+ mobilization from intracellular pools, and during activated secretion. Thus depolarization of MIN6 cell populations with high K+ increased [Ca2+] both in the bulk cytosol and close to the granules to approx. 4 microM, with near-identical kinetics of increase and recovery. Similarly, stimulation of PC12 cells with ATP provoked an increase in -Ca2+- in either domain to 1.3 microM. These data argue that, in MIN6 and PC12 neuroendocrine cells (i) significant mobilization of Ca2+ from most secretory granules probably does not occur during activated Ca2+ influx or mobilization of internal Ca2+ stores, and (ii) agonist-stimulated Ca2+-dependent secretion can occur without development of a large gradient of [Ca2+] between the surface of most secretory vesicles and the rest of the cytosol.

Pouli, A E; Karagenc, N; Wasmeier, C; Hutton, J C; Bright, N; Arden, S; Schofield, J G; Rutter, G A



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

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

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



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

SciTech Connect

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.

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



Mapping Dynamic Protein Interactions to Insulin Secretory Granule Behavior with TIRF-FRET  

PubMed Central

Biological processes are governed by extensive networks of dynamic molecular interactions. Yet, establishing a spatial and temporal map of these interactions and their direct relationship to specific cell functions has remained a challenge. Here, we implement sensitized emission Förster resonance energy transfer (FRET) stoichiometry under total internal reflection fluorescence (TIRF) microscopy. We demonstrate through quantitative analysis and modeling that evanescent fields must be precisely matched between FRET excitation wavelengths to isolate dynamic interactions between bimolecular FRET pairs that are not entirely membrane-delimited. We then use TIRF-FRET to monitor the behavior of individual insulin-containing secretory granules at the plasma membrane of living cells, while simultaneously tracking the dynamic interaction between the GTPase Rab27A and its effector Slp4A, on those same granules. Notably, insulin granules that underwent exocytosis demonstrated a specific increase in Rab27A-GTP/Slp4A FRET in the 5 s before membrane fusion, which coincided temporally with an increase in granule displacement and mobility. These results demonstrate an initial spatiotemporal mapping of a dynamic protein-protein interaction on individual secretory granules that is linked to a specific granule behavior in living cells.

Lam, Alice D.; Ismail, Sahar; Wu, Ray; Yizhar, Ofer; Passmore, Daniel R.; Ernst, Stephen A.; Stuenkel, Edward L.



Mapping dynamic protein interactions to insulin secretory granule behavior with TIRF-FRET.  


Biological processes are governed by extensive networks of dynamic molecular interactions. Yet, establishing a spatial and temporal map of these interactions and their direct relationship to specific cell functions has remained a challenge. Here, we implement sensitized emission Förster resonance energy transfer (FRET) stoichiometry under total internal reflection fluorescence (TIRF) microscopy. We demonstrate through quantitative analysis and modeling that evanescent fields must be precisely matched between FRET excitation wavelengths to isolate dynamic interactions between bimolecular FRET pairs that are not entirely membrane-delimited. We then use TIRF-FRET to monitor the behavior of individual insulin-containing secretory granules at the plasma membrane of living cells, while simultaneously tracking the dynamic interaction between the GTPase Rab27A and its effector Slp4A, on those same granules. Notably, insulin granules that underwent exocytosis demonstrated a specific increase in Rab27A-GTP/Slp4A FRET in the 5 s before membrane fusion, which coincided temporally with an increase in granule displacement and mobility. These results demonstrate an initial spatiotemporal mapping of a dynamic protein-protein interaction on individual secretory granules that is linked to a specific granule behavior in living cells. PMID:20713017

Lam, Alice D; Ismail, Sahar; Wu, Ray; Yizhar, Ofer; Passmore, Daniel R; Ernst, Stephen A; Stuenkel, Edward L



Glucose Transporter (GLUT-4) Is Targeted to Secretory Granules in Rat Atrial Cardiomyocytes  

PubMed Central

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the transGolgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulindependent movement to the cell surface. To examine the relationship between these GLUT-4–containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.

Slot, Jan W.; Garruti, Gabriella; Martin, Sally; Oorschot, Viola; Posthuma, George; Kraegen, Edward W.; Laybutt, Ross; Thibault, Gaetan; James, David E.



The release of Hsp70 from A431 carcinoma cells is mediated by secretory-like granules  

Microsoft Academic Search

In our earlier work we have demonstrated that the treatment of squamous carcinoma cell line A431 with a pharmacological inhibitor of phospholipase C activity, U73122, resulted in fast release of stress-inducible heat shock protein 70 (Hsp70) into the extracellular medium (Evdonin et al., Cancer Cell Int., 4, 2, 2004). The purpose of the present study was to identify cellular organelles

Anton L. Evdonin; Marina G. Martynova; Olga A. Bystrova; Irina V. Guzhova; Boris A. Margulis; Natalia D. Medvedeva



Ultrastructural and immunocytochemical studies on the cytoskeleton in the anterior pituitary of rats, with special regard to the relationship between actin filaments and secretory granules  

Microsoft Academic Search

As previously reported, in anterior pituitary cells of the rat, secretory granules are linked with adjacent granules, cytoorganelles, microtubules, and plasma membrane by thin filaments, 4–10 nm in diameter. The quick-freeze, deep-etching method revealed that some of the filaments linking adjacent secretory granules show 5 nm-spaced striations on their surface which are known to be characteristic of actin. Immunocytochemistry showed

T. Senda; H. Fujita; T. Ban; C. Zhong; K. Ishimura; K. Kanda; K. Sobue



Localization of Cyclo-Oxygenase and Prostaglandin E2 in the Secretory Granule of the Mast Cell.  

National Technical Information Service (NTIS)

The application of anti-cyclo-oxygenase and anti-prostaglandin E2 immunoglobulins to A23187-stimulated rat connective tissue mast cells has permitted the localization of cyclo-oxygenase activity(prostaglandin H2 synthetase) and the site of prostaglandin E...

E. A. Schmauder-Chock S. P. Chock



Rab3D, a Small GTPase, Is Localized on Mast Cell Secretory Granules and Translocates to the Plasma Membrane upon Exocytosis  

Microsoft Academic Search

Although mast cell secretion has been intensively studied because of its pivotal role in allergic reactions and its advantages as a physiologic model, the molecular composition of the secretory machine is virtually unknown. In view of the guanine-nucleotide dependency of mast cell exocytosis and the participation of Rab3 proteins in synaptic vesicle release, we hypothesized that a Rab3 isoform regulates

Michael J. Tuvim; Roberto Adachi; Jose F. Chocano; Robert H. Moore; Robert M. Lampert; Evelyn Zera; Elkin Romero; Brian J. Knoll; Burton F. Dickey


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

Microsoft Academic Search

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

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



Immunoelectron microscopical demonstration of endogenous avidin in secretory granules of the hen oviduct mucosa: a preliminary study  

Microsoft Academic Search

Summary  The localization of avidin in the oviduct of the laying hen was investigated using ultrastructural immunoperoxidase techniques. Endogenous avidin was localized in secretory granules of both tubular gland cells and non-ciliated single epithelial cells in the magnum mucosa. These immunospecific granules were electron-dense and heterogeneous with a patchy core and dense peripheral region, especially in acinar cells. The size varied

Koji Kami; Kenjiro Yasuda



The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules  

Microsoft Academic Search

Synopsis  A series of studies was performed to assess the optimum fixation conditions for staining of carbohydrate-containing constituents of rat salivary gland secretory granules. In the parotid and submandibular salivary glands of the rat, the reactivity of secretory granules, at both the light and electron microscopic level, with routine stains and with cytochemical reagents was highly dependent upon the nature of

J. A. V. Simson



A subset of 50 secretory granules in close contact with L-type Ca2+ channels accounts for first-phase insulin secretion in mouse beta-cells.  


Capacitance measurements were applied to mouse pancreatic beta-cells to elucidate the cellular mechanisms underlying biphasic insulin secretion. We report here that only <50 of the beta-cell's >10,000 granules are immediately available for release. The releasable granules tightly associate with the voltage-gated alpha(1C) Ca(2+) channels, and it is proposed that the release of these granules accounts for first-phase insulin secretion. Subsequent replenishment of the releasable pool by priming of previously nonreleasable granules is required for second-phase insulin secretion. The latter reaction depends on intragranular acidification due to the concerted action of granular bafilomycin-sensitive v-type H(+)-ATPase and 4,4-diisothiocyanostilbene-2,2-disulfonate--blockable ClC-3 Cl(-) channels. Lowering the cytoplasmic ATP/ADP ratio prevents granule acidification, granule priming, and refilling of the releasable pool. The latter finding provides an explanation to the transient nature of insulin secretion elicited by, for example, high extracellular K(+) in the absence of metabolizable fuels. PMID:11815462

Barg, Sebastian; Eliasson, Lena; Renström, Erik; Rorsman, Patrik



Sorting and storage during secretory granule biogenesis: looking backward and looking forward.  

PubMed Central

Secretory granules are specialized intracellular organelles that serve as a storage pool for selected secretory products. The exocytosis of secretory granules is markedly amplified under physiologically stimulated conditions. While granules have been recognized as post-Golgi carriers for almost 40 years, the molecular mechanisms involved in their formation from the trans-Golgi network are only beginning to be defined. This review summarizes and evaluates current information about how secretory proteins are thought to be sorted for the regulated secretory pathway and how these activities are positioned with respect to other post-Golgi sorting events that must occur in parallel. In the first half of the review, the emerging role of immature secretory granules in protein sorting is highlighted. The second half of the review summarizes what is known about the composition of granule membranes. The numerous similarities and relatively limited differences identified between granule membranes and other vesicular carriers that convey products to and from the plasmalemma, serve as a basis for examining how granule membrane composition might be established and how its unique functions interface with general post-Golgi membrane traffic. Studies of granule formation in vitro offer additional new insights, but also important challenges for future efforts to understand how regulated secretory pathways are constructed and maintained.

Arvan, P; Castle, D



Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway  

Microsoft Academic Search

Dense granules are Apicomplexa specific secretory organelles. In Toxoplasma gondii, the dense granules proteins, named GRA proteins, are massively secreted into the parasitophorous vacuole (PV) shortly after invasion. Despite the presence of hydrophobic membrane segments, they are stored as both soluble and aggregated forms within the dense granules and are secreted as soluble forms into the vacuolar space where they

Laurence Braun; Laetitia Travier; Sylvie Kieffer; Karine Musset; Jérôme Garin; Corinne Mercier; Marie-France Cesbron-Delauw



Purification, pH-dependent conformational change, aggregation, and secretory granule membrane binding property of secretogranin II (chromogranin C).  


Secretogranin II (SgII) is one of the three major proteins, the other two being chromogranins A (CGA) and B (CGB), of secretory granules of neuroendocrine cells. The Ca(2+) storage proteins CGA and CGB not only are coupled to the IP(3) receptor (IP(3)R)/Ca(2+) channels that exist on the secretory granule membrane but also are known to play key roles in secretory granule biogenesis. Unlike the better studied CGA and CGB, secretogranin II has never been completely purified in the native state and studied. We have therefore purified SgII in native form from bovine adrenal medulla and subjected it to biochemical characterization. Secretogranin II consisted of largely beta-sheet and random coil structures with a low level of alpha-helicity. Like CGA and CGB, it also underwent pH-dependent conformational changes, showing 9.5% alpha-helicity at pH 7.5 and 17.0% alpha-helicity at pH 5.5. Secretogranin II also underwent acidic pH- and Ca(2+)-dependent aggregation, and it was approximately 8-fold more sensitive than CGA to Ca(2+) in its pH-dependent aggregation but was 8-fold less sensitive than CGB. Further, similar to CGA and CGB that had interacted with the secretory granule membrane at the intragranular pH 5.5, SgII also interacted with the secretory granule membrane at pH 5.5 and dissociated from it at near-physiological pH 7.5, implying similar roles of SgII in the cell as those of CGA and CGB. Secretogranin II hence appeared to actively participate in secretory granule biogenesis as has been proposed for CGA and CGB. PMID:11802725

Park, Hee Yun; So, Seung Ho; Lee, Woo Bok; You, Soon Hee; Yoo, Seung Hyun



Secretion granules of the rabbit parotid. Selective removal of secretory contaminants from granule membranes  

PubMed Central

A membrane subfraction obtained from secretion granules isolated from rabbit parotid has been shown to be contaminated by residual secretory proteins to an estimated level of 25-30% of its total protein. In the present study an additional contaminant has been identified by improved mixing experiments and by comparative peptide mapping of specific polypeptides recovered from gels of membrane and content subfractions. This contaminant coelectrophoresis with (and probably comprises the bulk of) the majority component of the membrane subfraction (mol wt approximately 40,000). The contaminating polypeptides can be removed to a large extent by treating the membranes with low concentrations of saponin in the presence of 0.3 M Na2SO4. Although this treatment disrupts the typical bilayer structure of the granule membrane, it does not appear to cause dissociation of its phospholipids or bona fide membrane proteins.

Castle, J. D.; Palade, G. E.



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

PubMed Central

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

Bowman, G R; Turkewitz, A P



Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation  

Microsoft Academic Search

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold\\u000a electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein\\u000a is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations\\u000a revealed a marked accumulation of colloidal gold particles at

Enrico Crivellato; Luciana Travan; Luigi Candussio; Fiora Bartoli Klugmann; Giuliana Decorti



Secretory mechanisms in cell-mediated cytotoxicity.  


Cytotoxic T lymphocytes (CTLs) play a critical role in the immune system; they are able to recognize and destroy virally infected and tumorigenic cells. Specific recognition of MHC class I-peptide complexes by the T cell receptor (TcR) results in precise delivery of lytic granules to the target cell, sparing neighboring cells and the CTL itself. Over the past 10 years various studies have eludicated the mechanisms that lead to the rapid polarization of the secretory apparatus in CTLs. These studies highlight similarities and differences between polarity and secretory mechanisms seen in other cell types and developmental systems. This review focuses on recent advances in our understanding of the molecular basis of polarized secretion from CTLs and the novel mechanism used by these cells to deliver their lethal hit. PMID:17506701

Stinchcombe, Jane C; Griffiths, Gillian M



Ultrastructural Immunogold Cytochemistry with Autoimmune Human Sera and an Antibody to Uridine Implicate Human Mast Cell Granules in RNA Biology  

Microsoft Academic Search

Human mast cells are professional secretory cells that store synthetic products in large granules filling their cytoplasm. Unlike many secretory cells, the principal synthetic organelle, ribosome-rich endoplasmic reticulum, is a minor component of their cytoplasm. Sightings of nonmembrane-bound ribosomes in and near their secretory granules stimulated detailed ultrastructural studies of various RNA species to implicate secretory-storage granules in RNA biology.

Ann M. Dvorak; Ellen S. Morgan



Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules  

Microsoft Academic Search

The conversion of proglucagon and proinsulin by secretory granules isolatedfrom both prelabeled and unlabeled anglerfish isletswas investigated.Eithergranules isolatedfrom tissuelabeled with (3H)tryptophan and (14C)isoleucine or (\\




Evidence for defects in membrane traffic in Paramecium secretory mutants unable to produce functional storage granules  

PubMed Central

The ciliated protozoan Paramecium has a regulated secretory system amenable to genetic analysis. The secretory storage granules, known as trichocysts, enclose a crystalline matrix with a genetically determined shape whose biogenesis involves proteolytic maturation of a family of precursor molecules into a heterogeneous set of small acidic polypeptides that crystallize within the maturing vesicles. We have developed an original pulse-chase protocol for monoxenic Paramecium cultures using radiolabeled bacteria to study the processing of trichocyst matrix proteins in wild-type and mutant cells. In wild-type cells, proteolytic processing is blocked in the presence of monensin and otherwise rapidly completed after approximately 20 min of chase, suggesting that the conversion occurs in the trans-Golgi and/or in small vesicles soon after sorting to the regulated pathway, probably before crystallization begins. In trichless mutant cells, which contain no visible trichocysts, secretory proteins are synthesized but not processed and we report constitutive secretion of the uncleaved precursor molecules. The mutation thus appears to affect sorting to the regulated pathway and should prove useful for analysis of the sorting machinery and of the relationship between sorting and proteolytic processing of secretory proteins. In mutants bearing misshapen trichocysts with poorly crystallized contents (tam33, tam38, stubbyA), the proteolytic processing of the trichocyst matrix proteins appears to be normal, while both pulse-chase and morphological data indicate that intracellular transport is perturbed, probably between ER and Golgi. Precursor molecules are present in the mutant trichocysts but not in wild-type trichocysts and may account for the defective crystallization. Our analysis of these mutants suggests that the temporal coordination of intracellular traffic plays a regulatory role in granule maturation.



CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells  

PubMed Central

Measurements of cell capacitance were used to investigate the mechanisms by which acetylcholine (ACh) stimulates Ca2+-induced exocytosis in single insulin-secreting mouse pancreatic B-cells.ACh (250 ?M) increased exocytotic responses elicited by voltage-clamp depolarizations 2.3-fold. This effect was mediated by activation of muscarinic receptors and dependent on elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) attributable to mobilization of Ca2+ from intracellular stores. The latter action involved interference with the buffering of [Ca2+]i and the time constant (?) for the recovery of [Ca2+]i following a voltage-clamp depolarization increased 5-fold. As a result, Ca2+ was present at concentrations sufficient to promote the replenishment of the readily releasable pool of granules (RRP; > 0.2 ?M) for much longer periods in the presence than in the absence of the agonist.The effect of Ca2+ on exocytosis was mediated by activation of CaM kinase II, but not protein kinase C, and involved both an increased size of the RRP from 40 to 140 granules and a decrease in ? for the refilling of the RRP from 31 to 19 s.Collectively, the effects of ACh on the RRP and ? result in a > 10-fold stimulation of the rate at which granules are supplied for release.

Gromada, Jesper; H?y, Marianne; Renstrom, Erik; Bokvist, Krister; Eliasson, Lena; Gopel, Sven; Rorsman, Patrik



Immunogold labeling of carbonic anhydrase isozyme (CA-VI) in secretory granules of human parotid glands.  


Serous granules in the human parotid gland have a well-defined substructure, consisting of a dense spherule suspended in a moderately dense matrix. Immunogold labeling with an antibody against carbonic anhydrase VI revealed that this enzyme is localized within the matrix and is absent from the spherule. This location matches that of a number of other salivary gland proteins. Cell organelles involved in the secretory pathway are devoid of label. Labeling was not observed in any ductular component of the gland. PMID:21962635

Piras, Monica; Tandler, Bernard; Barbarossa, Iole Tomassini; Piludu, Marco



The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals.  


Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1-6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals. PMID:23418362

McGirr, Rebecca; Guizzetti, Leonardo; Dhanvantari, Savita



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

PubMed Central

The electron-dense cores of regulated secretory granules in the ciliate Tetrahymena thermophila are crystal lattices composed of multiple proteins. Granule synthesis involves a series of steps beginning with protein sorting, followed by the condensation and precise geometric assembly of the granule cargo. These steps may to various degrees be determined by the cargo proteins themselves. A prominent group of granule proteins, in ciliates as well as in vertebrate neuronal and endocrine cells, are acidic, heat-stable, and bind calcium. We focused on a protein with these characteristics named granule lattice protein 1 (Grl1p), which represents 16% of total granule contents, and we have now cloned the corresponding gene. Mutants in which the macronuclear copies of GRL1 have been disrupted continue to synthesize dense-core granules but are nonetheless defective in regulated protein secretion. To understand the nature of this defect, we characterized mutant and wild-type granules. In the absence of Grl1p, the sorting of the remaining granule proteins appears normal, and they condense to form a well-defined core. However, the condensed cores do not demonstrate a visible crystalline lattice, and are notably different from wild type in size and shape. The cellular secretion defect arises from failure of the aberrant granule cores to undergo rapid expansion and extrusion after exocytic fusion of the granule and plasma membranes. The results suggest that sorting, condensation, and precise granule assembly are distinct in their requirements for Grl1p.



Molecular cloning of a cDNA that encodes the peptide core of a mouse mast cell secretory granule proteoglycan and comparison with the analogous rat and human cDNA.  

PubMed Central

A cDNA that encodes a mouse secretory granule proteoglycan peptide core was isolated from a cDNA library prepared from nontransformed mouse bone marrow-derived mast cells (BMMC) using as a probe a 280-base-pair fragment of a rat cDNA that encodes the proteoglycan peptide core of rat basophilic leukemia (RBL)-1 cells. Based on the consensus nucleotide sequence and deduced amino acid sequence of the cDNA, the mouse BMMC proteoglycan peptide core is 16.7 kDa and contains a 21-amino acid glycosaminoglycan attachment region consisting of alternating serine and glycine residues. When the predicted amino acid sequence of the mouse BMMC proteoglycan peptide core was compared with the predicted amino acid sequences of the homologous molecules expressed in RBL-1 cells and in human promyelocytic leukemia HL-60 cells, the mouse-derived sequence was more closely homologous to the rat sequence than the human sequence except for the length of the serine-glycine repeat region. The N terminus was found to be a highly conserved region of the molecule in the three species, suggesting that this region is important for the structure, function, and/or metabolism of this family of proteoglycans. Nucleotide sequences within the 5' and 3' untranslated regions of the mouse, rat, and human proteoglycan cDNA were conserved. That similar sequences were also present in the corresponding regions of a cDNA that encodes a rat mast cell protease suggests that particular nucleotide sequences may be important for regulation of expression of those proteins that are destined to reside in secretory granules. Images

Avraham, S; Stevens, R L; Nicodemus, C F; Gartner, M C; Austen, K F; Weis, J H



Syntaxin-6 SNARE Involvement in Secretory and Endocytic Pathways of Cultured Pancreatic  Cells  

Microsoft Academic Search

In pancreatic -cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in

Regina Kuliawat; Elena Kalinina; Jason Bock; Lloyd Fricker; Timothy E. McGraw; Se Ryoung Kim; Jiayu Zhong; Richard Scheller; Peter Arvan



Increased exocytosis of secretory granules in contrast to reduced serum hormone levels in pituitary adenomas of humans and rats treated with dopamine agonist  

Microsoft Academic Search

. The release of secretory granules outside the cells increased in frequency, in inverse proportion to the marked decrease in serum prolactin (PRL) levels in human prolactin-secreting adenomas (PRLomas) treated with bromocriptine (CB), a dopamine agonist. Rat pituitary adenomas induced by diethylstilbestrol also showed an increase in exocytosis of the granules despite a reduction in serum PRL levels after CB

Hiroshi Mori; Youichi Saitoh; Tamaki Maeda; Yoshikatsu Okada; Hiroshi Hirano; Motomu Tsuji



Kinetics of release of serotonin from isolated secretory granules. II. Ion exchange determines the diffusivity of serotonin.  

PubMed Central

We measured the efflux of 5-hydroxytryptamine (5-HT, serotonin) from an intact secretory granule extracted from the mast cell of the beige mouse. The efflux was measured with amperometry after rupture of the granule membrane was triggered by electroporation. We determined the diffusivity of 5-HT within the secretory granule to be 2.0 x 10(-8) cm2 s(-1) when the granule is in contact with a physiological saline and found that this diffusivity depends on the valence of the cation in the external electrolyte. There is a fivefold increase in the diffusion coefficient of 5-HT determined in CsCl (150 mM, pH 7.2) at 3.7 x 10(-8) cm2 s(-1) compared to that determined in histamine dihydrochloride (Hi, 100 mM at pH 4.5) at 0.7 x 10(-8) cm2 s(-1). We found that the rate of expansion of the granule matrix observed in physiological medium correlates with the efflux of 5-HT, and that the rate of swelling of the matrix and the efflux depend on the microviscosity within the granule matrix and not the bulk viscosity of the external solution. The low diffusivity of 5-HT (approximately 500-fold less than in the bulk), the observation that the valence of the counterion affects this diffusivity, and the relationship between the volume changes of the matrix and the efflux suggest that 5-HT is released from the granule by ion exchange. We discuss the implications of this result for exocytotic release in mast cells and propose that an ion exchange mechanism could control the rate of release in other secretory systems. Images FIGURE 1

Marszalek, P E; Farrell, B; Verdugo, P; Fernandez, J M



Ultrastructural characteristics of molecular release of secretory products from thyroid mast cells induced by lipopolysaccharide.  


Ultrastructural characteristics of molecular release of secretory products from thyroid mast cells during different periods after LPS injection were studied in rats. The main morphological manifestation of this mechanism was progressing hydration of the hyaloplasm around secretory granules and dissolution and release of osmiophilic contents from the granules to the cytoplasm with the formation of a hydropic vacuole at the site of the granule. PMID:24131004

Yaglova, N V; Yaglov, V V



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

PubMed Central

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

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



Reconstruction of the Electron Transport Chain of Secretory Granule Membrane  

NASA Astrophysics Data System (ADS)

In the present study we identified full molecular assembly of the electron transport chain of chromaffin granule membrane and demonstrated a modulating effect of phospholipid microenvironment on this chain.

Boyajyan, Anna


Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin  

PubMed Central

Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca2+-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI–specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.

Tomatis, Vanesa M.; Papadopulos, Andreas; Malintan, Nancy T.; Martin, Sally; Wallis, Tristan; Gormal, Rachel S.; Kendrick-Jones, John; Buss, Folma



Functional Amyloids as Natural Storage of Peptide Hormones in Pituitary Secretory Granules  

PubMed Central

Amyloids are highly organized cross ?-sheet-rich protein or peptide aggregates that are associated with pathological conditions including Alzheimer’s disease and type II diabetes. However, amyloids may also have a normal biological function as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. Here, we show that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross ?-sheet-rich conformation. Thus, in contrast to the original association of amyloids with diseases, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology.

Maji, Samir K.; Perrin, Marilyn H.; Sawaya, Michael R.; Jessberger, Sebastian; Vadodaria, Krishna; Rissman, Robert A.; Singru, Praful S.; Nilsson, K Peter R; Simon, Rozalyn; Schubert, David; Eisenberg, David; Rivier, Jean; Sawchenko, Paul; Vale, Wylie; Riek, Roland



Mast cell immunohistochemistry: non-immunological immunostaining mediated by non-specific F(ab?) 2 mast cell secretory granule interaction  

Microsoft Academic Search

Summary  During investigations of murine and human mast cell immunoreactivity with potential anti-interleukin-4 antibodies, non-specific, non-immunological labelling of mouse and human mast cells became apparent. Non-specific, non-immunological labelling was identified by (i) immunolabelling of mast cells when using control isotype primary antibodies, (ii) ability of conjugated secondary antibodies to label mast cells without prior mast cell exposure to a primary antibody,

Patric M. Schiltz; Janet Lieber; Ralph C. Giorno; Henry N. Claman



Association between Endocrine Pancreatic Secretory Granules and In-vitro-assembled Microtubules IsDependent upon Microtubule-associated Proteins  

Microsoft Academic Search

By use of dark-fieldlightmicroscopy, secretorygranules isolatedfrom the anglerfish endocrine pancreas were observed to attach to and release from microtubules assembled in vitrofrom brain homogenates .Secretory granules only bound to microtubules assembled in the presence of microtubule-associated proteins (MAPS) and not to microtubules assembled from purified tubulin.The addition of a MAP fraction to purified tubulin restored secretory granule binding .The secretory



Tension in Secretory Granule Membranes Causes Extensive Membrane Transfer Through the Exocytotic Fusion Pore  

Microsoft Academic Search

For fusion to occur the repulsive forces between two interacting phospholipid bilayers must be reduced. In model systems, this can be achieved by increasing the surface tension of at least one of the membranes. However, there has so far been no evidence that the secretory granule membrane is under tension. We have been studying exocytosis by using the patch-clamp technique

Jonathan R. Monck; Guillermo Alvarez de Toledo; Julio M. Fernandez



Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic b Cells  

Microsoft Academic Search

In cells specialized for secretory granule exo- cytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 b -cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate recep- tors (MPRs), respectively. Interestingly,

Regina Kuliawat; Judith Klumperman; Thomas Ludwig; Peter Arvan


Microscopic nodes and ducts inside lymphatics and on the surface of internal organs are rich in granulocytes and secretory granules.  


The blood and lymphatic systems are the two well-established circulatory systems. The existence of a third circulatory system representing acupuncture meridians was claimed in the 1960s. The very existence and function of the system, however, remained uncertain. We have found that microscopic nodes and ducts inside lymphatics, as well as on the surface of internal organs of the rat. The nodes and ducts are covered by a layer of EMP-3-positive spindle-shaped epithelium with, below, a layer of vWF-positive but CD31-negative endothelium. The nodes contain a variety of immune cells, usually enriched with mast cells, eosinophils, neutrophils and histiocytes, as well as chromaffin cells, other granule-containing cells. Secretory granules originating from the mast cells in the nodes appear to pass along ductules, two or more of which make up a duct. Our results reveal a potential circulatory system whose anatomical structure and cellular content differ from the blood and lymph systems, and which may be involved in the transport of secretory granules. PMID:22884518

Kwon, Byoung S; Ha, Chang M; Yu, Sungsook; Lee, Byung-Cheon; Ro, Jae Y; Hwang, Sunhee



Ultrastructural Cytochemical, Immunocytochemical and in situ Hybridization Methods with Polyuridine Probes Detect mRNA in Human Mast Cell Granules  

Microsoft Academic Search

Mature human mast cells are classical secretory cells that are filled with secretory-storage granules but are poorly endowed with visible free or membrane-bound cytoplasmic ribosomes. We recently reported close associations of ribosomes and various components essential to RNA metabolism in and close to human mast cell granules using multiple ultrastructural imaging methods. In view of these findings and an increased

Ann M. Dvorak; Ellen S. Morgan



Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules  

PubMed Central

The conversion of proglucagon and proinsulin by secretory granules isolated from both prelabeled and unlabeled anglerfish islets was investigated. Either granules isolated from tissue labeled with [3H]tryptophan and [14C]isoleucine or [35S]cysteine, or lysed granules from unlabeled tissue to which exogenously labeled prohormones had been added were incubated under various conditions. Acetic acid extracts of these granule preparations were analyzed for prohormone and hormone content by gel filtration. Both prelabeled and lysed, unlabeled secretory granules converted radiolabeled precursor peptides (Mr 8,000- 15,000) to labeled insulin and glucagon. The accuracy of the cleavage process was established by demonstrating comigration of products obtained from in vitro cleavage with insulin and glucagon extracted from intact islets using electrophoresis and high-pressure liquid chromatography (HPLC). The pH optimum for granule-mediated conversion was found to be in the range of pH 4.5-5.5. Conversion of both proglucagon and proinsulin by secretory granules was significantly inhibited in the presence of antipain, leupeptin, p- chloromercuribenzoate (PCMB) or dithiodipyridine (DDP) but not chloroquine, diisopropyl fluorophosphate, EDTA, p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, or N-p-tosyl-L-lysine chloromethyl ketone HCl. The inhibitory action of PCMB and DDP was reversed in the presence of dithiothreitol. Both membranous and soluble components of the secretory granules possessed significant converting activity. HPLC and electrophoretic analysis of cleavage products demonstrated that the converting activities of the membranous and soluble components were indistinguishable. The amount of inhibition of proinsulin and proglucagon conversion caused by 600 micrograms/ml porcine proinsulin was significantly lower than that caused by the same concentration of unlabeled anglerfish precursor peptides. These results indicate that the proinsulin and proglucagon converting enzyme(s) in the anglerfish pancreatic islet is a unique intracellular thiol proteinase(s) that may be granule membrane-associated and may require the presence of prohormone sequences in addition to the dibasic residues at cleavage sites for substrate recognition and/or binding.



Effects of Sex Steroids on Secretory Granule Formation in Gonadotropes of Castrated Male Rats with Respect to Granin Expression  

Microsoft Academic Search

Pituitary gonadotropes show sex-related differences in their ul- trastructure. Typical gonadotropes of male rats exhibit both large granules, which contain chromogranin A (CgA), and small granules, which contain secretogranin II (SgII). In contrast, typical female rat gonadotropes show only a very few large granules among the numer- ous small granules. To clarify the nature of the biogenesis of these secretory




A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules  

Microsoft Academic Search

Exocrine acinar cells, like parotid cells, have difficulty in maintaining their functions in cell lines or in primary cultures. For this reason, molecular studies on exocrine cell functions are unsatisfactory. To examine the mechanisms whereby the functions of parotid acinar cells are maintained, we attempted to establish a system for primary culture and transfection of exogenous genes. Acinar cells were

Junko Fujita-Yoshigaki; Asako Tagashira; Tomoyoshi Yoshigaki; Shunsuke Furuyama; Hiroshi Sugiya



Molecular interpretation of ACTH-?-endorphin coaggregation: relevance to secretory granule biogenesis.  


Peptide/protein hormones could be stored as non-toxic amyloid-like structures in pituitary secretory granules. ACTH and ?-endorphin are two of the important peptide hormones that get co-stored in the pituitary secretory granules. Here, we study molecular interactions between ACTH and ?-endorphin and their colocalization in the form of amyloid aggregates. Although ACTH is known to be a part of ACTH-?-endorphin aggregate, ACTH alone cannot aggregate into amyloid under various plausible conditions. Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-?-endorphin system, ?-endorphin-only system and ACTH-only system. We find that ?-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system. Our data suggest that ACTH is not only involved in interaction with ?-endorphin but also enhances the stability of mixed oligomers of the entire system. PMID:22403619

Ranganathan, Srivastav; Singh, Pradeep K; Singh, Uday; Singru, Praful S; Padinhateeri, Ranjith; Maji, Samir K



AQPs and Control of Vesicle Volume in Secretory Cells  

Microsoft Academic Search

Aquaporins (AQPs) are a family of small, hydrophobic, integral membrane proteins. In mammals, they are expressed in many epithelia\\u000a and endothelia and function as channels that permit water or small solutes to pass. Although the AQPs reside constitutively\\u000a at the plasma membrane in most cell types, the presence of AQPs in intracellular organelles such as secretory granules and\\u000a vesicles has

H. Sugiya; M. Matsuki



beta2-Syntrophin Is a Cdk5 Substrate That Restrains the Motility of Insulin Secretory Granules  

Microsoft Academic Search

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic ?-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of ?2-syntrophin anchors granules to actin filaments and that the phosphorylation\\/dephosphorylation of ?2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-?2-syntrophin through

Sandra Schubert; Klaus-Peter Knoch; Joke Ouwendijk; Shabaz Mohammed; Yury Bodrov; Melanie Jäger; Anke Altkrüger; Carolin Wegbrod; Marvin E. Adams; Yong Kim; Stanley C. Froehner; Ole N. Jensen; Yannis Kalaidzidis; Michele Solimena; Kathrin Maedler



Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic ?-Cells  

PubMed Central

In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 ?-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature ?-granules, while immunolabeling for cathepsin L, but not B, persists in mature ?-granules. By contrast, in islets from normal male SpragueDawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulusdependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.

Kuliawat, Regina; Klumperman, Judith; Ludwig, Thomas; Arvan, Peter



Ultrastructural changes in the Golgi apparatus and secretory granules of HL60 cells treated with the imino sugar N-butyldeoxynojirimycin  

Microsoft Academic Search

The imino sugar N-butyldeoxynojirimycin inhibits the N-linked oligosaccharide processing enzymes ?-glucosidases I and II, and the ceramide specific glucosyltransferase which catalyses the first step in glucosphingolipid biosynthesis. We have studied the effects of this compound on the ultrastructure of HL-60 cells to identify novel activities of this compound. Treatment of HL-60 cells with this imino sugar results in several morphological

Gabrielle R Neises; Philip G Woodman; Terry D Butters; Richard L Ornberg; Frances M Platt



Reduced Number and LH Content of Marginated Secretory Granules in Pituitary Gonadotropes of Streptozocin-lnduced Diabetic Male Rats  

Microsoft Academic Search

The transport of secretory granules towards the subplasmalemmal (SPL) region of the luteinizing-hormone (LH) gonadotrope is controlled by the LH-releasing-hormone-dependent pathway. The SPL granules contain the most readily releasable LH. To test the effect of diabetes on both the number and LH content of marginated granules, we studied by indirect immunogold-labelling pituitaries from control and streptozocin (STZ)-treated male rats. On

G. E. Bestetti; R. Spycher; P. Brändli; G. L. Rossi



Porosome: the secretory NanoMachine in cells.  


Cells synthesize and store within membranous sacs products such as hormones, growth factors, neurotransmitters, or digestive enzymes, for release on demand. As recently as just 15 years ago, it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intravesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation, however, failed to explain the generation of partially empty vesicles observed in electron micrographs following secretion. Logically therefore, in a 1993 News and Views article in the journal Nature, Prof. Erwin Neher wrote "It seems terribly wasteful that, during the release of hormones and neurotransmitters from a cell, the membrane of a vesicle should merge with the plasma membrane to be retrieved for recycling only seconds or minutes later." The discovery of permanent secretory portals or nanomachines at the cell plasma membrane called POROSOMES, where membrane-bound secretory vesicles transiently dock and fuse to release intravesicular contents to the cell exterior, has finally resolved this conundrum. Following this discovery, the composition of the porosome, its structure and dynamics visualized with high-resolution imaging techniques atomic force and electron microscopy, and its functional reconstitution into artificial lipid membrane have provided a molecular understanding of cell secretion. In agreement, it has been demonstrated that "secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells" (Proc Natl Acad Sci U S A 100:2070-2075, 2003); that "single synaptic vesicles fuse transiently and successively without loss of identity" (Nature 423:643-647, 2003); and that "zymogen granule exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity" (Proc Natl Acad Sci U S A 101:6774-6779, 2004). It made no sense all these years to argue that mammalian cells possess an "all or none" mechanism of cell secretion resulting from complete vesicle merger at the cell plasma membrane, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such as the secretion apparatus of Toxoplasma gondii, contractile vacuoles in paramecium, and different types of secretory structures in bacteria. The discovery of the porosome and its functional reconstitution in artificial lipid membrane, and an understanding of its morphology, composition, and dynamics, has resulted in a paradigm shift in our understanding of the secretory process in cells. PMID:23027011

Jena, Bhanu P



Molecular mechanism of myosin Va recruitment to dense core secretory granules.  


The brain-spliced isoform of Myosin Va (BR-MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide-producing cells. The molecular composition of the protein complex that recruits BR-MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG-associated proteins granuphilin-a/b (Gran-a/b), BR-MyoVa and Rab27a, a member of the Rab family of GTPases. Gran-a/b-BR-MyoVa interaction is direct, involves regions downstream of the Rab27-binding domain, and the C-terminal part of Gran-a determines exon specificity. MyoVa and Gran-a/b are partially colocalised on SGs and disruption of Gran-a/b-BR-MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient-stimulated hormone secretion in pancreatic beta-cells. These results indicate the existence of at least another binding partner of BR-MyoVa that was identified as rabphilin-3A (Rph-3A). BR-MyoVa-Rph-3A interaction is also direct and enhanced when secretion is activated. The BR-MyoVa-Rph-3A and BR-MyoVa-Gran-a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR-MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells. PMID:21985333

Brozzi, Flora; Diraison, Frederique; Lajus, Sophie; Rajatileka, Shavanthi; Philips, Thomas; Regazzi, Romano; Fukuda, Mitsunori; Verkade, Paul; Molnár, Elek; Váradi, Anikó



Active caspase-3 is stored within secretory compartments of viable mast cells.  


Caspase-3 is a main executioner of apoptotic cell death. The general notion is that, in viable cells, caspase-3 is found as a cytosolic inactive proenzyme and that caspase-3 activation is largely confined to processes associated with cell death. In this study, we challenge this notion by showing that enzymatically active caspase-3 is stored in viable mast cells. The enzymatically active caspase-3 was undetectable in the cytosol of viable cells, but was recovered in subcellular fractions containing secretory granule-localized proteases. Moreover, active caspase-3 was rapidly released into the cytosolic compartment after permeabilization of the secretory granules. Using a cell-permeable substrate for caspase-3, the presence of active caspase-3-like activity in granule-like compartments close to the plasma membrane was demonstrated. Moreover, it was shown that mast cell activation caused release of the caspase-3 to the cell exterior. During the course of mast cell differentiation from bone marrow cells, procaspase-3 was present in cells of all stages of maturation. In contrast, active caspase-3 was undetectable in bone marrow precursor cells, but increased progressively during the process of mast cell maturation, its accumulation coinciding with that of a mast cell-specific secretory granule marker, mouse mast cell protease 6. Together, the current study suggests that active caspase-3 can be stored within secretory compartments of viable mast cells. PMID:23817418

Garcia-Faroldi, Gianni; Melo, Fabio R; Rönnberg, Elin; Grujic, Mirjana; Pejler, Gunnar



Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway.  


Dense granules are Apicomplexa specific secretory organelles. In Toxoplasma gondii, the dense granules proteins, named GRA proteins, are massively secreted into the parasitophorous vacuole (PV) shortly after invasion. Despite the presence of hydrophobic membrane segments, they are stored as both soluble and aggregated forms within the dense granules and are secreted as soluble forms into the vacuolar space where they further stably associate with PV membranes. In this study, we explored the unusual biochemical behavior of GRA proteins during their trafficking. Conventional chromatography indicated that the GRA proteins form high globular weight complexes within the parasite. To confirm these results, DeltaGRA knocked-out parasites were stably complemented with their respective HA-FLAG tagged GRA2 or GRA5. Purification of the tagged proteins by affinity chromatography showed that within the parasite and the PV soluble fraction, both the soluble GRA2-HA-FLAG and GRA5-HA-FLAG associate with several GRA proteins, the major ones being GRA3, GRA6 and GRA7. Following their insertion into the PV membranes, GRA2-HA-FLAG associated with GRA5 and GRA7 while GRA5-HA-FLAG associated with GRA7 only. Taken together, these data suggest that the GRA proteins form oligomeric complexes that may explain their solubility within the dense granules and the vacuolar matrix by sequestering their hydrophobic domains within the interior of the complex. Insertion into the PV membranes correlates with the decrease of the GRA partners number. PMID:17959262

Braun, Laurence; Travier, Laetitia; Kieffer, Sylvie; Musset, Karine; Garin, Jérôme; Mercier, Corinne; Cesbron-Delauw, Marie-France



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


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

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



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

PubMed Central

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.

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



?2-Syntrophin Is a Cdk5 Substrate That Restrains the Motility of Insulin Secretory Granules  

PubMed Central

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic ?-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of ?2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of ?2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-?2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that ?2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and ?2-syntrophin phosphomutants we found that phosphorylation of ?2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis.

Schubert, Sandra; Knoch, Klaus-Peter; Ouwendijk, Joke; Mohammed, Shabaz; Bodrov, Yury; Jager, Melanie; Altkruger, Anke; Wegbrod, Carolin; Adams, Marvin E.; Kim, Yong; Froehner, Stanley C.; Jensen, Ole N.; Kalaidzidis, Yannis; Solimena, Michele



The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-?-cyclodextrin  

Microsoft Academic Search

Glutamate transport in blood platelets resembles that in brain nerve terminals because platelets contain neuronal Na+-dependent glutamate transporters, glutamate receptors in the plasma membrane, vesicular glutamate transporters in secretory granules, which use the proton gradient as a driving force, and can release glutamate during aggregation\\/activation. The acidification of secretory granules and glutamate transport were assessed during acute treatment of isolated

Tatiana Borisova; Ludmila Kasatkina; Ludmila Ostapchenko



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

Microsoft Academic Search

The mouse pituitary cell line, AtT-20, packages the adrenocorticotropic hormone (ACTH) in secretory vesicles and releases it when the cell is stimulated with secretagogues. These cells have the capacity, after transfection with the appropriate DNA, to package heterologous peptide hormones into the regulated secretory vesicles (Moore, H. P. H., M. D. Walker, F. Lee, and R. B. Kelly, 1983, Cell,




Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules? †  

PubMed Central

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

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



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


Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, DeltaGRT1 DeltaGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in DeltaGRT1 DeltaGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from DeltaGRT1 DeltaGRT2 cells appear less adhesive than those from the wild type. PMID:19684282

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



Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump  

SciTech Connect

Isolated rat pancreatic islets were pulse-labeled for 5 min with (/sup 3/H)leucine then chased for 25 min, during which time endogenously labeled (/sup 3/H)proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled (/sup 3/H)proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer, proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect.

Rhodes, C.J.; Lucas, C.A.; Mutkoski, R.L.; Orci, L.; Halban, P.A.



POMC-related products in the intermediate pituitary of the amphibian, Bufo marinus: differential subcellular processing in the Golgi and secretory granules.  


In the intermediate pituitary of the anuran amphibian, Bufo marinus, the N-acetylation of ACTH(1-13)-NH2 to yield alpha-MSH occurs as a cosecretory processing event, whereas the N-acetylation of beta-endorphin occurs as a posttranslational processing event. To understand how these two N-acetylation reactions are segregated, B. marinus intermediate pituitary cells were analyzed by immunogold labeling electron microscopy, and by using an ultracentrifugation procedure. The immunogold labeling studies indicated that ACTH(1-13)-NH2-related immunoreactivity was colocalized with N-acetylated beta-endorphin-related immunoreactivity in secretory granules. Furthermore, ACTH(1-13)-NH2-related immunoreactivity was not detected in either the ER or the Golgi. N-Acetylated beta-endorphin-related immunoreactivity, however, was detected in the Golgi. Ultracentrifugation analysis revealed that in an ER/microsomal fraction, beta-LPH-sized and nonacetylated beta-endorphin-sized immunoreactive material were present in a molar ratio of 1:2. No N-acetylated forms of beta-endorphin were detected in the ER/microsomal fraction. In a Golgi/secretory granule fraction, the molar ratio of beta-LPH to beta-endorphin was 1:9 with 58% of the beta-endorphin being N-acetylated. Collectively, these data support the following hypotheses. The proteolytic cleavage of ACTH (1-39) to yield ACTH (1-13)-NH2 is a late processing event occurring in secretory granules. The cleavage of beta-LPH to yield nonacetylated beta-endorphin is an early processing event that may occur in the ER or the Golgi. Because N-acetylated beta-endorphin and nonacetylated ACTH(1-13)-NH2 are colocalized in secretory granules, it appears, therefore, that the N-acetylation of beta-endorphin is completed prior to loading into secretory granules. Thus, there is a spatial and temporal separation of the posttranslational processing events associated with the beta-LPH portion and ACTH portion of the POMC biosynthetic pathway in amphibian intermediate pituitary cells. PMID:8735969

Steveson, T C; Dores, R M




PubMed Central

We have examined, in the pancreatic exocrine cell, the metabolic requirements for the conversion of condensing vacuoles into zymogen granules and for the discharge of the contents of zymogen granules. To study condensing vacuole conversion, we pulse labeled guinea pig pancreatic slices for 4 min with leucine-3H and incubated them in chase medium for 20 min to allow labeled proteins to reach condensing vacuoles. Glycolytic and respiratory inhibitors were then added and incubation continued for 60 min to enable labeled proteins to reach granules in control slices. Electron microscope radioautography of cells or of zymogen granule pellets from treated slices showed that a large proportion of prelabeled condensing vacuoles underwent conversion in the presence of the combined inhibitors. Osmotic fragility studies on zymogen granule suspensions suggest that condensation may result from the aggregation of secretory proteins in an osmotically inactive form. Discharge was studied using an in vitro radioassay based on the finding that prelabeled zymogen granules can be induced to release their labeled contents to the incubation medium by carbamylcholine or pancreozymin. Induced discharge is not affected if protein synthesis is blocked by cycloheximide for up to 2 hr, but is strictly dependent on respiration. The data indicate that transport and discharge do not require the pari passu synthesis of secretory or nonsecretory proteins (e.g. membrane proteins), suggesting that the cell may reutilize its membranes during the secretory process. The energy requirements for zymogen discharge may be related to the fusion-fission of the granule membrane with the apical plasmalemma.

Jamieson, James D.; Palade, George E.



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

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.

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.



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

Microsoft Academic Search

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

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


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

PubMed Central

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

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



Dynamics of secretory granules in somatotrophs of rats after stimulation with growth hormone-releasing factor: a stereological analysis  

Microsoft Academic Search

The anterior pituitary tissue of male rats injected with growth hormone-releasing factor (GRF) was either processed for stereology at the light-and electron-microscopic levels, or homogenized for growth hormone (GH) assay 2–60 min after GRF injection. Secretory granules of somatotrophs became smaller but increased in numerical density 2 min after GRF injection. Their volume density began to increase at 5 min.

Jun-ichi Nakagawa; Hiroshi Mori; Tamaki Maeda; Takahiro Matsuo; Yoshikatsu Okada



Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment  

PubMed Central

We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C- peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Arvan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. J. Biol. Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (t1/2 approximately equal to 1.5 h) compared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20 degrees C temperature block reveals a t1/2 less than or equal to 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway.



Regulation and recruitment of phosphatidylinositol 4-kinase on immature secretory granules is independent of ADP-ribosylation factor 1.  

PubMed Central

Heterotrimeric G-proteins, as well as small GTPases of the Rho and ADP-ribosylation factor (ARF) family, are implicated in the regulation of lipid kinases, including PtdIns 4-kinases and PtdIns(4)P 5-kinases. Here, we describe a PtdIns 4-kinase activity on immature secretory granules (ISGs), regulated secretory organelles formed from the trans-Golgi network (TGN), and investigate the regulation of PtdIns4P levels on these membranes. Over 50% of the PtdIns 4-kinase activity on ISGs is inhibited by both a low concentration of adenosine and the monoclonal antibody 4C5G, a specific inhibitor of the type II PtdIns 4-kinase. Treatment of ISGs with mastoparan 7 (M7) stimulates the type II PtdIns 4-kinase via pertussis-toxin-sensitive G(i)/G(0) proteins, which, in contrast with previous results obtained with chromaffin granules [Gasman, Chasserot-Golaz, Hubert, Aunis and Bader (1998) J. Biol. Chem. 273, 16913-16920], does not require Rho A, B or C. M7 treatment also leads to an inhibition in the recruitment of ARF to ISG membranes: this inhibition is not dependent on G(i)/G(0) activation, and is not linked to the stimulation of PtdIns 4-kinase observed with M7. PtdIns 4-kinase activity on ISGs is not regulated by myristoylated ARF1-GTP, in contrast with results obtained with Golgi membranes [Godi, Pertile, Meyers, Marra, Di Tullio, Iurisci, Luini, Corda and De Matteis (1999) Nat. Cell Biol. 1, 280-287; Jones, Morris, Morgan, Kondo, Irvine and Cockcroft (2000) J. Biol. Chem. 275, 13962-13170], whereas ARF1-GTP does regulate the production of PtdIns(4,5)P(2). Our results suggest that the regulation of PtdIns 4-kinase on the ISGs differs in comparison with that on the TGN, and might be related to a specific requirement of ISG maturation.

Panaretou, Christina; Tooze, Sharon A



Regulation of Insulin Granule Turnover in Pancreatic ?-Cells by Cleaved ICA512*S?  

PubMed Central

Insulin maintains homeostasis of glucose by promoting its uptake into cells from the blood. Hyperglycemia triggers secretion of insulin from pancreatic ?-cells. This process is mediated by secretory granule exocytosis. However, how ?-cells keep granule stores relatively constant is still unknown. ICA512 is an intrinsic granule membrane protein, whose cytosolic domain binds ?2-syntrophin, an F-actin-associated protein, and is cleaved upon granule exocytosis. The resulting cleaved cytosolic fragment, ICA512-CCF, reaches the nucleus and up-regulates the transcription of granule genes, including insulin and ICA512. Here, we show that ICA512-CCF also dimerizes with intact ICA512 on granules, thereby displacing it from ?2-syntrophin. This leads to increased granule mobility and insulin release. Based on these findings, we propose a model whereby the generation of ICA512-CCF first amplifies insulin secretion. The ensuing reduction of granule stores would then increase the probability of newly generated ICA512-CCF to reach the nucleus and enhance granule biogenesis, thus allowing ?-cells to constantly adjust production of granules to their storage size and consumption. Pharmacological modulation of these feedback loops may alleviate deficient insulin release in diabetes.

Trajkovski, Mirko; Mziaut, Hassan; Schubert, Sandra; Kalaidzidis, Yannis; Altkruger, Anke; Solimena, Michele



Chronic Palmitate Exposure Inhibits Insulin Secretion by Dissociation of Ca2+ Channels from Secretory Granules  

PubMed Central

Summary Long-term (72 hr) exposure of pancreatic islets to palmitate inhibited glucose-induced insulin secretion by >50% with first- and second-phase secretion being equally suppressed. This inhibition correlated with the selective impairment of exocytosis evoked by brief (action potential-like) depolarizations, whereas that evoked by long (?250 ms) stimuli was unaffected. Under normal conditions, Ca2+ influx elicited by brief membrane depolarizations increases [Ca2+]i to high levels within discrete microdomains and triggers the exocytosis of closely associated insulin granules. We found that these domains of localized Ca2+ entry become dispersed by long-term (72 hr), but not by acute (2 hr), exposure to palmitate. Importantly, the release competence of the granules was not affected by palmitate. Thus, the location rather than the magnitude of the Ca2+ increase determines its capacity to evoke exocytosis. In both mouse and human islets, the palmitate-induced secretion defect was reversed when the ? cell action potential was pharmacologically prolonged.

Hoppa, Michael B.; Collins, Stephan; Ramracheya, Reshma; Hodson, Leanne; Amisten, Stefan; Zhang, Quan; Johnson, Paul; Ashcroft, Frances M.; Rorsman, Patrik



Localization of human intestinal defensin 5 in Paneth cell granules.  

PubMed Central

Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells but recently also found to be produced by epithelial cells. We biosynthesized recombinant human intestinal defensin 5 (rHD-5) using the baculovirus-insect cell expression system. Since insect cells process defensin incompletely and secrete the precursor proHD-5, we substituted a methionine for an alanine at a likely processing site to allow selective chemical cleavage with cyanogen bromide, and rHD-5 was used to elicit polyclonal antibodies. By the immunoperoxidase-staining technique, the antibodies selectively stained Paneth cells of the normal adult small intestine. Immunogold electron microscopy further localized HD-5 to the Paneth cell secretory granules. Since some defensins exert activity cytotoxic to mammalian cells, we assayed the effect of rHD-5 on the human intestinal cell lines Caco2 and Int407. proHD-5 did not exert cytotoxic activity, and rHD-5 showed only minimal activity against Int407 and was inert against Caco2. Since Paneth cells release their granules adjacent to the mitotic cells of the intestinal crypts, HD could protect this cell population against invasion and parasitization by microbes.

Porter, E M; Liu, L; Oren, A; Anton, P A; Ganz, T



Exocytosis of cytolytic granules may not be required for target cell lysis by cytotoxic T-lymphocytes  

Microsoft Academic Search

Secretory processes have been implicated in the mechanism of target-cell lysis by cytotoxic T-lymphocytes (CTL) (refs 1,2). Both CTL and cytotoxic large granular lymphocytes have cytolytic granules3,4, containing the cytolytic molecules 'perforin'3 and 'cytolysin'4; perforin and cytotoxic lymphocytes can damage target cells through the membrane assembly of pores3-5. The description of proteases which are cytotoxic cell-associated and granule-located has supported

Guido Trenn; Hajime Takayama; Michail V. Sitkovsky



Most nonparotid "acinic cell carcinomas" represent mammary analog secretory carcinomas.  


Acinic cell carcinoma (ACC) is a low-grade salivary gland malignancy characterized by serous acinar differentiation. Most ACCs arise in the parotid gland, but ACCs have been reported to originate in nonparotid salivary glands where serous acini are less abundant. Given the recent discovery of mammary analog secretory carcinoma (MASC)-a salivary malignancy that histologically mimics ACC-a retrospective reevaluation of nonparotid ACCs is warranted. The surgical pathology archives of The Johns Hopkins Hospital were searched for all ACCs arising outside of the parotid gland. For each case, the histologic slides were reviewed; immunohistochemical analysis (mammaglobin, S100 protein) was performed; and confirmatory ETV6 breakapart fluorescence in situ hybridization assay was completed. Demographic and clinical data were obtained from the medical records. Fourteen extraparotid tumors diagnosed as ACC were identified. Eleven of 14 (79%) tumors harbored the ETV6 translocation (oral cavity=9 of 11; submandibular gland=2 of 2). The translocation-positive tumors occurred in 7 women and 4 men ranging in age from 20 to 86 years (mean, 56 y) and usually presented as painless masses. Immunohistochemistry for mammaglobin and S100 was positive in all 11 translocation-positive tumors but negative in the 3 translocation-negative tumors. Histologically, the translocation-positive tumors exhibited uniform cells with vacuolated cytoplasm, microcystic/cystic and papillary architecture, and intraluminal secretions; however, the presence of basophilic cytoplasmic granules was conspicuously absent. Basophilic cytoplasmic granules, indicative of true serous acinar differentiation, were present in the 3 translocation-negative tumors. Of the translocation-positive tumors, only 1 locally recurred, and none metastasized. Most alleged ACCs of nonparotid origin actually represent misclassified MASCs. The impact of diagnostic error is mitigated by the low-grade nature of MASC that, like ACCs, do not appear to be clinically aggressive. PMID:23681074

Bishop, Justin A; Yonescu, Raluca; Batista, Denise; Eisele, David W; Westra, William H



Hilar mossy cell circuitry controlling dentate granule cell excitability.  


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

Jinde, Seiichiro; Zsiros, Veronika; Nakazawa, Kazu



Mechanisms of granule membrane recapture following exocytosis in intact mast cells.  


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

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



The Significance of Cytoskeleton in Exocytosis: Control of Apical Plasma Membrane-Evoked Amylase Release from Secretory Granules Isolated from the Rat Parotid Gland  

Microsoft Academic Search

To investigate the significance of cytoskeletal proteins in the exocytotic in vitro interaction system, we observed the effects of two types of inhibitors, colchicine and cytochalasin D, which depolymerize the microtuble structure consisting of tubulin and microfilament consisting of actins, respectively. The apical plasma membrane-evoked amylase release was increased approximately two times by colchicine. These findings suggest that secretory granules




P Granule Assembly and Function in Caenorhabditis elegans Germ Cells  

PubMed Central

Germ granules are large, non–membrane-bound, ribonucleoprotein (RNP) organelles found in the germ line cytoplasm of most, if not all, animals. The term germ granule is synonymous with the perinuclear nuage in mouse and human germ cells. These large RNPs are complexed with germ line–specific cytoplasmic structures such as the mitochondrial cloud, intermitochondrial cement, and chromatoid bodies. The widespread presence of germ granules across species and the associated germ line defects when germ granules are compromised suggest that germ granules are key determinants of the identity and special properties of germ cells. The nematode Caenorhabditis elegans has been a very fruitful model system for the study of germ granules, wherein they are referred to as P granules. P granules contain a heterogeneous mixture of RNAs and proteins. To date, most of the known germ granule proteins across species, and all of the known P granule components in C elegans, are associated with RNA metabolism, which suggests that a main function of germ granules is posttranscriptional regulation. Here we review P granule structure and localization, P granule composition, the genetic pathway of P granule assembly, and the consequences in the germ line when P granule components are lost. The findings in C elegans have important implications for the germ granule function during postnatal germ cell differentiation in mammals.




Co-localization of the adipokinetic hormones I and II in the same glandular cells and in the same secretory granules of corpus cardiacum of Locusta migratoria and Schistocerca gregaria  

Microsoft Academic Search

The immunocytochemical reactivity of the glandular cells of the corpus cardiacum (CCG-cells) of Locusta migratoria and Schistocerca gregaria was investigated at the electron-microscopic level, using the protein A-gold method, with three antisera against fragments of the adipokinetic hormones AKH I and AKH II. This combination of antisera permitted discrimination between anti-AKH I and anti-AKH II immunoreactivity. Fixation in a mixture

J. H. B. Diederen; H. A. Maas; H. J. Pel; H. Schooneveld; W. F. Jansen; H. G. B. Vullings



Quick-freeze, deep-etch visualization of exocytosis in anterior pituitary secretory cells: localization and possible roles of actin and annexin II  

Microsoft Academic Search

The exocytotic process in the anterior pituitary secretory cells was studied using quick-freeze deep-etch electron microscopy, fluorescein-isothiocyanate-phalloidin staining, heavy meromyosin decoration, and immuno-electron microscopy. The subcortical actin filaments are distributed unevenly in the peripheral cytoplasm. Few secretory granules are seen beneath the plasma membrane in the region where the peripheral cytoplasm is occupied by numerous subcortical actin filaments. On the

Takao Senda; Toshio Okabe; Morihiro Matsuda; Hisao Fujita



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

NASA Astrophysics Data System (ADS)

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

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



Identification of a membrane-bound, glycol-stimulated phospholipase A sub 2 located in the secretory granules of the adrenal medulla  

SciTech Connect

Chromaffin granule membranes prepared from bovine adrenal medullae showed Ca{sup 2+}-stimulated phospholipase A{sub 2} (PLA{sub 2}) activity when assayed at pH 9.0 with phosphatidylcholine containing an ({sup 14}C)-arachidonyl group in the 2-position. However, the activity occurred in both soluble and particulate subcellular fractions, and did not codistribute with markers for the secretory granule. PLA{sub 2} activity in the granule membrane preparation was stimulated dramatically by addition of glycerol, ethylene glycole, or poly(ethylene glycol). This glycol-stimulated PLA{sub 2} activity codistributed with membrane-bound dopamine {beta}-hydroxylase, a marker for the granule membranes, through the sequence of differential centrifugation steps employed to prepare the granule membrane fraction, as well as on a sucrose density gradient which resolved the granules from mitochondria, lysosomes, and plasma membrane. The glycol-stimulated PLA{sub 2} of the chromaffin granule was membrane-bound, exhibited a pH optimum of 7.8, retained activity in the presence of EDTA, and was inactivated by p-bromophenacyl bromide. When different {sup 14}C-labeled phospholipids were incorporated into diarachidonylphosphatidylcholine liposomes, 1-palmitoyl-2-arachidonylphosphatidylcholine was a better substrate for this enzyme than 1-palmitoyl-2-oleylphosphatidylcholine or 1-acyl-2-arachidonyl-phosphatidylethhanolamine, and distearoylphosphatidylcholine was not hydrolyzed.

Hildebrandt, E.; Albanesi, J.P. (Univ. of Texas Southwestern Medical Research Center, Dallas (USA))



Golgi apparatus, GERL, and secretory granule formation within neurons of the hypothalamo-neurohypophysial system of control and hyperosmotically stressed mice  

Microsoft Academic Search

The vasopressin-producing neurons of the hypothalamo-neurohypophysial system are a particularlygood model with which to consider the relationship between the Golgi apparatus and GERL and their roles in secretory granule production because these neurons increase theirsynthesisand secretionof vasopressin in response to hyperosmotic stress.Enzyme cytochemical techniques for acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) activitieswere used to distinguishGERL from the Golgi apparatus




Spatiotemporal resolution of mast cell granule exocytosis reveals correlation with Ca2+ wave initiation.  


Mast cell activation initiated by antigen-mediated crosslinking of IgE receptors results in stimulated exocytosis of secretory lysosomes in the process known as degranulation. Much has been learned about the molecular mechanisms important for this process, including the crucial role of Ca(2+) mobilization, but spatio-temporal relationships between stimulated Ca(2+) mobilization and granule exocytosis are incompletely understood. Here we use a novel imaging-based method that uses fluorescein isothiocyanate (FITC)-dextran as a reporter for granule exocytosis in RBL mast cells and takes advantage of the pH sensitivity of FITC. We demonstrate the selectivity of FITC-dextran, accumulated by fluid-phase uptake, as a marker for secretory lysosomes, and we characterize its capacity to delineate different exocytotic events, including full fusion, kiss-and-run transient fusion and compound exocytosis. Using this method, we find strong dependence of degranulation kinetics on the duration of cell to substrate attachment. We combine imaging of degranulation and Ca(2+) dynamics to demonstrate a spatial relationship between the sites of Ca(2+) wave initiation in extended cell protrusions and exocytosis under conditions of limited antigen stimulation. In addition, we find that the spatially proximal Ca(2+) signaling and secretory events correlate with participation of TRPC1 channels in Ca(2+) mobilization. PMID:22393234

Cohen, Roy; Corwith, Kathryn; Holowka, David; Baird, Barbara



Spatiotemporal resolution of mast cell granule exocytosis reveals correlation with Ca2+ wave initiation  

PubMed Central

Summary Mast cell activation initiated by antigen-mediated crosslinking of IgE receptors results in stimulated exocytosis of secretory lysosomes in the process known as degranulation. Much has been learned about the molecular mechanisms important for this process, including the crucial role of Ca2+ mobilization, but spatio-temporal relationships between stimulated Ca2+ mobilization and granule exocytosis are incompletely understood. Here we use a novel imaging-based method that uses fluorescein isothiocyanate (FITC)–dextran as a reporter for granule exocytosis in RBL mast cells and takes advantage of the pH sensitivity of FITC. We demonstrate the selectivity of FITC–dextran, accumulated by fluid-phase uptake, as a marker for secretory lysosomes, and we characterize its capacity to delineate different exocytotic events, including full fusion, kiss-and-run transient fusion and compound exocytosis. Using this method, we find strong dependence of degranulation kinetics on the duration of cell to substrate attachment. We combine imaging of degranulation and Ca2+ dynamics to demonstrate a spatial relationship between the sites of Ca2+ wave initiation in extended cell protrusions and exocytosis under conditions of limited antigen stimulation. In addition, we find that the spatially proximal Ca2+ signaling and secretory events correlate with participation of TRPC1 channels in Ca2+ mobilization.

Cohen, Roy; Corwith, Kathryn; Holowka, David; Baird, Barbara



Cell-specific analysis of tracheobronchial secretory cells and secretions  

SciTech Connect

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

Finkbeiner, W.E.



Rab3a ablation related changes in morphology of secretory vesicles in major endocrine pancreatic cells, pituitary melanotroph cells and adrenal gland chromaffin cells in mice.  


In this work we have compared the ultrastructural characteristics of major pancreatic endocrine cells, pituitary melanotrophs and adrenal chromaffin cells in the normal mouse strain (wild type, WT) and mice with a known secretory deficit, the Rab3a knockout strain (Rab3a KO). For this purpose, pancreata, pituitary glands and adrenal glands from the Rab3a KO and from the WT mice were analysed, using conventional transmission electron microscopy (TEM). In order to assess the significance of the presence of Rab3a proteins in the relevant cells, we focused primarily on their secretory vesicle morphology and distribution. Our results showed a comparable general morphology in Rab3a KO and WT in all assessed endocrine cell types. In all studied cell types, the distribution of secretory granules along the plasma membrane (number of docked and almost-docked vesicles) was comparable between Rab3a KO and WT mice. Specific differences were found in the diameters of their secretory vesicles, diameters of their electron-dense cores and the presence of autophagic structures in the cells of Rab3A KO mice only. Occasionally, individual electron-dense round vesicles were present inside autophagosome-like structures; these were possibly secretory vesicles or their remnants. The differences found in the diameters of the secretory vesicles confirm the key role of Rab3a proteins in controlling the balance between secretory vesicle biogenesis and degradation, and suggest that the ablation of this protein probably changes the nature of the reservoir of secretory vesicles available for regulated exocytosis. PMID:23399968

Lipovšek, Saška; Janžekovi?, Franc; Leitinger, Gerd; Rupnik, Marjan Slak



N-cadherin is dispensable for pancreas development but required for ?-cell granule turnover  

PubMed Central

Summary The cadherin family of cell adhesion molecules mediates adhesive interactions that are required for the formation and maintenance of tissues. Previously, we demonstrated that N-cadherin, which is required for numerous morphogenetic processes, is expressed in the pancreatic epithelium at E9.5, but later becomes restricted to endocrine aggregates in mice. To study the role of N-cadherin during pancreas formation and function we generated a tissue specific knockout of N-cadherin in the early pancreatic epithelium by inter-crossing N-cadherin-floxed mice with Pdx1Cre mice. Analysis of pancreas-specific ablation of N-cadherin demonstrates that N-cadherin is dispensable for pancreatic development, but required for ?-cell granule turnover. The number of insulin secretory granules is significantly reduced in N-cadherin-deficient ?-cells, and as a consequence insulin secretion is decreased.

Johansson, Jenny K; Voss, Ulrikke; Kesavan, Gokul; Kostetskii, Igor; Wierup, Nils; Radice, Glenn L.; Semb, Henrik



N-cadherin is dispensable for pancreas development but required for beta-cell granule turnover.  


The cadherin family of cell adhesion molecules mediates adhesive interactions that are required for the formation and maintenance of tissues. Previously, we demonstrated that N-cadherin, which is required for numerous morphogenetic processes, is expressed in the pancreatic epithelium at E9.5, but later becomes restricted to endocrine aggregates in mice. To study the role of N-cadherin during pancreas formation and function we generated a tissue-specific knockout of N-cadherin in the early pancreatic epithelium by inter-crossing N-cadherin-floxed mice with Pdx1Cre mice. Analysis of pancreas-specific ablation of N-cadherin demonstrates that N-cadherin is dispensable for pancreatic development, but required for beta-cell granule turnover. The number of insulin secretory granules is significantly reduced in N-cadherin-deficient beta-cells, and as a consequence insulin secretion is decreased. PMID:20533404

Johansson, Jenny K; Voss, Ulrikke; Kesavan, Gokul; Kostetskii, Igor; Wierup, Nils; Radice, Glenn L; Semb, Henrik



Involvement of Aquaporin5 Water Channel in Osmoregulation in Parotid Secretory Granules  

Microsoft Academic Search

Aquaporins (AQPs) are a family of channel proteins that allow water or very small solutes to pass, functioning in tissues where the rapid and regulated transport of fluid is necessary, such as the kidney, lung, and salivary glands. Aquaporin-5 (AQP5) has been demonstrated to localize on the luminal surface of the acinar cells of the salivary glands. In this paper,

M. Matsuki; S. Hashimoto; M. Shimono; M. Murakami; J. Fujita-Yoshigaki; S. Furuyama; H. Sugiya



Cytochemical Calcium Distribution in Secretory Ameloblasts of the Rat in Relation to Enamel Mineralization  

Microsoft Academic Search

Calcium distribution in secretory ameloblasts was studied in rat incisor enamel in which mineralization was temporarily disturbed by injection of either fluoride or cobalt. Pyroantimonate precipitates of calcium were analysed morphometrically in regions of the cell membranes, mitochondria and secretory granules. The disturbances in mineralization were characterized by accumulations of unmineralized enamel matrix at the secretory regions of Tomes’ process

S. Chen; D. R. Eisenmann; A. E. Zaki; S. H. Ashrafi



Comparison of cell deaths induced by transmembrane and secretory TNF-?  

Microsoft Academic Search

Summary  Our previous study showed that transmembrane TNF-? (TM-TNF-?) had broader tumoricidal spectrum than secretory TNF-? (s-TNF-?).\\u000a This study examined the difference between the two kinds of TNF-? in inducing cells and the relationship between the apoptosis\\u000a induced by TM-TNF-? and the cell cycle. Bioassay was employed to compare the cytotoxic effect of two kinds of TNF-? on cell\\u000a lines L-929

Lin Yang; Wenfang Shi; Jing Wang; Xiaodan Jiang; Wei Feng; Zuoya Li



Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells  

Microsoft Academic Search

Several types of cells store proteins in secretory vesicles from which they are released by an appropriate stimulus. It might be expected that the secretory vesicles in different cell types use similar molecular machinery. Here we describe a transmembrane glycoprotein (Mr ~100,000) that is present in secretory vesicles in all neurons and endocrine cells studied, in species from elasmobranch fish




Paraquat-induced apoptotic cell death in cerebellar granule cells  

Microsoft Academic Search

We examined the toxicity of paraquat, a possible environmental risk factor for neurodegenerative disorders like Parkinson's disease (PD). Paraquat is structurally similar to the neurotoxin MPP+ that can induce Parkinsonian-like features in rodents, non-human primates and human. Exposure of cerebellar granule cells to relatively low concentrations of paraquat (5 ?M) produces apoptotic cell death with a reduction in mitochondrial cytochrome

Rosa A González-Polo; Andrea Rodr??guez-Mart??n; José M Morán; Mireia Niso; Germán Soler; José M Fuentes



Bicarbonate transport in sheep parotid secretory cells.  


1. Intracellular pH (pH1) was measured by microfluorimetry in secretory endpieces isolated from sheep parotid glands and loaded with the pH-sensitive fluoroprobe 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). 2. Stimulation with 1 microM acetylcholine (ACh) caused a large, transient decrease in pH1 of 0.37 +/- 0.02 pH units followed by a slower recovery. The transient, which was reduced by 60% in the absence of HCO3-, could be attributed mainly to HCO3- efflux. During sustained stimulation, pH1 increased to a value that exceeded the resting value by 0.083 +/- 0.023 pH units after 20 min. 3. The anion channel blocker NPPB (0.1 mM) reduced the transient acidification in response to ACh by 48% and raised pH1 during sustained stimulation. Simultaneous application of NPPB and ACh accelerated the re-alkalinization following the initial acidification, indicating that NPPB inhibits HCO3- efflux. 4. The stilbene derivative H2DIDS (0.5 mM) reduced the transient acidification in response to ACh by 76% but caused a marked decrease in pH1 during sustained stimulation. Simultaneous application of H2DIDS and ACh slowed the re-alkalinization following the initial acidification, indicating that the main effect of H2DIDS was to inhibit HCO3- accumulation. 5. In the absence of HCO3-, the recovery from an acid load was unaffected by ACh stimulation. Acid extrusion, although dependent on Na+, was not inhibited by amiloride (1 mM), clonidine (1 mM) or H2DIDS (0.5 mM) and was therefore provisionally attributed to a Na(+)-H+ exchanger isoform other than NHE1 or NHE2. 6. In the presence of HCO3-, the rate of recovery from an acid load was reduced during ACh stimulation, probably as a result of the increased efflux of HCO3-. Acid extrusion was dependent on Na+ and was significantly inhibited by H2DIDS. 7. We conclude that ACh-evoked HCO3- secretion in the sheep parotid gland differs from that in many other salivary glands by being driven predominantly by basolateral Na(+)-HCO3- cotransport rather than by Na(+)-H+ exchange. PMID:8865077

Steward, M C; Poronnik, P; Cook, D I



The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma membrane  

PubMed Central

Granuphilin, an effector of the small GTPase Rab27a, mediates the stable attachment (docking) of insulin granules to the plasma membrane and inhibits subsequent fusion of docked granules, possibly through interaction with a fusion-inhibitory Munc18-1/syntaxin complex. However, phenotypes of insulin exocytosis differ considerably between Rab27a- and granuphilin-deficient pancreatic ? cells, suggesting that other Rab27a effectors function in those cells. We found that one of the putative Rab27a effector family proteins, exophilin7/JFC1/Slp1, is expressed in ? cells; however, unlike granuphilin, exophilin7 overexpressed in the ?-cell line MIN6 failed to show granule-docking or fusion-inhibitory activity. Furthermore, exophilin7 has no affinities to either Munc18-1 or Munc18-1–interacting syntaxin-1a, in contrast to granuphilin. Although ? cells of exophilin7-knockout mice show no apparent abnormalities in intracellular distribution or in ordinary glucose-induced exocytosis of insulin granules, they do show impaired fusion in response to some stronger stimuli, specifically from granules that have not been docked to the plasma membrane. Exophilin7 appears to mediate the fusion of undocked granules through the affinity of its C2A domain toward the plasma membrane phospholipids. These findings indicate that the two Rab27a effectors, granuphilin and exophilin7, differentially regulate the exocytosis of either stably or minimally docked granules, respectively.

Wang, Hao; Ishizaki, Ray; Xu, Jun; Kasai, Kazuo; Kobayashi, Eri; Gomi, Hiroshi; Izumi, Tetsuro



Abnormal apocrine secretory cell mitochondria in a Huntington disease patient.  


Over two decades, a 42-year old woman experienced the gradual onset of choreic involuntary movements, dystonia, and tics. Decreased caudate nucleus metabolism on 2-deoxyglucose PET scan and a heterozygous 49-CAG repeat expansion within the HTT gene established the diagnosis of HD, although no other family history was known. An axillary skin biopsy revealed a distinctive abnormality of mitochondria limited to the apocrine secretory cells on electron microscopy. All mitochondria were transformed into rounded structures with disrupted cristae and prominent myelin figures; many were enlarged up to 4 times the normal. Cytoplasm of apocrine secretory cells showed an abundance of lipid vacuoles, empty vesicles, and dense bodies. Biopsied skeletal muscle histology (light microscopy) was normal, as was a mitochondrial metabolism study. Biopsies from other HD patients have shown similar mitochondrial changes in cerebral neurons, muscle, fibroblasts, and lymphoblasts, adding to evidence for a systemic disturbance of mitochondria in HD. PMID:22985800

Sidiropoulos, Christos; LeWitt, Peter; Hashimoto, Ken



Visualization of the exocytosis/endocytosis secretory cycle in cultured adrenal chromaffin cells.  


Cultured bovine adrenal medullary chromaffin cells were stimulated to secrete catecholamines by addition of veratridine or nicotine. The formation of an exocytotic pit exposes a major secretory granule membrane antigen, the enzyme dopamine beta-hydroxylase, to the external medium. By including antiserum to this enzyme in the medium, we were able to visualize sites of exocytosis by decoration of bound antibody using a fluorescent second antibody. Internalization of this antibody-antigen complex was then followed in chase experiments: approximately half the surface complex was internalized in 15-30 min. In other experiments, secretion was triggered in the absence of antiserum, and surface enzyme was revealed by binding antibodies at various times after secretion had been halted by an antagonist. Surface patches of antigen remained discrete from the bulk of the plasma membrane for at least 30 min, although a substantial proportion of the antigen was internalized within this time. Cell surface concanavalin A receptors were internalized at a roughly similar rate, suggesting that mechanisms may be similar. After internalization, chromaffin granule membranes fused to larger structures, possibly lysosomes, and were transported over a few hours to the perinuclear region of the cell. PMID:6643581

Phillips, J H; Burridge, K; Wilson, S P; Kirshner, N



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

PubMed Central

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.

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



Densely granulated murine NK cells eradicate large solid tumors.  


Natural killer (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 interleukin 15 (IL-15) released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphologic feature of being densely loaded with periodic acid-Schiff-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 IL-15 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 IL-15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors. PMID:22374983

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



Densely Granulated Murine NK Cells Eradicate Large Solid Tumors  

PubMed Central

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.

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




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...


Isolating stromal stem cells from periodontal granulation tissues  

Microsoft Academic Search

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

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



Microsoft Academic Search

Synchronization of the secretory cycle in vivowas obtained by injecting isoprenaline as an inducer of secretion . A quantitative correlation between enzyme release, its subsequent reaccumulation, and the sequence of ultrastructural changes was found . At the ultra- structural level secretion was paralleled by depletion of zymogen granules through fusion of the granule membrane with the lumen membrane and discharge




Localization of human neutrophil interleukin-8 (CXCL-8) to organelle(s) distinct from the classical granules and secretory vesicles  

Microsoft Academic Search

Mature human neutrophils contain small amounts of interleukin-8 (CXC chemokine ligand 8 (CXCL-8)), which upon proinflammatory activation, increases significantly. It has been sug- gested that the CXCL-8 content of resting human neutrophils is stored in the secretory vesicles. Here, we have used a fractionation technique, which allows isolation of these vesicles, and we find that CXCL-8 neither colocalizes with the

Sara Pellme; Matthias Morgelin; Hans Tapper; Ulf-Henrik Mellqvist; Claes Dahlgren; Anna Karlsson



Young dentate granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion.  


Adult-born granule cells (GCs), a minor population of cells in the hippocampal dentate gyrus, are highly active during the first few weeks after functional integration into the neuronal network, distinguishing them from less active, older adult-born GCs and the major population of dentate GCs generated developmentally. To ascertain whether young and old GCs perform distinct memory functions, we created a transgenic mouse in which output of old GCs was specifically inhibited while leaving a substantial portion of young GCs intact. These mice exhibited enhanced or normal pattern separation between similar contexts, which was reduced following ablation of young GCs. Furthermore, these mutant mice exhibited deficits in rapid pattern completion. Therefore, pattern separation requires adult-born young GCs but not old GCs, and older GCs contribute to the rapid recall by pattern completion. Our data suggest that as adult-born GCs age, their function switches from pattern separation to rapid pattern completion. PMID:22365813

Nakashiba, Toshiaki; Cushman, Jesse D; Pelkey, Kenneth A; Renaudineau, Sophie; Buhl, Derek L; McHugh, Thomas J; Rodriguez Barrera, Vanessa; Chittajallu, Ramesh; Iwamoto, Keisuke S; McBain, Chris J; Fanselow, Michael S; Tonegawa, Susumu



Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells  

PubMed Central

Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087- 2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by preincubating the permeabilized cells with low concentrations of N- ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTP gamma S suggesting a role for GTP- binding proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed only by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the dialyzed salt extract. The formation of nascent secretory vesicles contrasts with prosomatostatin processing which required only ATP for efficient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processing can be uncoupled from the generation of nascent secretory vesicles.



The virtues of youth and maturity (in dentate granule cells).  


How does adult neurogenesis contribute to memory? Nakashiba and colleagues generated mutant mice in which synaptic output from older hippocampal granule cells was specifically blocked. Experiments with these mice reveal an unpredicted age-dependent specialization of function, demonstrating that recently born cells support pattern separation, whereas older cells support pattern completion. PMID:22464319

Lever, Colin; Burgess, Neil



Cholesterol accumulation increases insulin granule size and impairs membrane trafficking  

PubMed Central

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.

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



Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells  

SciTech Connect

Permeabilized cells treated with the adenosine triphosphate analog, (/sup 35/S)adenosine-5'-0-3(3-thiotriphosphate) ((..gamma..-/sup 35/S)ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated /sup 35/S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of /sup 35/S from the cells over a 20 min period. Treatment of permeabilized cells with ATP..gamma..S inhibited secretion and /sup 35/S incorporation into the cells. Pretreatment with ATP..gamma..S resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate /sup 35/S from (..gamma..-/sup 35/S)ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells. 15 references, 1 figure, 3 tables.

Brooks, J.C.; Brooks, M.



Eosinophil crystalloid granules: structure, function, and beyond  

PubMed Central

Eosinophils are granulocytes associated with host defense against parasitic helminths with allergic conditions and more recently, with immunoregulatory responses. Eosinophils are distinguished from leukocytes by their dominant population of cytoplasmic crystalloid (also termed secretory, specific, or secondary) granules that contain robust stores of diverse, preformed cationic proteins. Here, we provide an update on our knowledge about the unique and complex structure of human eosinophil crystalloid granules. We discuss their significance as rich sites of a variety of receptors and review our own recent research findings and those of others that highlight discoveries concerning the function of intracellular receptors and their potential implications in cell signaling. Special focus is provided on how eosinophils might use these intracellular receptors as mechanisms to secrete, selectively and rapidly, cytokines or chemokines and enable cell-free extracellular eosinophil granules to function as independent secretory structures. Potential roles of cell-free eosinophil granules as immune players in the absence of intact eosinophils will also be discussed.

Muniz, Valdirene S.; Weller, Peter F.; Neves, Josiane S.



Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes.  


Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules. PMID:19525969

Herz, Jasmin; Pardo, Julian; Kashkar, Hamid; Schramm, Michael; Kuzmenkina, Elza; Bos, Erik; Wiegmann, Katja; Wallich, Reinhard; Peters, Peter J; Herzig, Stefan; Schmelzer, Elmon; Krönke, Martin; Simon, Markus M; Utermöhlen, Olaf



Multiple modes of GABAergic inhibition of rat cerebellar granule cells  

PubMed Central

Cerebellar granule cells are inhibited phasically by GABA released synaptically from Golgi cells, but are inhibited more powerfully by tonic activity of high affinity ?6 subunit-containing GABAA receptors. During development the tonic activity is generated by the accumulation of GABA released by action potentials, but in the adult the tonic activity is independent of action potentials. Here we show that in adult rats the tonic activation of GABAA receptors is produced by non-vesicular transmitter release and is reduced by the activity of GAT-1 and GAT-3 GABA transporters, demonstrating that alterations of GABA uptake will modulate information flow through granule cells. Acetylcholine (ACh) evokes a large Ca2+-dependent but action potential-independent release of GABA, which activates ?6 subunit-containing GABAA receptors. These data show that three separate modes of transmitter release can activate GABAA receptors in adult cerebellar granule cells: action potential-evoked exocytotic GABA release, non-vesicular release, and ACh-evoked Ca2+-dependent release independent of action potentials. The relative magnitudes of the inhibitory charge transfers generated by action potential-evoked release (during high frequency stimulation of the mossy fibres), tonic inhibition and superfused ACh are 1:3:12, indicating that tonic and ACh-mediated inhibition may play a major role in regulating granule cell firing.

Rossi, David J; Hamann, Martine; Attwell, David



Granule exocytosis mediates immune surveillance of senescent cells  

PubMed Central

Senescence is a stable cell cycle arrest program that contributes to tumor suppression, organismal aging and certain wound healing responses. During liver fibrosis, for example, hepatic stellate cells initially proliferate and secrete extracellular matrix components that produce fibrosis; however, these cells eventually senesce and are cleared by immune cells, including natural killer (NK) cells. Here, we examine how NK cells target senescent cells and assess the impact of this process on liver fibrosis. We show that granule exocytosis, but not death-receptor-mediated apoptosis, is required for NK-cell-mediated killing of senescent cells. This pathway bias is due to upregulation of the decoy death receptor, Dcr2, an established senescence marker that attenuates NK-mediated cell death. Accordingly, mice with defects in granule exocytosis accumulate senescent stellate cells and display more liver fibrosis in response to a fibrogenic agent. Our results thus provide new insights into the immune surveillance of senescent cells and reveal how granule exocytosis has a protective role against liver fibrosis.

Sagiv, A; Biran, A; Yon, M; Simon, J; Lowe, S W; Krizhanovsky, V



Identification of the polyhydroxybutyrate granules in mammalian cultured cells.  


Poly-3-hydroxybutyrate (PHB) is a biological polyester present in bacteria and eukaryotic cells. Long-chain (or storage) sPHB (up to 100,000 residues) is typically present in PHB-accumulating bacteria and localized in specialized granules known as carbonosomes. In these organisms, sPHB plays a major role as carbon and energy storage. On the other hand, short-chain (or complexed) cPHB (10-100 residues) is present in eukaryotic organisms, including mammals as well as in many bacteria. Previous studies indicated that cPHB is localized in various subcellular compartments of the eukaryotic organisms. Here, we used fluorescent microscopy to directly investigate the localization of PHB in mammalian cells. PHB was visualized in cultured U87 cells using fluorescent probe BODIPY 493/503. Specificity of PHB staining was confirmed by markedly decreased fluorescence of samples treated with PHB-specific depolymerase (PhaZ7). We found that PHB is associated with granules, and that these PHB-enriched granules do not co-localized with mitochondria, lysosomes, or endoplasmic reticulum. These results suggest that, in mammalian cells, PHB can accumulate in the cytoplasm in granules similar to 'energy storage' carbonosomes found in PHB-accumulating bacteria. PMID:23161637

Elustondo, Pia; Zakharian, Eleonora; Pavlov, Evgeny



Dynamic functions of GABA signaling during granule cell maturation  

PubMed Central

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

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



Dynamic functions of GABA signaling during granule cell maturation.  


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

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



The cytochemical localization of lysozyme in Paneth cell granules  

Microsoft Academic Search

Synopsis  The breakdown products resulting from the hydrolysis of chitin by lysozyme stain with Alcian Blue. A method based upon this observation has been developed for the histochemical demonstration of lysozyme activity. The application of this method to the jejunal crypts of several animal species indicates that Paneth cell granules contain lysozyme. The binding of the hydrolysis products with Alcian Blue

Y. Ghoos; G. Vantrappen



ICA 512, an autoantigen of type I diabetes, is an intrinsic membrane protein of neurosecretory granules.  

PubMed Central

Islet cell autoantigen (ICA) 512 is a novel autoantigen of insulin-dependent diabetes mellitus (IDDM) which is homologous to receptor-type protein tyrosine phosphatases (++PTPases). We show that ICA 512 is an intrinsic membrane protein of secretory granules expressed in insulin-producing pancreatic beta-cells as well as in virtually all other peptide-secreting endocrine cells and neurons containing neurosecretory granules. ICA 512 is cleaved at its luminal domain and, following exposure at the cell surface, recycles to the Golgi complex region and is sorted into newly formed secretory granules. By immunoprecipitation, anti-ICA 512 autoantibodies were detected in 15/17 (88%) newly diagnosed IDDM patients, but not in 10/10 healthy subjects. These results suggest that tyrosine phosphorylation participates in some aspect of secretory granule function common to all neuroendocrine cells and that a subset of autoantibodies in IDDM is directed against an integral membrane protein of insulin-containing granules. Images

Solimena, M; Dirkx, R; Hermel, J M; Pleasic-Williams, S; Shapiro, J A; Caron, L; Rabin, D U



The Dual Glutamatergic\\/GABAergic Phenotype of Hippocampal Granule Cells  

Microsoft Academic Search

\\u000a Not only are markers of the glutamatergic and GABAergic phenotype both found in developing hippocampal granule cells but,\\u000a the activation of these cells simultaneously produces responses mediated by glutamate and GABA receptors in their postsynaptic\\u000a cells. In the adult, the markers of the GABAergic phenotype and as a consequence, GABAergic transmission disappears. However,\\u000a these elements can still be transiently expressed

R. Gutiérrez


Two activation states of the prohormone convertase PC1 in the secretory pathway.  


PC1, a neuroendocrine member of the prohormone convertase family of serine proteinases, is implicated in the processing of proproteins in the secretory pathway. PC1 is synthesized as a zymogen and cleaves not only its own profragment in the endoplasmic reticulum, but a subset of protein substrates in the Golgi apparatus and in the Golgi-distal compartments of the regulated secretory pathway. Likewise, mouse PC1 (mPC1) has previously been shown to cleave human prorenin in GH4 cells (that contain secretory granules) while being unable to cleave prorenin in cells, such as Chinese hamster ovary (CHO) or BSC-40, which are devoid of secretory granules. In the current study, we show that removal of a C-terminal tail of mPC1 allows the efficient cleavage of prorenin in the constitutive secretory pathway of CHO cells. The C-terminal tail thus appears to act as an inhibitor of PC1 activity against certain substrates in the endoplasmic reticulum and Golgi apparatus, and its removal, which occurs naturally in secretory granules, may explain the observed granule-specific processing of certain proproteins. These results also demonstrate that PC1 is present in a partially active state prior to the secretory granules where it is processed to a maximally active state. PMID:9182540

Jutras, I; Seidah, N G; Reudelhuber, T L; Brechler, V



Dendritic-cell activation by secretory phospholipase A2.  


Dendritic cells (DCs), also referred to as the sentinels of the immune system, induce and coordinate important functions of immune surveillance. DCs acquire immunity-initiating capacity only after a process of maturation usually induced by ligands that bind to members of the tumor necrosis factor (TNF) or toll-like receptor families. Secretory phospholipase A2 (sPLA2), which hydrolyzes the sn-2 ester bond of glycerophospholipids, regulates a variety of cellular functions including migration of endothelial cells and neurite outgrowth. In the present study we investigated the role of sPLA2 in DC biology. We report that human monocyte-derived DC cultures lack sPLA2 activity but respond to exogenous sPLA2. sPLA2 alone and in cooperation with TNF-alpha and interleukin 1 beta (IL-1beta) induced fatty acid release from DC membranes, which was accompanied by upregulation of surface markers and by an increase in the migratory and immunostimulatory capacity of the DCs. Our findings indicate that secreted enzymes such as sPLA2 can contribute to DC maturation and emphasize the role of lipid mediators in the regulation of immune responses. This observation may also have implications for DC-based vaccine development. PMID:15494434

Ramoner, Reinhold; Putz, Thomas; Gander, Hubert; Rahm, Andrea; Bartsch, Georg; Schaber, Claudia; Thurnher, Martin



A pivotal role of calcineurin signaling in development and maturation of postnatal cerebellar granule cells  

Microsoft Academic Search

Primary culture of postnatal cerebellar granule cells provides a model system that recapitulates many molecular events of developing granule cells in vivo. Depolarization of cultured granule cells increases intracellular Ca2+ and activates Ca2+\\/calmodulin-dependent calcineurin (CaN) phosphatase. This Ca2+ signaling mimics some of the signaling events for proliferation, migration, and differentiation of granule cellsin vivo. We investigated the genome-wide expression profiles

Masaaki Sato; Kazunori Suzuki; Hiroshi Yamazaki; Shigetada Nakanishi



Effects of chronic ethanol exposure on cultured cerebellar granule cells  

Microsoft Academic Search

The aim of this study was to investigate the lipid content and composition of rat cerebellar granule cells grown in the presence\\u000a of ethanol (40, 55, or 80 mM) during in vitro differentiation. Quantitative analyses showed no effects of 40 mM ethanol, whereas a significant increase of total cholesterol was observed at 55 mM. Cells exposed to the highest ethanol

Fausta Omodeo-Salé; Marina Pitto; Massimo Masserini; Paola Palestini



Cerebellar granule cell genesis in the hydrocortisone-treated rats.  


The development of the cerebellar cortex was studied by quantitative light microscopic methods in rats treated with hydrocortisone on postnatal days 7-18. This treatment resulted in a decreased number of cells in the external granular layer (EGL) and an early disappearance of the EGL. In the adult cerebellum of these neonatally treated animals, the total number of granule cells in lobule VIII was decreased by 41% and that of molecular layer microneurons by 28%. Autoradiographic determinations of the 'birthdays' (time of origin) of cerebellar microneurons showed that the peaks of cell formation in the internal granular and molecular layers occurred at earlier ages than in controls. In the granule cell population, this observation was the result of both decreased cell proliferation during the treatment and premature cessation of division in granule cell precursors in the EGL. The morphological effects of 'late' hydrocortisone treatment on the cerebellum observed in this study are compared with those of 'early' hydrocortisone treatment on days 1-4 and those of thyroid hormone from previous studies. It is suggested that endogenous levels of both thyroid and glucocorticoid hormones during the neonatal period may influence the rate of neurogenesis in the postnatal rat cerebellum. PMID:7418632

Bohn, M C; Lauder, J M



Postnatal development of the granule cell in the kitten cerebellum.  


Newborn to 9-week-old kittens were anesthetized and then sacrificed by inserting a scalpel into the thoracic cavity and severing the descending aorta. The cerebella were removed and processed according to Golgi-Cox and rapid Golgi modifications. The results indicate that the dendritic processes of the granule cells undergo a marked postnatal maturation even though there is little postmigratory change in the size and shape of the cell body. A club-shaped swelling of the distal portions of the dendrites is observed by the end of the first week; this "claw" becomes multilobated by three weeks and displays elaborate digitiform projections by nine weeks. The sequence of postnatal development in the kitten appears to follow a time course similar to that observed in other species. A possible correlation between the postnatal development of the granule cell and the postnatal development of the spincocerebellar system is discussed. PMID:646872

Smith, D E; Downs, I



Cytoskeleton-secretory vesicle interactions during the docking of secretory vesicles at the cell membrane in Paramecium tetraurelia cells  

PubMed Central

Stationary-phase cells of Paramecium tetraurelia have most of their many secretory vesicles ("trichocysts") attached to the cell surface. Log-phase cells contain numerous unoccupied potential docking sites for trichocysts and many free trichocysts in the cytoplasm. To study the possible involvement of cytoskeletal elements, notably of microtubules, in the process of positioning of trichocysts at the cell surface, we took advantage of these stages. Cells were stained with tannic acid and subsequently analyzed by electron microscopy. Semithin sections allowed the determination of structural connections over a range of up to 10 micrometer. Microtubules emanating from ciliary basal bodies are seen in contact with free trichocysts, which appear to be transported, with their tip first, to the cell surface. (This can account for the saltatory movement reported by others). It is noteworthy that the "rails" represented by the microtubules do not directly determine the final attachment site of a trichocyst. Unoccupied attachment sites are characterized by a "plug" of electron-dense material just below the plasma membrane; the "plug" seems to act as a recognition or anchoring site; this material is squeezed out all around the trichocyst attachment zone, once a trichocyst is inserted (Westphal and Plattner, in press. [53]). Slightly below this "plug" we observed fasciae of microfilaments (identified by immunocytochemistry using peroxidase labeled F(ab) fragments against P. tetraurelia actin). Their arrangement is not altered when a trichocyst is docked. These fasciae seem to form a loophole for the insertion of a trichocyst. Trichocyst remain attached to the microtubules originating from the ciliary basal bodies--at least for some time--even after they are firmly installed in the preformed attachment sites. Evidently, the regular arrangement of exocytotic organelles is controlled on three levels: one operating over a long distance from the exocytosis site proper (microtubules), one over a short distance (microfilament bundles), and one directly on the exocytosis site ("plug").



Neuroligin-1 Overexpression in Newborn Granule Cells In Vivo  

PubMed Central

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

Schnell, Eric; Bensen, AeSoon L.; Washburn, Eric K.; Westbrook, Gary L.



Generation of cerebellar granule neurons in vivo by transplantation of BMP-treated neural progenitor cells  

Microsoft Academic Search

Cerebellar granule neurons, the most abundant class of CNS neurons, have a critical role in cerebellar function. Granule neurons are generated at the dorsal border of the mesencephalon and metencephalon, the rhombic lip. In the mouse embryo, rhombic lip cells express a number of granule neuron markers, notably the bHLH transcription factor Math1. Dorsal midline cells adjacent to the rhombic

Janet Alder; Kevin J. Lee; Thomas M. Jessell; Mary E. Hatten



The use of lectins as markers for differentiated secretory cells in planarians  

PubMed Central

Freshwater planarians have reemerged as excellent models to investigate mechanisms underlying regeneration. The introduction of molecular tools has facilitated the study of planarians, but cell- and tissue-specific markers are still needed to examine differentiation of most cell types. Here we report the utility of fluorescent lectin-conjugates to label tissues in the planarian Schmidtea mediterranea. We show that 16 lectin-conjugates stain planarian cells or tissues; 13 primarily label the secretory cells, their cytoplasmic projections, and terminal pores. Thus, we examined regeneration of the secretory system using lectin markers and functionally characterized two genes expressed in the secretory cells: marginal adhesive gland-1 (mag-1) and Smed-reticulocalbin1 (Smed-rcn1). RNAi knockdown of these genes caused a dramatic reduction of secretory cell lectin staining, suggesting a role for mag-1 and Smed-rcn1 in secretory cell differentiation. Our results provide new insights into planarian secretory system regeneration and add new markers for labeling several planarian tissues.

Zayas, Ricardo M.; Cebria, Francesc; Guo, Tingxia; Feng, Junjie; Newmark, Phillip A.



Functional extracellular eosinophil granules: novel implications in eosinophil immunobiology  

PubMed Central

Human eosinophils contain within their cytoplasmic granules multiple preformed proteins, including over three dozen cytokines with nominal Th1, Th2 and immunoregulatory capabilities and four distinctive cationic proteins. The secretion of these granule-derived proteins within eosinophils occurs principally by a mechanism whereby selected proteins are mobilized into vesicles for transport to and release at the cell surface. In contrast, the enigmatic presence of membrane-bound cell free granules extruded from eosinophils has been long recognized in tissues associated with eosinophilia, including allergic diseases and responses to helminths. Functional capabilities for extracellular granules have recently been demonstrated. Eosinophil granules express cytokine receptors on their membranes and function, upon extrusion from eosinophils, as independent secretory organelles releasing granule constituents in response to activating cytokines and chemokines. We provide an update on the processes that mediate selective protein secretion from within eosinophil granules both as intracellular organelles and, as novelty demonstrated, as cell-free extracellular structures.

Neves, Josiane S.; Weller, Peter F.



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

Microsoft Academic Search

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)

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



Cytoplasmic granule formation in mouse pancreatic acinar cells. Evidence for formation of immature granules (condensing vacuoles) by aggregation and fusion of progranules of unit size, and for reductions in membrane surface area and immature granule volume during granule maturation  

Microsoft Academic Search

We used a computer-assisted morphometry approach to analyze quantitatively the process of cytoplasmic granule formation in mouse pancreatic acinar cells stimulated with pilocarpine to induce secretion. Our findings suggest that each condensing vacuole\\/immature granule of pancreatic acinar cells is formed by the progressive aggregation of 106 to 128 “unit progranules” of narrowly fixed volume, define a range of 7.7 to

Sylvia Lew; Ilan Hammel; Stephen J. Galli



Selective and Signal-dependent Recruitment of Membrane Proteins to Secretory Granules Formed by Heterologously Expressed von Willebrand Factor  

Microsoft Academic Search

von Willebrand factor (vWF) is a large, multimeric protein secreted by endothelial cells and involved in hemostasis. When expressed in AtT-20 cells, vWF leads to the de novo formation of cigar-shaped organelles similar in appearance to the Weibel-Palade bodies of endothelial cells in which vWF is normally stored before regulated secretion. The membranes of this vWF-induced organelle, termed the pseudogranule,

Anastasia D. Blagoveshchenskaya; Matthew J. Hannah; Simon Allen; Daniel F. Cutler



Plasticity of the ? cell insulin secretory competence: preparing the pancreatic ? cell for the next meal  

PubMed Central

It is well established that the acute rise in plasma glucose and in the incretin hormones glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (7–36) amide (GLP-1), as occurs during a meal, is of pivotal importance in regulating the minute-to-minute output of insulin from pancreatic ? cells. In addition to this well studied acute effect, both glucose and incretin hormones have been recently observed to determine the future secretory responsiveness of the cells. Such plasticity of the insulin secretory competence would imply that glucose and incretins not only act during the present meal, but also help to prepare the ? cells to function during the subsequent meal. Evidence supporting this hypothesis is growing as a result of physiological studies of cultured ? cells (either primary cells or ? cell lines), as well as from an increasing number of large-scale gene expression studies, exploring transcriptional and post-transcriptional events in genes regulated by glucose and incretins. On the basis of this hypothesis, one can speculate that genetic or environmental disturbances of plasticity of the insulin secretory competence is one aspect of ? cell dysfunction that can contribute to the aetiology of type 2 diabetes.

Hinke, Simon A; Hellemans, Karine; Schuit, Frans C



Development of secretory cells and crystal cells in Eichhornia crassipes ramet shoot apex.  


The distribution and development of secretory cells and crystal cells in young shoot apexes of water hyacinth were investigated through morphological and cytological analysis. The density of secretory cells and crystal cells were high in parenchyma tissues around the vascular bundles of shoot apexes. Three developmental stages of the secretory cells can be distinguished under transmission electron microscopy. Firstly, a large number of electron-dense vesicles formed in the cytoplasm, then fused with the tonoplast and released into the vacuole in the form of electron-dense droplets. As these droplets fused together, a large mass of dark material completely filled the vacuole. To this end, a secretion storage vacuole (SSV) formed. Secondly, an active secretion stage accompanied with degradation of the large electron-dense masses through an ill-defined autophagic process at periphery and in the limited internal regions of the SSV. Finally, after most storage substances were withdrawn, the materials remaining in the spent SSV consisted of an electron-dense network structure. The distribution and development of crystal cells in shoot apical tissue of water hyacinth were also studied by light and electron microscopy. Crystals initially formed at one site in the vacuole, where tube-like membrane structures formed crystal chambers. The chamber enlarged as the crystal grew in bidirectional manner and formed needle-shaped raphides. Most of these crystals finally occurred as raphide bundles, and the others appeared as block-like rhombohedral crystals in the vacuole. These results suggest that the formation of both secretory cells and crystal cells are involved in the metamorphosis of vacuoles and a role for vacuoles in water hyacinth rapid growth and tolerance. PMID:20461420

Xu, Guo Xin; Tan, Chao; Wei, Xiao Jing; Gao, Xiao Yan; Zheng, Hui Qiong



Chromogranin A promotes peptide hormone sorting to mobile granules in constitutively and regulated secreting cells: role of conserved N- and C-terminal peptides.  


Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N- and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N- and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA. PMID:19179339

Montero-Hadjadje, Maité; Elias, Salah; Chevalier, Laurence; Benard, Magalie; Tanguy, Yannick; Turquier, Valérie; Galas, Ludovic; Yon, Laurent; Malagon, Maria M; Driouich, Azeddine; Gasman, Stéphane; Anouar, Youssef



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

PubMed Central

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

Bertrand, Florie; Muller, Sabina; Roh, Kyung-Ho; Laurent, Camille; Dupre, Loic; Valitutti, Salvatore



Long-term plasticity of excitatory inputs to granule cells in the rat olfactory bulb  

PubMed Central

Using 2-photon-guided focal stimulation, we demonstrate spike timing-dependent plasticity of proximal excitatory inputs to olfactory bulb granule cells that originate, in part, from cortical feedback projections. The protocol that potentiated proximal inputs depressed distal, dendrodendritic inputs to granule cells. Granule cell EPSPs, and mitral cell inhibition, also were potentiated by theta-burst stimulation. Plasticity of cortical feedback inputs to interneurons provides a novel mechanism to encode information by modulating bulbar inhibition.

Gao, Yuan; Strowbridge, Ben W.



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

PubMed Central

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

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



Protein folding in the secretory pathway of animal cells  

Microsoft Academic Search

The exit of newly-synthesized proteins from the lumen of the endoplasmic reticulum (ER) is the rate-determining step in protein secretion. Only correctly-folded and fully-assembled proteins exit the ER and progress along the secretory pathway. Folding and assembly in the ER are mediated by a variety of factors including folding catalysts and molecular chaperones. The properties of these factors, and the

Robert B. Freedman; Carole Greenall; Nigel Jenkins; Mick F. Tuite



Spontaneous calcium waves in granule cells in cerebellar slice cultures.  


Multiple regions in the CNS display propagating correlated activity during embryonic and postnatal development. This activity can be recorded as waves of increased calcium concentrations in spiking neurons or glia cells, and have been suggested to be involved in patterning, axonal guidance and establishment of synaptic transmission. Here, we used calcium imaging in slice cultures of the postnatal cerebellum, and observe spontaneous propagating calcium waves in NeuN-positive granule-like cells. Wave formation was blocked by TTX and the AMPA antagonist NBQX, but persisted after NMDA receptor blockade with MK-801. Whole-cell recordings during wave formation showed cyclic EPSP barrages with an amplitude of 10-20mV concurrent with wave activity. Local non-propagating putative transglial waves were also present in the cultures, and could be reproduced by pressure application of ATP. We hypothesize, that the propagating wave activity is carried through the tissue by axonal collaterals formed by neighboring granule cells, and further suggest that the correlated activity may be related to processes that ensure correct postnatal wiring of the cerebellar circuits. PMID:23973304

Apuschkin, Mia; Ougaard, Maria; Rekling, Jens C



Stochastic Differential Equation Model for Cerebellar Granule Cell Excitability  

PubMed Central

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.

Yli-Harja, Olli



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

Microsoft Academic Search

Recurrent inhibition in olfactory bulb mitral cells is mediated via reciprocal dendrodendritic synapses with granule cells. Al- though GABAergic granule cells express both NMDA and non- NMDA glutamate receptors, dendrodendritic inhibition (DDI) relies on the activation of NMDA receptors. Using whole-cell recordings from rat olfactory bulb slices, we now show that olfactory NMDA receptors have a dual role; they depolarize

Brian Halabisky; Daniel Friedman; Milan Radojicic; Ben W. Strowbridge


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

PubMed Central

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.

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



Mechanisms of ethanol-induced death of cerebellar granule cells.  


Maternal ethanol exposure during pregnancy may cause fetal alcohol spectrum disorders (FASD). FASD is the leading cause of mental retardation. The most deleterious effect of fetal alcohol exposure is inducing neuroapoptosis in the developing brain. Ethanol-induced loss of neurons in the central nervous system underlies many of the behavioral deficits observed in FASD. The cerebellum is one of the brain areas that are most susceptible to ethanol during development. Ethanol exposure causes a loss of both cerebellar Purkinje cells and granule cells. This review focuses on the toxic effect of ethanol on cerebellar granule cells (CGC) and the underlying mechanisms. Both in vitro and in vivo studies indicate that ethanol induces apoptotic death of CGC. The vulnerability of CGC to ethanol-induced death diminishes over time as neurons mature. Several mechanisms for ethanol-induced apoptosis of CGC have been suggested. These include inhibition of N-methyl-D-aspartate receptors, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, disturbance of potassium channel currents, thiamine deficiency, and disruption of translational regulation. Cultures of CGC provide an excellent system to investigate cellular/molecular mechanisms of ethanol-induced neurodegeneration and to evaluate interventional strategies. This review will also discuss the approaches leading to neuroprotection against ethanol-induced neuroapoptosis. PMID:20927663

Luo, Jia



Mechanisms of Ethanol-induced Death of Cerebellar Granule Cells  

PubMed Central

Maternal ethanol exposure during pregnancy may cause fetal alcohol spectrum disorders (FASD). FASD is the leading cause of mental retardation. The most deleterious effect of fetal alcohol exposure is inducing neuroapoptosis in the developing brain. Ethanol-induced loss of neurons in the central nervous system (CNS) underlies many of the behavioral deficits observed in FASD. The cerebellum is one of the brain areas that is most susceptible to ethanol during development. Ethanol exposure causes a loss of both cerebellar Purkinje cells and granule cells. This review focuses on the toxic effect of ethanol on cerebellar granule cells (CGC) and the underlying mechanisms. Both in vitro and in vivo studies indicate that ethanol induces apoptotic death of CGC. The vulnerability of CGC to ethanol-induced death diminishes over time as neurons mature. Several mechanisms for ethanol-induced apoptosis of CGC have been suggested. These include inhibition of NMDA receptors, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, disturbance of potassium channel currents, thiamine deficiency, and disruption of translational regulation. Cultures of CGC provide an excellent system to investigate cellular/molecular mechanisms of ethanol-induced neurodegeneration and to evaluate interventional strategies. This review will also discuss the approaches leading to neuroprotection against ethanol-induced neuroapoptosis.

Luo, Jia



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

NASA Astrophysics Data System (ADS)

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

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



Maturation of glutamatergic neurotransmission in dentate gyrus granule cells.  


We studied the development of glutamatergic neurotransmission in dentate gyrus granule cells (GCs) in hippocampal slices from 5 to 12-day-old rats. The active postnatal neuronogenesis in dentate permits GCs with staggered birthdates to be studied in situ in a single preparation. We recorded evoked responses to medial perforant path stimulation using visually-guided whole-cell patch clamping to select immature GCs, and biocytin filling to correlate electrophysiologic responses with maturational stage. Even within this immature cell population we found four distinct electrophysiologic patterns. Type 1 cells had no glutamatergic current; Type 2 cells had only N-methyl-D-aspartate receptor (NMDA) current; Type 3 cells had both NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current although the NMDA component could be isolated at low stimulus intensity (NMDA thresholdcells had both AMPA and NMDA currents with NMDA threshold>/=AMPA threshold. Type 1 cells were least mature, and Type 4 cells most mature as assessed by cell properties, dendritic arborization, and penetration of dendrites into the molecular layer. Thus NMDA-mediated currents predominate early in GC development as is consistent with their role in processes that determine dentate architecture - neuronal migration, dendritic outgrowth and regression, and synapse stabilization. By analogy with 'silent synapses' (i.e. synapses that contain only NMDA receptors), Type 2 cells are candidate 'silent cells' that may undergo activity-dependent acquisition of functional fast-conducting AMPA receptors with maturation. PMID:11113509

Ye, G L; Song Liu, X; Pasternak, J F; Trommer, B L



Microglia-associated granule cell death in the normal adult dentate gyrus.  


Microglial cells are constantly monitoring the central nervous system for sick or dying cells and pathogens. Previous studies showed that the microglial cells in the dentate gyrus have a heterogeneous morphology with multipolar cells in the hilus and fusiform cells apposed to the granule cell layer both at the hilar and at the molecular layer borders. Although previous studies showed that the microglia in the dentate gyrus were not activated, the data in the present study show dying granule cells apposed by Iba1-immunolabeled microglial cell bodies and their processes both at hilar and at molecular layer borders of the granule cell layer. Initially, these Iba1-labeled microglial cells surround individual, intact granule cell bodies. When small openings in the plasma membrane of granule cells are observed, microglial cells are apposed to these openings. When larger openings in the plasma membrane occur at this site of apposition, the granule cells display watery perikaryal cytoplasm, watery nucleoplasm and damaged organelles. Such morphological features are characteristic of neuronal edema. The data also show that following this localized disintegration of the granule cell's plasma membrane, the Iba1-labeled microglial cell body is found within the electron-lucent perikaryal cytoplasm of the granule cell, where it is adjacent to the granule cell's nucleus which is deformed. We propose that granule cells are dying by a novel microglia-associated mechanism that involves lysis of their plasma membranes followed by neuronal edema and nuclear phagocytosis. Based on the morphological evidence, this type of cell death differs from either apoptosis or necrosis. PMID:19936784

Ribak, Charles E; Shapiro, Lee A; Perez, Zachary D; Spigelman, Igor



Hilar mossy cell degeneration causes transient dentate granule cell hyperexcitability and impaired pattern separation.  


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

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



Secretory cells in Piper umbellatum (Piperaceae) leaves: A new example for the development of idioblasts  

Microsoft Academic Search

This work aims to investigate the origins and development of secretory cells in Piper umbellatum (L.) Miq. (Piperaceae) leaves as well as the course and the nature of their secretion. The results were compared with studies in oil-secreting cells of several species. Fully expanded fresh leaves were sectioned and subjected to different histochemical tests. Leaves in different developmental stages were

Cristina Ribeiro Marinho; Adilson Ariza Zacaro; Marília Contin Ventrella


A pivotal role of calcineurin signaling in development and maturation of postnatal cerebellar granule cells.  


Primary culture of postnatal cerebellar granule cells provides a model system that recapitulates many molecular events of developing granule cells in vivo. Depolarization of cultured granule cells increases intracellular Ca(2+) and activates Ca(2+)/calmodulin-dependent calcineurin (CaN) phosphatase. This Ca(2+) signaling mimics some of the signaling events for proliferation, migration, and differentiation of granule cells in vivo. We investigated the genome-wide expression profiles of depolarization- and CaN-regulated genes in cultured mouse granule cells and addressed their relevance to gene regulation in developing granule cells in vivo. Granule cells were cultured under a nondepolarization condition (5 mM KCl) and a depolarization condition (25 mM KCl) with and without the CaN inhibitor FK506. Gene expression profiles between depolarization and nondepolarization and between FK506 treatment and untreatment were analyzed by microarray techniques. Both depolarization and FK506 treatment influence expression levels of a large number of genes, most of which are overlapping, however, are conversely regulated by these two treatments. Importantly, many of the FK506-responsive genes are up- or down-regulated in parallel with gene expression in postnatal granule cells in vivo. The FK506-down-regulated genes are highly expressed in proliferating/premigratory granule cells and many of these genes encode cellular components involved in cell proliferation, migration, and differentiation. In contrast, the FK506-up-regulated genes are predominantly expressed in postmigratory granule cells, including many functional molecules implicated in synaptic transmission and modulation. This investigation demonstrates that the CaN signaling plays a pivotal role in development and synaptic organization of granule cells during the postnatal period. PMID:15809415

Sato, Masaaki; Suzuki, Kazunori; Yamazaki, Hiroshi; Nakanishi, Shigetada



Novel actin rings within the secretory cells of honeybee royal jelly glands.  


We describe a novel cytoskeletal element within secretory cells of an arthropod gland system, the hypopharyngeal gland of the honeybee, Apis mellifera. The hypopharyngeal secretory cells are the source of royal jelly in nurse bees and enzymes in foragers. Each cell possesses an elongate invagination that is occupied by a tubular cuticular structure, the end-apparatus, that accumulates secretion and transfers it into a cuticular microtube and then into a collecting duct. Within the secretory cell, a conspicuous series of actin rings, about 3 ?m in diameter, follows the same path as the end-apparatus, surrounding it at spaced intervals. Transmission electron microscopy confirmed that the actin rings lie within septa of the secretory cell that are closely juxtaposed to the end-apparatus at regularly spaced intervals. We speculate that the function of the actin rings is to hold the end apparatus in place as secretion swells the extracellular compartments between the end apparatus and the cell membrane. To our knowledge, no such cytoskeletal component has been described in animal cells. © 2012 Wiley Periodicals, Inc. PMID:22903954

Kheyri, Homayoun; Cribb, Bronwen W; Reinhard, Judith; Claudianos, Charles; Merritt, David J



Contributions of mature granule cells to structural plasticity in temporal lobe epilepsy  

PubMed Central

During the development of epilepsy in adult animals, newly-generated granule cells integrate abnormally into the hippocampus. These new cells migrate to ectopic locations in the hilus, develop aberrant basal dendrites, contribute to mossy fiber sprouting and exhibit changes in apical dendrite structure and dendritic spine number. Mature granule cells do not appear to exhibit migration defects, basal dendrites and mossy fiber sprouting, but whether they exhibit apical dendrite abnormalities or spine changes is not known. To address these questions, we examined the apical dendritic structure of BrdU-birthdated, GFP-expressing granule cells born two months before pilocarpine-induced status epilepticus. In contrast to immature granule cells, exposing mature granule cells to status epilepticus did not significantly disrupt the branching structure of their apical dendrites. Mature granule cells did, however, exhibit significant reductions in spine density and spine number relative to age-matched cells from control animals. These data demonstrate that while mature granule cells are resistant to developing the gross structural abnormalities exhibited by younger granule cells, they show similar plastic rearrangement of their dendritic spines.

Santos, Victor R.; de Castro, Olagide Wagner; Pun, Raymund Y.K.; Hester, Michael S.; Murphy, Brian L.; Loepke, Andreas W.; Garcia-Cairasco, Norberto; Danzer, Steve C.



Myosin IIA is required for cytolytic granule exocytosis in human NK cells  

PubMed Central

Natural killer (NK) cell cytotoxicity involves the formation of an activating immunological synapse (IS) between the effector and target cell through which granzymes and perforin contained in lytic granules are delivered to the target cell via exocytosis. Inhibition of nonmuscle myosin II in human NK cells with blebbistatin or ML-9 impaired neither effector–target cell conjugation nor formation of a mature activating NK cell IS (NKIS; formation of an actin ring and polarization of the microtubule-organizing center and cytolytic granules to the center of the ring). However, membrane fusion of lytic granules, granzyme secretion, and NK cell cytotoxicity were all effectively blocked. Specific knockdown of the myosin IIA heavy chain by RNA interference impaired cytotoxicity, membrane fusion of lytic granules, and granzyme secretion. Thus, myosin IIA is required for a critical step between NKIS formation and granule exocytosis.

Andzelm, Milena M.; Chen, Xi; Krzewski, Konrad; Orange, Jordan S.; Strominger, Jack L.



GABA(A) receptor modulation in rat cerebellum granule cells.  


The inhibitory GABA(A) receptor is a key element in determining the pattern of nerve cell electrical activity. Thus, modulation of its function is of paramount impact in shaping neuronal functional activity under physiological and pathological conditions. This applies to cerebellar granule neurons as to all the other neurons in the brain. The culture of cerebellar granules from newborn rats is a convenient means by which to approach these cells for electrophysiological studies provided that they maintain, as far as GABA(A) receptors are concerned, the same characteristics as in situ. Thus, the regulation of GABA(A) receptor activity in these neurons has been studied by the patch-clamp technique, both in the whole-cell and outside-out configuration. An obvious first level of control of such receptors' activity is their desensitization under continued agonist application, with biphasic kinetics. The data do not allow one to conclude whether one is dealing with two different populations of receptors or with a single population with two desensitization phases; although the presence of two GABA(A) receptor populations is suggested by a host of observations. The granule cell GABA(A) receptors are modulated by changes in extracellular pH with lower pH resulting in an enhanced receptor activity. They display, under the conditions of whole-cell recording, a run-down phenomenon which is most probably due to a tyrosine phosphatase activity which is in turn under control by a protein serine kinase. Thus, in situ tyrosine phosphorylation is a key element in determining the efficiency of GABA mediated inhibition. Activation of protein kinase A or protein kinase G (PKG) down-regulates GABA(A) receptors' activity. This last event is involved in the depression of those receptors' activity by L-arginine via the production of nitric oxide. In addition, the activity of calmodulin-activated adenylate cyclase I is controlled by GABA(B) receptors. Dendritic GABA(A) receptor activity is partially blocked by previous activation of N-methyl-D-aspartate (NMDA) receptors via calcineurin mediated dephosphorylation/activation of protein tyrosine phosphatase and concomitant production of nitric oxide and PKG activation. The site phosphorylated by PKG is evidently not available for calcineurin-mediated serine dephosphorylation, due to calcineurin-specific membrane localization in respect of the GABA(A) receptor. Overall, a complex network of biochemical signals appear to keep granule cells GABA(A) receptors under a fine balance between up- and down-regulatory mechanisms. The overall data appear also to indicate the presence of two GABA(A) receptor populations: a dendritic one which can be modulated by Ca++ entering via NMDA receptors and a cell body one. The two populations are probably different in terms of desensitization kinetics and benzodiazepine sensitivity. PMID:10952091

Cupello, A; Robello, M



Sequestration of highly expressed mRNAs in cytoplasmic granules, P-bodies, and stress granules enhances cell viability.  


Transcriptome analyses indicate that a core 10%-15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress. PMID:22383896

Lavut, Anna; Raveh, Dina



Reliability of pattern separation by the cerebellar mossy fiber — granule cell system  

Microsoft Academic Search

As a simple example of a neuronal network in which synaptic connectivity among neurons is probabilistic, Marr's model for the granular layer of cat cerebellar cortex is examined. The mean and variance are computed for the fraction of granule cells activated, and for the extent of pattern separation by granule cells, for various mossy fiber inputs and various values of

Jay E. Mittenthal



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

Microsoft Academic Search

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).

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



Stress granules contribute to ?-globin homeostasis in differentiating erythroid cells.  


Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin proteins and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2? and inhibiting the translation of globin proteins when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2?-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced ?-globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells. PMID:22452989

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



The secretory synapse: the secrets of a serial killer.  


Cytotoxic T lymphocytes (CTLs) destroy their targets by a process involving secretion of specialized granules. The interactions between CTLs and target can be very brief; nevertheless, adhesion and signaling proteins segregate into an immunological synapse. Secretion occurs in a specialized secretory domain. Use of live and fixed cell microscopy allows this secretory synapse to be visualized both temporally and spatially. The combined use of confocal and electron microscopy has produced some surprising findings, which suggest that the secretory synapse may be important both in delivering the lethal hit and in facilitating membrane transfer from target to CTL. Studies on the secretory synapse in wild-type and mutant CTLs have been used to identify proteins involved in secretion. Further clues as to the signals required for secretion are emerging from comparisons of inhibitory and activating synapses formed by natural killer cells. PMID:12445272

Bossi, Giovanna; Trambas, Christina; Booth, Sarah; Clark, Richard; Stinchcombe, Jane; Griffiths, Gillian M



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


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

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



Calcium transients in cerebellar granule cell presynaptic terminals.  

PubMed Central

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

Regehr, W G; Atluri, P P



The Arg617-Arg618 cleavage site in the C-terminal domain of PC1 plays a major role in the processing and targeting of the enzyme within the regulated secretory pathway.  


The C-terminal domain of the prohormone convertase PC1 is involved in targeting of the enzyme to secretory granules in neuroendocrine cells and is subsequently processed in this compartment at an Arg617-Arg618 site. Three other dibasics are found in the C-terminal domain of mouse PC1. Here, we examined the role of the four dibasics in targeting PC1 to secretory granules. All 15 possible combinations of dibasic mutations were performed. Wild-type (WT) and mutant PC1 were stably expressed in neuroendocrine PC12 cells that lacked endogenous PC1. Processing, secretion and intracellular localization of PC1 and its mutants were analyzed. Leaving intact Arg617-Arg618 and mutating any combination of the three other dibasics yielded proteins that were stored and processed in secretory granules, similarly to WT PC1. Mutating Arg617-Arg618 alone or with any one of the three remaining dibasics generated proteins that were efficiently stored in secretory granules but were not processed further. Mutating Arg617-Arg618 with more than one of the remaining dibasics produced proteins that reached the TGN but were not stored in secretory granules and exited the cells through the constitutive secretory pathway. These data demonstrate that the Arg617-Arg618 plays a prominent role in targeting PC1 to secretory granules. PMID:12787078

Bernard, Natacha; Kitabgi, Patrick; Rovere-Jovene, Carole



Neuroendocrinelike (small granule) epithelial cells of the lung.  

PubMed Central

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.

DiAugustine, R P; Sonstegard, K S



Regulated and constitutive protein targeting can be distinguished by secretory polarity in thyroid epithelial cells  

PubMed Central

We have studied concurrent apical/basolateral and regulated/constitutive secretory targeting in filter-grown thyroid epithelial monolayers in vitro, by following the exocytotic routes of two newly synthesized endogenous secretory proteins, thyroglobulin (Tg) and p500. Tg is a regulated secretory protein as indicated by its acute secretory response to secretagogues. Without stimulation, pulse-labeled Tg exhibits primarily two kinetically distinct routes: less than or equal to 80% is released in an apical secretory phase which is largely complete by 6-10 h, with most of the remaining Tg retained in intracellular storage from which delayed apical discharge is seen. The rapid export observed for most Tg is unlikely to be because of default secretion, since its apical polarity is preserved even during the period (less than or equal to 10 h) when p500 is released basolaterally by a constitutive pathway unresponsive to secretagogues. p500 also exhibits a second, kinetically distinct secretory route: at chase times greater than 10 h, a residual fraction (less than or equal to 8%) of p500 is secreted with an apical preponderance similar to that of Tg. It appears that this fraction of p500 has failed to be excluded from the regulated pathway, which has a predetermined apical polarity. From these data we hypothesize that a targeting hierarchy may exist in thyroid epithelial cells such that initial sorting to the regulated pathway may be a way of insuring apical surface delivery from one of two possible exocytotic routes originating in the immature storage compartment.



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

Microsoft Academic Search

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

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


Use of transgenic mice to study the routing of secretory proteins in intestinal epithelial cells: analysis of human growth hormone compartmentalization as a function of cell type and differentiation [published erratum appears in J Cell Biol 1990 Jan;110(1):following 227  

PubMed Central

The intestinal epithelium is a heterogeneous cell monolayer that undergoes continuous renewal and differentiation along the crypt-villus axis. We have used transgenic mice to examine the compartmentalization of a regulated endocrine secretory protein, human growth hormone (hGH), in the four exocrine cells of the mouse intestinal epithelium (Paneth cells, intermediate cells, typical goblet cells, and granular goblet cells), as well as in its enteroendocrine and absorptive (enterocyte) cell populations. Nucleotides -596 to +21 of the rat liver fatty acid binding protein gene, when linked to the hGH gene (beginning at nucleotide +3) direct efficient synthesis of hGH in the gastrointestinal epithelium of transgenic animals (Sweetser, D. A., D. W. McKeel, E. F. Birkenmeier, P. C. Hoppe, and J. I. Gordon. 1988. Genes & Dev. 2:1318-1332). This provides a powerful in vivo model for analyzing protein sorting in diverse, differentiating, and polarized epithelial cells. Using EM immunocytochemical techniques, we demonstrated that this foreign polypeptide hormone entered the regulated basal granules of enteroendocrine cells as well as the apical secretory granules of exocrine Paneth cells, intermediate cells, and granular goblet cells. This suggests that common signals are recognized by the "sorting mechanisms" in regulated endocrine and exocrine cells. hGH was targeted to the electron-dense cores of secretory granules in granular goblet and intermediate cells, along with endogenous cell products. Thus, this polypeptide hormone contains domains that promote its segregation within certain exocrine granules. No expression of hGH was noted in typical goblet cells, suggesting that differences exist in the regulatory environments of granular and typical goblet cells. In enterocytes, hGH accumulated in dense-core granules located near apical and lateral cell surfaces, raising the possibility that these cells, which are known to conduct constitutive vesicular transport toward both apical and basolateral surfaces, also contain a previously unrecognized regulated pathway. Together our studies indicate that transgenic mice represent a valuable system for analyzing trafficking pathways and sorting mechanisms of secretory proteins in vivo.



Ultrastructure and cytochemistry of lipid granules in the many-celled magnetotactic prokaryote, ‘ Candidatus Magnetoglobus multicellularis’  

Microsoft Academic Search

Conspicuous cytoplasmic granules are reported in a magnetotactic multicellular prokaryote named ‘Candidatus Magnetoglobus multicellularis’. Unfortunately, this microorganism, which consists of an assembly of gram-negative bacterial cells, cannot yet be cultivated, limiting the biochemical analysis of the granules and preventing in vitro studies with starvation\\/excess of nutrients. In this scenario, light and electron microscopy techniques were used to partially address the

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



Intestinal Neurogenin 3 Directs Differentiation of a Bipotential Secretory Progenitor to Endocrine Cell Rather than Goblet Cell Fate  

PubMed Central

Neurogenin 3 is essential for enteroendocrine cell development; however, it is unknown whether this transcription factor is sufficient to induce an endocrine program in the intestine or how it affects the development of other epithelial cells originating from common progenitors. In this study, the mouse villin promoter was used to drive Neurogenin 3 expression throughout the developing epithelium to measure the affect on cell fate. Although the general morphology of the intestine was unchanged, transgenic founder embryos displayed increased numbers of cells expressing the pan-endocrine marker chromogranin A. Accordingly, expression of several hormones and pro-endocrine transcription factors were increased in the transgenics suggesting that Neurogenin 3 stimulated a program of terminal enteroendocrine cell development. To test whether increased endocrine cell differentiation affected the development of other secretory cell lineages, we quantified goblet cells, the only other secretory cell formed in embryonic intestine. The Neurogenin 3-expressing transgenics had decreased numbers of goblet cells in correspondence to the increase in endocrine cells, with no change in the total secretory cell numbers. Thus, our data suggest that Neurogenin 3 can redirect the differentiation of bipotential secretory progenitors to endocrine rather than goblet cell fate.

Lopez-Diaz, Lymari; Jain, Renu N.; Keeley, Theresa M.; VanDussen, Kelli L.; Brunkan, Cynthia S.; Gumucio, Deborah L.; Samuelson, Linda C.



Significant qualitative differences exist between thyrotropin and prolactin secretory dynamics induced by pituitary cell swelling.  


Cell swelling produced by a variety of techniques is a potent stimulus intensity-related inducer of an immediate secretory burst of thyroid-stimulating hormone (TSH) and prolaction (PRL) secretion from anterior pituitary cells. A 2-min "square wave" exposure to either hyposmolarity or isotonic urea induced stimulus intensity-correlated TSH and PRL secretory bursts peaking within 3 min, but the PRL zenith occurred 1 min later than that of TSH. With continuous exposure to these stimuli, TSH secretion rapidly decreased and remained only slightly above the unstimulated rate after 5 min. PRL secretion fell to and remained below the unstimulated level after 10 min. After stopping the stimulus, another secretory burst ("off" response) occurred with PRL, but not with TSH. A progressive "ramp" increase in stimulus intensity over 18 min induced a corresponding gradual increase in TSH secretion; there was a progressive depression, rather than increase, in PRL secretion during the stimulus ramp, with an off response secretory burst when the stimulus was discontinued. Removal of extracellular Ca2+ or addition of verapamil to the medium did not alter the dynamics of hyposmolarity-induced TSH secretion, but markedly altered those of PRL secretion; there was no off response PRL secretion and a hyposmolar ramp induced a corresponding gradual increase in PRL secretion, with a return to baseline after removing the stimulus. The dramatic qualitative differences in the response of the thyrotroph and lactotroph may reflect differences between the cell types in the size of secretory vesicles, membrane potential, the mechanism of exocytosis, and/or the role of Ca2+ influx across the plasmalemma. PMID:1909795

Wang, X B; Sato, N; Greer, M A; Greer, S E; McAdams, S



Protection against MPP+ neurotoxicity in cerebellar granule cells by antioxidants.  


The neuropathology associated with Parkinson's disease (PD) is thought to involve excessive production of free radicals, dopamine autoxidation, defects in glutathione peroxidase expression, attenuated levels of reduced glutathione, altered calcium homeostasis, excitotoxicity and genetic defects in mitochondrial complex I activity. While the neurotoxic mechanisms are vastly different for excitotoxins and 1-methyl-4-phenylpyridinium ion (MPP(+)), both are thought to involve free radical production, compromised mitochondrial activity and excessive lipid peroxidation. We show here that the levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) increased significantly after treatment of cultured cerebellar granule cells (CGCs) with 50 microM MPP(+). Co-treatment with antioxidants such as ascorbate (ASC), catalase, alpha-tocopherol (alpha-TOH), coenzyme Q(10) (CoQ(10)) or superoxide dismutase (SOD) rescued the cells from MPP(+)-induced death. MPP(+)-induced cell death was also abolished by co-treatment with nitric oxide synthase (NOS) inhibitors such as 7-nitroindazole (7-NI), 2-ethyl-2-thiopseudourea hydrobromide (EPTU) or S-methylisothiourea sulphate (MPTU). We also tested the protective effects of an iron chelator (deferoxamine mesylate, DFx) and a peroxynitrite scavenger (FeTTPS) and the results lend further support to the view that the free radical cytotoxicity plays an essential role in MPP(+)-induced death in primary cultures of CGC. PMID:15193280

González-Polo, Rosa A; Soler, Germán; Rodríguezmartín, Andrea; Morán, Jose M; Fuentes, José M



The major tyrosine-sulfated protein of the bovine anterior pituitary is a secretory protein present in gonadotrophs, thyrotrophs, mammotrophs, and corticotrophs  

PubMed Central

The anterior pituitary is a complex secretory tissue known to contain several sulfated macromolecules. In the present study, we identified the major tyrosine-sulfated protein of the bovine anterior pituitary and investigated its cellular and subcellular localization. This protein consisted of two tyrosine-sulfated polypeptides of molecular weight 86,000 and 84,000 that were highly homologous to each other. In agreement with previous biochemical studies, the tyrosine-sulfated protein of Mr 86,000/84,000 was found to be secretory, as it was observed in the matrix of secretory granules by immunoelectron microscopy. Immunofluorescence studies indicated that the tyrosine- sulfated, secretory protein of Mr 86,000/84,000, referred to as TSP 86/84, was present in all endocrine cells except for some somatotrophic cells. Higher levels of immunoreactivity for TSP 86/84 were observed in gonadotrophic and thyrotrophic than in mammotrophic and corticotrophic cells. This appeared to result from the occurrence of TSP 86/84 in all secretory granules of the former cells and in only some secretory granules of the latter cells. We discuss the possibility that TSP 86/84 may have a role in the packaging of several distinct peptides hormones into secretory granules. One, though not the only, possible function of tyrosine sulfation may concern the sorting of this protein in the Golgi complex.



Myosin Va Transports Dense Core Secretory Vesicles in Pancreatic MIN6  Cells  

Microsoft Academic Search

The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic -cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of

Aniko Varadi; Takashi Tsuboi; Guy A. Rutter



Purkinje cells in olivopontocerebellar atrophy and granule cell-type cerebellar degeneration: an immunohistochemical study  

Microsoft Academic Search

We carried out immunohistochemical studies on cerebellar Purkinje cells in sporadic olivopontocerebellar atrophy (OPCA) and\\u000a in granule cell-type cerebellar degeneration (gc-CD). The cell bodies, axons and dendrites including spiny branchlets and\\u000a dendritic spines of normal Purkinje cells were intensely stained by the antibody against P400 glycoprotein\\/inositol 1,4,5-trisphosphate receptor protein (P400\\/IP3R). The staining pattern of OPCA Purkinje cells was heterogeneous: some

Shinsuke Kato; Hiroko Hayashi; Katsuhiko Mikoshiba; Asao Hirano; Shu-Hui Yen; Eisaku Ohama



Vps33a mediates RANKL storage in secretory lysosomes in osteoblastic cells.  


Previous studies have indicated that the amount of RANKL expressed on the cell surface of osteoblasts or bone marrow stromal cells (BMSCs) is considered an important factor determining the extent of osteoclast activation. However, subcellular trafficking of RANKL and its regulatory mechanisms in osteoblastic cells is still unclear. In this study, we showed that RANKL is predominantly localized in lysosomal organelles, but little is found on the cell surface of osteoblastic cells. We also showed that RANKL is relocated to the plasma membrane in response to stimulation with RANK-Fc-coated beads, indicating that the lysosomal organelles where RANKL is localized function as secretory lysosomes. In addition, using a protein pull-down method, we identified vacuolar protein sorting (Vps)33a as interacting with the cytoplasmic tail of RANKL. Furthermore, knockdown of Vps33a expression reduced the lysosomal storage of RANKL and caused the accumulation of newly synthesized RANKL in the Golgi apparatus, indicating that Vps33a is involved in transporting RANKL from the Golgi apparatus to secretory lysosomes. We also showed that suppression of Vps33a affects the cell surface expression level of RANKL and disrupts the regulated behavior of RANKL. These results suggest that RANKL storage in secretory lysosomes is important to control osteoclast activation and to maintain bone homeostasis. PMID:19419298

Kariya, Yoshiaki; Honma, Masashi; Aoki, Shigeki; Chiba, Atsushi; Suzuki, Hiroshi



Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults  

SciTech Connect

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.

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



The Docking Protein Cas Links Tyrosine Phosphorylation Signaling to Elongation of Cerebellar Granule Cell Axons  

PubMed Central

Crk-associated substrate (Cas) is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of the actin cytoskeletal organization and cell migration in fibroblasts. The function of Cas in neurons, however, is poorly understood. Here we report that Cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. During cerebellar development, Cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. Cas coimmunoprecipitates with Src family protein tyrosine kinases, Crk, and cell adhesion molecules and colocalizes with these proteins in granule cells. The axon extension of granule cells is inhibited by either RNA interference knockdown of Cas or overexpression of the Cas mutant lacking the YDxP motifs, which are tyrosine phosphorylated and thereby interact with Crk. These findings demonstrate that Cas acts as a key scaffold that links the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation.

Huang, Jinhong; Sakai, Ryuichi



Secretory state regulates Zn2+ transport in gastric parietal cell of the rabbit  

PubMed Central

Secretory compartments of neurons, endocrine cells, and exocrine glands are acidic and contain high levels of labile Zn2+. Previously, we reported evidence that acidity is regulated, in part, by the content of Zn2+ in the secretory [i.e., tubulovesicle (TV)] compartment of the acid-secreting gastric parietal cell. Here we report studies focusing on the mechanisms of Zn2+ transport by the TV compartment in the mammalian (rabbit) gastric parietal cell. Uptake of Zn2+ by isolated TV structures was monitored with a novel application of the fluorescent Zn2+ reporter N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ). Uptake was suppressed by removal of external ATP or blockade of H+-K+-ATPase that mediates luminal acid secretion. Uptake was diminished with dissipation of the proton gradient across the TV membrane, suggesting Zn2+/H+ antiport as the connection between Zn2+ uptake and acidity in the TV lumen. In isolated gastric glands loaded with the reporter fluozin-3, inhibition of H+-K+-ATPase arrested the flow of Zn2+ from the cytoplasm to the TV compartment and secretory stimulation with forskolin enhanced vectorial movement of cytoplasmic Zn2+ into the tubulovesicle/lumen (TV/L) compartment. Our findings suggest that Zn2+ accumulation in the TV/L compartment is physiologically coupled to secretion of acid. These findings offer novel insight into mechanisms regulating Zn2+ homeostasis in the gastric parietal cell and potentially other cells in which acidic subcellular compartments serve signature functional roles.

Naik, Haley B.; Beshire, Melissa; Walsh, Breda M.; Liu, Jingjing



Granulated peripolar epithelial cells in the renal corpuscle of marine elasmobranch fish.  


Granulated epithelial cells at the vascular pole of the renal corpuscle, peripolar cells, have been found in the kidneys of five species of elasmobranchs, the little skate (Raja erinacea), the smooth dogfish shark (Mustelus canis), the Atlantic sharpnose shark (Rhizoprionodon terraenovae), the scalloped hammerhead shark (Sphyrna lewini), and the cow-nosed ray (Rhinoptera bonasus). In a sixth elasmobranch, the spiny dogfish shark (Squalus acanthias), the peripolar cells could not be identified among numerous other granulated epithelial cells. The peripolar cells are located at the transition between the parietal epithelium of Bowman's capsule and the visceral epithelium (podocytes) of the glomerulus, thus forming a cuff-like arrangement surrounding the hilar vessels of the renal corpuscle. These cells may have granules and/or vacuoles. Electron microscopy shows that the granules are membrane-bounded, and contain either a homogeneous material or a paracrystalline structure with a repeating period of about 18 nm. The vacuoles are electron lucent or may contain remnants of a granule. These epithelial cells lie close to the granulated cells of the glomerular afferent arteriole. They correspond to the granular peripolar cells of the mammalian, avian and amphibian kidney. The present study is the first reported occurrence of peripolar cells in a marine organism or in either bony or cartilagenous fish. PMID:2519933

Lacy, E R; Reale, E



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

PubMed Central

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

Sun, Jianjun; Spradling, Allan C.



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


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

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



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

PubMed Central

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

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



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


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

Insausti, T C; Casas, J



Killing by cytotoxic T cells and natural killer cells: Multiple granule serine proteases as initiators of DNA fragmentation  

Microsoft Academic Search

The vectorial secretion of the contents of highly specialized cytoplasmic granules is of pivotal importance to the killing by cytotoxic T cells and natural killer cells. The purification and biochemical characterization of some of the granule constituents, in particular the pore-forming protein perforin, had engendered the notion that the killing of cellular targets was largely an osmotic phenomenon analogous to

Joseph A Trapani; Mark J Smyth



Effects of placental secretory factors on cytokine production by endothelial cells.  


We studied the effects of soluble products of the placental tissue from women with normal pregnancy and gestosis on the cytokine secretion by endothelial EA.Hy926 cells. The secretory products of the placental tissue induced the production of angiogenin, bFGF, IL-8, MCP-1, and RANTES by endothelial cells. The secretion of bFGF by EA.Hy926 cells increased, while IL-8 secretion decreased under the effects of factors produced by the placental tissue in gestosis but not in normal pregnancy. This could be aimed at reduction of inflammation intensity in the placental tissue and maintenance of endothelial and trophoblast cells viability. PMID:23484204

Stepanova, O I; Safronova, N U; Furaeva, K N; Lvova, T U; Sokolov, D I; Selkov, S A



High-throughput trapping of secretory pathway genes in mouse embryonic stem cells.  


High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, approximately 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (, are freely available to the scientific community. PMID:16478711

De-Zolt, Silke; Schnütgen, Frank; Seisenberger, Claudia; Hansen, Jens; Hollatz, Melanie; Floss, Thomas; Ruiz, Patricia; Wurst, Wolfgang; von Melchner, Harald



Intracellular ion concentrations and cell volume during cholinergic stimulation of eccrine secretory coil cells  

SciTech Connect

Methacholine (MCh)-induced changes in intracellular concentrations of Na, K, and Cl (( Na)i, (K)i, and (Cl)i, respectively) and in cellular dry mass (a measure of cell shrinkage) were examined in isolated monkey eccrine sweat secretory coils by electron probe X-ray microanalysis using the peripheral standard method. To further confirm the occurrence of cell shrinkage during MCh stimulation, the change in cell volume of dissociated clear and dark cells were directly determined under a light microscope equipped with differential interference contrast (DIC) optics. X-ray microanalysis revealed a biphasic increase in cellular dry mass in clear cells during continuous MCh stimulation; an initial increase of dry mass to 158% (of control) followed by a plateau at 140%, which correspond to the decrease in cell volume of 37 and 29%, respectively. The latter agrees with the MCh-induced cell shrinkage of 29% in dissociated clear cells. The MCh-induced increase in dry mass in myoepithelial cells was less than half that of clear cells. During the steady state of MCh stimulation, both (K+)i and (Cl)i of clear cells decreased by about 45%, whereas (Na)i increased in such a way to maintain the sum of (Na) i + (K)i constant. There was a small (12-15 mM) increase in (Na)i and a decrease in (K)i in myoepithelial cells during stimulation with MCh. Dissociated dark cells failed to significantly shrink during MCh stimulation. The decrease in (Cl)i in the face of constant (Na)i + (K)i suggests the accumulation of unknown anion(s) inside the clear cell during MCh stimulation.

Takemura, T.; Sato, F.; Saga, K.; Suzuki, Y.; Sato, K. (Univ. of Iowa College of Medicine, Iowa City (USA))



Only high concentrations of ethanol affect GABAA receptors of rat cerebellum granule cells in culture.  


In the experiments described in the present report, we evaluated the effects of ethanol on the activity of GABAA receptors of cerebellar granule cells in culture. Only very high ethanol concentrations (100-300 mM) showed a clear and significant stimulatory effect on the activity of such receptors. This result was unexpected. In fact, previous reports from other groups would have suggested high ethanol sensitivity of at least one population of GABAA receptors expressed by granule cells. PMID:17234340

Casagrande, S; Cupello, A; Pellistri, F; Robello, M



Potentiation of monosynaptic EPSPs in the perforant path-dentate granule cell synapse  

Microsoft Academic Search

1.In rabbits, initially anaesthetized by urethane\\/chloralose and maintained on urethane alone, the perforant path, contacting the apical dendrites of the dentate granule cells by way of boutons en-passage, was activated by paired stimuli. The effect of the first conditioning stimulus was studied by recording the extracellular field response and the extra- and intracellular responses of single granule cells to a

T. Lřmo



Selective Release of Glutamate from Cerebellar Granule Cells Differentiating in Culture  

Microsoft Academic Search

The aim of the present study was to assess whether endogenous and newly synthesized glutamate can be released from differentiating cultured cerebellar granule cells in a way compatible with a neurotransmitter role. Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum for 2-12 days in vitro(DIV), then washed with Krebs-Ringer medium, and

V. Gallo; M. T. Ciotti; A. Coletti; F. Aloisi; G. Levi



Ultrastructure of lamellar granules coating glial cell basal lamina in the olfactory bulb and olfactory subtracts of goldfish.  


Lamellar granules, 35 to 80 nm in diameter, are observed in the pia-arachnoid tissue of regenerated olfactory bulb and olfactory subtracts of the goldfish. The granules correspond to multiple stacks of disks which cover basal lamina of both pial and ependymal cell layers and blood vessels. The granules are also in close association with collagen fibril bundles. The granules are observed only in young goldfish or after bilateral olfactory tract transection in 1 year postoperated animals. Our findings suggest that these granules are secreted by connective tissue cells in response to damage associated with bilateral olfactory tract transection. Coexisting extracellular free granules and the granules covering basal lamina are simultaneously found in regenerated olfactory bulb and olfactory tract. Many of our findings, as far as we know, have not been described previously. The composition and function of these granules are at present unknown. PMID:7983372

Popov, V I; Moisseeva, A A; Zippel, H P



Proliferation of Acid-Secretory Cells in the Kidney during Adaptive Remodelling of the Collecting Duct  

PubMed Central

The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H+-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H+-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH4Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH4Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions.

Welsh-Bacic, Desa; Nowik, Marta; Kaissling, Brigitte; Wagner, Carsten A.



Proliferation of acid-secretory cells in the kidney during adaptive remodelling of the collecting duct.  


The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H(+)-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H(+)-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH(4)Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH(4)Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions. PMID:22039408

Welsh-Bacic, Desa; Nowik, Marta; Kaissling, Brigitte; Wagner, Carsten A



Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length.  


Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model. PMID:11781333

Schott, Daniel H; Collins, Ruth N; Bretscher, Anthony



Ovulation in Drosophila is controlled by secretory cells of the female reproductive tract  

PubMed Central

How oocytes are transferred into an oviduct with a receptive environment remains poorly known. We found that glands of the Drosophila female reproductive tract, spermathecae and/or parovaria, are required for ovulation and to promote sperm storage. Reducing total secretory cell number by interferring with Notch signaling during development blocked ovulation. Knocking down expression after adult eclosion of the nuclear hormone receptor Hr39, a master regulator of gland development, slowed ovulation and blocked sperm storage. However, ovulation (but not sperm storage) continued when only canonical protein secretion was compromised in adult glands. Our results imply that proteins secreted during adulthood by the canonical secretory pathway from female reproductive glands are needed to store sperm, while a non-canonical glandular secretion stimulates ovulation. Our results suggest that the reproductive tract signals to the ovary using glandular secretions, and that this pathway has been conserved during evolution. DOI:

Sun, Jianjun; Spradling, Allan C



Secretory Mechanisms in Opticortin Cells during Cold Stress.  

National Technical Information Service (NTIS)

The goals of the study are to investigate the effects of acute exposure to cold or novel environment on the hypothalamic pituitary axis. The study focuses on one system. In the hypothalamus, the nerve cells that produce corticotropin releasing hormone (CR...

G. V. Childs



Nitric oxide as a secretory product of mammalian cells  

Microsoft Academic Search

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




Infected periodontal granulation tissue contains cells expressing embryonic stem cell markers. A pilot study.  


The commonly practiced removal of granulation tissue during periodontal surgery, aiming to eliminate infection and optimize healing conditions, may also remove progenitor stem cells that could otherwise support periodontal regeneration. The present study aimed to investigate if cells with embryonic stem cell properties are present in periodontal granulation tissue. During the course of flap surgery inflammatory granulation tissue was obtained from four patients and five periodontal defects. Tissues were processed in a collagenase/dispase solution to release the cells. Part of the resulting suspension was processed for bacteriological analysis (IAI PadoTest 4.5), whereas the remaining cell suspension was cultured and passaged once. Upon reaching confluence, total RNA was extracted, followed by cDNA synthesis. PCR was then performed (SYBR Green-based protocols) to measure gene expression levels of Collagen type I, and embryonic stem cell markers Nanog, Oct4, Rex-1 and Sox2. Results are expressed as 2??(Ct) values of the target gene, calibrated against a house-keeping gene (GAPDH). A high total bacterial load up to 20.6 ± 11.0×10(6) counts/mg of tissue was found. Collagen type I was strongly expressed, confirming the predominance of mesenchymal/fibroblastic cells. Among the studied embryonic stem cells markers, Nanog was most highly expressed (2.3 ± 1.2), followed by Oct4 (1.1 ± 0.5), Rex-1 (0.6 ± 0.2) and Sox2 (0.3 ± 0.2). This is the first study that demonstrates the presence of cells expressing embryonic stem cell markers among infected granulation tissue. This knowledge needs to be considered when devising future strategies to improve periodontal wound healing and regeneration. PMID:23426569

Ronay, Valerie; Belibasakis, Georgios N; Schmidlin, Patrick R; Bostanci, Nagihan



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

SciTech Connect

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

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



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

Microsoft Academic Search

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

Judy Lieberman



Labeling and exocytosis of secretory compartments in RBL mastocytes by polystyrene and mesoporous silica nanoparticles  

PubMed Central

Background For a safe ‘in vivo’ biomedical utilization of nanoparticles, it is essential to assess not only biocompatibility, but also the potential to trigger unwanted side effects at both cellular and tissue levels. Mastocytes (cells having secretory granules containing cytokines, vasoactive amine, and proteases) play a pivotal role in the immune and inflammatory responses against exogenous toxins. Mastocytes are also recruited in the tumor stroma and are involved in tumor vascularization and growth. Aim and methods In this work, mastocyte-like rat basophilic leukemia (RBL) cells were used to investigate whether carboxyl-modified 30 nm polystyrene (PS) nanoparticles (NPs) and naked mesoporous silica (MPS) 10 nm NPs are able to label the secretory inflammatory granules, and possibly induce exocytosis of these granules. Uptake, cellular retention and localization of fluorescent NPs were analyzed by cytofluorometry and microscope imaging. Results Our findings were that: (1) secretory granules of mastocytes are accessible by NPs via endocytosis; (2) PS and MPS silica NPs label two distinct subpopulations of inflammatory granules in RBL mastocytes; and (3) PS NPs induce calcium-dependent exocytosis of inflammatory granules. Conclusion These findings highlight the value of NPs for live imaging of inflammatory processes, and also have important implications for the clinical use of PS-based NPs, due to their potential to trigger the unwanted activation of mastocytes.

Ekkapongpisit, Maneerat; Giovia, Antonino; Nicotra, Giuseppina; Ozzano, Matteo; Caputo, Giuseppe; Isidoro, Ciro



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

PubMed Central

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

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



Mast Cells and Oral Inflammation  

Microsoft Academic Search

Mast cells are mobile granule-containing secretory cells that are distributed preferentially about the microvascular endothelium in oral mucosa and dental pulp. The enzyme profile of mast cells in oral tissues resembles that of skin, with most mast cells expressing the serine proteases tryptase and chymase. Mast cells in oral tissues contain the pro-inflammatory cytokine tumour necrosis factor-? in their granules,

Laurence J. Walsh



Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II.  


To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi-intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways. PMID:7962053

Carnell, L; Moore, H P



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

PubMed Central

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

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



Kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+.  

PubMed Central

The kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+ were studied by capacitance (Cm) measurements with millisecond time resolution. After elevation of the internal Ca2+ concentration ([Ca2+]i), Cm rises rapidly with one or more exponentials. The time constant of the fastest component decreases for higher [Ca2+]i (range 3-600 microM) over three orders of magnitude before it saturates at approximately 1 ms. The corresponding maximal rates of secretion can be as fast as 100,000 fF/s or 40,000 vesicles/s. There is a Ca(2+)-dependent delay before Cm rises, which may reflect the kinetics of multiple Ca2+ ions binding to the secretory apparatus. The initial rise in Cm is described by models containing a sequence of two to four single Ca(2+)-binding steps followed by a rate-limiting exocytosis step. The predicted Ca2+ dissociation constant (Kd) of a single Ca(2+)-binding site is between 7 and 21 microM. At [Ca2+]i > 30 microM clear indications of a fast endocytotic process complicate the analysis of the secretory response.

Heinemann, C; Chow, R H; Neher, E; Zucker, R S



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


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 Ca(2+) 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

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



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

PubMed Central

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.

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



Regulation of the chromaffin granule catecholamine transporter in cultured bovine adrenal medullary cells: stimulus-biosynthesis coupling.  


The transsynaptic induction of the monoamine transporter present on the membrane of chromaffin granules was studied in primary cultures of dissociated bovine adrenomedullary cells submitted to a chronic secretory stimulation. The amount of the vesicular monoamine transporter was assayed by binding of the specific ligand [3H]-dihydrotetrabenazine. After several days of incubation in the presence of high potassium, the concentration of [3H]-dihydrotetrabenazine binding sites was increased by a 1.5-2.5 factor. This increase was smaller in the presence of the cholinergic agonist carbachol. The long-term inductions of the vesicular monoamine transporter, of tyrosine hydroxylase, and of acetylcholinesterase were of similar magnitude. Under the same conditions, we found no variation in either the activities of other catecholamine biosynthetic enzymes (dopamine beta-hydroxylase and DOPA decarboxylase), or in metabolic enzymes such as lactate dehydrogenase and cytochrome c oxidase, and a decrease in the cellular content of chromogranin A and cytochrome b-561. The induction of the vesicular monoamine transporter was inhibited by the calcium channel antagonists, fluspirilene and nifedipine, and was increased by the agonist Bay K 8644. It was abolished by cycloheximide and actinomycin D. These results indicate that calcium entry into chromaffin cells increases the synthesis of the vesicular monoamine transporter, presumably by transcriptional activation. Elevation of intracellular cyclic AMP concentration or activation of protein kinase C also induced an increase in the expression of the vesicular monoamine transporter. Our results confirm that components of storage vesicle membranes are differentially regulated in response to secretory stimulation, as are several cytosolic or intravesicular soluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1279122

Desnos, C; Laran, M P; Scherman, D



Spatial partitioning of secretory cargo from Golgi resident proteins in live cells  

PubMed Central

Background To maintain organelle integrity, resident proteins must segregate from itinerant cargo during secretory transport. However, Golgi resident enzymes must have intimate access to secretory cargo in order to carry out glycosylation reactions. The amount of cargo and associated membrane may be significant compared to the amount of Golgi membrane and resident protein, but upon Golgi exit, cargo and resident are efficiently sorted. How this occurs in live cells is not known. Results We observed partitioning of the fluorescent Golgi resident T2-CFP and fluorescent cargo proteins VSVG3-YFP or VSVG3-SP-YFP upon Golgi exit after a synchronous pulse of cargo was released from the ER. Golgi elements remained stable in overall size, shape and relative position as cargo emptied. Cargo segregated from resident rapidly by blebbing into micron-sized domains that contained little or no detectable resident protein and that appeared to be continuous with the parent Golgi element. Post-Golgi transport carriers (TCs) exited repeatedly from these domains. Alternatively, entire cargo domains exited Golgi elements, forming large TCs that fused directly with the plasma membrane. However, domain formation did not appear to be an absolute prerequisite for TC exit, since TCs also exited directly from Golgi elements in the absence of large domains. Quantitative cargo-specific photobleaching experiments revealed transfer of cargo between Golgi regions, but no discrete intra-Golgi TCs were observed. Conclusions Our results establish domain formation via rapid lateral partitioning as a general cellular strategy for segregating different transmembrane proteins along the secretory pathway and provide a framework for consideration of molecular mechanisms of secretory transport.

White, Jamie; Keller, Patrick; Stelzer, Ernst HK



Cells Containing Langerhans Granules in Human Lymph Nodes of Dermatopathic Lymphadenopathy1  

Microsoft Academic Search

Cells containing Langerhans granules are found in human lymph nodes of dermato-pathic lymphadenopathy. The ultrastructural characteristics of these cells are identical to the epidermal Langerhans cells of the same patients. The possible development of Langerhans cells from mesenchymal tissues is discussed.

Kowichi Jimbow; Syozo Sato; Atsushi Kukita



Activation of cerebellar granule cells GABA(A) receptors by guanidinoacetate.  


The extracellular concentration of guanidinoacetate (GAA) in the brain increases in guanidino acetate methyl transferase (GAMT) deficiency, an inherited disorder. We tested whether the levels which this substance can reach in the brain in GAMT deficiency are able to activate GABA(A) receptors in key cerebellar neurons such as the cerebellar granules. GAA in fact activates these receptors in rat cerebellar granules in culture although at quite high concentrations, in the millimolar range. However, these millimolar GAA levels are not reached extracellularly in the brain in GAMT deficiency. In addition, GAA does not act as a partial agonist on granules' GABA(A) receptors. This appears to deny an effect by this molecule on cerebellar function in the disease via interference with granule cells' GABA(A) receptors. Study of partial blockage by furosemide of chloride currents activated by GABA and GAA in granule cells allowed us to distinguish two populations of GABA(A) receptors presumably involved in granule cells' tonic inhibition. One is devoid of alpha6 subunit and another one contains it. The latter when activated by GABA has a decay kinetics much slower than the former. GAA does not distinguish between these two populations. In any case, the very high extracellular GAA concentrations able to activate them are not likely to be reached in GAMT deficiency. PMID:18222046

Cupello, A; Balestrino, M; Gatta, E; Pellistri, F; Siano, S; Robello, M



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

PubMed Central

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

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



Foxa1 and Foxa2 Maintain the Metabolic and Secretory Features of the Mature ?-Cell  

PubMed Central

Foxa1 and Foxa2 play both redundant and distinct roles in early pancreas development. We demonstrate here that inducible ablation of both transcription factors in mature mouse ?-cells leads to impaired glucose homeostasis and insulin secretion. The defects in both glucose-stimulated insulin secretion and intracellular calcium oscillation are more pronounced than those in ?-cells lacking only Foxa2. Unexpectedly, in contrast to the severe reduction of ?-cell-enriched factors contributing to metabolic and secretory pathways, expression of a large number of genes that are involved in neural differentiation and function is significantly elevated. We further demonstrate that expression of carbohydrate response element-binding protein (ChREBP or Mlxipl), an important transcriptional regulator of carbohydrate metabolism, is significantly affected in compound Foxa1/a2 mutant ?-cells. ChREBP expression is directly controlled by Foxa1 and Foxa2 in both the fetal endocrine pancreas as well as mature islets. These data demonstrate that Foxa1 and Foxa2 play crucial roles in the development and maintenance of ?-cell-specific secretory and metabolic pathways.

Gao, Nan; Le Lay, John; Qin, Wei; Doliba, Nicolai; Schug, Jonathan; Fox, Alan J.; Smirnova, Olga; Matschinsky, Franz M.; Kaestner, Klaus H.



An LRRTM4-HSPG complex mediates excitatory synapse development on dentate gyrus granule cells.  


Selective synapse development determines how complex neuronal networks in the brain are formed. Complexes of postsynaptic neuroligins and LRRTMs with presynaptic neurexins contribute widely to excitatory synapse development, and mutations in these gene families increase the risk of developing psychiatric disorders. We find that LRRTM4 has distinct presynaptic binding partners, heparan sulfate proteoglycans (HSPGs). HSPGs are required to mediate the synaptogenic activity of LRRTM4. LRRTM4 shows highly selective expression in the brain. Within the hippocampus, we detected LRRTM4 specifically at excitatory postsynaptic sites on dentate gyrus granule cells. LRRTM4(-/-) dentate gyrus granule cells, but not CA1 pyramidal cells, exhibit reductions in excitatory synapse density and function. Furthermore, LRRTM4(-/-) dentate gyrus granule cells show impaired activity-regulated AMPA receptor trafficking. These results identifying cell-type-specific functions and multiple presynaptic binding partners for different LRRTM family members reveal an unexpected complexity in the design and function of synapse-organizing proteins. PMID:23911104

Siddiqui, Tabrez J; Tari, Parisa Karimi; Connor, Steven A; Zhang, Peng; Dobie, Frederick A; She, Kevin; Kawabe, Hiroshi; Wang, Yu Tian; Brose, Nils; Craig, Ann Marie



Role of LAT in the Granule-Mediated Cytotoxicity of CD8 T Cells  

PubMed Central

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is essential to bridge T cell receptor (TCR) engagement to downstream signaling events. The indispensable role of LAT in thymocyte development and T cell activation has been well characterized; however, the function of LAT in cytotoxic-T-lymphocyte (CTL) cytotoxicity remains unknown. We show here that LAT-deficient CTLs failed to upregulate FasL and produce gamma interferon after engagement with target cells and had impaired granule-mediated killing. We further dissected the effect of the LAT deletion on each step of granule exocytosis. LAT deficiency led to altered synapse formation, subsequently causing unstable T cell–antigen-presenting cell (APC) conjugates. Microtubule organizing center polarization and granule reorientation were also impaired by LAT deficiency, leading to reduced granule delivery. Despite these defects, granule release was still observed in LAT-deficient CTLs due to residual calcium flux and phospholipase C (PLC) activity. Our data demonstrated that LAT-mediated signaling intricately regulates CTL cytotoxicity at multiple steps.

Ou-Yang, Chih-wen; Zhu, Minghua; Fuller, Deirdre M.; Sullivan, Sarah A.; Chuck, Mariana I.; Ogden, Sarah



Suppression of GABA input by A1 adenosine receptor activation in rat cerebellar granule cells.  


Synaptic transmission has been shown to be modulated by purinergic receptors. In the cerebellum, spontaneous inhibitory input to Purkinje neurons is enhanced by ATP via P2 receptors, while evoked excitatory input via the granule cell parallel fibers is reduced by presynaptic P1 (A1) adenosine receptors. We have now studied the modulation of the complex GABAergic input to granule cells by the purinergic receptor agonists ATP and adenosine in acute rat cerebellar tissue slices using the whole-cell patch-clamp technique. Our experiments indicate that ATP and adenosine substantially reduce the bicuculline- and gabazine-sensitive GABAergic input to granule cells. Both phasic and tonic inhibitory components were reduced leading to an increased excitability of granule cells. The effect of ATP and adenosine could be blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), but not by other P1 and P2 receptor antagonists, indicating that it was mediated by activation of A1 adenosine receptors. Our results suggest that, in the cerebellar network, A1 receptor activation, known to decrease the excitatory output of granule cells, also increases their excitability by reducing their complex GABAergic input. These findings extend our knowledge on purinergic receptors, mediating multiple modulations at both inhibitory and excitatory input and output sites in the cerebellar network. PMID:19477241

Courjaret, R; Tröger, M; Deitmer, J W



Neuropeptidomic components generated by proteomic functions in secretory vesicles for cell-cell communication.  


Diverse neuropeptides participate in cell-cell communication to coordinate neuronal and endocrine regulation of physiological processes in health and disease. Neuropeptides are short peptides ranging in length from ~3 to 40 amino acid residues that are involved in biological functions of pain, stress, obesity, hypertension, mental disorders, cancer, and numerous health conditions. The unique neuropeptide sequences define their specific biological actions. Significantly, this review article discusses how the neuropeptide field is at the crest of expanding knowledge gained from mass-spectrometry-based neuropeptidomic studies, combined with proteomic analyses for understanding the biosynthesis of neuropeptidomes. The ongoing expansion in neuropeptide diversity lies in the unbiased and global mass-spectrometry-based approaches for identification and quantitation of peptides. Current mass spectrometry technology allows definition of neuropeptide amino acid sequence structures, profiling of multiple neuropeptides in normal and disease conditions, and quantitative peptide measures in biomarker applications to monitor therapeutic drug efficacies. Complementary proteomic studies of neuropeptide secretory vesicles provide valuable insight into the protein processes utilized for neuropeptide production, storage, and secretion. Furthermore, ongoing research in developing new computational tools will facilitate advancements in mass-spectrometry-based identification of small peptides. Knowledge of the entire repertoire of neuropeptides that regulate physiological systems will provide novel insight into regulatory mechanisms in health, disease, and therapeutics. PMID:20734175

Hook, Vivian; Bark, Steven; Gupta, Nitin; Lortie, Mark; Lu, Weiya D; Bandeira, Nuno; Funkelstein, Lydiane; Wegrzyn, Jill; O'Connor, Daniel T; Pevzner, Pavel



Direct effects of secretory products of immune cells on neurons and glia.  


This review summarizes recent research contributions by Dr. Robert Lisak in collaboration with Dr. Joyce Benjamins on direct effects of secretory products of immune cells on neurons and glia. Highlights from studies analyzing cytokine-induced changes in early gene expression in mixed CNS glial cultures focus on comparison of potential damaging effects of pro-inflammatory cytokines versus protective effects of downregulatory cytokines. The three categories of changes examined include (a) immune-related molecules, (b) neurotrophins, growth factors and structural proteins, and (c) molecules associated with metabolism, signaling and regulation. Subsequent studies in CNS neuronal cultures showed that early responses of neurons to cytokines were fewer in number and lower in magnitude than in glia, consistent with the idea that microglia and astroglia serve as "first responders" to inflammatory signals. To explore the hypothesis that B cells of patients with multiple sclerosis secrete soluble products damaging to oligodendroglia (OL), in collaboration with Dr. Bar-Or at Montreal Neurologic Institute, we compared secretory products of cultured B cells from relapsing remitting multiple sclerosis (RRMS) patients and healthy controls. In support of the hypothesis, 7 supernatants from RRMS B cells induced death of rat OL in vitro, while 3 of 4 control samples did not. PMID:23827826

Benjamins, Joyce A



Regulated and constitutive protein targeting can be distinguished by secretory polarity in thyroid epithelial cells  

Microsoft Academic Search

We have studied concurrent apical\\/basolateral and regulated\\/constitutive secretory targeting in filter- grown thyroid epithelial monolayers in vitro, by fol- lowing the exocytotic routes of two newly synthesized endogenous secretory proteins, thyroglobulin (Tg) and p500. Tg is a regulated secretory protein as indicated by its acute secretory response to secretagogues. With- out stimulation, pulse-labeled Tg exhibits primarily two kinetically distinct routes:

Peter Arvan; Juliane Lee



Microglia-associated granule cell death in the normal adult dentate gyrus  

Microsoft Academic Search

Microglial cells are constantly monitoring the central nervous system for sick or dying cells and pathogens. Previous studies\\u000a showed that the microglial cells in the dentate gyrus have a heterogeneous morphology with multipolar cells in the hilus and\\u000a fusiform cells apposed to the granule cell layer both at the hilar and at the molecular layer borders. Although previous studies\\u000a showed

Charles E. RibakLee; Lee A. Shapiro; Zachary D. Perez; Igor Spigelman



Astroglial cells in the external granular layer are precursors of cerebellar granule neurons in neonates  

PubMed Central

It is well established that cerebellar granule cell precursors (GCPs) initially derive from progenitors in the rhombic lip of the embryonic cerebellar primordium. GCPs proliferate and migrate tangentially across the cerebellum to form the external granule cell layer (EGL) in late embryogenesis and early postnatal development. It is unclear whether GCPs are specified exclusively in the embryonic rhombic lip or whether their precursor persists in the neonate. Using transgenic mice expressing DsRed under the human glial fibrillary acidic protein (hGFAP) promoter, we found 2 populations of DsRed+ cells in the EGL in the first postnatal week defined by bright and faint DsRed fluorescent signal. Bright DsRed+ cells have a protein expression profile and electrophysiological characteristics typical of astrocytes, but faint DsRed+ cells in the EGL and internal granule cell layer (IGL) express markers and physiological properties of immature neurons. To determine if these astroglial cells gave rise to GCPs, we genetically tagged them with EGFP or ?gal reporter genes at postnatal day (P)3-P5 using a hGFAP promoter driven inducible Cre recombinase. We found that GFAP promoter+ cells in the EGL are proliferative and express glial and neural stem cell markers. In addition, immature granule cells (GCs) en route to the IGL at P12 as well as GCs in the mature cerebellum, 30 days after recombination, express the reporter protein, suggesting that GFAP promoter+ cells in the EGL generate a subset of granule cells. The identification of glial cells which function as neuronal progenitor cells profoundly impacts our understanding of cellular plasticity in the developing cerebellum.

Silbereis, John; Heintz, Tristan; Taylor, Mary Morgan; Ganat, Yosif; Ment, Laura R.; Bordey, Angelique; Vaccarino, Flora



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

PubMed Central

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

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



Upregulation of inward rectifier K+ (Kir2) channels in dentate gyrus granule cells in temporal lobe epilepsy  

Microsoft Academic Search

In humans, temporal lobe epilepsy (TLE) is often associated with Ammon's horn sclerosis (AHS) characterized by hippocampal cell death, gliosis and granule cell dispersion (GCD) in the dentate gyrus. Granule cells surviving TLE have been proposed to be hyperexcitable and to play an important role in seizure generation. However, it is unclear whether this applies to conditions of AHS. We

Christina C. Young; M ichael Stegen; R. Bernard; M. Muller; Josef Bischofberger; R. W. Veh; C. A. Haas; J. Wolfart



Exocytosis from pancreatic ?-cells: mathematical modelling of the exit of low-molecular-weight granule content  

PubMed Central

Pancreatic ?-cells use Ca2+-dependent exocytosis of large dense core vesicles to release insulin. Exocytosis in ?-cells has been studied biochemically, biophysically and optically. We have previously developed a biophysical method to monitor release of endogenous intragranular constituents that are co-released with insulin. This technique involves the expression of ionotropic membrane receptors in the ?-cell plasma membrane and enables measurements of exocytosis of individual vesicles with sub-millisecond resolution. Like carbon fibre amperometry, this method allows fine details of the release process, like the expansion of the fusion pore (the narrow connection between the granule lumen and the extracellular space), to be monitored. Here, we discuss experimental data obtained with this method within the framework of a simple mathematical model that describes the release of low-molecular constituents during exocytosis of the insulin granules. Our findings suggest that the fusion pore functions as a molecular sieve, allowing differential release of low- and high-molecular-weight granule constituents.

Galvanovskis, Juris; Braun, Matthias; Rorsman, Patrik



Prevention by cycloheximide of neutral red-induced formation of autophagic vacuoles and krinom granules in mouse pancreatic acinar cells  

Microsoft Academic Search

Summary  The effect of cycloheximide on neutral red-induced autophagocytosis in pancreatic acinar cells of the mousein vivo was investigated. Administration of the dye resulted in an appearance of basophilic granules (Chlopin’s krinom granules)\\u000a in the cytoplasm. The cells containing krinom granules were always found to contain a great number of autophagic vacuoles\\u000a in various stages of degradation. It was concluded, that

G. Réz; J. Kovács



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

PubMed Central

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.

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



Enhanced synaptic plasticity in newly generated granule cells of the adult hippocampus  

Microsoft Academic Search

Neural stem cells in various regions of the vertebrate brain continuously generate neurons throughout life. In the mammalian hippocampus, a region important for spatial and episodic memory, thousands of new granule cells are produced per day, with the exact number depending on environmental conditions and physical exercise. The survival of these neurons is improved by learning and conversely learning may

Christoph Schmidt-Hieber; Peter Jonas; Josef Bischofberger



Granulated peripolar epithelial cells in the renal corpuscle of marine elasmobranch fish  

Microsoft Academic Search

Granulated epithelial cells at the vascular pole of the renal corpuscle, peripolar cells, have been found in the kidneys of five species of elasmobranchs, the little skate (Raja erinaced), the smooth dogfish shark (Mustelus canis), the Atlantic sharpnose shark (Rhizoprionodon terraenovae), the scalloped hammerhead shark (Sphryna lewini), and the cow-nosed ray (Rhinoptera bonasus). In a sixth elasmobranch, the spiny dogfish

E. R. Lacy; Enrico Reale



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

Microsoft Academic Search

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

M. Horisberger; Jaqueline Rosset; H. Bauer



Role of membrane oxidation in controlling the activity of human group IIa secretory phospholipase A(2) toward apoptotic lymphoma cells.  


The membranes of healthy lymphocytes normally resist hydrolysis by secretory phospholipase A(2). However, they become susceptible during the process of apoptosis. Previous experiments have demonstrated the importance of certain physical changes to the membrane during cell death such as a reduction in membrane lipid order and exposure of phosphatidylserine on the membrane surface. Nevertheless, those investigations also showed that at least one additional factor was required for rapid hydrolysis by the human group IIa phospholipase isozyme. This study was designed to test the possibility that oxidation of membrane lipids is the additional factor. Flow cytometry and confocal microscopy with a fluorescent probe of oxidative potential suggested that oxidation of the plasma membrane occurs during apoptosis stimulated by thapsigargin. When oxidative potential was high, the activity of human group IIa secretory phospholipase A(2) was enhanced 30- to 100-fold compared to that observed with conditions sufficient for maximal hydrolysis by other secretory phospholipase A(2) isoforms. Direct oxidation of cell membranes with either of two oxidizing agents also stimulated hydrolysis by secretory phospholipase A(2). Both oxidizers caused externalization of phosphatidylserine, but a change in lipid order did not always occur. These results demonstrated that membrane oxidation strongly stimulates human group IIa secretory phospholipase A(2) activity toward apoptotic cells. Interestingly, the change in membrane order, previously thought to be imperative for high rates of hydrolysis, was not required when membrane lipids were oxidized. Whether phosphatidylserine exposure is still necessary with oxidation remains unresolved since the two events could not be deconvoluted. PMID:22989727

Gibbons, Elizabeth; Nelson, Jennifer; Anderson, Lynn; Brewer, Kelly; Melchor, Stephanie; Judd, Allan M; Bell, John D



Regulating secretory lysosomes.  


Secretory lysosomes are lysosomes which are capable of undergoing regulated secretion in response to external stimuli. Many cells of the immune system use secretory lysosomes to release proteins involved in their specialised effector mechanisms. Precisely how lysosomal secretion is regulated in each of these cell types is now the study of much research as these mechanisms control the ability of each of these cells to function. Studies on a number of human genetic diseases have identified some key proteins in controlling secretory lysosome release, and now many interacting partners have been identified. The different regulatory components seem to vary from one cell type to another, providing a multitude of ways for fine tuning the release of secretory lysosomes. PMID:16877763

Holt, Oliver J; Gallo, Federico; Griffiths, Gillian M



Non-opioid dynorphin binding site on secretory vesicles of a pituitary-derived cell line.  


Accumulating evidence indicates that the endogenous opioid peptides dynorphinA-(1-17) and dynorphinA-(1-13) interact not only with opioid but also with yet poorly characterized non-opioid receptors. The latter have been implicated in a number of the effects of dynorphins including induction of ACTH release in sheep and in AtT 20 cells, a pituitary-derived mouse cell line. AtT 20 cells do not express opioid receptors and therefore are particularly suitable for search of non-opioid dynorphin receptors. We report here that 3H-dynorphinA-(1-13)-NH2 associates specifically with AtT 20 cells, apparently through an uptake process and a binding site. Within the cell, it binds preferentially to fractions containing secretory vesicles, with a Kd of about 100 nM. DynorphinA-(1-17), and several non-opioid fragments of dynorphin, including A-(2-17), A-(2-16) and A-(2-13), compete with 3H-dynorphinA-(1-13)-NH2 for that site with IC50s ranging from 200 nM to 2 microM. ACTH(1-39) also competes with 3H-dynorphinA-(1-13)-NH2 for the site with an IC50 of about 300 nM. DynorphinA-(2-17) at microM concentrations stimulates release of ACTH from the isolated vesicles. The results indicate the presence of a non-opioid dynorphin binding site on the secretory vesicle fractions of AtT20 cells that might be involved in ACTH release. The ability of ACTH itself to compete for the binding sites associated with the vesicles suggest that those sites may be involved in an autocrine loop. PMID:9593843

Yarygin, K N; Zhang, X H; Lee, N M



Novel Method for Isolation of Murine Clara Cell Secretory Protein-Expressing Cells with Traces of Stemness  

PubMed Central

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.

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



Ionic and secretory response of pancreatic islet cells to minoxidil sulfate  

SciTech Connect

Minoxidil sulfate is an antihypertensive agent belonging to the new class of vasodilators, the K+ channel openers. The present study was undertaken to characterize the effects of minoxidil sulfate on ionic and secretory events in rat pancreatic islets. The drug unexpectedly provoked a concentration-dependent decrease in 86Rb outflow. This inhibitory effect was reduced in a concentration-dependent manner by glucose and tolbutamide. Minoxidil sulfate did not affect 45Ca outflow from islets perfused in the presence of extracellular Ca++ and absence or presence of glucose. However, in islets exposed to a medium deprived of extracellular Ca++, the drug provoked a rise in 45Ca outflow. Whether in the absence or presence of extracellular Ca++, minoxidil sulfate increased the cytosolic free Ca++ concentration of islet cells. Lastly, minoxidil sulfate increased the release of insulin from glucose-stimulated pancreatic islets. These results suggest that minoxidil sulfate reduces the activity of the ATP-sensitive K+ channels and promotes an intracellular translocation of Ca++. The latter change might account for the effect of the drug on the insulin-releasing process. However, the secretory response to minoxidil sulfate could also be mediated, at least in part, by a modest Ca++ entry.

Antoine, M.H.; Hermann, M.; Herchuelz, A.; Lebrun, P. (Laboratory of Pharmacology, Brussels Free University School of Medicine (Belgium))



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

PubMed Central

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.

Weinkopff, Tiffany; Lammie, Patrick



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

Microsoft Academic Search

Recurrent mossy fiber synapses in the dentate gyrus of epileptic brain facilitate the synchronous firing of granule cells and may promote seizure propagation. Mossy fiber terminals contain and release zinc. Released zinc inhibits the activation of NMDA receptors and may therefore oppose the development of granule cell epileptiform activity. Hippocampal slices from rats that had experienced pilocarpine-induced status epilepticus and

Olga Timofeeva; J. Victor Nadler



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

SciTech Connect

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.

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



Quantitative analysis of granule cell axons and climbing fiber afferents in the turtle cerebellar cortex  

PubMed Central

The turtle cerebellar cortex is a single flat sheet of gray matter that greatly facilitates quantitative analysis of biotylinated dextran amine labeled granule cell and olivocerebellar axons and Nissl-stained granule and Purkinje neurons. On average, ascending granule cell axons are relatively thicker than their parallel fiber branches (mean±SD: 0.84±0.17 vs 0.64±0.12 µm, respectively). Numerous en passant swellings, the site of presynaptic contact, were present on both ascending and parallel fiber granule cell axons. The swellings on ascending axons (1.82±0.34 µm, n=52) were slightly larger than on parallel fibers (1.43±0.24 µm, n=430). In addition, per unit length (100 µm) there were more swellings on ascending axons (11.2±4.2) than on parallel fibers (9.7±4.2). Each parallel fiber branch from an ascending axon is approximately 1.5 mm long. Olivocerebellar climbing fiber axons followed the highly tortuous dendrites of Purkinje cells in the inner most 15–20% of the molecular layer. Climbing fibers displayed relatively fewer en passant swellings. The spatial perimeter of climbing fiber arbors (area) increased 72% from anteriorly (1797 µm˛) to posteriorly (3090 µm˛) and 104% from medially (1690 µm˛) to laterally (3450 µm˛). Differences in the size and spacing of en passant swellings on granule cell axons suggest that ascending axons may have a functionally more significant impact on the excitability of a limited number of radially overlying Purkinje cells than the single contacts by parallel fiber with multiple orthogonally aligned Purkinje cell dendrites. The spatially restricted distribution of climbing fibers to the inner most molecular layer, the paucity of en passant swellings, and different terminal arbor areas are enigmatic. Nevertheless, these finding provide important anatomical information for future optical imaging and electrophysiological experiments.

Tolbert, D. L.; Conoyer, B.; Ariel, M.



Quantitative analysis of granule cell axons and climbing fiber afferents in the turtle cerebellar cortex.  


The turtle cerebellar cortex is a single flat sheet of gray matter that greatly facilitates quantitative analysis of biotylinated dextran amine labeled granule cell and olivocerebellar axons and Nissl-stained granule and Purkinje neurons. On average, ascending granule cell axons are relatively thicker than their parallel fiber branches (mean +/- SD: 0.84 +/- 0.17 vs 0.64 +/- 0.12 microm, respectively). Numerous en passant swellings, the site of presynaptic contact, were present on both ascending and parallel fiber granule cell axons. The swellings on ascending axons (1.82 +/- 0.34 microm, n = 52) were slightly larger than on parallel fibers (1.43 +/- 0.24 microm, n = 430). In addition, per unit length (100 microm) there were more swellings on ascending axons (11.2 +/- 4.2) than on parallel fibers (9.7 +/- 4.2). Each parallel fiber branch from an ascending axon is approximately 1.5 mm long. Olivocerebellar climbing fiber axons followed the highly tortuous dendrites of Purkinje cells in the inner most 15-20% of the molecular layer. Climbing fibers displayed relatively fewer en passant swellings. The spatial perimeter of climbing fiber arbors (area) increased 72% from anteriorly (1797 microm2) to posteriorly (3090 microm2) and 104% from medially (1690 microm2) to laterally (3450 microm2). Differences in the size and spacing of en passant swellings on granule cell axons suggest that ascending axons may have a functionally more significant impact on the excitability of a limited number of radially overlying Purkinje cells than the single contacts by parallel fiber with multiple orthogonally aligned Purkinje cell dendrites. The spatially restricted distribution of climbing fibers to the inner most molecular layer, the paucity of en passant swellings, and different terminal arbor areas are enigmatic. Nevertheless, these finding provide important anatomical information for future optical imaging and electrophysiological experiments. PMID:15503131

Tolbert, D L; Conoyer, B; Ariel, M



Adult Neurogenesis Produces a Large Pool of New Granule Cells in the Dentate Gyrus  

Microsoft Academic Search

Knowing the rate of addition of new granule cells to the adult dentate gyrus is critical to understanding the function of adult neurogenesis. Despite the large number of studies of neurogenesis in the adult dentate gyrus, basic questions about the magnitude of this phenomenon have never been addressed. The S-phase marker bromodeoxyuridine (BrdU) has been extensively used in recent studies




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

PubMed Central

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.

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



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

SciTech Connect

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.

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



Flavonol kaempferol improves chronic hyperglycemia-impaired pancreatic beta-cell viability and insulin secretory function.  


Considerable evidence shows that chronic hyperglycemia can cause pancreatic beta-cell dysfunction, which contributes to progressive deterioration of glucose homeostasis and overt diabetes. In the present study, we found that kaempferol, a flavonol compound present in various Chinese medicinal herbs, has cytoprotective effects on cultured clonal beta-cells and pancreatic human islets. Kaempferol treatment dose-dependently promoted viability, inhibited cellular apoptosis, and reduced caspase-3 activity in beta-cells and human islets exposed to chronic high glucose, with 10 ?M kaempferol exerting the maximum effect. In addition, kaempferol treatment improved the expression of anti-apoptotic proteins Akt and Bcl-2 that was significantly reduced in beta-cells and human islets chronically exposed to hyperglycemia. Furthermore, exposure of beta-cells and human islets to kaempferol restored high glucose-attenuated intracellular cAMP and ATP production. Inhibition of protein kinase A or Akt activation ablated the anti-apoptotic effect of kaempferol. These cytoprotective effects of kaempferol were associated with improved insulin secretory function and synthesis in beta-cells and human islets. These findings provide evidence that kaempferol may be a naturally occurring anti-diabetic compound by protecting pancreatic beta-cell survival and function in a hostile environment that would otherwise lead to type 2 diabetes. PMID:21914439

Zhang, Yanling; Liu, Dongmin



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

Microsoft Academic Search

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

Gang Tong; Robert C Malenka; Roger A Nicoll



Sources of Reactive Oxygen Species Production in Excitotoxin-Stimulated Cerebellar Granule Cells  

Microsoft Academic Search

Reactive oxygen species (ROS) production in rat cerebellar granule cells in the presence of the excitotoxinsN-methyl-d-aspartate (NMDA) and kainic acid (KA) and by the protein kinase C activator phorbol myristate acetate (PMA) was Ca2+-dependent and resulted in decreased cell viability. Exposure of stimulated cells to rotenone (a respiratory chain inhibitor) did not decrease ROS levels and did not affect short-term

Alexander A. Boldyrev; David O. Carpenter; Matthew J. Huentelman; Craig M. Peters; Peter Johnson



Methylmercury Affects Multiple Subtypes of Calcium Channels in Rat Cerebellar Granule Cells  

Microsoft Academic Search

We tested the ability of methylmercury (MeHg) to block calcium channel current in cultures of neonatal cerebellar granule cells using whole-cell patch clamp techniques and Ba2+ as charge carrier. Low micromolar concentrations of MeHg (0.25–1 ?M) reduced the amplitude of whole cell Ba2+ current in a concentration- and time-dependent fashion; however, this effect was not voltage-dependent and the current–voltage relationship

Jay E. Sirois; William D. Atchison



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


The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i. PMID:11978799

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



Isoprenoid biosynthesis. Metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis.  


Two independent pathways operate in plants for the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of all isoprenoids. The mevalonate pathway is present in the cytosol, whereas the recently discovered mevalonate-independent pathway is localized to plastids. We have used isolated peppermint (Mentha piperita) oil gland secretory cells as an experimental model system to study the effects of the herbicides fosmidomycin, phosphonothrixin, methyl viologen, benzyl viologen, clomazone, 2-(dimethylamino)ethyl diphosphate, alendronate, and pamidronate on the pools of metabolites related to monoterpene biosynthesis via the mevalonate-independent pathway. A newly developed isolation protocol for polar metabolites together with an improved separation and detection method based on liquid chromatography-mass spectrometry have allowed assessment of the enzyme targets for a number of these herbicides. PMID:11553758

Lange, B M; Ketchum, R E; Croteau, R B



The unique ultrastructure of secretory membranes in gastric parietal cells depends upon the presence of H + , K + ATPase  

Microsoft Academic Search

Ion transporters play a central role in gastric acid secretion. To determine whether some of these transporters are necessary for the normal ultrastructure of secretory membranes in gastric parietal cells, mice lacking transporters for H+, K+, Cl-, and Na+ were examined for alterations in volume density (Vd) of basolateral, apical, tubulovesicular and canalicular membranes, microvillar dimensions, membrane flexibility, and ultrastructure.

Marian L. Miller; Louise M. Judd; Ian R. van Driel; Anastasia Andringa; Michael Flagella; Sheila M. Bell; Patrick J. Schultheis; Zachary Spicer; Gary E. Shull



Thrombopoietin regulates proliferation, apoptosis, secretory activity and intracellular messengers in porcine ovarian follicular cells: involvement of protein kinase A  

Microsoft Academic Search

Thrombopoietin (TPO) is known to be involved in megakariocytopoesis, but its role in the control of ovarian function is unknown. The aims of this study were to determine whether TPO can regulate the proliferation, apoptosis and secretory activity of ovarian cells, to identify possible intracellular mediators of TPO action, especially protein kinase A (PKA), and to define their inter- relationships

A V Sirotkin; P Sanislo; H-J Schaeffer; I Florkovicová; J Kotwica; J Bulla; L Hetényi



A Novel Function of Noc2 in Agonist-Induced Intracellular Ca2+ Increase during Zymogen-Granule Exocytosis in Pancreatic Acinar Cells  

PubMed Central

Noc2, a putative Rab effector, contributes to secretory-granule exocytosis in neuroendocrine and exocrine cells. Here, using two-photon excitation live-cell imaging, we investigated its role in Ca2+-dependent zymogen granule (ZG) exocytosis in pancreatic acinar cells from wild-type (WT) and Noc2-knockout (KO) mice. Imaging of a KO acinar cell revealed an expanded granular area, indicating ZG accumulation. In our spatiotemporal analysis of the ZG exocytosis induced by agonist (cholecystokinin or acetylcholine) stimulation, the location and rate of progress of ZG exocytosis did not differ significantly between the two strains. ZG exocytosis from KO acinar cells was seldom observed at physiological concentrations of agonists, but was normal (vs. WT) at high concentrations. Flash photolysis of a caged calcium compound confirmed the integrity of the fusion step of ZG exocytosis in KO acinar cells. The decreased ZG exocytosis present at physiological concentrations of agonists raised the possibility of impaired elicitation of calcium spikes. When calcium spikes were evoked in KO acinar cells by a high agonist concentration: (a) they always started at the apical portion and traveled to the basal portion, and (b) calcium oscillations over the 10 µM level were observed, as in WT acinar cells. At physiological concentrations of agonists, however, sufficient calcium spikes were not observed, suggesting an impaired [Ca2+]i-increase mechanism in KO acinar cells. We propose that in pancreatic acinar cells, Noc2 is not indispensable for the membrane fusion of ZG per se, but instead performs a novel function favoring agonist-induced physiological [Ca2+]i increases.

Ogata, Sho; Miki, Takashi; Seino, Susumu; Tamai, Seiichi; Kasai, Haruo; Nemoto, Tomomi



LIS1 Deficiency Promotes Dysfunctional Synaptic Integration of Granule Cells Generated in the Developing and Adult Dentate Gyrus  

PubMed Central

Type I lissencephaly, a neuronal migration disorder characterized by cognitive disability and refractory epilepsy, is often caused by heterozygous mutations in the LIS1 gene. Histopathologies of malformation-associated epilepsies have been well described, but it remains unclear whether hyperexcitability is attributable to disruptions in neuronal organization or abnormal circuit function. Here, we examined the effect of LIS1 deficiency on excitatory synaptic function in the dentate gyrus of hippocampus, a region believed to serve critical roles in seizure generation and learning and memory. Mice with heterozygous deletion of LIS1 exhibited robust granule cell layer dispersion, and adult-born granule cells labeled with enhanced green fluorescent protein were abnormally positioned in the molecular layer, hilus, and granule cell layer. In whole-cell patch-clamp recordings, reduced LIS1 function was associated with greater excitatory synaptic input to mature granule cells that was consistent with enhanced release probability at glutamatergic synapses. Adult-born granule cells that were ectopically positioned in the molecular layer displayed a more rapid functional maturation and integration into the synaptic network compared with newborn granule cells located in the hilus or granule cell layer or in wild-type controls. In a conditional knock-out mouse, induced LIS1 deficiency in adulthood also enhanced the excitatory input to granule cells in the absence of neuronal disorganization. These findings indicate that disruption of LIS1 has direct effects on excitatory synaptic transmission independent of laminar disorganization, and the ectopic position of adult-born granule cells within a malformed dentate gyrus critically influences their functional maturation and integration.

Dinday, Matthew T.; Hindle-Katel, William; Baraban, Scott C.



Caspase 6 activity initiates caspase 3 activation in cerebellar granule cell apoptosis  

Microsoft Academic Search

Using a well documented ex vivo system consisting of rodent cerebellar granule cells (CGCs) the activation of caspases 3 and 6 during apoptosis induced by withdrawal of trophic support was analyzed. At the time of deprivation, the addition of the irreversible, broad-spectrum caspase inhibitor zVADfmk or the cell permeable, caspase 6 inhibitor CP-VEID-cho can transiently suppress the appearance of apoptosis,

T E Allsopp; J McLuckie; L E Kerr; M Macleod; J Sharkey; J S Kelly



Target Cell Lysis by CTL Granule Exocytosis Is Independent of ICE\\/Ced3 Family Proteases  

Microsoft Academic Search

Activation of ICE\\/Ced-3 family proteases (caspases) has been proposed to mediate both the granule exocytosis and Fas–Fas ligand pathways of rapid target cell death by cytotoxic T lymphocytes. In agreement with this model, two peptide fluoromethyl ketone caspase inhibitors and baculovirus p35 blocked apoptotic nuclear damage and target cell lysis by the CTL-mediated Fas–Fas ligand pathway. The peptide caspase inhibitors

Apurva Sarin; Mark S. Williams; Martha A. Alexander-Miller; Jay A. Berzofsky; Charles M. Zacharchuk; Pierre A. Henkart



Interleukin10 Prevents Glutamate-Mediated Cerebellar Granule Cell Death by Blocking Caspase3Like Activity  

Microsoft Academic Search

Interleukin-10 (IL-10) has been shown to reduce neuronal de- generation after CNS injury. However, the molecular mecha- nisms underlying the neuroprotective properties of this cytokine are still under investigation. Glutamate exacerbates secondary injury caused by trauma. Thus, we examined whether IL-10 prevents glutamate-mediated cell death. We used rat cerebellar granule cells in culture because these neurons undergo apo- ptosis upon

Alessia Bachis; Anna M. Colangelo; Stefano Vicini; Pylord P. Doe; Maria A. De Bernardi; Gary Brooker; Italo Mocchetti



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

Microsoft Academic Search

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).

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



Studies on histamine-retaining granules obtained from isolated rat mast cells  

Microsoft Academic Search

Histamine-retaining granules were isolated from rat mast cells after sonication in either sucrose or Ficoll-Hypaque media. The preparations obtained were compared in regard to recovery and spontaneous loss of histamine. The effect of agents known to release histamine from intact rat mast cells (antigen, compound 48\\/80, decylamine, the ionophores A23187 and X537A as well as ATP) was studied on the

N. Grosman; B. Diamant



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


Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABA?6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage. PMID:23225629

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



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

PubMed Central

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.

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



An intracellular role for ABCG1-mediated cholesterol transport in the regulated secretory pathway of mouse pancreatic ? cells  

PubMed Central

Cholesterol is a critical component of cell membranes, and cellular cholesterol levels and distribution are tightly regulated in mammals. Recent evidence has revealed a critical role for pancreatic ? cell–specific cholesterol homeostasis in insulin secretion as well as in ? cell dysfunction in diabetes and the metabolic response to thiazolidinediones (TZDs), which are antidiabetic drugs. The ATP-binding cassette transporter G1 (ABCG1) has been shown to play a role in cholesterol efflux, but its role in ? cells is currently unknown. In other cell types, ABCG1 expression is downregulated in diabetes and upregulated by TZDs. Here we have demonstrated an intracellular role for ABCG1 in ? cells. Loss of ABCG1 expression impaired insulin secretion both in vivo and in vitro, but it had no effect on cellular cholesterol content or efflux. Subcellular localization studies showed the bulk of ABCG1 protein to be present in insulin granules. Loss of ABCG1 led to altered granule morphology and reduced granule cholesterol levels. Administration of exogenous cholesterol restored granule morphology and cholesterol content and rescued insulin secretion in ABCG1-deficient islets. These findings suggest that ABCG1 acts primarily to regulate subcellular cholesterol distribution in mouse ? cells. Furthermore, islet ABCG1 expression was reduced in diabetic mice and restored by TZDs, implicating a role for regulation of islet ABCG1 expression in diabetes pathogenesis and treatment.

Sturek, Jeffrey M.; Castle, J. David; Trace, Anthony P.; Page, Laura C.; Castle, Anna M.; Evans-Molina, Carmella; Parks, John S.; Mirmira, Raghavendra G.; Hedrick, Catherine C.



Methylmercury impairs motor function in early development and induces oxidative stress in cerebellar granule cells.  


Environmental toxicants such as methylmercury play a critical role in the pathogenesis of many neurodevelopmental disorders. Environmental exposure to methylmercury frequently occurs at low doses, most frequently through fish consumption. Although the general population is at risk for exposure, pregnant women and young children are the most vulnerable. A common symptom of perinatal exposure to methylmercury is increased sensory (visual) deficits, motor impairment, and an overall cognitive decline. Research has indicated that the developing cerebellum, specifically the cerebellar granular layer, is particularly vulnerable to methylmercury neurotoxicity. This review examines the effects of low-level methylmercury exposure on motor coordination. We specifically focus on the role of cerebellar granule cells in methylmercury neurotoxicity. We suggest that methylmercury induces oxidative stress in cerebellar granule cells, which subsequently results in apoptotic cell death. Understanding the mechanism by which methylmercury induces toxicity within the developing brain will allow for enhanced treatments and potential reversal of the detrimental effects. PMID:23948120

Patel, Eshan; Reynolds, Mindy



Cytokine signals through STAT3 promote expression of granulocyte secondary granule proteins in 32D cells  

PubMed Central

Objective In a previous study, we showed that activation of a transfected human erythropoietin receptor (EPOR) in the murine myeloid cell line 32D resulted in the development of morphologic features of granulocytic differentiation and expression of the neutrophil primary granule protein myeloperoxidase. We now studied if EPOR signaling could also mediate secondary granule protein gene expression and investigated the signal transduction requirements for induction of secondary granule gene expression in 32D cells. Materials and Methods Wild-type and variant 32D cells expressing normal or chimeric EPORs or receptors for granulocyte colony-stimulating factor (G-CSFRs) were stimulated with EPO or G-CSF and the expression of granulocyte-specific genes was analyzed by Northern blot analysis. To determine the signaling mechanisms required for secondary granule protein gene induction, the activation of STAT pathways following growth factor stimulation was studied by Western blot analysis. Results We found that EPO treatment of 32D cells engineered to express EPOR did not result in induction of the secondary granule protein genes encoding lactoferrin and 24p3 lipocalin, the mouse homolog of human N-Gal, or the myeloid transcription factor C/EBP?. Replacement of the intracellular domain of EPOR with the intracellular domain of G-CSFR in a chimeric receptor was associated with EPO-mediated induction of lactoferrin, 24p3 lipocalin, and C/EBP? genes. We found that STAT3 phosphorylation was mediated by the intracellular domain of G-CSFR, but not EPOR. Replacement of one or two of the STAT5 binding sites in the intracytoplasmic domain of the EPOR with STAT3 binding sites resulted in EPO-mediated STAT3 activation and a marked increase in the expression of the 24p3 lipocalin gene. Knockdown of STAT3 protein levels with siRNA caused significant decrease in 24p3 lipocalin gene induction. Conclusion These results indicate that EPOR signaling cannot substitute for G-CSFR signaling to stimulate secondary granule protein gene expression in 32D cells. In addition, STAT3 is a critical mediator of 24p3 lipocalin gene expression in these cells.

Wang, Lei; Arcasoy, Murat O.; Watowich, Stephanie S.; Forget, Bernard G.



Dynamic light-scattering study on changes in mobility of chromaffin granules in actin network with its assembly and Ca2+-dependent disassembly by gelsolin  

NASA Astrophysics Data System (ADS)

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. In order to elucidate dynamical behaviors of secretory granules in actin network, 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 medullae, and found that the entanglement of actin filaments rapidly formed cages which confined granules in them. We also studied the effect of gelsolin, one of the actin-severing proteins, on the network of actin filaments performed in the presence of chromaffin granules. It turned out that the cages which confined granules rapidly disappeared when gelsolin was added in the presence of free Ca2+ ions. Semiquantitative analyses of dynamic light-scattering spectra permitted us to estimate the changes in the mobility (or translational diffusion coefficient) of chromaffin granules in the actin network with its assembly and Ca2+-dependent disassembly by gelsolin. Based on the present results and some pieces of evidence in literature, a model is proposed for biophysical situations before, during, and after an exocytotic event.

Fujime, Satoru; Miyamoto, Shigeaki; Funatsu, Takashi; Ishiwata, S.



Secretory kinetics in the follicular cells of silkmoths during eggshell formation  

PubMed Central

Procedures for quantitative autoradiography were used for studying the process of secretion of eggshell (chorion) proteins in the follicular epithelium of silkmoths. The method was based on photometric measurements of the reflectance of vertically illuminated autoradiographic silver grains. Results were analyzed and plotted by computer. Secretory kinetics were also determined by analysis of labeled proteins in physically separated epithelium and chorion. Rapid accumulation of radioactivity into "clumps" visualized by light microscope autoradiography and evidence from preliminary electron microscope autoradiography indicate that, within 2 min from the time of synthesis, labeled chorion proteins move to Golgi regions scattered throughout the cytoplasm. The proteins begin to accumulate in the apical area 10-20 min later and to be discharged from the cell. The time for half-secretion is 20-25 min, and discharge is essentially complete 30-50 min after labeling. At the developmental stages examined, the kinetics of secretion appear to be similar for all proteins. Within the chorion the proteins rapidly assume a characteristic distribution, which varies for different developmental stages. Two relatively slow steps have been identified in secretion, associated with residence in Golgi regions and in the cell apex, respectively. By contrast, translocation of proteins across the cell and deposition of discharged proteins in the chorion are rapid steps.



POMC-related products in the intermediate pituitary of the amphibian, Bufo marinus: Differential subcellular processing in the Golgi and secretory granules  

Microsoft Academic Search

In the intermediate pituitary of the anuran amphibian, Bufo marinus, the N-acetylation of ACTH(1–13)-NH2 to yield ?-MSH occurss as a cosecretory processing event, whereas the N-acetylation of ?-endorphin occurs as a posttranslational processing event. To understand how these two N-acetylation reactions are segregated, B. marinus intermediate pituitary cells were analyzed by immunogold labeling electron microscopy, and by using an ultracentrifugation

Tami C. Steveson; Robert M. Dores



Evidence for the Existence of Secretory Granule (Dense-Core Vesicle)Based Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Signaling System in Astrocytes  

Microsoft Academic Search

BackgroundThe gliotransmitters released from astrocytes are deemed to play key roles in the glial cell-neuron communication for normal function of the brain. The gliotransmitters, such as glutamate, ATP, D-serine, neuropeptide Y, are stored in vesicles of astrocytes and secreted following the inositol 1,4,5-trisphosphate (IP3)-induced intracellular Ca2+ releases. Yet studies on the identity of the IP3-dependent intracellular Ca2+ stores remain virtually

Yong Suk Hur; Ki Deok Kim; Sun Ha Paek; Seung Hyun Yoo; Rafael Linden



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


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

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



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


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

Mathias, Amandine; Longet, Stéphanie; Corthésy, Blaise



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

Microsoft Academic Search

Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (Mr=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the

David McCaskill; Jonathan Gershenzon; Rodney Croteau



Effect of lysosomotropic amines on the secretory pathway and on the recycling of the asialoglycoprotein receptor in human hepatoma cells  

Microsoft Academic Search

We studied the intracellular transport of secretory and membrane proteins in the human hepatoma cell line HepG-2 infected with vesicular stomatitis virus. Cells were pulse- labeled in the presence of (3SS)methionine and chased in the presence of the lysosomotropic agent primaquine. At a concentration of 0.3 mM primaquine effectively inhibited the secretion of albumin and, to a lesser extent, that




Biochemical and microscopic evidence for the internalization of drug-containing mast cell granules by macrophages and smooth muscle cells.  


During mast cell degranulation the soluble component of the granule is released into extracellular fluid, whereas two neutral proteases and heparin proteoglycans form the extracellular granule remnants. These structures are negatively charged and bind with high affinity LDL and other basic molecules. In this study we show that granule remnants expelled into extracellular fluid are able to bind the aminoglycoside antibiotic gentamicin and the anticancer agent doxorubicin in a dose-dependent manner. In addition, granule remnants loaded with the two basic substances are subsequently phagocytosed by macrophages. Indeed, when cells are incubated for 24 h with 1 mg/ml gentamicin, the intracellular concentration of the drug, which in basal conditions is extremely low, increases significantly in the presence of degranulating mast cells (from 5.1 +/- 1.0 to 25.4 +/- 2.5 microg/mg protein) and a good correlation between histamine release and gentamicin uptake is evident. The antineoplastic agent doxorubicin can penetrate cells by passive diffusion; however, when mast cells are added to macrophage monolayer, incubated for 30 min with 50 microM of the antineoplastic agent, a significant increase in intracellular doxorubicin concentration is observed (from 3.5 +/- 0.2 to 4.7 +/- 0.2 microg/mg protein). Internalization of granule remnants carrying gentamicin or doxorubicin is also evident in smooth muscle cells of the synthetic phenotype. In particular, when smooth muscle cells are incubated for 24 h with 1 mg/ml gentamicin, addition of isolated granules increases the uptake from 2.4 +/- 0.2 to 4.8 +/- 0.4 microg/mg protein. Similar results are obtained in smooth muscle cells incubated for 4 h with doxorubicin 50 microM (from 3.3 +/- 0.2 to 4.8 +/- 0.5 microg/mg protein). Data are confirmed by microscopic experiments by means of fluorescence microscopy and electron microscopic studies. The study demonstrates that basic substances can enter phagocytic cells when loaded to granule remnants. The phenomenon can be of particular interest for substances like the aminoglycosides that do not cross biological membranes; indeed, the storage of these antibiotics in phagocytic cells could have important consequences on their antibacterial activity in vivo. Macrophages and smooth muscle cells can also act as a reservoir for doxorubicin. High concentrations of the antineoplastic agent in these cells could be responsible for toxicity, as well as play an important role in the transport of the drug to tumor cells. PMID:11133349

Decorti, G; Klugmann, F B; Crivellato, E; Malusŕ, N; Furlan, G; Candussio, L; Giraldi, T



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

PubMed Central

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.

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



N-acetyl cysteine improves affinity of beta-tricalcium phosphate granules for cultured osteoblast-like cells.  


Enhancement of bone substitute's biocompatibility may accelerate healing of surrounding bone. Although widely used as a biodegradable alloplastic bone substitute for alveolar bone augmentation, the osteocompatibility of beta-tricalcium phosphate (?-TCP) remains to be proven. The adverse cellular response to biomaterials is associated with oxidative stress. We hypothesized that commercially available ?-TCP granules for clinical use, caused oxidative stress and was not optimal in osteocompatibility and that application of antioxidant amino acid derivative N-acetyl cysteine (NAC) would improve osteoblastic responses to the material. Only 20% of rat calvarial osteoblasts cultured on ?-TCP granules remained viable at 24 h after seeding as opposed to 90% on polystyrene. Cell death on ?-TCP granules was characterized by necrosis. However, the percentage of viable osteoblasts cultured on ?-TCP granules showed a 100% increase with pre-treatment with NAC. NAC restored suppressed alkaline phosphatase activity on ?-TCP granules at day 5. Intracellular ROS level on ?-TCP granules was 16-fold greater than that on polystyrene, but decreased by half with pre-treatment with NAC. Cell death and intracellular ROS elevation were also induced in polystyrene culture under ?-TCP granules even when the osteoblasts were not in direct contact with the ?-TCP granules. NAC, however, prevented induction of cell death and elevation of intracellular ROS under ?-TCP granules. These results indicate that commercially available ?-TCP granules negatively affect cultured osteoblastic viability and function via oxidative stress and that NAC improves these negative responses to the material. This implies enhanced bone regeneration around biodegradable calcium phosphate-based bone substitute by NAC. PMID:20876635

Yamada, Masahiro; Minamikawa, Hajime; Ueno, Takeshi; Sakurai, Kaoru; Ogawa, Takahiro



Promiscuous T cell epitope prediction of Candida albicans secretory aspartyl protienase family of proteins.  


Candida albicans is one of the most important opportunistic dimorphic fungi responsible for hospital acquired fungal infection in humans. Candida infection rarely occurs in healthy individuals but it is frequently associated with patients who suffer from acquired immunodeficiency syndromes. To date, there is no effective vaccine against this fungal infection. Herein we demonstrated the use of immunomics to characterize promiscuous T cell epitope of C. albicans virulence factors by utilizing CandiVF, a C. albicans database previously constructed to be equipped with protein sequence analysis tool, three dimensional structure visualization software, sequence variable analysis program and Hotspot Hunter epitope prediction tool. Secretory aspartyl proteinase (Sap) family was chosen as a model to validate the Hotspot Hunter prediction. Analysis of Saps1-10 protein entries from CandiVF database revealed that a consensus T cell epitope was located at the C-terminal region of Saps1-10. The result of the in silico prediction was subsequently validated by conventional immunological methods. By using overlapping peptides span the predicted consensus T cell epitopes of Saps1-10 as stimulators, it was demonstrated that peptides S6 and S7 could stimulate PBMC proliferation in 9 of 12 blood donors. Interestingly, S2, the predicted T cell epitope of Sap2, was able to induce proliferation of all donors' PBMC. ELISpot assay for the detection of gamma-interferon producing clones confirmed that the peptide S2 actually stimulated T cell proliferation. The results suggest that S2 might be a potential candidate for vaccine development against C. albicans infection or to be utilized as an adjuvant to stimulate the pre-existing CD4+ T cell in other vaccine development. PMID:17974505

Tongchusak, Songsak; Brusic, Vladimir; Chaiyaroj, Sansanee C



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


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

Reistad, Trine; Fonnum, Frode; Mariussen, Espen



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

PubMed Central

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.

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.



Distinct pathways for basolateral targeting of membrane and secretory proteins in polarized epithelial cells.  

PubMed Central

Polarized epithelial cells target distinct sets of membrane and secretory proteins to their apical and basolateral domains. Here we examine whether constitutively secreted and membrane proteins that are bound for the same domain share the same carrier vesicles. To address the issue, differential effects of microtubule depolymerization on basolateral protein targeting in the polarized Madin-Darby canine kidney II cell line were studied. We find that the basolateral insertion of the active, ouabain-binding Na+,K(+)-ATPase and of a set of very late antigen integrins is little affected by microtubule disruption. Under equivalent conditions, the basolateral secretion of the basement membrane protein laminin is strongly suppressed. More specifically, it is demonstrated that microtubules are involved in targeting laminin, but not integrins, from the compartment related to the accumulation of newly synthesized proteins at 20 degrees C (trans-Golgi network) to the basolateral domain. Our study also reveals that laminin associated with basolateral binding sites interacts with those sites only secondarily to secretion. The data provide evidence for a branch in the basolateral targeting pathway, with secreted and membrane proteins loaded into distinct carrier vesicles. Images

Boll, W; Partin, J S; Katz, A I; Caplan, M J; Jamieson, J D



Suppression of dendritic cell maturation by Trichinella spiralis excretory/secretory products.  


Evidence from experimental studies indicates that during chronic infections with certain helminth species a regulatory network is induced that can down-modulate not only parasite-induced inflammation but also reduce other immunopathologies such as allergies and autoimmune diseases. The mechanisms however, and the molecules involved in this immunomodulation are unknown. Here, we focus on the effect of Trichinella spiralis excretory/secretory antigens (TspES) on the innate immune response by studying the effect of TspES on DC maturation in vitro. Bone marrow-derived DC from BALB/c mice were incubated with TspES either alone or in combination with LPS derived from two different bacteria. As indicators of DC maturation, the cytokine production (IL-1alpha, IL-6, IL-10, IL-12p70 and TNF-alpha) and the expression of various surface molecules (MHC-II, CD40, CD80 and CD86) were measured. Results indicate that while TspES alone did not change the expression of the different surface molecules or the cytokine production, it completely inhibited DC maturation induced by Escherichia coli LPS (E. coli LPS). In contrast, DC maturation induced by LPS from another bacterium, Neisseria meningitidis, was not affected by TspES. These results were confirmed using TLR4/MD2/CD14 transfected HEK 293 cells. In conclusion, T. spiralis ES antigens lead to suppression of DC maturation but this effect depends on the type of LPS used to activate these cells. PMID:19751476

Langelaar, M; Aranzamendi, C; Franssen, F; Van Der Giessen, J; Rutten, V; van der Ley, P; Pinelli, E



Single cell RT-PCR demonstrates differential expression of GABAC receptor rho subunits in rat hippocampal pyramidal and granule cells.  


Although gamma-aminobutyric acid (GABA)C receptor rho1, rho2 and rho3 subunits are reportedly expressed in pyramidal and granule cells in the hippocampus at various developmental stages, it is not clear whether these three rho subunits are coexpressed in a single neuron. To attempt to answer this question, we performed single-cell RT-PCR for rho subunits from neurons of rat brain hippocampus. In hippocampal cultures, pyramidal cells were positive for rho1 mRNA expression in 89%, rho2 in 94% and rho3 in 94%, while granule cells were positive for rho1 mRNA in only 6%, rho2 in 36% and rho3 in 91%. Intensive amplification of the RT-PCR products by the second PCR revealed that all the three rho subunits were coexpressed in a single pyramidal and granule cells from both of the cultures and the slices. These results suggest that all the three GABAC receptor rho1, rho2 and rho3 subunits are present probably in different compositions in pyramidal and granule cells in the rat hippocampus. PMID:15046860

Liu, Bing; Hattori, Naoki; Jiang, Baohong; Nakayama, Yasuhisa; Zhang, Nan-Yan; Wu, Bo; Kitagawa, Kaori; Taketo, Megumi; Matsuda, Hiroko; Inagaki, Chiyoko



Enlargement of secretory vesicles by protein tyrosine phosphatase PTP-MEG2 in rat basophilic leukemia mast cells and Jurkat T cells.  


Stimulus-induced secretion of bioactive polypeptides is a fundamental aspect of the immune system. Secretory proteins are synthesized in the endoplasmic reticulum and are transported through the Golgi apparatus to the trans-Golgi network, where they are sorted into transport vesicles that bud off and fuse into condensing vacuoles, which subsequently undergo an editing and concentration process to become mature secretory vesicles. In this study, we report that the PTP-MEG2 protein tyrosine phosphatase is located on these vesicles in mast cells. Expression of PTP-MEG2 caused a striking enlargement of these vesicles in both rat basophilic leukemia mast cells and Jurkat T leukemia cells into giant vesicles with diameters of up to several micrometers. The fused vesicles did not acquire markers for other compartments and were adjacent to the trans-Golgi network, contained carboxypeptidase E, chromogranin C, and IL-2, and had an electron-dense core typical of secretory vesicles. Expression of PTP-MEG2 also caused a reduction in the secretion of IL-2 from stimulated Jurkat cells. The effects of PTP-MEG2 on secretory vesicles required the catalytic activity of PTP-MEG2 and was rapidly reversed by pervanadate. We propose that PTP-MEG2 represents a novel connection between tyrosine dephosphorylation and the regulation of secretory vesicles in hematopoietic cells. PMID:11971009

Wang, Xiaodong; Huynh, Huong; Gjörloff-Wingren, Anette; Monosov, Edvard; Stridsberg, Mats; Fukuda, Minoru; Mustelin, Tomas



Strong paired pulse depression of dentate granule cells in slices from patients with temporal lobe epilepsy  

Microsoft Academic Search

Summary.   Recurrent, feedback excitation by sprouted mossy fibers may contribute to the hyperexcitability observed in human temporal\\u000a lobe epilepsy. Observations in rodent models of epilepsy mimic the findings in human tissue and reveal that dentate granule\\u000a cells sprout axons which innervate fibers in their own dendritic layer. However, recent evidence in rodents suggest that these\\u000a sprouted fibers may form connections

T. H. Swanson; M. R. Sperling; M. J. O'Connor



The absence of cerebellar granule cell necrosis in the mouse following L- 2-chloropropionic acid administration  

Microsoft Academic Search

Oral administration of L-2-chloropropionic acid (L-CPA) to rats either as a single dose (750 mg\\/kg) or daily doses (250 mg\\/kg per day for 3 days) produces selective necrosis to the granule cell layer of the cerebellum. As part of a study to understand the mechanism of this selective toxicity, we investigated the toxicity of L-CPA and a related analogue, DL-2-bromopropionic

Edward A. Lock; Andrew Gyte; Stephen Duffell; Ian Wyatt



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

PubMed Central

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

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



Prenatal ethanol exposure enhances glutamate release stimulated by quisqualate in rat cerebellar granule cell cultures  

Microsoft Academic Search

Effects of prenatal ethanol exposure on extracellular glutamate accumulation stimulated by glutamate receptor agonists were\\u000a studied in rat cerebellar granule cell cultures. The prenatal exposure to ethanol was achieved via maternal consumption of\\u000a a SustacalTM liquid diet containing either 5% ethanol or isocaloric sucrose (pair-fed) substituted for ethanol from gestation d 11 until\\u000a the day of parturition. Neither the basal

Philip G. Rhodes; Zhengwei Cai



Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat  

Microsoft Academic Search

Structural neuronal network plasticity is associated with epileptogenesis during limbic kindling, but the full extent of network changes is not well understood. We investigated whether dentate granule cell (DGC) neurogenesis, which continues into adulthood in the rodent, is altered in the amygdala kindling model of epileptogenesis. Adult rats were stimulated to either 4–6, 9–10 or 19–20 class 4\\/5 (generalized) kindled

Jack M Parent; Sridevi Janumpalli; James O McNamara; Daniel H Lowenstein



[Fibronectin fragmentation unmasks the activity stimulating DNA and RNA biosynthesis in granulation tissue cells in vitro].  


Human blood plasma fibronectin decreased slightly the incorporation of precursors into nucleic acids of granulation tissue culture cells. A slight fragmentation of fibronectin, where the fragments with 180-200 kD molecular mass were developed, led to occurrence of the activity 2-fold stimulating the DNA synthesis. After more effective proteolysis using plasmin and trypsin the stimulating effect of fibronectin fragments on synthesis of nucleic acids maintained and constituted 165 +/- 12% and 127 +/- 7% for DNA and RNA, respectively. PMID:2437701

Zlatopol'ski?, A D; Za?denberg, M A; Berman, A E; Mazurov, V I; Karelin, A A


Application of dendritic cells stimulated with Trichinella spiralis excretory-secretory antigens alleviates experimental autoimmune encephalomyelitis.  


The parasitic nematode, Trichinella spiralis (T. spiralis), exerts an immunomodulatory effect on the host immune response through excretory-secretory products (ES L1) released from encysted muscle larvae. Our model of combined T. spiralis infection and experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats demonstrated a significant reduction in EAE severity in infected animals. Recently, we have created an immune status characteristic for the live infection by in vivo application of dendritic cells (DCs) stimulated with ES L1 products of T. spiralis muscle larvae. Moreover, these cells were able to ameliorate EAE when applied 7 days before EAE induction. ES L1-stimulated DCs increased production of IL-4, IL-10 and TGF-?, and decreased production of IFN-? and IL-17, both at the systemic level and in target organs. A significant increase in the proportion of CD4+CD25+Foxp3+ T cells was found among spleen cells, and CNS infiltrates from DA rats treated with ES L1-stimulated DCs before EAE induction, compared to controls injected with unstimulated DCs. Regulatory T cells, together with elevated levels of IL-10 and TGF-?, are most likely involved in restraining the production of Th1 and Th17 cytokines responsible for autoimmunity and thus are responsible for the beneficial effect of ES L1-educated DCs on the course of EAE. Our results show that ES L1 antigen-stimulated DCs are able not only to provoke, but also to sustain anti-inflammatory and regulatory responses regardless of EAE induction, with subsequent amelioration of EAE, or even protection from the disease. PMID:23307236

Sofronic-Milosavljevic, L J; Radovic, I; Ilic, N; Majstorovic, I; Cvetkovic, J; Gruden-Movsesijan, A



Differential Expression of Members of the bcl-2 Gene Family in Proliferative and Secretory Human Endometrium: Glandular Epithelial Cell Apoptosis Is Associated with Increased Expression of bax  

Microsoft Academic Search

Glandular epithelial cells of the human endometrium initiate ap- optosis in the secretory phase of the cycle. To better understand the regulation of apoptosis in this paradigm of endocrine-regulated cell turnover, we studied the expression of the cell death regulatory genes, bax, bcl-2, and bcl-x, in human proliferative and secretory endometria relative to the absence or presence of apoptosis. As



Upregulation of inward rectifier K+ (Kir2) channels in dentate gyrus granule cells in temporal lobe epilepsy.  


In humans, temporal lobe epilepsy (TLE) is often associated with Ammon's horn sclerosis (AHS) characterized by hippocampal cell death, gliosis and granule cell dispersion (GCD) in the dentate gyrus. Granule cells surviving TLE have been proposed to be hyperexcitable and to play an important role in seizure generation. However, it is unclear whether this applies to conditions of AHS. We studied granule cells using the intrahippocampal kainate injection mouse model of TLE, brain slice patch-clamp recordings, morphological reconstructions and immunocytochemistry. With progressing AHS and GCD, 'epileptic' granule cells of the injected hippocampus displayed a decreased input resistance, a decreased membrane time constant and an increased rheobase. The resting leak conductance was doubled in epileptic granule cells and roughly 70-80% of this difference were sensitive to K(+) replacement. Of the increased K(+) leak, about 50% were sensitive to 1 mm Ba(2+). Approximately 20-30% of the pathological leak was mediated by a bicuculline-sensitive GABA(A) conductance. Epileptic granule cells had strongly enlarged inwardly rectifying currents with a low micromolar Ba(2+) IC(50), reminiscent of classic inward rectifier K(+) channels (Irk/Kir2). Indeed, protein expression of Kir2 subunits (Kir2.1, Kir2.2, Kir2.3, Kir2.4) was upregulated in epileptic granule cells. Immunolabelling for two-pore weak inward rectifier K(+) channels (Twik1/K2P1.1, Twik2/K2P6.1) was also increased. We conclude that the excitability of granule cells in the sclerotic focus of TLE is reduced due to an increased resting conductance mainly due to upregulated K(+) channel expression. These results point to a local adaptive mechanism that could counterbalance hyperexcitability in epilepsy. PMID:19564397

Young, Christina C; Stegen, Michael; Bernard, René; Müller, Martin; Bischofberger, Josef; Veh, Rüdiger W; Haas, Carola A; Wolfart, Jakob



Effect of perinatally administered ethanol on the development of the cerebellar granule cell.  


The effect of perinatally administered ethanol on the postnatal development of cerebellar granule cells was studied in mice which received 25% of their caloric intake as ethanol. Timed-pregnant females were given ethanol in a chocolate-flavored liquid from gestation day 12 until postnatal day 7, then the mice were returned to a chow and water regime. The pups were killed on postnatal day 14 and the tissue processed for Golgi analysis of the dendritic arbor. Camera lucida drawings were made of the granule cells and these drawings were subsequently analyzed using a Zeiss Videoplan. Examination of the granule cells revealed that there was a significant decrease in dendritic length and in the extent of the dendritic field. In addition, there was a delayed maturation of the terminal dendritic processes. These results are in accord with other reports in the literature which suggest that the altered dendritic patterns seen in ethanol toxicity may be one of the causative factors in the minimal brain dysfunction that is characteristic of the fetal alcohol syndrome. PMID:3709729

Smith, D E; Foundas, A; Canale, J



Expression and function of ganglioside 9-O-acetyl GD3 in postmitotic granule cell development.  


We have shown previously that the Jones monoclonal antibody (Jones mAb) recognizes 9-O-acetyl GD3 expressed during periods of neuronal migration and neurite outgrowth in the developing rat nervous system. In the present study we investigated the expression of this ganglioside in the developing cerebellum and correlated this expression with granule cell migration. Electron microscopic immunocytochemistry revealed that around the peak of cerebellar neuronal migration (7-day-old rat), 9-O-acetyl GD3 was localized at the contact sites between migrating granule cells and radial glia in the external granular layer and prospective molecular layer. In addition, using microexplant and slice cultures of the postnatal rat cerebellum, we tested whether the ganglioside detected by our antibody contribute to the regulation of neuronal migration in the cerebellar cortex. We have shown that the Jones mAb blocks the migration of neurons in a dose-dependent manner. These findings suggest strongly that 9-O-acetyl GD3 is involved in granule cell migration in the developing cerebellum. PMID:11273644

Santiago, M F; Berredo-Pinho, M; Costa, M R; Gandra, M; Cavalcante, L A; Mendez-Otero, R



A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes.  


Secretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein (EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane. We have now investigated in detail the degree of colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2-EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange, neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH(3)