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1

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

PubMed Central

Background Mast 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 components of mast cell secretory granules. Methodology/Principal Findings Spermidine was released by mouse bone marrow derived mast cells (BMMCs) after degranulation induced by IgE/anti-IgE or calcium ionophore A23187. Additionally, both spermidine and spermine were detected in isolated mouse mast cell granules. Further, depletion of polyamines by culturing BMMCs with ?-difluoromethylornithine (DFMO) caused aberrant secretory granule ultrastructure, impaired histamine storage, reduced serotonin levels and increased ?-hexosaminidase content. A proteomic approach revealed that DFMO-induced polyamine depletion caused an alteration in the levels of a number of proteins, many of which are connected either with the regulated exocytosis or with the endocytic system. Conclusions/Significance Taken together, our results show evidence that polyamines are present in mast cell secretory granules and, furthermore, indicate an essential role of these polycations during the biogenesis and homeostasis of these organelles. PMID:21151498

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

2010-01-01

2

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

Microsoft Academic Search

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

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

2009-01-01

3

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

PubMed

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

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

2009-02-01

4

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: edith.puchelle@univ-reims.fr; Balossier, G. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France)

2005-10-01

5

The Glycosylation Pattern of Secretory Granules in Binucleate Trophoblast Cells is Highly Conserved in Ruminants  

Microsoft Academic Search

The binucleate trophoblast cells (BNCs) in the ruminant placenta are a unique feature of this taxon. These cells produce several secretory proteins and transfer these across the fetomaternal barrier into the dam. We used lectin histochemistry with a panel of 24 lectins to characterise the glycosylation pattern of BNC secretory granules in a variety of ruminants. Seven species out of

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

2010-01-01

6

Secretory granules in rat submandibular acinar cells during experimental Chagas disease.  

PubMed

The fine structure of rat submandibular glands has been compared in control and infected-rats with Y strain of Trypanosoma cruzi at day 18 of infection. In the acinar cells of the infected rats the secretory granules exhibit a variety of morphological types suggesting and early form of secretion product or a new secretory protein. PMID:2134905

Alves, J B

1990-01-01

7

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

PubMed

Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge. PMID:23975861

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

2013-10-01

8

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

PubMed

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

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

2000-01-01

9

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

PubMed

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

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

2010-01-01

10

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

2005-01-01

11

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)

2006-04-01

12

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

PubMed Central

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

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

2014-01-01

13

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

PubMed Central

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

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

2011-01-01

14

A role for serglycin proteoglycan in mast cell apoptosis induced by a secretory granule-mediated pathway.  

PubMed

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

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

2011-02-18

15

Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells  

PubMed Central

Background Cytotoxic cells of the immune system have evolved a lysosomal compartment to store and mobilize effector molecules. In T lymphocytes and NK cells, the death factor FasL is one of the characteristic marker proteins of these so-called secretory lysosomes, which combine properties of conventional lysosomes and exocytotic vesicles. Although these vesicles are crucial for immune effector function, their protein content in T cells has so far not been investigated in detail. Results In the present study, intact membranous vesicles were enriched from homogenates of polyclonally activated T cells and initially characterized by Western blotting and electron microscopic inspection. The vesicular fraction that contained the marker proteins of secretory lysosomes was subsequently analyzed by 2D electrophoresis and mass spectrometry. The proteome analysis and data evaluation revealed that 70% of the 397 annotated proteins had been associated with different lysosome-related organelles in previous proteome studies. Conclusion We provide the first comprehensive proteome map of T cell-derived secretory lysosomes with only minor contaminations by cytosolic, nuclear or other proteins. This information will be useful to more precisely address the activation-dependent maturation and the specific distribution of effector organelles and proteins in individual T or NK cell populations in future studies. PMID:21255389

2011-01-01

16

Age-Dependent Labeling and Imaging of Insulin Secretory Granules  

PubMed Central

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

2013-01-01

17

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

SciTech Connect

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.

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

2006-05-26

18

Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells  

SciTech Connect

The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of {sup 35}S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although ({sup 35}S)heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate ({sup 35}S)sulfate into separate heparin and chondroitin sulfate proteoglycans in an {approx}2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin ({sup 35}S)sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin ({sup 35}S)sulfate E proteoglycans and the ({sup 35}S)heparin proteoglycans were exocytosed from the ({sup 35}S)sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of {sup 35}S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.

Stevens, R.L.; Austen, K.F. (Brigham and Women's Hospital, Boston, MA (USA)); Fox, C.C.; Lichtenstein, L.M. (Johns Hopkins School of Medicine, Baltimore, MD (USA))

1988-04-01

19

Neurexin-1? Contributes to Insulin-containing Secretory Granule Docking*  

PubMed Central

Neurexins are a family of transmembrane, synaptic adhesion molecules. In neurons, neurexins bind to both sub-plasma membrane and synaptic vesicle-associated constituents of the secretory machinery, play a key role in the organization and stabilization of the presynaptic active zone, and help mediate docking of synaptic vesicles. We have previously shown that neurexins, like many other protein constituents of the neurotransmitter exocytotic machinery, are expressed in pancreatic ? cells. We hypothesized that the role of neurexins in ? cells parallels their role in neurons, with ?-cell neurexins helping to mediate insulin granule docking and secretion. Here we demonstrate that ? cells express a more restricted pattern of neurexin transcripts than neurons, with a clear predominance of neurexin-1? expressed in isolated islets. Using INS-1E ? cells, we found that neurexin-1? interacts with membrane-bound components of the secretory granule-docking machinery and with the granule-associated protein granuphilin. Decreased expression of neurexin-1?, like decreased expression of granuphilin, reduces granule docking at the ?-cell membrane and improves insulin secretion. Perifusion of neurexin-1? KO mouse islets revealed a significant increase in second-phase insulin secretion with a trend toward increased first-phase secretion. Upon glucose stimulation, neurexin-1? protein levels decrease. This glucose-induced down-regulation may enhance glucose-stimulated insulin secretion. We conclude that neurexin-1? is a component of the ?-cell secretory machinery and contributes to secretory granule docking, most likely through interactions with granuphilin. Neurexin-1? is the only transmembrane component of the docking machinery identified thus far. Our findings provide new insights into the mechanisms of insulin granule docking and exocytosis. PMID:22235116

Mosedale, Merrie; Egodage, Sonya; Calma, Rei C.; Chi, Nai-Wen; Chessler, Steven D.

2012-01-01

20

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

Microsoft Academic Search

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

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

1996-01-01

21

Processing of Proopiornelanocortin by Insulin Secretory Granule Proinsulin Processing Endopeptidases*  

PubMed Central

A lysed preparation of isolated insulin secretory granules efficiently cleaved murine proopiomelanocortin (mPOMC) at physiologically important Lys-Arg processing sites. This processing was mostly attributed to an activity that co-eluted with the proinsulin processing type-II endopeptidase from anion exchange chromatography (Lys-Arg-directed; Davidson, H. W., Rhodes, C. J., and Hutton, J. C. (1988) Nature 333, 93–96). The principal peptide hormone products generated by the insulin secretory granule lysate were identified by specific radioimmunoassay and NH2-terminal microsequencing analysis of high performance liquid chromatography-separated products as ?-melanocyte-stimulating hormone, corticotropin-like intermediate, ?-lipotropin, ?-endorphin-(1–31), 18-kDa NH2-terminal fragment and, to a lesser extent, adrenocorticotrophin and ?-lipotropin. This processing had an acidic pH optimum (pH 5–5.5) and was Ca2+-dependent (K0.5 activation = 5–80 µm). With increasing Ca2+ concentrations there was an increase in the extent to which mPOMC was processed. The in vitro processing of mPOMC by the insulin secretory granule endopeptidase activity reported here is in excellent agreement with the in vivo processing of this prohormone by a combination of PC2 and PC3, candidates of prohormone endpeptidase, in gene transfer studies with cells that express the regulated secretory pathway PMID:8382698

Rhodes, Christopher J.; Thorne, Barbara A.; Lincoln, Beth; Nielsen, Egon; Hutton, John C.; Thomas, Gary

2015-01-01

22

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

PubMed Central

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

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

1999-01-01

23

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

PubMed Central

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

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

2011-01-01

24

Separation of rat pituitary secretory granules by continuous flow electrophoresis  

NASA Technical Reports Server (NTRS)

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

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

1990-01-01

25

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. PMID:10777760

Rohrbach, A

2000-01-01

26

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

PubMed

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 Hg²?, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²? (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 Hg²?, concentrations that activate AQP6. The Hg lysis was completely blocked by ?-mercaptoethanol which disrupts Hg²?-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 Hg²?-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

2013-03-01

27

A biosynthetic regulated secretory pathway in constitutive secretory cells  

PubMed Central

It has frequently been proposed that while the constitutive secretory pathway is present in all cells, the regulated secretory pathway is found only in specialized cells such as neuronal, endocrine, or exocrine types. In this study we provide evidence that suggests that this distinction is not as restrictive as proposed. We have identified a population of post-Golgi storage vesicles in several constitutive secretory cells using [35S]SO4-labeled glycosaminoglycan (GAG) chains as a marker. A fraction of this pool of vesicles can undergo exocytosis in response to stimuli such as cytoplasmic Ca2+ and phorbol esters. The effect of Ca2+ was demonstrated both in intact cells in the presence of the ionophore A23187 and in streptolysin-O-permeabilized semi-intact cells. N-ethylmaleiimide, under conditions known to block regulated and constitutive secretion, inhibited the stimulated secretion from these cells, suggesting that the observed release of labeled GAG chains was not due to a leakage artefact. Subcellular fractionation revealed that the stored GAG chains were in low-density membrane granules (d approximately 1.12 g/ml), whose size was greater than that of synaptic- like vesicles found in PC12 cells. In addition, in CHO cells that express epitope-tagged rab 3D, the labeled GAG chains were found to cofractionate with the exogenous rab protein. When expressed in the regulated cell line AtT-20, this tagged rab protein was found to colocalize with ACTH-containing dense-core granules by indirect immunofluorescence. Taken together, these results provide evidence for the presence of a cryptic regulated secretory pathway in "constitutive" cells and suggest that the regulated secretory pathway is more widespread amongst different cell types than previously believed. PMID:8682857

1996-01-01

28

Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland  

SciTech Connect

An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with ({sup 32}P)orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.

Marino, C.R.; Castle, J.D.; Gorelick, F.S. (Yale Univ. School of Medicine, New Haven, CT (USA))

1990-07-01

29

Redundant and Segregated Functions of Granule-Associated Heparin-Binding Group II Subfamily of Secretory  

E-print Network

Redundant and Segregated Functions of Granule-Associated Heparin-Binding Group II Subfamily of secretory phospholipase A2 (sPLA2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA2-IIA, -V, and -IID

Gelb, Michael

30

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

SciTech Connect

Purified human C3a was iodinated (/sup 125/I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of /sup 125/I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of /sup 125/I-C3a and the rate of dissociation from the cell were temperature dependent. At 0/sup 0/C, the binding of /sup 125/I-C3a was increased and the rate of dissociation reduced, as compared to 37/sup 0/C. Once /sup 125/I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of /sup 125/I precipitable by TCA and the appearance of /sup 125/I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of /sup 125/I-C3a. Treatment of RMC bearing /sup 125/I-C3a with Bis (sulfosuccinimidyl) suberate (BS/sup 3/) covalently crosslinked the /sup 125/I-Ca to chymase, the predominant enzyme found in the secretory granules. Indirect immunofluorescence of RMC using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. The results indicate that /sup 125/I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan. In addition, this proteolysis of /sup 125/I-C3a by chymase must be blocked in order to detect plasma membrane C3a binding components on RMC.

Gervasoni, J.E. Jr.

1986-01-01

31

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.

1985-06-01

32

Role of Adaptor Proteins in Secretory Granule Biogenesis and Maturation  

PubMed Central

In the regulated secretory pathway, secretory granules (SGs) store peptide hormones that are released on demand. SGs are formed at the trans-Golgi network and must undergo a maturation process to become responsive to secretagogues. The production of mature SGs requires concentrating newly synthesized soluble content proteins in granules whose membranes contain the appropriate integral membrane proteins. The mechanisms underlying the sorting of soluble and integral membrane proteins destined for SGs from other proteins are not yet well understood. For soluble proteins, luminal pH and divalent metals can affect aggregation and interaction with surrounding membranes. The trafficking of granule membrane proteins can be controlled by both luminal and cytosolic factors. Cytosolic adaptor proteins (APs), which recognize the cytosolic domains of proteins that span the SG membrane, have been shown to play essential roles in the assembly of functional SGs. Adaptor protein 1A (AP-1A) is known to interact with specific motifs in its cargo proteins and with the clathrin heavy chain, contributing to the formation of a clathrin coat. AP-1A is present in patches on immature SG membranes, where it removes cargo and facilitates SG maturation. AP-1A recruitment to membranes can be modulated by Phosphofurin Acidic Cluster Sorting protein 1 (PACS-1), a cytosolic protein which interacts with both AP-1A and cargo that has been phosphorylated by casein kinase II. A cargo/PACS-1/AP-1A complex is necessary to drive the appropriate transport of several cargo proteins within the regulated secretory pathway. The Golgi-localized, ?-ear containing, ADP-ribosylation factor binding (GGA) family of APs serve a similar role. We review the functions of AP-1A, PACS-1, and GGAs in facilitating the retrieval of proteins from immature SGs and review examples of cargo proteins whose trafficking within the regulated secretory pathway is governed by APs. PMID:23966980

Bonnemaison, Mathilde L.; Eipper, Betty A.; Mains, Richard E.

2013-01-01

33

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

PubMed Central

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

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

2002-01-01

34

Differential trafficking of soluble and integral membrane secretory granule-associated proteins  

Microsoft Academic Search

The posttranslational processing enzyme peptidylglycine a-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have com- pared the maturation, processing, secretion, and stor- age of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the

Sharon L. Milgram; Betty A. Eipper; Richard E. Mains

1994-01-01

35

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

1989-01-01

36

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

1977-01-01

37

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. PMID:9620860

Arvan, P; Castle, D

1998-01-01

38

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. PMID:11779800

Bowman, G R; Turkewitz, A P

2001-01-01

39

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

E-print Network

5 FRAP Analysis of Secretory Granule Lipids and Proteins in the Sea Urchin Egg Julian L. Wong and Gary M. Wessel Summary Cortical granules of the sea urchin are secreted at fertilization in response the egg cortex (reviewed in Refs. 1 and 2). In animals such as sea urchins, this transformation may take

Wessel, Gary M.

40

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

PubMed Central

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

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

2014-01-01

41

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

PubMed

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

Tsutusumi, T; Kurabuchi, S; Aiyama, S

1989-06-01

42

Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena  

PubMed Central

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

Briguglio, Joseph S.; Kumar, Santosh

2013-01-01

43

Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules.  

PubMed Central

We have investigated the relationship of the so-called small dense core vesicle (SDCV), the major catecholamine-containing neurosecretory vesicle of sympathetic neurons, to synaptic vesicles containing classic neurotransmitters and secretory granules containing neuropeptides. SDCVs contain membrane proteins characteristic of synaptic vesicles such as synaptophysin and synaptoporin. However, SDCVs also contain membrane proteins characteristic of certain secretory granules like the vesicular monoamine transporter and the membrane-bound form of dopamine beta-hydroxylase. In neurites of sympathetic neurons, synaptophysin and dopamine beta-hydroxylase are found in distinct vesicles, consistent with their transport from the trans-Golgi network to the site of SDCV formation in constitutive secretory vesicles and secretory granules, respectively. Hence, SDCVs constitute a distinct type of neurosecretory vesicle that is a hybrid of the synaptic vesicle and the secretory granule membranes and that originates from the contribution of both the constitutive and the regulated pathway of protein secretion. Images Fig. 1 Fig. 3 Fig. 4 PMID:7638193

Bauerfeind, R; Jelinek, R; Hellwig, A; Huttner, W B

1995-01-01

44

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. PMID:23382463

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

2013-01-01

45

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

PubMed

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 Ca(2+)-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. PMID:23382463

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

2013-02-01

46

A cement protein of the tick Rhipicephalus appendiculatus, located in the secretory e cell granules of the type III salivary gland acini, induces strong antibody responses in cattle.  

PubMed

Protein components of the cement cone of ixodid ticks are candidates for inclusion in vaccines against tick infestation, since they are essential for tick attachment and feeding. We describe here the cloning of a cDNA encoding a 36 kDa protein, designated Rhipicephalus Immuno-dominant Molecule 36 (RIM36), present in salivary glands and the cement cone material secreted by Rhipicephalus appendiculatus. The 334-amino-acid sequence of RIM36 has a high content of glycine, serine and proline. The protein contains a predicted N-terminal signal peptide and two classes of glycine-rich amino acid repeats, a GL[G/Y/S/F/L] tripeptide and a GSPLSGF septapeptide. Comparison of genomic and cDNA sequences reveals a 597 bp intron within the 3' end of the RIM36 gene. Immuno-electron microscopy demonstrates that RIM36 is predominantly located in the e cell granules of the type III salivary gland acini. An Escherichia coli recombinant form of the proline-rich C-terminal domain of RIM36 reacts with antisera from Bos indicus cattle, either experimentally infested with R. appendiculatus, or exposed to ticks in the field. The 36 kDa protein is strongly recognised on Western blots of salivary gland lysates and soluble extracts of purified R. appendiculatus cement cones by polyclonal antibodies generated against recombinant RIM36, and by antisera from cattle experimentally infested with ticks. The data indicate that this tick cement component is a target of strong antibody responses in cattle exposed to feeding ticks. PMID:12062554

Bishop, Richard; Lambson, Bronwen; Wells, Clive; Pandit, Pratibala; Osaso, Julius; Nkonge, Catherine; Morzaria, Subhash; Musoke, Antony; Nene, Vishvanath

2002-06-15

47

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

K. A. SUPRENANT; W. L. DENTLER

48

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

NASA Astrophysics Data System (ADS)

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

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

2004-01-01

49

Secretory Granule Proteases in Rat Mast Cells. Cloning of 10 Different Serine Proteases and a Carboxypeptidase A from Various Rat Mast Cell Populations  

PubMed Central

Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the rat mucosal MC line RBL-1. cDNA clones for 10 different serine proteases (RMCP-1-10), and the MC carboxypeptidase A were isolated and characterized. Six of these proteases have not been isolated previously. Based on their protease content, three separate subpopulations of MCs were identified. Connective tissue MCs (CTMCs) from the ear and peritoneum express the chymases RMCP-1 and -5, the tryptases RMCP-6, and -7 and the carboxypeptidase A. However, based on a large difference in the level of expression of RMCP-7, CTMCs of these two organs may be regarded as two separate subpopulations. RMCP-2 and the three closely related proteases of the RMCP-8 subfamily were identified as the major mucosal MC proteases in rat. In contrast to what has been reported for human MCs, no expression of cathepsin G or cathepsin G–like proteases was detected in any of the rat MC populations. To determine mRNA frequencies for the various proteases expressed by normal tissue MCs, an unamplified peritoneal MC cDNA library was screened with a panel of monospecific cDNA probes. These results showed that peritoneal MCs are highly specialized effector cells with mRNA frequencies for the major proteases in the range of several percent of the total mRNA pool. PMID:8996238

Lützelschwab, Claudia; Pejler, Gunnar; Aveskogh, Maria; Hellman, Lars

1997-01-01

50

The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation  

PubMed Central

After cell fate specification, differentiating cells must amplify the specific subcellular features required for their specialized function. How cells regulate such subcellular scaling is a fundamental unanswered question. Here, we show that the E3 ubiquitin ligase Mindbomb 1 (MIB1) is required for the apical secretory apparatus established by gastric zymogenic cells as they differentiate from their progenitors. When Mib1 was deleted, death-associated protein kinase–1 (DAPK1) was rerouted to the cell base, microtubule-associated protein 1B (MAP1B) was dephosphorylated, and the apical vesicles that normally support mature secretory granules were dispersed. Consequently, secretory granules did not mature. The transcription factor MIST1 bound the first intron of Mib1 and regulated its expression. We further showed that loss of MIB1 and dismantling of the apical secretory apparatus was the earliest quantifiable aberration in zymogenic cells undergoing transition to a precancerous metaplastic state in mouse and human stomach. Our results reveal a mechanistic pathway by which cells can scale up a specific, specialized subcellular compartment to alter function during differentiation and scale it down during disease. PMID:23478405

Capoccia, Benjamin J.; Jin, Ramon U.; Kong, Young-Yun; Peek, Richard M.; Fassan, Matteo; Rugge, Massimo; Mills, Jason C.

2013-01-01

51

The role of secretory granules in radiation-induced dysfunction of rat salivary glands  

SciTech Connect

To investigate the possible role of secretory granules in radiation-induced salivary gland dysfunction, rats were pretreated with isoproterenol (5 mg/kg intraperitoneally) to degranulate salivary gland acini. At maximal depletion, salivary glands were locally irradiated with a single dose of 15 Gy of X rays. Parotid and submandibular/sublingual saliva samples were collected before and 1-10 days after irradiation. The lag phase, flow rate, concentrations of potassium and sodium, and amylase secretion were determined. Sham-treated, isoproterenol-treated and irradiated animals provided reference data. In the parotid gland, but not in the submandibular gland, protection against radiation-induced changes in flow rate and composition of saliva occurred after pretreatment with isoproterenol. Combining morphological data from a previous study with data from the current study, it is suggested that improvement of parotid gland function is attributed predominantly to a proliferative stimulus on acinar cells by isoproterenol and not to its degranulation effect. After pretreatment with isoproterenol, an earlier expression of radiation-induced acinar cell damage leading to death was observed, followed by a faster tissue recovery. Thus the proliferative stimulus on acinar cells may accelerate the unmasking of latent lethal damage, resulting in the earlier replacement of dead cells by new, functionally intact cells. 33 refs., 2 figs.

Peter, B.; Van Waarde, M.A.W.H.; Konings, A.W.T. [Univ. of Groningen (Netherlands); Vissink, A. [Univ. of Groningen (Netherlands)]|[Univ. Hospital, Groningen (Netherlands); `s-Gravenmade, E.J. [Univ. Hospital, Groningen (Netherlands)

1995-02-01

52

Molecular Interpretation of ACTH-?-Endorphin Coaggregation: Relevance to Secretory Granule Biogenesis  

PubMed Central

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

Singh, Uday; Singru, Praful S.; Padinhateeri, Ranjith; Maji, Samir K.

2012-01-01

53

Heterogeneity in composition of mouse uterine natural killer cell granules.  

PubMed

uNK cells differ from cNK cells, as they produce angiogenic molecules critical for normal implantation site development. We evaluated heterogeneity among DBA(+)uNK cells for Prf, Gzma, and Vegfa. Ctsd and Srgn expression was used to assign intracellular sorting of these molecules on gd7, -9, and -14. Vegfa was present in small, granule-free DBA(+)uNK cells at gd7 and in large, granule-rich DBA(+)uNK cells at gd9 and -14. Prf and Gzma were only found in granulated DBA(+)uNK cells (gd9 and -14). All granule-rich Prf(+)DBA(+)uNK cells appeared to coexpress Vegfa. Thus, all DBA(+)uNK cells were Vegfa-producing cells. PC analysis and immunogold ultrastructure confirmed colocalization of Prf/Ctsd in secretory-lysosome granules (PC>0.5). Surprisingly, Gzma and Prf(+)Ctsd(+) were not colocalized (PC<0.5). Rather, Gzma colocalized with Srgn (PC>0.5) in small granules in cells with Vegfa expression (PC<0.5). NK1.1(+)sNK cells and DBA(+)uNK cells expressed genes regulating vesicular traffic (rab11, rab27a, snap23, vamp7), but uNK cells also expressed rab34 and vamp8, molecules associated with constitutive secretion. SEE activated the regulated secretory pathway of DBA(+)uNK cells in vivo, mobilizing Prf and Gzma but not Vegfa. Thus, DBA(+)uNK cells display constitutive and regulated secretion. Further, these results demonstrate that granule-free DBA(+)uNK cells are not quiescent immature cells, but they are cells with potentially significant angiogenic roles before and in addition to their initiation of spiral arterial remodeling. PMID:22566570

Lima, Patrícia D A; Croy, Barbara A; Degaki, Karina Y; Tayade, Chandrakant; Yamada, Aureo T

2012-07-01

54

A Dynamic Analysis of Secretory Granules Containing Proteins Involved In Learning  

NASA Astrophysics Data System (ADS)

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

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

2010-10-01

55

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. 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.

2012-01-01

56

Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells  

PubMed Central

Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7?Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. PMID:23484122

González, Claudia; Espinosa, Marisol; Sánchez, María Trinidad; Droguett, Karla; Ríos, Mariana; Fonseca, Ximena; Villalón, Manuel

2013-01-01

57

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. PMID:20127366

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

2013-01-01

58

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. PMID:1639842

1992-01-01

59

Proinsulin intermolecular interactions during secretory trafficking in pancreatic ? cells.  

PubMed

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

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

2013-01-18

60

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

SciTech Connect

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

Lorenson, M.Y.

1985-04-01

61

Biogenesis of secretory organelles during B cell differentiation  

Microsoft Academic Search

The differentiation of B cells into Ig-secreting plasma cells requires the expansion of secretory organelles to cope with the increased cargo load. To evaluate the timeline of this process, we have quantitated the kinet- ics of secretory organelle expansion relative to Ig se- cretion and examined regulatory components of secre- tory transport following in vitro activation of human B lymphocytes.

Semra J. Kirk; Jacqueline M. Cliff; J. Alero Thomas; Theresa H. Ward

2009-01-01

62

Granulated round cell tumor of cats.  

PubMed

Morphologic and biologic features of five feline granulated round cell tumours were compared with those previously reported to be of globule leukocyte and large granular lymphocyte origin. The five cats ranged from 6 to 9 years of age and presented with nonspecific gastrointestinal signs. Four of the five cats were tested for feline leukemia virus and were negative by enzyme-linked immunosorbent assay. The neoplastic process involved the abdominal cavity in all cases, with a predilection for the distal small intestine and mesentery. The liver and peripheral and thoracic lymphoid tissues were also sporadically affected. Neoplastic round cells contained 0.5-1.5-microns eosinophilic cytoplasmic granules that were difficult to discern on causal observation with hematoxylin and eosin stain but were deep blue and easily visualized when stained with phosphotungstic acid-hematoxylin. In two cases, epithelium in the affected ileum and liver contained unusually large numbers of apparently normal globule leukocytes. Ultrastructurally, the tumor granules tended to cluster at one nuclear pole and were spindle to round in shape with variably dense contents. Some granules contained a dense "cap" at one end or internal crystalloid bars that distorted the granule membrane. The tumors reported herein are similar to all three of the previously reported feline granulated round cell tumors and probably have a common cellular origin. PMID:8470340

McEntee, M F; Horton, S; Blue, J; Meuten, D J

1993-03-01

63

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

E-print Network

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 ...

Nakashiba, Toshiaki

64

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

PubMed

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

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

1988-09-01

65

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

PubMed

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

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

2015-03-15

66

Two modes of lytic granule fusion during degranulation by natural killer cells  

PubMed Central

Lytic granules in cytotoxic lymphocytes, which include T cells and natural killer (NK) cells, are secretory lysosomes that release their content upon fusion with the plasma membrane (PM), a process known as degranulation. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Here, we used total internal reflection fluorescence microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand (FasL) in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptor–ligand interactions. Two fusion modes were observed: complete fusion, characterized by loss of granule content and rapid diffusion of FasL at the PM; and incomplete fusion, characterized by transient fusion pore opening and retention of FasL at the fusion site. The pH-sensitive green fluorescence protein (pHluorin) fused to the lumenal domain of FasL was used to visualize fusion pore opening with a time resolution of 30?ms. Upon incomplete fusion, pHluorin emission lasted several seconds in the absence of noticeable diffusion. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the PM, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules. PMID:21483445

Liu, Dongfang; Martina, Jose A; Wu, Xufeng S; Hammer III, John A; Long, Eric O

2011-01-01

67

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

PubMed Central

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

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

2014-01-01

68

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.

1984-07-01

69

Cytokine secretion is distinct from secretion of cytotoxic granules in NK cells.  

PubMed

NK cells are renowned for their ability to kill virally infected or transformed host cells by release of cytotoxic granules containing granzymes and perforin. NK cells also have important regulatory capabilities chiefly mediated by secretion of cytokines, such as IFN-gamma and TNF. The secretory pathway for the release of cytokines in NK cells is unknown. In this study, we show localization and trafficking of IFN-gamma and TNF in human NK cells in compartments and vesicles that do not overlap with perforin or other late endosome granule markers. Cytokines in post-Golgi compartments colocalized with markers of the recycling endosome (RE). REs are functionally required for cytokine release because inactivation of REs or mutation of RE-associated proteins Rab11 and vesicle-associated membrane protein-3 blocked cytokine surface delivery and release. In contrast, REs are not needed for release of perforin from preformed granules but may be involved at earlier stages of granule maturation. These findings suggest a new role for REs in orchestrating secretion in NK cells. We show that the cytokines IFN-gamma and TNF are trafficked and secreted via a different pathway than perforin. Although perforin granules are released in a polarized fashion at lytic synapses, distinct carriers transport both IFN-gamma and TNF to points all over the cell surface, including within the synapse, for nonpolarized release. PMID:20368273

Reefman, Esther; Kay, Jason G; Wood, Stephanie M; Offenhäuser, Carolin; Brown, Darren L; Roy, Sandrine; Stanley, Amanda C; Low, Pei Ching; Manderson, Anthony P; Stow, Jennifer L

2010-05-01

70

Carboxypeptidase E Is a Regulated Secretory Pathway Sorting Receptor: Genetic Obliteration Leads to Endocrine Disorders in Cpe fat Mice  

Microsoft Academic Search

A proposed mechanism for sorting secretory proteins into granules for release via the regulated secretory pathway in endocrine-neuroendocrine cells involves binding the proteins to a sorting receptor at the trans-Golgi network, followed by budding and granule formation. We have identified such a sorting receptor as membrane-associated carboxypeptidase E (CPE) in pituitary Golgi-enriched and secretory granule membranes. CPE specifically bound regulated

David R Cool; Emmanuel Normant; Fu-sheng Shen; Hao-Chia Chen; Lewis Pannell; Ying Zhang; Y. Peng Loh

1997-01-01

71

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

PubMed Central

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

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

2013-01-01

72

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

KATHLEEN BUCKLEY; REGIS B. KELLY

1985-01-01

73

LAMP1/CD107a is required for efficient perforin delivery to lytic granules and NK-cell cytotoxicity  

PubMed Central

Secretory lysosomes of natural killer (NK) cells, containing perforin and granzymes, are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell apoptosis. Lysosome-associated membrane protein (LAMP) 1/CD107a is used as a marker for NK-cell degranulation, but its role in NK-cell biology is unknown. We show that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNA interference (RNAi) cells fail to deliver granzyme B to target cells. Reduction of LAMP1 expression affects the movement of lytic granules and results in decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi network–derived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity. PMID:23632890

Gil-Krzewska, Aleksandra; Nguyen, Victoria; Peruzzi, Giovanna

2013-01-01

74

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

PubMed

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

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

2004-12-01

75

Cerebellar granule cells elaborate neurites before mitosis.  

PubMed

Neuronal birth and neurite outgrowth have been regarded as discrete, sequential stages of development. However, we recently found that sympathetic neuroblasts often elaborate axons before mitosis, in culture [E. Wolf, I.B. Black, E. DiCicco-Bloom, Mitotic neuroblasts determine neuritic patterning of progeny, J. Comp. Neurol. 367 (1996) 623-635] and in vivo [E. Wolf, I.B. Black, E. DiCicco-Bloom, Central and peripheral neuroblasts elaborate neurites prior to division in vivo and in vitro, Soc. Neurosci. Abstr. 21 (1995) 785; E. Wolf, I.B. Black, E. DiCicco-Bloom, Mitotic neuroblasts engage in axonal outgrowth and pathfinding in vivo, Soc. Neurosci. Abstr. 22 (1996) 525]. Here, we report that cerebellar granule cells often divide with heritable neurites in vitro. Therefore, mitotic CNS precursors, in addition to peripheral neuroblasts, simultaneously undergo proliferation and process formation. Potentially, neurites on dividing precursors may allow target fields to influence directly the course of neurogenesis. PMID:9352115

Wolf, E; Wagner, J P; Black, I B; DiCicco-Bloom, E

1997-09-20

76

Is cerebellar granule cell migration regulated by an internal clock?  

PubMed

We have studied the time course of migratory behavior of cerebellar granule cells in the microwell tissue culture system. [3H]Thymidine served as a marker for particular granule cell generations. When cultured 4 hr after [3H]thymidine injection for 6 days in microwell cultures, labeled granule cells were seen to migrate along fiber bundles expanding between reaggregates called "cables" for 3 to 4 days. After 5 and 6 days in vitro the percentage of labeled non-migrating cells found in clusters in reaggregates and on cables increased considerably. Whereas unlabeled cells continued to migrate. Comparable results were obtained when granule cells developed in vivo for various times after label and their developmental state was determined in vitro. Cells from cerebellar populations labeled 1 to 4 days before culture maintained their ability to migrate in vitro, even after granule cells had entered the internal granule cell layer. In contrast, the percentage of migrating cells labeled 5 and 6 days before culture was reduced significantly. The results suggest that the time span of granule cell migration is predetermined intrinsically rather than by external signals. PMID:6502207

Trenkner, E; Smith, D; Segil, N

1984-11-01

77

Capturing protein interactions in the secretory pathway of living cells  

PubMed Central

The secretory pathway is composed of membrane compartments specialized in protein folding, modification, transport, and sorting. Numerous transient protein-protein interactions guide the transport-competent proteins through the secretory pathway. Here we have adapted the yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) to detect protein-protein interactions in the secretory pathway of living cells. Fragments of YFP were fused to the homooligomeric cargo-receptor lectin endoplasmic reticulum Golgi intermediate compartment (ERGIC)-53, to the ERGIC-53-interacting multicoagulation factor deficiency protein MCFD2, and to ERGIC-53's cargo glycoprotein cathepsin Z. YFP PCA analysis revealed the oligomerization of ERGIC-53 and its interaction with MCFD2, as well as its lectin-mediated interaction with cathepsin Z. Mutation of the lectin domain of ERGIC-53 selectively decreased YFP complementation with cathepsin Z. Using YFP PCA, we discovered a carbohydrate-mediated interaction between ERGIC-53 and cathepsin C. We conclude that YFP PCA can detect weak and transient protein interactions in the secretory pathway and hence is a powerful approach to study luminal processes involved in protein secretion. The study extends the application of PCA to carbohydrate-mediated protein-protein interactions of low affinity. PMID:15849265

Nyfeler, Beat; Michnick, Stephen W.; Hauri, Hans-Peter

2005-01-01

78

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

PubMed Central

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

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

2013-01-01

79

Bicarbonate transport in sheep parotid secretory cells.  

PubMed Central

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

1996-01-01

80

How helminths use excretory secretory fractions to modulate dendritic cells  

PubMed Central

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

White, Rhiannon R.; Artavanis-Tsakonas, Katerina

2012-01-01

81

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

EPA Science Inventory

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

82

UVC-Induced Stress Granules in Mammalian Cells  

PubMed Central

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

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

2014-01-01

83

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

PubMed

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

2012-03-30

84

Assessing the secretory capacity of pancreatic acinar cells.  

PubMed

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

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

2014-01-01

85

Functional channel formation associated with cytotoxic T-cell granules.  

PubMed Central

Lymphocyte granules from cytotoxic T-lymphocyte lines A2, A11, and R8 were enriched by subcellular fractionation using a Percoll gradient. Granule-enriched fractions showed potent hemolytic activity in the presence of Ca2+. Isolated granules induced rapid Ca2+-dependent membrane depolarization of J774 macrophage-like cells. When tested in planar bilayers, granules induced the formation of Ca2+-dependent functional ion channels of large conductance steps of 1-6 nS in 0.1 M NaCl. Granule-induced channels were resistant to closing by an increase in transmembrane potential, with few channels shifting to the closed state only at voltages of greater than 70 mV, following a Poisson process. These channels showed poor ion selectivity and were permeable to all monovalent and divalent ions (K+, Na+, Li+, Cl-, Ca2+, Mg2+, Zn2+, Ba2+). Ultrastructural examination of soluble granule proteins incubated for 48 hr at 37 degrees C in the presence of Ca2+ revealed ring-like structures of 150-200 A. Structural and functional channel formation may be involved in cytolysis induced by cytotoxic T lymphocytes. Images PMID:2417234

Young, J D; Nathan, C F; Podack, E R; Palladino, M A; Cohn, Z A

1986-01-01

86

Antibodies against mesangial cells and their secretory products in chronic renal allograft rejection in the rat.  

PubMed Central

Antibodies or cell-mediated immunity can cause chronic rejection of vascularized organ grafts, but the nature and specificity of the antigen(s) involved has remained elusive. We have previously demonstrated the presence of antibodies against cryptic glomerular basement membrane antigens and undefined antigens in the mesangial area in rats with chronic renal allograft rejection. Current experiments were designed to study the post-transplant antibody response against cultured mesangial and endothelial cells in rats with chronic rejection using flow cytometry, indirect immunofluorescent staining, immunoelectron microscopy, confocal microscopy, and Western blots. The results were compared with those obtained with alloantisera raised by immunization with cultured mesangial cells. Post-transplant and post-immunization sera contained IgG antibodies against trypsinized mesangial cells detected by flow cytometry. Indirect immunofluorescent studies using mesangial cells grown on coverslips showed autoantibody binding to cytoplasmic granules in cultures early after plating whereas staining of later cultures showed antibody binding in an interrupted, web-like pattern on the outside of the cells. Immunoelectron microscopy showed autoantibody binding to intracellular secretory granules and to cell surface focal adhesion plaques. The latter finding was confirmed in double-labeling experiments with an antiserum against vinculin. Western blots with mesangial cell culture supernatants demonstrated autoantibody reactivity with antigens in the 40-kd and 60- to 70-kd range, and immunoprecipitation identified these molecules as biglycan and decorin. Absorption of the sera with mesangial cell culture supernatant removed most of the antibodies except those that gave a punctate staining with the mesangial cell surface. However, not all immunostaining of mesangial cells could be explained by antibodies against biglycan and decorin. Post-transplant sera, furthermore, contained low-titered antibodies against endothelial cells. We conclude that rats with chronic renal transplant rejection produce a strong autoantibody response against mesangial cell focal adhesion plaques and proteins secreted by these cells in culture. Such antibodies may cause local damage and interfere in the tissue repair process after injury. Images Figure 2 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 12 Figure 13 Figure 14 PMID:9588890

Paul, L. C.; Muralidharan, J.; Muzaffar, S. A.; Manting, E. H.; Valentin, J. F.; de Heer, E.; Kashgarian, M.

1998-01-01

87

Sparse incomplete representations: a potential role of olfactory granule cells  

PubMed Central

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

Koulakov, Alexei A.; Rinberg, Dmitry

2011-01-01

88

Most Non-Parotid “Acinic Cell Carcinomas” Represent Mammary Analogue Secretory Carcinomas  

PubMed Central

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 non-parotid salivary glands where where serous acini are less abundant. Given the recent discovery of mammary analogue secretory carcinoma (MASC) – a salivary malignancy that histologically mimics ACC – a retrospective re-evaluation of non-parotid 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; immunohistochemistry (mammaglobin, S100 protein) was performed; and confirmatory ETV6 break-apart FISH assay was completed. Demographic and clinical data was obtained from the medical records. Fourteen extra-parotid 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 20–86 years (mean, 56), 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; but 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 one locally recurred, and none metastasized. Most alleged ACCs of non-parotid 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.

2013-01-01

89

Granule cells and cerebellar boundaries: analysis of Unc5h3 mutant chimeras  

Microsoft Academic Search

Mutations in the Unc5h3 gene, a receptor for the netrin 1 ligand, result in abnormal migrations of both Purkinje and granule cells to regions outside the cerebellum and of granule cells to regions within the cerebellum. Because both Purkinje and granule cells express this molecule we sought to determine whether one or both of these cell types are the primary

Dan Goldowitz; Kristin M. Hamre; Stefan A. Przyborski; Susan L. Ackerman

2000-01-01

90

The unfolded protein response in secretory cell function.  

PubMed

The endoplasmic reticulum (ER) controls many important aspects of cellular function, including processing of secreted and membrane proteins, synthesis of membranes, and calcium storage. Maintenance of ER function is controlled through a network of signaling pathways collectively known as the unfolded protein response (UPR). The UPR balances the load of incoming proteins with the folding capacity of the ER and allows cells to adapt to situations that disrupt this balance. This disruption is referred to as ER stress. Although ER stress often arises in pathological situations, the UPR plays a central role in the normal development and function of cells specializing in secretion. Many aspects of this response are conserved broadly across eukaryotes; most organisms use some subset of a group of ER transmembrane proteins to signal to the nucleus and induce a broad transcriptional upregulation of genes involved in ER function. However, new developments in metazoans, plants, and fungi illustrate interesting variations on this theme. Here, we summarize mechanisms for detecting and counteracting ER stress, the role of the UPR in normal secretory cell function, and how these pathways vary across organisms and among different tissues and cell types. PMID:22934644

Moore, Kristin A; Hollien, Julie

2012-01-01

91

Studies on ectopic granule cells in the cerebellar cortex  

Microsoft Academic Search

In order to strengthen a hypothesis concerning the occurrence of ectopic granule cells, one of the assumptions made was tested systematically. The reaction of the EGL to partial destruction by various single doses of hydroxyurea at various ages was followed. Under all conditions examined, re-population of the EGL takes place—rapidly after lower doses, slowly after high doses of HU. The

Ebel J. Ebels; Ieteke Peters; Aaltje Thijs

1975-01-01

92

Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells  

Microsoft Academic Search

Neuroligins (NLGs) are postsynaptic cell adhesion molecules that are thought to function in synaptogenesis. To investigate the role of NLGs on synaptic transmission once the synapse is formed, we transfected neuroligin-2 (NLG-2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABAA (?-aminobutyric acid) receptor mediated miniature postsynaptic currents (mIPSCs). NLG-2 transfected cells had mIPSCs with faster decay than matching

Zhanyan Fu; Stefano Vicini

2009-01-01

93

Exocyst complexes multiple functions in plant cells secretory pathways.  

PubMed

The exocyst is a complex of proteins mediating first contact (tethering) between secretory vesicles and the target membrane. Discovered in yeast as an effector of RAB and RHO small GTPases, it was also found to function in land plants. Plant cells and tissues rely on targeted exocytosis and this implies that the exocyst is involved in regulation of cell polarity and morphogenesis, including cytokinesis, plasma membrane protein recycling (including PINs, the auxin efflux carriers), cell wall biogenesis, fertilization, stress and biotic interactions including defence against pathogens. The dramatic expansion of the EXO70 subunit gene family, of which individual members are likely responsible for exocyst complex targeting, implies that there are specialized functions of different exocysts with different EXO70s. One of these functions comprises a role in autophagy-related Golgi independent membrane trafficking into the vacuole or apoplast. It is also possible, that some EXO70 paralogues have been recruited into exocyst independent functions. The exocyst has the potential to function as an important regulatory hub to coordinate endomembrane dynamics in plants. PMID:24246229

Zárský, Viktor; Kulich, Ivan; Fendrych, Matyáš; Pe?enková, Tamara

2013-12-01

94

[Primary culture of epithelial secretory cells from venom gland of the common adder Vipera berus].  

PubMed

A primary culture of epithelial secretory cells from the venom gland of Vipera berus was obtained. The cells adhered to collagen 1 and to a mixture of adhesion proteins (Matrigel), proliferated and retained the features of differentiation. Electron microscopy demonstrated the presence of all ultrastructures typical of these cells in vivo, a full complex of intercellular junctions, and cellular membrane polarity. The immunohistochemistry confirmed the capacity of secretory cells to synthesize venom in culture. We have studied the role of carbochole, an agonist of M-cholinoreceptor, in the initiation of the secretory cycle in cells in vitro. We propose that M-cholinoreceptors may play an important role in the initiation of the secretory cycle in vivo. PMID:11517661

Golubkov, V S; Lezhnev, E I; Bezgina, E N; Moshkov, D A

2001-01-01

95

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

E-print Network

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

van Oudenaarden, Alexander

96

Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell secretory lysosome.  

PubMed

Natural killer (NK) cells and cytotoxic T lymphocytes eliminate virally infected and transformed cells. Target cell killing is mediated by the regulated exocytosis of secretory lysosomes, which deliver perforin and proapoptotic granzymes to the infected or transformed cell. Yet despite the central role that secretory lysosome exocytosis plays in the immune response to viruses and tumors, little is known about the molecular machinery that regulates the docking and fusion of this organelle with the plasma membrane. To identify potential components of this exocytic machinery we used proteomics to define the protein composition of the NK cell secretory lysosome membrane. Secretory lysosomes were isolated from the NK cell line YTS by subcellular fractionation, integral membrane proteins and membrane-associated proteins were enriched using Triton X-114 and separated by SDS-PAGE, and tryptic peptides were identified by LC ESI-MS/MS. In total 221 proteins were identified unambiguously in the secretory lysosome membrane fraction of which 61% were predicted to be either integral membrane proteins or membrane-associated proteins. A significant proportion of the proteins identified play a role in vesicular trafficking, including members of both the Rab GTPase and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and protein families. These proteins include Rab27a and the SNARE vesicle-associated membrane protein-7, both of which were enriched in the secretory lysosome fraction and represent potential components of the machinery that regulates the exocytosis of this organelle in NK cells. PMID:17272266

Casey, Tammy M; Meade, Josephine L; Hewitt, Eric W

2007-05-01

97

FIB/SEM cell sectioning for intracellular metal granules characterization  

NASA Astrophysics Data System (ADS)

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

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

2009-05-01

98

Volume Density, Distribution, and Ultrastructure of Secretory and Basolateral Membranes and Mitochondria Predict Parietal Cell Secretory (Dys)function  

PubMed Central

Acid secretion in gastric parietal cells requires highly coordinated membrane transport and vesicle trafficking. Histologically, consensus defines acid secretion as the ratio of the volume density (Vd) of canalicular and apical membranes (CAMs) to tubulovesicular (TV) membranes, a value which varies widely under normal conditions. Examination of numerous achlorhydric mice made it clear that this paradigm is discrepant when used to assess most mice with genetic mutations affecting acid secretion. Vd of organelles in parietal cells of 6 genetically engineered mouse strains was obtained to identify a stable histological phenotype of acid secretion. We confirmed that CAM to TV ratio fairly represented secretory activity in untreated and secretion-inhibited wild-type (WT) mice and in NHE2?/? mice as well, though the response was significantly attenuated in the latter. However, high CAM to TV ratios wrongly posed as active acid secretion in AE2?/?, GHKA??/?, and NHE4?/? mice. Achlorhydric genotypes also had a significantly higher Vd of basolateral membrane than WT mice, and reduced Vd of mitochondria and canaliculi. The Vd of mitochondria, and ratio of the Vd of basolateral membranes/Vd of mitochondria were preferred predictors of the level of acid secretion. Alterations in acid secretion, then, cause significant changes not only in the Vd of secretory membranes but also in mitochondria and basolateral membranes. PMID:20339514

Miller, Marian L.; Andringa, Anastasia; Zavros, Yana; Bradford, Emily M.; Shull, Gary E.

2010-01-01

99

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland  

PubMed Central

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b?/? and Rab27ash/ash/Rab27b?/? mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release. PMID:21525430

Chiang, Lilian; Ngo, Julie; Schechter, Joel E.; Karvar, Serhan; Tolmachova, Tanya; Seabra, Miguel C.; Hume, Alistair N.

2011-01-01

100

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

PubMed Central

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

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

2010-01-01

101

Media composition modulates excitatory amino acid - induced death of rat cerebellar granule cells  

Microsoft Academic Search

This study examined the effects of maintaining cells in different media and the role of serum in glutamate and NMDA - induced neurotoxicity in rat cerebellar granule cells. Glutamate stimulated a concentration - dependent cell death with similar potency in cerebellar granule cells grown in BME and Neurobasal media without serum. However, the maximal cell death to glutamate and N-

Anita M Wood; Priyanka Tiwari; David R Bristow

1997-01-01

102

Response of eosinophilic granule cells of gilthead seabream (Sparus aurata, Teleostei) to bacteria and bacterial products.  

PubMed

Eosinophilic granule cells in the gills and peritoneal exudate of gilthead seabream (Sparus aurata L.) are characterized by the presence of prominent eosinophilic granules in their cytoplasm and are here described for the first time. The oval granules of these cells contain an electron-dense inclusion surrounded by a less dense filamentous matrix and are peroxidase- and acid phosphatase-negative. Unlike other granulocytes of gilthead seabream, eosinophilic granule cells do not ingest bacteria in vivo. The intraperitoneal injection of extracellular products of Pasteurella piscicida induces mobilization of eosinophilic granule cells to the blood and other tissues and causes changes in their structure. Shortly after injection, the granules of eosinophilic granule cells become swollen and some fuse with the cell membrane. From 7 h post-injection, many eosinophilic granule cells in the gills degenerate and are then phagocytosed by macrophages, which are especially abundant after 24 h. From 24 h to 72 h, eosinophilic granule cells from the gills contain abundant autolysosomes together with granules of a normal morphology. PMID:9011398

Noya, M; Lamas, J

1997-01-01

103

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

Microsoft Academic Search

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

Annie Meiniel

2007-01-01

104

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

PubMed Central

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 approximately 100,000) that is present in secretory vesicles in all neurons and endocrine cells studied, in species from elasmobranch fish to mammals, and in neural and endocrine cell lines. It was detected by cross-reactivity with monoclonal antibodies raised to highly purified cholinergic synaptic vesicles from the electric organ of fish. By immunoprecipitation of intact synaptic vesicles and electron microscopic immunoperoxidase labeling, we have shown that the antigenic determinant is on the cytoplasmic face of the synaptic vesicles. However, the electrophoretic mobility of the antigen synthesized in the presence of tunicamycin is reduced to Mr approximately 62,000, which suggests that the antigen is glycosylated and must therefore span the vesicle membrane. PMID:2579958

1985-01-01

105

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

Microsoft Academic Search

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

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

2009-01-01

106

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

PubMed

Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression. PMID:24373221

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

2014-03-01

107

Loss of Sonic Hedgehog Leads to Alterations in Intestinal Secretory Cell Maturation and Autophagy  

PubMed Central

Background Intestinal epithelial cells express the Sonic and Indian hedgehog ligands. Despite the strong interest in gut hedgehog signaling in GI diseases, no studies have specifically addressed the singular role of intestinal epithelial cell Sonic hedgehog signaling. The aim of this study was to investigate the specific role of Sonic hedgehog in adult ileal epithelial homeostasis. Methodology/Principal Findings A Sonic hedgehog intestinal epithelial conditional knockout mouse model was generated. Assessment of ileal histological abnormalities, crypt epithelial cell proliferation, epithelial cell fate, junctional proteins, signaling pathways, as well as ultrastructural analysis of intracellular organelles were performed in control and mutant mice. Mice lacking intestinal epithelial Sonic Hedgehog displayed decreased ileal crypt/villus length, decreased crypt proliferation as well as a decrease in the number of ileal mucin-secreting goblet cells and antimicrobial peptide-secreting Paneth cells during adult life. These secretory cells also exhibited disruption of their secretory products in mutant mice. Ultrastructural microscopy analysis revealed a dilated ER lumen in secretory cells. This phenotype was also associated with a decrease in autophagy. Conclusions/Significance Altogether, these findings indicate that the loss of Sonic hedgehog can lead to ileal secretory cell modifications indicative of endoplasmic reticulum stress, accompanied by a significant reduction in autophagy. PMID:24887421

Gagné-Sansfaçon, Jessica; Allaire, Joannie M.; Jones, Christine; Boudreau, François; Perreault, Nathalie

2014-01-01

108

Fine structure of the secretion granules in the mandibular gland of the echidna, Tachyglossus aculeatus (Monotremata).  

PubMed

The cells of the secretory tubules in the mandibular gland of the echidna are packed with fairly large birefringent granules, which show a lamellated structure consisting of alternating thin and thick layers or shells of protein. This presumably rigid substructure collapses during exocytosis and the shells unravel as sheets that form a tangled mass in the lumen of the secretory tubule. Relatively pure fractions were obtained of the relevant granules and protein sheets, which should allow a further study to be made on the secretory proteins in this gland. PMID:710670

van Lennep, E W; Kennerson, A R; Duck-Chong, C G; Pollak, J K

1978-10-01

109

Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells  

Microsoft Academic Search

Ca2+-triggered exocytosis was studied in single rat melanotrophs and bovine chromaffin cells by capacitance measurements. Sustained exocytosis required MgATP, but even in the absence of MgATP, Ca2+ could trigger exocytosis of 2700 granules in a typical melanotroph and of 840 granules in a chromaffin cell. Granules undergoing ATP-independent exocytosis were similar in number to those appearing docked to the plasmalemma

T. D. Parsons; J. R. Coorssen; H. Horstmann; W. Almers

1995-01-01

110

Loss-of-Function Mutations in ABCA1 and Enhanced ?-Cell Secretory Capacity in Young Adults.  

PubMed

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

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

2015-01-01

111

Granule cell survival is deficient in PAC1-/- mutant cerebellum.  

PubMed

PACAP exerts neuroprotective effects during development, especially in the cerebellum where PAC1 receptor and ligand are both expressed. However, while previous studies using PACAP injections in postnatal animals defined trophic effects of exogenous peptide, the role of endogenous PACAP remains unexplored. Here, we used PAC1(-/-) mice to investigate the role of PACAP receptor signaling in postnatal day 7 cerebellum. There was no difference in DNA synthesis in the cerebellar EGL of PAC1(-/-) compared to wild type animals, assessed using thymidine incorporation and BrdU immunohistochemistry. In contrast, we found that a significant proportion of newly generated neurons were eliminated before they successfully differentiated in the granule cell layer. In aggregate, these results suggest that endogenous PACAP plays an important role in cell survival during cerebellar development, through the activation of the PAC1 receptor. PMID:18409023

Falluel-Morel, Anthony; Tascau, Liana I; Sokolowski, Katie; Brabet, Philippe; DiCicco-Bloom, Emanuel

2008-11-01

112

Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells  

SciTech Connect

Incubation of (/sup 35/S)heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of /sup 35/S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of (/sup 35/S)heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular (/sup 35/S)heparin proteoglycan after 24 hours and the appearance of free (/sup 35/S)sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading (/sup 35/S)heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule.

Atkins, F.M.; Friedman, M.M.; Metcalfe, D.D.

1985-03-01

113

Clostridium difficile toxin B inhibits the secretory response of human mast cell line-1 (HMC-1) cells stimulated with high free-Ca(2+) and GTP?S.  

PubMed

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

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

2015-02-01

114

Glutamic acid: A strong candidate as the neurotransmitter of the cerebellar granule cell  

Microsoft Academic Search

Free amino acids and cholinergic enzymes were investigated in the cerebellum of reeler and weaver mice in an attempt to identify the neurotransmitter characteristic of the granule cell population and to clarify any neurotransmitter abnormalities of their pre- and postsynaptic neurons induced by their depletion. The data indicate that glutamic acid may be the neurotransmitter of the granule cells. Pre-

D. B. Hudson; T. Valcana; G. Bean; P. S. Timiras

1976-01-01

115

Delta1 Expression, Cell Cycle Exit, and Commitment to a Specific Secretory Fate Coincide within a Few Hours in the Mouse Intestinal Stem Cell System  

PubMed Central

The stem cells of the small intestine are multipotent: they give rise, via transit-amplifying cell divisions, to large numbers of columnar absorptive cells mixed with much smaller numbers of three different classes of secretory cells - mucus-secreting goblet cells, hormone-secreting enteroendocrine cells, and bactericide-secreting Paneth cells. Notch signaling is known to control commitment to a secretory fate, but why are the secretory cells such a small fraction of the population, and how does the diversity of secretory cell types arise? Using the mouse as our model organism, we find that secretory cells, and only secretory cells, pass through a phase of strong expression of the Notch ligand Delta1 (Dll1). Onset of this Dll1 expression coincides with a block to further cell division and is followed in much less than a cell cycle time by expression of Neurog3 – a marker of enteroendocrine fate – or Gfi1 – a marker of goblet or Paneth cell fate. By conditional knock-out of Dll1, we confirm that Delta-Notch signaling controls secretory commitment through lateral inhibition. We infer that cells stop dividing as they become committed to a secretory fate, while their neighbors continue dividing, explaining the final excess of absorptive over secretory cells. Our data rule out schemes in which cells first become committed to be secretory, and then diversify through subsequent cell divisions. A simple mathematical model shows how, instead, Notch signaling may simultaneously govern the commitment to be secretory and the choice between alternative modes of secretory differentiation. PMID:21915337

Hodgetts, Christine; Jeffery, Rosemary; Nye, Emma; Spencer-Dene, Bradley; Winton, Douglas J.; Lewis, Julian

2011-01-01

116

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

PubMed

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

2012-04-20

117

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

E-print Network

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

Terasaki, Mark

118

Multiple extra-synaptic spillover mechanisms regulate prolonged activity in cerebellar Golgi cell–granule cell loops  

PubMed Central

Abstract Despite a wealth of in vitro and modelling studies it remains unclear how neuronal populations in the cerebellum interact in vivo. We address the issue of how the cerebellar input layer processes sensory information, with particular focus on the granule cells (input relays) and their counterpart inhibitory interneurones, Golgi cells. Based on the textbook view, granule cells excite Golgi cells via glutamate forming a negative feedback loop. However, Golgi cells express inhibitory mGluR2 receptors suggesting an inhibitory role for glutamate. We set out to test this glutamatergic paradox in Golgi cells. Here we show that granule cells and Golgi cells interact through extra-synaptic signalling mechanisms during sensory information processing, as well as synaptic mechanisms. We demonstrate that such interactions depend on granule cell-derived glutamate acting via inhibitory mGluR2 receptors leading causally to the suppression of Golgi cell activity for several hundreds of milliseconds. We further show that granule cell-derived inhibition of Golgi cell activity is regulated by GABA-dependent extra-synaptic Golgi cell inhibition of granule cells, identifying a regulatory loop in which glutamate and GABA may be critical regulators of Golgi cell–granule cell functional activity. Thus, granule cells may promote their own prolonged activity via paradoxical feed-forward inhibition of Golgi cells, thereby enabling information processing over long timescales. PMID:21669981

Holtzman, Tahl; Sivam, Vanessa; Zhao, Tian; Frey, Oivier; van der Wal, Peter Dow; de Rooij, Nico F; Dalley, Jeffrey W; Edgley, Steve A

2011-01-01

119

Multiple extra-synaptic spillover mechanisms regulate prolonged activity in cerebellar Golgi cell-granule cell loops.  

PubMed

Despite a wealth of in vitro and modelling studies it remains unclear how neuronal populations in the cerebellum interact in vivo. We address the issue of how the cerebellar input layer processes sensory information, with particular focus on the granule cells (input relays) and their counterpart inhibitory interneurones, Golgi cells. Based on the textbook view, granule cells excite Golgi cells via glutamate forming a negative feedback loop. However, Golgi cells express inhibitory mGluR2 receptors suggesting an inhibitory role for glutamate. We set out to test this glutamatergic paradox in Golgi cells. Here we show that granule cells and Golgi cells interact through extra-synaptic signalling mechanisms during sensory information processing, as well as synaptic mechanisms. We demonstrate that such interactions depend on granule cell-derived glutamate acting via inhibitory mGluR2 receptors leading causally to the suppression of Golgi cell activity for several hundreds of milliseconds. We further show that granule cell-derived inhibition of Golgi cell activity is regulated by GABA-dependent extra-synaptic Golgi cell inhibition of granule cells, identifying a regulatory loop in which glutamate and GABA may be critical regulators of Golgi cell–granule cell functional activity. Thus, granule cells may promote their own prolonged activity via paradoxical feed-forward inhibition of Golgi cells, thereby enabling information processing over long timescales. PMID:21669981

Holtzman, Tahl; Sivam, Vanessa; Zhao, Tian; Frey, Oivier; van der Wal, Peter Dow; de Rooij, Nico F; Dalley, Jeffrey W; Edgley, Steve A

2011-08-01

120

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

PubMed

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

2008-12-01

121

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. PMID:1991788

1991-01-01

122

A signaling network stimulated by ?2 integrin promotes the polarization of lytic granules in cytotoxic cells.  

PubMed

Cytotoxic lymphocytes kill target cells through the polarized release of the contents of intracellular perforin-containing granules. In natural killer (NK) cells, the binding of ?2 integrin to members of the intercellular adhesion molecule family is sufficient to promote not only the adhesion of NK cells to target cells but also the polarization of intracellular lytic granules toward the target. We used NK cells in an experimental system designed to enable us to study the polarization of lytic granules in the absence of their release through degranulation, as well as ?2 integrin signaling independently of inside-out signals from other receptors. Through a proteomics approach, we identified a signaling network centered on an integrin-linked kinase (ILK)-Pyk2-paxillin core that was required for granule and microtubule-organizing center (MTOC) polarization. The conserved Cdc42-Par6 signaling pathway, which controls cell polarity, was also activated by ILK and was required for granule polarization toward the target cell. A subset of the signaling components required for polarization contributed also to the convergence of granules on the MTOC. These results delineate two connected signaling networks that are stimulated upon ?2 integrin engagement and control the polarization of the MTOC and associated lytic granules toward the site of contact with target cells to mediate cellular cytotoxicity. PMID:25292215

Zhang, Minggang; March, Michael E; Lane, William S; Long, Eric O

2014-10-01

123

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

PubMed

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

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

1985-01-01

124

Changes in Secretory Cells during Early Stages of Experimental Carcinogenesis in the Rat Submandibular Gland1  

Microsoft Academic Search

Previous studies have shown that the local injection of 7,12-dimethylbenz(a)anthracene induces largely squamous cell carcinoma in the rat submandibular gland. This study describes cytological changes that occur initially in the secretory cells of the rat submandibular gland following the injection of 7,12-dimethylbenz(a)anthracene. One of the most striking features noted early in injected glands is the appearance of duct-like structures, which

Sun Kee Kim; Herbert H. Spencer; Lee Weatherbee; Carlos E. Nasjleti

125

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

PubMed Central

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

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

2011-01-01

126

The position of mitochondria and ER in relation to that of the secretory sites in chromaffin cells.  

PubMed

Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca(2+) signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca(2+) microdomains described previously. PMID:25300794

Villanueva, José; Viniegra, Salvador; Gimenez-Molina, Yolanda; García-Martinez, Virginia; Expósito-Romero, Giovanna; Del Mar Frances, Maria; García-Sancho, Javier; Gutiérrez, Luis M

2014-12-01

127

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

PubMed

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

1989-07-01

128

Brain-derived neurotrophic factor suppresses programmed death of cerebellar granule cells through a posttranslational mechanism  

Microsoft Academic Search

Cerebellar granule cells isolated from 7-d-old rats have been shown to die in vitro unless they are continuously exposed to\\u000a elevated K+ (25 mM). Here we have characterized this neuronal death, and examined whether its major features are shared with those of sympathetic\\u000a neurons following nerve growth factor (NGF) deprivation. Granule cells underwent active cell death accompanied by morphological\\u000a features

Kazuhiko Suzuki; Tatsuro Koike

1997-01-01

129

Distinct Determinants of Sparse Activation during Granule Cell Maturation  

PubMed Central

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

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

2013-01-01

130

Effect of taxol on secretory cells: functional, morphological, and electrophysiological correlates  

Microsoft Academic Search

The effect of 0.5-1.0 #M taxol, a potent promoter of microtubule polymerization in vitro, was studied on the secretory activity of chromaffin cells of the adrenal medulla. Taxol was found to have a dual effect: the long-term effect (after a 1-h incubation) of taxol was to induce almost complete inhibition of catecholamine release, whereas after a short incubation (10 rain)

JOSETTE THURET-CARNAHAN; JEAN-LOUIS BOSSU; ANNE FELTZ; KEITH LANGLEY; DOMINIQUE AUNIS

1985-01-01

131

Secretory organelles of pathogenic protozoa.  

PubMed

Secretory processes play an important role on the biology and life cycles of parasitic protozoa. This review focus on basic aspects, from a cell biology perspective, of the secretion of (a) micronemes, rhoptries and dense granules in members of the Apicomplexa group, where these organelles are involved in the process of protozoan penetration into the host cell, survival within the parasitophorous vacuole and subsequent egress from the host cell, (b) the Maurer's cleft in Plasmodium, a structure involved in the secretion of proteins synthesized by the intravacuolar parasite and transported through vesicles to the erythrocyte surface, (c) the secretion of macromolecules into the flagellar pocket of trypanosomatids, and (d) the secretion of proteins which make the cyst wall of Giardia and Entamoeba, with the formation of encystation vesicles. PMID:16710566

Souza, Wanderley de

2006-06-01

132

Sustained granule cell activity disinhibits juvenile mouse cerebellar stellate cells through presynaptic mechanisms.  

PubMed

GABA release from cerebellar molecular layer interneurons can be modulated by presynaptic glutamate and/or GABA B receptors upon perfusing the respective agonists. However, it is unclear how release and potential spillover of endogenous transmitter lead to activation of presynaptic receptors. High frequency firing of granule cells, as observed in vivo upon sensory stimulation, could lead to glutamate and/or GABA spillover. Here, we established sustained glutamatergic activity in the granule cell layer of acute mouse cerebellar slices and performed 190 paired recordings from connected stellate cells. Train stimulation at 50 Hz reduced by about 30% the peak amplitude of IPSCs evoked by brief depolarization of the presynaptic cell in 2-week-old mice. A presynaptic mechanism was indicated by changes in failure rate, paired-pulse ratio and coefficient of variation of evoked IPSCs. Furthermore, two-photon Ca2+ imaging in identified Ca2+ hot spots of stellate cell axons confirmed reduced presynaptic Ca2+ influx after train stimulation within the granular layer. Pharmacological experiments indicated that glutamate released from parallel fibres activated AMPARs in stellate cells, evoking GABA release from surrounding cells. Consequential GABA spillover activated presynaptic GABA B Rs, which reduced the amplitude of eIPSCs. Two-thirds of the total disinhibitory effect were mediated by GABA B Rs, one-third being attributable to presynaptic AMPARs. This estimation was confirmed by the observation that bath applied baclofen induced a more pronounced reduction of evoked IPSCs than kainate. Granule cell-mediated disinhibition persisted at near-physiological temperature but was strongly diminished in 3-week-old mice. At this age, GABA release probability was not reduced and presynaptic GABA B Rs were still detectable, but GABA uptake appeared to be advanced, attenuating GABA spillover. Thus, sustained granule cell activity modulates stellate cell-to-stellate cell synapses, involving transmitter spillover during a developmentally restricted period. PMID:18033809

Astori, Simone; Köhr, Georg

2008-01-15

133

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. Lømo

1971-01-01

134

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.

1984-10-01

135

The biology and dynamics of mammalian cortical granules  

PubMed Central

Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals. PMID:22088197

2011-01-01

136

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. PMID:22672875

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

2012-01-01

137

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

PubMed Central

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

Chancey, Jessica H.; Poulsen, David J.

2014-01-01

138

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.

CARL NATHAN

1992-01-01

139

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

PubMed

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

Zacharogianni, Margarita; Rabouille, Catherine

2013-01-01

140

Real-Time Imaging of the Axonal Transport of Granules Containing a Tissue Plasminogen Activator/Green Fluorescent Protein Hybrid  

PubMed Central

A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation. PMID:9725906

Lochner, Janis E.; Kingma, Mary; Kuhn, Samuel; Meliza, C. Daniel; Cutler, Bryan; Scalettar, Bethe A.

1998-01-01

141

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 PMID:8863674

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

1996-01-01

142

Role of Cdc42 in neurite outgrowth of PC12 cells and cerebellar granule neurons.  

PubMed

Inactivation of Rho GTPases inhibited the neurite outgrowth of PC12 cells. The role of Cdc42 in neurite outgrowth was then studied by selective inhibition of Cdc42 signals. Overexpression of ACK42, Cdc42 binding domain of ACK-1, inhibited NGF-induced neurite outgrowth in PC12 cells. ACK42 also inhibited the neurite outgrowth of PC12 cells induced by constitutively activated mutant of Cdc42, but not Rac. These results suggest that Cdc42 plays an important role in mediating NGF-induced neurite outgrowth of PC12 cells. Inhibition of neurite outgrowth was also demonstrated using a cell permeable chimeric protein, penetratin-ACK42. A dominant negative mutant of Rac, RacN17 inhibited Cdc42-induced neurite outgrowth of PC12 cells suggesting that Rac acts downstream of Cdc42. Further studies, using primary-cultures of rat cerebellar granule neurons, showed that Cdc42 is also involved in the neurite outgrowth of cerebellar granule neurons. Both penetratin-ACK42 and Clostridium difficile toxin B, which inactivates all members of Rho GTPases strongly inhibited the neurite outgrowth of cerebellar granule neurons. These results show that Cdc42 plays a similar and essential role in the development of neurite outgrowth of PC12 cells and cerebellar granule neurons. These results provide evidence that Cdc42 produces signals that are essential for the neurite outgrowth of PC12 cells and cerebellar granule neurons. PMID:16328953

Ahmed, Ijaz; Calle, Yolanda; Iwashita, Shintaro; Nur-E-Kamal, Alam

2006-01-01

143

Expression profiling of pancreatic ? cells: Glucose regulation of secretory and metabolic pathway genes  

PubMed Central

Pancreatic ? cells respond to changes in blood glucose by secreting insulin and increasing insulin synthesis. To identify genes used in these responses, we have carried out expression profiling of ? cells exposed to high (25 mM) or low (5.5 mM) glucose by using oligonucleotide microarrays. Functional clustering of genes that averaged a 2.2-fold or greater change revealed large groups of secretory pathway components, enzymes of intermediary metabolism, cell-signaling components, and transcription factors. Many secretory pathway genes were up-regulated in high glucose, including seven members of the endoplasmic reticulum (ER) translocon. In agreement with array analysis, protein levels of translocon components were increased by high glucose. Most dramatically, the ? subunit of the signal recognition particle receptor was increased over 20-fold. These data indicate that the translocon and ribosome docking are major regulatory targets of glucose in the ? cell. Analysis of genes encoding enzymes of intermediary metabolism indicated that low glucose brought about greater utilization of amino acids as an energy source. This conclusion was supported by observations of increased urea production under low-glucose conditions. The above results demonstrate genome-wide integration of ?-cell functions at the level of transcript abundance and validate the efficacy of expression profiling in identifying genes involved in the ?-cell glucose response. PMID:10811900

Webb, Gene C.; Akbar, Murtaza S.; Zhao, Chongjian; Steiner, Donald F.

2000-01-01

144

Diphenylhydantoin induces apoptotic cell death of cultured rat cerebellar granule neurons.  

PubMed

Apoptosis is one form of physiological or programmed cell death responsible for the selective elimination of various cell types during development. We have observed and characterized a delayed-type of neurotoxicity induced in cultured cerebellar granule neurons by diphenylhydantoin. Diphenylhydantoin toxicity of cerebellar granule neurons is time and concentration dependent. Morphological studies using Nomarski optics and staining with the fluorescent dye Hoechst 33258 demonstrate that diphenylhydantoin-induced neurotoxicity of cerebellar granule neurons is associated with cytoplasmic blebbing, heterochromatic clumping and condensation of chromatin that precede cell death. Unlike glutamate toxicity (excitotoxicity) diphenylhydantoin-induced neurotoxicity of cerebellar granule neurons is attenuated by actinomycin D and cycloheximide, and is associated with nucleosomal size DNA fragmentation. Since we have previously reported that depolarization of cultured cerebellar granule neurons with high concentrations of K+ promotes the survival of these neurons by blocking apoptosis, we examined the effects of diphenylhydantoin on the K(+)-evoked increase in intracellular calcium. Using microfluorimetry and fura-2 to measure intracellular calcium we found that neurotoxic concentrations of diphenylhydantoin markedly reduce the increase in intracellular calcium associated with elevated extracellular potassium. Taken together, these data demonstrate that exposure of cultured cerebellar granule neurons to pharmacologically relevant concentrations of diphenylhydantoin results in a delayed type of neurotoxicity characterized by the biochemical and morphological features of apoptosis. PMID:7636763

Yan, G M; Irwin, R P; Lin, S Z; Weller, M; Wood, K A; Paul, S M

1995-08-01

145

Granule cell number, cell death and cell proliferation in the dentate gyrus of wild-living rodents.  

PubMed

Adult neurogenesis in the dentate gyrus occurs at species-specific levels. Wood mice (Apodemus flavicollis) show higher proliferation rates than laboratory mice and voles (Clethrionomys glareolus, Microtus subterraneus). We compare rates of cell death and proliferation and investigate if cell proliferation leads to the long-term recruitment of granule cells. Granule and pyknotic cell numbers were estimated in wild-living rodents in different age classes and compared with laboratory mice of mixed genetic background. All species differ significantly in their number of granule cells, except for the comparison of laboratory mice with European pine voles. Granule cell number is significantly higher in old bank voles and wood mice as compared to adults (23 and 37%, respectively). The number of pyknotic cells is highest in wood mice and lowest in laboratory mice. Across all species, the numbers of proliferating and pyknotic cells correlate. Despite differences in cell proliferation and cell death, the ratio of proliferating to pyknotic cells does not differ between adults of the wild-living species, but in laboratory mice a significantly lower proportion of cells die compared with the other species. In addition, the ratio of proliferating to pyknotic cells was significantly higher in old wood mice than in adults. We conclude (i) that cell proliferation can lead to an increase in granule cell number in wild-living rodents and (ii) that species- and age-specific changes of the ratio between proliferating and pyknotic cells occur as deviations from a close correlation of these two numbers across all species and age groups. PMID:15610166

Amrein, Irmgard; Slomianka, Lutz; Lipp, Hans-Peter

2004-12-01

146

LYST Controls the Biogenesis of the Endosomal Compartment Required for Secretory Lysosome Function.  

PubMed

Chediak-Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome-like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling-late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal-sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13-4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin-containing vesicles into secretory cytotoxic granules. PMID:25425525

Sepulveda, Fernando E; Burgess, Agathe; Heiligenstein, Xavier; Goudin, Nicolas; Ménager, Mickaël M; Romao, Maryse; Côte, Marjorie; Mahlaoui, Nizar; Fischer, Alain; Raposo, Graça; Ménasché, Gaël; de Saint Basile, Geneviève

2015-02-01

147

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

PubMed Central

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

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

2014-01-01

148

Improvement in ?-cell secretory capacity after human islet transplantation according to the CIT07 protocol.  

PubMed

The Clinical Islet Transplantation 07 (CIT07) protocol uses antithymocyte globulin and etanercept induction, islet culture, heparinization, and intensive insulin therapy with the same low-dose tacrolimus and sirolimus maintenance immunosuppression as in the Edmonton protocol. To determine whether CIT07 improves engrafted islet ?-cell mass, our center measured ?-cell secretory capacity from glucose-potentiated arginine tests at days 75 and 365 after transplantation and compared those results with the results previously achieved by our group using the Edmonton protocol and normal subjects. All subjects were insulin free, with CIT07 subjects receiving fewer islet equivalents from a median of one donor compared with two donors for Edmonton protocol subjects. The acute insulin response to glucose-potentiated arginine (AIRpot) was greater in the CIT07 protocol than in the Edmonton protocol and was less in both cohorts than in normal subjects, with similar findings for C-peptide. The CIT07 subjects who completed reassessment at day 365 exhibited increasing AIRpot by trend relative to that of day 75. These data indicate that engrafted islet ?-cell mass is markedly improved with the CIT07 protocol, especially given more frequent use of single islet donors. Although several peritransplant differences may have each contributed to this improvement, the lack of deterioration in ?-cell secretory capacity over time in the CIT07 protocol suggests that low-dose tacrolimus and sirolimus are not toxic to islets. PMID:23630300

Rickels, Michael R; Liu, Chengyang; Shlansky-Goldberg, Richard D; Soleimanpour, Scott A; Vivek, Kumar; Kamoun, Malek; Min, Zaw; Markmann, Eileen; Palangian, Maral; Dalton-Bakes, Cornelia; Fuller, Carissa; Chiou, Allen J; Barker, Clyde F; Luning Prak, Eline T; Naji, Ali

2013-08-01

149

Improvement in ?-Cell Secretory Capacity After Human Islet Transplantation According to the CIT07 Protocol  

PubMed Central

The Clinical Islet Transplantation 07 (CIT07) protocol uses antithymocyte globulin and etanercept induction, islet culture, heparinization, and intensive insulin therapy with the same low-dose tacrolimus and sirolimus maintenance immunosuppression as in the Edmonton protocol. To determine whether CIT07 improves engrafted islet ?-cell mass, our center measured ?-cell secretory capacity from glucose-potentiated arginine tests at days 75 and 365 after transplantation and compared those results with the results previously achieved by our group using the Edmonton protocol and normal subjects. All subjects were insulin free, with CIT07 subjects receiving fewer islet equivalents from a median of one donor compared with two donors for Edmonton protocol subjects. The acute insulin response to glucose-potentiated arginine (AIRpot) was greater in the CIT07 protocol than in the Edmonton protocol and was less in both cohorts than in normal subjects, with similar findings for C-peptide. The CIT07 subjects who completed reassessment at day 365 exhibited increasing AIRpot by trend relative to that of day 75. These data indicate that engrafted islet ?-cell mass is markedly improved with the CIT07 protocol, especially given more frequent use of single islet donors. Although several peritransplant differences may have each contributed to this improvement, the lack of deterioration in ?-cell secretory capacity over time in the CIT07 protocol suggests that low-dose tacrolimus and sirolimus are not toxic to islets. PMID:23630300

Rickels, Michael R.; Liu, Chengyang; Shlansky-Goldberg, Richard D.; Soleimanpour, Scott A.; Vivek, Kumar; Kamoun, Malek; Min, Zaw; Markmann, Eileen; Palangian, Maral; Dalton-Bakes, Cornelia; Fuller, Carissa; Chiou, Allen J.; Barker, Clyde F.; Luning Prak, Eline T.; Naji, Ali

2013-01-01

150

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

PubMed

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

Meiniel, Annie

2007-01-01

151

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

PubMed

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

Wada, Norio; Funabiki, Kazuo; Nakanishi, Shigetada

2014-04-01

152

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

PubMed Central

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

Wada, Norio; Funabiki, Kazuo; Nakanishi, Shigetada

2014-01-01

153

Myosins of secretory tissues  

PubMed Central

Myosin has been purified from the principal pancreatic islet of catfish, hog salivary gland, and hog pituitary. Use of the protease inhibitor Trasylol (FBA Pharmaceuticals, New York) was essential in the isolation of pituitary myosin. Secretory tissue myosins were very similar to smooth muscle myosin, having a heavy chain of 200,000 daltons and light chains of 14,000 and 19,000 daltons. Salivary gland myosin cross-reacted with antibodies directed toward both smooth muscle myosin and fibroblast myosin, but not with antiskeletal muscel myosin serum. The specific myosin ATPase activity measured in 0.6 M KCl was present. Tissues associated with secretion of hormone granules contained substantial amounts of this ATPase, rat pancreatic islets having 4.5 times that of rat liver. Activation of low ionic strength myosin ATPase by actin could not be demonstrated despite adequate binding of the myosin to muscle actin and elution by MgATP. The myosins were located primarily in the cytoplasm as determined by cell fractionation and were quite soluble in buffers of low ionic strength. PMID:150427

1978-01-01

154

Depolarization or glutamate receptor activation blocks apoptotic cell death of cultured cerebellar granule neurons.  

PubMed

Cerebellar granule neurons can be readily maintained in culture if depolarized with high concentrations of K+ or subtoxic concentrations of various excitatory amino acids. We now report that these depolarizing stimuli promote cerebellar granule neuron survival by blocking their programmed death via apoptosis. Cerebellar granule neurons maintained in depolarizing conditions and then changed to non-depolarizing conditions, exhibit the morphological and biochemical features of apoptosis, including cytoplasmic blebbing, condensation and aggregation of nuclear chromatin and internucleosomal DNA fragmentation. Inhibitors of RNA or protein synthesis greatly attenuate cell death induced by non-depolarizing culture conditions. In contrast, cerebellar granule neurons, when exposed to fresh serum-containing medium or to high concentrations of glutamate, exhibit a delayed-type of neurotoxicity which is non-apoptotic in nature. Given the actions of excitatory amino acid receptor agonists in preventing apoptosis of cultured cerebellar granule neurons, we hypothesize that the functional innervation of postmigratory granule neurons during cerebellar development may prevent further elimination of these neurons by blocking their programmed death. PMID:7804844

Yan, G M; Ni, B; Weller, M; Wood, K A; Paul, S M

1994-09-01

155

Bone Marrow Cells Expressing Clara Cell Secretory Protein Increase Epithelial Repair After Ablation of Pulmonary Clara Cells  

PubMed Central

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

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

2013-01-01

156

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

1989-01-01

157

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

1975-01-01

158

Glutamate Transporters Contribute to the Time Course of Synaptic Transmission in Cerebellar Granule Cells  

Microsoft Academic Search

Transporters are thought to assist in the termination of synaptic transmission at some synapses by removing neurotransmitter from the synapse. To investigate the role of glutamate transport in shaping the time course of excitatory transmission at the mossy fiber-granule cell synapse, the effects of transport impairment were studied using whole-cell voltage- and current- clamp recordings in slices of rat cerebellum.

Linda S. Overstreet; Gregory A. Kinney; Ying-Bing Liu; Daniela Billups; N. Traverse

159

Sperm-Storage Defects and Live Birth in Drosophila Females Lacking Spermathecal Secretory Cells  

PubMed Central

Male Drosophila flies secrete seminal-fluid proteins that mediate proper sperm storage and fertilization, and that induce changes in female behavior. Females also produce reproductive-tract secretions, yet their contributions to postmating physiology are poorly understood. Large secretory cells line the female's spermathecae, a pair of sperm-storage organs. We identified the regulatory regions controlling transcription of two genes exclusively expressed in these spermathecal secretory cells (SSC): Spermathecal endopeptidase 1 (Send1), which is expressed in both unmated and mated females, and Spermathecal endopeptidase 2 (Send2), which is induced by mating. We used these regulatory sequences to perform precise genetic ablations of the SSC at distinct time points relative to mating. We show that the SSC are required for recruiting sperm to the spermathecae, but not for retaining sperm there. The SSC also act at a distance in the reproductive tract, in that their ablation: (1) reduces sperm motility in the female's other sperm-storage organ, the seminal receptacle; and (2) causes ovoviviparity—the retention and internal development of fertilized eggs. These results establish the reproductive functions of the SSC, shed light on the evolution of live birth, and open new avenues for studying and manipulating female fertility in insects. PMID:22087073

Schnakenberg, Sandra L.; Matias, Wilfredo R.; Siegal, Mark L.

2011-01-01

160

The Origin and Role of Autophagy in the Formation of Cytoplasmic Granules in Canine Lingual Granular Cell Tumors.  

PubMed

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

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

2014-08-26

161

Spatial Relationships between Markers for Secretory and Endosomal Machinery in Human Cytomegalovirus-Infected Cells versus Those in Uninfected Cells?†  

PubMed Central

Human cytomegalovirus (HCMV) induces extensive remodeling of the secretory apparatus to form the cytoplasmic virion assembly compartment (cVAC), where virion tegumentation and envelopment take place. We studied the structure of the cVAC by confocal microscopy to assess the three-dimensional distribution of proteins specifically associated with individual secretory organelles. In infected cells, early endosome antigen 1 (EEA1)-positive vesicles are concentrated at the center of the cVAC and, as previously seen, are distinct from structures visualized by markers for the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network (TGN). EEA1-positive vesicles can be strongly associated with markers for recycling endosomes, to a lesser extent with markers associated with components of the endosomal sorting complex required for transport III (ESCRT III) machinery, and then with markers of late endosomes. In comparisons of uninfected and infected cells, we found significant changes in the structural associations and colocalization of organelle markers, as well as in net organelle volumes. These results provide new evidence that the HCMV-induced remodeling of the membrane transport apparatus involves much more than simple relocation and expansion of preexisting structures and are consistent with the hypothesis that the shift in identity of secretory organelles in HCMV-infected cells results in new functional profiles. PMID:21471245

Das, Subhendu; Pellett, Philip E.

2011-01-01

162

Autonomous Turning of Cerebellar Granule Cells in vitro by Intrinsic Programs  

PubMed Central

External guidance cues play a role in controlling neuronal cell turning in the developing brain, but little is known about whether intrinsic programs are also involved in controlling the turning. In this study, we examined whether granule cells undergo autonomous changes in the direction of migration in the microexplant cultures of the early postnatal mouse cerebellum. We found that granule cells exhibit spontaneous and periodical turning without cell-cell contact and in the absence of external guidance cues. The frequency of turning was increased by stimulating the Ca2+ influx and the internal Ca2+ release, or inhibiting the cAMP signaling pathway, while the frequency was reduced by inhibiting the Ca2+ influx. Granule cell turning in vitro was classified into four distinct modes, which were characterized by the morphological changes in the leading process and the trailing process, such as bifurcating, turning, withdrawing, and changing the polarity. The occurrence of the 1st and 2nd modes of turning was differentially affected by altering the Ca2+ and cAMP signaling pathways. Collectively, the results demonstrate that intrinsic programs regulate the autonomous turning of cerebellar granule cells in vitro. Furthermore, the results suggest that extrinsic signals play a role as essential modulators of intrinsic programs. PMID:19063877

Kumada, Tatsuro; Jiang, Yulan; Kawanami, Aya; Cameron, D. Bryant; Komuro, Hitoshi

2009-01-01

163

Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures  

Microsoft Academic Search

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. Although expression of GFP in plants has been widely reported, research on the use of GFP in plant cell cultures for bioprocess applications has been limited. In this study, the suitability of GFP as a secretory reporter and a useful tool in plant cell bioprocess optimization was demonstrated. GFP

S. Liu; R. C. Bugos; N. Dharmasiri; W. W. Su

2001-01-01

164

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

PubMed Central

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

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

2010-01-01

165

Kinetic analysis of the triggered exocytosis/endocytosis secretory cycle in cultured bovine adrenal medullary cells  

PubMed Central

Cultured bovine adrenal medullary cells are an excellent preparation for quantitative analysis of the secretory exocytosis/endocytosis cycle. In this paper we examine the kinetics of endocytosis after stimulation of secretion. Membrane retrieval was monitored by uptake of the fluid phase marker horseradish peroxidase. Horseradish peroxidase was found to be suitable because it can be washed off completely, assayed quantitatively, and its uptake increases linearly with concentration. If this marker is present during stimulation, the rate of uptake is initially slower than catecholamine secretion but faster at a later time, suggesting that the formation of endocytotic vesicles follows exocytosis. To monitor the time-dependent concentration of secretory vesicle-plasma membrane fusion product (omega-profiles), secretion was halted at various time intervals after stimulation and the excess membrane allowed to transform into endocytotic vesicles in the presence of horseradish peroxidase. By adding horseradish peroxidase at various times after inhibition of secretion, the time course of membrane retrieval could be measured directly. All our results are consistent with a two-step kinetic model in which exocytosis and membrane retrieval are consecutive events. The estimated volumes of the compartments involved are roughly equal. The rate of endocytosis is strongly temperature-dependent but unaffected by extracellular calcium in the range of 10(-8)-2.5 X 10(-3) M, suggesting that calcium is not required at the site of endocytotic membrane fusion. Membrane retrieval is also unaffected by Lanthanum (1 mM) but is slowed by hypertonic media. PMID:3782299

1986-01-01

166

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

PubMed Central

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

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

2014-01-01

167

Regulation of zymogen granule exocytosis by Ca 2+, cAMP, and PKC in pancreatic acinar cells  

Microsoft Academic Search

The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10?M), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a

Misun Lee; Sungkwon Chung; Dae Yong Uhm; Myoung Kyu Park

2005-01-01

168

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

E-print Network

honeybee (Apis mellifera L.) H. Raes W. Bohyn P.H. De Rycke F. Jacobs ! Labo/'afo/y!o/'Zoop/tys/o/ot! State of adult worker honeybees. By using different preparation techniques, we collected additional data). The presence of calciferous granules in midgut epithelial cells of the honeybee has been known for some time

Boyer, Edmond

169

Development/Plasticity/Repair BDNF-Mediated Cerebellar Granule Cell Development Is  

E-print Network

Development/Plasticity/Repair BDNF-Mediated Cerebellar Granule Cell Development Is Impaired in Mice (CREB) correlates with Bdnf transcription, which is required for normal development of cerebellar in decreased CREB phosphorylation (pCREB), Bdnf exon I and IV-containing mRNAs, and brain-derived neurotrophic

West, Anne

170

GABAA ?6-Containing Receptors Are Selectively Compromised in Cerebellar Granule Cells of the Ataxic Mouse, Stargazer*  

PubMed Central

Stargazer mice fail to express the ?2 isoform of transmembrane ?-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor regulatory proteins that has been shown to be absolutely required for the trafficking and synaptic targeting of excitatory AMPA receptors in adult murine cerebellar granule cells. Here we show that 30 ± 6% fewer inhibitory ?-aminobutyric acid, type A (GABAA), receptors were expressed in adult stargazer cerebellum compared with controls because of a specific loss of GABAA receptor expression in the cerebellar granule cell layer. Radioligand binding assays allied to in situ immunogold-EM analysis and furosemide-sensitive tonic current estimates revealed that expression of the extrasynaptic (?6?x?) ?6-containing GABAA receptor were markedly and selectively reduced in stargazer. These observations were compatible with a marked reduction in expression of GABAA receptor ?6, ? (mature cerebellar granule cell-specific proteins), and ?3 subunit expression in stargazer. The subunit composition of the residual ?6-containing GABAA receptors was unaffected by the stargazer mutation. However, we did find evidence of an ~4-fold up-regulation of ?1?? receptors that may compensate for the loss of ?6-containing GABAA receptors. PCR analysis identified a dramatic reduction in the steady-state level of ?6 mRNA, compatible with ?6 being the primary target of the stargazer mutation-mediated GABAA receptor abnormalities. We propose that some aspects of assembly, trafficking, targeting, and/or expression of extrasynaptic ?6-containing GABAA receptors in cerebellar granule cells are selectively regulated by AMPA receptor-mediated signaling. PMID:17646167

Payne, Helen L.; Connelly, William M.; Ives, Jane H.; Lehner, Reinhard; Furtmuller, Birgit; Sieghart, Werner; Tiwari, Priyanka; Lucocq, John M.; Lees, George; Thompson, Christopher L.

2010-01-01

171

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. PMID:21854518

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

2013-01-01

172

Proliferation of Granule Cell Precursors in the Dentate Gyrus of Adult Monkeys is Diminished by Stress  

Microsoft Academic Search

Although granule cells continue to be added to the dentate gyrus of adult rats and tree shrews, this phenomenon has not been demonstrated in the dentate gyrus of adult primates. To determine whether neurons are produced in the dentate gyrus of adult primates, adult marmoset monkeys (Callithrix jacchus) were injected with BrdU and perfused 2 hr or 3 weeks later.

Elizabeth Gould; Patima Tanapat; Bruce S. McEwen; Gabriele Flugge; Eberhard Fuchs

1998-01-01

173

Senescent Cells and Their Secretory Phenotype as Targets for Cancer Therapy  

PubMed Central

Cancer is a devastating disease that increases exponentially with age. Cancer arises from cells that proliferate in an unregulated manner, an attribute that is countered by cellular senescence. Cellular senescence is a potent tumor-suppressive process that halts the proliferation, essentially irreversibly, of cells at risk for malignant transformation. A number of anti-cancer drugs have emerged that induce tumor cells to undergo cellular senescence. However, although a senescence response can halt the proliferation of cancer cells, the presence of senescent cells in tissues has been associated with age-related diseases, including, ironically, late-life cancer. Thus, anti-cancer therapies that can induce senescence might also drive aging phenotypes and age-related pathology. The deleterious effects of senescent cells most likely derive from their senescence-associated secretory phenotype or SASP. The SASP entails the secretion of numerous inflammatory cytokines, growth factors and proteases that can render the tissue microenvironment favorable for tumor growth. Here, we discuss the beneficial and detrimental effects of inducing cellular senescence, and propose strategies for targeting senescent cells as a means to fight cancer. PMID:23503512

Velarde, Michael C.; Demaria, Marco; Campisi, Judith

2014-01-01

174

Sphingomyelin Patches on Pancreatic Beta-cells Are Indicative of Insulin Secretory Capacity  

PubMed Central

The establishment and validation of specific markers on the surfaces of pancreatic beta-cells would have a significant impact on the development of agents that specifically target these cells for imaging and/or image-guided therapy in diabetes patient samples. We have recently described unique, cholesterol-stabilized sphingomyelin (SM) patches on the surfaces of beta-cells using the IC2 antibody. To further investigate the utility of SM patches as a unique beta-cell biomarker, we embarked on the current study to correlate the expression of this antigen with the insulin secretory capacity of beta-cells in tissue samples from patients and animals with type 1 and type 2 diabetes and compared this with samples from normal subjects. We found that the locations of SM patches were consistent with the insulin status of islets in all tissues studied. Using immunohistochemistry and staining with an IC2 antibody, we demonstrated a direct correlation between the reduced expression of SM patches and insulin production in diabetic individuals, indicating that the former could potentially serve as a functional biomarker of beta-cells. We believe that our results have significant implications for the further development of ligands with SM specificity for the non-invasive functional assessment of beta-cells and/or for targeted therapeutic delivery in diabetic patients. PMID:23920110

Kavishwar, Amol

2013-01-01

175

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. PMID:22615885

Ogata, Sho; Miki, Takashi; Seino, Susumu; Tamai, Seiichi; Kasai, Haruo; Nemoto, Tomomi

2012-01-01

176

A novel function of Noc2 in agonist-induced intracellular Ca2+ increase during zymogen-granule exocytosis in pancreatic acinar cells.  

PubMed

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 Ca(2+)-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 [Ca(2+)](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 [Ca(2+)](i) increases. PMID:22615885

Ogata, Sho; Miki, Takashi; Seino, Susumu; Tamai, Seiichi; Kasai, Haruo; Nemoto, Tomomi

2012-01-01

177

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

Microsoft Academic Search

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

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

1985-01-01

178

Media composition modulates excitatory amino acid-induced death of rat cerebellar granule cells.  

PubMed

This study examined the effects of maintaining cells in different media and the role of serum in glutamate and NMDA-induced neurotoxicity in rat cerebellar granule cells. Glutamate stimulated a concentration-dependent cell death with similar potency in cerebellar granule cells grown in BME and Neurobasal media without serum. However, the maximal cell death to glutamate and N-methyl-D-aspartate (NMDA) varied in the different media compositions. In the presence of serum, glutamate and NMDA-induced excitotoxicity was abolished, suggesting a factor(s) in serum which influences glutamate-receptor mediated death. The protective effect of serum could be overcome by chronic stimulation with high doses of glutamate. The glutamate-stimulated increase in intracellular calcium load was attenuated in the presence of serum, resulting from an elevated basal calcium level, suggesting an association between raised basal calcium and neuroprotection. PMID:9257158

Wood, A M; Tiwari, P; Bristow, D R

1997-07-01

179

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

PubMed

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

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

2014-01-01

180

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

NASA Technical Reports Server (NTRS)

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.

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

2002-01-01

181

Fine morphology of the secretory mode of the tetragastrin-stimulated chief cells of human and dog gastric mucosa.  

PubMed

The chief cells of the gastrin-stimulated gastric mucosa of human and dog were observed under a light and electron microscope. Four microgram/kg AOC-tetragastrin were given parenterally by a single shot to a man and three dogs respectively. Pepsinogen in the gastric mucosa increased at the wash-out stage and at the following dynamic equibrium stage of the chief cell secretion cycle after the administration of AOC-tetragastrin. During those stages, the chief cells released zymogen granules intensively. As the main ultrastructural process for releasing the zymogen granules, the emiocytosis in man and the apical cytoplasm dissociation in dog were discussed. PMID:689336

Machino, M; Aoike, A; Ikeuchi, H; Sasaki, Z; Misaki, F; Kawai, K

1978-01-01

182

Ultrastructural studies on the secretory mechanism of goblet cells in the rat jejunal epithelium.  

PubMed

Goblet cells in the jejunal epithelium of adult and suckling rats were studied with transmission electron microscope. The use of triple fixation, i.e. osmium-aldehyde-osmium has some advantages in the preservation of mucous substances and some cell organelles. Cytochemical demonstration of polysaccharide and glycoprotein by methenamine silver as well as detection of TPPase and acid phosphatase (AcPase) were performed. Accumulation of secretory substances and combination with sugar moieties occurred in the internal sacs of Golgi lamellae, where TPPase activity was recognized. No rigid lamella usually positive to AcPase was found in goblet cells, though it was easily found in the columnar absorptive cells. AcPase was positive in some small immature mucus droplets as well as lysosomes. As the differentiation of goblet cells advances, mucus droplets come into contact and fuse to each other. As a result of the close contact of two adjacent membranes of mucus droplets, a pentalaminar structure is first formed. Then the central dense lamina disappears and the membrane becomes trilaminar. Disappearance of one of the apposed unit membranes, leaving another unit membrane, may take place. Finally the single unit membrane left between the adjacent droplets is also ruptured and complete fusion occurs. The mechanism of extrusion of mucous substance is complicated. Some mucus droplets located at the most superficial area may be released by the mechanisms of exocytosis. More frequently, however, deeply located droplets fuse to each other and a huge vacuole is made in the apical cytoplasm. The substance filling the vacuole contains debris of cytoplasmic matrix and droplet membranes along with mucous secretory substance. These substances derived from different sources are expelled into the lumen at the same time. This may be classified into the apocrine mechanism, because a part of the cytoplasm and membrane are lost. The typical apocrine secretion, i.e. pinching off of cytoplasmic projection containing mucus droplets, was also found in the goblet cells. Thus, two or three different mechanisms of secretion discharge may possibly occur simultaneously in goblet cells. PMID:6118110

Kurosumi, K; Shibuichi, I; Tosaka, H

1981-07-01

183

Inhibition of BACE2 counteracts hIAPP-induced insulin secretory defects in pancreatic ?-cells.  

PubMed

BACE2 (?-site APP-cleaving enzyme 2) is a protease localized in the brain, where it appears to play a role in the development of Alzheimer disease (AD). It is also found in the pancreas, although its biologic function is not fully known. Amyloidogenic diseases, including AD and type 2 diabetes mellitus (T2D), share the accumulation of abnormally folded and insoluble proteins that interfere with cell function. Islet amyloid polypeptide (IAPP) deposits are a key pathogenic feature of T2D. Within this context, we found by global gene expression profiling that BACE2 was up-regulated in the rat pancreatic ?-cell line INS1E stably transfected with human IAPP gene (hIAPP-INS1E). Glucose-stimulated insulin secretion (GSIS) in hIAPP-INS1E cells was 30% lower than in INS1E cells. Additionally, INS1E cells transfected with a transient overexpression of BACE2 showed a 60% decrease in proliferation, a 3-fold increase in reactive oxygen species production, and a 25% reduction in GSIS compared to control cells. Remarkably, silencing of endogenous BACE2 in hIAPP-INS1E cells resulted in a significant improvement in GSIS (3-fold increase vs. untransfected cells), revealing the significant role of BACE2 expression in ?-cell function. Thus, BACE2 inhibition may be useful to recover insulin secretion in hIAPP-INS1E defective cells and may be proposed as a therapeutic target for T2D.-Alcarraz-Vizán, G., Casini, P., Cadavez, L., Visa, M., Montane, J., Servitja, J.-M., Novials, A. Inhibition of BACE2 counteracts hIAPP-induced insulin secretory defects in pancreatic ?-cells. PMID:25342134

Alcarraz-Vizán, Gema; Casini, Paola; Cadavez, Lisa; Visa, Montse; Montane, Joel; Servitja, Joan-Marc; Novials, Anna

2015-01-01

184

Eosinophil Secretion of Granule-Derived Cytokines  

PubMed Central

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

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

2014-01-01

185

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

PubMed

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

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

2015-02-01

186

Interactions of PACAP and ceramides in the control of granule cell apoptosis during cerebellar development.  

PubMed

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal polypeptide superfamily. The PACAPergic system is actively expressed in the developing cerebellum of mammals. In particular, PACAP receptors are expressed by granule cell precursors suggesting a role of the peptide in neurogenesis of this cell type. Consistent with this hypothesis, several studies reported antiapoptotic effects of PACAP in the developing cerebellum. On the other hand, the sphingomyelin metabolites ceramides are recognized as important signaling molecules that play pivotal roles during neuronal development. Ceramides, which production can be induced by death factors such as FasL or TNFalpha, are involved in the control of cell survival during brain development through activation of caspase-dependent mechanisms. The present review focuses on the interactions between PACAP and ceramides in the control of granule cell survival and on the transduction mechanisms associated with the anti- and proapoptotic effects of PACAP and ceramides, respectively. PMID:18574733

Falluel-Morel, A; Aubert, N; Vaudry, D; Desfeux, A; Allais, A; Burel, D; Basille, M; Vaudry, H; Laudenbach, V; Gonzalez, B J

2008-11-01

187

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. PMID:22973010

Dinday, Matthew T.; Hindle-Katel, William; Baraban, Scott C.

2012-01-01

188

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

Microsoft Academic Search

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

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

1989-01-01

189

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

PubMed

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

Bruenner, U; Burnside, B

1986-11-01

190

Secretory responses of intact glomus cells in thin slices of rat carotid body to hypoxia and tetraethylammonium  

Microsoft Academic Search

We have developed a thin-slice preparation of whole rat carotid body that allows us to perform patch-clamp recording of membrane ionic currents and to monitor catecholamine secretion by amperometry in single glomus cells under direct visual control. In normoxic conditions (PO2 140 mmHg; 1 mmHg = 133 Pa), most glomus cells did not have measurable secretory activity, but exposure to

Ricardo Pardal; Uwe Ludewig; Julia García-Hirschfeld; José López-Barneo

2000-01-01

191

Insulin Granule Biogenesis, Trafficking and Exocytosis  

PubMed Central

It is becoming increasingly apparent that beta cell dysfunction resulting in abnormal insulin secretion is the essential element in the progression of patients from a state of impaired glucose tolerance to frank type 2 diabetes (Del Prato, 2003; Del Prato and Tiengo, 2001). Although extensive studies have examined the molecular, cellular and physiologic mechanisms of insulin granule biogenesis, sorting, and exocytosis the precise mechanisms controlling these processes and their dysregulation in the developed of diabetes remains an area of important investigation. We now know that insulin biogenesis initiates with the synthesis of preproinsulin in rough endoplastic reticulum and conversion of preproinsulin to proinsulin. Proinsulin begins to be packaged in the Trans-Golgi Network and is sorting into immature secretory granules. These immature granules become acidic via ATP-dependent proton pump and proinsulin undergoes proteolytic cleavage resulting the formation of insulin and C-peptide. During the granule maturation process, insulin is crystallized with zinc and calcium in the form of dense-core granules and unwanted cargo and membrane proteins undergo selective retrograde trafficking to either the constitutive trafficking pathway for secretion or to degradative pathways. The newly formed mature dense-core insulin granules populate two different intracellular pools, the readily releasable pools (RRP) and the reserved pool. These two distinct populations are thought to be responsible for the biphasic nature of insulin release in which the RRP granules are associated with the plasma membrane and undergo an acute calcium-dependent release accounting for first phase insulin secretion. In contrast, second phase insulin secretion requires the trafficking of the reserved granule pool to the plasma membrane. The initial trigger for insulin granule fusion with the plasma membrane is a rise in intracellular calcium and in the case of glucose stimulation results from increased production of ATP, closure of the ATP-sensitive potassium channel and cellular depolarization. In turn, this opens voltage-dependent calcium channels allowing increased influx of extracellular calcium. Calcium is thought to bind to members of the fusion regulatory proteins synaptogamin that functionally repressors the fusion inhibitory protein complexin. Both complexin and synaptogamin interact as well as several other regulatory proteins interact with the core fusion machinery composed of the Q- or t-SNARE proteins syntaxin 1 and SNAP25 in the plasmamembrane that assembles with the R- or v-SNARE protein VAMP2 in insulin granules. In this chapter we will review the current progress of insulin granule biogenesis, sorting, trafficking, exocytosis and signaling pathways that comprise the molecular basis of glucose-dependent insulin secretion. PMID:19251047

Hou, June Chunqiu; Min, Le; Pessin, Jeffrey E.

2015-01-01

192

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

PubMed Central

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

2013-01-01

193

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

PubMed Central

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

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

2013-01-01

194

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

PubMed Central

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

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

2011-01-01

195

N-acetyl cysteine improves affinity of beta-tricalcium phosphate granules for cultured osteoblast-like cells.  

PubMed

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

2012-07-01

196

Parallel Computational Subunits in Dentate Granule Cells Generate Multiple Place Fields  

PubMed Central

A fundamental question in understanding neuronal computations is how dendritic events influence the output of the neuron. Different forms of integration of neighbouring and distributed synaptic inputs, isolated dendritic spikes and local regulation of synaptic efficacy suggest that individual dendritic branches may function as independent computational subunits. In the present paper, we study how these local computations influence the output of the neuron. Using a simple cascade model, we demonstrate that triggering somatic firing by a relatively small dendritic branch requires the amplification of local events by dendritic spiking and synaptic plasticity. The moderately branching dendritic tree of granule cells seems optimal for this computation since larger dendritic trees favor local plasticity by isolating dendritic compartments, while reliable detection of individual dendritic spikes in the soma requires a low branch number. Finally, we demonstrate that these parallel dendritic computations could contribute to the generation of multiple independent place fields of hippocampal granule cells. PMID:19750211

Ujfalussy, Balázs; Kiss, Tamás; Érdi, Péter

2009-01-01

197

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. PMID:21299834

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

2011-01-01

198

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

PubMed

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. 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 ?-frequency bursts in the dentate gyrus and hilus, as well as increased synchronization of dentate neuron firing to these bursts. A similar increase in gamma burst amplitude was also found in animals in which adult neurogenesis was eliminated using the GFAP:TK pharmacogenetic ablation technique. These data suggest that young neurons may inhibit or destabilize recurrent network activity in the dentate and hilus. This unexpected result yields a new perspective on how a modest number of young adult-generated granule cells may modulate activity in the larger population of mature granule cells, rather than acting solely as independent encoding units. PMID:20882540

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

2012-01-01

199

Dynamic properties of sensory stimulation evoked responses in mouse cerebellar granule cell layer and molecular layer.  

PubMed

Sensory information coming from climbing fiber and mossy fiber-granule cell pathways, generates motor-related outputs according to internal rules of integration and computation in the cerebellar cortex. However, the dynamic properties of sensory information processing in mouse cerebellar cortex are less understood. Here, we studied the dynamic properties of sensory stimulation-evoked responses in the cerebellar granule cell layer (GCL) and molecular layer (ML) by electrophysiological recordings method. Our data showed that air-puff stimulation (5-10ms in duration) of the ipsilateral whisker pad evoked single-peak responses in the GCL and ML; whereas a duration of stimulation ?30ms in GCL and ?60ms in ML, evoked double-peak responses that corresponded with stimulation-on and -off responses via mossy fiber pathway. The highest frequency of stimulation train for evoking GCL responses was 33Hz. In contrast, the highest frequency of stimulation train for evoking ML responses was 4Hz. These results indicate that the cerebellar granule cells transfer the high-fidelity sensory information from mossy fibers, which is cut-off by molecular layer interneurons (MLIs). Our results suggest that the MLIs network acts as a low-pass filter during the processing of high-frequency sensory information. PMID:25434871

Bing, Yan-Hua; Zhang, Guang-Jian; Sun, Lei; Chu, Chun-Ping; Qiu, De-Lai

2015-01-12

200

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

PubMed Central

Pollination triggers not only embryo development but also the differentiation of the ovule integuments to form a specialized seed coat. The mucilage secretory cells of the Arabidopsis thaliana seed coat undergo a complex differentiation process in which cell growth is followed by the synthesis and secretion of pectinaceous mucilage. A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis. To identify additional genes acting in mucilage synthesis and secretion, a screen for enhancers of the mum4 phenotype was performed. Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61). Our results show that, in a mum4 background, mutations in MEN1, MEN4, and MEN5 lead to further reductions in mucilage compared to mum4 single mutants, suggesting that they are involved in mucilage synthesis or secretion. Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity. With the exception of men4, whose single mutant exhibits reduced mucilage, none of these genes have a single mutant phenotype, suggesting that they would not have been identified outside the compromised mum4 background. PMID:19401413

Arsovski, Andrej A.; Villota, Maria M.; Rowland, Owen; Subramaniam, Rajagopal; Western, Tamara L.

2009-01-01

201

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

PubMed Central

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

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

1999-01-01

202

Barhl1 regulates migration and survival of cerebellar granule cells by controlling expression of the neurotrophin-3 gene.  

PubMed

The neurons generated at the germinal rhombic lip undergo long distance migration along divergent pathways to settle in widely dispersed locations within the hindbrain, giving rise to cerebellar granule cells and precerebellar nuclei. Neurotrophin-3 (NT-3) signaling has been shown to be required for proper migration and survival of cerebellar granule cells. The molecular bases that govern NT-3 expression within the cerebellum, however, remain unknown at present. Here we report that, during early mouse neurogenesis, the Barhl1 homeobox gene is highly expressed by the rhombic lip and rhombic lip-derived migratory neurons. Its expression is later restricted to cerebellar granule cells and precerebellar neurons extending mossy fibers, two groups of neurons that synaptically connect in the adult cerebellar system. Loss of Barhl1 function causes cerebellar phenotypes with a striking similarity to those of NT-3 conditional null mice, which include attenuated cerebellar foliation as well as defective radial migration and increased apoptotic death of granule cells. Correlating with these defects, we find that NT-3 expression is dramatically downregulated in granule cells of the posterior lobe of Barhl1(-)/- cerebella. Moreover, in the precerebellar system of Barhl1(-/-) mice, all five nuclei that project mossy fibers fail to form correctly because of aberrant neuronal migration and elevated apoptosis. These results suggest that Barhl1 plays an essential role in the migration and survival of cerebellar granule cells and precerebellar neurons and functionally link Barhl1 to the NT-3 signaling pathway during cerebellar development. PMID:15044550

Li, Shengguo; Qiu, Feng; Xu, Anlong; Price, Sandy M; Xiang, Mengqing

2004-03-24

203

Perturbation of 1Integrin Function in Involuting Mammary Gland Results in Premature Dedifferentiation of Secretory Epithelial Cells  

Microsoft Academic Search

To study the mechanism of 1-integrin function in vivo, we have generated transgenic mouse expressing a dominant negative mutant of 1-integrin under the control of mouse mammary tumor virus (MMTV) promoter (MMTV-1-cyto). Mammary glands from MMTV-1-cyto trans- genic females present significant growth defects during pregnancy and lactation and impaired differentiation of secretory epithelial cells at the onset of lactation. We

Marisa M. Faraldo; Marie-Ange Deugnier; Sylvie Tlouzeau; Jean Paul Thiery; Marina A. Glukhova

2002-01-01

204

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

NASA Astrophysics Data System (ADS)

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

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

2012-03-01

205

Potentiation of Polarized Intestinal Caco-2 Cell Responsiveness to Probiotics Complexed with Secretory IgA*  

PubMed Central

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-?B and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria. PMID:20729211

Mathias, Amandine; Duc, Mélanie; Favre, Laurent; Benyacoub, Jalil; Blum, Stephanie; Corthésy, Blaise

2010-01-01

206

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

PubMed Central

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

Mbikay, M; Seidah, N G; Chrétien, M

2001-01-01

207

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

SciTech Connect

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

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

2009-12-04

208

The Secretory Pathway Calcium ATPase PMR-1/SPCA1 Has Essential Roles in Cell Migration during Caenorhabditis elegans Embryonic Development  

PubMed Central

Maintaining levels of calcium in the cytosol is important for many cellular events, including cell migration, where localized regions of high calcium are required to regulate cytoskeletal dynamics, contractility, and adhesion. Studies show inositol-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), which release calcium into the cytosol, are important regulators of cell migration. Similarly, proteins that return calcium to secretory stores are likely to be important for cell migration. The secretory protein calcium ATPase (SPCA) is a Golgi-localized protein that transports calcium from the cytosol into secretory stores. SPCA has established roles in protein processing, metal homeostasis, and inositol-trisphosphate signaling. Defects in the human SPCA1/ATP2C1 gene cause Hailey-Hailey disease (MIM# 169600), a genodermatosis characterized by cutaneous blisters and fissures as well as keratinocyte cell adhesion defects. We have determined that PMR-1, the Caenorhabditis elegans ortholog of SPCA1, plays an essential role in embryogenesis. Pmr-1 strains isolated from genetic screens show terminal phenotypes, such as ventral and anterior enclosure failures, body morphogenesis defects, and an unattached pharynx, which are caused by earlier defects during gastrulation. In Pmr-1 embryos, migration rates are significantly reduced for cells moving along the embryo surface, such as ventral neuroblasts, C-derived, and anterior-most blastomeres. Gene interaction experiments show changing the activity of itr-1/IP3R and unc-68/RyR modulates levels of embryonic lethality in Pmr-1 strains, indicating pmr-1 acts with these calcium channels to regulate cell migration. This analysis reveals novel genes involved in C. elegans cell migration, as well as a new role in cell migration for the highly conserved SPCA gene family. PMID:23696750

Praitis, Vida; Simske, Jeffrey; Kniss, Sarah; Mandt, Rebecca; Imlay, Leah; Feddersen, Charlotte; Miller, Michael B.; Mushi, Juliet; Liszewski, Walter; Weinstein, Rachel; Chakravorty, Adityarup; Ha, Dae-Gon; Schacht Farrell, Angela; Sullivan-Wilson, Alexander; Stock, Tyson

2013-01-01

209

PACAP MODULATION OF CALCIUM ION ACTIVITY IN DEVELOPING GRANULE CELLS OF THE MOUSE OLFACTORY BULB.  

PubMed

Ca(2+) activity in the CNS is critical for the establishment of developing neuronal circuitry prior to and during early sensory input. In developing olfactory bulb (OB), the neuromodulators that enhance network activity are largely unknown. Here we provide evidence that PACAP-specific PAC1 receptors expressed in P2-P5 mouse OB are functional and enhance network activity as measured by increases in calcium in genetically identified granule cells. We used confocal Ca(2+) imaging of OB slices from Dlx2-tdTomato mice to visualize GABAergic granule cells. To address whether the PACAP-induced Ca(2+) oscillations were direct or indirect effects of PAC1R activation, we used antagonists for the GABA receptors (GABARs) and/or glutamate receptors (GlutRs) in the presence and absence of PACAP. Combined block of GABARs and GlutRs yielded a 68% decrease in the numbers of PACAP responsive cells suggesting that 34% of OB neurons are directly activated by PACAP. Similarly, immunocytochemistry using anti-PAC1 antibody showed that 34% of OB neurons express PAC1R. Blocking either GlutRs or GABARs alone indirectly showed that PACAP stimulates release of both glutamate and GABA which activate GCs. The appearance of PACAP-induced Ca(2+) activity in immature GCs suggests a role for PACAP in GC maturation. To conclude, we find that PACAP has both direct and indirect effects on neonatal OB GABAergic cells and may enhance network activity by promoting glutamate and GABA release. Furthermore, the numbers of PACAP responsive granule cells significantly increased between postnatal day two and five suggesting PACAP-induced Ca(2+) activity contributes to neonatal OB development. PMID:25475351

Irwin, Mavis A; Greig, Ann; Tvrdik, Petr; Lucero, Mary T

2014-12-01

210

Measurement of GABAA receptor function in rat cultured cerebellar granule cells by the Cytosensor microphysiometer.  

PubMed

1. gamma-Aminobutyric acid (GABA), acting via the GABAA receptor, increased the extracellular acidification rate of rat primary cultured cerebellar granule cells, measured by the Cytosensor microphysiometer. 2. The optimal conditions for the measurement of GABAA receptor function in cerebellar granule cells by microphysiometry were: cells seeded at 9-12 x 10(5) cells/transwell cup and maintained in vitro for 8 days, GABA stimulation performed at 25 degrees C, with a stimulation time of 33 s. 3. GABA stimulated a concentration-dependent increase in the extracellular acidification rate with an EC50 of 2.0 +/- 0.2 microM (mean +/- s.e.mean, n = 7 experiments) and maximal increase (Emax) over basal response of 15.4 +/- 1.2%. 4. The sub-maximal GABA-stimulated increase in acidification rate could be potentiated by the 1,4-benzodiazepine, flunitrazepam (100 nM). The 10 nM GABA response showed the maximal benzodiazepine facilitation (GABA alone, 1.4 microV s-1, GABA + flunitrazepam, 3.8 microV s-1, mean increment over basal, n = 7). 5. The GABA-stimulated increase in acidification rate was inhibited by the GABAA antagonist, bicuculline (100 microM) (90% inhibition at 1 mM GABA). 6. The results of this study show that activation of GABAA receptors in rat cerebellar granule cells caused an increase in the extracellular acidification rate; an effect which was potentiated by benzodiazepines and inhibited by a GABAA receptor antagonist. This paper defines the conditions and confirms the feasibility of using microphysiometry to investigate GABAA receptor function in primary cultured CNS neurones. The microphysiometer provides a rapid and sensitive technique to investigate the regulation of the GABAA receptor in populations of neurones. PMID:9146889

Brown, M J; Wood, M D; Coldwell, M C; Bristow, D R

1997-05-01

211

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

PubMed Central

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

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

2014-01-01

212

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

NASA Astrophysics Data System (ADS)

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

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

2014-02-01

213

Reactive gliosis of immature Bergmann glia and microglial cell activation in response to cell death of granule cell precursors induced by methylazoxymethanol treatment in developing rat cerebellum  

Microsoft Academic Search

The morphology, organization and expression of proliferating cell nuclear antigen (PCNA) and the cytoskeletal proteins vimentin and GFAP in immature Bergmann glial cells were studied after a developmental injury induced by a single dose of the cytotoxic agent methylazoxymethanol (MAM) administered on postnatal day 5. This drug, which produces cell death of cerebellar granule cell precursors, did not induce apoptosis

M. Lafarga; M. A. Andres; E. Calle; M. T. Berciano

1998-01-01

214

Enriched Monolayer Precursor Cell Cultures from Micro-Dissected Adult Mouse Dentate Gyrus Yield Functional Granule Cell-Like Neurons  

PubMed Central

Background Stem cell cultures are key tools of basic and applied research in Regenerative Medicine. In the adult mammalian brain, lifelong neurogenesis originating from local precursor cells occurs in the neurogenic regions of the hippocampal dentate gyrus. Despite widespread interest in adult hippocampal neurogenesis and the use of mouse models to study it, no protocol existed for adult murine long-term precursor cell cultures with hippocampus-specific differentiation potential. Methodology/Principal Findings We describe a new strategy to obtain serum-free monolayer cultures of neural precursor cells from microdissected dentate gyrus of adult mice. Neurons generated from these adherent hippocampal precursor cell cultures expressed the characteristic markers like transcription factor Prox1 and showed the TTX-sensitive sodium currents of mature granule cells in vivo. Similar to granule cells in vivo, treatment with kainic acid or brain derived neurotrophic factor (BDNF) elicited the expression of GABAergic markers, further supporting the correspondence between the in vitro and in vivo phenotype. When plated as single cells (in individual wells) or at lowest density for two to three consecutive generations, a subset of the cells showed self-renewal and gave rise to cells with properties of neurons, astrocytes and oligodendrocytes. The precursor cell fate was sensitive to culture conditions with their phenotype highly influenced by factors within the media (sonic hedgehog, BMP, LIF) and externally applied growth factors (EGF, FGF2, BDNF, and NT3). Conclusions/Significance We report the conditions required to generate adult murine dentate gyrus precursor cell cultures and to analyze functional properties of precursor cells and their differentiated granule cell-like progeny in vitro. PMID:17460755

Babu, Harish; Cheung, Giselle; Kettenmann, Helmut; Palmer, Theo D.; Kempermann, Gerd

2007-01-01

215

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

PubMed Central

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

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

2014-01-01

216

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

PubMed

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

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

2013-01-01

217

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

SciTech Connect

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

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

2010-09-15

218

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

PubMed

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

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

2010-09-01

219

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

PubMed

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

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

2001-02-01

220

Secretory phospholipase A2 induces dendritic cell maturation Laure Perrin-Cocon1  

E-print Network

thus generate signals that influence immune responses. Here, group III secretory PLA2s were tested that transient increase in PLA2 activity generates signals that promote transition of innate to adaptive immunity of stress such as injury, physical trauma or infection and result in the release of inflammatory mediators

Paris-Sud XI, Université de

221

Prox1 Is Required for Granule Cell Maturation and Intermediate Progenitor Maintenance During Brain Neurogenesis  

PubMed Central

The dentate gyrus has an important role in learning and memory, and adult neurogenesis in the subgranular zone of the dentate gyrus may play a role in the acquisition of new memories. The homeobox gene Prox1 is expressed in the dentate gyrus during embryonic development and adult neurogenesis. Here we show that Prox1 is necessary for the maturation of granule cells in the dentate gyrus during development and for the maintenance of intermediate progenitors during adult neurogenesis. We also demonstrate that Prox1-expressing intermediate progenitors are required for adult neural stem cell self-maintenance in the subgranular zone; thus, we have identified a previously unknown non-cell autonomous regulatory feedback mechanism that controls adult neurogenesis in this region of the mammalian brain. Finally, we show that the ectopic expression of Prox1 induces premature differentiation of neural stem cells. PMID:20808958

Lavado, Alfonso; Lagutin, Oleg V.; Chow, Lionel M. L.; Baker, Suzanne J.; Oliver, Guillermo

2010-01-01

222

Comparative effects of two polychlorinated biphenyl congeners on calcium homeostasis in rat cerebellar granule cells.  

PubMed

Some polychlorinated biphenyls (PCBs) have been reported to alter locomotor activity and decrease brain dopamine function in laboratory animals. PCBs with ortho- and/or parachlorine substitutions and varying number of chlorinations are known to decrease cell dopamine content in vitro and have been detected in brains of animals exposed to PCBs, suggesting that the neurotoxicity could be mediated by ortho-substituted congeners. Dopamine or other neurotransmitter uptake and release phenomena are dependent on the maintenance of intracellular Ca2+ homeostasis, and perturbations in Ca2+ homeostasis could lead to altered cell function and/or death. We compared the effects of two PCB congeners on Ca2+ homeostasis in cerebellar granule cells: 2,2'-dichlorobiphenyl (DCBP), a putative neurotoxic congener, and 3,3',4,4',5-pentachlorobiphenyl (PCBP), a presumed nonneurotoxic congener. In cerebellar granule cells (6-8 days in vitro), DCBP was cytotoxic as indicated by a significant increase in LDH leakage at 200 microM after 2 hr of exposure and at 100 microM after 4 hr exposure. PCBP, on the other hand, did not affect LDH leakage even at 200 microM for up to 4 hr. Although both congeners increased cerebellar granule cell [Ca2+]i, DCPB was more effective in increasing [Ca2+]i to a greater extent than PCBP. The increase in [Ca2+]i produced by both congeners was not transient, but a steady rise was observed with time. To understand cellular Ca(2+)-buffering capacity, Ca2+ sequestration and Ca2+ extrusion were studied in mitochondria, microsomes, and synaptosomes, isolated from adult rat cerebellum. DCBP was a potent inhibitor of 45Ca2+ uptake by mitochondria (IC50 = 6.17 +/- 0.53 microM) and microsomes (IC50 = 7.61 +/- 0.35 microM). PCBP inhibited Ca2+ sequestration by mitochondria (68% of control) and microsomes (72% of control), but the effects were much less than those produced by equivalent concentrations of DCBP. Synaptosomal Ca(2+)-ATPase was inhibited by DCBP, but not by PCBP. These results indicate that at concentrations where cytotoxicity in cerebellar granule cells was not observed, DCBP increased intracellular [Ca2+]i, and at the same concentrations, Ca2+ sequestration by intracellular organelles and Ca(2+)-ATPase in synaptic plasma membrane were inhibited. Although PCBP increased [Ca2+]i in cerebellar granule cells to some extent, it was not potent in affecting Ca2+ sequestration or Ca2+ extrusion in adult cerebellar components. Hence, PCBP-induced slight increase of [Ca2+]i levels in the cells might have been associated with effective Ca2+ sequestration by intracellular organelles, as seen in cerebellar preparations. The results of this study support the hypothesis that the position of chlorine substitution on the biphenyl ring and/or number of chlorine substitutions may have significant implications for predicting potential effects of PCB congeners in the nervous system, and perturbations in Ca2+ homeostasis might play a significant role in the neuroactivity of PCBs. PMID:8236268

Kodavanti, P R; Shin, D S; Tilson, H A; Harry, G J

1993-11-01

223

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

PubMed Central

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

1975-01-01

224

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

PubMed Central

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

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

2014-01-01

225

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

NASA Astrophysics Data System (ADS)

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

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

226

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

PubMed Central

In most mammals the cochlear nuclear complex (CN) contains a distributed system of granule cells (GCS), whose parallel fiber axons innervate the dorsal cochlear nucleus (DCN). Like their counterpart in cerebellum, CN granules are innervated by mossy fibers of various origins. The GCS is complemented by unipolar brush (UBCs) and Golgi cells, and by stellate and cartwheel cells of the DCN. This cerebellum-like microcircuit modulates the activity of the DCN’s main projection neurons, the pyramidal, giant and tuberculoventral neurons, and is thought to improve auditory performance by integrating acoustic and proprioceptive information. In this paper, we focus on the UBCs, a chemically heterogeneous neuronal population, using antibodies to calretinin, mGluR1? epidermal growth factor substrate 8 (Eps8) and the transcription factor Tbr2. Eps8 and Tbr2 labeled most of the CN’s UBCs, if not the entire population, while calretinin and mGluR1? distinguished two largely separate subsets with overlapping distributions. By double labeling with antibodies to Tbr2 and the ?6 GABAA-receptor subunit, we found that UBCs populate all regions of the GCS and occur at remarkably high densities in the DCN and subpeduncular corner, but rarely in the lamina. Although GCS subregions likely share the same microcircuitry, their dissimilar UBC densities suggest they may be functionally distinct. UBCs and granules are also present in regions previously not included in the GCS, namely the rostrodorsal magnocellular portions of VCN, vestibular nerve root, trapezoid body, spinal tract and sensory and principal nuclei of the trigeminal nerve, and cerebellar peduncles. The UBC’s dendritic brush receives AMPA- and NMDA-mediated input from an individual mossy fiber, favoring singularity of input, and its axon most likely forms several mossy fiber-like endings that target numerous granule cells and other UBCs, as in the cerebellum. The UBCs therefore, may amplify afferent signals temporally and spatially, synchronizing pools of target neurons. PMID:18343594

Diño, Maria. R.; Mugnaini, Enrico

2009-01-01

227

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

SciTech Connect

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

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

2007-06-01

228

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

PubMed

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

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

2015-01-21

229

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

PubMed Central

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

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

2015-01-01

230

Platelet ?–granules: Basic biology and clinical correlates  

PubMed Central

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

Blair, Price; Flaumenhaft, Robert

2009-01-01

231

Ribozyme-Mediated Inhibition of Caspase3 Protects Cerebellar Granule Cells from Apoptosis Induced by Serum-Potassium Deprivation  

Microsoft Academic Search

Apoptosis is an important mechanism of physiological and pathological cell death. It is regulated by several gene products, including caspases and the bcl-2-like proteins, whose roles have been demonstrated in numerous systems. One of these is a model of cerebellar granule cells (CGCs) in which apoptosis is induced by acute removal of serum and depolarizing concen- trations of potassium. Previous

Basil A. Eldadah; Renee F. Ren; Alan I. Faden

2000-01-01

232

Vesicular uptake of eosinophil peroxidase by guinea pig basophils and by cloned mouse mast cells and granule-containing lymphoid cells.  

PubMed Central

Guinea pig basophils, cloned mouse mast cells, and cloned mouse granule-containing lymphoid cells were found to utilize a vesicular transport system to internalize eosinophil peroxidase (EPO) added in vitro. Kinetic analysis indicated that EPO internalization involved the binding of EPO to the plasma membrane, the formation of complex surface invaginations, and the movement of EPO-laden vesicles, tubules, and vacuoles toward the center of the cells. EPO became associated with multivesicular bodies in granule-containing lymphoid cells and mast cells, with immature granules in mast cells, and with mature granules in basophils. In other cells, the endogenous production of granule peroxidases (neutrophils and eosinophils) or the prior uptake of exogenous peroxidatic substances (some basophils) precluded cytochemical analysis of granules for EPO. Vesicular transport of EPO provides a possible explanation for the variable detection of peroxidase activity in mast cells or basophils. It also provides a mechanism for sequestration of this potentially toxic material or for its storage for possible future use. Images Figure 1 p430-a Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:3976846

Dvorak, A. M.; Klebanoff, S. J.; Henderson, W. R.; Monahan, R. A.; Pyne, K.; Galli, S. J.

1985-01-01

233

Methods to evaluate Zinc transport into and out of the secretory and endosomal-lysosomal compartments in DT40 cells.  

PubMed

Zinc plays crucial roles in diverse biological processes. Recently, in addition to zinc mobilization into and out of the cell, zinc mobilization into and out of intracellular organelles, including the secretory and endosomal-lysosomal compartments, has received growing interest. In vertebrate cells, the Zrt/Irt-like proteins (ZIPs) and Zn transporters (ZnTs) are the two major families of zinc transport proteins involved in zinc mobilization across cellular membranes. Importantly, nearly half of them are localized to subcellular compartments. Thus, to elucidate the numerous zinc-related cellular events, understanding those ZIP and ZnT functions is critical. This chapter describes advanced methods used in our laboratory to examine zinc mobilization by them. Specifically, genetic and molecular approaches using chicken DT40 cells deficient in multiple ZIPs and ZnTs are described. Moreover, procedures to evaluate zinc-related phenotypes caused by the impairment of zinc mobilization into and out of the secretory and endosomal-lysosomal compartments are also described. These methods should be useful in characterizing the roles of zinc in diverse cellular events including endosomal signaling. PMID:24359949

Kambe, Taiho

2014-01-01

234

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

PubMed Central

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

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

2013-01-01

235

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

PubMed

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

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

2013-01-01

236

Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells.  

PubMed

The binding and signaling properties of neuronal NTS2 neurotensin (NT) receptors were examined in cultured rat cerebellar granule cells. As shown by reverse transcription-PCR, receptor autoradiography, and confocal microscopic localization of fluorescent NT, these cells selectively express the NTS2 receptor subtype. Accordingly, a single apparent class of (125)I-NT-binding sites, with an affinity of 3.1 nm, was detected in cerebellar granule cell cultures. This binding was competed for with high affinity (IC(50) = 5.7 nm) by the NTS2 ligand levocabastine and with low affinity (IC(50) = 203 nm) by the NTS1 antagonist SR48692. Hypertonic acid stripping of surface-bound ligand and hyperosmolar sucrose treatment revealed that 64% of specifically bound (125)I-NT was internalized at equilibrium via a clathrin-dependent pathway. In cells loaded with the Ca(2+)-sensitive fluorescent dye Fluo4, SR48692, but neither NT nor levocabastine, triggered a marked increase in cytosolic [Ca(2+)](i). By contrast, both NT and levocabastine, but not SR48692, induced a sustained (>60 min) activation of the mitogen-activated protein kinases, p42/p44, indicating functional coupling of NTS2 receptors. Complementary experiments carried out on synaptosomes from adult rat cerebellum demonstrated the presence of presynaptic NTS2 receptors. However, in contrast to perikaryal NTS2 sites, these presynaptic receptors did not internalize in response to NT stimulation. Taken together, the present results demonstrate that NTS2 receptors are present both presynaptically and postsynaptically in central neurons and that NT and levocabastine act as agonists on these receptors. PMID:12084713

Sarret, Philippe; Gendron, Louis; Kilian, Peter; Nguyen, Ha Minh Ky; Gallo-Payet, Nicole; Payet, Marcel-Daniel; Beaudet, Alain

2002-09-27

237

Delineation of glutamate pathways and secretory responses in pancreatic islets with ?-cell–specific abrogation of the glutamate dehydrogenase  

PubMed Central

In pancreatic ?-cells, glutamate dehydrogenase (GDH) modulates insulin secretion, although its function regarding specific secretagogues is unclear. This study investigated the role of GDH using a ?-cell–specific GDH knockout mouse model, called ?Glud1?/?. The absence of GDH in islets isolated from ?Glud1–/– mice resulted in abrogation of insulin release evoked by glutamine combined with 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid or l-leucine. Reintroduction of GDH in ?Glud1–/– islets fully restored the secretory response. Regarding glucose stimulation, insulin secretion in islets isolated from ?Glud1–/– mice exhibited half of the response measured in control islets. The amplifying pathway, tested at stimulatory glucose concentrations in the presence of KCl and diazoxide, was markedly inhibited in ?Glud1–/– islets. On glucose stimulation, net synthesis of glutamate from ?-ketoglutarate was impaired in GDH-deficient islets. Accordingly, glucose-induced elevation of glutamate levels observed in control islets was absent in ?Glud1–/– islets. Parallel biochemical pathways, namely alanine and aspartate aminotransferases, could not compensate for the lack of GDH. However, the secretory response to glucose was fully restored by the provision of cellular glutamate when ?Glud1–/– islets were exposed to dimethyl glutamate. This shows that permissive levels of glutamate are required for the full development of glucose-stimulated insulin secretion and that GDH plays an indispensable role in this process. PMID:22875990

Vetterli, Laurène; Carobbio, Stefania; Pournourmohammadi, Shirin; Martin-del-Rio, Rafael; Skytt, Dorte M.; Waagepetersen, Helle S.; Tamarit-Rodriguez, Jorge; Maechler, Pierre

2012-01-01

238

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

ERIC Educational Resources Information Center

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

Hood-DeGrenier, Jennifer K.

2008-01-01

239

Mast Cell Proteoglycans  

PubMed Central

Mast cells are versatile effector cells of the immune system, contributing to both innate and adaptive immunity toward pathogens but also having profound detrimental activities in the context of inflammatory disease. A hallmark morphological feature of mast cells is their large content of cytoplasmic secretory granules, filled with numerous secretory compounds, including highly negatively charged heparin or chondroitin sulfate proteoglycans of serglycin type. These anionic proteoglycans provide the basis for the strong metachromatic staining properties of mast cells seen when applying various cationic dyes. Functionally, the mast cell proteoglycans have been shown to have an essential role in promoting the storage of other granule-contained compounds, including bioactive monoamines and different mast cell-specific proteases. Moreover, granule proteoglycans have been shown to regulate the enzymatic activities of mast cell proteases and to promote apoptosis. Here, the current knowledge of mast cell proteoglycans is reviewed. PMID:22899859

Rönnberg, Elin; Melo, Fabio R.

2012-01-01

240

Stress granules and cell signaling: more than just a passing phase?  

PubMed Central

Stress granules (SGs) contain translationally-stalled mRNAs, associated preinitiation factors and specific RNA-binding proteins. In addition, many signaling proteins are recruited to SGs and/or influence their assembly, which is transient, lasting only until the cells adapt to stress or die. Beyond their role as mRNA triage centers, we posit that SGs constitute RNA-centric signaling hubs analogous to classical multiprotein signaling domains such as transmembrane receptor complexes. As signaling centers, SG formation communicates a “state of emergency”, and their transient existence alters multiple signaling pathways by intercepting and sequestering signaling components. SG assembly and downstream signaling functions may require a cytosolic phase transition facilitated by intrinsically disordered, aggregation-prone protein regions shared by RNA-binding and signaling proteins. PMID:24029419

Kedersha, Nancy; Ivanov, Pavel; Anderson, Paul

2013-01-01

241

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

SciTech Connect

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

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

2008-10-24

242

Observer-independent quantification of insulin granule exocytosis and pre-exocytotic mobility by TIRF microscopy.  

PubMed

Total internal reflection fluorescence microscopy of fluorescently labeled secretory granules permits monitoring of exocytosis and the preceding granule behavior in one experiment. While observer-dependent evaluation may be sufficient to quantify exocytosis, most of the other information contained in the video files cannot be accessed this way. The present program performs observer-independent detection of exocytosis and tracking of the entire submembrane population of insulin granules. A precondition is the exact localization of the peak of the granule fluorescence. Tracking is based on the peak base radius, peak intensity, and the precrossing itineraries. Robustness of the tracking was shown by simulated tracks of original granule patterns. Mobility in the X-Y dimension is described by the caging diameter which in contrast to the widely used mean square displacement has an inherent time resolution. Observer-independent detection of exocytosis in MIN6 cells labeled with insulin-EGFP is based on the maximal decrease in fluorescence intensity and position of the centroid of the dissipating cloud of released material. Combining the quantification of KCl-induced insulin exocytosis with the analysis of prefusion mobility showed that during the last 3 s pre-exocytotic granules had a smaller caging diameter than control granules and that it increased significantly immediately before fusion. PMID:24230985

Matz, Magnus; Schumacher, Kirstin; Hatlapatka, Kathrin; Lorenz, Dirk; Baumann, Knut; Rustenbeck, Ingo

2014-02-01

243

Endocrine cells in the gastrointestinal tract of a stomachless teleostean fish.  

PubMed

Endocrine cells in the gastrointestinal tract of the stomachless teleostean fish, Notemigonus crysoleucas, were studied using electron microscopy. Located between the absorptive cells of the intestinal epithelium, the enteroendocrine cells were very few in number. While some of the cells had their secretory granules located basally and a long narrow part extending toward the lumen, many appeared rounder and the plane of the section did not indicate that they extended to the lumen. Based upon size and shape of secretory granules, there appear to be several different types of cells: those with the smallest granules distributed throughout the intestine, those with intermediate sized granules more commonly found in the middle and distal segments and a few with large granules seen most often in the distal intestine. PMID:3223592

Reifel, C W

1988-01-01

244

Tonic inhibition sets the state of excitability in olfactory bulb granule cells  

PubMed Central

GABAergic granule cells (GCs) regulate, via mitral cells, the final output from the olfactory bulb to piriform cortex and are central for the speed and accuracy of odour discrimination. However, little is known about the local circuits in which GCs are embedded and how GCs respond during functional network activity. We recorded inhibitory and excitatory currents evoked during a single sniff-like odour presentation in GCs in vivo. We found that synaptic excitation was extensively activated across cells, whereas phasic inhibition was rare. Furthermore, our analysis indicates that GCs are innervated by a persistent firing of deep short axon cells that mediated the inhibitory evoked responses. Blockade of GABAergic synaptic input onto GCs revealed a tonic inhibitory current mediated by furosemide-sensitive GABAA receptors. The average current associated with this tonic GABAergic conductance was 3-fold larger than that of phasic inhibitory postsynaptic currents. We show that the pharmacological blockage of tonic inhibition markedly increased the occurrence of supra-threshold responses during an odour-stimulated sniff. Our findings suggest that GCs mediate recurrent or lateral inhibition, depending on the ambient level of extracellular GABA. PMID:23318869

Labarrera, Christina; London, Michael; Angelo, Kamilla

2013-01-01

245

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

PubMed Central

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

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

2014-01-01

246

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation.  

PubMed

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1? dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3? may work in concert with CK1? to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3? and GSK3?, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3? depletion reduced Wee1 turnover. Given Wee1's central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3? mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3? or Wee1 are dysregulated. PMID:25616418

Penas, Clara; Mishra, Jitendra K; Wood, Spencer D; Schürer, Stephan C; Roush, William R; Ayad, Nagi G

2015-02-01

247

Cystic fibrosis transmembrane conductance regulator is expressed in mucin granules from Calu-3 and primary human airway epithelial cells.  

PubMed

Cystic fibrosis (CF) is caused by mutations in the tightly regulated anion channel cystic fibrosis transmembrane conductance regulator (CFTR), yet much of the pathology in this disease results from mucus obstruction of the small airways and other organs. Mucus stasis has been attributed to the abnormal luminal environment of CF airways, which results from dehydration of the mucus gel or low bicarbonate concentration. We show here that CFTR and MUC5AC are present in single mucin-containing granules isolated from a human airway epithelial cell line and from highly differentiated airway primary cell cultures. CFTR was not detected in MUC5AC granules from CFTR knockdown cells or CF primary cells. The results suggest a direct link between CFTR and the mucus defect. PMID:23742042

LeSimple, Pierre; Goepp, Julie; Palmer, Melissa L; Fahrenkrug, Scott C; O'Grady, Scott M; Ferraro, Pasquale; Robert, Renaud; Hanrahan, John W

2013-10-01

248

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

PubMed Central

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity. PMID:24088571

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

2013-01-01

249

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

PubMed

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity. PMID:24088571

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

2013-12-01

250

Adult-born granule cells mature through two functionally distinct states  

PubMed Central

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

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

2014-01-01

251

Silencing the majority of cerebellar granule cells uncovers their essential role in motor learning and consolidation.  

PubMed

Cerebellar granule cells (GCs) account for more than half of all neurons in the CNS of vertebrates. Theoretical work has suggested that the abundance of GCs is advantageous for sparse coding during memory formation. Here, we minimized the output of the majority of GCs by selectively eliminating their CaV2.1 (P/Q-type) Ca(2+) channels, which mediate the bulk of their neurotransmitter release. This resulted in reduced GC output to Purkinje cells (PCs) and stellate cells (SCs) as well as in impaired long-term plasticity at GC-PC synapses. As a consequence modulation amplitude and regularity of simple spike (SS) output were affected. Surprisingly, the overall motor performance was intact, whereas demanding motor learning and memory consolidation tasks were compromised. Our findings indicate that a minority of functionally intact GCs is sufficient for the maintenance of basic motor performance, whereas acquisition and stabilization of sophisticated memories require higher numbers of normal GCs controlling PC firing. PMID:23583179

Galliano, Elisa; Gao, Zhenyu; Schonewille, Martijn; Todorov, Boyan; Simons, Esther; Pop, Andreea S; D'Angelo, Egidio; van den Maagdenberg, Arn M J M; Hoebeek, Freek E; De Zeeuw, Chris I

2013-04-25

252

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

PubMed

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

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

2014-10-01

253

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

PubMed Central

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

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

2013-01-01

254

Altered accessory cell function of alveolar macrophages: a possible mechanism for induction of Th2 secretory profile in idiopathic pulmonary fibrosis  

Microsoft Academic Search

Altered accessory cell function of alveolar macrophages: a possible mechanism for induction of Th2 secretory profile in idiopathic pulmonary fibrosis. H. Furuie, H. Yamasaki, M. Suga, M. Ando. ?ERS Journals Ltd 1997. ABSTRACT: Alveolar macrophages (AMs) are considered to play a central role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Recent studies have re- vealed a predominance of the

H. Furuie; H. Yamasaki; M. Suga; M. Ando

255

Formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors  

Microsoft Academic Search

Recent evidence suggests that secretory vesi- cle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-binding pro- teins, heterotrimeric G proteins, inositol phospholipid metabolism, and protein serine\\/threonine phosphoryla- tion. At the cell surface, protein phosphorylation and dephosphorylation on tyrosine residues can rapidly modulate cytosolic signaling pathways in response to extracellular stimuli and have been implicated in

Cary D. Austin; Dennis Shields

1996-01-01

256

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

PubMed Central

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

Taniuchi, Keisuke; Furihata, Mutsuo; Saibara, Toshiji

2014-01-01

257

Lack of MHC class I surface expression on neoplastic cells and poor activation of the secretory pathway of cytotoxic cells in oral squamous cell carcinomas  

PubMed Central

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the secretory pathway of perforin/granzymes to kill their target cells. In contrast to NK cells, CTL responses are MHC class I restricted. In this study we analysed the relative activation of CTL and NK cells in relation with MHC class I expression on oral squamous cell carcinomas (OSCCs). MHC class I expression was investigated in 47 OSCCs by immunohistochemistry using HCA2, HC10 and ?2-m antibodies. The presence of CTLs, NK cells, and its activation, was investigated in 21 of these OSCCs using respectively, CD8, CD57 and GrB7 antibodies. The Q-Prodit measuring system was used for quantification of cytotoxic cells. All OSCCs showed weak or absent staining of ?2-m on the cell surface. The absence of ?2-m was significantly associated with absent expression of MHC class I heavy chain as detected by HC10 antibody (P = 0.004). In tumour infiltrates CTLs always outnumbered NK cells, as reflected by the ratio CD57/CD8 being always inferior to one (mean: 0.19; SD: 0.15). The proportion of activated cytotoxic cells as detected by granzyme B expression was generally low (mean: 8.6%; SD 8.9). A clear correlation between MHC class I expression and the relative proportion of NK cells/CTLs was not found. This study shows that the majority of OSCCs show weak or absent expression of MHC class I molecules on the cell surface, possibly due to alterations in the normal ?2-m pathway. The low proportion of granzyme B-positive CTLs/NK cells indicates that the secretory pathway of cytotoxicity is poor in these patients. The lack of correlation between MHC class I expression and CTL/NK cell activation as detected by granzyme B expression suggests that, next to poor antigen presentation, also local factors seem to determine the final outcome of the cytotoxic immune response. © 1999 Cancer Research Campaign PMID:10555762

Cruz, I; Meijer, C J L M; Walboomers, J M M; Snijders, P J F; Waal, I Van der

1999-01-01

258

Chromospheric granulation  

Microsoft Academic Search

Photographs obtained in the core of H-alpha, free of parasitic continuum radiation, reveal a granular structure in supergranule centers wherever not obscured by mottles or fibrils. Granulation is seen well in the wings out to plus or minus 0.5 A from line center, the contrasts being largely reversed in opposite wings. The granule diameters (from boundary to boundary) are observed

R. G. Giovanelli

1974-01-01

259

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

PubMed Central

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

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

2014-01-01

260

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

PubMed Central

Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain development is unknown. In this study, we show that mouse Sema5A negatively regulates synaptogenesis in early, developmentally born, hippocampal dentate granule cells (GCs). Sema5A is strongly expressed by GCs and regulates dendritic spine density in a cell-autonomous manner. In the adult mouse brain, newly born Sema5A?/? GCs show an increase in dendritic spine density and increased AMPA-type synaptic responses. Sema5A signals through PlexinA2 co-expressed by GCs, and the PlexinA2-RasGAP activity is necessary to suppress spinogenesis. Like Sema5A?/? mutants, PlexinA2?/? mice show an increase in GC glutamatergic synapses, and we show that Sema5A and PlexinA2 genetically interact with respect to GC spine phenotypes. Sema5A?/? mice display deficits in social interaction, a hallmark of autism-spectrum-disorders. These experiments identify novel intra-dendritic Sema5A/PlexinA2 interactions that inhibit excitatory synapse formation in developmentally born and adult-born GCs, and they provide support for SEMA5A contributions to autism-spectrum-disorders. DOI: http://dx.doi.org/10.7554/eLife.04390.001 PMID:25313870

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

2014-01-01

261

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

PubMed

Age-related declines in spatial memory have been linked to abnormal functional properties and connectivity of newborn granule cells. However, the relationship between adult neurogenesis, aging, and cognitive performance seems more complex than previously anticipated, likely due to the difficulty of disentangling alterations related to training as such and those associated with cognitive performance. Here, we investigated how different aspects of adult neurogenesis might be related to training, age and cognitive performance amongst aged subjects by comparing behaviourally naïve and tested rats of 3, 6, 24mo of age. We separated aged rats into learning-impaired and -unimpaired groups based on their performance in the Morris water maze to investigate neurogenesis-related morphological and neurochemical changes. We report an age-related decline in cell proliferation and maturation independent of cognitive performance and testing. We confirm an age-related altered differentiation of newborn neurons which was particularly prominent in learning-impaired rats. This was associated with an abnormally prolonged expression of the early progenitor marker Nestin, potentially also affecting maturation, survival/integration of newborn neurons into existing neuronal networks, which might underlie the individual differences in cognitive performance during aging. PMID:19100662

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

2010-11-01

262

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

PubMed

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

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

2015-02-01

263

Outgoing synapses of small granule-containing cells in the rat superior cervical ganglion after post-ganglionic axotomy.  

PubMed Central

Small granule-containing cells are intrinsic and interneurone-like in the rat superior cervical ganglion, being innervated by preganglionic axons and giving outgoing synapses of asymmetrical type to the principal neurones. A quantitative ultrastructural investigation has been made of the effect on these outgoing synapses of axotomy of the major post-ganglionic nerve trunks 18.5 h-390 days previously. Cutting, or cutting and ligating, the internal and external carotid nerves 2-3 mm from the ganglion in rats aged 1.5-5.5 months resulted in a statistically significant mean loss of up to 85% of the asymmetrical synapses given by small granule-containing cells in the injured ganglion. The reduction of synapses was maximal 5-9 days post-operatively, and thereafter the incidence of synapses showed significant signs of progressive recovery. The time course and magnitude of the change in incidence of these synapses resembled those found earlier (Matthews & Nelson, 1975) for the loss of preganglionic synapses to principal neurones in the same ganglia, and after an identical post-ganglionic lesion. Control experiments showed that there was no loss of outgoing synapses from the small granule-containing cells as a result of surgical stress or of simple ageing. Older rats (5.5 and 13 months) showed a small but significant increase in the incidence of these synapses. Unilateral post-ganglionic axotomy produced the same reaction in the injured ganglia as did bilateral lesions. Uninjured ganglia contralateral to unilateral axotomies, however, also showed some deficit of outgoing synapses from small granule-containing cells, but this was slight, amounting to 9.9% over-all in comparison to normal values in young rats, and this difference did not reach statistical significance. Cutting the cervical sympathetic trunk to produce preganglionic denervation 2 days before surgical removal of ganglia for analysis did not alter the incidence of outgoing synapses of the small granule-containing cells, either in ganglia post-ganglionically axotomized 5-128 days earlier or in contralateral ganglia, indicating that at no stage was any significant proportion of these synapses given to preganglionic axons. These findings suggest that most of the outgoing synapses from the intra-ganglionic small granule-containing cells are directed to principal neurones whose axons leave with the injured branches, the internal and external carotid nerves.(ABSTRACT TRUNCATED AT 400 WORDS) Images Plate 1 Plate 2 Plate 3 PMID:3746684

Case, C P; Matthews, M R

1986-01-01

264

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

PubMed Central

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

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

2014-01-01

265

Nanocrystalline spherical hydroxyapatite granules for bone repair: in vitro evaluation with osteoblast-like cells and osteoclasts.  

PubMed

Conventionally sintered hydroxyapatite-based materials for bone repair show poor resorbability due to the loss of nanocrystallinity. The present study describes a method to establish nanocrystalline hydroxyapatite granules. The material was prepared by ionotropic gelation of an alginate sol containing hydroxyapatite (HA) powder. Subsequent thermal elimination of alginate at 650 °C yielded non-sintered, but unexpectedly stable hydroxyapatite granules. By adding stearic acid as an organic filler to the alginate/HA suspension, the granules exhibited macropores after thermal treatment. A third type of material was achieved by additional coating of the granules with silica particles. Microstructure and specific surface area of the different materials were characterized in comparison to the already established granular calcium phosphate material Cerasorb M(®). Cytocompatibility and potential for bone regeneration of the materials was evaluated by in vitro examinations with osteosarcoma cells and osteoclasts. Osteoblast-like SaOS-2 cells proliferated on all examined materials and showed the typical increase of alkaline phosphatase (ALP) activity during cultivation. Expression of bone-related genes coding for ALP, osteonectin, osteopontin, osteocalcin and bone sialoprotein II on the materials was proven by RT-PCR. Human monocytes were seeded onto the different granules and osteoclastogenesis was examined by activity measurement of tartrate-specific acid phosphatase (TRAP). Gene expression analysis after 23 days of cultivation revealed an increased expression of osteoclast-related genes TRAP, vitronectin receptor and cathepsin K, which was on the same level for all examined materials. These results indicate, that the nanocrystalline granular materials are of clinical interest, especially for bone regeneration. PMID:23625348

Bernhardt, A; Dittrich, R; Lode, A; Despang, F; Gelinsky, M

2013-07-01

266

Effect of macrophage secretory products on elaboration of virulence factors by planktonic and biofilm cells of Pseudomonas aeruginosa  

Microsoft Academic Search

Macrophages, which constitute the first line of defense, pour their secretions in the mileu following stimulation with pathogens. These secretory products, referred to as macrophage secretory products (MSPs), can influence ultimate outcome of an infection. In the present investigation, it was observed that different strains of Pseudomonas aeruginosa vary in their ability to stimulate macrophages leading to variability in generation

Rahul Mittal; Saroj Sharma; Sanjay Chhibber; Kusum Harjai

2006-01-01

267

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

PubMed

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

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

2014-11-19

268

Digoxin net secretory transport in bronchial epithelial cell layers is not exclusively mediated by P-glycoprotein/MDR1.  

PubMed

The impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe (3)H-digoxin in air-liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin-Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. (3)H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER=11.4 or 3.0 were measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on (3)H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in (3)H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs. PMID:23816640

Hutter, Victoria; Chau, David Y S; Hilgendorf, Constanze; Brown, Alan; Cooper, Anne; Zann, Vanessa; Pritchard, David I; Bosquillon, Cynthia

2014-01-01

269

Secretory type of recombinant thioredoxin h induces ER stress in endosperm cells of transgenic rice.  

PubMed

Thioredoxin h (TRX h) functions as a reducing protein and is present in all organisms. As a new approach for inducing the endoplasmic reticulum (ER) stress, TRX h (OsTRX23) was expressed as a secretory protein using the endosperm-specific glutelin GluB-1 promoter and a signal peptide. In transgenic rice seeds, the majority of the recombinant TRX h accumulated in the ER but some was also localized to the protein body IIs (PB-IIs). The rice grain quality was dependent on the TRX h accumulation level. Increased TRX h expression resulted in aberrant phenotypes, such as chalky and shriveled features, lower seed weight and lower seed protein content. Furthermore, the accumulation of some seed storage proteins (SSPs) was significantly suppressed and the morphology of the protein bodies (PB-Is and PB-IIs) changed according to the level of TRX h. SSPs, such as 13kDa prolamin and GluA, were specifically modified via the reducing action of TRX h. These changes led to the activation of the ER stress response, which was accompanied by the expression of several chaperone proteins. Specifically, the ER stress markers BiP4 and BiP5 were significantly up-regulated by an increase in the level of TRX h. These results suggest that changes in the conformation of certain SSPs via the action of recombinant TRX h lead to an induced ER stress response in transgenic rice seeds. PMID:23043988

Wakasa, Yuhya; Yasuda, Hiroshi; Takaiwa, Fumio

2013-01-15

270

The lysosomal membrane glycoproteins Lamp-1 and Lamp-2 are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils.  

PubMed Central

The subcellular localization of two members of a highly glycosylated protein group present in lysosomal membranes in most cells, the lysosome-associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2), was examined in human neutrophil granulocytes. Antibodies that were raised against purified Lamp-1 adn Lamp-2 gave a distinct granular staining of the cytoplasm upon immunostaining of neutrophils. Subcellular fractionation was used to separate the azurophil and specific granules from a light-membrane fraction containing plasma membranes and secretory vesicles, and Western blotting was used to determine the presence of the Lamps in these fractions. The results show that Lamp-1 and Lamp-2 are present in the specific-granule-enriched fraction and in the light-membrane fraction, but not in the azurophil granules. Separation of secretory vesicles from plasma membranes disclosed that the light-membrane Lamps were present primarily in the secretory-vesicle-enriched fraction. During phagocytosis both Lamp-1 and Lamp-2 became markedly concentrated around the ingested particle and they both appear on the cell surface when the secretory organelles are mobilized. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 8 PMID:7487911

Dahlgren, C; Carlsson, S R; Karlsson, A; Lundqvist, H; Sjölin, C

1995-01-01

271

Metabotropic Glutamate Receptors in the Main Olfactory Bulb Drive Granule Cell-Mediated Inhibition  

PubMed Central

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

Heinbockel, Thomas; Laaris, Nora; Ennis, Matthew

2009-01-01

272

Proteomic identification of potential Clonorchis sinensis excretory/secretory products capable of binding and activating human hepatic stellate cells.  

PubMed

Epidemiological and experimental evidence demonstrated that Clonorchis sinensis is an important risk factor of hepatic fibrosis and cholangiocarcinoma. C. sinensis excretory/secretory products (CsESPs) are protein complex including proteases, antioxidant enzymes, and metabolic enzymes, which may contribute to pathogenesis of liver fluke-associated hepatobiliary diseases. However, potential CsESP candidates involved into hepatic fibrosis and cholangiocarcinoma still remain to be elucidated. In the present study, we performed proteomic identification of CsESP candidates capable of binding and activating human hepatic stellate cell line LX-2. Immunofluorescence analysis confirmed the interaction of CsESPs with LX-2 cell membrane. LX-2 cells could be stimulated by CsESPs from 24 h post incubation (p?cell proliferation in methyl thiazolyl tetrazolium (MTT) assay which could also be demonstrated by flow cytometry analysis (p?cells treated with CsESPs was significantly higher than that in control cells measured by molecular beacon and semiquantitative reverse transcription (RT)-PCR approaches (p?cells were subjected to two-dimensional gel electrophoresis (2-DE) analysis and matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Nine proteins with abundance change above threefold were Rho GTPase-activating protein, mitochondrial cytochrome c oxidase subunit Va, ?-enolase, phospholipase C, interleukin-15, insect-derived growth factor, cytochrome c oxidase subunit VI, DNAH1 protein, and kinesin light chain. Taken together, we identified potential CsESP candidates capable of binding and activating human hepatic stellate cells, providing more direct evidences that are previously unknown to accelerate strategies for C. sinensis prevention. PMID:24894083

Wang, Xiaoyun; Hu, Fengyu; Hu, Xuchu; Chen, Wenjun; Huang, Yan; Yu, Xinbing

2014-08-01

273

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

PubMed

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

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

2014-01-01

274

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

PubMed Central

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

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

2014-01-01

275

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

PubMed

Epidemiology studies have clearly documented that the central nervous system is highly susceptible to methylmercury toxicity, and exposure to this neurotoxicant in humans primarily results from consumption of contaminated fish. While the effects of methylmercury exposure have been studied in great detail, comparatively little is known about the effects of moderate to low dose methylmercury toxicity in the aging central nervous system. We examined the toxic effects of a moderate dose of methylmercury on the aging mouse cerebellum. Male and female C57BL/6 mice at 16-20?months of age were exposed to methylmercury by feeding a total dose of 5.0?mg?kg(-1) body weight and assessed using four behavioral tests. Methylmercury-treated aged mice performed significantly worse in open field, footprint analysis and the vertical pole test compared with age-matched control mice. Isolated cerebellar granule cells from methylmercury-treated aged mice exhibited higher levels of reactive oxygen species and reduced mitochondrial membrane potentials, but no differences in basal intracellular calcium ion levels compared with age-matched control mice. When aged mice were exposed to a moderate dose of methylmercury, they exhibited a similar degree of impairment when compared with young adult mice exposed to the same moderate dose of methylmercury, as reported in earlier studies from this laboratory. Thus, at least in mice, exposure of the aged brain to moderate concentrations methylmercury does not pose greater risk compared with the young adult brain exposed to similar concentrations of methylmercury. PMID:22886740

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

2013-09-01

276

Input integration around the dendritic branches in hippocampal dentate granule cells.  

PubMed

Recent studies have shown that the dendrites of several neurons are not simple translators but are crucial facilitators of excitatory postsynaptic potential (EPSP) propagation and summation of synaptic inputs to compensate for inherent voltage attenuation. Granule cells (GCs)are located at the gateway for valuable information arriving at the hippocampus from the entorhinal cortex. However, the underlying mechanisms of information integration along the dendrites of GCs in the hippocampus are still unclear. In this study, we investigated the input integration around dendritic branches of GCs in the rat hippocampus. We applied differential spatiotemporal stimulations to the dendrites using a high-speed glutamate-uncaging laser. Our results showed that when two sites close to and equidistant from a branching point were simultaneously stimulated, a nonlinear summation of EPSPs was observed at the soma. In addition, nonlinear summation (facilitation) depended on the stimulus location and was significantly blocked by the application of a voltage-dependent Ca(2+) channel antagonist. These findings suggest that the nonlinear summation of EPSPs around the dendritic branches of hippocampal GCs is a result of voltage-dependent Ca(2+) channel activation and may play a crucial role in the integration of input information. PMID:25009669

Kamijo, Tadanobu Chuyo; Hayakawa, Hirofumi; Fukushima, Yasuhiro; Kubota, Yoshiyuki; Isomura, Yoshikazu; Tsukada, Minoru; Aihara, Takeshi

2014-08-01

277

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

PubMed Central

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

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

2013-01-01

278

Exocytosis of Secretory Organelles from Blood Platelets Incubated with Cationic Polypeptides  

PubMed Central

The blood platelet release reaction involves the secretion, in parallel, of specific chemical constituents stored in intracellular organelles to the plasma without loss of substances suggestive of cell damage. The present investigation has employed an unusual effect of cationic polypeptides to follow the platelet secretory process. When platelets were incubated with polybrene and polylysine, the agents were taken up and deposited in cytoplasmic organelles. The matrix of granules developed a lattice-like substructure not produced by other chemical agents, and dark zones of granules and dense bodies became more electron opaque. Subsequently, the platelets exposed to cationic polypeptides underwent the shape change and internal transformation similar to that produced by potent aggregating agents. Granules and dense bodies appeared in the open canalicular system and were extruded in a relatively intact state from the altered platelets. No differences were observed in the response of normal, afibrinogenemic or thrombasthenic platelets to incubation with polybrene and polylysine. The results indicate that cationic polypeptides can stimulate the extrusion of secretory organelles, and support the previous reports suggesting that channels of the open canalicular system serve as conduits for discharging the products of the release reaction. ImagesFigs 1A-1EFigs 2A-2FFigs 3A-3F PMID:5080705

White, James G.

1972-01-01

279

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

PubMed

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

Robinson, N; Brattsand, R; Dahlbäck, M

1990-03-01

280

Dentate granule cell GABA(A) receptors in epileptic hippocampus: enhanced synaptic efficacy and altered pharmacology.  

PubMed

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

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

2003-04-01

281

Influence of prostaglandin F?? analogues on the secretory function of bovine luteal cells and ovarian arterial contractility in vitro.  

PubMed

Although prostaglandin (PG) F2? analogues are routinely used for oestrus synchronisation in cattle, their effects on the function of the bovine corpus luteum (CL), and on ovarian arterial contractility, may not reflect the physiological effects of endogenous PGF2?. In the first of two related experiments, the effects of different analogues of PGF2? (aPGF2?) on the secretory function and apoptosis of cultured bovine cells of the CL were assessed. Enzymatically-isolated bovine luteal cells (from between days 8 and 12 of the oestrous cycle), were stimulated for 24h with naturally-occurring PGF2? or aPGF2? (dinoprost, cloprostenol or luprostiol). Secretion of progesterone (P4) was determined and cellular [Ca(2+)]i mobilisation, as well as cell viability and apoptosis were measured. Naturally-occurring PGF2? and dinoprost stimulated P4 secretion (P<0.05), whereas cloprostenol and luprostiol did not influence P4 synthesis. The greatest cytotoxic and pro-apoptotic effects were observed in the luprostiol-treated cells, at 37.3% and 202%, respectively (P<0.001). The greatest effect on [Ca(2+)]i mobilisation in luteal cells was observed post-luprostiol treatment (200%; P<0.001). In a second experiment, the influence of naturally-occurring PGF2? and aPGF2? on ovarian arterial contraction in vitro, were examined. No differences in the effects of dinoprost or naturally-occurring PGF2? were found across the studied parameters. The effects of cloprostenol and luprostiol on luteal cell death, in addition to their effects on ovarian arterial contractility, were much greater than those produced by treatment with naturally-occurring PGF2?. PMID:24268486

Korzekwa, A J; Lukasik, K; Pilawski, W; Piotrowska-Tomala, K K; Jaroszewski, J J; Yoshioka, S; Okuda, K; Skarzynski, D J

2014-01-01

282

Polarized but differential localization and recruitment of STIM1/Orai1 and STIM1/TRPC channels in secretory cells  

PubMed Central

Polarized Ca2+ signals in secretory epithelial cells are determined by compartmentalized localization of Ca2+ signaling proteins at the apical pole. Recently the ER Ca2+ sensor STIM1 and the Orai channels were shown to play a critical role in store-dependent Ca2+ influx. STIM1 also gates the TRPC channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca2+ influx and frequency of Ca2+ oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% co-localization. Consequently, STIM1 shows higher co-localization with the basolateral membrane marker E-cadherin than does Orai1, while Orai1 showed higher co-localization with the tight junction protein ZO1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co-IP of STIM1/Orai1/IP3Rs/TRPCs is enhanced by cell stimulation and disrupted by 2APB. The polarized localization and recruitment of these proteins results in preferred Ca2+ entry that is initiated at the apical pole. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca2+ permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca2+] oscillations and thus impact critical cellular functions. PMID:21054717

Hong, Jeong Hee; Li, Qin; Kim, Min Seuk; Shin, Dong Min; Feske, Stefan; Birnbaumer, Lutz; Cheng, Kwong Tai; Ambudkar, Indu S.; Muallem, Shmuel

2011-01-01

283

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

Microsoft Academic Search

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

OLA B. REITE

1996-01-01

284

Increasing ?-Cell Mass Requires Additional Stimulation for Adaptation to Secretory Demand.  

PubMed

Type 2 diabetes mellitus (T2DM) is caused by relative insulin deficiency, subsequent to both reduced ?-cell mass and insufficient insulin secretion, and both augmenting ?-cell mass and ?-cell function are therapeutic strategies for treating T2DM. However, the relative significance of increasing ?-cell mass vs improving ?-cell stimulus secretion coupling remains unclear. We have developed a mouse model that allows proliferation of ?-cells in adult mice without affecting ?-cell function by inducible expression of the positive cell cycle regulator cyclin A2 specifically in ?-cells. In these mice, when kept on a standard diet, doubling of ?-cell mass does not result in altered glucose tolerance or glucose-stimulated circulating insulin levels. Notably, a doubling of ?-cell mass also does not confer improved glycemic control and ability of ?-cells to respond to diabetogenic high-fat diet-induced glucose intolerance. However, in high-fat diet-exposed mice, an increase in endogenous ?-cell mass confers increased potentiation of in vivo glucose-stimulated rise in circulating insulin in response to acute pharmacologic treatment with the incretin glucagon-like peptide-1 receptor agonist exendin-4. These observations indicate that increasing endogenous ?-cell mass may not be sufficient to improve glycemic control in T2DM without additional strategies to increase ?-cell stimulus secretion coupling. PMID:25387052

Mondal, Prosenjit; Song, Woo-Jin; Li, Yuanyuan; Yang, Kil S; Hussain, Mehboob A

2015-01-01

285

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

PubMed

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. PMID:23601110

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

2013-04-17

286

Phylogenetic considerations of neurosecretory granule contents: role of nucleotides and basic hormone/transmitter packaging mechanisms.  

PubMed

The characteristics of neurosecretory granules include the presence of an acidic interior, a hyperosmolar concentration of granule solutes, the presence of chromogranin (CG) or CG-like soluble acidic proteins and a high content of nucleotides, predominantly ATP. The identification of "nucleotides" within the neuroendocrine "stem cells" of coelenterates (e.g. Hydra) has raised some interesting evolutionary questions as to the function of intragranular nucleotides. The chromaffin granules of adrenal medullary cells have been studied extensively, and are representative of the neurohormone/neurotransmitter packaging problems encountered in neurosecretory granules, in general. At the acid pH (5.7) of the interior of the chromaffin granule, ATP has three negative charges based on the pK value of the gamma-phosphate group. ATP can therefore interact with positively charged amines, acetylcholine and divalent cations, forming binary and ternary complexes. The results of nuclear magnetic resonance (NMR) spectroscopy indicate that the hyperosmolar solutes within the chromaffin granule exist in a viscous, but fluid state; one function of ATP could be to help lower the osmotic pressure of the granule contents through extensive, but weak, intermolecular bonding. In addition, ATP is an excellent buffer to help maintain a pH of 5.7 within the interior of the chromaffin granule. An acidic milieu contributes to neurohormone/neurotransmitter packaging and granule stability. The presence of nucleotides within neurosecretory granules cannot, however, be explained on the basis of the ability of ATP to simply reduce osmotic pressure, since insulin molecules exist in a crystalline phase, a condition which, by itself, could substantially reduce osmotic pressure; nucleotides, nevertheless, co-exist in these insulin cores. ATP and ATP metabolites such as ADP, AMP and adenosine, formed as a result of the action of ectonucleotidases, can have extensive extracellular trophic and feedback effects after secretion. Extracellular nucleotides and adenosine can function as neuromodulators, agonists and antagonists to inflammatory cells, and regulators of blood flow, etc. It is possible that intragranular nucleotides were retained through a billion or more years of evolution because of the importance of these trophic and feedback effects. Parts of the neuroscretory granule, such as the F1 subunit of the proton-translocating ATPase, can be traced back to the aerobic bacteria, vacuolar amine transport to yeast and a CG-like acidic protein to protozoan secretory granules (i.e., the trichocysts of Paramecia). PMID:2573383

Payne, C M

1989-01-01

287

In vivo 7 Tesla imaging of the dentate granule cell layer in Schizophrenia  

PubMed Central

PURPOSE The hippocampus is central to the pathophysiology of schizophrenia. Histology shows abnormalities in the dentate granule cell layer (DGCL), but its small size (~100 micron thickness) has precluded in vivo human studies. We used ultra high field magnetic resonance imaging (MRI) to compare DGCL morphology of schizophrenic patients to matched controls’. METHOD Bilateral hippocampi of 16 schizophrenia patients (10 male) 40.7±10.6 years old (mean ±standard deviation) were imaged at 7 Tesla MRI with heavily T2*-weighted gradient-echo sequence at 232 micron in-plane resolution (0.08 ?L image voxels). Fifteen matched controls (8 male, 35.6±9.4 years old) and one ex vivo post mortem hippocampus (that also underwent histopathology) were scanned with same protocol. Three blinded neuroradiologists rated each DGCL on a qualitative scale of 1 to 6 (from “not discernible” to “easily visible, appearing dark gray or black”) and mean left and right DGCL scores were compared using a non-parametric Mann-Whitney test. RESULTS MRI identification of the DGCL was validated with histopathology. Mean right and left DGCL ratings in patients (3.2±1.0 and 3.5±1.2) were not statistically different from controls’ (3.9±1.1 and 3.8±0.8), but patients’ had a trend for lower right DGCL score (p=0.07), which was significantly associated with patient diagnosis (p=0.05). The optimal 48% sensitivity and 80% specificity for schizophrenia was achieved with a DGCL rating of ?2. CONCLUSION Decreased contrast in the right DGCL in schizophrenia was predictive of schizophrenia diagnosis. Better utility of this metric as a schizophrenia biomarker may be achieved in future studies of patients with homogeneous disease subtypes and progression rates. PMID:23664589

Kirov, Ivan I.; Hardy, Caitlin J.; Matsuda, Kant; Messinger, Julie; Cankurtaran, Ceylan Z.; Warren, Melina; Wiggins, Graham C.; Perry, Nissa N.; Babb, James S.; Goetz, Raymond R.; George, Ajax; Malaspina, Dolores; Gonen, Oded

2013-01-01

288

GABAB receptor subtypes differentially modulate synaptic inhibition in the dentate gyrus to enhance granule cell output  

PubMed Central

Background and Purpose Activation of GABAB receptors in the dentate gyrus (DG) enhances granule cell (GC) activity by reducing synaptic inhibition imposed by hilar interneurons. This disinhibitory action facilitates signal transfer from the perforant path to the hippocampus. However, as the two main molecular subtypes, GABAB(1a,2) and GABAB(1b,2) receptors, prefer axonal terminal and dendritic compartments, respectively, they may modulate the hilar pathways at different synaptic localizations. We examined their relative expression and functions in the DG. Experimental Approach The localization of GABAB subtypes was revealed immunohistochemically using subunit-selective antibodies in GABAB1a–/– and GABAB1b–/– mice. Effects of subtype activation by the GABAB receptor agonist, baclofen, were examined on the perforant path-stimulated GC population activities in brain slices. Key Results GABAB(1a,2) receptors were concentrated in the inner molecular layer, the neuropil of the hilus and hilar neurons at the border zone; while GABAB(1b,2) receptors dominated the outer molecular layer and hilar neurons in the deep layer, showing their differential localization on GC dendrite and in the hilus. Baclofen enhanced the GC population spike to a larger extent in the GABAB1b–/– mice, demonstrating exclusively disinhibitory roles of the GABAB(1a,2) receptors. Conversely, in the GABAB1a–/– mice baclofen not only enhanced but also inhibited the population spike during GABAA blockade, revealing both disinhibitory and inhibitory effects of GABAB(1b,2) receptors. Conclusions and Implications The GABAB(1a,2) and GABAB(1b,2) receptor subtypes differentially modulate GC outputs via selective axonal terminal and dendritic locations in the hilar pathways. The GABAB(1a,2) receptors exclusively mediate disinhibition, thereby playing a greater role in gating signal transfer for hippocampal spatial and pattern learning. PMID:23186302

Foster, Joshua D; Kitchen, Ian; Bettler, Bernhard; Chen, Ying

2013-01-01

289

Synaptosomal-associated protein 25 mutation induces immaturity of the dentate granule cells of adult mice  

PubMed Central

Background Synaptosomal-associated protein, 25 kDa (SNAP-25) regulates the exocytosis of neurotransmitters. Growing evidence suggests that SNAP-25 is involved in neuropsychiatric disorders, such as schizophrenia, attention-deficit/hyperactivity disorder, and epilepsy. Recently, increases in anxiety-related behaviors and epilepsy have been observed in SNAP-25 knock-in (KI) mice, which have a single amino acid substitution of Ala for Ser187. However, the molecular and cellular mechanisms underlying the abnormalities in this mutant remain unknown. Results In this study, we found that a significant number of dentate gyrus (DG) granule cells was histologically and electrophysiologically similar to immature DG neurons in the dentate gyrus of the adult mutants, a phenomenon termed the “immature DG” (iDG). SNAP-25 KI mice and other mice possessing the iDG phenotype, i.e., alpha-calcium/calmodulin-dependent protein kinase II heterozygous mice, Schnurri-2 knockout mice, and mice treated with the antidepressant fluoxetine, showed similar molecular expression patterns, with over 100 genes similarly altered. A working memory deficit was also identified in mutant mice during a spontaneous forced alternation task using a modified T-maze, a behavioral task known to be dependent on hippocampal function. Chronic treatments with the antiepileptic drug valproate abolished the iDG phenotype and the working memory deficit in mutants. Conclusions These findings suggest that the substitution of Ala for Ser187 in SNAP-25 induces the iDG phenotype, which can also be caused by epilepsy, and led to a severe working memory deficit. In addition, the iDG phenotype in adulthood is likely an endophenotype for at least a part of some common psychiatric disorders. PMID:23497716

2013-01-01

290

L-type voltage-gated calcium channels modulate kainic acid neurotoxicity in cerebellar granule cells.  

PubMed

This study reports on the regulation of kainate neurotoxicity in cerebellar granule cells by calcium entry through voltage-gated calcium channels and by calcium release from internal cellular stores. Kainate neurotoxicity was prevented by the AMPA selective antagonist LY 303070 (10 microM). Kainate neurotoxicity was potentiated by cadmium, a general voltage-gated calcium channel blocker, and the L-type voltage-gated calcium channel blocker nifedipine. The antagonists of intracellular Ca2+ ([Ca2+]i) release, thapsigargin and ryanodine, were also able to potentiate kainate neurotoxicity. Kainate treatment elevated [Ca2+]i concentration with a rapid initial increase that peaked at 1543 nM and then declined to plateau at approximately 400 nM. Nifedipine lowered the peak response to 764 nM and the plateau response to approximately 90 nM. Thapsigargin also lowered the kainate-induced increase in [Ca2+]i (640 nM peak, 125 nM plateau). The ryanodine receptor agonist caffeine eliminated the kainate-induced increase in [Ca2+]i, and reduced kainate neurotoxicity. Kainate neurotoxicity potentiated by nifedipine was not prevented by RNA or protein synthesis inhibitors, nor by the caspase inhibitors YVAD-CHO and DEVD-CHO. Neither DNA laddering nor the number of apoptotic nuclei were increased following treatment with kainate and nifedipine. Increased nuclear staining with the membrane impermeable dye propidium iodide was observed immediately following kainate treatment, indicating a loss of plasma membrane integrity. Thus, kainate neurotoxicity is prevented by calcium entry through L-type calcium channels. PMID:10320722

Leski, M L; Valentine, S L; Coyle, J T

1999-05-15

291

Identification of endothelin 1 and big endothelin 1 in secretory vesicles isolated from bovine aortic endothelial cells.  

PubMed

Vesicles containing endothelin 1 (ET-1) were isolated from bovine aortic endothelial cells (BAECs) by fractionation of homogenates on sucrose density gradients by ultracentrifugation. The vesicles were localized at the 1.0/1.2 M sucrose interface using a specific anti-ET-1-(16-21) RIA. Identification of ET-1 and big ET-1 in this fraction was confirmed by HPLC analysis combined with RIA. Morphological examination of the ET-1-enriched fraction by electron microscopy identified clusters of vesicles approximately 100 nm in diameter. Immunostaining of ultrathin cryosections prepared from the vesicle fraction for ET-1 or big ET-1 showed clusters of 15-nm gold particles attached to or within vesicles. Immunofluorescence staining of whole BAECs using a specific ET-1-(16-21) IgG purified by affinity chromatography revealed punctate granulation of the cell cytoplasm viewed under light microscopy. This distinct pattern of staining was shown by confocal light microscopy to be intracellular. Immunofluorescence staining of whole cells with a polyclonal antiserum for big ET-1-(22-39) showed a defined perinuclear localization of precursor molecule. Hence, several different approaches have demonstrated that ET-1 and big ET-1 are localized within intracellular vesicles in BAECs, suggesting that these subcellular compartments are an important site for processing of big ET-1 by endothelin-converting enzyme. PMID:7603993

Harrison, V J; Barnes, K; Turner, A J; Wood, E; Corder, R; Vane, J R

1995-07-01

292

Cholinergic Stimulation of Lacrimal Acinar Cells Promotes Redistribution of Membrane-associated Kinesin and the Secretory Protein, ?-hexosaminidase, and Increases Kinesin Motor Activity  

Microsoft Academic Search

The role of the microtubule-based motor, kinesin, in membrane trafficking has been investigated in resting and stimulated acinar cells from rabbit lacrimal gland, a cholinergically controlled secretory tissue. Microtubule-dependent motors from extracts of control and carbachol-treated acini were isolated by microtubule-affinity purification and their activity was determined using a video-enhanced differential interference contrast microscopy assay for microtubule gliding. The observation

SARAH F. HAMM-ALVAREZ; SILVIA DA COSTA; TAO YANG; XINHUA WEI; PETER J. GIEROW; AUSTIN K. MIRCHEFF

1997-01-01

293

MicroRNA-34a Induces Vascular Smooth Muscle Cells Senescence by SIRT1 Downregulation and Promotes the Expression of Age-Associated Pro-inflammatory Secretory Factors.  

PubMed

Arterial aging is a major risk factor for the occurrence of cardiovascular diseases. The aged artery is characterized by endothelial dysfunction and vascular smooth muscle cells altered physiology together with low-grade chronic inflammation. MicroRNA-34a (miR-34a) has been recently implicated in cardiac, endothelial, and endothelial progenitor cell senescence; however, its contribution to aging-associated vascular smooth muscle cells phenotype has not been explored so far. We found that miR-34a was highly expressed in aortas isolated from old mice. Moreover, its well-known target, the longevity-associated protein SIRT1, was significantly downregulated during aging in both endothelial cells and vascular smooth muscle cells. Increased miR-34a as well as decreased SIRT1 expression was also observed in replicative-senescent human aortic smooth muscle cells. miR-34a overexpression in proliferative human aortic smooth muscle cells caused cell cycle arrest along with enhanced p21 protein levels and evidence of cell senescence. Furthermore, miR-34a ectopic expression induced pro-inflammatory senescence-associated secretory phenotype molecules. Finally, SIRT1 protein significantly decreased upon miR-34a overexpression and restoration of its levels rescued miR-34a-dependent human aortic smooth muscle cells senescence, but not senescence-associated secretory phenotype factors upregulation. Taken together, our findings suggest that aging-associated increase of miR-34a expression levels, by promoting vascular smooth muscle cells senescence and inflammation through SIRT1 downregulation and senescence-associated secretory phenotype factors induction, respectively, may lead to arterial dysfunctions. PMID:25352462

Badi, Ileana; Burba, Ilaria; Ruggeri, Clarissa; Zeni, Filippo; Bertolotti, Matteo; Scopece, Alessandro; Pompilio, Giulio; Raucci, Angela

2014-10-28

294

Phosducin Regulates Secretory Activity in TT Line of Thyroid Parafollicular C Cells.  

PubMed

The endocrine activity of the thyroid gland is accomplished by its follicular and parafollicular cells. In these cells, numerous G proteins-dependent pathways are active and potentially could be regulated by a 33-kDa cytoplasmic protein phosducin, which interacts with the G? subunit and may compete with G? or G?? dimer effectors. Significant expression of phosducin has been shown in the retina, pineal gland, and some neurons. Here, we studied postoperative thyroid tissue samples collected from patients with nodular goiter and 2 thyroid-derived cell lines for the presence of phosducin. Using reverse transcription PCR with product sequencing and highly sensitive immunodetection we identified phosducin mRNA and protein in the thyroid gland and parafollicular C TT cells, but not in the follicular Nthy-ori 3-1 cell line. We also observed that siRNA-mediated silencing of phosducin gene expression decreased Ca(2+)-stimulated secretion of calcitonin and serotonin by TT cells. PMID:25153685

Piotrowska, U; Adler, G; Kozicki, I

2014-08-25

295

RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses  

PubMed Central

Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genome-wide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of VSV infection, such as entry/uncoating, gene expression, and assembly/release, were identified. Depletion of the proteins of the coatomer complex I or its upstream effectors ARF1 or GBF1 led to detection of reduced levels of VSV RNA. Coatomer complex I was also required for infection of lymphocytic choriomeningitis virus and human parainfluenza virus type 3. These results highlight the evolutionarily conserved requirements for gene expression of diverse families of NS RNA viruses and demonstrate the involvement of host cell secretory pathway in the process. PMID:22065774

Panda, Debasis; Das, Anshuman; Dinh, Phat X.; Subramaniam, Sakthivel; Nayak, Debasis; Barrows, Nicholas J.; Pearson, James L.; Thompson, Jesse; Kelly, David L.; Ladunga, Istvan; Pattnaik, Asit K.

2011-01-01

296

Differentiation of Mucilage Secretory Cells of the Arabidopsis Seed Coat1  

E-print Network

mucilage (pectin). Such seed coat mucilage cells are necessary for neither viability nor germination under in germination and dispersal. Tissues of the seed coat are derived from cells of the ovule integuments/or protection of the emerging seedling during imbibition and germination. In addition to the seed coat

Western, Tamara L.

297

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product  

PubMed Central

AIM: To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product. METHODS: NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR. RESULTS: Among a total of 15?000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr?, jak 1, eps 8, tgf? 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgf? 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-? (TGF-?) showed statistical significance (P < 0.05). CONCLUSION: O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-? and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation. PMID:16773716

Thuwajit, Chanitra; Thuwajit, Peti; Uchida, Kazuhiko; Daorueang, Daoyot; Kaewkes, Sasithorn; Wongkham, Sopit; Miwa, Masanao

2006-01-01

298

Enhanced bursts of IPSCs in dentate granule cells in mice with regionally inhibited long-term potentiation.  

PubMed Central

Until recently, most studies on the synaptic-cellular basis of learning and memory concentrated on the activity-dependent changes occurring in principal cells such as hippocampal pyramidal cells and dentate granule cells. However, the ability of the inhibitory interneurons to regulate synaptic plasticity remains less understood. This study tested the hypothesis that the gamma-aminobutyric-acid (GABA)-mediated inhibitory neurotransmission is enhanced in mice that show no detectable long-term potentiation in the dentate gyrus in the absence of the GABAA receptor antagonist bicuculline. Patch clamp recordings were made from dentate granule cells in brain slices from wild-type and Thy-1 knockout (KO) mice. The frequency, amplitude and kinetics of miniature inhibitory postsynaptic currents (mIPSCs, generated by the action potential-independent release of GABA) was not different between animals. However, bursts of spontaneous IPSCs (sIPSCs, generated by both action potential-independent and -dependent GABA release) in KO mice were associated with larger synaptic charge transfers and increased durations. When pairs of IPSCs were evoked at varying intervals, the amplitude of the second response with respect to the first was significantly larger in KO animals. These results further support the concept that enhancement of interneuronal functions in cortical structures can have profound effects on the activity-dependent synaptic plasticity observed in principal cells. PMID:9470216

Hollrigel, G S; Morris, R J; Soltesz, I

1998-01-01

299

Specific immunomodulatory and secretory activities of stevioside and steviol in intestinal cells.  

PubMed

Stevioside, isolated from Stevia rebaudiana, is a commercial sweetener. It was previously demonstrated that stevioside attenuates NF-kappaB-dependent TNF-alpha and IL-1beta synthesis in LPS-stimulated monocytes. The present study examined the effects of stevioside and its metabolite, steviol, on human colon carcinoma cell lines. High concentrations of stevioside (2-5 mM) and steviol (0.2-0.8 mM) decreased cell viability in T84, Caco-2, and HT29 cells. Stevioside (2 mM) potentiated TNF-alpha-mediated IL-8 release in T84 cells. However, steviol (0.01-0.2 mM) significantly suppressed TNF-alpha-induced IL-8 release in all three cell lines. In T84 cells, steviol attenuated TNF-alpha-stimulated IkappaB --> NF-kappaB signaling. Chloride transport was stimulated by steviol (0.1 mM) > stevioside (1 mM) at 30 min. Two biological effects of steviol in the colon are demonstrated for the first time: stimulation of Cl(-) secretion and attenuation of TNF-alpha-stimulated IL-8 production. The immunomodulatory effects of steviol appear to involve NF-kappaB signaling. In contrast, at nontoxic concentrations stevioside affects only Cl(-) secretion. PMID:18433103

Boonkaewwan, Chaiwat; Ao, Mei; Toskulkao, Chaivat; Rao, Mrinalini C

2008-05-28

300

Munc13-3 superprimes synaptic vesicles at granule cell-to-basket cell synapses in the mouse cerebellum.  

PubMed

Munc13-3 is a presynaptic protein implicated in vesicle priming that is strongly expressed in cerebellar granule cells (GCs). Mice deficient of Munc13-3 (Munc13-3(-/-)) show an increased paired-pulse ratio (PPR), which led to the hypothesis that Munc13-3 increases the release probability (pr) of vesicles. In the present study, we analyzed unitary synaptic connections between GCs and basket cells in acute cerebellar slices from wild-type and Munc13-3(-/-) mice. Unitary EPSCs recorded from Munc13-3(-/-) GCs showed normal kinetics and synaptic latency but a significantly increased PPR and fraction of synaptic failures. A quantal analysis revealed that neither the charge of single quanta nor the binominal parameter N were affected by loss of Munc13-3 but that pr was almost halved in Munc13-3(-/-). Neither presynaptic Ca(2+) influx was affected by deletion of Munc13-3 nor replenishment of the readily releasable vesicle pool. However, a high concentration of EGTA led to a reduction in EPSCs that was significantly stronger in Munc13-3(-/-). We conclude that Munc13-3 is responsible for an additional step of molecular and/or positional "superpriming" that substantially increases the efficacy of Ca(2+)-triggered release. PMID:25355221

Ishiyama, Shimpei; Schmidt, Hartmut; Cooper, Benjamin H; Brose, Nils; Eilers, Jens

2014-10-29

301

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

PubMed Central

The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor. PMID:18316814

2008-01-01

302

Secretory competence in a gateway endocrine cell conferred by the nuclear receptor ?FTZ-F1 enables stage-specific ecdysone responses throughout development in Drosophila.  

PubMed

Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor ?FTZ-F1. Selective RNA silencing of ?ftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in ?ftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted ?FTZ-F1 expression. Moreover, premature ?ftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a "gateway cell", whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed ?FTZ-F1 expression late in the molt as steroids decline. PMID:24247008

Cho, Kook-Ho; Daubnerová, Ivana; Park, Yoonseong; Zitnan, Dusan; Adams, Michael E

2014-01-15

303

Insufficient ER-stress response causes selective mouse cerebellar granule cell degeneration resembling that seen in congenital disorders of glycosylation  

PubMed Central

Background Congenital disorders of glycosylation (CDGs) are inherited diseases caused by glycosylation defects. Incorrectly glycosylated proteins induce protein misfolding and endoplasmic reticulum (ER) stress. The most common form of CDG, PMM2-CDG, is caused by deficiency in the cytosolic enzyme phosphomannomutase 2 (PMM2). Patients with PMM2-CDG exhibit a significantly reduced number of cerebellar Purkinje cells and granule cells. The molecular mechanism underlying the specific cerebellar neurodegeneration in PMM2-CDG, however, remains elusive. Results Herein, we report that cerebellar granule cells (CGCs) are more sensitive to tunicamycin (TM)-induced inhibition of total N-glycan synthesis than cortical neurons (CNs). When glycan synthesis was inhibited to a comparable degree, CGCs exhibited more cell death than CNs. Furthermore, downregulation of PMM2 caused more CGCs to die than CNs. Importantly, we found that upon PMM2 downregulation or TM treatment, ER-stress response proteins were elevated less significantly in CGCs than in CNs, with the GRP78/BiP level showing the most significant difference. We further demonstrate that overexpression of GRP78/BiP rescues the death of CGCs resulting from either TM-treatment or PMM2 downregulation. Conclusions Our results indicate that the selective susceptibility of cerebellar neurons to N-glycosylation defects is due to these neurons’ inefficient response to ER stress, providing important insight into the mechanisms of selective neurodegeneration observed in CDG patients. PMID:24305089

2013-01-01

304

Differential effects of polychlorinated biphenyl congeners on phosphoinositide hydrolysis and protein kinase C translocation in rat cerebellar granule cells.  

PubMed

Previous reports from our laboratory have suggested that the neuroactivity of some polychlorinated biphenyl (PCB) congeners is associated with perturbations in cellular Ca(2+)-homeostasis. We have characterized further the neurochemical effects of PCBs on signal transduction in primary cultures of cerebellar granule cells. The present experiments found that neither 2,2'-dichlorobiphenyl (DCBP), an ortho-substituted congener, nor 3,3',4,4',5-pentachlorobiphenyl (PCBP), a non-ortho-substituted congener, affected basal phosphoinositide (PI) hydrolysis in cerebellar granule cells. However, at concentrations up to 50 microM, DCBP potentiated carbachol-stimulated PI hydrolysis, while decreasing it at 100 microM. PCBP, on the other hand, had no effect on carbachol-stimulated PI hydrolysis in concentrations up to 100 microM. [3H]Phorbol ester ([3H]PDBu) binding was used to determine protein kinase C (PKC) translocation. DCBP increased [3H]PDBu binding in a concentration-dependent manner and a twofold increase was observed at 100 microM in cerebellar granule cells. PCBP had no effect on [3H]PDBu binding at concentrations up to 100 microM. The effect of DCBP on [3H]PDBu binding was time-dependent and was also dependent on the presence of external Ca2+ in the medium. To test the hypothesis that DCBP increases [3H]PDBu binding by acting on receptor-activated calcium channels, the effects of DCBP were compared to those of L-glutamate. The effects of DCBP (50 microM) and glutamate (20 microM) were additive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7859093

Kodavanti, P R; Shafer, T J; Ward, T R; Mundy, W R; Freudenrich, T; Harry, G J; Tilson, H A

1994-10-31

305

Systematic Single-Cell Analysis of Pichia pastoris Reveals Secretory Capacity Limits Productivity  

Microsoft Academic Search

Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of

Kerry Routenberg Love; Timothy J. Politano; Vasiliki Panagiotou; Bo Jiang; Terrance A. Stadheim; J. Christopher Love

2012-01-01

306

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

SciTech Connect

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

Ohara-Imaizumi, Mica; Aoyagi, Kyota; Nakamichi, Yoko; Nishiwaki, Chiyono [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan); Sakurai, Takashi [Photon Medical Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192 Shizuoka (Japan)] [Photon Medical Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192 Shizuoka (Japan); Nagamatsu, Shinya, E-mail: shinya@kyorin-u.ac.jp [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)

2009-07-31

307

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

PubMed Central

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

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

2009-01-01

308

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

SciTech Connect

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

Dreiem, Anne [Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, Oslo (Norway); Norwegian Defence Research Establishment, Department for Protection, Kjeller (Norway)], E-mail: axd17@health.state.ny.us; Rykken, Sidsel [Norwegian Defence Research Establishment, Department for Protection, Kjeller (Norway); Lehmler, Hans-Joachim; Robertson, Larry W. [Department of Occupational and Environmental Health, University of Iowa, Iowa (United States); Fonnum, Frode [Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, Oslo (Norway)

2009-10-15

309

Differential interaction of splicing snRNPs with coiled bodies and interchromatin granules during mitosis and assembly of daughter cell nuclei  

PubMed Central

In the interphase nucleus of mammalian cells the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs), which are subunits of spliceosomes, associate with specific subnuclear domains including interchromatin granules and coiled bodies. Here, we analyze the association of splicing snRNPs with these structures during mitosis and reassembly of daughter nuclei. At the onset of mitosis snRNPs are predominantly diffuse in the cytoplasm, although a subset remain associated with remnants of coiled bodies and clusters of mitotic interchromatin granules, respectively. The number and size of mitotic coiled bodies remain approximately unchanged from metaphase to early telophase while snRNP-containing clusters of mitotic interchromatin granules increase in size and number as cells progress from anaphase to telophase. During telophase snRNPs are transported into daughter nuclei while the clusters of mitotic interchromatin granules remain in the cytoplasm. The timing of nuclear import of splicing snRNPs closely correlates with the onset of transcriptional activity in daughter nuclei. When transcription restarts in telophase cells snRNPs have a diffuse nucleoplasmic distribution. As cells progress to G1 snRNP- containing clusters of interchromatin granules reappear in the nucleus. Coiled bodies appear later in G1, although the coiled body antigen, p80 coilin, enters early into telophase nuclei. After inhibition of transcription we still observe nuclear import of snRNPs and the subsequent appearance of snRNP-containing clusters of interchromatin granules, but not coiled body formation. These data demonstrate that snRNP associations with coiled bodies and interchromatin granules are differentially regulated during the cell division cycle and suggest that these structures play distinct roles connected with snRNP structure, transport, and/or function. PMID:8027171

1994-01-01

310

Polarized TIRFM Reveals Changes in Plasma Membrane Topology Before and During Granule Fusion  

PubMed Central

We have recently developed a combination of polarization and total internal reflection fluorescence microscopy (pTIRFM) to monitor changes in plasma membrane topology occurring after fusion of chromaffin granules. In this report, pTIRFM is further exploited to reveal two major findings in regards to the secretory pathway in bovine chromaffin cells. First, we show that changes in membrane topology are sometimes detected even prior to fusion. This occurs with high probability in a small subset of granules that appear in the evanescent field during the experiment. On these occasions, the plasma membrane invaginates with the movement just preceding the appearance of a granule in the evanescent field. Such events may represent a direct interaction of the granule with the plasma membrane. Second, we show that the topological fate of the post-fusion, granule/plasma membrane intermediate is regulated by divalent cation. When Sr2+ is used instead of Ca2+ to trigger exocytosis, membrane topology in the exocytotic region is stabilized with significant curvature and indentation. PMID:21061164

Anantharam, Arun; Axelrod, Daniel; Holz, Ronald W.

2011-01-01

311

Yizhijiannao Granule and a combination of its effective monomers, icariin and Panax notoginseng saponins, inhibit early PC12 cell apoptosis induced by beta-amyloid (25–35)?  

PubMed Central

One of our previous studies showed that Yizhijiannao Granule, a compound Chinese medicine, effectively improved the clinical symptoms of Alzheimer's disease. In the present study, we established a model of Alzheimer's disease using beta-amyloid (25–35) in PC12 cells, and treated the cells with Yizhijiannao Granule and its four monomers, i.e., icariin, catechin, Panax notoginseng saponins, and eleutheroside E. Flow cytometry showed that Yizhijiannao Granule-containing serum, icariin, Panax notoginseng saponins, and icariin + Panax notoginseng saponins were protective against beta-amyloid (25–35)-induced injury in PC12 cells. Icariin in combination with Panax notoginseng saponins significantly inhibited early apoptosis of PC12 cells with beta-amyloid (25–35)-induced injury compared to icariin or Panax notoginseng saponins alone. The effects of icariin + Panax notoginseng saponins were similar to the effects of Yizhijiannao Granule. The findings indicate that two of the effective monomers of Yizhijiannao Granule, icariin and Panax notoginseng saponins, can synergistically inhibit early apoptosis of PC12 cells induced by beta-amyloid (25–35).

Zhang, Ting; Zhang, Zhanwei; Dong, Keli; Li, Guangcheng; Zhu, Hong

2012-01-01

312

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway  

PubMed Central

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

Barlowe, Charles K.; Miller, Elizabeth A.

2013-01-01

313

Regulation of lipid synthesis genes and milk fat production in human mammary epithelial cells during secretory activation  

PubMed Central

Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our goal was to study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretory activation. Gene expression from mRNA isolated from milk fat globule (MFG) and milk FA composition were measured from 6 h to 42 days postpartum in seven normal women. Over the first 96 h postpartum, daily milk fat output increased severalfold and mirrored expression of genes for all aspects of lipid metabolism and milk FA production, including lipolysis at the MEC membrane, FA uptake from blood, intracellular FA transport, de novo FA synthesis, FA and glycerol activation, FA elongation, FA desaturation, triglyceride synthesis, cholesterol synthesis, and lipid droplet formation. Expression of the gene for a key lipid synthesis regulator, sterol regulatory element-binding transcription factor 1 (SREBF1), increased 2.0-fold by 36 h and remained elevated over the study duration. Expression of genes for estrogen receptor 1, thyroid hormone-responsive protein, and insulin-induced 2 increased progressively to plateau by 96 h. In contrast, mRNA of peroxisome proliferator-activated receptor-? decreased severalfold. With onset of lactation, increased de novo synthesis of FA was the most prominent change in milk FA composition and mirrored the expression of FA synthesis genes. In conclusion, milk lipid synthesis and secretion in humans is a complex process requiring the orchestration of a wide variety of pathways of which SREBF1 may play a primary role. PMID:23880316

Mohammad, Mahmoud A.

2013-01-01

314

Secretory pathway of trypanosomatid parasites.  

PubMed

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

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

2002-03-01

315

Ultrastructural distribution of glycinergic and GABAergic neurons and axon terminals in the rat dorsal cochlear nucleus, with emphasis on granule cell areas  

PubMed Central

A knowledge of neurotransmitters in the neurons of the rat cochlear nuclear complex is of importance in understanding the function of auditory circuits. Using post-embedding ultrastructural immunogold labelling, the distribution of glycinergic and GABAergic neurons and axonal terminals has been studied in the molecular, fusiform and polymorphic layers of the rat dorsal cochlear nucleus (DCN). This technique is not limited by the penetration of antibodies into the nervous tissue as in pre-embedding methods, and allows a fine neurochemical mapping of the nervous tissue. Numerous glycinergic and GABAergic axon terminals contain pleomorphic and flat synaptic vesicles, and are present in all layers (1, 2, 3) of the dorsal cochlear nucleus. Glycine and GABA-negative large terminals (mossy fibres) are mainly seen in granule cell areas of layer 2 (fusiform layer). Mossy fibres contact the dendrites of GABA- and glycine-negative granule cells and of the few unipolar brush cells (excitatory neurons). The least common cells in the granule cell areas are GABAergic and glycinergic Golgi-stellate neurons. In unipolar brush cells, aggregations of vesicles seem to be the origin of their characteristic ringlet-bodies. Golgi-stellate cells send their inhibitory terminals to the dendrites of granule and unipolar brush cells, occasionally directly to mossy fibres. Small or (less frequently) large GABAergic terminals contact the soma or the main dendrite of unipolar brush cells. The circuit of a hypothetical functional unit of neurons in the DCN is proposed. The inputs from auditory tonotopic or non-auditory non-tonotopic mossy fibres eventually reach pyramidal cells through axons from the granule cells or unipolar brush cells. Pyramidal cells convey an excitatory signal from the DCN to higher mesencephalic nuclei for further elaboration of the acoustic signal. PMID:12892405

Alibardi, Lorenzo

2003-01-01

316

The weaver mutation of GIRK2 results in a loss of inwardly rectifying K+ current in cerebellar granule cells.  

PubMed Central

The weaver mutation in mice results in a severe ataxia that is attributable to the degeneration of cerebellar granule cells and dopaminergic neurons in the substantia nigra. Recent genetic studies indicate that the GIRK2 gene is altered in weaver. This gene codes for a G-protein-activated, inwardly rectifying K+ channel protein (8). The mutation results in a single amino acid substitution (glycine-->serine) in the pore-forming H5 region of the channel. The functional consequences of this mutation appear to depend upon the co-expression of other GIRK subunits--leading to either a gain or loss of function. Here, we show that G-protein-activated inwardly rectifying K+ currents are significantly reduced in cerebellar granule cells from animals carrying the mutant allele. The reduction is most pronounced in homozygous neurons. These findings suggest that the death of neurons in weaver is attributable to the loss of GIRK2-mediated currents, not to the expression of a nonspecific cation current. Images Fig. 3 PMID:8855331

Surmeier, D J; Mermelstein, P G; Goldowitz, D

1996-01-01

317

Heterogeneous imaging characteristics of JC virus granule cell neuronopathy (GCN): a case series and review of the literature.  

PubMed

Granule cell neuronopathy (GCN) is a rare JC virus (JCV)-related disease in immunocompromised patients, characterized by lytic infection of the cerebellar granule cell layer. To enable early diagnosis and intervention, we identify features of GCN and describe possible aspects of disease heterogeneity. We report on two new cases of GCN in HIV-infected patients of whom we retrospectively assessed clinical and radiologic data. In addition, we carried out a literature search and review of clinical, radiologic and histopathologic findings of all published GCN cases. Including the two new cases reported here, a total of 18 GCN cases were included in this study. HIV infection, present in 12 of the cases, was the most common underlying condition, followed by monoclonal antibody treatment which was present in three cases. Cerebellar atrophy was detected in all except two cases. In 12 patients a heterogeneous distribution pattern of white matter changes in the cerebellum and brainstem was observed. Imaging findings in GCN are remarkably heterogeneous; exhibiting cerebellar atrophy, as well as white matter pathology, particularly in the adjacent infratentorial white matter. This suggests an overlap of GCN with other JCV-related diseases, such as progressive multifocal leukoencephalopathy. PMID:25297924

Wijburg, Martijn T; van Oosten, Bob W; Murk, Jean-Luc; Karimi, Ouafae; Killestein, Joep; Wattjes, Mike P

2015-01-01

318

The QKI-6 RNA Binding Protein Localizes with the MBP mRNAs in Stress Granules of Glial Cells  

PubMed Central

Background The quaking viable (qkv) mouse has several developmental defects that result in rapid tremors in the hind limbs. The qkI gene expresses three major alternatively spliced mRNAs (5, 6 and 7 kb) that encode the QKI-5, QKI-6 and QKI-7 RNA binding proteins that differ in their C-terminal 30 amino acids. The QKI isoforms are known to regulate RNA metabolism within oligodendrocytes, however, little is known about their roles during cellular stress. Methodology/Principal Findings In this study, we report an interaction between the QKI-6 isoform and a component of the RNA induced silencing complex (RISC), argonaute 2 (Ago2). We show in glial cells that QKI-6 co-localizes with Ago2 and the myelin basic protein mRNA in cytoplasmic stress granules. Conclusions Our findings define the QKI isoforms as Ago2-interacting proteins. We also identify the QKI-6 isoform as a new component of stress granules in glial cells. PMID:20862255

Wang, Yunling; Lacroix, Geneviève; Haines, Jeffery; Doukhanine, Evgueni; Almazan, Guillermina; Richard, Stéphane

2010-01-01

319

The dendrites of granule cell layer neurons are the primary injury sites in the "Brain Diabetes" rat.  

PubMed

We previously demonstrated that rats that receive dorsal third ventricle (3V) streptozotocin (STZ) injections (STZ-3V-rats) exhibit cognitive decline as measured by the Morris Water Maze (MWM) and can be used as an animal model of Alzheimer's disease (AD). Immunohistochemical studies of the hippocampal formations of these animals have revealed significant changes in cerebral insulin signalling pathways, as well as marked increases of amyloid beta (Ab) deposition. Here, we performed Sholl analyses of granule cell layer dendrites and measured dendrite spine densities to assess the effect of STZ on hippocampal morphology. In STZ-3V rats as the results, more branching, complex dendrite arborisation, and increased soma size of the granule cells were observed, while spine densities were decreased in all three spine types. An intraventricular injection of a long-acting insulin analogue improved STZ-induced behavioural and immunohistochemical changes. Nevertheless, dendrite spine densities remained diminished, presumably due to overall null changes since new spine formation due to insulin stimulation has been compensated by loss of old spines. It is concluded that cognitive decline in the "Brain Diabetes" rats is primarily due to impaired intracerebral insulin signalling and the ultimate results were injured excitatory inputs through the perforant pathway. PMID:25476563

Shingo, Akiko Sheala; Mervis, Ronald F; Kanabayashi, Tomomichi; Kito, Shozo; Murase, Toshio

2015-03-01

320

siRNA knock-down of mutant torsinA restores processing through secretory pathway in DYT1 dystonia cells  

PubMed Central

Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the C-terminal region (torsinA?E). TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinA?E when compared with control fibroblasts expressing only torsinA. In this study, small interfering RNA (siRNA) oligonucleotides were identified, which downregulate the levels of torsinA or torsinA?E mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared with the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinA?E message in DYT fibroblasts increased luciferase secretion when compared with cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinA?E acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinA?E to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia. PMID:18258738

Hewett, Jeffrey W.; Nery, Flávia C.; Niland, Brian; Ge, Pei; Tan, Pamela; Hadwiger, Philipp; Tannous, Bakhos A.; Sah, Dinah W.Y.; Breakefield, Xandra O.

2008-01-01

321

Russell bodies: a general response of secretory cells to synthesis of a mutant immunoglobulin which can neither exit from, nor be degraded in, the endoplasmic reticulum  

PubMed Central

Dilated cisternae of the ER resembling Russell Bodies (RBs) are induced in light (L) chain producing myeloma cell lines by transfection of a mu heavy (H) chain gene lacking the first constant domain (mu delta CH1). RBs do not appear to be tissue specific, since they are also induced in a rat glioma cell line transfected with mu delta CH1 and L chain genes. Efficient RB biogenesis requires H-L assembly and polymerization. The mutant Ig is partially degraded in a pre-Golgi compartment. The remnant, however, becomes an insoluble lattice when intersubunit disulphide bonds are formed. The resulting insoluble aggregate accumulates in RBs. Replacing the COOH-terminal cysteine of mu delta CH1 chains with alanine reverses the RB-phenotype: the double mutant mu ala delta CH1 chains assemble noncovalently with L and are secreted as H2L2 complexes. Similarly, secretion of mu delta CH1 chains can be induced by culturing transfectant cells in the presence of reducing agents. The presence of RBs does not alter transport of other secretory or membrane molecules, nor does it affect cell division. Resident proteins of the ER and other secretory proteins are not concentrated in RBs, implying sorting at the ER level. Sorting could be the result of the specific molecular structure of the insoluble lattice. We propose that RBs represent a general response of the cell to the accumulation of abundant, nondegradable protein(s) that fail to exit from the ER. PMID:1955467

1991-01-01

322

Particle size of hydroxyapatite granules calcified from red algae affects the osteogenic potential of human mesenchymal stem cells in vitro.  

PubMed

Hydroxyapatite (HA) microparticles as a carrier in an injectable tissue-engineered bone filler are considered promising candidates for the treatment of small bone defects in the craniomaxillofacial region. HA granules calcified from red algae, varying in size, were evaluated in vitro for their suitability to be used as a carrier for human mesenchymal stem cells (hMSCs). Three groups of granules were produced in grain sizes of 10-100, 200-500 and 600-1,000 mum. After seeding and culturing hMSCs under osteogenic differentiation conditions onto HA particles for 3, 6 and 9 days, cellular proliferation (tetrazolium salt, XTT), alkaline phosphatase (ALP)-specific activity and total protein synthesis were investigated. The osteoblastic phenotype of the cells was evaluated by assaying the bone-specific genes osteocalcin, osteopontin and collagen type I. XTT assay revealed significantly higher (p < 0.01) proliferation of cells grown on the smallest grain size after 9 days of culture. Regarding ALP-specific activity, significantly higher levels of activity were detected in cells grown on the smallest grain size. Different grain sizes had no significant effects on the secretion of osteocalcin and osteopontin. Collagen type I production was significantly higher (p < 0.05) in cells grown on the biggest grain size in comparison with the two other grain sizes. These results show that the particle size of HA microparticles affects the osteogenic potential of cultured hMSCs and lead to the conclusion that particle size has differential effects on ALP-specific activity and collagen type I production. PMID:16804298

Weissenboeck, Martina; Stein, Elisabeth; Undt, Gerhard; Ewers, Rolf; Lauer, Gunter; Turhani, Dritan

2006-01-01

323

Monocyte-derived dendritic cells have a phenotype comparable to that of dermal dendritic cells and display ultrastructural granules distinct from Birbeck granules  

Microsoft Academic Search

Most monocyte-derived dendritic cells (DC) display CD1a, like Langerhans cells (LC) and some dermal DC, but their relationship with these skin DC remains unclear. To address this issue, we studied the expression of different antigens charac- teristic of skin DC and of monocyte\\/macrophages in CD1a1 and CD1a2 monocyte-derived DC. Their phenotype indicated that they may be related to dermal DC

Fernanda Grassi; Colette Dezutter-Dambuyant; Dorian McIlroy; Christelle Jacquet; Kozo Yoneda; Sadao Imamura; Laurence Boumsell; Daniel Schmitt; Brigitte Autran; Patrice Debre; Anne Hosmalin

324

Conditional deletion of ?-CaMKII impairs integration of adult-generated granule cells into dentate gyrus circuits and hippocampus-dependent learning.  

PubMed

New granule cells are continuously integrated into hippocampal circuits throughout adulthood, and the fine-tuning of this process is likely important for efficient hippocampal function. During development, this integration process is critically regulated by the ?-calcium/calmodulin-dependent protein kinase II (?-CaMKII), and here we ask whether this role is conserved in the adult brain. To do this, we developed a transgenic strategy to conditionally delete ?-CaMKII from neural progenitor cells and their progeny in adult mice. First, we found that the selective deletion of ?-CaMKII from newly generated dentate granule cells led to an increase in dendritic complexity. Second, ?-CaMKII deletion led to a reduction in number of mature synapses and cell survival. Third, consistent with altered morphological and synaptic development, acquisition of one-trial contextual fear conditioning was impaired after deletion of ?-CaMKII from newly generated dentate granule cells. Previous work in Xenopus identified ?-CaMKII as playing a key role in the stabilization of dendritic and synaptic structure during development. The current study indicates that ?-CaMKII plays a plays a similar, cell-autonomous role in the adult hippocampus and, in addition, reveals that the loss of ?-CaMKII from adult-generated granule cells is associated with impaired hippocampus-dependent learning. PMID:25186740

Arruda-Carvalho, Maithe; Restivo, Leonardo; Guskjolen, Axel; Epp, Jonathan R; Elgersma, Ype; Josselyn, Sheena A; Frankland, Paul W

2014-09-01

325

Theta-Gamma-Modulated Synaptic Currents in Hippocampal Granule Cells In Vivo Define a Mechanism for Network Oscillations  

PubMed Central

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

Pernía-Andrade, Alejandro Javier; Jonas, Peter

2014-01-01

326

Enrichment and analysis of secretory lysosomes from lymphocyte populations  

Microsoft Academic Search

BACKGROUND: In specialized cells, such as mast cells, macrophages, T lymphocytes and Natural Killer cells in the immune system and for instance melanocytes in the skin, secretory lysosomes (SL) have evolved as bifunctional organelles that combine degradative and secretory properties. Mutations in lysosomal storage, transport or sorting molecules are associated with severe immunodeficiencies, autoimmunity and (partial) albinism. In order to

Hendrik Schmidt; Christoph Gelhaus; Ralph Lucius; Melanie Nebendahl; Matthias Leippe; Ottmar Janssen

2009-01-01

327

Mutant Human FUS Is Ubiquitously Mislocalized and Generates Persistent Stress Granules in Primary Cultured Transgenic Zebrafish Cells  

PubMed Central

FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS), a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C) human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG) inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons. PMID:24912067

Acosta, Jamie Rae; Goldsbury, Claire; Winnick, Claire; Badrock, Andrew P.; Fraser, Stuart T.; Laird, Angela S.; Hall, Thomas E.; Don, Emily K.; Fifita, Jennifer A.; Blair, Ian P.; Nicholson, Garth A.; Cole, Nicholas J.

2014-01-01

328

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

Microsoft Academic Search

We describe an in vitro system with all components derived from the yeast Saccharomyces cerevisiae that can translocate a yeast secretory pro- tein across microsomal membranes. In vitro tran- scribed prepro-a-factor mRNA served to program a membrane-deplete d yeast translation system. Translo- cation and core glycosylation of prepro-a-factor were observed when yeast microsomal membranes were added during or after translation.

M. Gerard Waters; Giinter Blobel

1986-01-01

329

Ectopic TGF?1 Expression in the Secretory Mammary Epithelium Induces Early Senescence of the Epithelial Stem Cell Population  

Microsoft Academic Search

An important feature of the mammary gland is the regenerative capacity of its epithelium which is demonstrated upon successive cycles of lactation and involution. Pregnant mice expressing a whey-acidic protein (WAP) promoter-driven transforming growth factor-?1 (TGF?1) cDNA are unable either to generate a secretory mammary epithelium or to lactate. Here we investigate whether ectopic TGF?1 induces this phenotype by affecting

Edith C. Kordon; Robert A. McKnight; Chamelli Jhappan; Lothar Hennighausen; Glenn Merlino; Gilbert H. Smith

1995-01-01

330

Radiation-induced alterations in synaptic neurotransmission of dentate granule cells depend on the dose and species of charged particles.  

PubMed

The evaluation of potential health risks associated with neuronal exposure to space radiation is critical for future long duration space travel. The purpose of this study was to evaluate and compare the effects of low-dose proton and high-energy charged particle (HZE) radiation on electrophysiological parameters of the granule cells in the dentate gyrus (DG) of the hippocampus and its associated functional consequences. We examined excitatory and inhibitory neurotransmission in DG granule cells (DGCs) in dorsal hippocampal slices from male C57BL/6 mice at 3 months after whole body irradiation with accelerated proton, silicon or iron particles. Multielectrode arrays were used to investigate evoked field synaptic potentials, an extracellular measurement of synaptic excitability in the perforant path to DG synaptic pathway. Whole-cell patch clamp recordings were used to measure miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) in DGCs. Exposure to proton radiation increased synaptic excitability and produced dose-dependent decreases in amplitude and charge transfer of mIPSCs, without affecting the expression of ?-aminobutyric acid type A receptor ?2, ?3 and ?2 subunits determined by Western blotting. Exposure to silicon radiation had no significant effects on synaptic excitability, mEPSCs or mIPSCs of DGCs. Exposure to iron radiation had no effect on synaptic excitability and mIPSCs, but significantly increased mEPSC frequency at 1 Gy, without changes in mEPSC kinetics, suggesting a presynaptic mechanism. Overall, the data suggest that proton and HZE exposure results in radiation dose- and species-dependent long-lasting alterations in synaptic neurotransmission, which could cause radiation-induced impairment of hippocampal-dependent cognitive functions. PMID:25402556

Marty, V N; Vlkolinsky, R; Minassian, N; Cohen, T; Nelson, G A; Spigelman, I

2014-12-01

331

Amyloid ?-Mediated Zn2+ Influx into Dentate Granule Cells Transiently Induces a Short-Term Cognitive Deficit  

PubMed Central

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn2+ transport system due to a local increase in amyloid-? (A?) concentration. A single injection of A? (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. A?-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn2+ is involved in A? action. When A? was injected into the dentate gyrus, intracellular Zn2+ levels were increased only in the injected area in the dentate gyrus, suggesting that A? induces the influx of Zn2+ into cells in the injected area. When A? was added to hippocampal slices, A? did not increase intracellular Zn2+ levels in the dentate granule cell layer in ACSF without Zn2+, but in ACSF containing Zn2+. The increase in intracellular Zn2+ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that A?-mediated Zn2+ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn2+ may play a key role for transiently A?-induced cognition deficits. PMID:25536033

Takeda, Atsushi; Nakamura, Masatoshi; Fujii, Hiroaki; Uematsu, Chihiro; Minamino, Tatsuya; Adlard, Paul A.; Bush, Ashley I.; Tamano, Haruna

2014-01-01

332

SUR1 Regulates PKA-independent cAMP-induced Granule Priming in Mouse Pancreatic B-cells  

PubMed Central

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS–insensitive) component correlated with a rapid increase in membrane capacitance of ?80 fF that plateaued within ?200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the Kd values were 6 and 29 ?M for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1?/? mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1?/? mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl? into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane KATP-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca2+-induced exocytosis. PMID:12601083

Eliasson, Lena; Ma, Xiaosong; Renström, Erik; Barg, Sebastian; Berggren, Per-Olof; Galvanovskis, Juris; Gromada, Jesper; Jing, Xingjun; Lundquist, Ingmar; Salehi, Albert; Sewing, Sabine; Rorsman, Patrik

2003-01-01

333

A novel alternate secretory pathway for the export of Plasmodium proteins into the?host?erythrocyte  

PubMed Central

The malarial parasite dramatically alters its host cell by exporting and targeting proteins to specific locations within the erythrocyte. Little is known about the mechanisms by which the parasite is able to carry out this extraparasite transport. The fungal metabolite brefeldin A (BFA) has been used to study the secretory pathway in eukaryotes. BFA treatment of infected erythrocytes inhibits protein export and results in the accumulation of exported Plasmodium proteins into a compartment that is at the parasite periphery. Parasite proteins that are normally localized to the erythrocyte membrane, to nonmembrane bound inclusions in the erythrocyte cytoplasm, or to the parasitophorous vacuolar membrane accumulate in this BFA-induced compartment. A single BFA-induced compartment is detected per parasite and the various exported proteins colocalize to this compartment regardless of their final destinations. Parasite membrane proteins do not accumulate in this novel compartment, but accumulate in the endoplasmic reticulum (ER), suggesting that the parasite has two secretory pathways. This alternate secretory pathway is established immediately after merozoite invasion and at least some dense granule proteins also use the alternate pathway. The BFA-induced compartment exhibits properties that are similar to the ER, but it is clearly distinct from the ER. We propose to call this new organelle the secondary ER of apicomplexa. This ER-like organelle is an early, if not the first, step in the export of Plasmodium proteins into the host erythrocyte. PMID:9256443

Wiser, Mark F.; Lanners, H. Norbert; Bafford, Richard A.; Favaloro, Jenny M.

1997-01-01

334

The story of cell secretion: events leading to the discovery of the 'porosome' - the universal secretory machinery in cells  

Microsoft Academic Search

Cell secretion has come of age, and a century old quest has been elegantly solved. We have come a long way since earlier observations of what appeared to be 'fibrillar regions' at the cell plasma membrane, and electrophysiological studies suggesting the presence of 'fusion pores' at the cell plasma membrane where secretion occurs. Finally, the fusion pore or 'porosome' has

S. Jeftinija

2006-01-01

335

Delayed Coupling to Feedback Inhibition during a Critical Period for the Integration of Adult-Born Granule Cells.  

PubMed

Developing granule cells (GCs) of the adult dentate gyrus undergo a critical period of enhanced activity and synaptic plasticity before becoming mature. The impact of developing GCs on the activity of preexisting dentate circuits remains unknown. Here we combine optogenetics, acute slice electrophysiology, and in vivo chemogenetics to activate GCs at different stages of maturation to study the recruitment of local target networks. We show that immature (4-week-old) GCs can efficiently drive distal CA3 targets but poorly activate proximal interneurons responsible for feedback inhibition (FBI). As new GCs transition toward maturity, they reliably recruit GABAergic feedback loops that restrict spiking of neighbor GCs, a mechanism that would promote sparse coding. Such inhibitory loop impinges only weakly in new cohorts of young GCs. A computational model reveals that the delayed coupling of new GCs to FBI could be crucial to achieve a fine-grain representation of novel inputs in the dentate gyrus. PMID:25533485

Temprana, Silvio G; Mongiat, Lucas A; Yang, Sung M; Trinchero, Mariela F; Alvarez, Diego D; Kropff, Emilio; Giacomini, Damiana; Beltramone, Natalia; Lanuza, Guillermo M; Schinder, Alejandro F

2015-01-01

336

Granule-dependent cytolysis of Mycobacterium tuberculosis-infected macrophages by human ??+ T cells has no effect on intracellular mycobacterial viability  

PubMed Central

One of the most important effector functions of activated ??+ T cells in tuberculosis is their strong cytolytic activity against a variety of target cells, including M. tuberculosis-infected macrophages. In the present study, we investigated the relationship between the mechanism of cytolysis utilized by ??+ CTL and intracellular M. tuberculosis survival using a panel of cytolytic human M. tuberculosis-specific ??+ CTL clones. Cytolysis mediated by the ??+ T-cell clones was found to be Ca2+-dependent, sensitive to Cyclosporin A, and was completely abrogated following Sr2+-induced de-granulation of the ??+ T cell effectors. These data demonstrate that ??+ T-cell-mediated cytoxicity was mediated via the granule exocytosis/perforin pathway. Despite significant cytolytic activity against mycobacteria infected U937 cells, the ??+ CTL clones had no impact on the survival of intracellular M. tuberculosis. PMID:11678902

Passmore, J S; Glashoff, R H; Lukey, P T; Ress, S R

2001-01-01

337

Toxoplasma gondii Rhoptry Discharge Correlates with Activation of the Early Growth Response 2 Host Cell Transcription Factor  

Microsoft Academic Search

Toxoplasma gondii is a ubiquitous apicomplexan parasite that can cause severe disease in fetuses and immune-compromised patients. Rhoptries, micronemes, and dense granules, which are secretory or- ganelles unique to Toxoplasma and other apicomplexan parasites, play critical roles in parasite growth and virulence. To understand how these organelles modulate infected host cells, we sought to identify host cell transcription factors triggered

Eric D. Phelps; Kristin R. Sweeney; Ira J. Blader

2008-01-01

338

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

PubMed Central

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

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

2013-01-01

339

The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair  

PubMed Central

Cell-based therapies, using multipotent mesenchymal stem cells (MSCs) for organ regeneration, are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. We compared MSCs isolated from MRL/MpJ mice, known to demonstrate enhanced regenerative capacity, to those from C57BL/6 (WT) mice. Compared with WT-MSCs, MRL-MSCs demonstrated increased proliferation, in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity in a murine model of repair stimulation. The MRL-MSCs also reduced infarct size and improved function in a murine myocardial infarct model compared with WT-MSCs. Genomic and functional analysis indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized by significant up-regulation of specific secreted frizzled-related proteins (sFRPs). Specific knockdown of sFRP2 by shRNA in MRL-MSCs decreased their proliferation and their engraftment in and the vascular density of MRL-MSC-generated experimental granulation tissue. These results led us to generate WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and, when implanted in vivo, recapitulated the MRL phenotype. Peri-infarct intramyocardial injection of sFRP2-MSCs resulted in enhanced engraftment, vascular density, reduced infarct size, and increased cardiac function after myocardial injury in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype in MSCs. PMID:19017790

Alfaro, Maria P.; Pagni, Matthew; Vincent, Alicia; Atkinson, James; Hill, Michael F.; Cates, Justin; Davidson, Jeffrey M.; Rottman, Jeffrey; Lee, Ethan; Young, Pampee P.

2008-01-01

340

Arsenite-Activated JNK Signaling Enhances CPEB4-Vinexin Interaction to Facilitate Stress Granule Assembly and Cell Survival  

PubMed Central

Stress granules (SGs) are compartmentalized messenger ribonucleoprotein particles (mRNPs) where translationally repressed mRNAs are stored when cells encounter environmental stress. Cytoplasmic polyadenylation element-binding protein (CPEB)4 is a sequence-specific RNA-binding protein and translational regulator. In keeping with the results obtained from the study of other RNA-binding proteins, we found CPEB4 localized in SGs in various arsenite-treated cells. In this study, we identified that Vinexin, a CPEB4-interacting protein, is a novel component of SGs. Vinexin is a SH3-domain-containing adaptor protein and affects cell migration through its association with Vinculin to localize at focal adhesions (FAs). Unexpectedly, Vinexin is translocated from FAs to SGs under arsenite-induced stress. The recruitment of Vinexin to SGs depends on its interaction with CPEB4 and influences SG formation and cell survival. Arsenite-activated c-Jun N-terminal kinase (JNK) signaling enhances the association between CPEB4 and Vinexin, which consequently facilitates SG localization of Vinexin. Taken together, this study uncovers a novel interaction between a translational regulator and an adaptor protein to influence SG assembly and cell survival. PMID:25237887

Chang, Yu-Wei; Huang, Yi-Shuian

2014-01-01

341

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

PubMed Central

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

Long, Eric O.

2010-01-01

342

Microbial communities of aerobic granules: granulation mechanisms.  

PubMed

Aerobic granulation is an advanced biological wastewater treatment technology. This study for the first time identified the microbial communities of sliced samples of mature granules by polymerase chain reaction (PCR) amplification and denaturing gradient gel electrophoresis (DGGE) technique and those of whole growing granules by high-throughput sequencing technique. The sliced sample study revealed that mature granules have a spherical core with anaerobic Rhodocyclaceae covered by an outer spherical shell with both aerobic and anaerobic strains. The growing granule study showed that the flocculated flocs were first transited to young granules with increased abundances of Flavobacteriaceae, Xanthomonadaceae, Rhodobacteraceae and Microbacteriaceae, then the abundances of anaerobic strains were increased owing to the formation of anaerobic core. Since the present granules were cultivated from flocculated flocs, the microbial community data suggested that granules were formed via a deterministic rather than via a random aggregation-disintegration mechanism. PMID:25063977

Lv, Yi; Wan, Chunli; Lee, Duu-Jong; Liu, Xiang; Tay, Joo-Hwa

2014-10-01

343

Twin screw granulation: steps in granule growth.  

PubMed

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

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

2012-11-15

344

Circadian Gene Expression Regulates Pulsatile Gonadotropin-Releasing Hormone (GnRH) Secretory Patterns in the Hypothalamic GnRH-Secreting GT1–7 Cell Line  

PubMed Central

Although it has long been established that episodic secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus is required for normal gonadotropin release, the molecular and cellular mechanisms underlying the synchronous release of GnRH are primarily unknown. We used the GT1–7 mouse hypothalamic cell line as a model for GnRH secretion, because these cells release GnRH in a pulsatile pattern similar to that observed in vivo. To explore possible molecular mechanisms governing secretory timing, we investigated the role of the molecular circadian clock in regulation of GnRH secretion. GT1–7 cells express many known core circadian clock genes, and we demonstrate that oscillations of these components can be induced by stimuli such as serum and the adenylyl cyclase activator forskolin, similar to effects observed in fibroblasts. Strikingly, perturbation of circadian clock function in GT1–7 cells by transient expression of the dominant-negative Clock-?19 gene disrupts normal ultradian patterns of GnRH secretion, significantly decreasing mean pulse frequency. Additionally, overexpression of the negative limb clock gene mCry1 in GT1–7 cells substantially increases GnRH pulse amplitude without a commensurate change in pulse frequency, demonstrating that an endogenous biological clock is coupled to the mechanism of neurosecretion in these cells and can regulate multiple secretory parameters. Finally, mice harboring a somatic mutation in the Clock gene are subfertile and exhibit a substantial increase in estrous cycle duration as revealed by examination of vaginal cytology. This effect persists in normal light/dark (LD) cycles, suggesting that a suprachiasmatic nucleus-independent endogenous clock in GnRH neurons is required for eliciting normal pulsatile patterns of GnRH secretion. PMID:14657179

Chappell, Patrick E.; White, Rachel S.; Mellon, Pamela L.

2010-01-01

345

Selective disruption of Tcf7l2 in the pancreatic ? cell impairs secretory function and lowers ? cell mass.  

PubMed

Type 2 diabetes (T2D) is characterized by ? cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired ? cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the ? cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in ? cells from the earliest expression of the Ins1 gene (?E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2(fl/fl)::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks, respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcfl2(fl/fl)::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 (?20%) and Glp1r (?40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca(2+) increases, and connectivity between individual ? cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed ?30% decrease in ? cell mass in pancreata from Tcfl2(fl/fl)::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of ? cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed. PMID:25355422

Mitchell, Ryan K; Mondragon, Angeles; Chen, Lingling; Mcginty, James A; French, Paul M; Ferrer, Jorge; Thorens, Bernard; Hodson, David J; Rutter, Guy A; Da Silva Xavier, Gabriela

2015-03-01

346

Ca2+-induced exocytosis in individual human neutrophils: high- and low-affinity granule populations and submaximal responses.  

PubMed Central

We have investigated Ca2+-induced exocytosis from human neutrophils using the whole cell patch-clamp capacitance technique. Microperfusion of Ca2+ buffer solutions (<30 nM to 5 mM free Ca2+) through the patch-clamp pipette revealed a biphasic activation of exocytosis by Ca2+. The first phase was characterized by high affinity (1.5-5 microM) and low apparent cooperativity (<=2) for Ca2+, and the second phase by low affinity (approximately 100 microM) and high cooperativity (>6). Only the second phase was accompanied by loss of myeloperoxidase, suggesting that the low-affinity exocytosis reflected release of peroxidase-positive (primary) granules, while the high-affinity exocytosis reflected release of peroxidase-negative (secondary and tertiary) granules. At submaximal Ca2+ concentrations, only a fraction of a given granule population was released. This submaximal release cannot simply be explained by Ca2+ modulation of the rate of exocytosis, and it suggests that the secretory response of individual cells is adjusted to the strength of the stimulus. The Ca2+ dependence of the high- and low-affinity phases of neutrophil exocytosis bears a resemblance to endocrine and neuronal exocytosis, respectively. The occurrence of such high- and low-affinity exocytosis in the same cell is novel, and suggests that the Ca2+ sensitivity of secretion is granule-, rather than cell-specific. PMID:9482725

Nüsse, O; Serrander, L; Lew, D P; Krause, K H

1998-01-01

347

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

PubMed

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca(2+) to directly measure the Ca(2+) sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca(2+) sensitivity of release in that a small proportion of granules makes up a highly Ca(2+)-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca(2+). A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca(2+) are not located close to Ca(2+) channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca(2+)-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca(2+), suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca(2+) sensor that is poised to respond to modest, global elevations of [Ca(2+)](i). PMID:15572344

Yang, Yan; Gillis, Kevin D

2004-12-01

348

Interaction of collagen with hydrophobic protein granules in the egg capsule of the dogfish scyliorhinus canicula.  

PubMed

The egg capsule of the dogfish is a composite material containing collagenous fibrils and 2 mum spherical hydrophobic protein granules. The latter appear to owe much of their hydrophobicity to an exceptionally high tyrosine content (approximately 20% of total amino acid residues). The hydrophobic component appears to form as an emulsion in the secretory granules of the D and E zone gland cells of the nidamental gland. Droplets of the hydrophobic material appear to become coated with remarkably regular layers of radially-arranged collagen molecules which form a series of concentric, evenly spaced layers around each hydrophobic granule. Numerous disclinations were seen where the layers around adjacent granules interfered with one another. The layers are thought to represent a lamellar liquid crystalline phase previously described for this collagen (Knight et al., 1993). The fine structural appearance of the concentric layers and evidence for radial arrangement of collagen molecules within them is compatible with the suggestion that the layers are built from a dumbbell-shaped unit approximately 35 nm long with hydrophobic groups concentrated at the ends. This unit may represent a dumbbell-shaped molecule or an oligomer of two or more molecules lying parallel with one another in a head-to-tail arrangement. Such a unit can be readily incorporated into models for the micellar, hexagonal columnar and final fibrillar phases previously described for this collagen (Knight et al., 1993). Evidence from the TEM study of stretched egg capsule wall suggests that there is a mechanical interaction between the hydrophobic granules and the collagen fibrils in the fully formed material. We suggest that the radial, concentric layered arrangement of collagen molecules is established by hydrophobic interactions within the liquid crystalline material and locked into place by oxidative covalent cross-linking to give a 3-dimensional cross-linked meshwork of collagen fibrils and hydrophobic granules. The latter arrangement helps to account for the high tensilestrength and toughness of this material. PMID:18621266

Knight, D P; Feng, D

1994-04-01

349

Growth control of A431 cells in protein-free medium: Secretory products do not affect cell growth  

Microsoft Academic Search

Summary  A431 cells grew at similar rates in protein-free Coon's modified Ham's F12 medium (PF-C-F12) with and without added bovine\\u000a calf serum. The cells secreted a heparin-binding growth factor and a type-? transforming growth factor, but their growth in\\u000a PF-C-F12 was not affected by these factors, or by DNA synthesis factor from Rhodamine fibrosarcoma, basic fibroblast growth\\u000a factor, insulin, human transferrin,

Yoshishige Masuda; Yoshino Yoshitake; Katsuzo Nishikawa

1988-01-01

350

Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein  

Microsoft Academic Search

Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB- GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with

Irene Wacker; Christoph Kaether; Andreas Krömer; Andrea Migala; Wolf Almers; Hans-Hermann Gerdes

1997-01-01

351

Molecular characterization of mouse lens epithelial cell lines and their suitability to study RNA granules and cataract associated genes.  

PubMed

The discovery of cytosolic RNA granule (RG) component proteins associated with human cataract has initiated investigations on post-transcriptional mechanisms of gene expression control in the lens. Application of established mouse lens epithelial cell lines (LECs) can provide rapid insights on RG function in lens cells, especially because mouse mutants in several RG components are not available. However, although these LECs represent potential reagents for such analyses, they are uncharacterized for lens gene expression or RG formation. Therefore, a detailed molecular and cellular characterization of three permanent mouse LECs 17EM15, 21EM15 and ?TN4 is performed in this study. Comparative analysis between microarray gene expression datasets on LEC 21EM15 and iSyTE lens tissue demonstrates that 30% of top 200 iSyTE identified lens-enriched genes are expressed in these cells. Majority of these candidates are independently validated to either have lens expression, function or linkage to cataract. Moreover, analysis of microarray data with genes described in Cat-Map, an online database of cataract associated genes and loci, demonstrates that 131 genes linked to cataract loci are expressed in 21EM15 cells. Furthermore, gene expression in LECs is compared to isolated lens epithelium or fiber cells by qRT-PCR and by comparative analyses with publically available epithelium or fiber-specific microarray and RNA-seq (sequencing) datasets. Expression of select candidate genes was validated by regular and real-time quantitative RT-PCR. Expression of lens epithelium-enriched genes Foxe3, Pax6, Anxa4 and Mcm4 is up-regulated in LEC lines, compared to isolated lens fiber cells. Moreover, similar to isolated lens epithelium, all three LECs exhibit down-regulation of fiber cell-expressed genes Crybb1, Mip and Prox1 when compared to fiber cells. These data indicate that the LEC lines exhibit greater similarity to lens epithelium than to fiber cells. Compared to non-lens cell line NIH3T3, LECs exhibit significantly enriched expression of transcription factors with important function in the lens, namely Pax6, Foxe3 and Prox1. In addition to these genes, all three LECs also express key lens- and cataract-associated genes, namely Dkk3, Epha2, Hsf4, Jag1, Mab21l1, Meis1, Pknox1, Pou2f1, Sfrp1, Sparc, Tdrd7 and Trpm3. Additionally, 21EM15 microarrays indicate expression of Chmp4b, Cryab and Tcfap2a among others important genes. Immunostaining with makers for Processing bodies (P-bodies) and Stress granules (SGs) demonstrates that these classes of RGs are robustly expressed in all three LECs. Moreover, under conditions of stress, 17EM15 and ?TN4 exhibit significantly higher numbers of P-bodies and SGs compared to NIH3T3 cells. In sum, these data indicate that mouse LECs 21EM15, 17EM15 and ?TN4 express key lens or cataract genes, are similar to lens epithelium than fiber cells, and exhibit high levels of P-bodies and SGs, indicating their suitability for investigating gene expression control and RG function in lens-derived cells. PMID:25530357

Terrell, Anne M; Anand, Deepti; Smith, Sylvie F; Dang, Christine A; Waters, Stephanie M; Pathania, Mallika; Beebe, David C; Lachke, Salil A

2015-02-01

352

Late Steps in Secretory Lysosome Exocytosis in Cytotoxic Lymphocytes  

PubMed Central

Natural Killer cells are a subset of cytotoxic lymphocytes that are important in host defense against infections and transformed cells. They exert this function through recognition of target cells by cell surface receptors, which triggers a signaling program that results in a re-orientation of the microtubule organizing center and secretory lysosomes toward the target cell. Upon movement of secretory lysosomes to the plasma membrane and subsequent fusion, toxic proteins are released by secretory lysosomes in the immunological synapse which then enter and kill the target cell. In this minireview we highlight recent progress in our knowledge of late steps in this specialized secretion pathway and address important open questions. PMID:24302923

van der Sluijs, Peter; Zibouche, Mallik; van Kerkhof, Peter

2013-01-01

353

Boric acid induces cytoplasmic stress granule formation, eIF2? phosphorylation, and ATF4 in prostate DU-145 cells.  

PubMed

Dietary boron intake is associated with reduced prostate and lung cancer risk and increased bone mass. Boron is absorbed and circulated as boric acid (BA) and at physiological concentrations is a reversible competitive inhibitor of cyclic ADP ribose, the endogenous agonist of the ryanodine receptor calcium (Ca(+2)) channel, and lowers endoplasmic reticulum (ER) [Ca(2+)]. Low ER [Ca(2+)] has been reported to induce ER stress and activate the eIF2?/ATF4 pathway. Here we report that treatment of DU-145 prostate cells with physiological levels of BA induces ER stress with the formation of stress granules and mild activation of eIF2?, GRP78/BiP, and ATF4. Mild activation of eIF2? and its downstream transcription factor, ATF4, enables cells to reconfigure gene expression to manage stress conditions and mild activation of ATF4 is also required for the differentiation of osteoblast cells. Our results using physiological levels of boric acid identify the eIF2?/ATF pathway as a plausible mode of action that underpins the reported health effects of dietary boron. PMID:25425213

Henderson, Kimberly A; Kobylewski, Sarah E; Yamada, Kristin E; Eckhert, Curtis D

2015-02-01

354

AtBXL1 Encodes a Bifunctional ?-d-Xylosidase/?-l-Arabinofuranosidase Required for Pectic Arabinan Modification in Arabidopsis Mucilage Secretory Cells1[C][W][OA  

PubMed Central

Following pollination, the epidermal cells of the Arabidopsis (Arabidopsis thaliana) ovule undergo a complex differentiation process that includes the synthesis and polar secretion of pectinaceous mucilage followed by the production of a secondary cell wall. Wetting of mature seeds leads to the rapid bursting of these mucilage secretory cells to release a hydrophilic gel that surrounds the seed and is believed to aid in seed hydration and germination. A novel mutant is identified where mucilage release is both patchy and slow and whose seeds display delayed germination. While developmental analysis of mutant seeds reveals no change in mucilage secretory cell morphology, changes in monosaccharide quantities are detected, suggesting the mucilage release defect results from altered mucilage composition. Plasmid rescue and cloning of the mutant locus revealed a T-DNA insertion in AtBXL1, which encodes a putative bifunctional ?-d-xylosidase/?-l-arabinofuranosidase that has been implicated as a ?-d-xylosidase acting during vascular development. Chemical and immunological analyses of mucilage extracted from bxl1 mutant seeds and antibody staining of developing seed coats reveal an increase in (1?5)-linked arabinans, suggesting that BXL1 is acting as an ?-l-arabinofuranosidase in the seed coat. This implication is supported by the ability to rescue mucilage release through treatment of bxl1 seeds with exogenous ?-l-arabinofuranosidases. Together, these results suggest that trimming of rhamnogalacturonan I arabinan side chains is required for correct mucilage release and reveal a new role for BXL1 as an ?-l-arabinofuranosidase acting in seed coat development. PMID:19458117

Arsovski, Andrej A.; Popma, Theodore M.; Haughn, George W.; Carpita, Nicholas C.; McCann, Maureen C.; Western, Tamara L.

2009-01-01

355

Human microRNA-24 modulates highly pathogenic avian-origin H5N1 influenza A virus infection in A549 cells by targeting secretory pathway furin.  

PubMed

A common critical cellular event that many human enveloped viruses share is the requirement for proteolytic cleavage of the viral glycoprotein by furin in the host secretory pathway. For example, the furin-dependent proteolytic activation of highly pathogenic (HP) influenza A (infA) H5 and H7 haemagglutinin precursor (HA0) subtypes is critical for yielding fusion-competent infectious virions. In this study, we hypothesized that viral hijacking of the furin pathway by HP infA viruses to permit cleavage of HA0 could represent a novel molecular mechanism controlling the dynamic production of fusion-competent infectious virus particles during the viral life cycle. We explored the biological role of a newly identified furin-directed human microRNA, miR-24, in this process as a potential post-transcriptional regulator of the furin-mediated activation of HA0 and production of fusion-competent virions in the host secretory pathway. We report that miR-24 and furin are differentially expressed in human A549 cells infected with HP avian-origin infA H5N1. Using miR-24 mimics, we demonstrated a robust decrease in both furin mRNA levels and intracellular furin activity in A549 cells. Importantly, pretreatment of A549 cells with miR-24 mimicked these results: a robust decrease of H5N1 infectious virions and a complete block of H5N1 virus spread that was not observed in A549 cells infected with low-pathogenicity swine-origin infA H1N1 virus. Our results suggest that viral-specific downregulation of furin-directed microRNAs such as miR-24 during the life cycle of HP infA viruses may represent a novel regulatory mechanism that governs furin-mediated proteolytic activation of HA0 glycoproteins and production of infectious virions. PMID:25234642

Loveday, Emma-Kate; Diederich, Sandra; Pasick, John; Jean, François

2015-01-01

356

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

Microsoft Academic Search

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

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

1980-01-01

357

Gamma-aminobutyric acidA receptor function is desensitised in rat cultured cerebellar granule cells following chronic flunitrazepam treatment.  

PubMed

This study examined gamma-aminobutyric acidA (GABA(A)) receptor function in cultured rat cerebellar granule cells by using microphysiometry following chronic flunitrazepam exposure, and correlated the findings with the alpha1 and beta2/3 subunit protein expression and [3H]muscimol binding after the same treatment paradigm. Flunitrazepam treatment reduced (p < 0.05) the maximal GABA-stimulated increase in extracellular acidification rate (Emax) (16.5 +/- 1.2% and 11.3 +/- 1.0%, 2-day control and treated cells, respectively; 17.4 +/- 1.0% and 9.9 +/- 0.7%, 7-day control and treated cells, respectively; best-fit Emax +/- SEM, n = 7), without affecting the GABA concentration required to elicit 50% of maximal response (EC50) (1.2 +/- 1.7 and 2.3 +/- 1.8 microM, 2-day control and treated cells, respectively; 1.7 +/- 1.5 and 1.5 +/- 1.5 microM, 7-day control and treated cells, respectively; best-fit EC50 +/- SEM, n = 7). Flunitrazepam exposure also abolished the flunitrazepam potentiation of the GABA response, caused a transient reduction of the GABA(A) receptor alpha1 and beta2/3 subunit proteins over the initial 2 days, but did not alter [3H]muscimol binding compared with vehicle-treated cells. The results suggest that changes in GABA(A) receptor subunit protein expression, rather than loss of [3H]muscimol binding sites, underlie the chronic flunitrazepam-mediated desensitisation of GABA(A) receptor function. PMID:9721749

Brown, M J; Wood, M D; Coldwell, M C; Bristow, D R

1998-09-01

358

Secretory parvocellular neurons in the rostral hypothalamus and in the tuberal complex of Passer domesticus  

Microsoft Academic Search

Numerous secretory perikarya were found in the suprachiasmatic, medial preoptio and anterior hypothalamic nuclei of Passer domesticus. The secretory granules of these parvocellular neurons fall into the following ranges: 1000 Å; 1300–1500 Å; 1800–2000 Å. The specialized parvocellular neurons of the rostral hypothalamus form unit-like clusters. They are embedded in a neuropil rich in synaptic structures. Many presynaptic terminals contain

A. Oksche; H. Kirschstein; H. G. Hartwig; H. J. Oehmke; D. S. Farner

1974-01-01

359

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

NASA Astrophysics Data System (ADS)

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

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

1999-10-01

360

Glycolytic enzyme upregulation and numbness of mitochondrial activity characterize the early phase of apoptosis in cerebellar granule cells.  

PubMed

Alzheimer's disease (AD) and cancer proceed via one or more common molecular mechanisms: a metabolic shift from oxidative phosphorylation to glycolysis-corresponding to the activation of the Warburg effect-occurs in both diseases. The findings reported in this paper demonstrate that, in the early phase of apoptosis, glucose metabolism is enhanced, i.e. key proteins which internalize and metabolize glucose-glucose transporter, hexokinase and phosphofructokinase-are up-regulated, in concomitance with a parallel decrease in oxygen consumption by mitochondria and increase of L-lactate accumulation. Reversal of the glycolytic phenotype occurs in the presence of dichloroacetate, inhibitor of the pyruvate dehydrogenase kinase enzyme, which speeds up apoptosis of cerebellar granule cells, reawakening mitochondria and then modulating glycolytic enzymes. Loss of the adaptive advantage afforded by aerobic glycolysis, which occurs in the late phase of apoptosis, exacerbates the pathological processes underlying neurodegeneration, leading inevitably the cell to death. In conclusion, the data propose that both aerobic, i.e. Warburg effect, essentially due to the protective numbness of mitochondria, and anaerobic glycolysis, rather due to the mitochondrial impairment, characterize the entire time frame of apoptosis, from the early to the late phase, which mimics the development of AD. PMID:25351440

Bobba, A; Amadoro, G; La Piana, G; Calissano, P; Atlante, A

2015-01-01

361

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

PubMed

The dentate gyrus (DG) is thought to enable efficient hippocampal memory acquisition via pattern separation. With patterns defined as spatiotemporally distributed action potential sequences, the principal DG output neurons (granule cells, GCs), presumably sparsen and separate similar input patterns from the perforant path (PP). In electrophysiological experiments, we have demonstrated that during temporal lobe epilepsy (TLE), GCs downscale their excitability by transcriptional upregulation of "leak" channels. Here we studied whether this cell type-specific intrinsic plasticity is in a position to homeostatically adjust DG network function. We modified an established conductance-based computer model of the DG network such that it realizes a spatiotemporal pattern separation task, and quantified its performance with and without the experimentally constrained leaky GC phenotype. Two proposed TLE seizure mechanisms were implemented in various degrees and combinations: recurrent GC excitation via mossy fiber sprouting and increased PP input. While increasing PP strength degraded pattern separation only gradually, already the slight elevation of sprouting drastically (non-linearly) impaired pattern separation. In most tested hyperexcitable networks, leaky GCs ameliorated pattern separation. However, in some sprouting situations with all-or-none seizure behavior, pattern separation was disabled with and without leaky GCs. In the mild sprouting (and PP increase) region of non-linear impairment, leaky GCs were particularly effective in restoring pattern separation performance. These results are compatible with the hypothesis that the experimentally observed intrinsic rescaling of GCs serves to maintain the physiological function of the DG network. © 2014 Wiley Periodicals, Inc. PMID:25269417

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

2014-10-01

362

Ultrastructural Study on Colocalization of Glucagon-Like Peptide (GLP)-1 with GLP-2 in Chicken Intestinal L-Cells  

PubMed Central

ABSTRACT Colocalization of glucagon-like peptide (GLP)-1 with GLP-2 in L-cells was investigated in the chicken ileum by using double immunofluorescent and immunocytochemical techniques. Ultrastructural features of L-cells were also clarified in this study. L-cells showing immunoreactivity for both GLP-1 and GLP-2 were distributed in the whole ileum. They showed comma-like or flask-like shape and were located in epithelium of crypts and lower part of intestinal villi. L-cells showing GLP-1-immunoreactivity only were found in epithelium of lower and middle parts of intestinal villi. Transmission electron microscopy indicated that L-cells identified by colloidal gold-labeled immunocytochemistry were covered apically with microvilli, open-type and contained many secretory granules in their perikarya. These secretory granules without halo were round to oval in shape and showed moderate electron density. The longest and shortest diameters of secretory granules were 355 ± 62 nm (mean ± SD) and 287 ± 48 nm, respectively. Double labeling immunocytochemistry using two different sizes of particles (6 and 12 nm in diameter) of colloidal gold revealed that GLP-1 colocalized with GLP-2 in the same secretory granules. This study advances new morphological data about the endocrine system of the chicken small intestine. PMID:23759686

NISHIMURA, Kei; HIRAMATSU, Kohzy; MONIR, Mohammad M.; TAKEMOTO, Chihiro; WATANABE, Takafumi

2013-01-01

363

Souffle/Spastizin Controls Secretory Vesicle Maturation during Zebrafish Oogenesis  

PubMed Central

During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research. PMID:24967841

Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

2014-01-01

364

Cocultures of GFP(+) -granule cells with GFP(-) -pyramidal cells and interneurons for the study of mossy fiber neurotransmission with paired recordings.  

PubMed

Synaptic transmission of the granule cells (GCs) via their axons, the mossy fibers (MFs), is traditionally studied on acutely prepared or cultured slices. Usually, extracellular, bulk or minimal stimulation is used to evoke transmitter release from MF terminals, while recording from their postsynaptic target cells, the pyramidal cells and interneurons of CA3. However, the ideal method to assess MF neurotransmission, the simultaneous recording of a presynaptic GC and one of its target cells, is extremely difficult to achieve using slices. Alternatively, cultures of GCs establishing autapses have been developed, but in these, GCs do not contact their natural targets. We developed cocultures of GCs, dissociated from transgenic GFP(+) rats, with pyramidal cells and interneurons of CA3, dissociated from wild-type rats, and confirmed the expression of cell-specific markers by immunofluorescence. We conducted recordings of GFP(+) -GCs synaptically connected with their GFP(-) -target cells, and demonstrate that synaptic transmission and its plasticity have the signature of transmission of MF. Besides being strongly depressed by activation of mGluRs, high frequency activation of GC-to-pyramidal cells synapses undergo LTP, while GC-to-interneuron synapses undergo LTD. This coculture method allows a high reproducibility of recording connected pairs of identified cells, constituting a valuable tool to study MF transmission, as well as different combinations of identifiable pre- and postsynaptic cells. PMID:23436451

Osorio, Beatriz; León, Uriel; Galván, Emilio J; Gutiérrez, Rafael

2013-04-01

365

Diverse Effects of Eosinophil Cationic Granule Proteins on IMR32 Nerve Cell Signaling and Survival  

Microsoft Academic Search

Activatedeosinophilsreleasepotentiallytoxiccationicgranularpro- teins, including the major basic proteins (MBP) and eosinophil- derived neurotoxin (EDN). However, in inflammatory conditions including asthma and inflammatory bowel disease, localization of eosinophils to nerves is associated with nerve plasticity, specifically remodeling. In previous in vitro studies, we have shown that eosin- ophil adhesion to IMR-32 nerve cells, via nerve cell intercellular adhesion molecule-1, results in an

Ross K. Morgan; Richard W. Costello; Niamh Durcan; Paul J. Kingham; Gerald J. Gleich; W. Graham McLean; Marie-Therese Walsh

2005-01-01

366

Increased cell proliferation of mouse fibroblast NIH-3T3 in vitro induced by excretory/secretory product(s) from Opisthorchis viverrini.  

PubMed

Infection by Opisthorchis viverrini is a strong risk factor for cholangiocarcinoma. However, the mechanism by which the parasite is involved in carcinogenesis is not clear. In addition to the direct damage of the bile duct epithelium via direct contact with O. viverrini, the excretory/secretory (ES) product(s) released from the parasites may play important roles in this process. We therefore investigated the responses of a fibroblast cell line, NIH-3T3, to ES product(s) released from O. viverrini by using a non-contact co-culture technique. In this culture system, the parasites in the upper chamber had no direct contact with the NIH-3T3 cells in the lower chamber of the culture plate. The results indicated a marked increase in NIH-3T3 cell proliferation in the non-contact co-culture condition with either 0% or 10% calf serum in the medium compared with that without parasites. ES product(s) increased cell proliferation by stimulating the expression of phosphorylated retinoblastoma (pRB) and cyclin D1, the key proteins in driving cells through the G1/S transition point of the cell cycle. This led to the induction of cells going into the S-phase of the cell cycle. ES product(s) also changed the morphology of NIH-3T3 cells to a refractive and narrow shape, which allowed the cells to proliferate in the limited culture area. For the first time, we have been able to demonstrate increased cell proliferation induced by the ES product(s) from O. viverrini; this finding may clarify how O. viverrini ES product(s) affect human bile duct epithelium during cholangiocarcinogenesis. PMID:15521634

Thuwajit, C; Thuwajit, P; Kaewkes, S; Sripa, B; Uchida, K; Miwa, M; Wongkham, S

2004-10-01

367

An ultrastructural study of goblet cells in rat nasal mucosa as revealed by the quick-freezing method.  

PubMed

In order to clarify the natural ultrastructure of goblet cells in the rat nasal mucosa, they were examined by the quick-freezing and freeze-substitution (QF-FS) or deep-etching (QF-DE) methods for comparison with conventional fixation methods. Some nasal mucosal tissues were unstimulated; others were stimulated with acetylcholine or substance P. The QF-FS method yielded fewer artefacts on transmission electron microscopy than conventional fixation methods. In the stimulated goblet cells, most of the secretory granules appeared to be loose in the matrix and more distorted in shape. By the QF-DE method, they were observed 3-dimensionally to be larger in size and aggregated together. In contrast, the secretory granules in the unstimulated goblet cells were mostly round and small, and separate from each other. It is concluded that the ultrastructure of secretory granules is artefactually modified by conventional fixation methods and that granule structure in goblet cells alters during the secretory process. PMID:8763482

Shimomura, S; Hisamatsu, K; Fujii, Y; Ohno, S

1996-06-01

368