Science.gov

Sample records for cell secretory granules

  1. The use of permeabilized cells to investigate secretory granule biogenesis.

    PubMed

    Ling, W L; Siddhanta, A; Shields, D

    1998-10-01

    To investigate the mechanism of secretory granule biogenesis in endocrine cells, our laboratory used rat anterior pituitary GH3 cells which secrete growth hormone and prolactin. Here we describe a simple and rapid procedure for generating permeabilized cells to dissect molecular mechanisms involved in nascent secretory vesicle budding from the trans-Golgi network (TGN). Using this system, we demonstrate that vesicle budding is temperature, energy, and cytosol dependent; in addition, cytosol from a variety of cells, including yeast (Saccharomyces cerevisiae), can support vesicle release. The budding of nascent secretory vesicles from the TGN is stimulated by a phospholipase D activity that is associated with Golgi membranes. Our results suggest that phospholipid metabolism plays an important role in the release of nascent secretory vesicles from the TGN. PMID:9790860

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

    SciTech Connect

    Baconnais, S.; Delavoie, F. |; Zahm, J.M.; Milliot, M.; Castillon, N.; Terryn, C.; Banchet, V.; Michel, J.; Danos, O.; Merten, M.; Chinet, T.; Zierold, K.; Bonnet, N.; Puchelle, E. , E-Mail: edith.puchelle@univ-reims.fr; Balossier, G.

    2005-10-01

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

  3. Rab3D Is Critical for Secretory Granule Maturation in PC12 Cells

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2010-01-01

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

  5. Pasteurella multocida toxin: Targeting mast cell secretory granules during kiss-and-run secretion.

    PubMed

    Danielsen, Elisabeth M; Christiansen, Nina; Danielsen, E Michael

    2016-02-01

    Pasteurella multocida toxin (PMT), a virulence factor of the pathogenic Gram-negative bacterium P. multocida, is a 146kDa protein belonging to the A-B class of toxins. Once inside a target cell, the A domain deamidates the ?-subunit of heterotrimeric G-proteins, thereby activating downstream signaling cascades. However, little is known about how PMT selects and enters its cellular targets. We therefore studied PMT binding and uptake in porcine cultured intestinal mucosal explants to identify susceptible cells in the epithelium and underlying lamina propria. In comparison with Vibrio cholera B-subunit, a well-known enterotoxin taken up by receptor-mediated endocytosis, PMT binding to the epithelial brush border was scarce, and no uptake into enterocytes was detected by 2h, implying that none of the glycolipids in the brush border are a functional receptor for PMT. However, in the lamina propria, PMT distinctly accumulated in the secretory granules of mast cells. This also occurred at 4C, ruling out endocytosis, but suggestive of uptake via pores that connect the granules to the cell surface. Mast cell granules are known to secrete their contents by a "kiss-and-run" mechanism, and we propose that PMT may exploit this secretory mechanism to gain entry into this particular cell type. PMID:26763205

  6. Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity.

    PubMed

    Sutton, Vivien R; Brennan, Amelia J; Ellis, Sarah; Danne, Jill; Thia, Kevin; Jenkins, Misty R; Voskoboinik, Ilia; Pejler, Gunnar; Johnstone, Ricky W; Andrews, Daniel M; Trapani, Joseph A

    2016-03-01

    The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means. PMID:26756195

  7. BACE2 is stored in secretory granules of mouse and rat pancreatic beta cells.

    PubMed

    Finzi, Giovanna; Franzi, Francesca; Placidi, Claudia; Acquati, Francesco; Palumbo, Elisa; Russo, Antonella; Taramelli, Roberto; Sessa, Fausto; La Rosa, Stefano

    2008-01-01

    BACE2 is a protease homologous to BACE1 protein, an enzyme involved in the amyloid formation of Alzheimer disease (AD). However, despite the high homology between these two proteins, the biological role of BACE2 is still controversial, even though a few studies have suggested a pathogenetic role in sporadic inclusion-body myositis and hereditary inclusion-body myopathy, which are characterized by vacuolization of muscular fibers with intracellular deposits of proteins similar to those found in the brain of AD patients. Although BACE2 has also been identified in the pancreas, its function remains unknown and its specific localization in different pancreatic cell types has not been definitively ascertained. For these reasons, the authors have investigated the cellular and subcellular localization of BACE2 in normal rodent pancreases. BACE2 immunoreactivity was found in secretory granules of beta cells, co-stored with insulin and IAPP, while it was lacking in the other endocrine and exocrine cell types. The presence of BACE2 in secretory granules of beta cells suggests that it may play a role in diabetes-associated amyloidogenesis. PMID:19117266

  8. PROX1 Promotes Secretory Granule Formation in Medullary Thyroid Cancer Cells.

    PubMed

    Ishii, Jun; Yazawa, Takuya; Chiba, Tomohiro; Shishido-Hara, Yukiko; Arimasu, Yuu; Sato, Hanako; Kamma, Hiroshi

    2016-03-01

    Mechanisms of endocrine secretory granule (SG) formation in thyroid C cells and medullary thyroid cancer (MTC) cells have not been fully elucidated. Here we directly demonstrated that PROX1, a developmental homeobox gene, is transcriptionally involved in SG formation in MTC, which is derived from C cells. Analyses using gene expression databases on web sites revealed that, among thyroid cancer cells, MTC cells specifically and highly express PROX1 as well as several SG-forming molecule genes. Immunohistochemical analyses showed that in vivo MTC and C cells expressed PROX1, although follicular thyroid cancer and papillary thyroid cancer cells, normal follicular cells did not. Knockdown of PROX1 in an MTC cells reduced SGs detected by electron microscopy, and decreased expression of SG-related genes (chromogranin A, chromogranin B, secretogranin II, secretogranin III, synaptophysin, and carboxypeptidase E). Conversely, the introduction of a PROX1 transgene into a papillary thyroid cancer and anaplastic thyroid cancer cells induced the expression of SG-related genes. Reporter assays using the promoter sequence of chromogranin A showed that PROX1 activates the chromogranin A gene in addition to the known regulatory mechanisms, which are mediated via the cAMP response element binding protein and the repressor element 1-silencing transcription factor. Furthermore, chromatin immunoprecipitation-PCR assays demonstrated that PROX1 binds to the transcriptional regulatory element of the chromogranin A gene. In conclusion, PROX1 is an important regulator of endocrine SG formation in MTC cells. PMID:26760117

  9. Evidence for a Ca2+ pool associated with secretory granules in rat submandibular acinar cells.

    PubMed Central

    Martinez, J R; Willis, S; Puente, S; Wells, J; Helmke, R; Zhang, G H

    1996-01-01

    Intracellular Ca2+ stores in rat submandibular acinar cells were characterized using the Ca(2+)-sensitive fluorescent indicator fura 2 and the radiotracer 45Ca2+. Acetylcholine induced a rapid Ca2+ release from a store sensitive to inositol 1,4,5-trisphosphate (IP3) and to thapsigargin (TG). After this store was presumably depleted, ionomycin caused a further increase in cytosolic free Ca2+ concentration ([Ca2+]i), suggesting the presence of an IP3-insensitive Ca2+ release from a store that is more extensive and heterogeneous than the IP3-sensitive one and includes a small mitochondrial component. After both of these stores had been discharged, exposure to monensin caused an additional release of Ca2+ from a third store. This store appears to be associated with secretory granules, since Ca2+ release was significantly reduced when degranulation was induced by isoprenaline. This third store appears to be insensitive to IP3, discharges Ca2+ when the pH gradient across the limiting membrane is collapsed with monensin and only in the presence of both ionomycin and monensin. Ca2+ release from this store is not by Na+/Ca2+ exchange, since simply altering [Na+]i did not cause significant Ca2+ release. In permeabilized cells, IP3 and TG released approx. 35% of 45Ca2+, and ionomycin released an additional 57%, whereas monensin only caused a small additional release, suggesting that only IP3- and ionomycin-sensitive stores are loaded with 45Ca2+ under these conditions. The absence of significant isotope uptake into the ionomycin+monensin-sensitive store may result from a low rate of tracer accumulation or from the lack of Ca2+ pumps in the store. The pattern of response was similar in the presence and absence of mitochondrial inhibitors, indicating that the store is not located in mitochondria. In summary, these results suggest that a substantial IP3-insensitive Ca2+ store is present in secretory granules in rat submandibular acinar cells. PMID:8973577

  10. Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells

    PubMed Central

    Holroyd, Phillip; Lang, Thorsten; Wenzel, Dirk; De Camilli, Pietro; Jahn, Reinhard

    2002-01-01

    During exocytosis, secretory granules fuse with the plasma membrane and discharge their content into the extracellular space. The exocytosed membrane is then reinternalized in a coordinated fashion. A role of clathrin-coated vesicles in this process is well established, whereas the involvement of a direct retrieval mechanism (often called kiss and run) is still debated. Here we report that a significant population of docked secretory granules in the neuroendocrine cell line PC12 fuses with the plasma membrane, takes up fluid-phase markers, and is retrieved at the same position. Fusion allows for complete discharge of small molecules, whereas GFP-labeled neuropeptide Y (molecular mass ?35 kDa) is only partially released. Retrieved granules were preferentially associated with dynamin. Furthermore, recapture is inhibited by guanosine 5?-[?-thio]triphosphate and peptides known to block dynamin function. We conclude that secretory granules can be recaptured immediately after formation of an exocytotic opening by an endocytic reaction that is spatially and temporally coupled to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent fusion, but is not a reversal of the fusion reaction. PMID:12486251

  11. A method for detailed analysis of the structure of mast cell secretory granules by negative contrast imaging

    PubMed Central

    Tanaka, Shotaro; Takakuwa, Yuichi

    2016-01-01

    Secretory granules (SGs) in mast cells contain various molecules that elicit allergy symptoms and are generally considered therapeutic targets. However, the biogenesis, maintenance, regulation, and recycling of these granules remain controversial, mainly due to the lack of suitable live-cell imaging methods. In this study, we applied negative contrast imaging with soluble green fluorescent protein (GFP) expressed in the cytoplasm as a method to validate structural information of mast cell SGs. We evaluated the accuracy of the method in detail, and we demonstrated that it can be used for quantitative analysis. Using this technique, secretory granules, the nucleus, mitochondria, and the cell body were visualized in individual RBL-2H3 mast cells without any influence. When combined with conventional multicolor fluorescence imaging, visualization of SG-associated proteins and SG–SG fusion was achieved. Moreover, 3D images were constructed based on this method, and detailed information on the number, size, and shape of individual SGs was obtained. We found that cell volume was correlated with SG number. In summary, the technique provides valuable and unique data, and will therefore advance SG research. PMID:26997316

  12. A method for detailed analysis of the structure of mast cell secretory granules by negative contrast imaging.

    PubMed

    Tanaka, Shotaro; Takakuwa, Yuichi

    2016-01-01

    Secretory granules (SGs) in mast cells contain various molecules that elicit allergy symptoms and are generally considered therapeutic targets. However, the biogenesis, maintenance, regulation, and recycling of these granules remain controversial, mainly due to the lack of suitable live-cell imaging methods. In this study, we applied negative contrast imaging with soluble green fluorescent protein (GFP) expressed in the cytoplasm as a method to validate structural information of mast cell SGs. We evaluated the accuracy of the method in detail, and we demonstrated that it can be used for quantitative analysis. Using this technique, secretory granules, the nucleus, mitochondria, and the cell body were visualized in individual RBL-2H3 mast cells without any influence. When combined with conventional multicolor fluorescence imaging, visualization of SG-associated proteins and SG-SG fusion was achieved. Moreover, 3D images were constructed based on this method, and detailed information on the number, size, and shape of individual SGs was obtained. We found that cell volume was correlated with SG number. In summary, the technique provides valuable and unique data, and will therefore advance SG research. PMID:26997316

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

    SciTech Connect

    Fujita-Yoshigaki, Junko . E-mail: yoshigaki.junko@nihon-u.ac.jp; Katsumata, Osamu; Matsuki, Miwako; Yoshigaki, Tomoyoshi; Furuyama, Shunsuke; Sugiya, Hiroshi

    2006-05-26

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

  14. AP-1A controls secretory granule biogenesis and trafficking of membrane secretory granule proteins

    PubMed Central

    Bonnemaison, Mathilde; Bäck, Nils; Lin, Yimo; Bonifacino, Juan S.; Mains, Richard; Eipper, Betty

    2014-01-01

    The adaptor protein 1A complex (AP-1A) transports cargo between the trans-Golgi network (TGN) and endosomes. In professional secretory cells, AP-1A also retrieves material from immature secretory granules (SGs). The role of AP-1A in SG biogenesis was explored using AtT-20 corticotrope tumor cells expressing reduced levels of the AP-1A μ1A subunit. A two-fold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non-condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue-stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α-amidating monooxygenase-1 (PAM-1), integral membrane enzymes that enter immature SGs. The non-condensing SGs contained POMC products and PAM-1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM-1 into PHM was unaltered, but PHM basal secretion was increased in sh-μ1A PAM-1 cells. Despite lacking a canonical AP-1A binding motif, yeast two-hybrid studies demonstrated an interaction between the PAM-1 cytosolic domain and AP-1A. Co-immunoprecipitation experiments with PAM-1 mutants revealed an influence of the luminal domains of PAM-1 on this interaction. Thus, AP-1A is crucial for normal SG biogenesis, function and composition. PMID:25040637

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

    SciTech Connect

    Stevens, R.L.; Austen, K.F. ); Fox, C.C.; Lichtenstein, L.M. )

    1988-04-01

    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.

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

    PubMed Central

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

    2007-01-01

    The molecular mechanisms involved in the maturation of secretory granules, organelles that store hormones and neuropeptides, are poorly understood. As granule content proteins are processed, the composition of granule membranes changes, yielding constitutive-like secretion of immature content proteins and producing secretagogue-responsive mature granules. Constitutive-like secretion was not previously recognized as a process subject to regulation. We show that Kalirin and Trio, homologous Rho guanine nucleotide exchange factors (GEFs), which interact with a secretory granule resident protein, modulate cargo secretion from immature granules. Some of the Kalirin and Trio isoforms expressed in neuroendocrine cells colocalize with immature granules. Overexpression of their N-terminal GEF domain (GEF1) enhances secretion from immature granules, depleting cells of secretory cargo in the absence of secretagogue. This response requires GEF1 activity and is mimicked by Kalirin/Trio substrates Rac1 and RhoG. Accordingly, selective pharmacological inhibition of endogenous GEF1 activity decreases secretagogue-independent release of hormone precursors, accumulating product peptide in mature secretory granules. Kalirin/Trio modulation of cargo secretion from immature granules provides secretory cells with an extra layer of control over the sets of peptides released. Control of this step enhances the range of physiological responses that can be elicited, whereas lack of control could have pathological consequences. PMID:17881726

  17. Novel secretory granule morphology in physically fixed pancreatic islets.

    PubMed

    Dudek, R W; Boyne, A F; Charles, T M

    1984-09-01

    Protein A-gold immunocytochemistry has been applied to physically fixed beta cells from rat islets of Langerhans. The punctate nature of the gold particles permits improved resolution of the antigenic sites without obscuring the fine ultrastructural preservation obtained by physical fixation. There is a filamentous material within the halo of the secretory granules that is not preserved by aqueous, chemical fixation. When viewed in stereo the filaments appear as an annular cobweb or a series of wheel spokes attached to a centrally located hub (the dense core of the granule). The filaments demonstrate insulin-like immunoreactivity using the protein A-gold technique. The immunoreactivity appears to be restricted to the filaments and the surface of the dense cores. This may be a consequence of the preservation of a solid, insolubilized core state that resists penetration by the antibody and/or the protein A-gold complex. However, the evidence that there is a halo pool of insulin which is separate from the massive core aggregate suggests that i) correspondingly massive exocytotic pits may not be as mandatory for insulin release as has been assumed and ii) the complex kinetics of insulin secretion may be, in part, a reflection of multiple insulin compartments within secretory granules. PMID:6379039

  18. Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins.

    PubMed

    Thvenod, Frank

    2002-09-01

    Regulated secretion in exocrine and neuroendocrine cells occurs through exocytosis of secretory granules and the subsequent release of stored small molecules and proteins. The introduction of biophysical techniques with high temporal and spatial resolution, and the identification of Ca(2+)-dependent and -independent "docking" and "fusion" proteins, has greatly enhanced our understanding of exocytosis. The cloning of families of ion channel proteins, including intracellular ion channels, has also revived interest in the role of secretory granule ion channels in exocytotic secretion. Thus secretory granules of pancreatic acinar cell express a ClC-2 Cl(-) channel, a HCO-permeable member of the CLCA Ca(2+)-dependent anion channel family, and a KCNQ1 K(+) channel. Evidence suggests that these channels may facilitate the release of digestive enzymes and/or prevent exocytosed granules from collapsing during "kiss and run" recycling. In pancreatic beta-cells, a granular ClC-3 Cl(-) channel provides a shunt pathway for a vacuolar-type H(+)-ATPase. Acidification "primes" the granules for Ca(2+)-dependent exocytosis and release of insulin. In summary, secretory granules are equipped with specific sets of ion channels, which modulate regulated exocytosis and the release of macromolecules. These channels could represent excellent targets for therapeutic interventions to control exocytotic secretion in relevant diseases, such as pancreatitis, cystic fibrosis, or diabetes mellitus. PMID:12176723

  19. Lumenal Protein within Secretory Granules Affects Fusion Pore Expansion

    PubMed Central

    Weiss, AnnitaNgatchou; Anantharam, Arun; Bittner, MaryA.; Axelrod, Daniel; Holz, RonaldW.

    2014-01-01

    It is often assumed that upon fusion of the secretory granule membrane with the plasma membrane, lumenal contents are rapidly discharged and dispersed into the extracellular medium. Although this is the case for low-molecular-weight neurotransmitters and some proteins, there are numerous examples of the dispersal of a protein being delayed for many seconds after fusion. We have investigated the role of fusion-pore expansion in determining the contrasting discharge rates of fluorescent-tagged neuropeptide-Y (NPY) (within 200ms) and tissue plasminogen activator (tPA) (over many seconds) in adrenal chromaffin cells. The endogenous proteins are expressed in separate chromaffin cell subpopulations. Fusion pore expansion was measured by two independent methods, orientation of a fluorescent probe within the plasma membrane using polarized total internal reflection fluorescence microscopy and amperometry of released catecholamine. Together, they probe the continuum of the fusion-pore duration, from milliseconds to many seconds after fusion. Polarized total internal reflection fluorescence microscopy revealed that 71% of the fusion events of tPA-cer-containing granules maintained curvature for >10 s, with approximately half of the structures likely connected to the plasma membrane by a short narrow neck. Such events were not commonly observed upon fusion of NPY-cer-containing granules. Amperometry revealed that the expression of tPA-green fluorescent protein (GFP) prolonged the duration of the prespike foot ?2.5-fold compared to NPY-GFP-expressing cells and nontransfected cells, indicating that expansion of the initial fusion pore in tPA granules was delayed. The t1/2 of the main catecholamine spike was also increased, consistent with a prolonged delay of fusion-pore expansion. tPA added extracellularly bound to the lumenal surface of fused granules. We propose that tPA within the granule lumen controls its own discharge. Its intrinsic biochemistry determines not only its extracellular action but also the characteristics of its presentation to the extracellular milieu. PMID:24988338

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

    PubMed Central

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

    2011-01-01

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

  1. Giant secretory granules in the ducts of the parotid and submandibular glands of the slow loris.

    PubMed

    Tandler, B; Pinkstaff, C A; Nagato, T; Phillips, C J

    1996-06-01

    The parotid and submandibular glands of a slow loris, a rare Southeast Asian primate, were obtained after the head had been perfused by fixative for a study of the brain. These tissues were processed by conventional means for electron microscopy. Glands also were obtained at autopsy from 2 other lorises, fixed by immersion in formalin, and subjected to a battery of tests for glycoconjugates. In the parotid gland, a short segment of the proximal striated duct lacks both basal striations and any sign of secretory activity. The major portion of the striated duct consists of tall cells that contain a spectrum of secretory granules, some larger than the nuclei (many granules are > 9 mum in diameter). These granules, which are delimited by a single membrane, are capable of chain exocytosis. Many of the giant granules have bundles of cytofilaments (4.5-6.5 nm) in apparent association with their surface. Occasional cells contain numerous small granules. Duct cells with or without granules lack basal striations. The granules contain neutral glycoconjugates but no acidic glycoconjugates. Some, but not all, interlobular excretory ducts also have secretory granules that run the gamut from tiny to giant. Exactly the same situation occurs in the submandibular gland. Unlike other primates, which may have duct cells that contain only a few tiny granules in their apices, the cells in both the striated and excretory ducts in the slow loris appear to be specialized for secretion rather than for transport. The biofunction of the giant granules is unknown. PMID:18621331

  2. Secretory Granule Membrane Protein Recycles Through Multivesicular Bodies

    PubMed Central

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

    2010-01-01

    The recycling of secretory granule membrane proteins that reach the plasma membrane following exocytosis is poorly understood. As a model, peptidylglycine ?-amidating monooxygenase (PAM), a granule membrane protein that catalyzes a final step in peptide processing was examined. Ultrastructural analysis of antibody internalized by PAM and surface biotinylation demonstrated efficient return of plasma membrane PAM to secretory granules. Electron microscopy revealed the rapid movement of PAM from early endosomes to the limiting membranes of multivesicular bodies and then into intralumenal vesicles. Wheat germ agglutinin and PAM antibody internalized simultaneously were largely segregated when they reached multivesicular bodies. Mutation of basally phosphorylated residues (Thr946, Ser949) in the cytoplasmic domain of PAM to Asp (TS/DD) substantially slowed its entry into intralumenal vesicles. Mutation of the same sites to Ala (TS/AA) facilitated the entry of internalized PAM into intralumenal vesicles and its subsequent return to secretory granules. Entry of PAM into intralumenal vesicles is also associated with a juxtamembrane endoproteolytic cleavage that releases a 100 kDa soluble PAM fragment that can be returned to secretory granules. Controlled entry into the intralumenal vesicles of multivesicular bodies plays a key role in the recycling of secretory granule membrane proteins. PMID:20374556

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

    PubMed Central

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

    1996-01-01

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

  4. A hydrophobic patch in a charged alpha-helix is sufficient to target proteins to dense core secretory granules.

    PubMed

    Dikeakos, Jimmy D; Lacombe, Marie-Jose; Mercure, Chantal; Mireuta, Matei; Reudelhuber, Timothy L

    2007-01-12

    Many endocrine and neuroendocrine cells contain specialized secretory organelles called dense core secretory granules. These organelles are the repository of proteins and peptides that are secreted in a regulated manner when the cell receives a physiological stimulus. The targeting of proteins to these secretory granules is crucial for the generation of certain peptide hormones, including insulin and ACTH. Although previous work has demonstrated that proteins destined to a variety of cellular locations, including secretory granules, contain targeting sequences, no single consensus sequence for secretory granule-sorting signals has emerged. We have shown previously that alpha-helical domains in the C-terminal tail of the prohormone convertase PC1/3 play an important role in the ability of this region of the protein to direct secretory granule targeting (Jutras, I. Seidah, N. G., and Reudelhuber, T. L. (2000) J. Biol. Chem. 275, 40337-40343). In this study, we show that a variety of alpha-helical domains are capable of directing a heterologous secretory protein to granules. By testing a series of synthetic alpha-helices, we also demonstrate that the presence of charged (either positive or negative) amino acids spatially segregated from a hydrophobic patch in the alpha-helices of secretory proteins likely plays a critical role in the ability of these structures to direct secretory granule sorting. PMID:17092937

  5. Platelet secretory behaviour: as diverse as the granules or not?

    PubMed

    Heijnen, H; van der Sluijs, P

    2015-12-01

    Platelets play a central role in the arrest of bleeding after damage to a blood vessel and in the development of thrombosis. Platelets rapidly respond after interaction with sub-endothelial components and release cargo from their storage granules. The three principal granule types of platelets are ?-granules, dense granules and lysosomes. Timed release of granule contents and regulated expression of critical receptors are essential for maintenance of the platelet thrombus, yet also have important functions beyond hemostasis (i.e. inflammatory reactions and immune responses). ?-granules store adhesive molecules such as von Willebrand factor and fibrinogen, growth factors and inflammatory and angiogenic mediators, which play crucial roles in inflammatory responses and tumor genesis. The ?-granules comprise a group of subcellular compartments with a unique composition and ultrastructure. Recent studies have suggested that differential secretory kinetics of ?-granule subtypes is responsible for a thematic release of adhesive and inflammatory mediators. In addition, new results indicate that activation-dependent synthesis and release of cytokines also contribute to the inflammatory role of platelets. We will discuss the various methods that platelets use to regulate secretory processes and how these relate to potential differential secretion patterns, thereby promoting adhesiveness and/or inflammatory functions. We will focus on the heterogenic granule population, open canalicular system (OCS) plasticity, the role of contractile and mechanobiological forces, and the fusogenic machinery. PMID:26391322

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

    PubMed

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

    2015-05-01

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

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

    SciTech Connect

    Marino, C.R.; Castle, J.D.; Gorelick, F.S. )

    1990-07-01

    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.

  8. The heterotrimeric G-protein Gi is localized to the insulin secretory granules of beta-cells and is involved in insulin exocytosis.

    PubMed

    Konrad, R J; Young, R A; Record, R D; Smith, R M; Butkerait, P; Manning, D; Jarett, L; Wolf, B A

    1995-05-26

    Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from beta-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting beta-cell line beta-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized beta-TC3 cells. Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion. Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized beta-TC3 cells. Mastoparan-induced insulin secretion from permeabilized beta-TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin-sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western immunoblotting performed on beta-TC3 homogenate and ISG demonstrated that G alpha i was dramatically enriched in ISG. Levels of G alpha o and G alpha q were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of G alpha i to ISG was confirmed with electron microscopic immunocytochemistry in beta-TC3 cells and beta-cells from rat pancreas. In contrast to G alpha o and G alpha q, G alpha was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein G alpha i located in the ISG of beta-cells to stimulate insulin secretion. PMID:7759545

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

    PubMed Central

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

    1994-01-01

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

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

    SciTech Connect

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

    1985-06-01

    X-ray microanalysis of freeze-dried labial gland cryosections revealed that Na concentration was doubled and the Ca/S concentration ratio was decreased in secretory granules of labial glands from patients with cystic fibrosis (CF) when compared with glands from normal subjects. Other results suggested that the decrease in the Ca/S concentration ratio resulted from an increase in S concentration. These findings imply that mucous granules in labial saliva showed a CF-related increase in Na and S content, and such changes would be expected to affect the rheology of the mucus after exocytosis. In contrast with a previous study in human parotid glands, no evidence was found for CF-related changes in cytoplasmic or nuclear Na, K, and Ca concentrations. Significant elemental differences were found between secretory granules and nuclei and cytoplasm of control cells.

  11. α-Synuclein binds the KATP channel at insulin-secretory granules and inhibits insulin secretion

    PubMed Central

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

    2011-01-01

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

  12. Porosome: The Universal Secretory Portal in Cells

    NASA Astrophysics Data System (ADS)

    Jena, Bhanu

    2012-10-01

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

  13. Modulation of secretory granule-targeting efficiency by cis and trans compounding of sorting signals.

    PubMed

    Lacombe, Marie-Jose; Mercure, Chantal; Dikeakos, Jimmy D; Reudelhuber, Timothy L

    2005-02-11

    Several protein domains acting through seemingly different mechanisms have been reported to have the capacity to target proteins to dense core secretory granules. Because proteins enter secretory granules with different efficiencies and because some of these proteins contain more than one granule-targeting motif, we have investigated whether compounding sorting signals could alter the efficiency of protein entry into secretory granules. In the current study we demonstrate that a paired basic cleavage site from human prorenin and an alpha-helix-containing secretory granule-sorting signal from the prohormone convertase PC1/3 can synergize to increase granule-sorting efficiency not only when located on the same protein, but also when located on distinct proteins that associate in the secretory pathway. PMID:15569678

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

    PubMed Central

    Arvan, P; Castle, D

    1998-01-01

    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

  15. Rapid primary microwave-aldehyde and microwave-osmium fixation: improved detection of rat parotid acinar cell secretory granule alpha-amylase using a post-embedding immunogold ultrastructural morphometric analysis.

    PubMed

    Login, G R; Ku, T C; Dvorak, A M

    1995-05-01

    Studies of methods for improved fixation are becoming increasingly important in the field of quantitative immunocytochemistry. We used microwave (MW)-assisted chemical fixation to show improved retention of salivary gland acinar cell secretory granule alpha-amylase detected by a quantitative immunogold method. Blocks (4-mm3) of rat parotid gland were fixed by the following methods: (a) MW irradiation in an aldehyde fixative (AF) for 6 sec; (b) immersion in AF for 1.5 hr; (c) MW irradiation in osmium tetroxide (OT) for 9 sec; (d) immersion in OT for 1.5 hr; or (e) Sequential MW AF, 10 sec, MW OT rapid treatment (SMAORT), 10 sec. Specimens were processed routinely for transmission electron microscopy. Thin sections of Epon-embedded tissues were exposed first to rabbit IgG anti-human salivary alpha-amylase and second to gold-conjugated goat anti-rabbit IgG. Granule area was obtained by a point counting method. Labeling density was calculated as the number of gold particles/micron 2 +/- SD. Specimens fixed in seconds by MW-AF, MW-OT, or SMAORT showed ultrastructural preservation similar to immersion fixation in AF or OT for 1.5 hr. Immunogold labeling density of granule alpha-amylase was highest for SMAORT (874 microns 2) compared to MW-AF (295 microns 2), MW-OT (248 microns 2), routine sequential immersion in AF and OT (229 microns 2), or immersion in OT (no aldehyde) (190 microns 2). This study establishes the improved retention of salivary gland acinar cell secretory granule alpha-amylase and markedly enhanced fixation speed for ultrastructural studies made possible by MW-chemical fixation protocols that use aldehydes and osmium. PMID:7730590

  16. Functional amyloids as natural storage of peptide hormones in pituitary secretory granules.

    PubMed

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

    2009-07-17

    Amyloids are highly organized cross-beta-sheet-rich protein or peptide aggregates that are associated with pathological conditions including Alzheimer's disease and type II diabetes. However, amyloids may also have a normal biological function, as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. We found that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross-beta-sheet-rich conformation. Thus, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology. PMID:19541956

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

    PubMed Central

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

    2014-01-01

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

  18. Amyloid formation of growth hormone in presence of zinc: Relevance to its storage in secretory granules

    PubMed Central

    Jacob, Reeba S.; Das, Subhadeep; Ghosh, Saikat; Anoop, Arunagiri; Jha, Narendra Nath; Khan, Tuhin; Singru, Praful; Kumar, Ashutosh; Maji, Samir K.

    2016-01-01

    Amyloids are cross-β-sheet fibrillar aggregates, associated with various human diseases and native functions such as protein/peptide hormone storage inside secretory granules of neuroendocrine cells. In the current study, using amyloid detecting agents, we show that growth hormone (GH) could be stored as amyloid in the pituitary of rat. Moreover, to demonstrate the formation of GH amyloid in vitro, we studied various conditions (solvents, glycosaminoglycans, salts and metal ions) and found that in presence of zinc metal ions (Zn(II)), GH formed short curvy fibrils. The amyloidogenic nature of these fibrils was examined by Thioflavin T binding, Congo Red binding, transmission electron microscopy and X-ray diffraction. Our biophysical studies also suggest that Zn(II) initiates the early oligomerization of GH that eventually facilitates the fibrillation process. Furthermore, using immunofluorescence study of pituitary tissue, we show that GH in pituitary significantly co-localizes with Zn(II), suggesting the probable role of zinc in GH aggregation within secretory granules. We also found that GH amyloid formed in vitro is capable of releasing monomers. The study will help to understand the possible mechanism of GH storage, its regulation and monomer release from the somatotrophs of anterior pituitary. PMID:27004850

  19. FLOW CYTOMETRY-ASSISTED PURIFICATION AND PROTEOMIC ANALYSIS OF THE CORTICOTROPES DENSE-CORE SECRETORY GRANULES

    PubMed Central

    Gauthier, Daniel J; Sobota, Jacqueline A.; Ferraro, Francesco; Mains, Richard E; Lazure, Claude

    2010-01-01

    The field of organellar proteomics has emerged as an attempt to minimize the complexity of the proteomics data obtained from whole cell and tissue extracts while maximizing the resolution on the protein composition of a single subcellular compartment. Standard methods involve lengthy density-based gradient and/or immunoaffinity purification steps followed by extraction, one-dimensional or two-dimensional gel electrophoresis, gel staining, in-gel tryptic digestion and protein identification by mass spectrometry. In this paper, we present an alternate approach to purify subcellular organelles containing a fluorescent reporter molecule. The gel-free procedure involves fluorescence-assisted sorting of the secretory granules followed by gentle extraction in a buffer compatible with tryptic digestion and mass-spectrometry. Once the subcellular organelle labeled, this procedure can be done in a single day, requires no major modification to any instrumentation and can be readily adapted to the study of other organelles. When applied to corticotrope secretory granules, it led to a much enriched granular fraction from which numerous proteins could be identified through mass spectrometry. PMID:18704904

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

    PubMed Central

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

    1997-01-01

    We measured the efflux of 5-hydroxytryptamine (5-HT, serotonin) from an intact secretory granule extracted from the mast cell of the beige mouse. The efflux was measured with amperometry after rupture of the granule membrane was triggered by electroporation. We determined the diffusivity of 5-HT within the secretory granule to be 2.0 x 10(-8) cm2 s(-1) when the granule is in contact with a physiological saline and found that this diffusivity depends on the valence of the cation in the external electrolyte. There is a fivefold increase in the diffusion coefficient of 5-HT determined in CsCl (150 mM, pH 7.2) at 3.7 x 10(-8) cm2 s(-1) compared to that determined in histamine dihydrochloride (Hi, 100 mM at pH 4.5) at 0.7 x 10(-8) cm2 s(-1). We found that the rate of expansion of the granule matrix observed in physiological medium correlates with the efflux of 5-HT, and that the rate of swelling of the matrix and the efflux depend on the microviscosity within the granule matrix and not the bulk viscosity of the external solution. The low diffusivity of 5-HT (approximately 500-fold less than in the bulk), the observation that the valence of the counterion affects this diffusivity, and the relationship between the volume changes of the matrix and the efflux suggest that 5-HT is released from the granule by ion exchange. We discuss the implications of this result for exocytotic release in mast cells and propose that an ion exchange mechanism could control the rate of release in other secretory systems. Images FIGURE 1 PMID:9284284

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed Central

    Briguglio, Joseph S.; Kumar, Santosh

    2013-01-01

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

  3. Investigation of the protein-peptide composition of the secretory granules of bovine lactogenic hormone

    SciTech Connect

    Nagornaya, L.V.; Kizim, E.A.; Kravtsov, G.M.; Tsibezov, V.V.; Vinogradov, V.A.

    1987-02-20

    By a combination of methods of gel chromatography, fluorometric analysis, and electrophoresis in polyacrylamide gel with immunochemical identification, they investigated the protein-peptide composition of the secretory granules of lactogenic hormone (LTH), isolated from the bovine anterior pituitary. It was established that the content of peptides in the granules is less than 3% of the content of immunoreactive LTH. Using gel chromatography, the monomer of the hormone and two immunoreactive forms with molecular weight 42 and 65 kilodaltons were detected in the secondary granules. The content of the forms with respect to immunoreactivity was 90, 3, and 7%, respectively. Four immunoreactive forms of LTH were detected in the secretory granules by the method of electrophoresis in polyacrylamide gel followed by immunochemical identification.

  4. A functioning artificial secretory cell

    PubMed Central

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

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  6. Immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells.

    PubMed

    Kraehenbuhl, J P; Racine, L; Jamieson, J D

    1977-02-01

    The bovine exocrine pancreatic cell produces a variety of enzymes and proenzymes for export. Biochemical studies by Greene L.J., C.H. Hirs, and G.E. Palade (J. Biol. Chem. 1963. 238:2054) have shown that the mass proportions of several of these proteins in resting pancreatic juice and zymogen granule fractions are identical. In this study we have used immunocytochemical techniques at the electron microscope level to determine whether regional differences exist in the bovine gland with regard to production of individual secretory proteins and whether specialization of product handling occurs at the subcellular level. The technique used is a modification of one previously reported (McLean, J.D., and S.J. Singer. 1970. Proc. Natl. Acad. Sci U.S.A. 69:1771) in which immunocytochemical reagents are applied to thin sections of bovine serum albumin-imbedded tissue and zymogen granule fractions. A double antibody technique was used in which the first step consisted of rabbit F(ab')2 antibovine secretory protein and the detection step consisted of sheep (F(ab')2 antirabbit F(ab')2 conjugated to ferritin. The results showed that all exocrine cells in the gland, and all zymogen granules and Golgi cisternae in each cell, were qualitatively alike with regard to their content of secretory proteins examined (trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggest that these secretory proteins are transported through the cisternae of the Golgi complex where they are intermixed before copackaging in zymogen granules; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins, and is independent of extent of glycosylation, e.g., trypsinogen, a nonglycoprotein vs. DNase, a glycoprotein. PMID:319100

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

    SciTech Connect

    Peter, B.; Van Waarde, M.A.W.H.; Konings, A.W.T.; Vissink, A. |; `s-Gravenmade, E.J.

    1995-02-01

    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.

  8. Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles

    PubMed Central

    Dias, Felipe F.; Amaral, Ktia B.; Carmo, Lvia A. S.; Shamri, Revital; Dvorak, Ann M.; Weller, Peter F.

    2014-01-01

    Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes. PMID:24670795

  9. The ionogenic nature of the secretory-granule membrane. Electrokinetic properties of isolated chromaffin granules.

    PubMed

    Matthews, E K; Evans, R J; Dean, P M

    1972-12-01

    1. Chromaffin granules isolated from the bovine adrenal medulla possess an electrophoretic mobility of -1.12mum.s(-1).cm.V(-1), corresponding to a surface zeta potential of -14.4mV and surface charge density of 1.38x10(-6)C.cm(-2). 2. The mobility of chromaffin granules is pH-dependent, indicating an amphoteric surface with an isoelectric point at pH3.0 and acidic groups with a pK(a) of 3.11. 3. Addition of bi- and ter-valent cations decreased the mobility of chromaffin granules in a dose-dependent fashion with a relative potency of La(3+)>Mn(2+)>Ca(2+) >Sr(2+)>Mg(2+)>Ba(2+). 4. Treatment with neuraminidase decreased the mobility of erythrocytes by 84%, whereas chromaffin-granule mobility was decreased by only 14%. This correlates well with the small complement of neuraminic acid present in the granule membrane. 5. The nature, origin and significance of the anionic surface charge of the chromaffin granule is discussed. It is concluded that the net negative charge at the surface of shear derives chiefly from a single type of chemical group, namely -CO(2) (-), contributed by the alpha-carboxyl group of constituent proteins, the phospholipid phosphatidylserine and, to a lesser extent, the sialic acid component of glycoproteins. PMID:4664936

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

    NASA Astrophysics Data System (ADS)

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

    2010-10-01

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

  11. Porosome: the secretory NanoMachine in cells.

    PubMed

    Jena, Bhanu P

    2013-01-01

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

  12. The econobiology of pancreatic acinar cells granule inventory and the stealthy nano-machine behind it.

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2016-03-01

    The pancreatic gland secretes most of the enzymes and many other macromolecules needed for food digestion in the gastrointestinal tract. These molecules play an important role in digestion, host defense and lubrication. The secretion of pancreatic proteins ensures the availability of the correct mix of proteins when needed. This review describes model systems available for the study of the econobiology of secretory granule content. The secretory pancreatic molecules are stored in large dense-core secretory granules that may undergo either constitutive or evoked secretion, and constitute the granule inventory of the cell. It is proposed that the Golgi complex functions as a distribution center for secretory proteins in pancreatic acinar cells, packing the newly formed secretory molecules into maturing secretory granules, also known functionally as condensing vacuoles. Mathematical modelling brings forward a process underlying granule inventory maintenance at various physiological states of condensation and aggregation by homotypic fusion. These models suggest unique but simple mechanisms accountable for inventory buildup and size, as well as for the distribution of secretory molecules into different secretory pathways in pancreatic acinar cells. PMID:26702787

  13. Proteoglycans support proper granule formation in pancreatic acinar cells.

    PubMed

    Aroso, Miguel; Agricola, Brigitte; Hacker, Christian; Schrader, Michael

    2015-10-01

    Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas. PMID:26105026

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

    PubMed Central

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

    2012-01-01

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

  15. Molecular Interpretation of ACTH-β-Endorphin Coaggregation: Relevance to Secretory Granule Biogenesis

    PubMed Central

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

    2012-01-01

    Peptide/protein hormones could be stored as non-toxic amyloid-like structures in pituitary secretory granules. ACTH and β-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 β-endorphin and their colocalization in the form of amyloid aggregates. Although ACTH is known to be a part of ACTH-β-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-β-endorphin system, β-endorphin-only system and ACTH-only system. We find that β-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 β-endorphin but also enhances the stability of mixed oligomers of the entire system. PMID:22403619

  16. PC1/3, PC2 and PC5/6A are targeted to dense core secretory granules by a common mechanism.

    PubMed

    Dikeakos, Jimmy D; Mercure, Chantal; Lacombe, Marie-Jose; Seidah, Nabil G; Reudelhuber, Timothy L

    2007-08-01

    There are seven members of the proprotein convertase (PC) family of secreted serine proteases that cleave their substrates at basic amino acids, thereby activating a variety of hormones, growth factors, and viruses. PC1/3, PC2 and PC5/6A are the only members of the PC family that are targeted to dense core secretory granules, where they carry out the processing of proteins that are secreted from the cell in a regulated manner. Previous studies have identified alpha-helices in the C-termini of the PC1/3 and PC2 proteases that are required for this subcellular targeting. In the current study, we demonstrate that a predicted alpha-helix in the C-terminus of PC5/6A is also critical for the ability of this domain to target a heterologous protein to the regulated secretory pathway of mouse endocrine AtT-20 cells. Analysis of the subcellular distribution of fusion proteins containing the C-terminal domains of PC1/3, PC2 and PC5/6A confirmed that all three domains have the capacity to redirect a constitutively secreted protein to the granule-containing cytoplasmic extensions. Analysis of the predicted structures formed by these three granule-sorting helices shows a correlation between their granule-sorting efficiency and the clustering of hydrophobic amino acids in their granule-targeting helices. PMID:17645548

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

    PubMed Central

    Gonzlez, Claudia; Espinosa, Marisol; Snchez, Mara Trinidad; Droguett, Karla; Ros, Mariana; Fonseca, Ximena; Villaln, Manuel

    2013-01-01

    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

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

    PubMed Central

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

    2009-01-01

    The activity of the cystic fibrosis transmembrane conductance regulator (CFTR) can be mediated by surface G protein-coupled receptors such as the beta2-adrenergic receptor. In this study, we explored the effect of a long-acting beta2-adrenergic agonist, salmeterol, on the CFTR-dependent secretory capacity of a human CF tracheal gland serous cell line (CF-KM4), homozygous for the delF508 mutation. We showed that, compared to the untreated CF serous cells, a 24 h pre-incubation period with 200 nM salmeterol induced an 83 % increase in delF508-CFTR-mediated chloride efflux. The restoration of the bioelectric properties is associated with increased apical surface pool of delF508-CFTR. Salmeterol induced a decrease in ion concentration and an increase in the level of hydration of the mucus packaged inside the CF secretory granules. The effects of salmeterol are not associated with a persistent production of cAMP. Western blotting on isolated secretory granules demonstrated immunoreactivity for CFTR and lysozyme. In parallel, we measured by AFM an increased size of secretory granules isolated from CF serous cells compared to non-CF serous cells (MM39 cell line) and showed that salmeterol was able to restore a CF cell granule size similar to that of non-CF cells. To demonstrate that the salmeterol effect was a CFTR-dependent mechanism, we showed that the incubation of salmeterol-treated CF serous cells with CFTR-inh172 suppressed the restoration of normal secretory functions. The capacity of salmeterol to restore the secretory capacity of glandular serous cells suggests that it could also improve the airway mucociliary clearance in patients with CF. PMID:18931328

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

    PubMed

    Fitch-Tewfik, Jennifer L; Flaumenhaft, Robert

    2013-01-01

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

  20. Further characterization of the peptidyl alpha-amidating enzyme in rat anterior pituitary secretory granules.

    PubMed

    Glembotski, C C

    1985-09-01

    In previous studies we have demonstrated a secretory granule-associated peptide alpha-amidation activity in rat anterior, intermediate, and posterior pituitary. This activity is capable of converting 125I-labeled synthetic D-Tyr-Val-Gly to labeled D-Tyr-Val-NH2, and requires ascorbic acid, CuSO4, and molecular oxygen for optimal activity. Because of the requirement for peptides with COOH-terminal glycine residues, and cofactor requirements similar to monooxygenases such as dopamine beta-monooxygenase, we have proposed that the alpha-amidating enzyme be named peptidylglycine alpha-amidating monooxygenase, or PAM. The present study focused on (i) verifying that PAM could utilize a physiologically relevant peptide substrate, and (ii) demonstrating the retention of the cofactor requirements with purification of PAM. PAM (Mr = 50,000) was partially purified from rat anterior pituitary secretory granules and was shown to be capable of converting alpha-N-acetyl-ACTH(1-14) to alpha-N-acetyl-ACTH(1-13)NH2 (alpha-melanocyte stimulating hormone) and ACTH(9-14) to ACTH(9-13)NH2. The optimal rates for these conversions were dependent on ascorbic acid and CuSO4. Kinetic analyses, using the model compound D-Tyr-Val-Gly as the peptide substrate, demonstrated that, compared to the crude granule extract, the partially purified enzyme displayed increased apparent affinities for both the peptide substrate and ascorbate. These analyses also showed that the Km for D-Tyr-Val-Gly was dependent on the concentration of ascorbate, while the Km for ascorbate was constant over a wide range of D-Tyr-Val-Gly concentrations. The results presented here indicate that PAM can alpha-amidate physiologically relevant peptides related to alpha MSH, and performs the reaction in an ascorbate-dependent fashion. Retention of the ascorbate and copper requirements with purification further support the hypothesis that these cofactors are important requirements for the COOH-terminal alpha-amidation of neuro and endocrine peptides. PMID:2994573

  1. The organization of the secretory machinery in chromaffin cells as a major factor in modeling exocytosis

    PubMed Central

    Villanueva, José; Torregrosa-Hetland, Cristina J.; Gil, Amparo; González-Vélez, Virginia; Segura, Javier; Viniegra, Salvador; Gutiérrez, Luis M.

    2010-01-01

    The organization of cytoplasm in excitable cells was a largely ignored factor when mathematical models were developed to understand intracellular calcium and secretory behavior. Here we employed a combination of fluorescent evanescent and transmitted light microscopy to explore the F-actin cytoskeletal organization in the vicinity of secretory sites in cultured bovine chromaffin cells. This technique and confocal fluorescent microscopy show chromaffin granules associated with the borders of cortical cytoskeletal cages forming an intricate tridimensional network. Furthermore, the overexpression of SNAP-25 in these cells also reveals the association of secretory machinery clusters with the borders of these cytoskeletal cages. The importance of these F-actin cage borders is stressed when granules appear to interact and remain associated during exocytosis visualized in acridin orange loaded vesicles. These results will prompt us to propose a model of cytoskeletal cages, where the secretory machinery is associated with its borders. Both the calcium level and the secretory response are enhanced in this geometrical arrangement when compared with a random distribution of the secretory machinery that is not restricted to the borders of the cage. PMID:20885775

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

    SciTech Connect

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

    2009-06-03

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

  3. Biogenesis of the secretory granule: chromogranin A coiled-coil structure results in unusual physical properties and suggests a mechanism for granule core condensation.

    PubMed

    Mosley, Coleman A; Taupenot, Laurent; Biswas, Nilima; Taulane, Joseph P; Olson, Norman H; Vaingankar, Sucheta M; Wen, Gen; Schork, Nicholas J; Ziegler, Michael G; Mahata, Sushil K; O'Connor, Daniel T

    2007-09-25

    The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein "packing" in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a binding complex by the CHGA core. Inhibition of CHGA expression, by siRNA, disrupted regulated secretory protein traffic by approximately 65%, while targeted ablation of the CHGA gene in the mouse reduced chromaffin granule cotransmitter concentrations by approximately 40-80%. These results suggest new roles for secretory protein tertiary structure in hormone and transmitter storage, with implications for secretory cargo condensation (or dense core "packing" structure) within the regulated pathway. PMID:17718510

  4. Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

    PubMed Central

    Gabel, Marion; Delavoie, Franck; Demais, Valérie; Royer, Cathy; Bailly, Yannick; Vitale, Nicolas; Bader, Marie-France

    2015-01-01

    Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament–bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2–induced actin bundling is apparently essential for generating active exocytotic sites. PMID:26323692

  5. The Color of Lactotroph Secretory Granules Stained with FM1-43 Depends on Dye Concentration

    PubMed Central

    Johnson, Joseph M.; Betz, William J.

    2008-01-01

    When pituitary lactotroph granules undergo exocytosis in the presence of FM1-43, their cores absorb dye and fluoresce brightly. We report that different granules fluoresce with different colors, despite being stained with a single fluorescent dye; emission spectra from individual granules show up to a 25 nm difference between the greenest and reddest granules. We found a correlation between granule color and average fluorescence intensity, suggesting that granule color depends upon dye concentration. We confirmed this in two ways: by increasing FM dye concentration in granules, which red shifted granule color, and by partially photobleaching the FM dye in granules, which green shifted granule color. Increasing stimulation intensity (by increasing KCl concentration) increased the proportion of red granules, indicating that granules exocytosing during intense stimulation bound more dye. This, perhaps, reflects differences in granule core maturation and condensation in which mature granules with condensed cores bind more FM dye but require more intense stimulation to be released. Concentration-dependent color shifts of FM dyes may be useful for monitoring aggregation processes occurring on a size scale smaller than the optical limit. PMID:18065476

  6. Evidence for two secretory cell types in the Stannius bodies of the teleosts Fundulus heteroclitus and Carassius auratus.

    PubMed

    Wendelaar Bonga, S E; van der Meij, J C; Pang, P K

    1980-01-01

    The Stannius bodies of killifish and goldfish were ultrastructurally investigated after exposure of the fish to media of different osmolality and calcium content. In both species two structurally different secretory cell types are found. Type-1 cells predominate. They contain an extensive granular endoplasmic reticulum, some large Golgi areas, and secretory granules with a mean diameter of about 0.4 micron. These cells are activated by transfer of freshwater fish to seawater, but not by transfer to low-calcium seawater. Type-2 cells often contain cytoplasmic processes that penetrate between the type-1 cells and are ending on the basal lamina. In this contact area granule release takes place. Type-2 cells contain some strands of granular endoplasmic reticulum, several small Golgi areas, and secretory granules with a mean diameter between 0.15 and 0.20 micron. Type-2 cells are not activated after transfer of fish to seawater. In killifish seawater exposure leads to a reduction of type-2 cells. The differences between the reactions of both cell types to different media point to functional differences between their secretory products. Type-1 cells may produce hypocalcemic factor. It is concluded that type-2 cells are typical for freshwater fish or euryhaline fish spending part of their life cycle in freshwater. The proper function of these cells is unclear. PMID:7428033

  7. Studies on the pH gradient and histamine uptake of isolated mast cell granules

    SciTech Connect

    De Young, M.B.; Nemeth, E.F.; Scarpa, A.

    1986-05-01

    A purified preparation of mast cell granules with intact perigranular membranes was obtained using a method involving probe sonication of rat serosal mast cells followed by differential centrifugation and Percoll gradient separation of the granules. Purification was assessed with histamine and mast cell granule protease assays. Granule integrity was demonstrated by light and electron microscopy and quantitated with a ruthenium red binding assay. The low yield of granules (20 ..mu..g protein/4 rats) necessitated the development of two microanalytical techniques to demonstrate the existence of a pH gradient across the membrane: 9-aminoacridine fluorescence studies in a cuvet with 50 ..mu..l capacity and /sup 14/C-methylamine distribution studies on microgram quantities of granule protein. Quantitation of results from isotope studies were confounded by the presence of oil used for separating granules from the aqueous phase. Nonetheless, an extrapolation procedure calibrated by external pH yielded an internal pH value of 5.46 +/- .03 (n = 4), consistent with values observed in granules obtained from other secretory cells. Collapse of the pH gradient by NH/sub 4//sup +/ or nigericin/KCl was demonstrated using either technique. Addition of histamine depressed intragranular pH, suggesting that histamine transport may utilize the ..delta..pH as a driving force.

  8. Condensing vacuole conversion and zymogen granule discharge in pancreatic exocrine cells: metabolic studies.

    PubMed

    Jamieson, J D; Palade, G E

    1971-03-01

    We have examined, in the pancreatic exocrine cell, the metabolic requirements for the conversion of condensing vacuoles into zymogen granules and for the discharge of the contents of zymogen granules. To study condensing vacuole conversion, we pulse labeled guinea pig pancreatic slices for 4 min with leucine-(3)H and incubated them in chase medium for 20 min to allow labeled proteins to reach condensing vacuoles. Glycolytic and respiratory inhibitors were then added and incubation continued for 60 min to enable labeled proteins to reach granules in control slices. Electron microscope radioautography of cells or of zymogen granule pellets from treated slices showed that a large proportion of prelabeled condensing vacuoles underwent conversion in the presence of the combined inhibitors. Osmotic fragility studies on zymogen granule suspensions suggest that condensation may result from the aggregation of secretory proteins in an osmotically inactive form. Discharge was studied using an in vitro radioassay based on the finding that prelabeled zymogen granules can be induced to release their labeled contents to the incubation medium by carbamylcholine or pancreozymin. Induced discharge is not affected if protein synthesis is blocked by cycloheximide for up to 2 hr, but is strictly dependent on respiration. The data indicate that transport and discharge do not require the pari passu synthesis of secretory or nonsecretory proteins (e.g. membrane proteins), suggesting that the cell may reutilize its membranes during the secretory process. The energy requirements for zymogen discharge may be related to the fusion-fission of the granule membrane with the apical plasmalemma. PMID:5547590

  9. Rapid Lytic Granule Convergence to the MTOC in Natural Killer Cells Is Dependent on Dynein But Not Cytolytic Commitment

    PubMed Central

    Mentlik, Ashley N.; Sanborn, Keri B.; Holzbaur, Erika L.

    2010-01-01

    Natural killer cells are lymphocytes specialized to participate in host defense through their innate ability to mediate cytotoxicity by secreting the contents of preformed secretory lysosomes (lytic granules) directly onto a target cell. This form of directed secretion requires the formation of an immunological synapse and occurs stepwise with actin reorganization preceding microtubule-organizing center (MTOC) polarization to the synapse. Because MTOC polarization to the synapse is required for polarization of lytic granules, we attempted to define their interrelationship. We found that compared with the time required for MTOC polarization, lytic granules converged to the MTOC rapidly. The MTOC-directed movement of lytic granules was independent of actin and microtubule reorganization, dependent on dynein motor function, occurred before MTOC polarization, and did not require a commitment to cytotoxicity. This defines a novel paradigm for rapid MTOC-directed transport as a prerequisite for directed secretion, one that may prepare, but not commit cells for precision secretory function. PMID:20444980

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

    PubMed

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

    1988-09-01

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

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

    PubMed Central

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

    2013-01-01

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

  12. Orai-STIMmediated Ca2+ release from secretory granules revealed by a targeted Ca2+ and pH probe

    PubMed Central

    Dickson, Eamonn J.; Duman, Joseph G.; Moody, Mark W.; Chen, Liangyi; Hille, Bertil

    2012-01-01

    Secretory granules (SGs) sequester significant calcium. Understanding roles for this calcium and potential mechanisms of release is hampered by the difficulty of measuring SG calcium directly in living cells. We adapted the Frster resonance energy transfer-based D1-endoplasmic reticulum (ER) probe to develop a unique probe (D1-SG) to measure calcium and pH in secretory granules. It significantly localizes to SGs and reports resting free Ca2+ of 69 15 ?M and a pH of 5.8. Application of extracellular ATP to activate P2Y receptors resulted in a slow monotonic decrease in SG Ca2+ temporally correlated with the occurrence of store-operated calcium entry (SOCE). Further investigation revealed a unique receptor-mediated mechanism of calcium release from SGs that involves SG store-operated Orai channels activated by their regulator stromal interaction molecule 1 (STIM1) on the ER. SG Ca2+ release is completely antagonized by a SOCE antagonist, by switching to Ca2+-free medium, and by overexpression of a dominant-negative Orai1(E106A). Overexpression of the CRAC activation domain (CAD) of STIM1 resulted in a decrease of resting SG Ca2+ by ?75% and completely abolished the ATP-mediated release of Ca2+ from SGs. Overexpression of a dominant-negative CAD construct (CAD-A376K) induced no significant changes in SG Ca2+. Colocalization analysis suggests that, like the plasma membrane, SG membranes also possess Orai1 channels and that during SG Ca2+ release, colocalization between SGs and STIM1 increases. We propose Orai channel opening on SG membranes as a potential mode of calcium release from SGs that may serve to raise local cytoplasmic calcium concentrations and aid in refilling intracellular calcium stores of the ER and exocytosis. PMID:23184982

  13. Vaccine adjuvants: Tailor-made mast-cell granules

    NASA Astrophysics Data System (ADS)

    Gunzer, Matthias

    2012-03-01

    Mast cells induce protective immune responses through secretion of stimulatory granules. Microparticles modelled after mast-cell granules are now shown to replicate and enhance the functions of their natural counterparts and to direct the character of the resulting immunity.

  14. Secretory protein trafficking and organelle dynamics in living cells.

    PubMed Central

    Lippincott-Schwartz, J; Roberts, T H; Hirschberg, K

    2000-01-01

    Green fluorescent protein chimerae acting as reporters for protein localization and trafficking within the secretory membrane system of living cells have been used in a wide variety of applications, including time-lapse imaging, double-labeling, energy transfer, quantitation, and photobleaching experiments. Results from this work are clarifying the steps involved in the formation, translocation, and fusion of transport intermediates; the organization and biogenesis of organelles; and the mechanisms of protein retention, sorting, and recycling in the secretory pathway. In so doing, they are broadening our thinking about the temporal and spatial relationships among secretory organelles and the membrane trafficking pathways that operate between them. PMID:11031247

  15. Identification of human cysteine-rich secretory protein 3 (CRISP-3) as a matrix protein in a subset of peroxidase-negative granules of neutrophils and in the granules of eosinophils.

    PubMed

    Udby, Lene; Calafat, Jero; Sørensen, Ole E; Borregaard, Niels; Kjeldsen, Lars

    2002-09-01

    Cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) was originally discovered in human neutrophilic granulocytes. We have recently developed a sensitive sandwich enzyme-linked immunosorbent assay for CRISP-3 and demonstrated the presence of CRISP-3 in exocrine secretions. To investigate the subcellular localization and mobilization of CRISP-3 in human neutrophils, we performed subcellular fractionation of resting and activated neutrophils on three-layer Percoll density gradients, release-studies of granule proteins in response to different secretagogues, and double-labeling immunogold electron microscopy. CRISP-3 was found to be localized in a subset of granules with overlapping characteristics of specific and gelatinase granules and mobilized accordingly, thus confirming the hypothesis that peroxidase-negative granules exist as a continuum from specific to gelatinase granules regarding protein content and mobilization. CRISP-3 was found to be a matrix protein, which is stored in granules as glycosylated and as unglycosylated protein. The subcellular distribution of the two forms of CRISP-3 was identical. In addition, CRISP-3 was found as a granule protein in eosinophilic granulocytes. The presence of CRISP-3 in peroxidase-negative granules of neutrophils, in granules of eosinophils, and in exocrine secretions indicates a role in the innate host defense. PMID:12223513

  16. PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure

    PubMed Central

    LaSarge, Candi L.; Santos, Victor R; Danzer, Steve C.

    2015-01-01

    Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offset the reduction in boutons per axon length. These morphological changes predicts a net increase in granule cell >> CA3 innervation. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell >> CA3 communication. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy. PMID:25600212

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2012-06-15

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

  19. Rapid biogenesis and sensitization of secretory lysosomes in NK cells mediated by target-cell recognition

    PubMed Central

    Liu, Dongfang; Xu, Liang; Yang, Fan; Li, Dongdong; Gong, Feili; Xu, Tao

    2005-01-01

    Natural killer (NK) cells play important roles in defense against tumor, viral infection, and cell-mediated xenograft rejection through secretion of secretory lysosomes. In this study, we used high time-resolution membrane capacitance measurement and fluorescence-imaging techniques to study the biogenesis and exocytosis of secretory lysosomes in a human NK cell line. We demonstrated a high-affinity Ca2+-dependent exocytosis of secretory lysosomes, which is sensitized further after target-cell stimulation. Our data also suggest an unusual rapid and dramatic de novo formation of secretory lysosomes after target-cell recognition. The rapid biogenesis of secretory lysosomes was blocked by specific protein kinase C inhibitor but not by brefeldin A. We propose that target-cell recognition triggers rapid biogenesis and sensitization of secretory lysosomes in NK cells through activation of PKC. PMID:15618404

  20. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    PubMed

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D. PMID:26845357

  1. COMPARABLE OUTCOMES IN NON-SECRETORY AND SECRETORY MULTIPLE MYELOMA AFTER AUTOLOGOUS STEM CELL TRANSPLANTATION

    PubMed Central

    Kumar, Shaji; Prez, Waleska S.; Zhang, Mei-Jie; Ballen, Karen; Bashey, Asad; To, L. Bik; Bredeson, Christopher N.; Cairo, Mitchell S.; Elfenbein, Gerald J.; Freytes, Csar O.; Gale, Robert Peter; Gibson, John; Kyle, Robert A.; Lacy, Martha Q.; Lazarus, Hillard M.; McCarthy, Philip L.; Milone, Gustavo A.; Moreb, Jan S.; Pavlovsky, Santiago; Reece, Donna E.; Vesole, David H.; Wiernik, Peter H.; Hari, Parameswaran

    2008-01-01

    Non-secretory myeloma (NSM) accounts for <5% of cases of multiple myeloma (MM). The outcome of these patients following autologous stem cell transplantation (ASCT) has not been evaluated in clinical trials. We compared the outcomes after ASCT for patients with NSM reported to the CIBMTR between 1989 and 2003, to a matched group of 438 patients (4 controls for each patient) with secretory myeloma (SM). The patients were matched using propensity scores calculated using age, Durie-Salmon stage, sensitivity to pre-transplant therapy, time from diagnosis to transplant and year of transplant. Disease characteristics were similar in both groups at diagnosis and at transplant except higher risk of anemia, hypoalbuminemia and marrow plasmacytosis (in SM) and plasmacytoma (more in NSM). Cumulative incidence of TRM, relapse, PFS and OS were similar between the groups. In multivariate analysis, based on a Cox model stratified on matched pairs and adjusted for covariates not considered in the propensity score, we found no difference in outcome between the NSM and SM groups. In this large cohort of patients undergoing ASCT, we found no difference in outcomes of patients with NSM compared to those with SM. PMID:18804043

  2. Acquisition of Lubrol insolubility, a common step for growth hormone and prolactin in the secretory pathway of neuroendocrine cells.

    PubMed

    Lee, M S; Zhu, Y L; Chang, J E; Dannies, P S

    2001-01-01

    Rat prolactin in the dense cores of secretory granules of the pituitary gland is a Lubrol-insoluble aggregate. In GH(4)C(1) cells, newly synthesized rat prolactin and growth hormone were soluble, but after 30 min about 40% converted to a Lubrol-insoluble form. Transport from the endoplasmic reticulum is necessary for conversion to Lubrol insolubility, since incubating cells with brefeldin A or at 15 degrees C reduced formation of insoluble rat (35)S-prolactin. Formation of Lubrol-insoluble aggregates has protein and cell specificity; newly synthesized human growth hormone expressed in AtT20 cells underwent a 40% conversion to Lubrol insolubility with time, but albumin did not, and human growth hormone expressed in COS cells underwent less than 10% conversion to Lubrol insolubility. del32-46 growth hormone, a naturally occurring form of growth hormone, and P89L growth hormone underwent conversion, although they were secreted more slowly, indicating that there is some tolerance in structural requirements for aggregation. An intracellular compartment with an acidic pH is not necessary for conversion to Lubrol insolubility, because incubation with chloroquine or bafilomycin slowed, but did not prevent, the conversion. GH(4)C(1) cells treated with estradiol, insulin, and epidermal growth factor accumulate more secretory granules and store more prolactin, but not more growth hormone, than untreated cells; Lubrol-insoluble aggregates of prolactin and growth hormone formed to the same extent in hormone-treated or untreated GH(4)C(1) cells, but prolactin was retained longer in hormone-treated cells. These findings indicate that aggregation alone is not sufficient to cause retention of secretory granule proteins, and there is an additional selective process. PMID:11024038

  3. Pancreatic β-Cell Adaptive Plasticity in Obesity Increases Insulin Production but Adversely Affects Secretory Function.

    PubMed

    Alarcon, Cristina; Boland, Brandon B; Uchizono, Yuji; Moore, Patrick C; Peterson, Bryan; Rajan, Suryalekha; Rhodes, Olivia S; Noske, Andrew B; Haataja, Leena; Arvan, Peter; Marsh, Bradly J; Austin, Jotham; Rhodes, Christopher J

    2016-02-01

    Pancreatic β-cells normally produce adequate insulin to control glucose homeostasis, but in obesity-related diabetes, there is a presumed deficit in insulin production and secretory capacity. In this study, insulin production was assessed directly in obese diabetic mouse models, and proinsulin biosynthesis was found to be contrastingly increased, coupled with a significant expansion of the rough endoplasmic reticulum (without endoplasmic reticulum stress) and Golgi apparatus, increased vesicular trafficking, and a depletion of mature β-granules. As such, β-cells have a remarkable capacity to produce substantial quantities of insulin in obesity, which are then made available for immediate secretion to meet increased metabolic demand, but this comes at the price of insulin secretory dysfunction. Notwithstanding, it can be restored. Upon exposing isolated pancreatic islets of obese mice to normal glucose concentrations, β-cells revert back to their typical morphology with restoration of regulated insulin secretion. These data demonstrate an unrealized dynamic adaptive plasticity of pancreatic β-cells and underscore the rationale for transient β-cell rest as a treatment strategy for obesity-linked diabetes. PMID:26307586

  4. Gastric neuroendocrine cells and secretory products.

    PubMed Central

    Oberg, K.

    1998-01-01

    The ECL cell is the most common cell type in the oxyntic mucosa of the stomach. It is producing a number of peptides and amines where histamine and chromogranin A seems to be the most important and abundant products. Recent data indicate a direct correlation between ECL-cell mass and circulating chromogranin A levels. Chromogranin A and its splice products might serve as growth promoting agents in ECL-cell hyperplasia or gastric carcinoids. PMID:10461347

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

    PubMed

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

    2004-12-01

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

  6. Selective defect in myeloid cell lactoferrin gene expression in neutrophil specific granule deficiency.

    PubMed Central

    Lomax, K J; Gallin, J I; Rotrosen, D; Raphael, G D; Kaliner, M A; Benz, E J; Boxer, L A; Malech, H L

    1989-01-01

    Neutrophil specific granule deficiency (SGD) is a congenital disorder associated with an impaired inflammatory response and a deficiency of several granule proteins. The underlying abnormality causing the deficiencies is unknown. We examined mRNA transcription and protein synthesis of two neutrophil granule proteins, lactoferrin and myeloperoxidase in SGD. Metabolically labeled SGD nucleated marrow cells produced normal amounts of myeloperoxidase, but there was no detectable synthesis of lactoferrin. Transcripts of the expected size for lactoferrin were detectable in the nucleated marrow cells of two SGD patients, but were markedly diminished in abundance when compared with normal nucleated marrow cell RNA. Because lactoferrin is secreted by the glandular epithelia of several tissues, we also assessed lactoferrin in the nasal secretions of one SGD patient by ELISA and immunoblotting. Nasal secretory lactoferrin was the same molecular weight as neutrophil lactoferrin and was secreted in normal amounts. From these data, we conclude that lactoferrin deficiency in SGD neutrophils is tissue specific and is secondary to an abnormality of RNA production. We speculate that the deficiency of several granule proteins is due to a common defect in regulation of transcription that is responsible for the abnormal myeloid differentiation seen in SGD patients. Images PMID:2536400

  7. Evidence for recognition of novel islet T cell antigens by granule-specific T cell lines from new onset type 1 diabetic patients

    PubMed Central

    Tree, T I M; O'Byrne, D; Tremble, J M; Macfarlane, W M; Haskins, K; James, R F L; Docherty, K; Hutton, J C; Banga, J P

    2000-01-01

    Type 1 diabetes is a T cell-mediated autoimmune disease where a number of islet ?-cell target autoantigens have been characterized on the basis of reactivity with autoantibodies. Nevertheless, there remains uncertainty of the nature of another group of autoantigens associated with the secretory granule fraction of islet ?-cells that appear to be targeted predominantly by autoreactive T cells. We have previously characterized CD4+, HLA-DR-restricted T cell lines from new onset type 1 diabetic patients that are specific for the secretory granule fraction of rat tumour insulinoma, RIN. The T cell line from the first patient, HS, proliferates in response to crude microsomal membranes prepared from a recently established, pure human islet ?-cell line NES2Y. In addition, the HS line also responds to secretory granule fractions prepared from a murine tumour insulinoma grown in RIP-Tag mice, showing the recognition of species-conserved antigen(s) in ?-cells. Using partially matched antigen-presenting cells, the HS T cells and another line derived from a second patient, MR, were shown to be restricted by disease-associated DRB1*0101 and DRB1*0404 alleles, respectively. Neither the HS or MR T cell lines proliferate in response to a large panel of candidate islet cell antigens, including insulin, proinsulin, glutamic acid decarboxylase, the protein tyrosine phosphatase IA-2/phogrin, imogen-38, ICA69 or hsp60. Our data provide compelling evidence of the presence of a group of antigens associated with the secretory granule fraction of islet ?-cells recognized by the T cell lines, whose definition may contribute to our knowledge of disease induction as well as to diagnosis. PMID:10886245

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

    PubMed Central

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

    2014-01-01

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

  9. Patterns of dentate granule cell responses to perforant path stimulation in epileptic mice with granule cell dispersion.

    PubMed

    Rougier, A; Arthaud, S; Zombre, N; La Salle, G Le Gal

    2005-02-01

    In adult mice, intrahippocampal administration of kainic acid induces a structural modification of the granule cell layer reminiscent of granule cell dispersion (GCD) seen in humans with temporal lobe epilepsy. We tested that GCD might be involved in the patterns of granule cell responses to perforant path stimulation by recording field potentials in vivo after kainic acid-induced status epilepticus until the phase of chronic seizure activity in presence of GCD or after its alteration by K252a co-treatment, an inhibitor of tyrosine kinase activities. Stimulation triggered bursts of multiple population spikes, the number of which progressively increased with time whereas their amplitude decreased in parallel with the progressive decrease in granule cell density. The population spike threshold was reached for a lower excitatory synaptic drive than in controls, as assessed by the initial slope of the field excitatory post-synaptic potential. This indicates that, for identical synaptic responses, granule cells were closer to the firing threshold. Fast inhibition, assessed by paired pulse stimulation, was compromised immediately after the initial status epilepticus, consistent with the rapid loss of most hilar cells. Neither the epileptic course nor the epileptiform responses of the granule cells were modified and manipulation by alteration following GCD manipulation while granule cell neuropeptide-Y immunostaining was substantially decreased. In this mouse model of TLE, granule cells display a progressive increase in epileptiform responses to afferent input until the occurrence of spontaneous seizures. The population spike amplitude decreases in parallel with GCD while the granule cell excitability is enhanced. Consequently, data from field potentials in epilepsy experiments should be interpreted with care, taking into account the possible variations in the neuronal density in the recorded area. PMID:15777666

  10. Multimodal sensory integration in single cerebellar granule cells in vivo

    PubMed Central

    Ishikawa, Taro; Shimuta, Misa; Häusser, Michael

    2015-01-01

    The mammalian cerebellum is a highly multimodal structure, receiving inputs from multiple sensory modalities and integrating them during complex sensorimotor coordination tasks. Previously, using cell-type-specific anatomical projection mapping, it was shown that multimodal pathways converge onto individual cerebellar granule cells (Huang et al., 2013). Here we directly measure synaptic currents using in vivo patch-clamp recordings and confirm that a subset of single granule cells receive convergent functional multimodal (somatosensory, auditory, and visual) inputs via separate mossy fibers. Furthermore, we show that the integration of multimodal signals by granule cells can enhance action potential output. These recordings directly demonstrate functional convergence of multimodal signals onto single granule cells. DOI: http://dx.doi.org/10.7554/eLife.12916.001 PMID:26714108

  11. 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...

  12. Human NK cell lytic granules and regulation of their exocytosis

    PubMed Central

    Krzewski, Konrad; Coligan, John E.

    2012-01-01

    Natural killer (NK) cells form a subset of lymphocytes that play a key role in immuno-surveillance and host defense against cancer and viral infections. They recognize stressed cells through a variety of germline-encoded activating cell surface receptors and utilize their cytotoxic ability to eliminate abnormal cells. Killing of target cells is a complex, multi-stage process that concludes in the directed secretion of lytic granules, containing perforin and granzymes, at the immunological synapse. Upon delivery to a target cell, perforin mediates generation of pores in membranes of target cells, allowing granzymes to access target cell cytoplasm and induce apoptosis. Therefore, lytic granules of NK cells are indispensable for normal NK cell cytolytic function. Indeed, defects in lytic granule secretion lead or are related to serious and often fatal diseases, such as familial hemophagocytic lymphohistiocytosis (FHL) type 25 or Griscelli syndrome type 2. A number of reports highlight the role of several proteins involved in lytic granule release and NK cell-mediated killing of tumor cells. This review focuses on lytic granules of human NK cells and the advancements in understanding the mechanisms controlling their exocytosis. PMID:23162553

  13. UVC-induced stress granules in mammalian cells.

    PubMed

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

    2014-01-01

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

  14. UVC-Induced Stress Granules in Mammalian Cells

    PubMed Central

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

    2014-01-01

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

  15. Acid-induced secretory cell metaplasia in hamster bronchi

    SciTech Connect

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

    1988-02-01

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

  16. Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum.

    PubMed

    Haldipur, Parthiv; Sivaprakasam, Iswariya; Periasamy, Vinod; Govindan, Subashika; Mani, Shyamala

    2015-01-01

    The plane of division of granule neuron progenitors (GNPs) was analysed with respect to the pial surface in P0 to P14 cerebellum and the results showed that there was a significant bias towards the plane of cell division being parallel to pial surface across this developmental window. In addition, the distribution of ?-Catenin in anaphase cells was analysed, which showed that there was a significant asymmetry in the distribution of ?-Catenin in dividing GNPs. Further, inhibition of Sonic Hedgehog (Shh) signalling had an effect on plane of cell division. Asymmetric distribution of ?-Catenin was shown to occur towards the source of a localized extracellular cue. PMID:25979710

  17. Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum

    PubMed Central

    Haldipur, Parthiv; Sivaprakasam, Iswariya; Periasamy, Vinod; Govindan, Subashika; Mani, Shyamala

    2015-01-01

    ABSTRACT The plane of division of granule neuron progenitors (GNPs) was analysed with respect to the pial surface in P0 to P14 cerebellum and the results showed that there was a significant bias towards the plane of cell division being parallel to pial surface across this developmental window. In addition, the distribution of ?-Catenin in anaphase cells was analysed, which showed that there was a significant asymmetry in the distribution of ?-Catenin in dividing GNPs. Further, inhibition of Sonic Hedgehog (Shh) signalling had an effect on plane of cell division. Asymmetric distribution of ?-Catenin was shown to occur towards the source of a localized extracellular cue. PMID:25979710

  18. Carboxypeptidase E and Secretogranin III Coordinately Facilitate Efficient Sorting of Proopiomelanocortin to the Regulated Secretory Pathway in AtT20 Cells.

    PubMed

    Cawley, Niamh X; Rathod, Trushar; Young, Sigrid; Lou, Hong; Birch, Nigel; Loh, Y Peng

    2016-01-01

    Proopiomelanocortin (POMC) is a multivalent prohormone that can be processed into at least 7 biologically active peptide hormones. Processing can begin in the trans-Golgi network (TGN) and continues in the secretory granules of the regulated secretory pathway (RSP). Sorting of POMC into these granules is a complex process. Previously, a membrane-associated form of carboxypeptidase E (CPE) was shown to bind to POMC and facilitate its trafficking into these granules. More recently, secretogranin III (SgIII) was also found to affect POMC trafficking. Here, we show by RNA silencing that CPE and SgIII play a synergistic role in the trafficking of POMC to granules of the RSP in AtT20 cells. Reduction of either protein resulted in increased constitutive secretion of POMC and chromogranin A, which was increased even further when both proteins were reduced together, indicative of missorting at the TGN. In SgIII-reduced cells, POMC accumulated in a compartment that cofractionated and colocalized with syntaxin 6, a marker of the TGN, on sucrose density gradients and in immunocytochemistry, respectively, indicating an accumulation of this protein in the presumed sorting compartment. Regulated secretion of ACTH, as a measure of sorting and processing of POMC in mature granules, was reduced in the SgIII down-regulated cells but was increased in the CPE down-regulated cells. These results suggest that multiple sorting systems exist, providing redundancy to ensure the important task of continuous and accurate trafficking of prohormones to the granules of the RSP for the production of peptide hormones. PMID:26646096

  19. Fallopian tube secretory cell expansion: a sensitive biomarker for ovarian serous carcinogenesis

    PubMed Central

    Wang, Yiying; Li, Li; Wang, Yue; Tang, Sarah Ngocvi; Zheng, Wenxin

    2015-01-01

    Recent advances suggest that precancerous lesions of pelvic serous carcinoma originate from tubal secretory cells. The purpose of our study was to determine if an increased number of secretory cells varies with age or location in the fallopian tube and to examine its association with serous neoplasia. Three groups (benign control, high-risk, and pelvic serous carcinoma) of age-matched patients were studied. The age data were stratified into 10-year intervals ranging from 20-29 to older than 80. The number of secretory and ciliated cells from both tubal fimbria and ampulla segments was counted by microscopy and immunohistochemical staining methods. The data were analyzed by standard contingency table and Poisson distribution methods after age justification. We found that the absolute number of tubal secretory cells increased significantly with age in all three groups. But a more dramatic increase of secretory cells was observed in high-risk and pelvic serous carcinoma patients. Secretory cell expansion is more prevalent than secretory cell outgrowth in both fimbria and ampulla tubal segments and is significantly associated with serous neoplasia (P < 0.001). Furthermore, age remained a significant risk factor for serous neoplasia after age adjustment. These findings suggest that secretory cell expansion could serve as a potential sensitive biomarker for early serous carcinogenesis within the fallopian tube. The study also supports a relationship between serous neoplasia and increased secretory to ciliated cell ratios, and the relationship between frequency of secretory cell expansion within the fallopian tube and increasing age and-more significantly-presence of high-risk factors or co-existing serous cancers. PMID:26692952

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

    PubMed

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

    2006-09-01

    The major mechanism for NK cell lysis of tumor cells is granule-mediated cytotoxicity. Polarization of granules is a prelude to the release of their cytotoxic contents in response to target-cell binding. We describe the novel observation of constitutive granule polarization in the cytotoxic NK cell line, KHYG-1. Continuous degranulation of KHYG-1 cells, however, does not occur and still requires target-cell contact. Disruption of microtubules with colcemid is sufficient to disperse the granules in KHYG-1 and significantly decreases cytotoxicity. A similar effect is not obtained by inhibiting extracellular signal-related kinase 2 (ERK2), the most distal kinase investigated in the cytolytic pathway. Disruption of microtubules significantly down-regulates activation receptors, NKp44 and NKG2D, implicating them as potential microtubule-trafficking receptors. Such changes in upstream receptor expression may have caused deactivation of ERK2, since NKG2D cross-linking also leads to receptor down-regulation and diminished ERK phosphorylation. Thus, a functional role for NKG2D in KHYG-1 cytotoxicity is demonstrated. Moreover, the novel primed state may contribute to the high cytotoxicity exhibited by KHYG-1. PMID:16849396

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

    PubMed Central

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

    2014-01-01

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

  2. Secretory autophagy.

    PubMed

    Ponpuak, Marisa; Mandell, Michael A; Kimura, Tomonori; Chauhan, Santosh; Cleyrat, Cdric; Deretic, Vojo

    2015-08-01

    Autophagy, once viewed exclusively as a cytoplasmic auto-digestive process, has its less intuitive but biologically distinct non-degradative roles. One manifestation of these functions of the autophagic machinery is the process termed secretory autophagy. Secretory autophagy facilitates unconventional secretion of the cytosolic cargo such as leaderless cytosolic proteins, which unlike proteins endowed with the leader (N-terminal signal) peptides cannot enter the conventional secretory pathway normally operating via the endoplasmic reticulum and the Golgi apparatus. Secretory autophagy may also export more complex cytoplasmic cargo and help excrete particulate substrates. Autophagic machinery and autophagy as a process also affect conventional secretory pathways, including the constitutive and regulated secretion, as well as promote alternative routes for trafficking of integral membrane proteins to the plasma membrane. Thus, autophagy and autophagic factors are intimately intertwined at many levels with secretion and polarized sorting in eukaryotic cells. PMID:25988755

  3. A 20-nm Step toward the Cell Membrane Preceding Exocytosis May Correspond to Docking of Tethered Granules

    PubMed Central

    Karatekin, Erdem; Tran, Viet Samuel; Huet, Sbastien; Fanget, Isabelle; Cribier, Sophie; Henry, Jean-Pierre

    2008-01-01

    In endocrine cells, plasma membrane (PM)-bound secretory granules must undergo a number of maturation stages (i.e., priming) to become fusion-competent. Despite identification of several molecules involved in binding granules to the PM and priming them, the exact nature of events occurring at the PM still largely remains a mystery. In stimulated BON cells, we used evanescent wave microscopy to study trajectories of granules shortly before their exocytoses, which provided a physical description of vesicle-PM interactions at an unprecedented level of detail, and directly lead to an original mechanistic model. In these cells, tethered (T), nonfusogenic, vesicles are prevented from converting to fusogenic, docked (D) ones in resting conditions. Upon elevation of calcium, T-vesicles perform a 21-nm step toward the PM to become D, and fuse ?3 s thereafter. Our ability to directly visualize different modes of PM-attachment paves the way for clarifying the exact role of various molecules implicated in attachment and priming of granules in future studies. PMID:18178647

  4. Syncollin inhibits regulated corticotropin secretion from AtT-20 cells through a reduction in the secretory vesicle population.

    PubMed Central

    Wsle, Barbara; Hays, Lori B; Rhodes, Christopher J; Edwardson, J Michael

    2004-01-01

    Syncollin is a 13 kDa protein that is highly expressed in the exocrine pancreas. Syncollin normally exists as a doughnut-shaped homo-oligomer (quite probably a hexamer) in close association with the luminal surface of the zymogen granule membrane. In the present study, we examine the effect of expression of syncollin in AtT-20 neuroendocrine cells, which do not normally express this protein. Efficient expression was achieved by infection of the cells with adenoviral constructs encoding either untagged or GFP (green fluorescent protein)-tagged syncollin. Both forms of the protein were sorted into corticotropin (ACTH)-positive secretory vesicles present mainly at the tips of cell processes. Neither form affected basal corticotropin secretion or the constitutive secretion of exogenously expressed secreted alkaline phosphatase. In contrast, regulated secretion of corticotropin was inhibited (by 49%) by untagged but not by GFP-tagged syncollin. In parallel, untagged syncollin caused a 46% reduction in the number of secretory vesicles present at the tips of the cell processes. Syncollin-GFP was without effect. We could also show that native syncollin purified from rat pancreas was capable of permeabilizing erythrocytes. We suggest that syncollin may induce uncontrolled permeabilization of corticotropin-containing vesicles and subsequently destabilize them. Both forms of syncollin were tightly membrane-associated and appeared to exist as homooligomers. Hence, the lack of effect of syncollin-GFP on regulated exocytosis suggests that the GFP tag interferes in a subtler manner with the properties of the assembled protein. PMID:15040787

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

    PubMed

    Plattner, H; Westphal, C; Tiggemann, R

    1982-02-01

    Stationary-phase cells of Paramecium tetraurelia have most of their many secretory vesicles ("trichocysts") attached to the cell surface. Log-phase cells contain numerous unoccupied potential docking sites for trichocysts and many free trichocysts in the cytoplasm. To study the possible involvement of cytoskeletal elements, notably of microtubules, in the process of positioning of trichocysts at the cell surface, we took advantage of these stages. Cells were stained with tannic acid and subsequently analyzed by electron microscopy. Semithin sections allowed the determination of structural connections over a range of up to 10 micrometer. Microtubules emanating from ciliary basal bodies are seen in contact with free trichocysts, which appear to be transported, with their tip first, to the cell surface. (This can account for the saltatory movement reported by others). It is noteworthy that the "rails" represented by the microtubules do not directly determine the final attachment site of a trichocyst. Unoccupied attachment sites are characterized by a "plug" of electron-dense material just below the plasma membrane; the "plug" seems to act as a recognition or anchoring site; this material is squeezed out all around the trichocyst attachment zone, once a trichocyst is inserted (Westphal and Plattner, in press. [53]). Slightly below this "plug" we observed fasciae of microfilaments (identified by immunocytochemistry using peroxidase labeled F(ab) fragments against P. tetraurelia actin). Their arrangement is not altered when a trichocyst is docked. These fasciae seem to form a loophole for the insertion of a trichocyst. Trichocyst remain attached to the microtubules originating from the ciliary basal bodies--at least for some time--even after they are firmly installed in the preformed attachment sites. Evidently, the regular arrangement of exocytotic organelles is controlled on three levels: one operating over a long distance from the exocytosis site proper (microtubules), one over a short distance (microfilament bundles), and one directly on the exocytosis site ("plug"). PMID:7199530

  6. Impact of Chromogranin A deficiency on catecholamine storage, catecholamine granule morphology and chromaffin cell energy metabolism in vivo.

    PubMed

    Pasqua, Teresa; Mahata, Sumana; Bandyopadhyay, Gautam K; Biswas, Angshuman; Perkins, Guy A; Sinha-Hikim, Amiya P; Goldstein, David S; Eiden, Lee E; Mahata, Sushil K

    2016-03-01

    Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40 % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200 nm) than in WT mice (200-350 nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47 % increase in DCV/DC ratio, implying vesicle swelling due to increased osmotically active free catecholamines. Upon challenge with 2 U/kg insulin, there was a diminution in adrenomedullary EPI, no change in NE and a very large increase in the EPI and NE precursor dopamine (DA), consistent with increased catecholamine biosynthesis during prolonged secretion. We found dilated mitochondrial cristae, endoplasmic reticulum and Golgi complex, as well as increased synaptic mitochondria, synaptic vesicles and glycogen granules in Chga-KO mice compared to WT mice, suggesting that decreased granulogenesis and catecholamine storage in CgA-deficient mouse adrenal medulla is compensated by increased VMAT-dependent catecholamine update into storage vesicles, at the expense of enhanced energy expenditure by the chromaffin cell. PMID:26572539

  7. Segmental identity and cerebellar granule cell induction in rhombomere 1

    PubMed Central

    Eddison, Mark; Toole, Leah; Bell, Esther; Wingate, Richard JT

    2004-01-01

    Background Cerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment? Results We show that a Gbx2-positive, Otx2-/Hoxa2-negative territory corresponding to rhombomere 1 forms prior to an identifiable isthmic organiser. Early global overexpression of Hoxa2 at embryonic day 0 has no effect on the expression of isthmic signalling molecules or the allocation of rhombomere 1 territory, but selectively results in the loss of granule cell markers at embryonic day 6 and the depletion of cell bodies from the external granule cell layer. By comparison the trochlear nucleus and locus coeruleus form normally in ventral rhombomere 1 under these conditions. Microsurgery, coupled with electroporation, to target Hoxa2 overexpression to rhombic lip precursors, reveals a profound, autonomous respecification of migration. Rhombic lip derivatives, normally destined to occupy the external granule cell layer, violate the cerebellar boundary to form a ventrolateral nucleus in a position comparable to that occupied by rhombic lip derived neurons in rhombomere 2. Conclusions Different overexpression strategies reveal that the recognition of migration cues by granule cell precursors is dependent on their identity as rhombomere 1 derivatives. Segmental patterning cues operate autonomously within the rhombic lip precursor pool. By contrast, a subset of coextensive nuclei is refractory to ectopic Hoxa2 and is presumably induced solely by isthmic organiser activity. Thus, graded (isthmic) and segmental mechanisms may operate exclusively of one another in the specification of different neuronal populations within rhombomere 1. The early designation of an Otx2-negative, Hoxa2-negative region, prior to the appearance of the isthmic organiser, is a key initial step in the specification of the cerebellum. PMID:15198802

  8. Behavioral experience induces zif268 expression in mature granule cells but suppresses its expression in immature granule cells

    PubMed Central

    Huckleberry, Kylie A.; Kane, Gary A.; Mathis, Rita J.; Cook, Sarah G.; Clutton, Jonathan E.; Drew, Michael R.

    2015-01-01

    Thousands of neurons are born each day in the dentate gyrus (DG), but many of these cells die before reaching maturity. Both death and survival of adult-born neurons are regulated by neuronal activity in the DG. The immediate-early gene (IEG) zif268 appears to be an important mediator of these effects, as its expression can be induced by neural activity and knockout of zif268 impairs survival of adult-born neurons (Richardson et al., 1992; Veyrac et al., 2013). Despite the apparent importance of zif268 for adult neurogenesis, its behavior-induced expression has not been fully characterized in adult-born neurons. Here we characterize behavior-evoked expression of zif268 in mature and newborn dentate granule cells (DGCs). We first quantified zif268 expression in doublecortin-positive (DCX+) immature neurons and in the general granule cell population after brief exposure to a novel environment (NE). In the general granule cell population, zif268 expression peaked 1 h after NE exposure and returned to baseline by 8 h post-exposure. However, in the DCX+ cells, zif268 expression was suppressed relative to home cage for at least 8 h post-exposure. We next asked whether suppression of zif268 in DCX+ immature cells occurs in other behavioral paradigms that recruit the hippocampus. Exposure to Morris water maze (MWM) training, an enriched environment, or a NE caused approximately equal suppression of zif268 expression in DCX+ cells and approximately equal activation of zif268 expression among the general granule cell population. The same behavioral procedures activated zif268 expression in 6-week-old BrdU-labeled adult-born neurons, indicating that zif268 suppression is specific to immature neurons. Finally, we asked whether zif268 suppression varied as a function of age within the DCX+ population, which ranges in age from 0 to approximately 4 weeks. NE exposure had no significant effect on zif268 expression in 2- or 4-week-old BrdU-labeled neurons, but it significantly suppressed zif268 expression in 3-week-old neurons. In summary, behavioral experience transiently activated expression of zif268 in mature granule cells but caused a more long-lasting suppression of zif268 expression in immature, adult-born granule cells. We hypothesize that zif268 suppression inhibits memory-related synaptic plasticity in immature neurons or mediates learning-induced apoptosis of immature adult-born neurons. PMID:26347620

  9. Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

    PubMed

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation. PMID:24489879

  10. Mapping Organelle Motion Reveals a Vesicular Conveyor Belt Spatially Replenishing Secretory Vesicles in Stimulated Chromaffin Cells

    PubMed Central

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A.; Rubinsztein-Dunlop, Halina; Meunier, Frederic A.

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this conveyor belt towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation. PMID:24489879

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

    NASA Astrophysics Data System (ADS)

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

    2009-05-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion.

    PubMed

    Verkhratsky, Alexei; Matteoli, Michela; Parpura, Vladimir; Mothet, Jean-Pierre; Zorec, Robert

    2016-01-01

    Astrocytes are housekeepers of the central nervous system (CNS) and are important for CNS development, homeostasis and defence. They communicate with neurones and other glial cells through the release of signalling molecules. Astrocytes secrete a wide array of classic neurotransmitters, neuromodulators and hormones, as well as metabolic, trophic and plastic factors, all of which contribute to the gliocrine system. The release of neuroactive substances from astrocytes occurs through several distinct pathways that include diffusion through plasmalemmal channels, translocation by multiple transporters and regulated exocytosis. As in other eukaryotic cells, exocytotic secretion from astrocytes involves divergent secretory organelles (synaptic-like microvesicles, dense-core vesicles, lysosomes, exosomes and ectosomes), which differ in size, origin, cargo, membrane composition, dynamics and functions. In this review, we summarize the features and functions of secretory organelles in astrocytes. We focus on the biogenesis and trafficking of secretory organelles and on the regulation of the exocytotic secretory system in the context of healthy and diseased astrocytes. PMID:26758544

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed Central

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

    2011-01-01

    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

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

    PubMed

    Chiang, Lilian; Ngo, Julie; Schechter, Joel E; Karvar, Serhan; Tolmachova, Tanya; Seabra, Miguel C; Hume, Alistair N; Hamm-Alvarez, Sarah F

    2011-08-01

    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 Rab27(ash/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

  18. Disinhibition of olfactory bulb granule cells accelerates odour discrimination in mice.

    PubMed

    Nunes, Daniel; Kuner, Thomas

    2015-01-01

    Granule cells are the dominant cell type of the olfactory bulb inhibiting mitral and tufted cells via dendrodendritic synapses; yet the factors regulating the strength of their inhibitory output, and, therefore, their impact on odour discrimination, remain unknown. Here we show that GABAAR β3-subunits are distributed in a somatodendritic pattern, mostly sparing the large granule cell spines also known as gemmules. Granule cell-selective deletion of β3-subunits nearly abolishes spontaneous and muscimol-induced currents mediated by GABAA receptors in granule cells, yet recurrent inhibition of mitral cells is strongly enhanced. Mice with disinhibited granule cells require less time to discriminate both dissimilar as well as highly similar odourants, while discrimination learning remains unaffected. Hence, granule cells are controlled by an inhibitory drive that in turn tunes mitral cell inhibition. As a consequence, the olfactory bulb inhibitory network adjusts the speed of early sensory processing. PMID:26592770

  19. Disinhibition of olfactory bulb granule cells accelerates odour discrimination in mice

    PubMed Central

    Nunes, Daniel; Kuner, Thomas

    2015-01-01

    Granule cells are the dominant cell type of the olfactory bulb inhibiting mitral and tufted cells via dendrodendritic synapses; yet the factors regulating the strength of their inhibitory output, and, therefore, their impact on odour discrimination, remain unknown. Here we show that GABAAR β3-subunits are distributed in a somatodendritic pattern, mostly sparing the large granule cell spines also known as gemmules. Granule cell-selective deletion of β3-subunits nearly abolishes spontaneous and muscimol-induced currents mediated by GABAA receptors in granule cells, yet recurrent inhibition of mitral cells is strongly enhanced. Mice with disinhibited granule cells require less time to discriminate both dissimilar as well as highly similar odourants, while discrimination learning remains unaffected. Hence, granule cells are controlled by an inhibitory drive that in turn tunes mitral cell inhibition. As a consequence, the olfactory bulb inhibitory network adjusts the speed of early sensory processing. PMID:26592770

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

    PubMed Central

    Wegrzyn, Jill L.; Bark, Steven J.; Funkelstein, Lydiane; Mosier, Charles; Yap, Angel; Kazemi-Esfarjani, Parasa; La Spada, Albert; Sigurdson, Christina; OConnor, Daniel T.; Hook, Vivian

    2010-01-01

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

  1. Phospholipase C γ1 regulates early secretory trafficking and cell migration via interaction with p115

    PubMed Central

    Millarte, Valentina; Boncompain, Gaelle; Tillmann, Kerstin; Perez, Franck; Sztul, Elizabeth; Farhan, Hesso

    2015-01-01

    The role of early secretory trafficking in the regulation of cell motility remains incompletely understood. Here we used a small interfering RNA screen to monitor the effects on structure of the Golgi apparatus and cell migration. Two major Golgi phenotypes were observed—fragmented and small Golgi. The latter exhibited a stronger correlation with a defect in cell migration. Among the small Golgi hits, we focused on phospholipase C γ1 (PLCγ1). We show that PLCγ1 regulates Golgi structure and cell migration independently of its catalytic activity but in a manner that depends on interaction with the tethering protein p115. PLCγ1 regulates the dynamics of p115 in the early secretory pathway, thereby controlling trafficking from the endoplasmic reticulum to the Golgi. Our results uncover a new function of PLCγ1 that is independent of its catalytic function and link early secretory trafficking to the regulation of cell migration. PMID:25904324

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

    PubMed Central

    Bertrand, Florie; Mller, Sabina; Roh, Kyung-Ho; Laurent, Camille; Dupr, Loc; Valitutti, Salvatore

    2013-01-01

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

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

    PubMed

    Hammel, Ilan; Anaby, Debbie

    2007-09-01

    The introduction of wet SEM imaging technology permits electron microscopy of wet samples. Samples are placed in sealed specimen capsules and are insulated from the vacuum in the SEM chamber by an impermeable, electron-transparent membrane. The complete insulation of the sample from the vacuum allows direct imaging of fully hydrated, whole-mount tissue. In the current work, we demonstrate direct inspection of thick pancreatic tissue slices (above 400 mum). In the case of scanning of the pancreatic surface, the boundaries of intracellular features are seen directly. Thus no unfolding is required to ascertain the actual particle size distribution based on the sizes of the sections. This method enabled us to investigate the true granule size distribution and confirm early studies of improved conformity to a Poisson-like distribution, suggesting that the homotypic granule growth results from a mechanism, which favors the addition of a single unit granule to mature granules. PMID:17557275

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

    PubMed Central

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

    2008-01-01

    Background Zebrafish germ cells contain granular-like structures, organized around the cell nucleus. These structures share common features with polar granules in Drosophila, germinal granules in Xenopus and chromatoid bodies in mice germ cells, such as the localization of the zebrafish Vasa, Piwi and Nanos proteins, among others. Little is known about the structure of these granules as well as their segregation in mitosis during early germ-cell development. Results Using transgenic fish expressing a fluorescently labeled novel component of Zebrafish germ cell granules termed Granulito, we followed the morphology and distribution of the granules. We show that whereas these granules initially exhibit a wide size variation, by the end of the first day of development they become a homogeneous population of medium size granules. We investigated this resizing event and demonstrated the role of microtubules and the minus-end microtubule dependent motor protein Dynein in the process. Last, we show that the function of the germ cell granule resident protein the Tudor domain containing protein-7 (Tdrd7) is required for determination of granule morphology and number. Conclusion Our results suggest that Zebrafish germ cell granules undergo a transformation process, which involves germ cell specific proteins as well as the microtubular network. PMID:18507824

  5. Dentate granule cells form hilar basal dendrites in a rat model of hypoxia-ischemia

    PubMed Central

    Díaz-Cintra, Sofia; Xue, Baogang; Spigelman, Igor; Van, K.; Wong, Alan M.; Obenaus, Andre; Ribak, Charles E.

    2009-01-01

    Hilar basal dendrites form on dentate granule cells following seizures. To determine whether other brain insults cause the formation of hilar basal dendrites, a model of global cerebral hypoxia/ischemia was used. Rats underwent a transient induction of ischemia by occlusion of both common carotid arteries followed by reperfusion. Hippocampal slices were prepared from these animals 1 month after the ischemic insult, and granule cells were labeled with a retrograde tracing technique after biocytin injections into stratum lucidum of CA3b. Ischemic rats had numerous biocytin-labeled granule cells with hilar basal dendrites located at the hilar border of the granule cell layer. Quantitative analysis of ischemic rats compared to controls showed a significant increase in the percentage of biocytin-labeled granule cells with hilar basal dendrites. These data demonstrate that other brain insults in addition to epilepsy may result in the formation of hilar basal dendrites on granule cells. PMID:19539612

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

    PubMed

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

    2015-01-01

    The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1(Atoh1) CKO) to investigate the function of LKB1 in cerebellar development. The LKB1(Atoh1) CKO mice displayed motor dysfunction. In the LKB1(Atoh1) CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1(Atoh1) CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development. PMID:26549569

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

    PubMed Central

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

    2015-01-01

    The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1Atoh1 CKO) to investigate the function of LKB1 in cerebellar development. The LKB1Atoh1 CKO mice displayed motor dysfunction. In the LKB1Atoh1 CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1Atoh1 CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development. PMID:26549569

  8. Competitive Interactions of Cancer Cells and Normal Cells via Secretory MicroRNAs*

    PubMed Central

    Kosaka, Nobuyoshi; Iguchi, Haruhisa; Yoshioka, Yusuke; Hagiwara, Keitaro; Takeshita, Fumitaka; Ochiya, Takahiro

    2012-01-01

    Normal epithelial cells regulate the secretion of autocrine and paracrine factors that prevent aberrant growth of neighboring cells, leading to healthy development and normal metabolism. One reason for tumor initiation is considered to be a failure of this homeostatic cell competitive system. Here we identify tumor-suppressive microRNAs (miRNAs) secreted by normal cells as anti-proliferative signal entities. Culture supernatant of normal epithelial prostate PNT-2 cells attenuated proliferation of PC-3M-luc cells, prostate cancer cells. Global analysis of miRNA expression signature revealed that a variety of tumor-suppressive miRNAs are released from PNT-2 cells. Of these miRNAs, secretory miR-143 could induce growth inhibition exclusively in cancer cells in vitro and in vivo. These results suggest that secretory tumor-suppressive miRNAs can act as a death signal in a cell competitive process. This study provides a novel insight into a tumor initiation mechanism. PMID:22123823

  9. AB081. Study on the secretory function of Sertoli cells and testicular damage in diabetic rats

    PubMed Central

    Liu, Baoxing; Zhang, Xiuping; Qin, Mao; Song, Aiping; Cui, Tianwei

    2015-01-01

    Objective To investigate the changes of secretory function of Sertoli cells and testicular damage in diabetic rats. Methods Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 55 mg/kg/i.p.) in adult rats. Eight weeks later testicular tissue was harvested; serum INHB and testosterone (T) levels were measured by ELISA. The secretory proteins such as ABP, TF were measured by western blot. Testicular damage was detected by using hematoxylin and eosin (HE) staining and apoptosis was identified by terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL). Seminiferous tubular sperm formation was evaluated using Johnsens score. Results In diabetic rats, ABP protein levels, serum INHB and T levels were found to be decreased greatly. Meanwhile, TF protein levels decreased without significantly. Additionally, TUNEL-positive cells were increased and Johnsens score was decreased in diabetic testes. Conclusions These results suggest that the secretory function of Sertoli cell in diabetic rats was damaged. The altered secretory function on Sertoli cell may be responsible for the disturbed spermatogenesis.

  10. Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells*?

    PubMed Central

    Kosaka, Nobuyoshi; Iguchi, Haruhisa; Yoshioka, Yusuke; Takeshita, Fumitaka; Matsuki, Yasushi; Ochiya, Takahiro

    2010-01-01

    The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs. PMID:20353945

  11. Programmed cell death: a mechanism for the lysigenous formation of secretory cavities in leaves of Dictamnus dasycarpus.

    PubMed

    Zhou, Ya-Fu; Mao, Shao-Li; Li, Si-Feng; Ni, Xi-Lu; Li, Bin; Liu, Wen-Zhe

    2014-08-01

    The formation of secretory cavities in Rutaceae has been the subject of great interest. In this study, cytological events that are involved in the lysigenous formation of the secretory cavities in the leaves of Dictamnus dasycarpus are characterized by an interesting pattern of programmed cell death (PCD). During the developmental process, clusters of cells from a single protoepidermal cell embark on different trajectories and undergo different cell death fates: the cell walls of the secretory cells have characteristics of thinning or complete breakdown, while the sheath cells present a predominantly thick-walled feature. A DAPI assay shows deformed nuclei that are further confirmed to be TUNEL-positive. Gel electrophoresis indicates that DNA cleavage is random and does not result in ladder-like DNA fragmentation. Ultrastructurally, several remarkable features of PCD have been determined, such as misshapen nuclei with condensed chromatin and a significantly diffused membrane, degenerated mitochondria and plastids with disturbed membrane systems, multivesicular bodies, plastolysomes, vacuole disruption and lysis of the center secretory cell. Cytological evidence and Nile red stains exhibit abundant essential oils accumulated in degenerated outer secretory cells after the dissolution of the center secretory cell. In addition, explanations of taxonomic importance and the relationship between PCD and oil droplet accumulation in the secretory cavities are also discussed. PMID:25017170

  12. 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

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

    2011-01-01

    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

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

    PubMed Central

    1991-01-01

    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

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

    PubMed

    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

    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 cellgranule 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

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

    PubMed

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

    1985-01-01

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

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

    PubMed Central

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

    2015-01-01

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The ?7?nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional ?7?nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 23 week-old Wistar rats, and 29 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of ?7?nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric ?7?nicotinic receptor modulator, which were blocked by a specific ?7?nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic ?7?nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating ?7?nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain. PMID:25553616

  17. Non-cell autonomous or secretory tumor suppression.

    PubMed

    Chua, Christelle En Lin; Chan, Shu Ning; Tang, Bor Luen

    2014-10-01

    Many malignancies result from deletions or loss-of-function mutations in one or more tumor suppressor genes, the products of which curb unrestrained growth or induce cell death in those with dysregulated proliferative capacities. Most tumor suppressors act in a cell autonomous manner, and only very few proteins are shown to exert a non-cell autonomous tumor suppressor function on other cells. Examples of these include members of the secreted frizzled-related protein (SFRP) family and the secreted protein acidic and rich in cysteine (SPARC)-related proteins. Very recent findings have, however, considerably expanded our appreciation of non-cell autonomous tumor suppressor functions. Broadly, this may occur in two ways. Intracellular tumor suppressor proteins within cells could in principle inhibit aberrant growth of neighboring cells by conditioning an antitumor microenvironment through secreted factors. This is demonstrated by an apparent non-cell autonomous tumor suppressing property of p53. On the other hand, a tumor suppressor produced by a cell may be secreted extracellularly, and taken up by another cell with its activity intact. Intriguingly, this has been recently shown to occur for the phosphatase and tensin homolog (PTEN) by both conventional and unconventional modes of secretion. These recent findings would aid the development of therapeutic strategies that seek to reinstate tumor suppression activity in therapeutically recalcitrant tumor cells, which have lost it in the first place. PMID:24752809

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

    PubMed

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

    2012-08-01

    Temporal lobe epilepsy (TLE) is accompanied by an abnormal location of granule cells in the dentate gyrus. Using a rat model of complex febrile seizures, which are thought to be a precipitating insult of TLE later in life, we report that aberrant migration of neonatal-generated granule cells results in granule cell ectopia that persists into adulthood. Febrile seizures induced an upregulation of GABA(A) receptors (GABA(A)-Rs) in neonatally generated granule cells, and hyperactivation of excitatory GABA(A)-Rs caused a reversal in the direction of granule cell migration. This abnormal migration was prevented by RNAi-mediated knockdown of the Na(+)K(+)2Cl(-) co-transporter (NKCC1), which regulates the excitatory action of GABA. NKCC1 inhibition with bumetanide after febrile seizures rescued the granule cell ectopia, susceptibility to limbic seizures and development of epilepsy. Thus, this work identifies a previously unknown pathogenic role of excitatory GABA(A)-R signaling and highlights NKCC1 as a potential therapeutic target for preventing granule cell ectopia and the development of epilepsy after febrile seizures. PMID:22797810

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

    SciTech Connect

    Gianotti, A.J.; Clark, D.T.; Dash, J. )

    1991-04-01

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

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

    PubMed

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

    2004-02-01

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

  1. Phospholipase D2 Modulates the Secretory Pathway in RBL-2H3 Mast Cells

    PubMed Central

    Marchini-Alves, Claudia Maria Meirelles; Barbosa Lorenzi, Valeria Cintra; da Silva, Elaine Zayas Marcelino; Mazucato, Vivian Marino; Jamur, Maria Celia; Oliver, Constance

    2015-01-01

    Phospholipase D (PLD) hydrolyses phosphatidylcholine to produce phosphatidic acid (PA) and choline. It has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type. In mast cells it plays an important role in signal transduction. The aim of the present study was to clarify the role of PLD2 in the secretory pathway. RBL-2H3 cells, a mast cell line, transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2 were used. Previous observations showed that the Golgi complex was well organized in CA cells, but was disorganized and dispersed in CI cells. Furthermore, in CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized. These previous observations demonstrated that PLD2 is important for maintaining the morphology and organization of the Golgi complex. To further understand the role of PLD2 in secretory and vesicular trafficking, the role of PLD2 in the secretory process was investigated. Incorporation of sialic acid was used to follow the synthesis and transport of glycoconjugates in the cell lines. The modified sialic acid was subsequently detected by labeling with a fluorophore or biotin to visualize the localization of the molecule after a pulse-chase for various times. Glycoconjugate trafficking was slower in the CI cells and labeled glycans took longer to reach the plasma membrane. Furthermore, in CI cells sialic acid glycans remained at the plasma membrane for longer periods of time compared to RBL-2H3 cells. These results suggest that PLD2 activity plays an important role in regulating glycoconjugate trafficking in mast cells. PMID:26492088

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

    PubMed

    Lacy, E R; Reale, E

    1989-07-01

    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

  3. Plasticity of intrinsic excitability in mature granule cells of the dentate gyrus

    PubMed Central

    Lopez-Rojas, Jeffrey; Heine, Martin; Kreutz, Michael R.

    2016-01-01

    The dentate gyrus is the main entry gate for cortical input to the hippocampus and one of the few brain areas where adult neurogenesis occurs. Several studies have shown that it is relatively difficult to induce synaptic plasticity in mature but not in newborn dentate granule cells. In the present work we have systematically addressed how classical protocols to induce synaptic plasticity affect action potential firing and intrinsic excitability in mature granule cells. We found that stimulation paradigms considered to be relevant for learning processes consistently modified the probability to generate action potentials in response to a given synaptic input in mature cells, in some paradigms even without any modification of synaptic strength. Collectively the results suggest that plasticity of intrinsic dendritic excitability has a lower induction-threshold than synaptic plasticity in mature granule cells and that this form of plasticity might be an important mechanism by which mature granule cells contribute to hippocampal function. PMID:26857841

  4. Plasticity of intrinsic excitability in mature granule cells of the dentate gyrus.

    PubMed

    Lopez-Rojas, Jeffrey; Heine, Martin; Kreutz, Michael R

    2016-01-01

    The dentate gyrus is the main entry gate for cortical input to the hippocampus and one of the few brain areas where adult neurogenesis occurs. Several studies have shown that it is relatively difficult to induce synaptic plasticity in mature but not in newborn dentate granule cells. In the present work we have systematically addressed how classical protocols to induce synaptic plasticity affect action potential firing and intrinsic excitability in mature granule cells. We found that stimulation paradigms considered to be relevant for learning processes consistently modified the probability to generate action potentials in response to a given synaptic input in mature cells, in some paradigms even without any modification of synaptic strength. Collectively the results suggest that plasticity of intrinsic dendritic excitability has a lower induction-threshold than synaptic plasticity in mature granule cells and that this form of plasticity might be an important mechanism by which mature granule cells contribute to hippocampal function. PMID:26857841

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

    PubMed

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

    2012-04-25

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

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

    SciTech Connect

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

    2012-02-03

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

  7. Secretory organelles of pathogenic protozoa.

    PubMed

    Souza, Wanderley de

    2006-06-01

    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

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

    PubMed Central

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

    1994-01-01

    The kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+ were studied by capacitance (Cm) measurements with millisecond time resolution. After elevation of the internal Ca2+ concentration ([Ca2+]i), Cm rises rapidly with one or more exponentials. The time constant of the fastest component decreases for higher [Ca2+]i (range 3-600 microM) over three orders of magnitude before it saturates at approximately 1 ms. The corresponding maximal rates of secretion can be as fast as 100,000 fF/s or 40,000 vesicles/s. There is a Ca(2+)-dependent delay before Cm rises, which may reflect the kinetics of multiple Ca2+ ions binding to the secretory apparatus. The initial rise in Cm is described by models containing a sequence of two to four single Ca(2+)-binding steps followed by a rate-limiting exocytosis step. The predicted Ca2+ dissociation constant (Kd) of a single Ca(2+)-binding site is between 7 and 21 microM. At [Ca2+]i > 30 microM clear indications of a fast endocytotic process complicate the analysis of the secretory response. PMID:7696493

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

    PubMed Central

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

    2013-01-01

    While it has been hypothesized that adult neurogenesis (NG) plays a role in the encoding of temporal information at long time-scales, the temporal relationship of immature cells to the highly rhythmic network activity of the hippocampus has been largely unexplored. Here, we present a theory for how the activity of immature adult-born granule cells relates to hippocampal oscillations. Our hypothesis is that theta rhythmic (510 Hz) excitatory and inhibitory inputs into the hippocampus could differentially affect young and mature granule cells due to differences in intrinsic physiology and synaptic inhibition between the two cell populations. Consequently, immature cell activity may occur at broader ranges of theta phase than the activity of their mature counterparts. We describe how this differential influence on young and mature granule cells could separate the activity of differently aged neurons in a temporal coding regime. Notably, this process could have considerable implications on how the downstream CA3 region interprets the information conveyed by young and mature granule cells. To begin to investigate the phasic behavior of granule cells, we analyzed in vivo recordings of the rat dentate gyrus (DG), observing that the temporal behavior of granule cells with respect to the theta rhythm is different between rats with normal and impaired levels of NG. Specifically, in control animals, granule cells exhibit both strong and weak coupling to the phase of the theta rhythm. In contrast, the distribution of phase relationships in NG-impaired rats is shifted such that they are significantly stronger. These preliminary data support our hypothesis that immature neurons could distinctly affect the temporal dynamics of hippocampal encoding. PMID:23717259

  10. The biology and dynamics of mammalian cortical granules

    PubMed Central

    2011-01-01

    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

  11. Relationship between calcium mobilization and platelet ?- and ?-granule secretion. A role for TRPC6 in thrombin-evoked ?-granule exocytosis.

    PubMed

    Lopez, E; Bermejo, N; Berna-Erro, A; Alonso, N; Salido, G M; Redondo, P C; Rosado, J A

    2015-11-01

    Changes in cytosolic Ca(2+) concentration ([Ca(2+)]c) regulate granule secretion in different cell types. Thrombin activates PAR1 and PAR4 receptors and promotes release of Ca(2+) from distinct intracellular stores, which, in turn, activates store-operated Ca(2+) entry (SOCE). A crucial step during platelet function is the release of physiological agonists stored in secretory granules to the extracellular compartment during activation. We aim to study the role of Ca(2+) mobilization from the extracellular compartment or from different intracellular stores in platelet granule secretion. By using flow cytometry, we have found that ?- and ?-granules are secreted in thrombin-stimulated platelets in the absence of extracellular Ca(2+), and in a concentration-dependent manner. Our findings show that thrombin-stimulated granule secretion depends on Ca(2+) mobilization from intracellular stores. Analysis of the kinetics of granule secretion reveals that platelet stimulation with thrombin results in rapid release of ?-granules which precedes the secretion of ?-granules. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 573-586 of TRPC6, inhibited thrombin-evoked ?-granule exocytosis. Our results indicate that the mechanisms underlying thrombin-induced ?- and ?-granule secretion show differences in dependency on Ca(2+) mobilization. PMID:26386308

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

    Jefingija, S

    2006-01-01

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

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

    PubMed Central

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

    2012-01-01

    Background For a safe ‘in vivo’ biomedical utilization of nanoparticles, it is essential to assess not only biocompatibility, but also the potential to trigger unwanted side effects at both cellular and tissue levels. Mastocytes (cells having secretory granules containing cytokines, vasoactive amine, and proteases) play a pivotal role in the immune and inflammatory responses against exogenous toxins. Mastocytes are also recruited in the tumor stroma and are involved in tumor vascularization and growth. Aim and methods In this work, mastocyte-like rat basophilic leukemia (RBL) cells were used to investigate whether carboxyl-modified 30 nm polystyrene (PS) nanoparticles (NPs) and naked mesoporous silica (MPS) 10 nm NPs are able to label the secretory inflammatory granules, and possibly induce exocytosis of these granules. Uptake, cellular retention and localization of fluorescent NPs were analyzed by cytofluorometry and microscope imaging. Results Our findings were that: (1) secretory granules of mastocytes are accessible by NPs via endocytosis; (2) PS and MPS silica NPs label two distinct subpopulations of inflammatory granules in RBL mastocytes; and (3) PS NPs induce calcium-dependent exocytosis of inflammatory granules. Conclusion These findings highlight the value of NPs for live imaging of inflammatory processes, and also have important implications for the clinical use of PS-based NPs, due to their potential to trigger the unwanted activation of mastocytes. PMID:22605932

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

    SciTech Connect

    Adrian, T.E.; Goldenring, J.R.; Oddsdottir, M.; Zdon, M.J.; Zucker, K.A.; Lewis, J.J.; Modlin, I.M. )

    1989-11-01

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

  16. Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation

    SciTech Connect

    Choi, Bongkun; Kang, Soon-Suk; Kang, Sang-Wook; Min, Bon-Hong; Lee, Eun-Jin; Song, Da-Hyun; Kim, Sang-Min; Song, Youngsup; Yoon, Seung-Yong; Chang, Eun-Ju

    2014-07-18

    Highlights: • We describe the expression and secretion of clusterin in osteoclasts. • Endogenous clusterin deficiency does not affect osteoclast formation. • Exogenous treatment with secretory clusterin decreases osteoclast differentiation. • Secretory clusterin attenuates osteoclast precursor cell proliferation by inhibiting M-CSF-mediated ERK activation. - Abstract: Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU{sup −/−} mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.

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

    PubMed

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

    1988-08-01

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

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

    PubMed Central

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

    1998-01-01

    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

  19. In vitro three-dimensional modeling of fallopian tube secretory epithelial cells

    PubMed Central

    2013-01-01

    Background Fallopian tube secretory epithelial cells (FTSECs) have been implicated as a cell-of-origin for high-grade serous epithelial ovarian cancer. However, there are relatively few in vitro models of this tissue type available for use in studies of FTSEC biology and malignant transformation. In vitro three-dimensional (3D) cell culture models aim to recreate the architecture and geometry of tissues in vivo and restore the complex network of cell-cell/cell-matrix interactions that occur throughout the surface of the cell membrane. Results We have established and characterized 3D spheroid culture models of primary FTSECs. FTSEC spheroids contain central cores of hyaline matrix surrounded by mono- or multi-layer epithelial sheets. We found that 3D culturing alters the molecular characteristics of FTSECs compared to 2D cultures of the same cells. Gene expression profiling identified more than a thousand differentially expressed genes between 3D and 2D cultures of the same FTSEC lines. Pathways significantly under-represented in 3D FTSEC cultures were associated with cell cycle progression and DNA replication. This was also reflected in the reduced proliferative indices observed in 3D spheroids stained for the proliferation marker MIB1. Comparisons with gene expression profiles of fresh fallopian tube tissues revealed that 2D FTSEC cultures clustered with follicular phase tubal epithelium, whereas 3D FTSEC cultures clustered with luteal phase samples. Conclusions This 3D model of fallopian tube secretory epithelial cells will advance our ability to study the underlying biology and etiology of fallopian tube tissues and the pathogenesis of high-grade serous epithelial ovarian cancer. PMID:24070420

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

    PubMed Central

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

    2014-01-01

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

  1. Cancer Stem Cell Phenotype Is Supported by Secretory Phospholipase A2 in Human Lung Cancer Cells

    PubMed Central

    Bennett, Daine T.; Deng, Xin-Sheng; Yu, Jessica A.; Bell, Marshall T.; Mauchley, David C.; Meng, Xianzhong; Reece, T. Brett; Fullerton, David A.; Weyant, Michael J.

    2016-01-01

    Background Lung cancer stem cells (CSCs) are a sub-population of cells that drive growth, invasiveness, and resistance to therapy. Inflammatory eicosanoids are critical to maintain this malignant subpopulation. Secretory phospholipase A2 group IIa (sPLA2) is an important mediator of the growth and invasive potential of human lung cancer cells and regulates eicosanoid production. We hypothesized that sPLA2 plays a role in the maintenance of lung CSCs. Methods Cancer stem cells from lung adenocarcinoma cell lines H125 and A549 were isolated using aldehyde dehydrogenase activity and flow cytometry. Protein and mRNA levels for sPLA2 were compared between sorted cells using Western blotting and quantitative reverse transcriptasepolymerase chain reaction techniques. Chemical inhibition of sPLA2 and short-hairpin RNA knockdown of sPLA2 were used to evaluate effects on tumorsphere formation. Results Lung CSCs were isolated in 8.9% 4.1% (mean SD) and 4.1% 1.6% of H125 and A549 cells respectively. Both sPLA2 protein and mRNA expression were significantly elevated in the CSC subpopulation of H125 (p = 0.002) and A549 (p = 0.005; n = 4). Knockdown of sPLA2 significantly reduced tumorsphere formation in H125 (p = 0.026) and A549 (p = 0.001; n = 3). Chemical inhibition of sPLA2 resulted in dose-dependent reduction in tumorsphere formation in H125 (p = 0.003) and A549 (p = 0.076; n = 3). Conclusions Lung CSCs express higher levels of sPLA2 than the nonstem cell population. Our findings that viral knockdown and chemical inhibition of sPLA2 reduce tumorsphere formation in lung cancer cells demonstrate for the first time that sPLA2 plays an important role in CSCs. These findings suggest that sPLA2 may be an important therapeutic target for human lung cancer. PMID:24928671

  2. Primary culture and immortalization of human fallopian tube secretory epithelial cells.

    PubMed

    Karst, Alison M; Drapkin, Ronny

    2012-09-01

    Primary human fallopian tube secretory epithelial cell (FTSEC) cultures are useful for studying normal fallopian tube epithelial biology, as well as for developing models of fallopian tube disease, such as cancer. Because of the limited ability of primary human FTSECs to proliferate in vitro, it is necessary to immortalize them in order to establish a cell line that is suitable for long-term culture and large-scale in vitro experimentation. This protocol describes the isolation of FTSECs from human fallopian tube tissue, conditions for primary FTSEC culture and techniques for establishing immortal FTSEC lines. The entire process, from primary cell isolation to establishment of an immortal cell line, may take up to 2 months. Once established, immortal FTSECs can typically be maintained for at least 30 passages. PMID:22936217

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

    PubMed

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

    2015-08-01

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

  4. LYST controls the biogenesis of the endosomal compartment required for secretory lysosome function.

    PubMed

    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

    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

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

    PubMed

    Constantin, Lena; Wainwright, Brandon J

    2015-12-01

    MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development. PMID:25910616

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

    PubMed Central

    Wada, Norio; Funabiki, Kazuo; Nakanishi, Shigetada

    2014-01-01

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

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

    PubMed

    Wada, Norio; Funabiki, Kazuo; Nakanishi, Shigetada

    2014-04-01

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

  8. Identification of miRNAs differentially expressed in human epilepsy with or without granule cell pathology.

    PubMed

    Zucchini, Silvia; Marucci, Gianluca; Paradiso, Beatrice; Lanza, Giovanni; Roncon, Paolo; Cifelli, Pierangelo; Ferracin, Manuela; Giulioni, Marco; Michelucci, Roberto; Rubboli, Guido; Simonato, Michele

    2014-01-01

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

  9. Dentate Gyrus Granule Cell Firing Patterns Can Induce Mossy Fiber Long-Term Potentiation In Vitro

    PubMed Central

    Mistry, Rajen; Dennis, Siobhan; Frerking, Matthew; Mellor, Jack R.

    2010-01-01

    Hippocampal granule cells transmit information about behaviourally-relevant stimuli to CA3 pyramidal cells via mossy fiber synapses. These synapses express a form of long-term potentiation (mfLTP) that is non-Hebbian and does not require NMDA receptors. mfLTP is thought to be induced and expressed presynaptically, hence, the major determinant of whether mfLTP occurs is activity in the granule cells. However, it remains unclear whether mfLTP can be induced by activity patterns that granule cells exhibit in vivo, and if so what context generates these patterns. To address these issues, we examined granule cell activity from in vivo recordings from rats during performance of a delayed nonmatch-to-sample (DNMS) task and found that granule cells exhibit a wide range of spike patterns. In vitro slice experiments in mice demonstrated that some, but not all, of these patterns of activity could induce mfLTP. By further defining the activity thresholds for mfLTP in hippocampal slices, we found that mfLTP can only be induced by spike patterns that fire in high frequency bursts with a low average firing frequency. Using this information, we then screened for supra-threshold bursts of activity during the DNMS task. In a subset of cells, supra-threshold bursts occurred preferentially during the sampling phase of the task. If supra-threshold bursting took place later, during the delay phase, task performance was disrupted. We conclude that mfLTP can be induced by granule cell spike patterns during a memory task, and that the timing of mfLTP induction can predict task performance. PMID:20635414

  10. Intestinal label-retaining cells are secretory precursors expressing Lgr5.

    PubMed

    Buczacki, Simon J A; Zecchini, Heather Ireland; Nicholson, Anna M; Russell, Roslin; Vermeulen, Louis; Kemp, Richard; Winton, Douglas J

    2013-03-01

    The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation. PMID:23446353

  11. Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

    PubMed Central

    Bnard, Magalie; Lebon, Alexis; Komuro, Hitoshi; Vaudry, David; Galas, Ludovic

    2015-01-01

    During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration. PMID:25992599

  12. Conditioned eyeblink learning is formed and stored without cerebellar granule cell transmission.

    PubMed

    Wada, Norio; Kishimoto, Yasushi; Watanabe, Dai; Kano, Masanobu; Hirano, Tomoo; Funabiki, Kazuo; Nakanishi, Shigetada

    2007-10-16

    Classical conditioning of the eyeblink reflex is elicited by paired presentation of a conditioned stimulus and an unconditioned stimulus and represents a basic form of cerebellum-dependent motor learning. Purkinje cells and the deep nuclei receive convergent information of conditioned stimulus and unconditioned stimulus through the mossy fiber and climbing fiber projections, respectively. To explore the relative importance of these neural circuits and the underlying mechanism in associative eyeblink learning, we adopted a novel gene-manipulating technique, termed reversible neurotransmission blocking (RNB). In this technology, cerebellar granule cells specifically expressed neurotransmission-blocking tetanus toxin in a doxycycline (DOX)-dependent manner. Extracellular recording of Purkinje cells in awake RNB mice revealed that DOX treatment and withdrawal reversibly turned off and on simple spikes elicited by granule cell inputs, respectively, without interference with complex spikes evoked by climbing fiber inputs. Blockade of granule cell inputs to Purkinje cells abolished eyeblink conditioned responses (CRs) in a DOX-dependent manner. Importantly, when granule cell inputs recovered by removal of DOX, normal CRs were immediately produced in the DOX-treated, CR-negative RNB mice from the beginning of reconditioning. This learning process in RNB mice during DOX treatment was completely abolished by bilateral lesion of the interpositus nucleus before eyeblink conditioning. These results indicate that the convergent information at the interpositus nucleus is critical for acquisition and storage of learning in intimate association with the Purkinje cell circuit for expression of CRs in eyeblink conditioning. PMID:17923666

  13. Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.

    PubMed

    Hanzel, Michalina; Wingate, Richard J T; Butts, Thomas

    2015-01-01

    The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease. PMID:26709704

  14. Secretory transport of ranitidine and famotidine across Caco-2 cell monolayers.

    PubMed

    Lee, Kiho; Ng, Chee; Brouwer, Kim L R; Thakker, Dhiren R

    2002-11-01

    The secretory transport of the H(2)-antagonists, ranitidine and famotidine, across Caco-2 cell monolayers was found to be a saturable process. Both drugs exhibited greater permeability in the basolateral (BL) to apical (AP) direction than in the AP to BL direction, indicating apically directed secretion; BL to AP transport was inhibited by P-glycoprotein (P-gp) inhibitors verapamil and cyclosporin A. The cellular uptake of ranitidine across the BL membrane was saturable and temperature dependent, indicative of carrier-mediated transport. The K(m) and V(max) for the uptake process were estimated to be 66.9 mM and 20.9 nmol/mg of protein/min, respectively. The uptake of [(14)C]ranitidine across the BL membrane was inhibited by unlabeled ranitidine and structurally diverse organic cations. The tetraethylammonium (TEA)-sensitive organic cation transporters are not involved in the uptake of ranitidine and famotidine across the BL membrane of Caco-2. This conclusion was based on the evidence that functionally active TEA-sensitive organic cation transporters did not exist in the BL membranes of the Caco-2 cells, whereas the functionally active TEA-sensitive organic cation transporter(s) in LLC-PK(1) cells did not contribute to the transport of ranitidine or famotidine across the cell monolayers. Thus, we conclude that the secretory transport of ranitidine and famotidine across Caco-2 cell monolayers is mediated by 1) a carrier in the BL membrane that is distinct from the TEA-sensitive organic cation transporter(s) and 2) P-gp in the apical membrane. PMID:12388638

  15. Ionic and secretory response of pancreatic islet cells to minoxidil sulfate

    SciTech Connect

    Antoine, M.H.; Hermann, M.; Herchuelz, A.; Lebrun, P. )

    1991-07-01

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

  16. Group IIa secretory phospholipase expression correlates with group IIa secretory phospholipase inhibitionmediated cell death in K-ras mutant lung cancer cells

    PubMed Central

    Yu, Jessica A.; Li, Howard; Meng, Xianzhong; Fullerton, David A.; Nemenoff, Raphael A.; Mitchell, John D.; Weyant, Michael J.

    2013-01-01

    Objective There are currently no targeted therapies against lung tumors with oncogenic K-ras mutations that are found in 25% to ?40% of lung cancers and are characterized by their resistance to epidermal growth factor receptor inhibitors. The isozyme group IIa secretory phospholipase A2 (sPLA2IIa) is a potential biomarker and regulator of lung cancer cell invasion; however, the relationship between K-ras mutations and sPLA2IIa has yet to be investigated. We hypothesize that sPLA2IIa modulates lung cancer cell growth in K-ras mutant cells and that sPLA2IIa expression in human lung tumors is increased in K-ras mutant tumors. Methods Baseline sPLA2IIa expression in K-ras mutant lung cancer cell lines (A549, SW1573, H358, H2009) was assessed. Cells were treated with a specific sPLA2IIa inhibitor and evaluated for apoptosis and cell viability. Nuclear factor kappa-b (NF-?B) and extracellular signal-regulated kinase 1/2 activity were detected by Western blot. Human tumor samples were evaluated for sPLA2IIa mRNA expression by quantitative reverse-transcription polymerase chain reaction. Results Cytotoxicity of sPLA2IIa inhibition correlates with sPLA2IIa expression. Apoptosis in response to sPLA2 inhibition parallels attenuation in NF-?B activity. In addition, sPLA2IIa expression in human tumors correlates with squamous cell pathology and increasing stage of K-ras mutant lung tumors. Conclusions Baseline sPLA2IIa expression predicts response to sPLA2IIa inhibition in some K-ras mutant lung cancer cells. This finding is independent of p53 mutation status. Furthermore, squamous tumors and advanced-stage K-ras mutant tumors express more sPLA2IIa. These data support a role for sPLA2IIa as a potential global therapeutic target in the treatment of lung cancer. PMID:23026567

  17. High-affinity kainate-type ion channels in rat cerebellar granule cells

    PubMed Central

    Pemberton, Karen E; Belcher, Scott M; Ripellino, James A; Howe, James R

    1998-01-01

    Patch-clamp recordings were made from rat cerebellar granule cells in primary culture. In cells pre-exposed to concanavalin A (ConA) to remove kainate receptor desensitization, concentration-response data for kainate showed two components. The EC50 value for the high-affinity component (4 μM) was consistent with activation of kainate-type channels. ConA enhanced the apparent potency of the kainate receptor ligand SYM 2081 by 100-fold. In ConA-treated granule cells, currents evoked by 10 μM kainate were not significantly reduced by the AMPA receptor antagonist GYKI 53655, nor were these currents significantly reduced by the co-application of 100 μM AMPA. Currents activated by low concentrations of kainate in the presence of AMPA were completely inhibited by 10 μM La3+. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that granule cells express both unedited (Q) and edited (R) versions of GluR5, with the majority of the GluR5 transcripts being unedited. In contrast, GluR6(R) was detected in seven cells and GluR6(Q) was detected in one granule cell. Whole-cell current-voltage curves for kainate-type currents in granule cells were measured and the ratio of the slope conductances at +40 mV and −40 mV was used as an index of rectification. The mean +40 mV/-40 mV ratio determined from thirty-six granule cells was 1.3 ± 0.1. Spectral density analysis of kainate-evoked whole-cell current noise gave values for the apparent single-channel conductance, γnoise, that were on average about 1 pS. To compare further the properties of recombinant kainate channels with the native kainate-type channels in granule cells, we determined EC50 and γnoise values for SYM 2081 in stable cell lines expressing either GluR6(R) or GluR6(R) and KA2. Co-expression of KA2 with GluR6(R) shifts the EC50 and γnoise values determined for SYM 2081 closer to the values typically found for native kainate-type channels in granule cells. The results demonstrate that cerebellar granule cells in culture express functional kainate-type channels and that in most cells these channels show properties that are similar to those determined for heteromeric channels formed from GluR6(R) and KA2. However, the results also suggest that different granule cells express different repertoires of kainate-type channels with different, and perhaps variable, subunit composition. PMID:9705992

  18. Dengue virus infection induces formation of G3BP1 granules in human lung epithelial cells.

    PubMed

    Xia, Jun; Chen, Xiaoyan; Xu, Feng; Wang, Yi; Shi, Yongxia; Li, Yuye; He, Junfang; Zhang, Ping

    2015-12-01

    Cells reprogram ongoing translation in response to viral infection, resulting in formation of stress granules (SGs), while viruses have evolved a variety of strategies to antagonize the host SG response. Previous literature reported that in BHK-1 cells, infection with dengue virus (DENV) interfered with the SG formation. In the current study, we further investigated SG formation in human epithelial A549 cells by detecting subcellular localization of two SG hallmarks, TIA-1 and G3BP1. In response to DENV type 2 (DENV2) and type 3 (DENV3) infection, G3BP1, but not TIA-1, was recruited into cytoplasmic granules in some cells, and viral protein synthesis was significantly impaired in the G3BP1-granule-containing cells. Knockdown of G3BP1 significantly rescued the dsRNA-mediated suppression of DENV2 replication. Furthermore, our data showed that the phosphorylation of protein kinase regulated by dsRNA (PKR) and eIF2?, as well as accumulation of dsRNA, mainly occurred at the late stage of viral infection. This work revealed that in DENV-infected A549 cells, G3BP1 granules were assembled independently of TIA-1 and had a negative impact on viral replication. This extends our understanding of the antagonistic relationship between the SG response and dengue virus infection. PMID:26350772

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

    PubMed Central

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

    2013-01-01

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

  20. Degranulation of eosinophilic granule cells in neurofibromas and gastrointestinal tract in the bicolor damselfish.

    PubMed

    Schmale, Michael C; Vicha, Dale; Cacal, Saul M

    2004-07-01

    Damselfish neurofibromatosis (DNF) is a neoplastic disease affecting bicolor damselfish (Stegastes partitus Poey) on Florida reefs. Previous studies have demonstrated high densities of eosinophilic granule containing cells (EGC), the proposed equivalent of mast cells in fishes, in neurofibromas and malignant peripheral nerve sheath tumors (mpnst) in DNF. These lesions are similar to those in the disease neurofibromatosis type 1 (NF1) in humans, which contain large numbers of mast cells. In the present study, experiments were conducted to measure the response of EGC in these tumors as well as in the submucosa of the digestive tract to the mast cell degranulating agent compound 48/80. Degranulation of these cells was visible by light microscopy and characterized by conspicuous swelling of granules and often by the presence of free granules adjacent to the EGC. Degranulation occurred by release of intact granules (diacytosis), as reported in other fishes, rather than by fusion of granules with the cell membrane (exocytosis) as reported in mast cells in mammals. Baseline levels of EGC exhibiting degranulation ranged from 20-26% in the submucosa to 30% in tumors. Within 1-2h of exposure to compound 48/80, significant increases in average levels of degranulation were observed, to 67% in the gut and 72% in tumors. Degranulation was significantly more extensive in the tumors than in the gut. The outermost edges of the tumors contained significantly higher densities of EGC but these cells exhibited lower rates of degranulation than those in the inner regions of tumors. These observations support the hypothesis that the EGC present in neurofibromas and mpnst in DNF are equivalent to the mast cell component in neurofibromas in NF1. PMID:15145417

  1. Physiological properties of GABAA receptors from acutely dissociated rat dentate granule cells.

    PubMed

    Kapur, J; Haas, K F; Macdonald, R L

    1999-05-01

    Physiological properties of GABAA receptors from acutely dissociated rat dentate granule cells. Study of fast, GABAA receptor-mediated, inhibitory postsynaptic currents (IPSCs) in hippocampal dentate granule cells has suggested that properties of GABAA receptors influence the amplitude and time course of the IPSCs. This study describes the physiological properties of GABAA receptors present on hippocampal dentate granule cells acutely isolated from 18- to 35-day-old rats. Rapid application of 1 mM GABA to outside-out macropatches excised from granule cells produced GABAA receptor currents with rapid rise time and biexponential decay of current after removal of GABA. After activation, granule cell GABAA receptor currents desensitized incompletely. During a 400-ms application of 1 mM GABA, peak current only desensitized approximately 40%. In symmetrical chloride solutions there was no outward rectification of whole cell current. Activation rates and peak currents elicited by rapid application of GABA to macropatches were also similar at positive and negative holding potentials. However, deactivation of GABAA receptor currents was slower at positive holding potentials. When whole cell currents were recorded without ATP in the pipette, current run-down was not apparent for 30 min in 50% of neurons, but run-down appeared to start soon after access was established in the remaining neurons. When 2 mM ATP was included in the recording pipette no run-down was apparent in 30 min of recording. The efficacy and potency of GABA were lower in cells recorded with no ATP in the pipette and during run-down compared with those recorded with 2 mM ATP and no run-down. PMID:10322081

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

    PubMed Central

    Kavishwar, Amol

    2013-01-01

    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

  3. Axonal Na+ Channels Ensure Fast Spike Activation and Back-Propagation in Cerebellar Granule Cells

    PubMed Central

    Diwakar, Shyam; Magistretti, Jacopo; Goldfarb, Mitchell; Naldi, Giovanni; D'Angelo, Egidio

    2009-01-01

    In most neurons, Na+ channels in the axon are complemented by others localized in the soma and dendrites to ensure spike back-propagation. However, cerebellar granule cells are neurons with simplified architecture in which the dendrites are short and unbranched and a single thin ascending axon travels toward the molecular layer before bifurcating into parallel fibers. Here we show that in cerebellar granule cells, Na+ channels are enriched in the axon, especially in the hillock, but almost absent from soma and dendrites. The impact of this channel distribution on neuronal electroresponsiveness was investigated by multi-compartmental modeling. Numerical simulations indicated that granule cells have a compact electrotonic structure allowing excitatory postsynaptic potentials to diffuse with little attenuation from dendrites to axon. The spike arose almost simultaneously along the whole axonal ascending branch and invaded the hillock the activation of which promoted spike back-propagation with marginal delay (<200 ?s) and attenuation (<20 mV) into the somato-dendritic compartment. These properties allow granule cells to perform sub-millisecond coincidence detection of pre- and postsynaptic activity and to rapidly activate Purkinje cells contacted by the axonal ascending branch. PMID:19073816

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

    PubMed

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

    2013-05-01

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

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

    PubMed

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

    2014-01-01

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

  6. Senescent Vascular Smooth Muscle Cells Drive Inflammation Through an Interleukin-1α–Dependent Senescence-Associated Secretory Phenotype

    PubMed Central

    Gardner, Sarah E.; Humphry, Melanie; Bennett, Martin R.

    2015-01-01

    Objective— Vascular smooth muscle cells (VSMCs) that become senescent are both present within atherosclerotic plaques and thought to be important to the disease process. However, senescent VSMCs are generally considered to only contribute through inaction, with failure to proliferate resulting in VSMC- and collagen-poor unstable fibrous caps. Whether senescent VSMCs can actively contribute to atherogenic processes, such as inflammation, is unknown. Approach and Results— We find that senescent human VSMCs develop a proinflammatory state known as a senescence-associated secretory phenotype. Senescent human VSMCs release high levels of multiple cytokines and chemokines driven by secreted interleukin-1α acting in an autocrine manner. Consequently, the VSMC senescence-associated secretory phenotype promotes chemotaxis of mononuclear cells in vitro and in vivo. In addition, senescent VSMCs release active matrix metalloproteinase-9, secrete less collagen, upregulate multiple inflammasome components, and prime adjacent endothelial cells and VSMCs to a proadhesive and proinflammatory state. Importantly, maintaining the senescence-associated secretory phenotype places a large metabolic burden on senescent VSMCs, such that they can be selectively killed by inhibiting glucose utilization. Conclusions— Senescent VSMCs may actively contribute toward the chronic inflammation associated with atherosclerosis through the interleukin-1α–driven senescence-associated secretory phenotype and the priming of adjacent cells to a proatherosclerotic state. These data also suggest that inhibition of this potentially important source of chronic inflammation in atherosclerosis requires blockade of interleukin-1α and not interleukin-1β. PMID:26139463

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

    SciTech Connect

    Huang, C.M.; Liu, G.; Huang, R.H. )

    1991-03-11

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

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

    PubMed Central

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

    2014-01-01

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

  9. Heat-killed Lactobacillus acidophilus La205 enhances NK cell cytotoxicity through increased granule exocytosis.

    PubMed

    Cheon, Soyoung; Lee, Ki Woong; Kim, Kyung Eun; Park, Jung Kyu; Park, Sunyoung; Kim, Chul-hyun; Kim, Daejin; Lee, Hyong Joo; Cho, Daeho

    2011-05-01

    Heat-killed lactic acid bacteria (LAB) are known to be important immunomodulators that stimulate tumor necrosis factor-? (TNF-?) and nitric oxide (NO) production as well as increase phagocytic activity in macrophages. NK cells play a critical role in innate immune response and induce spontaneous killing of tumor cells and virus-infected cells. However, the effect of heat-killed LAB on NK cells is still unclear. In this study, we investigated the effect of heat-killed Lactobacillus acidophilus La205 (La205) on NK cytolytic activity. We found that heat-killed La205 directly stimulated NK cytolytic activity in dose- and time-dependent manners. To determine the mechanism underlying heat-killed La205-enhanced NK cytotoxicity, the expression of NK activating receptors was tested. Heat-killed La205 did not affect the expression of NK activating receptors. To investigate whether NK degranulation is related to heat-killed La205-enhanced NK cytotoxicity, NK degranulation inhibitor concanamycin A (CMA) was used. CMA effectively blocked heat-killed La205-induced NK cytotoxicity, and an assay for detection of a degranulation marker, CD107a, showed that heat-killed La205 increased granule exocytosis approximately 2-fold in comparison to non-treated NK cells. In addition, heat-killed La205 dramatically elevated mRNA expression of granulysin, a component of the cytolytic granule contents, in NK cells. However, other granule contents, including perforin and granzymes, were not changed by heat-killed La205. From these data, we concluded that heat-killed La205 stimulated NK cytolytic activity through enhancement of granule exocytosis, and granulysin may be a critical mediator in heat-killed La205-induced granule exocytosis. PMID:21256158

  10. Expression of ODC Antizyme Inhibitor 2 (AZIN2) in Human Secretory Cells and Tissues

    PubMed Central

    Rasila, Tiina; Lehtonen, Alexandra; Kanerva, Kristiina; Mäkitie, Laura T.; Haglund, Caj; Andersson, Leif C.

    2016-01-01

    Ornithine decarboxylase (ODC) antizyme inhibitor 2 (AZIN2), originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s) of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3) to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated. PMID:26963840

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  14. Clostridium difficile toxin B inhibits the secretory response of human mast cell line-1 (HMC-1) cells stimulated with high free-Ca²⁺ and GTPγS.

    PubMed

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

    2015-02-01

    Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho, Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency. Inactivation of Rho-family GTPases by this toxin in our experimental conditions induced morphological changes and reduction of electron-dense mast cell-specific granules in human mast cell line-1 (HMC-1) cells, but not cell death or permeabilisation of plasma-membranes. Previously reported patch-clamp dialysis experiments revealed that high intracellular free-Ca(2+) and GTPγS 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 GTPγS 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

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    1994-09-01

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

  17. Secretory production system of bionanocapsules using a stably transfected insect cell line.

    PubMed

    Shishido, Takuya; Muraoka, Masaru; Ueda, Masakazu; Seno, Masaharu; Tanizawa, Katsuyuki; Kuroda, Shun'ichi; Fukuda, Hideki; Kondo, Akihiko

    2006-12-01

    Bionanocapsules (BNCs) are hollow nanoscale particles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNCs can transfer genes and drugs into human hepatocytes efficiently and specifically. BNC can be expressed in yeast cells. In this study, we developed a new L particle production system using a stably transfected insect cell line. For this purpose, we established a host-vector system using the Trichoplusia ni insect cell line. L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 microg/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The production of L particles was maintained for at least 75 days. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was demonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC. PMID:16847600

  18. Porosome: the secretory portal.

    PubMed

    Jena, Bhanu P

    2012-07-01

    'It seems terribly wasteful that, during the release of hormones and neurotransmitters from a cell, the membrane of a vesicle should merge with the plasma membrane to be retrieved for recycling only seconds or minutes later.' - Erwin Neher, Nature 1993;363:497-498. This insightful statement so appropriately put, clearly reflected on the perception that secretory vesicles completely merge at the cell plasma membrane, failing to justify the generation of partially empty secretory vesicles in cells following secretion. A rational cellular mechanism would employ the transient fusion of secretory vesicles at the cell plasma membrane without compromising vesicle integrity, combined with vesicle retrieval following partial discharge of contents, to generate such partially empty vesicles following secretion. This hypothesis was finally confirmed with the serendipitous discovery of the porosome almost 16 years ago. The porosome has been demonstrated to be the universal secretory portal in cells and is present at the cell plasma membrane. In the past decade, the composition of the porosome, its dynamics, its structure at nanometer resolution in realtime using atomic force and electron microscopy, and its functional reconstitution into artificial lipid membrane, has resulted in a paradigm shift and a molecular understanding of the secretory process in cells. A brief background on porosome discovery, and our current understanding of its structure and function is summarized in this Minireview. PMID:22859740

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

    PubMed Central

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

    2010-01-01

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

  20. Electroconvulsive Seizure Promotes Spine Maturation in Newborn Dentate Granule Cells in Adult Rat

    PubMed Central

    Zhao, Chunmei; Warner-Schmidt, Jennifer; Duman, Ronald S.; Gage, Fred H.

    2013-01-01

    Neurogenesis continues in the dentate gyrus of the hippocampus throughout life in mammals. This process is influenced by daily activities such as exercise, learning, and stress and may contribute to certain forms of hippocampus-dependent learning and memory. Adult hippocampal neurogenesis is also subject to regulation by anti depressant treatment, including chronic treatment with antidepressant drugs or electroconvulsive seizure (ECS) therapy. Here we investigated how the connectivity of newborn and mature granule cells is influenced by ECS administration in rats. Specifically, we examined the dendritic spine morphology of newborn and mature granule cells in rats and found that ECS administration promoted the maturation of dendritic spines in newborn cells and increased spine density in mature cells. These changes could potentially lead to alteration in dentate circuitry and may partially contribute to the functional effects of ECS. PMID:21976455

  1. Electroconvulsive seizure promotes spine maturation in newborn dentate granule cells in adult rat.

    PubMed

    Zhao, Chunmei; Warner-Schmidt, Jennifer; Duman, Ronald S; Gage, Fred H

    2012-06-01

    Neurogenesis continues in the dentate gyrus of the hippocampus throughout life in mammals. This process is influenced by daily activities such as exercise, learning, and stress and may contribute to certain forms of hippocampus-dependent learning and memory. Adult hippocampal neurogenesis is also subject to regulation by antidepressant treatment, including chronic treatment with antidepressant drugs or electroconvulsive seizure (ECS) therapy. Here we investigated how the connectivity of newborn and mature granule cells is influenced by ECS administration in rats. Specifically, we examined the dendritic spine morphology of newborn and mature granule cells in rats and found that ECS administration promoted the maturation of dendritic spines in newborn cells and increased spine density in mature cells. These changes could potentially lead to alteration in dentate circuitry and may partially contribute to the functional effects of ECS. PMID:21976455

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

    PubMed

    Bywalez, Wolfgang G; Patirniche, Dinu; Rupprecht, Vanessa; Stemmler, Martin; Herz, Andreas V M; Plfi, Dnes; Rzsa, Balzs; Egger, Veronica

    2015-02-01

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

  3. Clonal Analysis of Newborn Hippocampal Dentate Granule Cell Proliferation and Development in Temporal Lobe Epilepsy(1,2,3).

    PubMed

    Singh, Shatrunjai P; LaSarge, Candi L; An, Amen; McAuliffe, John J; Danzer, Steve C

    2015-01-01

    Hippocampal dentate granule cells are among the few neuronal cell types generated throughout adult life in mammals. In the normal brain, new granule cells are generated from progenitors in the subgranular zone and integrate in a typical fashion. During the development of epilepsy, granule cell integration is profoundly altered. The new cells migrate to ectopic locations and develop misoriented "basal" dendrites. Although it has been established that these abnormal cells are newly generated, it is not known whether they arise ubiquitously throughout the progenitor cell pool or are derived from a smaller number of "bad actor" progenitors. To explore this question, we conducted a clonal analysis study in mice expressing the Brainbow fluorescent protein reporter construct in dentate granule cell progenitors. Mice were examined 2 months after pilocarpine-induced status epilepticus, a treatment that leads to the development of epilepsy. Brain sections were rendered translucent so that entire hippocampi could be reconstructed and all fluorescently labeled cells identified. Our findings reveal that a small number of progenitors produce the majority of ectopic cells following status epilepticus, indicating that either the affected progenitors or their local microenvironments have become pathological. By contrast, granule cells with "basal" dendrites were equally distributed among clonal groups. This indicates that these progenitors can produce normal cells and suggests that global factors sporadically disrupt the dendritic development of some new cells. Together, these findings strongly predict that distinct mechanisms regulate different aspects of granule cell pathology in epilepsy. PMID:26756038

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

    PubMed Central

    LaSarge, Candi L.; McAuliffe, John J.

    2015-01-01

    Abstract Hippocampal dentate granule cells are among the few neuronal cell types generated throughout adult life in mammals. In the normal brain, new granule cells are generated from progenitors in the subgranular zone and integrate in a typical fashion. During the development of epilepsy, granule cell integration is profoundly altered. The new cells migrate to ectopic locations and develop misoriented “basal” dendrites. Although it has been established that these abnormal cells are newly generated, it is not known whether they arise ubiquitously throughout the progenitor cell pool or are derived from a smaller number of “bad actor” progenitors. To explore this question, we conducted a clonal analysis study in mice expressing the Brainbow fluorescent protein reporter construct in dentate granule cell progenitors. Mice were examined 2 months after pilocarpine-induced status epilepticus, a treatment that leads to the development of epilepsy. Brain sections were rendered translucent so that entire hippocampi could be reconstructed and all fluorescently labeled cells identified. Our findings reveal that a small number of progenitors produce the majority of ectopic cells following status epilepticus, indicating that either the affected progenitors or their local microenvironments have become pathological. By contrast, granule cells with “basal” dendrites were equally distributed among clonal groups. This indicates that these progenitors can produce normal cells and suggests that global factors sporadically disrupt the dendritic development of some new cells. Together, these findings strongly predict that distinct mechanisms regulate different aspects of granule cell pathology in epilepsy. PMID:26756038

  5. Eosinophil secretion of granule-derived cytokines.

    PubMed

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

    2014-01-01

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

  6. Eosinophil Secretion of Granule-Derived Cytokines

    PubMed Central

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

    2014-01-01

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

  7. Bronchiolar nonciliated secretory (Clara) cells: source of guanylin in the mammalian lung.

    PubMed Central

    Cetin, Y; Kulaksiz, H; Redecker, P; Bargsten, G; Adermann, K; Grube, D; Forssmann, W G

    1995-01-01

    The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7597055

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

    PubMed Central

    Mathias, Amandine; Longet, Stéphanie

    2013-01-01

    Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-κB 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

  9. Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells.

    PubMed

    Madroñal, Noelia; Delgado-García, José M; Fernández-Guizán, Azahara; Chatterjee, Jayanta; Köhn, Maja; Mattucci, Camilla; Jain, Apar; Tsetsenis, Theodoros; Illarionova, Anna; Grinevich, Valery; Gross, Cornelius T; Gruart, Agnès

    2016-01-01

    The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories. PMID:26988806

  10. Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells

    PubMed Central

    Madroñal, Noelia; Delgado-García, José M.; Fernández-Guizán, Azahara; Chatterjee, Jayanta; Köhn, Maja; Mattucci, Camilla; Jain, Apar; Tsetsenis, Theodoros; Illarionova, Anna; Grinevich, Valery; Gross, Cornelius T.; Gruart, Agnès

    2016-01-01

    The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories. PMID:26988806

  11. Dendritic morphology, synaptic transmission, and activity of mature granule cells born following pilocarpine-induced status epilepticus in the rat

    PubMed Central

    Gao, Fei; Song, Xueying; Zhu, Dexiao; Wang, Xiaochen; Hao, Aijun; Nadler, J. Victor; Zhan, Ren-Zhi

    2015-01-01

    To understand the potential role of enhanced hippocampal neurogenesis after pilocarpine-induced status epilepticus (SE) in the development of epilepsy, we quantitatively analyzed the geometry of apical dendrites, synaptic transmission, and activation levels of normotopically distributed mature newborn granule cells in the rat. SE in male Sprague-Dawley rats (between 6 and 7 weeks old) lasting for more than 2 h was induced by an intraperitoneal injection of pilocarpine. The complexity, spine density, miniature post-synaptic currents, and activity-regulated cytoskeleton-associated protein (Arc) expression of granule cells born 5 days after SE were studied between 10 and 17 weeks after CAG-GFP retroviral vector-mediated labeling. Mature granule cells born after SE had dendritic complexity similar to that of granule cells born naturally, but with denser mushroom-like spines in dendritic segments located in the outer molecular layer. Miniature inhibitory post-synaptic currents (mIPSCs) were similar between the controls and rats subjected to SE; however, smaller miniature excitatory post-synaptic current (mEPSC) amplitude with a trend toward less frequent was found in mature granule cells born after SE. After maturation, granule cells born after SE did not show denser Arc expression in the resting condition or 2 h after being activated by pentylenetetrazol-induced transient seizure activity than vicinal GFP-unlabeled granule cells. Thus our results suggest that normotopic granule cells born after pilocarpine-induced SE are no more active when mature than age-matched, naturally born granule cells. PMID:26500490

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  14. Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells.

    PubMed

    Jeong, Ji Young; Son, Younghae; Kim, Bo-Young; Eo, Seong-Kug; Rhim, Byung-Yong; Kim, Koanhoi

    2015-11-01

    We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype. PMID:26557022

  15. SNAP23 is selectively expressed in airway secretory cells and mediates baseline and stimulated mucin secretion

    PubMed Central

    Ren, Binhui; Azzegagh, Zoulikha; Jaramillo, AnaM.; Zhu, Yunxiang; Pardo-Saganta, Ana; Bagirzadeh, Rustam; Flores, JoseR.; Han, Wei; Tang, Yong-jun; Tu, Jing; Alanis, DeniseM.; Evans, ChristopherM.; Guindani, Michele; Roche, PaulA.; Rajagopal, Jayaraj; Chen, Jichao; Davis, C.William; Tuvim, MichaelJ.; Dickey, BurtonF.

    2015-01-01

    Airway mucin secretion is important pathophysiologically and as a model of polarized epithelial regulated exocytosis. We find the trafficking protein, SNAP23 (23-kDa paralogue of synaptosome-associated protein of 25kDa), selectively expressed in secretory cells compared with ciliated and basal cells of airway epithelium by immunohistochemistry and FACS, suggesting that SNAP23 functions in regulated but not constitutive epithelial secretion. Heterozygous SNAP23 deletant mutant mice show spontaneous accumulation of intracellular mucin, indicating a defect in baseline secretion. However mucins are released from perfused tracheas of mutant and wild-type (WT) mice at the same rate, suggesting that increased intracellular stores balance reduced release efficiency to yield a fully compensated baseline steady state. In contrast, acute stimulated release of intracellular mucin from mutant mice is impaired whether measured by a static imaging assay 5min after exposure to the secretagogue ATP or by kinetic analysis of mucins released from perfused tracheas during the first 10min of ATP exposure. Together, these data indicate that increased intracellular stores cannot fully compensate for the defect in release efficiency during intense stimulation. The lungs of mutant mice develop normally and clear bacteria and instilled polystyrene beads comparable to WT mice, consistent with these functions depending on baseline secretion that is fully compensated. PMID:26182382

  16. Sphingomyelin derivatives increase the frequency of microvesicle and granule fusion in chromaffin cells.

    PubMed

    Garca-Martnez, V; Montes, M A; Villanueva, J; Gimenez-Molina, Y; de Toledo, G A; Gutirrez, L M

    2015-06-01

    Sphingomyelin derivatives like sphingosine have been shown to enhance secretion in a variety of systems, including neuroendocrine and neuronal cells. By studying the mechanisms underlying this effect, we demonstrate here that sphingomyelin rafts co-localize strongly with synaptosomal-associated protein of 25Kda (SNAP-25) clusters in cultured bovine chromaffin cells and that they appear to be linked in a dynamic manner. In functional terms, when cultured rat chromaffin cells are treated with sphingomyelinase (SMase), producing sphingomyelin derivatives, the secretion elicited by repetitive depolarizations is enhanced. This increase was independent of cell size and it was significant 15min after initiating stimulation. Interestingly, by evaluating the membrane capacitance we found that the events in control untreated cells corresponded to two populations of microvesicles and granules, and the fusion of both these populations is clearly enhanced after treatment with SMase. Furthermore, SMase does not increase the size of chromaffin granules. Together, these results strongly suggest that SNARE-mediated exocytosis is enhanced by the generation of SMase derivatives, reflecting an increase in the frequency of fusion of both microvesicles and chromaffin granules rather than an increase in the size of these vesicles. PMID:25813703

  17. Structural Plasticity of Dentate Granule Cell Mossy Fibers During the Development of Limbic Epilepsy

    PubMed Central

    Danzer, Steve C.; He, Xiaoping; Loepke, Andreas W.; McNamara, James O.

    2009-01-01

    Altered granule cell?CA3 pyramidal cell synaptic connectivity may contribute to the development of limbic epilepsy. To explore this possibility, granule cell giant mossy fiber bouton plasticity was examined in the kindling and pilocarpine models of epilepsy using green fluorescent protein-expressing transgenic mice. These studies revealed significant increases in the frequency of giant boutons with satellite boutons 2 days and 1 month after pilocarpine status epilepticus, and increases in giant bouton area at 1 month. Similar increases in giant bouton area were observed shortly after kindling. Finally, both models exhibited plasticity of mossy fiber giant bouton filopodia, which contact GABAergic interneurons mediating feedforward inhibition of CA3 pyramids. In the kindling model, however, all changes were fleeting, having resolved by 1 month after the last evoked seizure. Together, these findings demonstrate striking structural plasticity of granule cell mossy fiber synaptic terminal structure in two distinct models of adult limbic epileptogenesis. We suggest that these plasticities modify local connectivities between individual mossy fiber terminals and their targets, inhibitory interneurons, and CA3 pyramidal cells potentially altering the balance of excitation and inhibition during the development of epilepsy. PMID:19294647

  18. Leptin directly controls proliferation, apoptosis and secretory activity of cultured chicken ovarian cells.

    PubMed

    Sirotkin, A V; Grossmann, R

    2007-10-01

    The aim of our in-vitro experiments was to examine, whether leptin can directly control functions of avian ovarian cells and to outline potential intracellular mediators of its effects. Granulosa cells or fragments of ovarian follicular wall were cultured with leptin (0, 1, 10 or 100 ng/mL medium). The expression of peptides involved in apoptosis (TdT, bax, its binding protein, bcl-2, ASK-1 and p53), cell cycle-related peptides (PCNA and cyclin B1), release of hormones (progesterone, testosterone, estradiol, arginine-vasotocin), as well as the expression of protein kinases (PKA, MAPK/ERK1,2 and CDK/p34) in the ovarian cells were examined by using immunocytochemistry, TUNEL, SDS-PAGE-Western immunoblotting, EIA and RIA. It was found that leptin inhibited expression of all markers of cytoplasmic apoptosis (bax, ASK-1 and p53), stimulated expression of anti-apoptotic peptide bcl-2, but did not affect nuclear DNA fragmentation (TdT). Furthermore, leptin inhibited expression of PCNA (marker of S-phase of mitosis), but not of cyclin B1 (marker of G phase of cell cycle). Moreover, it promoted release of progesterone and estradiol, suppressed release of testosterone, but did not affect arginine-vasotocin. Finally, leptin inhibited expression of MAPK/ERK1,2 and CDK/p34 and stimulated expression of PKA. The present observations demonstrate that leptin can directly control basic chicken ovarian functions - inhibit cytoplasmic apoptosis and proliferation (S-phase, but not G-phases of mitosis), regulate secretory activity (release of steroids, but not nonapeptide hormone) and expression of MAPK, PKA and CDC2, which might be potential intracellular mediators of leptin action. PMID:17604668

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

    PubMed

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

    2014-06-01

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

  20. Granule proteinases define mast cell heterogeneity in the serosa and the gastrointestinal mucosa of the mouse.

    PubMed Central

    Miller, H R; Huntley, J F; Newlands, G F; Mackellar, A; Lammas, D A; Wakelin, D

    1988-01-01

    In order to define further mast cell heterogeneity in the mouse, affinity-purified antibodies against a 28,000 MW serine proteinase from mouse intestinal mast cells (IMCP) and against rat mast cell proteinase I (RMCPI) were used to characterize mast cell cytoplasmic granules immunohistochemically. On Western blot, anti-IMCP cross-reacted with RMCPI and with a 25,000 MW antigen from isolated mouse serosal mast cells (SMC). Anti-RMCPI did not react with IMCP, although it identified the same 25,000 MW antigen from SMC. Isolated SMC (85-90% pure) lacked the 28,000 MW IMCP on Western blot, even though, immunohistochemically, the cells were stained with both anti-RMCPI and anti-IMCP. Anti-IMCP stained the granules of more than 85% of all mast cells detected with toluidine blue in the tongue or gastrointestinal mucosa. The specificity of anti-RMCPI which, in the rat, detects very few mucosal mast cells was almost identical to that of anti-IMCP for murine tongue and gastric and large intestinal mucosae, but a significant proportion of cells in distal jejunal, ileal and caecal mucosae were not stained with this antibody. The immunohistochemistry of the large numbers of mast cells recruited to jejunum following infection 10 days previously with 300 Trichinella spiralis muscle larvae was similar to that of uninfected control mice. The results show that considerable mast cell heterogeneity exists within the gastrointestinal mucosa of the mouse and indicate that there are both similarities and differences between mouse and rat in the distribution of mast cells and of their granule proteinases. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:3065218

  1. Insulin Granule Biogenesis, Trafficking and Exocytosis

    PubMed Central

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

    2015-01-01

    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

  2. Insulin granule biogenesis, trafficking and exocytosis.

    PubMed

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

    2009-01-01

    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 plasma membrane 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

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

    PubMed

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

    2015-01-01

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

  4. Dynamic light-scattering study on changes in mobility of chromaffin granules in actin network with its assembly and Ca2+-dependent disassembly by gelsolin

    NASA Astrophysics Data System (ADS)

    Fujime, Satoru; Miyamoto, Shigeaki; Funatsu, Takashi; Ishiwata, S.

    1993-06-01

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

  5. Fine Tuning of Synaptic Plasticity and Filtering by GABA Released from Hippocampal Autaptic Granule Cells.

    PubMed

    Valente, Pierluigi; Orlando, Marta; Raimondi, Andrea; Benfenati, Fabio; Baldelli, Pietro

    2016-03-01

    The functional consequence of ?-aminobutyric acid (GABA) release at mossy fiber terminals is still a debated topic. Here, we provide multiple evidence of GABA release in cultured autaptic hippocampal granule cells. In ?50% of the excitatory autaptic neurons, GABA, VGAT, or GAD67 colocalized with vesicular glutamate transporter 1-positive puncta, where both GABAB and GABAA receptors (Rs) were present. Patch-clamp recordings showed a clear enhancement of autaptic excitatory postsynaptic currents in response to the application of the GABABR antagonist CGP58845 only in neurons positive to the selective granule cell marker Prox1, and expressing low levels of GAD67. Indeed, GCP non-responsive excitatory autaptic neurons were both Prox1- and GAD67-negative. Although the amount of released GABA was not sufficient to activate functional postsynaptic GABAARs, it effectively activated presynaptic GABABRs that maintain a tonic "brake" on the probability of release and on the size of the readily releasable pool and contributed to resting potential hyperpolarization possibly through extrasynaptic GABAAR activation. The autocrine inhibition exerted by GABABRs on glutamate release enhanced both paired-pulse facilitation and post-tetanic potentiation. Such GABABR-mediated changes in short-term plasticity confer to immature granule cells the capability to modulate their filtering properties in an activity-dependent fashion, with remarkable consequences on the dynamic behavior of neural circuits. PMID:25576534

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

    PubMed

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

    2014-03-01

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

  7. Decrease in an Inwardly Rectifying Potassium Conductance in Mouse Mammary Secretory Cells after Forced Weaning

    PubMed Central

    Kamikawa, Akihiro; Sugimoto, Shota; Ichii, Osamu; Kondoh, Daisuke

    2015-01-01

    Mammary glands are physiologically active in female mammals only during nursing. Immediately after weaning, most lactation-related genes are downregulated and milk production ceases. In our previous study, we have detected an inwardly rectifying potassium channel (Kir) 2.1-like current in mammary secretory (MS) cells freshly isolated from lactating mice. This current is highly sensitive to external Ba2+. The potassium permeability of the Kir channels may contribute to the secretion and/or preservation of ions in milk. We hypothesized that the functions of the Kir channels in MS cells are regulated after weaning. To test this hypothesis, we examined the effect of forced weaning on the Ba2+-sensitive Kir current and Kir2.1 expression in the mouse mammary glands. Twenty-four hours after weaning, the lumina of mammary acini were histologically enlarged by milk accumulation. The whole-cell patch-clamp analyses showed that the Ba2+-sensitive Kir current in the post-weaning MS cells was smaller than in the lactating MS cells. The inward conductances of the current in the lactating and post-weaning cells were 4.25 0.77 and 0.93 0.34 nS, respectively. Furthermore, real-time PCR and Western blot analyses showed that Kir2.1 mRNA and protein expression decreased in the post-weaning mammary gland (mRNA, 90% reduction; protein, 47% reduction). Moreover, the local milk accumulation caused by teat sealing decreased Kir conductance in MS cells (2.74 0.45 and 0.36 0.27 nS for control and sealed mammary glands, respectively). This was concomitant with the reduction in the Kir2.1 mRNA expression. Our results suggest that milk stasis after weaning immediately decreases the Kir conductance in MS cells. This decrease in the Kir conductance may be partly caused by the reduction in the Kir2.1 mRNA and protein expression. These alterations during the post-weaning period may be involved in the cessation of ion secretion and/or preservation in the milk. PMID:26484867

  8. Targeting vaccinia virus-expressed secretory beta subunit of human chorionic gonadotropin to the cell surface induces antibodies.

    PubMed Central

    Srinivasan, J; Singh, O; Chakrabarti, S; Talwar, G P

    1995-01-01

    We carried out experiments designed to study the effect of a protein's localization on its immunogenicity. A novel cell-surface protein was generated from a small, glycosylated secretory protein. The DNA sequence encoding the entire precursor of the human chorionic gonadotropin beta (beta hCG) subunit was fused in the correct reading frame to the DNA sequence encoding the transmembrane and cytoplasmic domains of vesicular stomatitis virus glycoprotein. This chimeric gene was introduced into the vaccinia virus genome to generate a recombinant virus. The recombinant virus, when used to infect animal cells, expressed a 135-amino-acid beta hCG subunit anchored in cellular membranes by the 48 carboxy-terminal amino acids of vesicular stomatitis virus glycoprotein. The immunogenicity of this recombinant virus with respect to its ability to generate anti-hCG antibodies was compared with that of a second recombinant vaccinia virus expressing the native secretory form of beta hCG. All animals immunized with the vaccinia virus expressing beta hCG on the cell surface elicited high titers of anti-hCG antibodies. Even after a single immunization with the recombinant vaccinia virus, the anti-hCG antibody titers persisted for a long period of time (more than 6 months). None of the animals immunized with vaccinia virus expressing the native secretory form of beta hCG showed any hCG-specific antibody response. PMID:7591154

  9. The histone deacetylase Hos2 forms an Hsp42-dependent cytoplasmic granule in quiescent yeast cells

    PubMed Central

    Liu, I-Chun; Chiu, Sheng-Wen; Lee, Hsin-Yi; Leu, Jun-Yi

    2012-01-01

    One of many physiological adjustments in quiescent cells is spatial regulation of specific proteins and RNA important for the entry to or exit from the stationary phase. By examining the localization of epigenetic-related proteins in Saccharomyces cerevisiae, we observed the formation of a reversible cytosolic stationary-phase granule (SPG) by Hos2, a nuclear histone deacetylase. In the stationary phase, hos2 mutants display reduced viability. Additionally, they exhibit a significant delay when recovering from stationary phase. Hos2 SPGs also contained Hst2, a Sir2 homologue, and several stress-related proteins, including Set3, Yca1, Hsp26, Hsp42, and some known components of stress granules. However, Hos2 SPG formation does not depend on the formation of stress granules or processing bodies. The absence or presence of glucose is sufficient to trigger assembly or disassembly of Hos2 SPGs. Among the identified components of Hos2 SPGs, Hsp42 is the first and last member observed in the Hos2 SPG assembly and disassembly processes. Hsp42 is also vital for the relocalization of the other components to Hos2 SPGs, suggesting that Hsp42 plays a central role in spatial regulation of proteins in quiescent cells. PMID:22337769

  10. Study of Mast Cells and Granules from Primo Nodes Using Scanning Ionic Conductance Microscopy.

    PubMed

    Yoo, Yeong-Yung; Jung, Goo-Eun; Kwon, Hee-Min; Bae, Kyoung-Hee; Cho, Sang-Joon; Soh, Kwang-Sup

    2015-12-01

    Acupuncture points have a notable characteristic in that they have a higher density of mast cells (MCs) compared with nonacupoints in the skin, which is consistent with the augmentation of the immune function by acupuncture treatment. The primo vascular system, which was proposed as the anatomical structure of the acupuncture points and meridians, also has a high density of MCs. We isolated the primo nodes from the surfaces of internal abdominal organs, and the harvested primo nodes were stained with toluidine blue. The MCs were easily recognized by their stained color and their characteristic granules. The MCs were classified into four stages according to the degranulation of histamine granules in the MCs. Using conventional optical microscopes details of the degranulation state of MCs in each stage were not observable. However, we were able to investigate the distribution of the granules on the surfaces of the MCs in each stage, and to demonstrate the height profiles and three-dimensional structures of the MCs without disturbance of the cell membrane by using the scanning ion conductance microscopy. PMID:26742911

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  12. The calcium-sensing receptor and integrins modulate cerebellar granule cell precursor differentiation and migration.

    PubMed

    Tharmalingam, Sujeenthar; Wu, Chiping; Hampson, David R

    2016-04-01

    In the developing cerebellum granule cell precursors (GCPs) proliferate in the external granule cell layer before differentiating and migrating to the inner granule cell layer. Aberrant GCP proliferation leads to medulloblastoma, the most prevalent form of childhood brain cancer. Here, we demonstrate that the calcium-sensing receptor (CaSR), a homodimeric G-protein coupled receptor, functions in conjunction with cell adhesion proteins, the integrins, to enhance GCP migration and cell homing by promoting GCP differentiation. During the second postnatal week a robust peak in CaSR expression was observed in GCPs; reciprocal immunoprecipitation experiments conducted during this period established that the CaSR and β1 integrins are present together in a macromolecular protein complex. Analysis of cell-surface proteins demonstrated that activation of the CaSR by positive allosteric modulators promoted plasma membrane expression of β1 integrins via ERK2 and AKT phosphorylation and resulted in increased GCP migration toward an extracellular matrix protein. The results of in vivo experiments whereby CaSR modulators were injected i.c.v. revealed that CaSR activation promoted radial migration of GCPs by enhancing GCP differentiation, and conversely, a CaSR inhibitor disrupted GCP differentiation and promoted GCP proliferation. Our results demonstrate that an ion-sensing G-protein coupled receptor acts to promote neuronal differentiation and homing during cerebellar maturation. These findings together with those of others also suggest that CaSR/integrin complexes act to transduce extracellular calcium signals into cellular movement, and may function in this capacity as a universal cell migration/homing complex in the developing brain. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 375-389, 2016. PMID:26138678

  13. Selective autophagic degradation of maternally-loaded germline P granule components in somatic cells during C. elegans embryogenesis.

    PubMed

    Zhao, Yu; Tian, E; Zhang, Hong

    2009-07-01

    Germline P granules are specialized protein/RNA aggregates that are found exclusively in germ cells in C. elegans. During the early embryonic divisions that generate germ blastomeres, aggregate-prone P granule components PGL-1 and PGL-3 that remain in the cytoplasm destined for somatic daughters are selectively removed by autophagy. Loss-of-function of components of the autophagy pathway, including the VPS-34/BEC-1 complex, causes accumulation of PGL-1 and PGL-3 into aggregates in somatic cells (termed PGL granules). Formation of PGL granules depends on SEPA-1, which is an integral component of these granules. SEPA-1 is preferentially degraded by autophagy and is also required for the autophagic degradation of PGL-1 and PGL-3. SEPA-1 functions as a bridging molecule in mediating degradation of P granule components by directly interacting with PGL-3 and also with the autophagy protein LGG-1/Atg8. The defect in embryonic development in autophagy mutants is suppressed by mutation of sepa-1, suggesting that autophagic degradation of PGL granule components may provide nutrients for embryogenesis and/or also prevent the formation of aggregates that could be toxic for animal development. Our study reveals a specific physiological function of selective autophagic degradation during C. elegans development. PMID:19372764

  14. Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production.

    PubMed

    Chaiyadet, Sujittra; Smout, Michael; Johnson, Michael; Whitchurch, Cynthia; Turnbull, Lynne; Kaewkes, Sasithorn; Sotillo, Javier; Loukas, Alex; Sripa, Banchob

    2015-10-01

    Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products (OvES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south-eastern Asia. PMID:26187786

  15. Participation of endogenous fatty acids in the secretory activity of the pancreatic B-cell.

    PubMed Central

    Malaisse, W J; Malaisse-Lagae, F; Sener, A; Hellerstrm, C

    1985-01-01

    The pancreatic B-cell may represent a fuel-sensor organ, the release of insulin evoked by nutrient secretagogues being attributable to an increased oxidation of exogenous and/or endogenous substrates. The participation of endogenous fatty acids in the secretory response of isolated rat pancreatic islets was investigated. Methyl palmoxirate (McN-3716, 0.1 mM), an inhibitor of long-chain-fatty-acid oxidation, suppressed the oxidation of exogenous [U-14C]palmitate and inhibited 14CO2 output from islets prelabelled with [U-14C]palmitate. Methyl palmoxirate failed to affect the oxidation of exogenous D-[U-14C]glucose or L-[U-14C]glutamine, the production of NH4+ and the output of 14CO2 from islets prelabelled with L-[U-14C]glutamine. In the absence of exogenous nutrient and after a lag period of about 60 min, methyl palmoxirate decreased O2 uptake to 69% of the control value. Methyl palmoxirate inhibited insulin release evoked by D-glucose, D-glyceraldehyde, 2-oxoisohexanoate, L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylate or 3-phenylpyruvate. However, methyl palmoxirate failed to affect insulin release when the oxidation of endogenous fatty acids was already suppressed, e.g. in the presence of pyruvate or L-glutamine. These findings support the view that insulin release evoked by nutrient secretagogues tightly depends on the overall rate of nutrient oxidation, including that of endogenous fatty acids. PMID:3924031

  16. Protein-targeting determinants in the secretory pathway of apicomplexan parasites.

    PubMed

    Kaasch, A J; Joiner, K A

    2000-08-01

    Apicomplexan parasites possess a highly specialized secretory apparatus. The timed secretion of proteins from three different organelles--micronemes, rhoptries and dense granules--serves to establish and maintain a parasitophorous vacuole inside the host cell in which the parasites can divide. Recent efforts have identified components that sort apicomplexan proteins to these unusual secretory organelles and have shown that this machinery is evolutionarily conserved across species. Concise amino acid sequences (e.g. tyrosine-based motifs) within the targeted protein determine their destination in Apicomplexa in a way similar to mammalian cells. Additionally, the parasite exploits new or unusual mechanisms of protein targeting (e.g. post-secretory membrane insertion). PMID:10972505

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

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro; Nakamichi, Yoko; Nishiwaki, Chiyono; Kawakami, Hayato; Nagamatsu, Shinya

    2009-12-04

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

  18. Morphological Alterations in Newly Born Dentate Gyrus Granule Cells That Emerge after Status Epilepticus Contribute to Make Them Less Excitable

    PubMed Central

    Tejada, Julián; Arisi, Gabriel M.; García-Cairasco, Norberto; Roque, Antonio C.

    2012-01-01

    Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting. PMID:22811762

  19. Morphological alterations in newly born dentate gyrus granule cells that emerge after status epilepticus contribute to make them less excitable.

    PubMed

    Tejada, Julián; Arisi, Gabriel M; García-Cairasco, Norberto; Roque, Antonio C

    2012-01-01

    Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting. PMID:22811762

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

    PubMed Central

    Le, Hoa H.; Belcher, Scott M.

    2010-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

  2. Lectin binding sites in Paramecium tetraurelia cells. I. Labeling analysis predominantly of secretory components.

    PubMed

    Lthe, N; Plattner, H; Haacke, B; Walther, P; Mller, M

    1986-01-01

    Though all three lectins tested (ConA, RCA II, WGA) bound to the entire cell membrane, none bound selectively to the docking site of secretory organelles (trichocysts); the same results were achieved with FITC-conjugates, or, on the EM level, with peroxidase- or gold-labeling. Only WGA triggered the release of trichocysts and none of the lectins tested inhibited AED-induced synchronous exocytosis. When exocytosis was triggered synchronously in the presence of any of these three lectins (FITC-conjugates), the resulting ghosts trapped the FITC-lectins and the cell surface was immediately afterwards studded with regularly spaced dots (corresponding to the ghosts located on the regularly spaced exocytosis sites). These disappeared within about 10 min from the cell surface (thus reflecting ghost internalization with a half life of 3 min) and fluorescent label was then found in approximately 6-10 vacuoles, which are several microns in diameter, stain for acid phosphatase and, on the EM level, contain numerous membrane fragments (otherwise not found in this form in digesting vacuoles). We conclude that synchronous massive exocytosis involves lysosomal breakdown rather than reutilization of internalized trichocyst membranes and that these contain lectin binding sites (given the fact free fluorescent probes did not efficiently stain ghosts). Trichocyst contents were analyzed for their lectin binding capacity in situ and on polyacrylamide gels. RCA II yielded intense staining (particularly of "tips"), while ConA (fluorescence concentrated over "bodies") and WGA yielded less staining of trichocyst contents on the light and electron microscopic level. Only ConA- and WGA-staining was inhibitable by an excess of specific sugars, while RCA II binding was not. ConA binding was also confirmed on polyacrylamide gels which also allowed us to assess the rather low degree of glycosylation (approximately 1% by comparison with known glycoprotein standards) of the main trichocyst proteins contained in their expandable "matrix". Since RCA II binding could be due to its own glycosylation residues we looked for an endogenous lectin. The conjecture was substantiated by the binding of FITC-lactose-albumin (inhibitable by a mixture of glucose-galactose). This preliminary new finding may be important for the elucidation of trichocyst function. PMID:3781884

  3. Altered patterning of dentate granule cell mossy fiber inputs onto CA3 pyramidal cells in limbic epilepsy

    PubMed Central

    McAuliffe, John J.; Bronson, Stefanie L.; Hester, Michael S.; Murphy, Brian L.; Dahlquist-Topal, Rene; Richards, David A.; Danzer, Steve C.

    2009-01-01

    Impaired gating by hippocampal dentate granule cells may promote the development of limbic epilepsy by facilitating seizure spread through the hippocampal trisynaptic circuit. The second synapse in this circuit, the dentate granule cell?CA3 pyramidal cell connection, may be of particular importance because pathological changes occurring within the dentate likely exert their principal effect on downstream CA3 pyramids. Here, we utilized GFP-expressing mice and immunolabeling for the zinc transporter ZnT-3 to reveal the pre- and postsynaptic components of granule cell?CA3 pyramidal cell synapses following pilocarpine-epileptogenesis. Confocal analyses of these terminals revealed that while granule cell presynaptic giant boutons increased in size and complexity one month after status epilepticus, individual thorns making up the postsynaptic thorny excrescences of the CA3 pyramidal cells were reduced in number. This reduction, however, was transient, and three months after status, thorn density recovered. This recovery was accompanied by a significant change in the distribution of thorns along pyramidal cells dendrites. While thorns in control animals tended to be tightly clustered, thorns in epileptic animals were more evenly distributed. Computational modeling of thorn distributions predicted an increase in the number of boutons required to cover equivalent numbers of thorns in epileptic vs. control mice. Confirming this prediction, ZnT-3 labeling of presynaptic giant boutons apposed to GFP-expressing thorns revealed a near doubling in bouton density, while the number of individual thorns per bouton was reduced by half. Together, these data provide clear evidence of novel plastic changes occurring within the epileptic hippocampus. PMID:20014385

  4. [Fusion of frog cerebellar granule cells induced by toxic effects of glutamate and NO-generating compound].

    PubMed

    Samosudova, N V; Reutov, V P; Larionova, N P

    2011-01-01

    The ultrastructural changes of cerebellar granule cells were studied under stroke modeling conditions, after the toxic effects of glutamate (Glu) and NO-generating compound. Glu toxic doses were shown to induce two types of nuclear chromatin changes. In some cases, the appearance of practically completely decondensed nuclear chromatin was detected, while in the others the nuclei contained partially decondensed chromatin. Pathological fusion of granule cells was observed in both cases. The toxic effect of NO-generating compound on granule cells also caused the appearance of the cells with both completely and partially decondensed (flocculent) chromatin. Granule cells with different chromatin type were able to fuse with each other. Thus, Glu and NO, causing changes in nuclear chromatin, activate the processes of cell clustering with the following cytoplasmic fusion and the formation of multinuclear conglomerates. The possible physiological role of granule cells fusion induced by high concentrations of Glu and NO-generating compound is discussed. This process is considered as a realization of the compensatory-adaptive reactions under extreme conditions observed in the stroke and oxidative stress. PMID:22171426

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

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

    Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.; Paulsen, Ragnhild E.

    2010-09-15

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

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

    PubMed

    Berlanga-Acosta, Jorge; Schultz, Gregory S; Lpez-Mola, Ernesto; Guillen-Nieto, Gerardo; Garca-Siverio, Marianela; Herrera-Martnez, Luis

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

  9. Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells.

    PubMed Central

    Ortega, E; Schweitzer-Stenner, R; Pecht, I

    1988-01-01

    Three biologically active monoclonal antibodies (mAbs) specific for the monovalent, high-affinity membrane receptor for IgE (Fc epsilon R) were employed in analysing the secretory response of mast cells of the RBL-2H3 line to crosslinking of their Fc epsilon R. All three mAbs (designated F4, H10 and J17) compete with each other and with IgE for binding to the Fc epsilon R. Their stoichiometry of binding is 1 Fab:1 Fc epsilon R, hence, the intact mAbs can aggregate the Fc epsilon Rs to dimers only. Since all three mAbs induce secretion, we conclude that Fc epsilon R dimers constitute a sufficient 'signal element' for secretion of mediators for RBL-2H3 cells. The secretory dose-response of the cells to these three mAbs are, however, markedly different: F4 caused rather high secretion, reaching almost 80% of the cells' content, while J17 and H10 induced release of only 30-40% mediators content. Both the intrinsic affinities and equilibrium constants for the receptor dimerization were derived from analysis of binding data of the Fab fragments and intact mAbs. These parameters were used to compute the extent of Fc epsilon R dimerization caused by each of the antibodies. However, the different secretory responses to the three mAbs could not be rationalized simply in terms of the extent of Fc epsilon R dimerization which they produce. This suggests that it is not only the number of crosslinked Fc epsilon Rs which determines the magnitude of secretion-causing signal, but rather other constraints imposed by each individual mAb are also important.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2977332

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

    SciTech Connect

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

    1988-07-01

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

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

    SciTech Connect

    Plopper, C.G. . E-mail: cgplopper@ucdavis.edu; Mango, G.W.; Hatch, G.E.; Wong, V.J.; Toskala, E.; Reynolds, S.D.; Tarkington, B.K.; Stripp, B.R.

    2006-05-15

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

  12. Entorhinal denervation induces homeostatic synaptic scaling of excitatory postsynapses of dentate granule cells in mouse organotypic slice cultures.

    PubMed

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

    2012-01-01

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

  13. NT-4/5 protects against adrenalectomy-induced apoptosis of rat hippocampal granule cells.

    PubMed

    Qiao, X; Hughes, P E; Venero, J L; Dugich-Djordjevic, M M; Nichols, N R; Hefti, F; Knusel, B

    1996-01-31

    Adrenalectomy (ADX) in rats has been shown to induce apoptosis of hippocampal granule cells. We tested whether neurotrophins are able to protect hippocampal neurons in this neurodegeneration model. Acid fucshsin stain was used to identify pyknotic cells in ADX rats treated for 4 days with NT-3, NT-4-5 or cytochrome-C, as a control protein. Cytochrome-C injections slightly decreased cell death on the ipsilateral side. NT-3 did not further promote this effect. Significantly less cell death was observed bilaterally in hippocampus treated with NT-4/5. TUNEL end labeling also confirmed the results. Our results demonstrated that NT-4/5, but not NT-3, promotes hippocampal neuron survival in adrenalectomized rats. They further show that injections of a control solution can induce a local protective effect. PMID:8730856

  14. TIA1 oxidation inhibits stress granule assembly and sensitizes cells to stress-induced apoptosis

    PubMed Central

    Arimoto-Matsuzaki, Kyoko; Saito, Haruo; Takekawa, Mutsuhiro

    2016-01-01

    Cytoplasmic stress granules (SGs) are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of misfolded proteins, and that are formed in response to certain types of stress including ER stress. SG formation contributes to cell survival not only by suppressing translation but also by sequestering some apoptosis regulatory factors. Because cells can be exposed to various stresses simultaneously in vivo, the regulation of SG assembly under multiple stress conditions is important but unknown. Here we report that reactive oxygen species (ROS) such as H2O2 oxidize the SG-nucleating protein TIA1, thereby inhibiting SG assembly. Thus, when cells are confronted with a SG-inducing stress such as ER stress caused by protein misfolding, together with ROS-induced oxidative stress, they cannot form SGs, resulting in the promotion of apoptosis. We demonstrate that the suppression of SG formation by oxidative stress may underlie the neuronal cell death seen in neurodegenerative diseases. PMID:26738979

  15. Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells.

    PubMed Central

    Smith, G. H.; Sharp, R.; Kordon, E. C.; Jhappan, C.; Merlino, G.

    1995-01-01

    Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and prolonging survival during involution. These points support the notion that TGF-alpha can act as a mitogen and also as a differentiation factor in mammary epithelium. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7573353

  16. Different subsets of newborn granule cells: a possible role in epileptogenesis?

    PubMed

    Bielefeld, Pascal; van Vliet, Erwin A; Gorter, Jan A; Lucassen, Paul J; Fitzsimons, Carlos P

    2014-01-01

    Several factors, including epileptic seizures, can strongly stimulate ongoing neurogenesis in the adult hippocampus. Although adult-born granule cells generated after seizure activity have different physiological properties from their normal counterparts, they integrate into the existing, mature network of the adult hippocampal dentate gyrus. However, the exact role of the neurogenic response during epilepsy and its possible involvement in epileptogenesis have remained elusive. Here, we discuss recent studies shedding new light on the interplay between epilepsy and neurogenesis, and try to explain discrepancies in this literature by proposing seizure severity-dependent induction of two subsets of newborn cells with different properties. We hypothesise that a low seizure intensity would stimulate neurogenesis to a 'physiological plasticity' level and have few pathological consequences. In contrast, a high initial seizure intensity may induce a specific subset of altered and/or ectopically located new granule cells with different electrophysiological properties that could initiate hyperexcitatory recurrent networks that could, in turn, contribute to chronic epilepsy. This hypothesis may clarify previously contradictory data in the literature, and could thereby aid in our understanding of the role of neurogenesis in epileptogenesis, and open up promising avenues for therapeutic intervention. PMID:24387591

  17. Secretory immunoglobulins in colonic neoplasms.

    PubMed Central

    Weisz-Carrington, P.; Poger, M. E.; Lamm, M. E.

    1976-01-01

    Secretory immunoglobulins are found in nongoblet columnar cells of normal intestinal epithelium. These molecules consist of a secretory component portion, which is synthesized in the columnar cells, and an immunoglobulin portion which enters the columnar cells from plasma cells in the adjacent lamina propria. In the present work, the synthesis and transport of these various subunits have been studied by immunofluorescence in benign polyps and cancers of the colon. In both the epithelium and plasma cells of benign and malignant tumors, as well as in normal tissue, IgA is the principal immunoglobulin, followed by IgM. However, when compared to normal tissue, neoplastic epithelium contains less immunoglobulin and also less secretory component; the decrement usually inversely parallels the degree of differentiation. Thus, benign polyps closely resemble normal colonic mucosa in so far as the secretory immunoglobulin system is concerned. In contrast, atypical areas of benign polyps and carcinomas exhibit greatly decreased or absent synthesis and transport of secretory IgA. Plasma cells tend to be markedly decreased in the stroma of carcinomas, suggestive of an alteration in the normal mechanism for attracting the circulating precursors of local IgA plasma cells. Whenever neoplastic epithelium contained IgA, plasma cells with IgA could be observed in the vicinity; this is in keeping with the concept of local synthesis of secretory IgA. In some instances in which local plasma cells were plentiful, neoplastic cells were deficient in secretory component and IgA, which suggested impairment of the mechanisms for transporting IgA across epithelium. The possible role of secretory component in such transport and in attracting lymphoblasts to mucous membranes is dicussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:998723

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  20. Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells, bind Tudor and protect from transposable elements.

    PubMed

    Gao, Ming; Thomson, Travis C; Creed, T Michael; Tu, Shikui; Loganathan, Sudan N; Jackson, Christina A; McCluskey, Patrick; Lin, Yanyan; Collier, Scott E; Weng, Zhiping; Lasko, Paul; Ohi, Melanie D; Arkov, Alexey L

    2015-03-01

    Germ cells give rise to all cell lineages in the next-generation and are responsible for the continuity of life. In a variety of organisms, germ cells and stem cells contain large ribonucleoprotein granules. Although these particles were discovered more than 100 years ago, their assembly and functions are not well understood. Here we report that glycolytic enzymes are components of these granules in Drosophila germ cells and both their mRNAs and the enzymes themselves are enriched in germ cells. We show that these enzymes are specifically required for germ cell development and that they protect their genomes from transposable elements, providing the first link between metabolism and transposon silencing. We further demonstrate that in the granules, glycolytic enzymes associate with the evolutionarily conserved Tudor protein. Our biochemical and single-particle EM structural analyses of purified Tudor show a flexible molecule and suggest a mechanism for the recruitment of glycolytic enzymes to the granules. Our data indicate that germ cells, similarly to stem cells and tumor cells, might prefer to produce energy through the glycolytic pathway, thus linking a particular metabolism to pluripotency. PMID:25600116

  1. Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells, bind Tudor and protect from transposable elements

    PubMed Central

    Gao, Ming; Thomson, Travis C; Creed, T Michael; Tu, Shikui; Loganathan, Sudan N; Jackson, Christina A; McCluskey, Patrick; Lin, Yanyan; Collier, Scott E; Weng, Zhiping; Lasko, Paul; Ohi, Melanie D; Arkov, Alexey L

    2015-01-01

    Germ cells give rise to all cell lineages in the next-generation and are responsible for the continuity of life. In a variety of organisms, germ cells and stem cells contain large ribonucleoprotein granules. Although these particles were discovered more than 100years ago, their assembly and functions are not well understood. Here we report that glycolytic enzymes are components of these granules in Drosophila germ cells and both their mRNAs and the enzymes themselves are enriched in germ cells. We show that these enzymes are specifically required for germ cell development and that they protect their genomes from transposable elements, providing the first link between metabolism and transposon silencing. We further demonstrate that in the granules, glycolytic enzymes associate with the evolutionarily conserved Tudor protein. Our biochemical and single-particle EM structural analyses of purified Tudor show a flexible molecule and suggest a mechanism for the recruitment of glycolytic enzymes to the granules. Our data indicate that germ cells, similarly to stem cells and tumor cells, might prefer to produce energy through the glycolytic pathway, thus linking a particular metabolism to pluripotency. PMID:25600116

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

    PubMed Central

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

    2014-01-01

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

  3. Novel mRNA-containing cytoplasmic granules in ALK-transformed cells

    PubMed Central

    Fawal, Mohamad; Jean-Jean, Olivier; Vanzo, Nathalie; Morello, Dominique

    2011-01-01

    In mammalian cells, nontranslating messenger RNAs (mRNAs) are concentrated in different cytoplasmic foci, such as processing bodies (PBs) and stress granules (SGs), where they are either degraded or stored. In the present study, we have thoroughly characterized cytoplasmic foci, hereafter called AGs for ALK granules that form in transformed cells expressing the constitutively active anaplastic lymphoma kinase (ALK). AGs contain polyadenylated mRNAs and a unique combination of several RNA binding proteins that so far has not been described in mammalian foci, including AUF1, HuR, and the poly (A+) binding protein PABP. AGs shelter neither components of the mRNA degradation machinery present in PBs nor known markers of SGs, such as translation initiation factors or TIA/TIAR, showing that they are distinct from PBs or SGs. AGs and PBs, however, both move on microtubules with similar dynamics and frequently establish close contacts. In addition, in conditions in which mRNA metabolism is perturbed, AGs concentrate PB components with the noticeable exception of the 5? to 3? exonuclease XRN1. Altogether, we show that AGs constitute novel mRNA-containing cytoplasmic foci and we propose that they could protect translatable mRNAs from degradation, contributing thus to ALK-mediated oncogenicity. PMID:21233286

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

    PubMed

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

    2015-01-21

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

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

    SciTech Connect

    Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B. . E-mail: jbarnett@hsc.wvu.edu

    2007-06-01

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

  6. Parallel Expression of Enzyme Inhibitors of CD8T Cell Activity in Tumor Microenvironments and Secretory Endometrium.

    PubMed

    Ibana, Joyce A; Cutay, Sandra Jelyn; Romero, Maevel; Schust, Danny Joseph

    2016-03-01

    The divergent requirement for tolerance to support conception and protective response against sexually transmitted infections defines the unique immunological dynamics in the female reproductive tract (FRT). In part, these requirements are achieved by the cyclic modulation of cytolytic CD8T cell function in the FRT that underlie the respective immunosuppressive and immunocompetent milieus during the secretory and proliferative phases of the menstrual cycle. The CD8T cell function can be dampened by exposure to indoleamine 2,3-dioxygenase and/or arginase enzymes. Indeed, these 2 enzymes are known as primary inducers of immune suppression in tumor microenvironments. This review discusses the intriguing parallel expression of these 2 enzymes in tumor microenvironments and in the secretory endometrium. We surmise that investigating the underlying natural mechanisms that suppress and restore the immunocompetence of CD8T cells in the FRT each month may provide valuable insights into ways to artificially recapitulate these mechanisms and inhibit immune suppression in tumor microenvironments. PMID:26335176

  7. Increased excitatory synaptic input to granule cells from hilar and CA3 regions in a rat model of temporal lobe epilepsy

    PubMed Central

    Zhang, Wei; Huguenard, John R.; Buckmaster, Paul S.

    2012-01-01

    One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked excitatory postsynaptic currents (EPSCs) in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared to controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in temporal lobe epilepsy. PMID:22279204

  8. Morphology of the granular secretory glands in skin of poison-dart frogs (Dendrobatidae).

    PubMed

    Neuwirth, M; Daly, J W; Myers, C W; Tice, L W

    1979-01-01

    The granular glands of nine species of dendrobatid frogs were examined using light and electron microscopy. The glands are surrounded by a discontinuous layer of smooth muscle cells. Within the glands proper the secretory cells form a true syncytium. Multiple flattened nuclei lie at the periphery of the gland. The peripheral cytoplasm also contains mitochondria, rough surfaced endoplasmic reticulum, the Golgi apparatus, and an abundance of smooth endoplasmic reticulum. Centrally, most of the gland is filled with membrane-bound granules surrounded by amorphous cytoplasm. Few other organelles are found in this region. Early in the secretory cycle, the central part of the gland is filled with flocculent material which appears to be progressively partitioned off by membranes to form the droplet anlage. As granules form, the structure of the contents becomes progressively more vesicular. Dense vesicles, which bud off from the Golgi apparatus, fuse with the granular membrane during the development of granules, and might contain enzymes involved in toxin synthesis. The granules at this point resemble multivesicular bodies. Their structure is similar in all species of dendrobatid frogs even though the different frogs secrete substances of different chemical structure and toxicity. PMID:524345

  9. Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

    NASA Astrophysics Data System (ADS)

    Rietdorf, Jens; Stephens, David J.; Squire, Anthony; Simpson, Jeremy; Shima, David T.; Paccaud, Jean-Pierre; Bastiaens, Philippe I.; Pepperkok, Rainer

    2000-04-01

    Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.

  10. Bidirectional GABAergic control of action potential firing in newborn hippocampal granule cells.

    PubMed

    Heigele, Stefanie; Sultan, Sbastien; Toni, Nicolas; Bischofberger, Josef

    2016-02-01

    Newly generated young neurons in the adult hippocampus receive GABAergic synaptic inputs, which are crucial for activity-dependent survival and functional maturation between 1-3 weeks after mitosis. We found synaptically driven action potential (AP) firing in these newborn young cells in adult mice. Although glutamatergic synaptic inputs remained subthreshold, activation of GABAergic synaptic inputs depolarized young neurons and reliably evoked APs. Furthermore, pairing of subthreshold excitatory postsynaptic potentials or somatic current injection with brief bursts of GABAergic inputs revealed efficient GABAergic excitation at conductances of ?1.5 nS, corresponding to the activity of only three or four interneurons. Stronger GABAergic inputs (>4 nS) effectively blocked AP firing via shunting inhibition, which might be important to dynamically control spiking output in both directions. Taken together, GABAergic interneurons differentially recruit newborn young granule cells by supporting either AP generation or shunting inhibition dependent on hippocampal network activity. PMID:26752162

  11. Maturation and Functional Integration of New Granule Cells into the Adult Hippocampus.

    PubMed

    Toni, Nicolas; Schinder, Alejandro F

    2015-01-01

    The adult hippocampus generates functional dentate granule cells (GCs) that release glutamate onto target cells in the hilus and cornus ammonis (CA)3 region, and receive glutamatergic and ?-aminobutyric acid (GABA)ergic inputs that tightly control their spiking activity. The slow and sequential development of their excitatory and inhibitory inputs makes them particularly relevant for information processing. Although they are still immature, new neurons are recruited by afferent activity and display increased excitability, enhanced activity-dependent plasticity of their input and output connections, and a high rate of synaptogenesis. Once fully mature, new GCs show all the hallmarks of neurons generated during development. In this review, we focus on how developing neurons remodel the adult dentate gyrus and discuss key aspects that illustrate the potential of neurogenesis as a mechanism for circuit plasticity and function. PMID:26637288

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

    PubMed Central

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

    2014-01-01

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

  13. NMDA receptor-dependent plasticity of granule cell spiking in the dentate gyrus of normal and epileptic rats.

    PubMed

    Lynch, M; Sayin, U; Golarai, G; Sutula, T

    2000-12-01

    Because granule cells in the dentate gyrus provide a major synaptic input to pyramidal neurons in the CA3 region of the hippocampus, spike generation by granule cells is likely to have a significant role in hippocampal information processing. Granule cells normally fire in a single-spike mode even when inhibition is blocked and provide single-spike output to CA3 when afferent activity converging into the entorhinal cortex from neocortex, brainstem, and other limbic regions increases. The effects of enhancement of N-methyl-D-aspartate (NMDA) receptor-dependent excitatory synaptic transmission and reduction in gamma-aminobutyric acid-A (GABA(A)) receptor-dependent inhibition on spike generation were examined in granule cells of the dentate gyrus. In contrast to the single-spike mode observed in normal bathing conditions, perforant path stimulation in Mg(2+)-free bathing conditions evoked graded burst discharges in granule cells which increased in duration, amplitude, and number of spikes as a function of stimulus intensity. After burst discharges were evoked during transient exposure to bathing conditions that relieve the Mg(2+) block of the NMDA receptor, there was a marked increase in the NMDA receptor-dependent component of the EPSP, but no significant increase in the non-NMDA receptor-dependent component of the EPSP in normal bathing medium. Supramaximal perforant path stimulation still evoked only a single spike, but granule cell spike generation was immediately converted from a single-spike firing mode to a graded burst discharge mode when inhibition was then reduced. The induction of graded burst discharges in Mg(2+)-free conditions and the expression of burst discharges evoked in normal bathing medium with subsequent disinhibition were both blocked by DL-2-amino-4-phosphonovaleric acid (APV) and were therefore NMDA receptor dependent, in contrast to long-term potentiation (LTP) in the perforant path, which is induced by NMDA receptors and is also expressed by alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors. The graded burst discharge mode was also observed in granule cells when inhibition was reduced after a single epileptic afterdischarge, which enhances the NMDA receptor-dependent component of evoked synaptic response, and in the dentate gyrus reorganized by mossy fiber sprouting in kindled and kainic acid-treated rats. NMDA receptor-dependent plasticity of granule cell spike generation, which can be distinguished from LTP and induces long-term susceptibility to epileptic burst discharge under conditions of reduced inhibition, could modify information processing in the hippocampus and promote epileptic synchronization by increasing excitatory input into CA3. PMID:11110816

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-04-01

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

  17. Granulator Selection

    SciTech Connect

    Gould, T H; Armantrout, G

    1999-08-02

    Following our detailed review of the granulation reports and additional conversations with process and development personnel, we have reached a consensus position regarding granulator selection. At this time, we recommend going forward with implementation of the tumbling granulator approach (GEMCO) based on our assessment of the tested granulation techniques using the established criteria. The basis for this selection is summarized in the following sections, followed by our recommendations for proceeding with implementation of the tumbling granulation approach. All five granulation technologies produced granulated products that can be made into acceptable sintered pucks. A possible exception is the product from the fluidized bed granulator. This material has been more difficult to press into uniform pucks without subsequent cracking of the puck during the sintering cycle for the pucks in this series of tests. This problem may be an artifact of the conditions of the particular granulation demonstration run involved, but earlier results have also been mixed. All granulators made acceptable granulated feed from the standpoint of transfer and press feeding, though the roller compactor and fluidized bed products were dustier than the rest. There was also differentiation among the granulators in the operational areas of (1) potential for process upset, (2) plant implementation and operational complexity, and (3) maintenance concerns. These considerations will be discussed further in the next section. Note that concerns also exist regarding the extension of the granulation processes to powders containing actinides. Only the method that involves tumbling and moisture addition has been tested with uranium, and in that instance, significant differences were found in the granulation behavior of the powders.

  18. Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

    PubMed Central

    Kuznetsov, G; Bush, K T; Zhang, P L; Nigam, S K

    1996-01-01

    The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72. Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8710914

  19. Stress Granules Modulate SYK to Cause Microglial Cell Dysfunction in Alzheimer's Disease

    PubMed Central

    Ghosh, Soumitra; Geahlen, Robert L.

    2015-01-01

    Microglial cells in the brains of Alzheimer's patients are known to be recruited to amyloid-beta (A?) plaques where they exhibit an activated phenotype, but are defective for plaque removal by phagocytosis. In this study, we show that microglia stressed by exposure to sodium arsenite or A?(142) peptides or fibrils form extensive stress granules (SGs) to which the tyrosine kinase, SYK, is recruited. SYK enhances the formation of SGs, is active within the resulting SGs and stimulates the production of reactive oxygen and nitrogen species that are toxic to neuronal cells. This sequestration of SYK inhibits the ability of microglial cells to phagocytose Escherichia coli or A? fibrils. We find that aged microglial cells are more susceptible to the formation of SGs; and SGs containing SYK and phosphotyrosine are prevalent in the brains of patients with severe Alzheimer's disease. Phagocytic activity can be restored to stressed microglial cells by treatment with IgG, suggesting a mechanism to explain the therapeutic efficacy of intravenous IgG. These studies describe a mechanism by which stress, including exposure to A?, compromises the function of microglial cells in Alzheimer's disease and suggest approaches to restore activity to dysfunctional microglial cells. PMID:26870803

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

    PubMed

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

    2007-05-01

    Neurogenesis in the dentate gyrus of the hippocampus follows a unique temporal pattern that begins during embryonic development, peaks during the early postnatal stages and persists through adult life. We have recently shown that dentate granule cells born in early postnatal and adult mice acquire a remarkably similar afferent connectivity and firing behavior, suggesting that they constitute a homogeneous functional population [Laplagne et al. (2006)PLoS Biol., 4, e409]. Here we extend our previous study by comparing mature neurons born in the embryonic and adult hippocampus, with a focus on intrinsic membrane properties and gamma-aminobutyric acid (GABA)ergic synaptic inputs. For this purpose, dividing neuroblasts of the ventricular wall were retrovirally labeled with green fluorescent protein at embryonic day 15 (E15), and progenitor cells of the subgranular zone were labeled with red fluorescent protein in the same mice at postnatal day 42 (P42, adulthood). Electrophysiological properties of mature neurons born at either stage were then compared in the same brain slices. Evoked and spontaneous GABAergic postsynaptic responses of perisomatic and dendritic origin displayed similar characteristics in both neuronal populations. Miniature GABAergic inputs also showed similar functional properties and pharmacological profile. A comparative analysis of the present data with our previous observations rendered no significant differences among GABAergic inputs recorded from neurons born in the embryonic, early postnatal and adult mice. Yet, embryo-born neurons showed a reduced membrane excitability, suggesting a lower engagement in network activity. Our results demonstrate that granule cells of different age, location and degree of excitability receive GABAergic inputs of equivalent functional characteristics. PMID:17509085

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

    ERIC Educational Resources Information Center

    Hood-DeGrenier, Jennifer K.

    2008-01-01

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

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

    ERIC Educational Resources Information Center

    Hood-DeGrenier, Jennifer K.

    2008-01-01

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

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

    PubMed

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

    2015-06-01

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

  4. Reconstituted Human Polyclonal Plasma-derived Secretory-like IgM and IgA Maintain the Barrier Function of Epithelial Cells Infected with an Enteropathogen*

    PubMed Central

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

    2014-01-01

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

  5. Sorafenib, a multikinase inhibitor, induces formation of stress granules in hepatocarcinoma cells

    PubMed Central

    Adjibade, Pauline; St-Sauveur, Valérie Grenier; Huberdeau, Miguel Quevillon; Fournier, Marie-Josée; Savard, Andreanne; Coudert, Laetitia; Khandjian, Edouard W.; Mazroui, Rachid

    2015-01-01

    Stress granules (SGs) are cytoplasmic RNA multimeric bodies that form under stress conditions known to inhibit translation initiation. In most reported stress cases, the formation of SGs was associated with the cell recovery from stress and survival. In cells derived from cancer, SGs formation was shown to promote resistance to either proteasome inhibitors or 5-Fluorouracil used as chemotherapeutic agents. Despite these studies, the induction of SGs by chemotherapeutic drugs contributing to cancer cells resistance is still understudied. Here we identified sorafenib, a tyrosine kinase inhibitor used to treat hepatocarcinoma, as a potent chemotherapeutic inducer of SGs. The formation of SGs in sorafenib-treated hepatocarcionoma cells correlates with inhibition of translation initiation; both events requiring the phosphorylation of the translation initiation factor eIF2α. Further characterisation of the mechanism of sorafenib-induced SGs revealed PERK as the main eIF2α kinase responsible for SGs formation. Depletion experiments support the implication of PERK-eIF2α-SGs pathway in hepatocarcinoma cells resistance to sorafenib. This study also suggests the existence of an unexpected complex regulatory balance between SGs and phospho-eIF2α where SGs dampen the activation of the phospho-eIF2α-downstream ATF4 cell death pathway. PMID:26556863

  6. Sorafenib, a multikinase inhibitor, induces formation of stress granules in hepatocarcinoma cells.

    PubMed

    Adjibade, Pauline; St-Sauveur, Valrie Grenier; Quevillon Huberdeau, Miguel; Fournier, Marie-Jose; Savard, Andreanne; Coudert, Laetitia; Khandjian, Edouard W; Mazroui, Rachid

    2015-12-22

    Stress granules (SGs) are cytoplasmic RNA multimeric bodies that form under stress conditions known to inhibit translation initiation. In most reported stress cases, the formation of SGs was associated with the cell recovery from stress and survival. In cells derived from cancer, SGs formation was shown to promote resistance to either proteasome inhibitors or 5-Fluorouracil used as chemotherapeutic agents. Despite these studies, the induction of SGs by chemotherapeutic drugs contributing to cancer cells resistance is still understudied. Here we identified sorafenib, a tyrosine kinase inhibitor used to treat hepatocarcinoma, as a potent chemotherapeutic inducer of SGs. The formation of SGs in sorafenib-treated hepatocarcionoma cells correlates with inhibition of translation initiation; both events requiring the phosphorylation of the translation initiation factor eIF2?. Further characterisation of the mechanism of sorafenib-induced SGs revealed PERK as the main eIF2? kinase responsible for SGs formation. Depletion experiments support the implication of PERK-eIF2?-SGs pathway in hepatocarcinoma cells resistance to sorafenib. This study also suggests the existence of an unexpected complex regulatory balance between SGs and phospho-eIF2? where SGs dampen the activation of the phospho-eIF2?-downstream ATF4 cell death pathway. PMID:26556863

  7. Iron oxide nanoparticles suppress the production of IL-1beta via the secretory lysosomal pathway in murine microglial cells

    PubMed Central

    2013-01-01

    Background Superparamagnetic iron oxide nanoparticles (IONPs) have been used as magnetic resonance imaging contrast agents for various research and diagnostic purposes, such as the detection of neuroinflammation and blood-brain-barrier integrity. As the central resident macrophage-like cells, microglia are responsible for managing foreign agents invading the CNS. The present study investigated the direct effect of IONPs on the production of pro-inflammatory cytokines by murine microglia stimulated with lipopolysaccharide (LPS). Methods Primary murine microglial cells were pretreated with IONPs (1–50 μg Fe/mL) for 30 min and then stimulated with LPS (100 ng/mL) for 24 h. Confocal microscopy is used to visualize the intracellular IONP distribution and secretory lysosomes after staining with LysoTracker and Rab27a, respectively. The production of interleukin (IL)-1β and tumor necrosis factor (TNF)-α was quantified by ELISA. The activity of IL-1β converting enzyme (ICE) and TNF-α converting enzyme (TACE) was measured by fluorescent microplate assay using specific substrates. The lysosomal number, alkalinity, permeability and cathepsin B activity were determined by flow cytometry with ectodermal dysplasia-1, lysosensor and acridine orange staining, and using cathepsin B specific substrate, respectively. Results Confocal imaging revealed that IONPs were markedly engulfed by microglia. Exposure to IONPs attenuated the production of IL-1β, but not TNF-α. Concordantly, the activity of ICE, but not the TACE, was suppressed in IONP-treated cells. Mechanistic studies showed that IONPs accumulated in lysosomes and the number of lysosomes was increased in IONP-treated cells. In addition, exposure to IONPs increased lysosomal permeability and alkalinity, but decreased the activity of cathepsin B, a secretory lysosomal enzyme involved in the activation of ICE. Conclusions Our results demonstrated a contrasting effect of IONPs on the production of IL-1β and TNF-α by LPS-stimulated microglia, in which the attenuation of IL-1β by IONPs was mediated by inhibiting the secretory lysosomal pathway of cytokine processing. PMID:24047432

  8. Protective Effect of Edaravone in Primary Cerebellar Granule Neurons against Iodoacetic Acid-Induced Cell Injury

    PubMed Central

    Zhou, Xinhua; Zhu, Longjun; Wang, Liang; Guo, Baojian; Zhang, Gaoxiao; Sun, Yewei; Zhang, Zaijun; Lee, Simon Ming-Yuen; Yu, Pei; Wang, Yuqiang

    2015-01-01

    Edaravone (EDA) is clinically used for treatment of acute ischemic stroke in Japan and China due to its potent free radical-scavenging effect. However, it has yet to be determined whether EDA can attenuate iodoacetic acid- (IAA-) induced neuronal death in vitro. In the present study, we investigated the effect of EDA on damage of IAA-induced primary cerebellar granule neurons (CGNs) and its possible underlying mechanisms. We found that EDA attenuated IAA-induced cell injury in CGNs. Moreover, EDA significantly reduced intracellular reactive oxidative stress production, loss of mitochondrial membrane potential, and caspase 3 activity induced by IAA. Taken together, EDA protected CGNs against IAA-induced neuronal damage, which may be attributed to its antiapoptotic and antioxidative activities. PMID:26557222

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

    PubMed Central

    Kheirbek, Mazen A.; Tannenholz, Lindsay; Hen, Ren

    2012-01-01

    Adult generated granule cells (GCs) in the dentate gyrus (DG) exhibit a period of heightened plasticity 46 weeks post-mitosis. However, the functional contribution of this critical window of plasticity to hippocampal neurogenesis and behavior remains unknown. Here, we show that deletion of NR2B-containing NMDA receptors from adult born GCs impairs a neurogenesis-dependent form of LTP in the DG, reduces dendritic complexity of adult born GCs, but does not impact their survival. Mice in which the NR2B-containing NMDA receptor was deleted from adult born GCs did not differ from controls in baseline anxiety-like behavior or discrimination of very different contexts, but were impaired in discrimination of highly similar contexts. These results indicate that NR2B-dependent plasticity of adult born GCs is necessary for fine contextual discrimination and is consistent with their proposed role in pattern separation. PMID:22723709

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

    SciTech Connect

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

    2008-10-24

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

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

    Copp, Jean-Philippe; Sun, Yu; Muoz, Denise P; Goldstein, Joshua; Nelson, Peter S; Desprez, Pierre-Yves; Campisi, Judith

    2008-01-01

    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 epithelialmesenchyme 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. PMID:19053174

  13. A Human-Like Senescence-Associated Secretory Phenotype Is Conserved in Mouse Cells Dependent on Physiological Oxygen

    PubMed Central

    Copp, Jean-Philippe; Krtolica, Ana; Beausjour, Christian M.; Parrinello, Simona; Hodgson, J. Graeme; Chin, Koei; Desprez, Pierre-Yves; Campisi, Judith

    2010-01-01

    Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP), which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that senescent mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen), do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3%) oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-?. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP. PMID:20169192

  14. A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen.

    PubMed

    Copp, Jean-Philippe; Patil, Christopher K; Rodier, Francis; Krtolica, Ana; Beausjour, Christian M; Parrinello, Simona; Hodgson, J Graeme; Chin, Koei; Desprez, Pierre-Yves; Campisi, Judith

    2010-01-01

    Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP), which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that "senescent" mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen), do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3%) oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-alpha. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP. PMID:20169192

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

    PubMed

    Młynarczuk, J; Wróbel, M H; Kotwica, J

    2013-07-01

    Coumestrol is one of a few biologically active substances present in leguminous plants, which are widely used as fodder for ruminants. Depending on the doses, coumestrol acts on the reproductive processes as an estrogen-like factor or antiestrogen to evoke a decrease in ovulation frequency, elongation of estrous cycle duration. The aim of the current investigations was to study the influence of coumestrol on secretory function of luteal cells obtained from first trimester of pregnant cows. Luteal cells (2.5 × 10(5) /mL) from 3rd to 5th, 6th to 8th, and 9th to 12th week of pregnancy were preincubated for 24 h and incubated with coumestrol (1 × 10(-6) M) for successive 48 h and the medium concentrations of progesterone (P4), oxytocin (OT), prostaglandin (PG) E2 and F2α were determined. Moreover, the expression of mRNA for neurophysin-I/oxytocin (NP-I/OT; precursor of OT) and peptidyl-glycine-α-amidating mono-oxygenase (PGA, an enzyme responsible for post-translational OT synthesis) was determined after 8 h of treatment. Coumestrol did not affect P4 secretion but increased the secretion of OT from the cells collected at all stages of gestation studied. Hence, the ratio of P4 to OT was markedly decreased. Simultaneously, coumestrol increased the expression of NP-I/OT mRNA during 9th to 12th weeks of pregnancy, and mRNA for PGA during 3rd to 5th and 9th to 12th weeks of gestation. Furthermore, coumestrol decreased PGE2 secretion from luteal cells in all studied stages of pregnancy, while it affected PGF2α metabolite (PGFM) concentration only from week 3 to 5 of pregnancy. Obtained results suggest that coumestrol impairs secretory function of the corpus luteum (CL) and this way it can affect the maintenance of pregnancy in the cow. PMID:21656645

  16. Expression of the AMPA Receptor Subunits GluR1 and GluR2 is Associated with Granule Cell Maturation in the Dentate Gyrus

    PubMed Central

    Hagihara, Hideo; Ohira, Koji; Toyama, Keiko; Miyakawa, Tsuyoshi

    2011-01-01

    The dentate gyrus produces new granule neurons throughout adulthood in mammals from rodents to humans. During granule cell maturation, defined markers are expressed in a highly regulated sequential process, which is necessary for directed neuronal differentiation. In the present study, we show that ?-amino-3-hydroxy-5-methy-4-isoxazole propionate (AMPA) receptor subunits GluR1 and GluR2 are expressed in differentiated granule cells, but not in stem cells, in neonatal, and adult dentate gyrus. Using markers for neural progenitors, immature and mature granule cells, we found that GluR1 and GluR2 were expressed mainly in mature cells and in some immature cells. A time-course analysis of 5-bromo-2?-deoxyuridine staining revealed that granule cells express GluR1 around 3?weeks after being generated. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, a putative animal model of schizophrenia and bipolar disorder in which dentate gyrus granule cells fail to mature normally, GluR1 and GluR2 immunoreactivities were substantially downregulated in the dentate gyrus granule cells. In the granule cells of mutant mice, the expression of both presynaptic and postsynaptic markers was decreased, suggesting that GluR1 and GluR2 are also associated with synaptic maturation. Moreover, GluR1 and GluR2 were also expressed in mature granule cells of the neonatal dentate gyrus. Taken together, these findings indicate that GluR1 and GluR2 expression closely correlates with the neuronal maturation state, and that GluR1 and GluR2 are useful markers for mature granule cells in the dentate gyrus. PMID:21927594

  17. Proteomic Analysis of Hippocampal Dentate Granule Cells in Frontotemporal Lobar Degeneration: Application of Laser Capture Technology

    PubMed Central

    Gozal, Yair M.; Dammer, Eric B.; Duong, Duc M.; Cheng, Dongmei; Gearing, Marla; Rees, Howard D.; Peng, Junmin; Lah, James J.; Levey, Allan I.

    2011-01-01

    Frontotemporal lobar degeneration (FTLD) is the most common cause of dementia with pre-senile onset, accounting for as many as 20% of cases. A common subset of FTLD cases is characterized by the presence of ubiquitinated inclusions in vulnerable neurons (FTLD-U). While the pathophysiological mechanisms underlying neurodegeneration in FTLD-U have not yet been elucidated, the presence of inclusions in this disease indicates enhanced aggregation of one or several proteins. Moreover, these inclusions suggest altered expression, processing, or degradation of proteins during FTLD-U pathogenesis. Thus, one approach to understanding disease mechanisms is to delineate the molecular changes in protein composition in FTLD-U brain. Using a combined approach consisting of laser capture microdissection (LCM) and high-resolution liquid chromatography-tandem mass spectrometry (LCMS/MS), we identified 1252 proteins in hippocampal dentate granule cells excised from three post-mortem FTLD-U and three unaffected control cases processed in parallel. Additionally, we employed a labeling-free quantification technique to compare the abundance of the identified proteins between FTLD-U and control cases. Quantification revealed 54 proteins with selective enrichment in FTLD-U, including TARDNA binding protein 43 (TDP-43), a recently identified component of ubiquitinated inclusions. Moreover, 19 proteins were selectively decreased in FTLD-U. Subsequent immunohistochemical analysis of TDP-43 and three additional protein candidates suggests that our proteomic profiling of FTLD-U dentate granule cells reveals both inclusion-associated proteins and non-aggregated disease-specific proteins. Application of LCM is a valuable tool in the molecular analysis of complex tissues, and its application in the proteomic characterization of neurodegenerative disorders such as FTLD-U may be used to identify proteins altered in disease. PMID:21577247

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

    PubMed

    Rhodes, P G; Cai, Z; Zhu, N

    1994-09-01

    Prenatal ethanol exposure-induced alteration in poly-phosphoinositide (PPI) hydrolysis stimulated by excitatory amino acids (EAA) was studied in rat cerebellar granule cells previously labeled with [3H]myoinositol. The prenatal exposure to ethanol was achieved via maternal consumption of a Sustacal (chocolate flavored) liquid diet containing either 5% ethanol (w/v, 35% of calories) or isocaloric sucrose (pair-fed) substituted for ethanol from gestation d 11 until the day of parturition. The ionotropic glutamate receptor agonists, N-methyl-D-aspartate, kainate or (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (100 microM each) induced a two- to four-fold increase in PPI hydrolysis over the basal level, regardless of the liquid dietary treatment. Stimulation with quisqualate (QA), an agonist activating both metabotropic and ionotropic glutamate receptors, resulted in a much stronger and dose-dependent response in PPI hydrolysis and exposure in utero to ethanol significantly reduced this response. Tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or (+/-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) had no effect on QA-stimulated PPI hydrolysis nor on the suppression of this hydrolysis by ethanol. Exposure in utero to ethanol did not affect PPI hydrolysis stimulated by a selective metabotropic glutamate receptor agonist, trans-(+/-)-l-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD). Although the PPI hydrolysis stimulated by t-ACPD could be blocked by (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG), an antagonist of the metabotropic glutamate receptor, MCPG was incapable of affecting QA-induced PPI hydrolysis and the suppressive effects of prenatal ethanol exposure on this hydrolysis. Taken together, the data suggest that the long-lasting suppressive effects of prenatal ethanol exposure on QA-stimulated PPI hydrolysis in cerebellar granule cell cultures is through a metabotropic QA receptor pathway that may be different from the one activated by t-ACPD. PMID:7893331

  19. Calnuc plays a role in dynamic distribution of Gαi but not Gβ subunits and modulates ACTH secretion in AtT-20 neuroendocrine secretory cells

    PubMed Central

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

    2009-01-01

    In AtT-20 cells ACTH secretion is regulated by both Ca2+ and G proteins. We previously demonstrated that calnuc, an EF-hand Ca2+ binding protein which regulates Alzheimer's β-amyloid precursor protein (APP) biogenesis, binds both Ca2+ as well as Gα subunits. Here we investigate calnuc's role in G protein-mediated regulation of ACTH secretion in AtT-20 neuroendocrine secretory cells stably overexpressing calnuc-GFP. Similar to endogenous calnuc, calnuc-GFP is mainly found in the Golgi, on the plasma membrane (PM), and associated with regulated secretion granules (RSG). By deconvolution immunofluorescence, calnuc-GFP partially colocalizes with Gαi1/2 and Gαi3 at the PM and on RSG. Cytosolic calnuc(ΔSS)-CFP with the signal sequence deleted also partially colocalizes with RSG and partially cosediments with Gαi1/2 in fractions enriched in RSG. Overexpression of calnuc-GFP specifically increases the distribution of Gαi1/2 on the PM whereas the distribution of Gβ subunits and synaptobrevin 2 (Vamp 2) is unchanged. Overexpression of calnuc-GFP or cytosolic calnuc(ΔSS)-CFP enhances ACTH secretion two-fold triggered by mastoparan or GTPγS but does not significantly affect glycosaminoglycan (GAG) chain secretion along the constitutive pathway or basal secretion of ACTH. Calnuc's facilitating effects on ACTH secretion are decreased after introducing anti-Gαi1/2, Gαi3, Gβ or calnuc IgG into permeabilized cells but not when Gα12 or preimmune IgG is introduced. The results suggest that calnuc binds to Gα subunits on the Golgi and on RSG and that overexpression of calnuc causes redistribution of Gαi subunits to the PM and RSG, indicating that calnuc plays a role in dynamic distribution of only Gα but not Gβ subunits. Thus calnuc may connect G protein signaling and calcium signaling during regulated secretion. PMID:19320978

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

    PubMed

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

    2015-05-01

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

  1. Identification of MMP-2 as a novel enhancer of cerebellar granule cell proliferation.

    PubMed

    Verslegers, Mieke; Van Hove, Inge; Buyens, Tom; Dekeyster, Eline; Knevels, Ellen; Moons, Lieve

    2013-11-01

    During the first postnatal days in the mouse, granule cells (GCs) undergo massive proliferation, which then gradually decreases. Matrix metalloproteinase-2 (MMP-2), a Zn(2+)-dependent proteolytic enzyme, is involved in a wide variety of pathological and physiological pathways. Evidence for a role of this proteinase in cell proliferation is emerging, reporting its involvement in pathological proliferation, as well as during neurogenesis and developmental proliferation of non-CNS tissues. In this study, MMP-2 protein expression was observed within the early postnatal cerebellar cortex, predominantly in Purkinje cells and within the GC proliferative zone, i.e. the superficial external granular layer (EGL). Consistently, the spatiotemporal MMP-2 mRNA and protein profiles highly correlated with the peak of GC precursor (GCP) proliferation and detailed morphometric analyses of MMP-2 deficient cerebella revealed a thinner EGL due to a decreased GCP proliferation. BrdU cumulative experiments, performed to measure the length of different cell cycle phases, further disclosed a transiently prolonged S-phase in MMP-2 deficient GCPs during early cerebellar development. In consequence, MMP-2 deficient animals displayed a transient delay in GC migration towards the IGL. In conclusion, our findings provide important evidence for a role for MMP-2 in neuronal proliferation and cell cycle kinetics in the developing CNS. PMID:24141049

  2. N-methyl-D-aspartate receptor-mediated neuroprotection in cerebellar granule cells requires new RNA and protein synthesis.

    PubMed Central

    Marini, A M; Paul, S M

    1992-01-01

    Cerebellar granule cells are susceptible to the excitotoxin glutamate, which acts at N-methyl-D-aspartate (NMDA) receptors, as well as the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+), the active cytotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Paradoxically, preincubation of cultured cerebellar granule cells with low concentrations of NMDA or glutamate markedly antagonizes the neurotoxicity resulting from subsequent exposure to toxic concentrations of either MPP+ or glutamate. The neuroprotective effects of NMDA and glutamate against MPP+ toxicity are observed at agonist concentrations as low as 1 microM, are blocked by specific NMDA receptor antagonists, and require at least 30 min to develop fully. Moreover, NMDA receptor-mediated neuroprotection is prevented by the RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide. Thus, in cerebellar granule cells activation of NMDA receptors by glutamate can result in either neurotoxicity or neuroprotection, depending on the apparent degree of receptor stimulation. NMDA receptor-mediated neuroprotection requires new RNA and protein synthesis and therefore appears to be mediated by the expression of a neuroprotective protein(s). These data demonstrate the presence of an active NMDA receptor-mediated and transcriptionally directed neuroprotective mechanism in cerebellar granule cells. Images PMID:1385875

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

    ERIC Educational Resources Information Center

    Spanswick, Simon C.; Sutherland, Robert J.

    2010-01-01

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

  4. Detection of chromogranin in neuroendocrine cells with a monoclonal antibody.

    PubMed Central

    Wilson, B. S.; Lloyd, R. V.

    1984-01-01

    A monoclonal antibody ( LK2H10 ) produced against a human pheochromocytoma reacted immunohistochemically with 126 normal and neoplastic endocrine tissues with secretory granules which were formalin-fixed and paraffin-embedded. Antibody LK2H10 did not react with 46 other endocrine tissues or tumors without secretory granules nor with 113 normal and neoplastic nonendocrine cells and tumors. Tumors with abundant secretory granules showed intense and diffuse staining, and tumors with few granules, such as Merkel cell carcinomas, neuroblastomas, and small cell carcinomas of lung, showed focal staining. Antibody LK2H10 did not react with melanomas, nevi, posterior pituitary, peripheral nerve tissues, or neurons. The target structure of LK2H10 was identified as human chromogranin, of which the major fraction was chromogranin A (mol wt 68,000 daltons). Preabsorption with purified chromogranin A blocked immunoperoxidase staining by LK2H10 in normal adrenal medulla, in the anterior pituitary, and in a pheochromocytoma. Ultrastructural immunohistochemistry with LK2H10 showed that chromogranin was present in cytoplasmic secretory granules. These results indicate that chromogranin is widely distributed in the secretory granules of most polypeptide-producing endocrine tissues, and it is readily detected with the use of monoclonal antibody LK2H10 . The detection of this marker can be very helpful as a diagnostic aid for neuroendocrine cells and tumors. Images Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 1 Figure 2 Figure 3 Figure 10 Figure 11 PMID:6375394

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

    PubMed Central

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

    2009-01-01

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

  6. Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis

    PubMed Central

    Zou, Tongtong; Rao, Jaladanki N.; Liu, Lan; Xiao, Lan; Cui, Yu-Hong; Jiang, Zhengran; Ouyang, Miao; Donahue, James M.

    2012-01-01

    Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with ?-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-?/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis. PMID:22555848

  7. High-efficiency secretory expression of human neutrophil gelatinase-associated lipocalin from mammalian cell lines with human serum albumin signal peptide.

    PubMed

    Chen, Wei; Zhao, Xiaozhi; Zhang, Mingxin; Yuan, Yimin; Ge, Liyuan; Tang, Bo; Xu, Xiaoyu; Cao, Lin; Guo, Hongqian

    2016-02-01

    Human neutrophil gelatinase associated lipocalin (NGAL) is a secretory glycoprotein initially isolated from neutrophils. It is thought to be involved in the incidence and development of immunological diseases and cancers. Urinary and serum levels of NGAL have been investigated as a new biomarker of acute kidney injury (AKI), for an earlier and more accurate detection method than with creatinine level. However, expressing high-quality recombinant NGAL is difficult both in Escherichia coli and mammalian cells for the low yield. Here, we cloned and fused NGAL to the C-terminus of signal peptides of human NGAL, human interleukin-2 (IL2), gaussia luciferase (Gluc), human serum albumin preproprotein (HSA) or an hidden Markov model-generated signal sequence (HMM38) respectively for transient expression in Expi293F suspension cells to screen for their ability to improve the secretory expression of recombinant NGAL. The best results were obtained with signal peptide derived from HSA. The secretory recombinant protein could react specifically with NGAL antibody. For scaled production, we used HSA signal peptide to establish stable Chinese hamster ovary cell lines. Then we developed a convenient colony-selection system to select high-expression, stable cell lines. Moreover, we purified the NGAL with Ni-Sepharose column. The recombinant human NGAL displayed full biological activity. We provide a method to enhance the secretory expression of recombinant human NGAL by using the HSA signal peptide and produce the glycoprotein in mammalian cells. PMID:26518367

  8. MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development.

    PubMed

    Constantin, Lena; Constantin, Myrna; Wainwright, Brandon J

    2016-03-01

    The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies. PMID:26773048

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-05-15

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

  11. The Exosome Secretory Pathway Transports Amyloid Precursor Protein Carboxyl-terminal Fragments from the Cell into the Brain Extracellular Space*

    PubMed Central

    Perez-Gonzalez, Rocio; Gauthier, Sebastien A.; Kumar, Asok; Levy, Efrat

    2012-01-01

    In vitro studies have shown that neuronal cell cultures secrete exosomes containing amyloid-? precursor protein (APP) and the APP-processing products, C-terminal fragments (CTFs) and amyloid-? (A?). We investigated the secretion of full-length APP (flAPP) and APP CTFs via the exosome secretory pathway in vivo. To this end, we developed a novel protocol designed to isolate exosomes secreted into mouse brain extracellular space. Exosomes with typical morphology were isolated from freshly removed mouse brains and from frozen mouse and human brain tissues, demonstrating that exosomes can be isolated from post-mortem tissue frozen for long periods of time. flAPP, APP CTFs, and enzymes that cleave both flAPP and APP CTFs were identified in brain exosomes. Although higher levels of both flAPP and APP CTFs were observed in exosomes isolated from the brains of transgenic mice overexpressing human APP (Tg2576) compared with wild-type control mice, there was no difference in the number of secreted brain exosomes. These data indicate that the levels of flAPP and APP CTFs associated with exosomes mirror the cellular levels of flAPP and APP CTFs. Interestingly, exosomes isolated from the brains of both Tg2576 and wild-type mice are enriched with APP CTFs relative to flAPP. Thus, we hypothesize that the exosome secretory pathway plays a pleiotropic role in the brain: exosome secretion is beneficial to the cell, acting as a specific releasing system of neurotoxic APP CTFs and A?, but the secretion of exosomes enriched with APP CTFs, neurotoxic proteins that are also a source of secreted A?, is harmful to the brain. PMID:23129776

  12. UNC-45A Is a Nonmuscle Myosin IIA Chaperone Required for NK Cell Cytotoxicity via Control of Lytic Granule Secretion.

    PubMed

    Iizuka, Yoshie; Cichocki, Frank; Sieben, Andrew; Sforza, Fabio; Karim, Razaul; Coughlin, Kathleen; Isaksson Vogel, Rachel; Gavioli, Riccardo; McCullar, Valarie; Lenvik, Todd; Lee, Michael; Miller, Jeffrey; Bazzaro, Martina

    2015-11-15

    NK cell's killing is a tightly regulated process under the control of specific cytoskeletal proteins. This includes Wiskott-Aldrich syndrome protein, Wiskott-Aldrich syndrome protein-interacting protein, cofilin, Munc13-4, and nonmuscle myosin IIA (NMIIA). These proteins play a key role in controlling NK-mediated cytotoxicity either via regulating the attachment of lytic granules to the actin-based cytoskeleton or via promoting the cytoskeletal reorganization that is requisite for lytic granule release. UNC-45A is a highly conserved member of the UNC-45/CRO1/She4p family of proteins that act as chaperones for both conventional and nonconventional myosin. Although we and others have shown that in lower organisms and in mammalian cells NMIIA-associated functions, such as cytokinesis, cell motility, and organelle trafficking, are dependent upon the presence of UNC-45A, its role in NK-mediated functions is largely unknown. In this article, we describe UNC-45A as a key regulator of NK-mediated cell toxicity. Specifically we show that, in human NK cells, UNC-45A localize at the NK cell immunological synapse of activated NK cells and is part of the multiprotein complex formed during NK cell activation. Furthermore, we show that UNC-45A is disposable for NK cell immunological synapse formation and lytic granules reorientation but crucial for lytic granule exocytosis. Lastly, loss of UNC-45A leads to reduced NMIIA binding to actin, suggesting that UNC-45A is a crucial component in regulating human NK cell cytoskeletal dynamics via promoting the formation of actomyosin complexes. PMID:26438524

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

    PubMed

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

    2014-11-01

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

  14. Potential implications of a monosynaptic pathway from mossy cells to adult-born granule cells of the dentate gyrus

    PubMed Central

    Scharfman, Helen E.; Bernstein, Hannah L.

    2015-01-01

    The dentate gyrus (DG) is important to many aspects of hippocampal function, but there are many aspects of the DG that are incompletely understood. One example is the role of mossy cells (MCs), a major DG cell type that is glutamatergic and innervates the primary output cells of the DG, the granule cells (GCs). MCs innervate the GCs as well as local circuit neurons that make GABAergic synapses on GCs, so the net effect of MCs on GCs – and therefore the output of the DG – is unclear. Here we first review fundamental information about MCs and the current hypotheses for their role in the normal DG and in diseases that involve the DG. Then we review previously published data which suggest that MCs are a source of input to a subset of GCs that are born in adulthood (adult-born GCs). In addition, we discuss the evidence that adult-born GCs may support the normal inhibitory ‘gate’ functions of the DG, where the GCs are a filter or gate for information from the entorhinal cortical input to area CA3. The implications are then discussed in the context of seizures and temporal lobe epilepsy (TLE). In TLE, it has been suggested that the DG inhibitory gate is weak or broken and MC loss leads to insufficient activation of inhibitory neurons, causing hyperexcitability. That idea was called the “dormant basket cell hypothesis.” Recent data suggest that loss of normal adult-born GCs may also cause disinhibition, and seizure susceptibility. Therefore, we propose a reconsideration of the dormant basket cell hypothesis with an intervening adult-born GC between the MC and basket cell and call this hypothesis the “dormant immature granule cell hypothesis.” PMID:26347618

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

    PubMed

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

    2015-01-01

    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

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

    PubMed Central

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

    2012-01-01

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

  17. Restricted distribution of mrg-1 mRNA in C. elegans primordial germ cells through germ granule-independent regulation.

    PubMed

    Miwa, Takashi; Takasaki, Teruaki; Inoue, Kunio; Sakamoto, Hiroshi

    2015-11-01

    The chromodomain protein MRG-1 is an essential maternal factor for proper germline development that protects germ cells from cell death in C. elegans. Unlike germ granules, which are exclusively segregated to the germline blastomeres at each cell division from the first cleavage of the embryo, MRG-1 is abundant in all cells in early embryos and is then gradually restricted to the primordial germ cells (PGCs) by the morphogenesis stage. Here, we show that this characteristic spatiotemporal expression pattern is dictated by the mrg-1 3'UTR and is differentially regulated at the RNA level between germline and somatic cells. Asymmetric segregation of germ granules is not necessary to localize MRG-1 to the PGCs. We found that MES-4, an essential chromatin regulator in germ cells, also accumulates in the PGCs in a germ granule-independent manner. We propose that C.elegans PGCs have a novel mechanism to accumulate at least some chromatin-associated proteins that are essential for germline immortality. PMID:26537333

  18. 2-ketoglutarate generation in pancreatic B-cell mitochondria regulates insulin secretory action of amino acids and 2-keto acids.

    PubMed

    Lenzen, S; Schmidt, W; Rustenbeck, I; Panten, U

    1986-02-01

    The various neutral amino acids and aliphatic 2-keto acids exhibit differential effects on insulin secretion. The common denominator for all these effects is the 2-ketoglutarate generation in the pancreatic B-cell mitochondria. The neutral amino acids L-leucine and L-norvaline and the aliphatic ketomonocarboxylic acids 2-ketoisocaproate, 2-ketocaproate, 2-ketovalerate, and 2-keto-3-methylvalerate all stimulate insulin secretion and increase 2-ketoglutarate generation in pancreatic B-cell mitochondria through activation of glutamate dehydrogenase and transamination with L-glutamate and L-glutamine, respectively. The neutral amino acids L-valine, L-norleucine, and L-alanine and the aliphatic 2-keto acids 2-ketoisovalerate and pyruvate do not stimulate insulin secretion and do not increase 2-ketoglutarate generation in pancreatic B-cell mitochondria. Inhibition of 2-keto acid induced insulin secretion by L-valine and L-isoleucine is accompanied by reduced 2-ketoglutarate generation in pancreatic B-cell mitochondria. Thus intramitochondrial 2-ketoglutarate generation in pancreatic B-cells may regulate the insulin secretory potency of amino acids and 2-keto acids. PMID:3521757

  19. Eighteen New Candidate Effectors of the Phytonematode Heterodera glycines Produced Specifically in the Secretory Esophageal Gland Cells During Parasitism.

    PubMed

    Noon, Jason B; Hewezi, Tarek; Maier, Thomas R; Simmons, Carl; Wei, Jun-Zhi; Wu, Gusui; Llaca, Victor; Deschamps, Stéphane; Davis, Eric L; Mitchum, Melissa G; Hussey, Richard S; Baum, Thomas J

    2015-10-01

    Heterodera glycines, the soybean cyst nematode, is the number one pathogen of soybean (Glycine max). This nematode infects soybean roots and forms an elaborate feeding site in the vascular cylinder. H. glycines produces an arsenal of effector proteins in the secretory esophageal gland cells. More than 60 H. glycines candidate effectors were identified in previous gland-cell-mining projects. However, it is likely that additional candidate effectors remained unidentified. With the goal of identifying remaining H. glycines candidate effectors, we constructed and sequenced a large gland cell cDNA library resulting in 11,814 expressed sequence tags. After bioinformatic filtering for candidate effectors using a number of criteria, in situ hybridizations were performed in H. glycines whole-mount specimens to identify candidate effectors whose mRNA exclusively accumulated in the esophageal gland cells, which is a hallmark of many nematode effectors. This approach resulted in the identification of 18 new H. glycines esophageal gland-cell-specific candidate effectors. Of these candidate effectors, 11 sequences were pioneers without similarities to known proteins while 7 sequences had similarities to functionally annotated proteins in databases. These putative homologies provided the bases for the development of hypotheses about potential functions in the parasitism process. PMID:25871857

  20. Cyclooxygenase-2 Inhibition Protects Cultured Cerebellar Granule Neurons from Glutamate-Mediated Cell Death

    PubMed Central

    MARINI, ANN M.

    2006-01-01

    Primary insults to the brain can initiate glutamate release that may result in excitotoxicity followed by neuronal cell death. This secondary process is mediated by both N-methyl-d-aspartate (NMDA) and non-NMDA receptors in vivo and requires new gene expression. Neuronal cyclooxygenase-2 (COX2) expression is upregulated following brain insults, via glutamatergic and inflammatory mechanisms. The products of COX2 are bioactive prostanoids and reactive oxygen species that may play a role in neuronal survival. This study explores the role of neuronal COX2 in glutamate excitotoxicity using cultured cerebellar granule neurons (day 8 in vitro). Treatment with excitotoxic concentrations of glutamate or kainate transiently induced COX2 mRNA (two- and threefold at 6 h, respectively, p < 0.05, Dunnett) and prostaglandin production (five- and sixfold at 30 min, respectively, p < 0.05, Dunnett). COX2 induction peaked at toxic concentrations of these excitatory amino acids. Surprisingly, NMDA, l-quisqualate, and trans-ACPD did not induce COX2 mRNA at any concentration tested. The glutamate receptor antagonist NBQX (5 ?M, AMPA/kainate receptor) completely inhibited kainate-induced COX2 mRNA and partially inhibited glutamate-induced COX2 (p < 0.05, Dunnett). Other glutamate receptor antagonists, such as MK-801 (1 ?M, NMDA receptor) or MCPG (500 ?M, class 1 metabotropic receptors), partially attenuated glutamate-induced COX2 mRNA. These antagonists all reduced steady-state COX2 mRNA (p < 0.05, Dunnett). To determine whether COX2 might be an effector of excitotoxic cell death, cerebellar granule cells were pretreated (24 h) with the COX2-specific enzyme inhibitor, DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methyl-sulphonyl) phenyl-2(5H)-furanone) prior to glutamate challenge. DFU (1 to 1000 nM) completely protected cultured neurons from glutamate-mediated neurotoxicity. Approximately 50% protection from NMDA-mediated neurotoxicity, and no protection from kainate-mediated neurotoxicity was observed. Therefore, glutamate-mediated COX2 induction contributes to excitotoxic neuronal death. These results suggest that glutamate, NMDA, and kainate neurotoxicity involve distinct excitotoxic pathways, and that the glutamate and NMDA pathways may intersect at the level of COX2. PMID:12042097

  1. 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

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

    1999-11-01

    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 beta2-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 beta2-m on the cell surface. The absence of beta2-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 beta2-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. PMID:10555762

  2. Kruppel-Like Factor 4 Regulates Granule Cell Pax6 Expression and Cell Proliferation in Early Cerebellar Development

    PubMed Central

    Zhang, Peter; Ha, Thomas; Larouche, Matt; Swanson, Douglas; Goldowitz, Dan

    2015-01-01

    Kruppel-like factor 4 (Klf4) is a transcription factor that regulates many important cellular processes in stem cell biology, cancer, and development. We used histological and molecular methods to study the expression of Klf4 in embryonic development of the normal and Klf4 knockout cerebellum. We find that Klf4 is expressed strongly in early granule cell progenitor development but tails-off considerably by the end of embryonic development. Klf4 is also co-expressed with Pax6 in these cells. In the Klf4-null mouse, which is perinatal lethal, Klf4 positively regulates Pax6 expression and regulates the proliferation of neuronal progenitors in the rhombic lip, external granular layer and the neuroepithelium. This paper is the first to describe a role for Klf4 in the cerebellum and provides insight into this genes function in neuronal development. PMID:26226504

  3. Mitochondrial and secretory human cytomegalovirus UL37 proteins traffic into mitochondrion-associated membranes of human cells.

    PubMed

    Bozidis, Petros; Williamson, Chad D; Colberg-Poley, Anamaris M

    2008-03-01

    The human cytomegalovirus (HCMV) UL37 exon 1 protein (pUL37x1), also known as vMIA, is the predominant UL37 isoform during permissive infection. pUL37x1 is a potent antiapoptotic protein, which prevents cytochrome c release from mitochondria. The UL37x1 NH(2)-terminal bipartite localization signal, which remains uncleaved, targets UL37 proteins to the endoplasmic reticulum (ER) and then to mitochondria. Based upon our findings, we hypothesized that pUL37x1 traffics from the ER to mitochondria through direct contacts between the two organelles, provided by mitochondrion-associated membranes (MAMs). To facilitate its identification, we cloned and tagged the human phosphatidylserine synthase 1 (huPSS-1) cDNA, whose mouse homologue localizes almost exclusively in the MAM. Using subcellular fractionation of stable HeLa cell transfectants expressing mEGFP-huPSS-1, we found that HCMV pUL37x1 is present in purified microsomes, mitochondria, and MAM fractions. We further examined the trafficking of the full-length UL37 glycoprotein cleavage products, which divergently traffic either through the secretory apparatus or into mitochondria. Surprisingly, pUL37(NH2) and gpUL37(COOH) were both detected in the ER and MAM fraction, even though only pUL37(NH2) is preferentially imported into mitochondria but gpUL37(COOH) is not. To determine the sequences required for MAM importation, we examined pUL37x1 mutants that were partially defective for mitochondrial importation. Deletion mutants of the NH(2)-terminal UL37x1 mitochondrial localization signal were reduced in trafficking into the MAM, indicating partial overlap of MAM and mitochondrial targeting signals. Taken together, these results suggest that HCMV UL37 proteins traffic from the ER into the MAM, where they are sorted into either the secretory pathway or to mitochondrial importation. PMID:18199645

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

    PubMed

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

    2015-02-01

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

  5. Age-Dependent Degeneration of Mature Dentate Gyrus Granule Cells Following NMDA Receptor Ablation

    PubMed Central

    Watanabe, Yasuhito; Müller, Michaela K.; von Engelhardt, Jakob; Sprengel, Rolf; Seeburg, Peter H.; Monyer, Hannah

    2016-01-01

    N-methyl-D-aspartate receptors (NMDARs) in all hippocampal areas play an essential role in distinct processes of memory formation as well as in sustaining cell survival of postnatally generated neurons in the dentate gyrus (DG). In contrast to the beneficial effects, over-activation of NMDARs has been implicated in many acute and chronic neurological diseases, reason why therapeutic approaches and clinical trials involving receptor blockade have been envisaged for decades. Here we employed genetically engineered mice to study the long-term effect of NMDAR ablation on selective hippocampal neuronal populations. Ablation of either GluN1 or GluN2B causes degeneration of the DG. The neuronal demise affects mature neurons specifically in the dorsal DG and is NMDAR subunit-dependent. Most importantly, the degenerative process exacerbates with increasing age of the animals. These results lead us to conclude that mature granule cells in the dorsal DG undergo neurodegeneration following NMDAR ablation in aged mouse. Thus, caution needs to be exerted when considering long-term administration of NMDAR antagonists for therapeutic purposes. PMID:26793056

  6. Nicotinamide and 1-methylnicotinamide reduce homocysteine neurotoxicity in primary cultures of rat cerebellar granule cells.

    PubMed

    Slomka, Marta; Zieminska, Elzbieta; Lazarewicz, Jerzy

    2008-01-01

    Nicotinamide is an important cofactor in many metabolic pathways and a known neuroprotective substance, while its methylated product, 1-methylnicotinamide, is a suspected neurotoxin. Homocysteine is a risk factor in Alzheimer's disease and neurodegeneration, causing inhibition of methylation processes and inducing excitotoxicity. In this study, using primary cultures of rat cerebellar granule cells and propidium iodide staining, we investigated the neurotoxicity of nicotinamide and 1-methylnicotinamide, and their neuroprotective potential in acute and sub-acute homocysteine neurotoxicity. Our results demonstrated that nicotinamide and 1-methylnicotinamide applied for 24 h to cultures at concentrations of up to 25 mM had no effect on neuronal viability. Moreover, nicotinamide at concentrations of 5-20 mM and 1-methylnicotinamide at 1-10 mM applied to cells 24 h before, and for 24 h after an acute 30 min application of 25 mM D,L homocysteine, reduced neuronal damage. 1-Methylnicotinamide at concentrations of 250 and 500 muM showed neuroprotective activity during a sub-acute 24-h exposure to 2.5 mM D,L-homocysteine, while 5 and 25 mM nicotinamide also evoked neuroprotection. These findings do not support suggestions that 1-methylnicotinamide may act as an endogenous neurotoxic agent; rather, they indicate the neuroprotective ability of nicotinamide and 1-methylnicotinamide in homocysteine neurotoxicity. The exact mechanisms of this neuroprotection are unclear and require further investigation. PMID:18389009

  7. Forward transport of proteins in the plasma membrane of migrating cerebellar granule cells

    PubMed Central

    Wang, Dong; She, Liang; Sui, Ya-nan; Yuan, Xiao-bing; Wen, Yunqing; Poo, Mu-ming

    2012-01-01

    Directional flow of membrane components has been detected at the leading front of fibroblasts and the growth cone of neuronal processes, but whether there exists global directional flow of plasma membrane components over the entire migrating neuron remains largely unknown. By analyzing the trajectories of antibody-coated single quantum dots (QDs) bound to two membrane proteins, overexpressed myc-tagged synaptic vesicle-associated membrane protein VAMP2 and endogenous neurotrophin receptor TrkB, we found that these two proteins exhibited net forward transport, which is superimposed upon Brownian motion, in both leading and trailing processes of migrating cerebellar granule cells in culture. Furthermore, no net directional transport of membrane proteins was observed in nonmigrating cells with either growing or stalling leading processes. Analysis of the correlation of motion direction between two QDs on the same process in migrating neurons also showed a higher frequency of correlated forward than rearward movements. Such correlated QD movements were markedly reduced in the presence of myosin II inhibitor blebbistatin, suggesting the involvement of myosin II-dependent active transport processes. Thus, a net forward transport of plasma membrane proteins exists in the leading and trailing processes of migrating neurons, in line with the translocation of the soma. PMID:23213239

  8. Forward transport of proteins in the plasma membrane of migrating cerebellar granule cells.

    PubMed

    Wang, Dong; She, Liang; Sui, Ya-nan; Yuan, Xiao-bing; Wen, Yunqing; Poo, Mu-ming

    2012-12-18

    Directional flow of membrane components has been detected at the leading front of fibroblasts and the growth cone of neuronal processes, but whether there exists global directional flow of plasma membrane components over the entire migrating neuron remains largely unknown. By analyzing the trajectories of antibody-coated single quantum dots (QDs) bound to two membrane proteins, overexpressed myc-tagged synaptic vesicle-associated membrane protein VAMP2 and endogenous neurotrophin receptor TrkB, we found that these two proteins exhibited net forward transport, which is superimposed upon Brownian motion, in both leading and trailing processes of migrating cerebellar granule cells in culture. Furthermore, no net directional transport of membrane proteins was observed in nonmigrating cells with either growing or stalling leading processes. Analysis of the correlation of motion direction between two QDs on the same process in migrating neurons also showed a higher frequency of correlated forward than rearward movements. Such correlated QD movements were markedly reduced in the presence of myosin II inhibitor blebbistatin,suggesting the involvement of myosin II-dependent active transport processes. Thus, a net forward transport of plasma membrane proteins exists in the leading and trailing processes of migrating neurons, in line with the translocation of the soma. PMID:23213239

  9. Distinct roles of NMDA receptors at different stages of granule cell development in the adult brain.

    PubMed

    Mu, Yangling; Zhao, Chunmei; Toni, Nicolas; Yao, Jun; Gage, Fred H

    2015-01-01

    NMDA receptor (NMDAR)-dependent forms of synaptic plasticity are thought to underlie the assembly of developing neuronal circuits and to play a crucial role in learning and memory. It remains unclear how NMDAR might contribute to the wiring of adult-born granule cells (GCs). Here we demonstrate that nascent GCs lacking NMDARs but rescued from apoptosis by overexpressing the pro-survival protein Bcl2 were deficient in spine formation. Insufficient spinogenesis might be a general cause of cell death restricted within the NMDAR-dependent critical time window for GC survival. NMDAR loss also led to enhanced mushroom spine formation and synaptic AMPAR activity throughout the development of newborn GCs. Moreover, similar elevated synapse maturation in the absence of NMDARs was observed in neonate-generated GCs and CA1 pyramidal neurons. Together, these data suggest that NMDAR operates as a molecular monitor for controlling the activity-dependent establishment and maturation rate of synaptic connections between newborn neurons and others. PMID:26473971

  10. Development of a Cell-Based Fluorescence Polarization Biosensor Using Preproinsulin to Identify Compounds That Alter Insulin Granule Dynamics.

    PubMed

    Yi, Na Young; He, Qingping; Caligan, Thomas B; Smith, Ginger R; Forsberg, Lawrence J; Brenman, Jay E; Sexton, Jonathan Z

    2015-11-01

    Diabetes currently affects 9.3% of the U.S. population totaling $245 billion annually in U.S. direct and indirect healthcare costs. Current therapies for diabetes are limited in their ability to control blood glucose and/or enhance insulin sensitivity. Therefore, innovative and efficacious therapies for diabetes are urgently needed. Herein we describe a fluorescent insulin reporter system (preproinsulin-mCherry, PPI-mCherry) that tracks live-cell insulin dynamics and secretion in pancreatic ?-cells with utility for high-content assessment of real-time insulin dynamics. Additionally, we report a new modality for sensing insulin granule packaging in conventional high-throughput screening (HTS), using a hybrid cell-based fluorescence polarization (FP)/internal FRET biosensor using the PPI-mCherry reporter system. We observed that bafilomycin, a vacuolar H(+) ATPase inhibitor and inhibitor of insulin granule formation, significantly increased mCherry FP in INS-1 cells with PPI-mCherry. Partial least squares regression analysis demonstrated that an increase of FP by bafilomycin is significantly correlated with a decrease in granularity of PPI-mCherry signal in the cells. The increased FP by bafilomycin is due to inhibition of self-Frster resonant energy transfer (homo-FRET) caused by the increased mCherry intermolecular distance. FP substantially decreases when insulin is tightly packaged in the granules, and the homo-FRET decreases when insulin granule packaging is inhibited, resulting in increased FP. We performed pilot HTS of 1782 FDA-approved small molecules and natural products from Prestwick and Enzo chemical libraries resulting in an overall Z'-factor of 0.52??0.03, indicating the suitability of this biosensor for HTS. This novel biosensor enables live-cell assessment of protein-protein interaction/protein aggregation in live cells and is compatible with conventional FP plate readers. PMID:26505612

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

    PubMed

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

    2014-01-01

    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

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

    PubMed

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

    2014-11-19

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

  13. PICK1 and ICA69 control insulin granule trafficking and their deficiencies lead to impaired glucose tolerance.

    PubMed

    Cao, Mian; Mao, Zhuo; Kam, Chuen; Xiao, Nan; Cao, Xiaoxing; Shen, Chong; Cheng, Kenneth K Y; Xu, Aimin; Lee, Kwong-Man; Jiang, Liwen; Xia, Jun

    2013-01-01

    Diabetes is a metabolic disorder characterized by hyperglycemia. Insulin, which is secreted by pancreatic beta cells, is recognized as the critical regulator of blood glucose, but the molecular machinery responsible for insulin trafficking remains poorly defined. In particular, the roles of cytosolic factors that govern the formation and maturation of insulin granules are unclear. Here we report that PICK1 and ICA69, two cytosolic lipid-binding proteins, formed heteromeric BAR-domain complexes that associated with insulin granules at different stages of their maturation. PICK1-ICA69 heteromeric complexes associated with immature secretory granules near the trans-Golgi network (TGN). A brief treatment of Brefeldin A, which blocks vesicle budding from the Golgi, increased the amount of PICK1 and ICA69 at TGN. On the other hand, mature secretory granules were associated with PICK1 only, not ICA69. PICK1 deficiency in mice caused the complete loss of ICA69 and led to increased food and water intake but lower body weight. Glucose tolerance tests demonstrated that these mutant mice had high blood glucose, a consequence of insufficient insulin. Importantly, while the total insulin level was reduced in PICK1-deficient beta cells, proinsulin was increased. Lastly, ICA69 knockout mice also displayed similar phenotype as the mice deficient in PICK1. Together, our results indicate that PICK1 and ICA69 are key regulators of the formation and maturation of insulin granules. PMID:23630453

  14. Proteomic analysis of differentially expressed proteins in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products.

    PubMed

    Pak, Jhang Ho; Moon, Ju Hyun; Hwang, Seung-Jun; Cho, Shin-Hyeong; Seo, Sang-Beom; Kim, Tong-Soo

    2009-12-15

    Severe Clonorchis sinensis infection is a significant risk factor for malignant changes in bile ducts and surrounding liver tissues occurring as a result of direct contact with C. sinensis worms and their excretory-secretory products (ESP). However, the intrinsic molecular mechanisms involved in these processes remain obscure. To determine the effects of C. sinensis infection on protein expression in host bile duct epithelium, we examined proteomic profile changes in the human cholangiocarcinoma cell line (HuCCT1) treated with ESP at 24 h. Using a combination of 2-DE, quantitative image and MALDI-TOF MS analysis, we identified 83 proteins that were translationally modulated in response to ESP, among which 49 were up-regulated and 34 down-regulated. These proteins were classified under various biological categories, including metabolism, cell structure and architecture, proteolysis, protein modification, transport, signal transduction, and reactive oxygen species (ROS) detoxification. In particular, ESP induced the expression of redox-regulating proteins, including peroxiredoxins (Prdx 2, 3, and 6) and thioredoxin 1 (Trx 1), possibly via intracellular ROS generation. Application of the proteomic approach to identify ESP response proteins should be a prerequisite before further investigation to clarify the molecular pathways and mechanisms involved in C. sinensis infection of host cells. PMID:19798681

  15. JAM-A promotes wound healing by enhancing both homing and secretory activities of mesenchymal stem cells.

    PubMed

    Wu, Minjuan; Ji, Shizhao; Xiao, Shichu; Kong, Zhengdong; Fang, He; Zhang, Yunqing; Ji, Kaihong; Zheng, Yongjun; Liu, Houqi; Xia, Zhaofan

    2015-10-01

    The homing ability and secretory function of mesenchymal stem cells (MSCs) are key factors that influence cell involvement in wound repair. These factors are controlled by multilayer regulatory circuitry, including adhesion molecules, core transcription factors (TFs) and certain other regulators. However, the role of adhesion molecules in this regulatory circuitry and their underlying mechanism remain undefined. In the present paper, we demonstrate that an adhesion molecule, junction adhesion molecule A (JAM-A), may function as a key promoter molecule to regulate skin wound healing by MSCs. In invivo experiments, we show that JAM-A up-regulation promoted both MSC homing to full-thickness skin wounds and wound healing-related cytokine secretion by MSCs. Invitro experiments also showed that JAM-A promoted MSC proliferation and migration by activating T-cell lymphoma invasion and metastasis 1 (Tiam1). We suggest that JAM-A up-regulation can increase the proliferation, cytokine secretion and wound-homing ability of MSCs, thus accelerating the repair rate of full-thickness skin defects. These results may provide insights into a novel and potentially effective approach to improve the efficacy of MSC treatment. PMID:25994236

  16. Immunomodulatory effects of adult Haemonchus contortus excretory/secretory products on human monocyte-derived dendritic cells.

    PubMed

    Rehman, Z U; Knight, J S; Koolaard, J; Simpson, H V; Pernthaner, A

    2015-12-01

    The levels of expression of surface molecules and release of cytokines and chemokines of human monocyte-derived dendritic cells were determined after their exposure to adult H.contortus excretory/secretory (ES) products or a combination of ES products and bacterial lipopolysaccharide (LPS). Worm products provoked a weak response and only partial maturation of the dendritic cells, consistent with the hyporesponsiveness and more tolerogenic immune environment present in parasitized animals and humans. Co-stimulation with LPS demonstrated that H.contortus secretions, like those of other helminths, contain immunomodulators capable of reducing some aspects of the strong TH 1/TH 2 response evoked by bacterial LPS. There were significant reductions in the release of some cytokine/chemokines by LPS-stimulated mdDCs and a trend (although not significant at P<005) for reduced expression levels of CD40, CD80 and HLA-DR. A prominent feature was the variability in responses of dendritic cells from the four donors, even on different days in repeat experiments, suggesting that generalized conclusions may be difficult to make, except in genetically related animals. Such observations may therefore be applicable only to restricted populations. In addition, previous exposure to parasites in a target population for immunomodulatory therapy may be an important factor in assessing the likelihood of adverse reactions or failures in the treatment to worm therapy. PMID:26457886

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

    PubMed

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

    2015-06-01

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

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

    PubMed Central

    Delvendahl, Igor; Straub, Isabelle; Hallermann, Stefan

    2015-01-01

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

  19. Somatosensory projections of cerebellar granule cell layer of giant bushbaby, Galago crassicaudatus.

    PubMed

    Welker, W; Blair, C; Shambes, G M

    1988-01-01

    Recent neurophysiological studies of the granule cell (GC) layer in opossums and rats revealed extensive somatosensory projections to the cerebellar hemispheres and caudal vermis. These projections are organized as asomatotopic mosaics that are species-specific. To determine whether similar projections exist in a primate with a relatively small and simple cerebellum, we explored the GC layer of exposed folial crowns of anterior and posterior lobe cerebellar cortex of anesthetized giant galagos using juxtathreshold natural stimulation of mechanoreceptors and in-depth microelectrode micromapping techniques. We found (1) that stimulation of somatosensory mechanoreceptors by gentle touch, deep pressure, muscle stretch and joint movement revealed projections to the GC layer throughout the mediolateral extent of crus II, paramedian lobule, pyramis and rostral uvula (crus I was unresponsive); (2) that mosaic patterns of peripheral sources and submodality of projections were different for each lobule, and (3) that there were intraspecies and individual differences in subfoliation and in details of projections. Except for differences in mosaic pattern and relative size of different projections, these findings are similar to those in opossums and rats. These data suggest that somatosensory inputs to the cerebellum are not only functionally significant, but that they exist widely among mammals. PMID:2836016

  20. Synthetic bastadins modify the activity of ryanodine receptors in cultured cerebellar granule cells.

    PubMed

    Zieminska, Elzbieta; Stafiej, Aleksandra; Pitsinos, Emmanuel N; Couladouros, Elias A; Moutsos, Vassilios; Kozlowska, Hanna; Toczylowska, Beata; Lazarewicz, Jerzy W

    Although the interactions of several natural bastadins with the RyR1 isoform of the ryanodine receptor in sarcoplasmic reticulum has been described, their structure-dependent interference with the RyR2 isoform, mainly expressed in cardiac muscle and brain neurons, has not been studied. In this work, we examined calcium transients induced by natural bastadin 10 and several synthetic bastadins in cultured cerebellar granule cells known to contain RyR2. The fluorescent calcium indicator fluo-3 and confocal microscopy were used to evaluate changes in the intracellular Ca(2+) concentration (Ca(i)), and the involvement of ryanodine receptors was assessed using pharmacological tools. Our results demonstrate that apart from the inactive BAST218F6 (a bisdebromo analogue of bastadin 10), synthetic bastadin 5, and synthetic analogues BAST217B, BAST240 and BAST268 (at concentrations >20 microM) increased Ca(i) in a concentration-dependent, ryanodine- and FK-506-sensitive way, with a potency significantly exceeding that of 20 mM caffeine. Moreover, the same active bastadins at a concentration of 5 muM in the presence of ryanodine prevented a thapsigargin-induced increase in Ca(i). These results indicate that bastadins, acting in a structure-dependent manner, modify the activity of RyR2 in primary neuronal culture and provide new information about structure-related pharmacological properties of bastadins. PMID:17726341

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

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

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

    PubMed Central

    Heinbockel, Thomas; Laaris, Nora; Ennis, Matthew

    2009-01-01

    Main olfactory bulb (MOB) granule cells (GCs) express high levels of the group I metabotropic glutamate receptor (mGluR), mGluR5. We investigated the role of mGluRs in regulating GC activity in rodent MOB slices using whole cell patch-clamp electrophysiology. The group I/II mGluR agonist ()-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) or the selective group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) depolarized (~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

  4. GABA/sub A/ receptor-controlled /sup 36/Cl/sup -/ influx in cultured rat cerebellar granule cells

    SciTech Connect

    Kardos, J.; Maderspach, K.

    1987-07-20

    ..gamma..-Aminobutyric acid (GABA)-mediated and bicuculline-sensitive /sup 36/Cl/sup -/ influx and bicuculline-sensitive (/sup 3/H) GABA binding were demonstrated in cultures of rat cerebellar granule cells. The addition of 10/sup -5/ M GABA produced a two-fold increase in /sup 36/Cl/sup -/ influx over the basal level and the maximal increase was observed after approximately 20 sec. Progressive occupation of GABA/sub A/ receptor by (/sup 3/H)-(1S-9R)-bicuculline methiodide decreased /sup 36/Cl/sup -/ influx activated by 10 ..mu..M GABA. The above results suggest that primary cultures of rat cerebellar granule cells provide a new and reliable model for studying the GABA activated chloride fluxes. 41 references, 4 figures.

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

    PubMed Central

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

    2008-01-01

    It is believed that gene/environment interaction (GEI) plays a pivotal role in the development of motor skills, which are acquired via practicing or motor training. However, the underlying molecular/neuronal mechanisms are still unclear. Here, we reported that the expression of NR2B, a subunit of NMDA receptors, in cerebellar granule cells specifically enhanced the effect of voluntary motor training on motor learning in the mouse. Moreover, this effect was characterized as motor learning-specific and developmental stage-dependent, because neither emotional/spatial memory was affected nor was the enhanced motor learning observed when the motor training was conducted starting at the age of 3 months old in these transgenic mice. These results indicate that changes in the expression of gene(s) that are involved in regulating synaptic plasticity in cerebellar granule cells may constitute a molecular basis for the cerebellum to be involved in the GEI by facilitating motor skill learning. PMID:18301761

  6. Initiation of human lactation: secretory differentiation and secretory activation.

    PubMed

    Pang, Wei Wei; Hartmann, Peter E

    2007-12-01

    Theories for the origin of milk have been recorded since the time of Ancient Greeks. In those times it was believed that milk was derived from special vessels that connected the uterus to the breasts. The "chyle theory" on the origin of milk was another prominent theory which persisted well into the nineteenth century before the realisation that milk components were derived from blood and some milk constituents were actually synthesized within the breasts. The demonstration that milk ejection was the expulsion of milk that had already been secreted and that milk secretion was a separate continuous process, set the background for the development for the current understanding of milk synthesis and secretion. Today we know that there are two stages in the initiation of lactation- secretory differentiation and secretory activation. Secretory differentiation represents the stage of pregnancy when the mammary epithelial cells differentiate into lactocytes with the capacity to synthesize unique milk constituents such as lactose. This process requires the presence of a 'lactogenic hormone complex' of the reproductive hormones, estrogen, progesterone, prolactin and some metabolic hormones. Secretory activation on the other hand, is the initiation of copious milk secretion and is associated with major changes in the concentrations of many milk constituents. The withdrawal of progesterone triggers the onset of secretory activation but prolactin, insulin and cortisol must also be present. This review describes the works of pioneers that have led to our current understanding of the biochemical and endocrinological processes involved in the initiation of human lactation. PMID:18027076

  7. Studies on the Regulation of Secretion in Clara Cells with Evidence for Chemical Nonautonomic Mediation of the Secretory Response to Increased Ventilation in Rat Lungs

    PubMed Central

    Massaro, Gloria D.; Amado, Ceferina; Clerch, Linda; Massaro, Donald

    1982-01-01

    Using electron microscopy and morphometric methods to assess secretion, we previously found that two times tidal volume ventilation of isolated perfused rat lung stimulates secretion by bronchiolar Clara cells; this effect is not prevented by ?-adrenergic blockade (J. Clin. Invest. 1981. 67: 345-351.). In this study we used the isolated perfused rat lung and the anesthetized mechanically ventilated rat, to further study the mechanism by which large tidal volumes stimulate secretion by Clara cells. With the perfused lung we found (a) ?-adrenergic inhibition did not block the secretory effect of ventilation at two times normal tidal volume; (b) indomethacin completely blocked the secretory action of two times tidal volume ventilation; (c) medium previously used to perfuse lungs ventilated at two times tidal volume, but not medium previously used to ventilate lungs at normal tidal volume, stimulated secretion by Clara cells when used to perfuse fresh lungs ventilated at tidal volume; (d) addition of prostacyclin to the fresh perfusate increased secretion by Clara cells of lungs ventilated at normal tidal volume. In anesthetized mechanically ventilated rats, sighs stimulated secretion by Clara cells; this increased secretion was inhibited by indomethacin but not by cholinergic blockade (bilateral vagotomy). These studies indicate that increased volume ventilation stimulates secretion by Clara cells in vivo and in vitro; they provide evidence that chemical nonadrenergic, noncholinergic mechanisms are involved in this secretion, and that prostaglandins may be the chemical messenger coupling the mechanico-secretory events. PMID:7050173

  8. Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells.

    PubMed

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

    2012-01-01

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

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

    PubMed

    Robinson, N; Brattsand, R; Dahlbäck, M

    1990-03-01

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

  10. Cytotoxicity of hexachlorocyclohexane isomers and cyclodienes in primary cultures of cerebellar granule cells.

    PubMed

    Rosa, R; Rodriguez-Farr, E; Sanfeliu, C

    1996-07-01

    The cytotoxicity of the neurotoxic hexachlorocyclohexane (HCH) isomers alpha, beta, gamma (lindane) and delta and of the cyclodienes aldrin, endrin and alpha-endosulfan were studied in primary neuronal cultures of cerebellar granule cells. Disruption of cell membrane integrity, as indicative of cytotoxicity, was measured by propidium iodide staining. Additionally, the effects of these xenobiotic agents on three endpoints of the cellular status, concentration of intracellular-free calcium, intracellular oxygen-free radical formation and mitochondrial transmembrane potential were analyzed in parallel cultures to understand better the mechanisms of their neurocytotoxic action. To measure these parameters, the probes of the acetoxymethyl ester of fluo-3, 2',7'-dichlorofluorescin diacetate and rhodamine 123, respectively, were used. The order of cytotoxic potency of the HCH-isomers and the cyclodienes (delta-HCH > gamma-HCH > alpha-HCH approximately equal to aldrin approximately equal to alpha-endosulfan > endrin approximately equal to beta-HCH) was very different from their in vivo LD50 order. delta-, gamma- and alpha-HCH increased the concentration of intracellular-free calcium, whereas delta- and gamma-HCH and alpha-endosulfan increased mitochondrial transmembrane potential, but none of the compounds generated oxygen-free radicals. The inhibition of delta- and gamma-HCH effects by several specific pharmacological agents suggests that delta-HCH causes its cytotoxic effects in part through intracellular Ca++ mobilization from intracellular pools sensitive to neomycin, whereas gamma-HCH acts through Ca++ influx and dantrolene-sensitive intracellular Ca++ mobilization. The use of selected endpoints of the cellular status proved to be a valuable tool to study the mechanisms of cytotoxicity. PMID:8764347

  11. Key role of mast cells and their major secretory products in inflammatory bowel disease

    PubMed Central

    He, Shao-Heng

    2004-01-01

    Historically, mast cells were known as a key cell type involved in type I hypersensitivity. Until last two decades, this cell type was recognized to be widely involved in a number of non-allergic diseases including inflammatory bowel disease (IBD). Markedly increased numbers of mast cells were observed in the mucosa of the ileum and colon of patients with IBD, which was accompanied by great changes of the content in mast cells such as dramatically increased expression of TNF-?, IL-16 and substance P. The evidence of mast cell degranulation was found in the wall of intestine from patients with IBD with immunohistochemistry technique. The highly elevated histamine and tryptase levels were detected in mucosa of patients with IBD, strongly suggesting that mast cell degranulation is involved in the pathogenesis of IBD. However, little is known of the actions of histamine, tryptase, chymase and carboxypeptidase in IBD. Over the last decade, heparin has been used to treat IBD in clinical practice. The low molecular weight heparin (LMWH) was effective as adjuvant therapy, and the patients showed good clinical and laboratory response with no serious adverse effects. The roles of PGD2, LTC4, PAF and mast cell cytokines in IBD were also discussed. Recently, a series of experiments with dispersed colon mast cells suggested there should be at least two pathways in man for mast cells to amplify their own activation-degranulation signals in an autocrine or paracrine manner. The hypothesis is that mast cell secretogogues induce mast cell degranulation, release histamine, then stimulate the adjacent mast cells or positively feedback to further stimulate its host mast cells through H1 receptor. Whereas released tryptase acts similarly to histamine, but activates mast cells through its receptor PAR-2. The connections between current anti-IBD therapies or potential therapies for IBD with mast cells were discussed, implicating further that mast cell is a key cell type that is involved in the pathogenesis of IBD. In conclusion, while pathogenesis of IBD remains unclear, the key role of mast cells in this group of diseases demonstrated in the current review implicates strongly that IBD is a mast cell associated disease. Therefore, close attentions should be paid to the role of mast cells in IBD. PMID:14760748

  12. [Rapid biosynthesis and release of 35 kD granzyme B by NK92 cells bypassing secretory lysosomes].

    PubMed

    Wang, Lei; Jiang, Shaowei; Xiao, Ling; Chen, Lin; Li, Anzheng; Zheng, Fang

    2016-02-01

    Objective To investigate the possibility of the biosynthesis and release of granzyme B (GZB) by NK92 cells bypassing the way of secretory lysosomes (SLs) and the possible mechanism. Methods As cell models, NK92 cells were activated by the phorbol myristate acetate (PMA) and ionomycin (ION). Within 4 hours following the activation, immuno- fluorescence and electron microscopy were used to detect the content and distribution of 35 000 (Mr) and 32 000 (Mr) GZB in the cytoplasm of NK92 before and after the protein synthesis was inhibited; Western blotting was performed to detect GZB inside and outside the SLs. After blocking the release of 32 000 (Mr) GZB by inhibiting the exocytosis of SLs with EDTA, we tested the content of Mr 35 000 GZB in activated NK92 supernatant. Activated NK92 cells were co-cultured with K562 cells to observe whether the Mr 35 000 GZB could enter the K562 cells. Activated NK92 cell death rate was determined and the enzyme activity of secreted Mr 35 000 GZB was examined. Results Four hours after stimulated by PMA/ION, NK92 cells generated large amount of Mr 35 000 GZB in the cytoplasm outside SLs where Mr 32 000 GZB was located. Immunoelectron microscope and immunofluorescence further approved that Mr 35 000 GZB outside SLs was located in vesicles. In addition, Mr 35 000 GZB could be secreted outside NK92 cells. Further investigation found that GZB/Serpinb9 composite and Mr 35 000 GZB could simultaneously emerge in the cytoplasm outside SLs. However, activated NK92 cell death rate did not rise. Mr 32 000 GZB inside SLs had enzyme activity in contrast with the Mr 35 000 GZB in zymogen form outside SLs, which suggested that Mr 35 000 GZB was not originated from the SLs. Conclusion The activated human NK cell lines could secreted rapidly inactive Mr 35 000 GZB outside SLs, and the GZB could enter the extracellular matrix or target cells bypassing SLs, which provides a part of the extracellular GZB. PMID:26927382

  13. Histological characterization of the special venom secretory cells in the stinger of rays in the northern waters of Persian Gulf and Oman Sea.

    PubMed

    Dehghani, Hadi; Sajjadi, Mir Masoud; Parto, Paria; Rajaian, Hamid; Mokhlesi, Amin

    2010-06-01

    Rays are common elasmobranches in the northern waters of Persian Gulf and Oman Sea that may have one or more mineralized serrated stingers on the whip-like tail. The stingers are covered by epidermal cells among which some can produce venom. When these animals are dorsally touched, the stinger can be introduced into the aggressor by a whip reflex mechanism of the tail when the pectoral fins are touched, causing severe mechanical injuries and inoculating the venom. The exact localization of the venom secretory cells in the stinger of different species is controversial, but it is known that the cells are preferentially located in the ventro-lateral grooves in marine stingrays. A comparative morphological characterization of the stinger epidermal tissue of different ray species in the northern part of Persian Gulf and Oman Sea was carried out in this study. EDTA was used for decalcification of stings and conventional histological processes were subsequently employed. The results indicated that structure of dermis and epidermis layers of stings in all species are similar to the structure of corresponding layers in other parts of fish's body. The results of the present study have shown that all examined species of Dasyatidae family, but not Myliobatidae and Gymnuridae families, had venom secretory cells. Distribution of venom secretory cells varies in each species and is often located around or inside the stinger ventro-lateral grooves. These differences among the stingers of various species may explain the envenomation severity in these species. PMID:20080118

  14. Exocytosis of Secretory Organelles from Blood Platelets Incubated with Cationic Polypeptides

    PubMed Central

    White, James G.

    1972-01-01

    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

  15. 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

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

    1995-01-01

    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

  16. Cell adhesion, ammonia removal and granulation of autotrophic nitrifying sludge facilitated by N-acyl-homoserine lactones.

    PubMed

    Li, An-Jie; Hou, Bao-Lian; Li, Mei-Xi

    2015-11-01

    In this study, six N-acyl-homoserine lactone (AHL) molecules (C6-HSL, C8-HSL, C10-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL and 3-oxo-C10-HSL) were each dosed into a bioreactor and seeded using autotrophic nitrifying sludge (ANS). The effects of the AHLs on cell adhesion, nitrification and sludge granulation were investigated. The results indicated that the efficiencies of cell adhesion and ammonia removal both had a close correlation with the side chain length and ? position substituent group of the AHLs. The best-performing AHL in terms of accelerating bacterial attached-growth was 3-oxo-C6-HSL, whereas C6-HSL outperformed the others in terms of the ammonia degradation rate. The addition of 3-oxo-C6-HSL or C6-HSL increased the biomass growth rate, microbial activity, extracellular proteins and nitrifying bacteria, which can accelerate the formation of nitrifying granules. Consequently, selecting AHL molecules that could improve bacteria in attached-growth mode and nitrification efficiency simultaneously will most likely facilitate the rapid granulation of nitrifying sludge. PMID:26295441

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

    PubMed Central

    2013-01-01

    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

  18. Retrograde endocannabinoid regulation of GABAergic inhibition in the rat dentate gyrus granule cell.

    PubMed

    Isokawa, Masako; Alger, Bradley E

    2005-09-15

    The dentate gyrus is a key input gateway for the hippocampus, and dentate function is potently regulated by GABAergic inhibition. GABAergic inhibition is plastic and modulated by many factors. Cytoplasmic calcium ([Ca(+)](i)) is one of these factors, and its elevation inhibits GABA-mediated transmission in the hippocampus including the dentate gyrus granule cells (DGCs). We examined whether the [Ca(+)](i)-dependent decrease of GABA(A) receptor-mediated inhibitory postsynaptic current (IPSC) is explained by the retrograde suppression of GABA release caused by the depolarization-induced elevation of [Ca(+)](i) in DGCs (DSI: depolarization-induced suppression of inhibition). Repeated brief depolarizations or a single long depolarization inhibited spontaneous IPSCs with amplitudes over 25 pA for up to a minute, and reduced the amplitude of IPSCs evoked by direct stimulation in the molecular layer, suggesting that DGCs are susceptible to DSI. The magnitude of DSI correlated linearly with the duration of depolarization, and so did the increase of [Ca(+)](i). DSI was blocked by intrapipette application of BAPTA. In addition, bath application of thapsigargin and ryanodine, and intrapipette application of ryanodine and ruthenium red reduced the [Ca(+)](i) increase caused by the DSI-inducing depolarization, and substantially reduced the magnitude of DSI. Finally, the cannabinoid receptor agonists, CP55,942 and WIN55,212-2, mimicked DSI and prevented further IPSC reduction by DSI. DSI was blocked by the antagonist, SR141716A. We conclude that GABAergic inhibition in DGCs is subject to endogenous cannabinoid (eCB)-mediated retrograde regulation, and this process involves a depolarization-initiated release of Ca(+) from ryanodine-sensitive stores. Our findings suggest eCBs probably have physiological functions in the regulation of GABAergic plasticity in the dentate gyrus. PMID:16037085

  19. Excretory/Secretory-Products of Echinococcus multilocularis Larvae Induce Apoptosis and Tolerogenic Properties in Dendritic Cells In Vitro

    PubMed Central

    Nono, Justin Komguep; Pletinckx, Katrien; Lutz, Manfred B.; Brehm, Klaus

    2012-01-01

    Background Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E.multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action. Methodology/Principal Findings We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and nave T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro. Conclusions This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis. PMID:22363826

  20. From Beaumont to poison ivy: marine sponge cell aggregation and the secretory basis of inflammation.

    PubMed

    Dunham, P B; Vosshall, L B; Bayer, C A; Rich, A M; Weissmann, G

    1985-11-01

    We have studied Microciona prolifera cells as a model for inflammation and secretion. Dissociated in Ca-, Mg-free seawater with 2.5 mM EDTA, the cells aggregate when exposed to Ca (greater than 5 mM) and Ca ionophores. Extracellular Ca is not required over the course of aggregation; brief pulses of Ca suffice. Aggregation was induced by A23187 in excess EDTA after cells were prepared by pulse Ca. It appeared that Ca ionophore stimulated the secretion of Microciona aggregation factor (MAF) to a locus or in a form inaccessible to external EDTA. Pulse-induced aggregation depended on MAF because it was inhibited by MAF fragments, which are ligands for MAF-binding sites. Sponge cells were preloaded with three fluorescent dyes that monitor aspects of stimulus-secretion coupling: 1) 3,3'-dipropylthiadicarbocyanine iodide (dis-C3-(5)), a carbocyanine dye presumed to report changes in membrane potential; 2) 9-aminoacridine (9AA), which presumably reports secretion from acid vesicles; and 3) chlortetracycline (CTC), presumed to report mobilization of membrane-associated Ca. Exposure of cells either to constant Ca or to pulse Ca stimuli caused prompt decreases in the fluorescence of cells with diS-C3-(5) and increases in fluorescence of cells with 9AA. In contrast, although constant Ca provoked decreases in fluorescence of cells with CTC, a pulse Ca was without effect. Moreover, inhibitors of stimulus-response coupling (e.g., aspirin, sodium salicylate, 5 mM; diclofenac, 100 microM) inhibited sponge aggregation induced by either constant or pulse stimuli. In contrast, like the endogenous mediator of inflammation, leukotriene B4, trienoic alkyl catechols (urushiol) from poison ivy provoked aggregation. These studies suggest the utility of this marine model for analysis of stimulus-response coupling in cells of higher species that also respond to secretagogues in the absence of external Ca. PMID:3932096

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

    PubMed Central

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

    2012-01-01

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

  2. Differential expression of markers for endothelial cells, pericytes, and basal lamina in the microvasculature of tumors and granulation tissue.

    PubMed Central

    Schlingemann, R. O.; Rietveld, F. J.; Kwaspen, F.; van de Kerkhof, P. C.; de Waal, R. M.; Ruiter, D. J.

    1991-01-01

    The structure and function of the tumor microvasculature is of great interest for cancer biology, diagnosis, and therapy. The distribution of endothelial cells, pericytes, and basal lamina in tumors is not well documented. In this study, the authors investigated the distribution of markers for these different components in a series of malignant human tumors and in human granulation tissue, both situations with extensive angiogenesis. Their results show a striking heterogeneity in the expression of markers for pericytes and endothelial cells between different tumors, but also within a single tumor lesion. To be able to distinguish between these two adjacent cell types decisively, all marker studies were carried out both on the light and the electron microscopical level and compared with staining results in granulation tissue of cutaneous wounds in healthy volunteers and of decubitus lesions. In granulation tissue of decubitus lesions, well-defined zones with increasing levels of maturation can be delineated. It was found that antibodies recognizing von Willebrand factor often failed to stain the tumor capillaries. Of the pericyte markers, alpha-smooth muscle actin was only locally expressed by pericytes in the tumor vasculature, whereas the high-molecular-weight melanoma-associated antigen, a chondroitin sulfate proteoglycan, stained the microvasculature broadly. Staining of the basal lamina components collagen type IV and laminin was, within the tumor, not restricted to the microvasculature. From their findings the authors conclude that 1) for the visualization of the tumor vasculature, antibodies recognizing endothelial markers, especially monoclonal antibodies PAL-E and BMA 120, are preferable to those recognizing pericytes or basal lamina; 2) within the microvasculature of tumors and granulation tissue, a heterogeneity of expression of endothelial and pericyte markers is observed; 3) during the formation of granulation tissue, all three microvascular components can be demonstrated already in the histologically earliest stage, suggesting not only an involvement of endothelial cells but also of pericytes and basal lamina in the initial steps of angiogenesis in wound healing. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 PMID:1711288

  3. Antioxidants J811 and 17beta-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death.

    PubMed

    Dar, E; Gtz, M E; Zhivotovsky, B; Manzo, L; Ceccatelli, S

    2000-11-15

    Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methylmercury (MeHg), a well-known neurotoxicant contaminating the environment. In the present study we report that doses of MeHg ranging from 0.1 microM to 1.5 microM activated apoptosis, as shown by cell shrinkage, nuclear condensation, and formation of high-molecular-weight DNA fragments. Nevertheless, caspase-3-like activity was not significantly induced, and the broad caspase inhibitor Z-VAD-FMK was not capable of protecting the cells. This argues for a minor role of caspases in the intracellular pathways leading to MeHg-induced cell death in CGC. Instead, proteolytic fragments obtained by specific calpain cleavage of procaspase-3 and alpha-fodrin were increased consistently in samples exposed to MeHg, pointing to a substantial activation of calpain. Notably, two antioxidants, 17beta-estradiol (10 microM) and the Delta(8,9)-dehydro derivative of 17alpha-estradiol J811 (10 microM), protected from MeHg damage, preventing morphological alterations, chromatin fragmentation, and activation of calpain. These findings underscore the key role of oxidative stress in MeHg toxicity, placing it upstream of calpain activation. The shielding effect of the 17beta-estradiol and the radical scavenger J811 is potentially relevant for the development of therapeutic strategies for MeHg intoxication. PMID:11070499

  4. Investigation into the Role of Phosphatidylserine in Modifying the Susceptibility of Human Lymphocytes to Secretory Phospholipase A2 using Cells Deficient in the Expression of Scramblase

    PubMed Central

    Nelson, Jennifer; Francom, Lyndee L.; Anderson, Lynn; Damm, Kelly; Baker, Ryan; Chen, Joseph; Franklin, Sarah; Hamaker, Amy; Izidoro, Izadora; Moss, Eric; Orton, Mikayla; Stevens, Evan; Yeung, Celestine; Judd, Allan M.; Bell, John D.

    2012-01-01

    Summary Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitts lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. PMID:22266334

  5. Phosducin regulates secretory activity in TT line of thyroid parafollicular C cells.

    PubMed

    Piotrowska, U; Adler, G; Kozicki, I

    2015-02-01

    The endocrine activity of the thyroid gland is accomplished by its follicular and parafollicular cells. In these cells, numerous G proteins-dependent pathways are active and potentially could be regulated by a 33-kDa cytoplasmic protein phosducin, which interacts with the Gβ subunit and may compete with Gα or Gβγ 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

  6. Regulation of renin processing and secretion: chemiosmotic control and novel secretory pathway.

    PubMed

    King, J A; Lush, D J; Fray, J C

    1993-08-01

    The renin-angiotensin-aldosterone system (RAAS) plays an important role in cardiovascular and electrolyte regulation in health and disease. Juxtaglomerular cells in the kidney regulate endocrine RAAS by physiologically controlling conversion of prorenin and secretion of renin. The classical baroceptor, neurogenic, and macula densa mechanisms regulate renin expression at the cellular level by Ca2+, adenosine 3',5'-cyclic monophosphate (cAMP), and chemiosmotic forces (K+, Cl-, and water flux coupled to H+ movement). The baroceptor mechanism (through Ca2+) activates K+ and Cl- channels in the surface membrane and deactivates a KCl-H+ exchange chemiosmotic transporter in the secretory granular membrane. The neurogenic mechanism (through cAMP) promotes prorenin processing to renin. The macula densa mechanism (through K+ and Cl-) involves the processing of prorenin to renin. Ca2+, by inhibiting the KCl-H+ exchange transporter, prevents secretory granules from engaging in chemiosmotically mediated exocytosis. cAMP, on the other hand, by stimulating H+ influx, provides the acidic granular environment for prorenin processing to renin. It is concluded that, in the presence of a favorable chemiosmotic environment, prorenin is processed to renin, which may then be secreted by regulative degranulation or divergence translocation, a novel secretory pathway used by several secretory proteins, including renin. PMID:7690183

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

    PubMed

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

    2015-11-01

    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

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

    PubMed Central

    Das, Subhendu; Pellett, Philip E.

    2011-01-01

    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

  9. The Secretory System of Arabidopsis

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

    Edwards, I. J.; Wagner, W. D.; Owens, R. T.

    1990-01-01

    Arterial dermatan sulfate proteoglycan has been shown to increase with atherosclerosis progression, but factors responsible for this increase are unknown. To test the hypothesis that smooth muscle cell proteoglycan synthesis may be modified by macrophage products, pigeon arterial smooth muscle cells were exposed to the media of either cholesteryl ester-loaded pigeon peritoneal macrophages or a macrophage cell line P388D1. Proteoglycans radiolabeled with [35S]sulfate and [3H]serine were isolated from culture media and smooth muscle cells and purified following precipitation with 1-hexadecylpyridinium chloride and chromatography. Increasing concentrations of macrophage-conditioned media were associated with a dose-response increase in [35S]sulfate incorporation into secreted proteoglycans, but there was no change in cell-associated proteoglycans. Incorporation of [3H]serine into total proteoglycan core proteins was not significantly different (5.2 X 10(5) dpm and 5.5 X 10(5) disintegrations per minute (dpm) in control and conditioned media-treated cultures, respectively), but selective effects were observed on individual proteoglycan types. Twofold increases in dermatan sulfate proteoglycan and limited degradation of chondroitin sulfate proteoglycan were apparent based on core proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoinhibition studies indicated that interleukin-1 was involved in the modulation of proteoglycan synthesis by macrophage-conditioned media. These data provide support for the role of macrophages in alteration of the matrix proteoglycans synthesized by smooth muscle cells and provide a mechanism to account for the reported increased dermatan sulfate/chondroitin sulfate ratios in the developing atherosclerotic lesion. Images Figure 6 PMID:2316626

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

    SciTech Connect

    Edwards, I.J.; Wagner, W.D.; Owens, R.T. )

    1990-03-01

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

  12. Proteomic analysis reveals that MAEL, a component of nuage, interacts with stress granule proteins in cancer cells.

    PubMed

    Yuan, Liqin; Xiao, Yuzhong; Zhou, Qiuzhi; Yuan, Dongmei; Wu, Baiping; Chen, Gannong; Zhou, Jianlin

    2014-01-01

    The Maelstrom (MAEL) gene is a cancer-testis (or cancer-germline) gene, which is predominantly expressed in germline cells under normal conditions, but is aberrantly expressed in a range of human cancer cells. In germline cells, MAEL is found predominantly in the nuage, where it plays an essential role in piRNA biogenesis and piRNA-mediated silencing of transposons. However, the role of MAEL in cancer has not been elucidated. We performed immunoprecipitation and Nano-LC-MS/MS analysis to investigate the interactome of MAEL, and identified 14 components of stress granules (SGs) as potential binding partners of MAEL in MDA-MB-231 human breast cancer and SW480 colorectal cancer cells. The interactions between MAEL and 8 of these SG components (PABPC1, YBX1, KHSRP, SYNCRIP, DDX39, ELAV1, EIF4A1 and EIF3F) were confirmed by anti-tag immunoprecipitation. Immunofluorescence analysis showed that MAEL co-localizes with the SG marker PABPC1 in SGs during oxidative stress. Nuages and SGs are the cytoplasmic RNA granules of germline cells and stressed somatic cells, respectively, and both serve as a platform for small RNA-mediated gene silencing. It is, therefore, suggested that MAEL may be involved in miRNA-mediated gene silencing in SGs, as it does in the nuage. This finding should be valuable toward understanding the function of MAEL in carcinogenesis. PMID:24189637

  13. Cell Division Mode Change Mediates the Regulation of Cerebellar Granule Neurogenesis Controlled by the Sonic Hedgehog Signaling

    PubMed Central

    Yang, Rong; Wang, Minglei; Wang, Jia; Huang, Xingxu; Yang, Ru; Gao, Wei-Qiang

    2015-01-01

    Summary Symmetric and asymmetric divisions are important for self-renewal and differentiation of stem cells during neurogenesis. Although cerebellar granule neurogenesis is controlled by sonic hedgehog (SHH) signaling, whether and how this process is mediated by regulation of cell division modes have not been determined. Here, using time-lapse imaging and cell culture from neuronal progenitor-specific and differentiated neuron-specific reporter mouse lines (Math1-GFP and Dcx-DsRed) and Patched+/− mice in which SHH signaling is activated, we find evidence for the existence of symmetric and asymmetric divisions that are closely associated with progenitor proliferation and differentiation. While activation of the SHH pathway enhances symmetric progenitor cell divisions, blockade of the SHH pathway reverses the cell division mode change in Math1-GFP;Dcx-DsRed;Patched+/− mice by promoting asymmetric divisions or terminal neuronal symmetric divisions. Thus, cell division mode change mediates the regulation of cerebellar granule neurogenesis controlled by SHH signaling. PMID:26527387

  14. Histochemical and Ultrastructural Modification of Mucosal Mast Cell Granules in Parasitized Mice Lacking the ?-Chymase, Mouse Mast Cell Protease-1

    PubMed Central

    Wastling, Jonathan M.; Knight, Pamela; Ure, Jan; Wright, Steven; Thornton, Elisabeth M.; Scudamore, Cheryl L.; Mason, John; Smith, Austin; Miller, Hugh R. P.

    1998-01-01

    The soluble ?-chymases mouse mast cell protease-1 (mMCP-1) and rat mast cell protease-II are predominantly expressed by intestinal mucosal mast cells (IMMCs) and may promote mucosal epithelial permeability when released during intestinal allergic hypersensitivity responses. To study the function of these chymases, we generated mice with a homozygous null mutation of the mMCP-1 gene and investigated their response to infection with the intestinal nematode Nippostrongylus brasiliensis. Whereas mMCP-2, -4, and -5 were transcribed normally, there was no transcription of the mMCP-1 gene in null (?/?) mice, nor was mature mMCP-1 protein detected in (?/?) jejunal mucosa. In contrast, levels of mMCP-1 in wild-type (+/+) jejunal mucosa increased 200- to 350-fold from 0.66 ?g mMCP-1/g wet weight in uninfected mice to 129 and 229 ?g/g wet weight on days 8 and 10 of infection, respectively. The kinetics of IMMC recruitment differed in ?/? mice compared with +/+ controls on days 8 (P < 0.05) and 10 (P < 0.03) of infection. The IMMCs in infected ?/? mice stained poorly, if at all, for esterase with naphthol AS-D chloroacetate compared with the intense staining observed in +/+ controls. Ultrastructurally, the prominent crystal intragranular structures that are found in intraepithelial +/+ IMMCs were absent from ?/? IMMCs. These data show that disruption of the mMCP-1 gene leads to profound histochemical and ultrastructural changes in IMMC granules. PMID:9708809

  15. Bisphenol A modulates receptivity and secretory function of human decidual cells: an in vitro study.

    PubMed

    Mannelli, Chiara; Szstek, Anna Z; Lukasik, Karolina; Carotenuto, Claudiopietro; Ietta, Francesca; Romagnoli, Roberta; Ferretti, Cristina; Paulesu, Luana; Wo?czynski, Slawomir; Skarzynski, Dariusz Jan

    2015-08-01

    The human endometrium is a fertility-determining tissue and a target of steroid hormones' action. Endocrine disruptors (EDs) can exert adverse effects on the physiological function of the decidua at the maternal-fetal interface. We examined the potential effects of an ED, bisphenol A (BPA), on endometrial maturation/decidualization, receptivity, and secretion of decidual factors (biomarkers). In vitro decidualized, endometrial stromal cells from six hysterectomy specimens were treated with 1? pM-1 ??M of BPA, for 24 ?h and assessed for cell viability and proliferation. Three non-toxic concentrations of BPA (1 ??M, 1 ?nM, and 1 ?pM) were selected to study its influence on secretion of cell decidualization biomarkers (IGF-binding protein and decidual prolactin (dPRL)), macrophage migration inhibitory factor (MIF) secretion, and hormone receptors' expression (estrogen receptors (ER? and ER?); progesterone receptors (PRA and PRB); and human chorionic gonadotropin (hCG)/LH receptor (LH-R)). The results showed a decrease in cell viability (P<0.001) in response to BPA at the level of 1? mM. At the non-toxic concentrations used, BPA perturbed the expression of ER?, ER?, PRA, PRB, and hCG/LH-R (P<0.05). Furthermore, 1 ??M of BPA reduced the mRNA transcription of dPRL (P<0.05). Secretion of MIF was stimulated by all BPA treatments, the lowest concentration (1 ?pM) being the most effective (P<0.001). The multi-targeted disruption of BPA on decidual cells, at concentrations commonly detected in the human population, raises great concern about the possible consequences of exposure to BPA on the function of decidua and thus its potential deleterious effect on pregnancy. PMID:26021997

  16. Complementary postsynaptic activity patterns elicited in olfactory bulb by stimulation of mitral/tufted and centrifugal fiber inputs to granule cells.

    PubMed

    Laaris, Nora; Puche, Adam; Ennis, Matthew

    2007-01-01

    Main olfactory bulb (MOB) granule cells receive spatially segregated glutamatergic synaptic inputs from the dendrites of mitral/tufted cells as well as from the axons of centrifugal fibers (CFFs) originating in olfactory cortical areas. Dendrodendritic synapses from mitral/tufted cells occur on granule cell distal dendrites in the external plexiform layer (EPL), whereas CFFs preferentially target the somata/proximal dendrites of granule cells in the granule cell layer (GCL). In the present study, tract tracing, and recordings of field potentials and voltage-sensitive dye optical signals were used to map activity patterns elicited by activation of these two inputs to granule cells in mouse olfactory bulb slices. Stimulation of the lateral olfactory tract (LOT) produced a negative field potential in the EPL and a positivity in the GCL. CFF stimulation produced field potentials of opposite polarity in the EPL and GCL to those elicited by LOT. LOT-evoked optical signals appeared in the EPL and spread subsequently to deeper layers, whereas CFF-evoked responses appeared in the GCL and then spread superficially. Evoked responses were reduced by N-methyl-d-aspartate (NMDA) receptor antagonists and completely suppressed by AMPA receptor antagonists. Reduction of extracellular Mg(2+) enhanced the strength and spatiotemporal extent of the evoked responses. These and additional findings indicate that LOT- and CFF-evoked field potentials and optical signals reflect postsynaptic activity in granule cells, with moderate NMDA and dominant AMPA receptor components. Taken together, these results demonstrate that LOT and CFF stimulation in MOB slices selectively activate glutamatergic inputs to the distal dendrites versus somata/proximal dendrites of granule cells. PMID:17035366

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

    PubMed Central

    Laaris, Nora; Puche, Adam; Ennis, Matthew

    2009-01-01

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

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

    SciTech Connect

    Arvan, P.; Castle, J.D.

    1986-10-01

    Secretion granules have been isolated from the parotid glands of rats that have been chronically stimulated with the ..beta..-adrenergic agonist, isoproterenol. These granules are of interest because they package a quantitatively different set of secretory proteins in comparison with granules from the normal gland. Polypeptides enriched in proline, glycine, and glutamine, which are known to have pI's >10, replace ..cap alpha..-amylase (pI's = 6.8) as the principal content species. The internal pH of granules from the treated rats changes from 7.8 in a potassium sulfate medium to 6.9 in a choline chloride medium. The increased pH over that of normal parotid granules (approx.6.8) appears to protect the change in composition of the secretory contents. Whereas normal mature parotide granules have practically negligible levels of H/sup +/ pumping ATPase activity, the isolated granules from isoproterenol-treated rats undergo a time-dependent internal acidification that requires the presence of ATP and is abolished by an H/sup +/ ionophore. Additionally, an inside-positive granule transmembrane potential develops after ATP addition that depends upon ATP hydrolysis. Two independent methods have been used that exclude the possibility that contaminating organelles are the source of the H/sup +/-ATPase activity. Together these data provide clear evidence for the presence of an H/sup +/ pump in the membranes of parotid granules from chronically stimulated rats. However, despite the presence of H/sup +/-pump activity, fluorescence microscopy with the weak base, acridine orange, reveals that the intragranular pH in live cells is greater than that of the cytoplasm.

  19. Melanoma cell lysosome secretory burst neutralizes the CTL-mediated cytotoxicity at the lytic synapse.

    PubMed

    Khazen, Roxana; Müller, Sabina; Gaudenzio, Nicolas; Espinosa, Eric; Puissegur, Marie-Pierre; Valitutti, Salvatore

    2016-01-01

    Human melanoma cells express various tumour antigens that are recognized by CD8(+) cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that, on conjugation with CTL, human melanoma cells undergo an active late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance, we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. PMID:26940455

  20. Melanoma cell lysosome secretory burst neutralizes the CTL-mediated cytotoxicity at the lytic synapse

    PubMed Central

    Khazen, Roxana; Müller, Sabina; Gaudenzio, Nicolas; Espinosa, Eric; Puissegur, Marie-Pierre; Valitutti, Salvatore

    2016-01-01

    Human melanoma cells express various tumour antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that, on conjugation with CTL, human melanoma cells undergo an active late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance, we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. PMID:26940455

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

    PubMed

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

    2015-06-01

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

  2. Transcriptional Profiling of Newly Generated Dentate Granule Cells Using TU Tagging Reveals Pattern Shifts in Gene Expression during Circuit Integration1,2

    PubMed Central

    Chatzi, Christina; Shen, Rongkun; Goodman, Richard H.

    2016-01-01

    Abstract Despite representing only a small fraction of hippocampal granule cells, adult-generated newborn granule cells have been implicated in learning and memory (Aimone et al., 2011). Newborn granule cells undergo functional maturation and circuit integration over a period of weeks. However, it is difficult to assess the accompanying gene expression profiles in vivo with high spatial and temporal resolution using traditional methods. Here we used a novel method [“thiouracil (TU) tagging”] to map the profiles of nascent mRNAs in mouse immature newborn granule cells compared with mature granule cells. We targeted a nonmammalian uracil salvage enzyme, uracil phosphoribosyltransferase, to newborn neurons and mature granule cells using retroviral and lentiviral constructs, respectively. Subsequent injection of 4-TU tagged nascent RNAs for analysis by RNA sequencing. Several hundred genes were significantly enhanced in the retroviral dataset compared with the lentiviral dataset. We compared a selection of the enriched genes with steady-state levels of mRNAs using quantitative PCR. Ontology analysis revealed distinct patterns of nascent mRNA expression, with newly generated immature neurons showing enhanced expression for genes involved in synaptic function, and neural differentiation and development, as well as genes not previously associated with granule cell maturation. Surprisingly, the nascent mRNAs enriched in mature cells were related to energy homeostasis and metabolism, presumably indicative of the increased energy demands of synaptic transmission and their complex dendritic architecture. The high spatial and temporal resolution of our modified TU-tagging method provides a foundation for comparison with steady-state RNA analyses by traditional transcriptomic approaches in defining the functional roles of newborn neurons. PMID:27011954

  3. A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype

    PubMed Central

    Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

    2015-01-01

    DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance. PMID:26503466

  4. A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype.

    PubMed

    Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

    2015-11-24

    DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance. PMID:26503466

  5. Two distinct secretory vesiclepriming steps in adrenal chromaffin cells

    PubMed Central

    Liu, Yuanyuan; Schirra, Claudia; Edelmann, Ludwig; Matti, Ulf; Rhee, JeongSeop; Hof, Detlef; Bruns, Dieter; Brose, Nils; Rieger, Heiko; Stevens, David R.

    2010-01-01

    Priming of large dense-core vesicles (LDCVs) is a Ca2+-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca2+ concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process. PMID:20855507

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

    PubMed Central

    Barlowe, Charles K.; Miller, Elizabeth A.

    2013-01-01

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

  7. Stimulation of the N-methyl-D-aspartate receptor has a trophic effect on differentiating cerebellar granule cells.

    PubMed

    Balázs, R; Hack, N; Jørgensen, O S

    1988-04-22

    N-methyl-D-aspartate (NMDA) supplementation of cerebellar cultures enriched in granule neurones (about 90%) prevented the extensive cell loss which occurs when cultivation takes place, in serum containing media, in the presence of 'low' K+ (5-15 mM). Estimation of tetanus toxin receptors and N-CAM contents indicated that NMDA rescued primarily nerve cells. The influence of NMDA in promoting cell survival was blocked by the receptor antagonist, 2-amino-5-phosphonovalerate. The effect depended both on the concentration of NMDA and on the degree of depolarization of cells, the affinity in the presence of 15 mM K+ being similar to that of NMDA receptor binding. The results attest a new role for excitatory amino acid transmitters by showing that they can exert a stage-dependent trophic action on developing nerve cells. PMID:2837687

  8. Stimulation of the N-methyl-D-aspartate receptor has a trophic effect on differentiating cerebellar granule cells.

    TOXLINE Toxicology Bibliographic Information

    Balázs R; Hack N; Jørgensen OS

    1988-04-22

    N-methyl-D-aspartate (NMDA) supplementation of cerebellar cultures enriched in granule neurones (about 90%) prevented the extensive cell loss which occurs when cultivation takes place, in serum containing media, in the presence of 'low' K+ (5-15 mM). Estimation of tetanus toxin receptors and N-CAM contents indicated that NMDA rescued primarily nerve cells. The influence of NMDA in promoting cell survival was blocked by the receptor antagonist, 2-amino-5-phosphonovalerate. The effect depended both on the concentration of NMDA and on the degree of depolarization of cells, the affinity in the presence of 15 mM K+ being similar to that of NMDA receptor binding. The results attest a new role for excitatory amino acid transmitters by showing that they can exert a stage-dependent trophic action on developing nerve cells.

  9. Chronic ethanol administration decreases phosphorylation of cyclic AMP response element-binding protein in granule cells of rat cerebellum.

    PubMed

    Yang, X; Horn, K; Baraban, J M; Wand, G S

    1998-01-01

    To help define the molecular basis of ethanol's actions on the nervous system, we have in previous studies demonstrated that ethanol administration triggers a robust increase in cyclic AMP-response element-binding protein (CREB) phosphorylation in the cerebellum. The purpose of the present study was to compare the effects of acute and chronic ethanol exposure on the phosphorylation of CREB in rat cerebellum and to determine which cell types in the cerebellum display this response to ethanol. An acute ethanol challenge (3.0 g/kg of body weight) induced a rapid increase in content of the phosphorylated form of CREB, peaking at 30 min and declining to basal levels within 2 h. Immunocytochemical studies revealed prominent ethanol-induced changes in phosphoCREB in the granule cell layer, with little phosphoCREB apparent in Purkinje cells. Following chronic ethanol exposure (5 weeks), induction of CREB phosphorylation by a subsequent acute ethanol challenge was markedly attenuated. The attenuation in CREB phosphorylation was associated with a significant reduction in the levels of the catalytic unit of protein kinase A and calcium/calmodulin-dependent protein kinase IV. In summary, induction of CREB phosphorylation in cerebellum is most prominent in the granule cell layer. Neuroadaptation to chronic ethanol exposure includes a reduction in nuclear protein kinase A and calcium/calmodulin-dependent protein kinase IV levels, an event associated with impaired CREB phosphorylation. PMID:9422366

  10. Endothelin-1 stimulates the release of preloaded ( sup 3 H)D-aspartate from cultured cerebellar granule cells

    SciTech Connect

    Lin, W.W.; Lee, C.Y.; Chuang, D.M. )

    1990-03-16

    We have recently reported that endothelin-1 (ET) induces phosphoinositide hydrolysis in primary cultures of rat cerebellar granule cells. Here we found that ET in a dose-dependent manner (1-30 nM) stimulated the release of preloaded ({sup 3}H)D-aspartate from granule cells. The ET-induced aspartate release was completely blocked in the absence of extracellular Ca{sup 2+}, but was unaffected by 1 mM Co{sup 2+} or 1 microM dihydropyridine derivatives (nisoldipine and nimodipine). At higher concentration (10 microM) of nisoldipine and nimodipine, the release was partially inhibited. Short-term pretreatment of cells with phorbol 12,13-dibutyrate (PDBu) potentiated the ET-induced aspartate release, while long-term pretreatment with PDBu attenuated the release. Long-term exposure of cells to pertussis toxin (PTX), on the other hand, potentiated the ET-induced effects. Our results suggest that ET has a neuromodulatory function in the central nervous system.

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

    SciTech Connect

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

    2009-10-15

    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.

  12. Interactions between Inhibitory Interneurons and Excitatory Associational Circuitry in Determining Spatio-Temporal Dynamics of Hippocampal Dentate Granule Cells: A Large-Scale Computational Study

    PubMed Central

    Hendrickson, Phillip J.; Yu, Gene J.; Song, Dong; Berger, Theodore W.

    2015-01-01

    This paper reports on findings from a million-cell granule cell model of the rat dentate gyrus that was used to explore the contributions of local interneuronal and associational circuits to network-level activity. The model contains experimentally derived morphological parameters for granule cells, which each contain approximately 200 compartments, and biophysical parameters for granule cells, basket cells, and mossy cells that were based both on electrophysiological data and previously published models. Synaptic input to cells in the model consisted of glutamatergic AMPA-like EPSPs and GABAergic-like IPSPs from excitatory and inhibitory neurons, respectively. The main source of input to the model was from layer II entorhinal cortical neurons. Network connectivity was constrained by the topography of the system, and was derived from axonal transport studies, which provided details about the spatial spread of axonal terminal fields, as well as how subregions of the medial and lateral entorhinal cortices project to subregions of the dentate gyrus. Results of this study show that strong feedback inhibition from the basket cell population can cause high-frequency rhythmicity in granule cells, while the strength of feedforward inhibition serves to scale the total amount of granule cell activity. Results furthermore show that the topography of local interneuronal circuits can have just as strong an impact on the development of spatio-temporal clusters in the granule cell population as the perforant path topography does, both sharpening existing clusters and introducing new ones with a greater spatial extent. Finally, results show that the interactions between the inhibitory and associational loops can cause high frequency oscillations that are modulated by a low-frequency oscillatory signal. These results serve to further illustrate the importance of topographical constraints on a global signal processing feature of a neural network, while also illustrating how rich spatio-temporal and oscillatory dynamics can evolve from a relatively small number of interacting local circuits. PMID:26635545

  13. Production of specific-protein secretion granules by fat body cells of the blowfly, Calliphora erythrocephala. Substitution of an ovarian key factor by beta-ecdysone.

    PubMed

    Thomsen, E; Thomsen, M

    1978-10-01

    During the first four days of the imaginal stage the fat cells of ovariectomized females of Calliphora develop a protein synthetic apparatus, and produce dense bodies (lysosomes) as do the fat cells of normal females, but apparently they cannot synthesize the protein secretion granules that characterize the productive phase of the fat cells of normal females and that we believe to represent vitellogenin. Injection of ovariectomized females with beta-ecdysone restored the ability of the fat cells to produce the secretion granules. It is suggested that the ovary gives off a factor which induces the production of the protein secretion granules by the fat cells, and that the factor from the ovary can be substituted by beta-ecdysone. This, we believe, is the first ultrastructural evidence for an effect of the ovary and of beta-ecdysone on the synthesis of specific protein. PMID:719713

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

    PubMed

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

    2004-12-01

    Thrombopoietin (TPO) is known to be involved in megakariocytopoesis, but its role in the control of ovarian function is unknown. The aims of this study were to determine whether TPO can regulate the proliferation, apoptosis and secretory activity of ovarian cells, to identify possible intracellular mediators of TPO action, especially protein kinase A (PKA), and to define their interrelationships within ovarian cells. We investigated the effect of TPO treatment (0, 1, 10 or 100 ng/ml) on the following characteristics of cultured porcine ovarian follicles, determined using SDS-PAGE and Western blotting, immunocytochemistry, RIA and ELISA: the expression of intracellular peptides associated with proliferation (PCNA), apoptosis (Bax), tyrosine kinase (TK, phosphotyrosine), Cdc2/p34 kinase, PKA and the transcription factor CREB-1, and the secretion of progesterone, androstenedione, estradiol-17beta, oxytocin, inhibin A, inhibin B, IGF-I, transforming growth factor-2beta (TGF-2beta) and IGF-binding protein 3 (IGFBP-3). The involvement of PKA-dependent pathways was examined by evaluating the effect of a PKA blocker (KT5720, 1 microg/ml), either alone or in combination with TPO, on the parameters listed above. A TPO-induced increase in expression of PCNA, Bax, PKA, TK, Cdc2/p34 and CREB was observed. Furthermore, TPO was able to inhibit androstenedione, estradiol, TGF-2beta and IGFBP-3 secretion, and to stimulate oxytocin, inhibin A, inhibin B and IGF-I secretion. Progesterone secretion was not stimulated. The PKA blocker KT5720, when given alone, reduced the expression of Bax and TGF-2beta, augmented the expression of PKA, CREB and oxytocin, but did not influence the secretion of progesterone, androstenedione, estradiol, IGFBP-3, inhibins A and B or IGF-I. When given together with TPO, the PKA blocker prevented or reversed the action of TPO on PKA, CREB, androstenedione, estradiol, IGFBP-3, oxytocin, but not its effect on Bax, TGF-2beta or inhibin B. On the other hand, treatment with KT5720 augmented the effect of TPO on progesterone, inhibin A and IGF-I. These results provide the first evidence that TPO may be a potent regulator of ovarian function (e.g. proliferation, apoptosis and the secretion of peptide hormones, steroids, growth factors and growth factor-binding protein, as well as of the expression of some intracellular messengers). Furthermore, they demonstrated the importance of PKA in controlling these functions and in mediating the effects of TPO on ovarian cells. It remains possible that other (TK- and Cdc2/p34-dependent) intracellular mechanisms are also involved in mediating TPO action on the ovary. PMID:15590985

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

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Nakamichi, Yoko; Nishiwaki, Chiyono; Sakurai, Takashi; Nagamatsu, Shinya

    2009-07-31

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

  16. N-myc is a key switch regulating the proliferation cycle of postnatal cerebellar granule cell progenitors.

    PubMed

    Ma, Ming; Wu, Wenting; Li, Qing; Li, Jinya; Sheng, Zhejin; Shi, Jiahao; Zhang, Mengjie; Yang, Hua; Wang, Zhugang; Sun, Ruilin; Fei, Jian

    2015-01-01

    N-myc plays an important role in early cerebellar development; however, the role of N-myc in postnatal cerebellar development is still unknown. In this study, inducible and reversible N-myc mouse models (Nmyc(TRE/TRE):tTS and Nmyc(EGFP/TRE):tTS) are used to regulate and track the expression of endogenous N-myc in vivo. Loss of N-myc at the neonatal stage results in reduced proliferation of granule cell precursors (GCPs) and reduced cerebellar volume/mass. Restoration of N-myc expression no later than postnatal day 4 can rescue the cerebellar developmental defect caused by the absence of N-myc after birth. During cerebellar postnatal development, N-myc acts as a key switch, regulating the proliferation cycle of postnatal granule cell progenitors. Loss of N-myc significantly impairs the Sonic hedgehog signalling pathway, and disrupts the expression of cell cycle effectors with a significant reduction of Ccnd2. More importantly, N-myc negatively regulates the expression of microRNA-9 during postnatal cerebellar development. Our findings demonstrate that over-expression of miR-9 can inhibit the proliferation of GCPs. The regulation of these factors by N-myc is at least partly responsible for the switch role of N-myc in the proliferation cycle of GCPs. PMID:26238256

  17. N-myc is a key switch regulating the proliferation cycle of postnatal cerebellar granule cell progenitors

    PubMed Central

    Ma, Ming; Wu, Wenting; Li, Qing; Li, Jinya; Sheng, Zhejin; Shi, Jiahao; Zhang, Mengjie; Yang, Hua; Wang, Zhugang; Sun, Ruilin; Fei, Jian

    2015-01-01

    N-myc plays an important role in early cerebellar development; however, the role of N-myc in postnatal cerebellar development is still unknown. In this study, inducible and reversible N-myc mouse models (NmycTRE/TRE:tTS and NmycEGFP/TRE:tTS) are used to regulate and track the expression of endogenous N-myc in vivo. Loss of N-myc at the neonatal stage results in reduced proliferation of granule cell precursors (GCPs) and reduced cerebellar volume/mass. Restoration of N-myc expression no later than postnatal day 4 can rescue the cerebellar developmental defect caused by the absence of N-myc after birth. During cerebellar postnatal development, N-myc acts as a key switch, regulating the proliferation cycle of postnatal granule cell progenitors. Loss of N-myc significantly impairs the Sonic hedgehog signalling pathway, and disrupts the expression of cell cycle effectors with a significant reduction of Ccnd2. More importantly, N-myc negatively regulates the expression of microRNA-9 during postnatal cerebellar development. Our findings demonstrate that over-expression of miR-9 can inhibit the proliferation of GCPs. The regulation of these factors by N-myc is at least partly responsible for the switch role of N-myc in the proliferation cycle of GCPs. PMID:26238256

  18. Calcium signalling and secretory epithelia.

    PubMed

    Petersen, O H

    2014-06-01

    Ca(2+) is now firmly established as the most important intracellular regulator of physiological and pathological events in a vast number of different cell types, including secretory epithelia. In these tissues, Ca(2+) signalling is crucially important for the control of both fluid secretion and electrolyte secretion as well as the regulation of macromolecule secretion. In this overview article, I shall attempt to give some general background to the concepts underlying our current thinking about Ca(2+) signalling in epithelia and its roles in regulating secretion. It is outside the scope of this review to provide a comprehensive account of Ca(2+) signalling and the many different processes in the many different secretory epithelia that are controlled by Ca(2+) signals. It is my aim to draw attention to some general features of Ca(2+) signalling processes in secretory epithelia, which are rather different from those in, for example, endocrine glands. The principal examples will be taken from studies of exocrine cells and, in particular, pancreatic acinar cells, as they are the pioneer cells with regard to investigations of Ca(2+) signalling due to primary intracellular Ca(2+) release. They also represent the cell type which has been characterized in most detail with regard to Ca(2+) transport events and mechanisms. PMID:24508392

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

    PubMed

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

    2015-03-01

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

  20. Delayed Dendritic Development in Newly Generated Dentate Granule Cells by Cell-Autonomous Expression of the Amyloid Precursor Protein

    PubMed Central

    Castao, Eduardo M.; Schinder, Alejandro F.

    2013-01-01

    Neuronal connectivity and synaptic remodeling are fundamental substrates for higher brain functions. Understanding their dynamics in the mammalian allocortex emerges as a critical step to tackle the cellular basis of cognitive decline that occurs during normal aging and in neurodegenerative disorders. In this work we have designed a novel approach to assess alterations in the dynamics of functional and structural connectivity elicited by chronic cell-autonomous overexpression of the human amyloid precursor protein (hAPP). We have taken advantage of the fact that the hippocampus continuously generates new dentate granule cells (GCs) to probe morphofunctional development of GCs expressing different variants of hAPP in a healthy background. hAPP was expressed together with a fluorescent reporter in neural progenitor cells of the dentate gyrus of juvenile mice by retroviral delivery. Neuronal progeny was analyzed several days post infection (dpi). Amyloidogenic cleavage products of hAPP such as the ?-C terminal fragment (?-CTF) induced a substantial reduction in glutamatergic connectivity at 21 dpi, at which time new GCs undergo active growth and synaptogenesis. Interestingly, this effect was transient, since the strength of glutamatergic inputs was normal by 35 dpi. This delay in glutamatergic synaptogenesis was paralleled by a decrease in dendritic length with no changes in spine density, consistent with a protracted dendritic development without alterations in synapse formation. Finally, similar defects in newborn GC development were observed by overexpression of ?-CTF, a non-amyloidogenic cleavage product of hAPP. These results indicate that hAPP can elicit protracted dendritic development independently of the amyloidogenic processing pathway. PMID:23851186

  1. Preventing effect of L-type calcium channel blockade on electrophysiological alterations in dentate gyrus granule cells induced by entorhinal amyloid pathology.

    PubMed

    Pourbadie, Hamid Gholami; Naderi, Nima; Mehranfard, Nasrin; Janahmadi, Mahyar; Khodagholi, Fariba; Motamedi, Fereshteh

    2015-01-01

    The entorhinal cortex (EC) is one of the earliest affected brain regions in Alzheimer's disease (AD). EC-amyloid pathology induces synaptic failure in the dentate gyrus (DG) with resultant behavioral impairment, but there is little known about its impact on neuronal properties in the DG. It is believed that calcium dyshomeostasis plays a pivotal role in the etiology of AD. Here, the effect of the EC amyloid pathogenesis on cellular properties of DG granule cells and also possible neuroprotective role of L-type calcium channel blockers (CCBs), nimodipine and isradipine, were investigated. The amyloid beta (A?) 1-42 was injected bilaterally into the EC of male rats and one week later, electrophysiological properties of DG granule cells were assessed. Voltage clamp recording revealed appearance of giant sIPSC in combination with a decrease in sEPSC frequency which was partially reversed by CCBs in granule cells from A? treated rats. EC amyloid pathogenesis induced a significant reduction of input resistance (Rin) accompanied by a profound decreased excitability in the DG granule cells. However, daily administration of CCBs, isradipine or nimodipine (i.c.v. for 6 days), almost preserved the normal excitability against A?. In conclusion, lower tendency to fire AP along with reduced Rin suggest that DG granule cells might undergo an alteration in the membrane ion channel activities which finally lead to the behavioral deficits observed in animal models and patients with early-stage Alzheimer's disease. PMID:25689857

  2. Preventing Effect of L-Type Calcium Channel Blockade on Electrophysiological Alterations in Dentate Gyrus Granule Cells Induced by Entorhinal Amyloid Pathology

    PubMed Central

    Pourbadie, Hamid Gholami; Naderi, Nima; Mehranfard, Nasrin; Janahmadi, Mahyar; Khodagholi, Fariba; Motamedi, Fereshteh

    2015-01-01

    The entorhinal cortex (EC) is one of the earliest affected brain regions in Alzheimer’s disease (AD). EC-amyloid pathology induces synaptic failure in the dentate gyrus (DG) with resultant behavioral impairment, but there is little known about its impact on neuronal properties in the DG. It is believed that calcium dyshomeostasis plays a pivotal role in the etiology of AD. Here, the effect of the EC amyloid pathogenesis on cellular properties of DG granule cells and also possible neuroprotective role of L-type calcium channel blockers (CCBs), nimodipine and isradipine, were investigated. The amyloid beta (Aβ) 1–42 was injected bilaterally into the EC of male rats and one week later, electrophysiological properties of DG granule cells were assessed. Voltage clamp recording revealed appearance of giant sIPSC in combination with a decrease in sEPSC frequency which was partially reversed by CCBs in granule cells from Aβ treated rats. EC amyloid pathogenesis induced a significant reduction of input resistance (Rin) accompanied by a profound decreased excitability in the DG granule cells. However, daily administration of CCBs, isradipine or nimodipine (i.c.v. for 6 days), almost preserved the normal excitability against Aβ. In conclusion, lower tendency to fire AP along with reduced Rin suggest that DG granule cells might undergo an alteration in the membrane ion channel activities which finally lead to the behavioral deficits observed in animal models and patients with early-stage Alzheimer’s disease. PMID:25689857

  3. Morphological observations of small granule-containing (chromaffin) cells in the celiac ganglion of the guinea pig, with emphasis on cell contacts.

    PubMed

    Mascorro, J A; Breaux, T F; Yates, R D

    1994-10-01

    Utilizing electron microscopic observation, several contacts between small, granule-containing cells (SGC) and postganglionic neurons (PGN) in the celiac ganglion of the guinea pig have been observed. A SGC in very close association with a PGN was seen to receive a distinct synaptic contact that contained many vesicles with dense cores. This contact was morphologically unlike cholinergic synapses previously reported on chromaffin cells. Because the SGC and PGN were clearly separated by a thin rim of satellite cell cytoplasm mutual to both cells, it is not known how or if the SGC would possibly exert a synaptic or paracrine effect on the PGN. Also, intraganglion SGC existed as large well-vascularized islands within the celiac ganglion. These intraganglion clusters sometimes contained more than 50 cells and perhaps could be considered to function as localized neuroendocrine components within the ganglion by secreting granule products into the nearby blood vessels for local or distant effects, although this certainly is not known. This work reports a unique synaptic ending upon a single-occurring SGC, which, in turn, closely approximates a ganglion neuron in a soma-somatic relationship. In addition, a very close association (but no actual contact) was observed between granule-containing processes, presumably emanating from the intraganglion clusters, and PGN. Whatever the function of ganglionic SGC may be, the exact relationship between SGC and PGN presumably would be of great interest and potential importance. PMID:7812038

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

    PubMed Central

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

    2008-01-01

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

  5. ZD7288 Enhances Long-Term Depression at Early Postnatal Medial Perforant Path-Granule Cell Synapses

    PubMed Central

    Guli, Xiati; Tokay, Tursonjan; Rohde, Marco; Bender, Roland A.; Khling, Rdiger; Kirschstein, Timo

    2012-01-01

    Hyperpolarization-activated, cyclic nucleotide-gated nonselective (HCN) channels modulate both membrane potential and resistance and play a significant role in synaptic plasticity. We compared the influence of HCN channels on long-term depression (LTD) at the medial perforant path-granule cell synapse in early postnatal (P915) and adult (P3060) rats. LTD was elicited in P915 slices using low-frequency stimulation (LFS, 900 pulses, 1?Hz; 80 4% of baseline). Application of the specific HCN channel blocker ZD7288 (10??M) before LFS significantly enhanced LTD (62 4%; P < 0.01), showing HCN channels restrain LTD induction. However, when ZD7288 was applied after LFS, LTD was similar to control values and significantly different from the values obtained with ZD7288 application before LFS (81 5%; P < 0.01), indicating that HCN channels do not modulate LTD expression. LTD in slices from adult rats were only marginally lower compared to those in P915 slices (85 6%), but bath application of ZD7288 prior to LFS resulted in the same amount of LTD (85 5%). HCN channels in adult tissue hence lose their modulatory effect. In conclusion, we found that HCN channels at the medial perforant path-granule cell synapse compromise LFS-associated induction, but not expression of LTD in early postnatal, but not in adult, rats. PMID:22792490

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

    PubMed Central

    Tani, Kunihiko; Iyo, Masaomi; Matsumoto, Hideo; Kawai, Masayoshi; Suzuki, Katsuaki; Iwata, Yasuhide; Won, Taketoshi; Tsukamoto, Toshio; Sekine, Yoshimoto; Sakanoue, Masatsuna; Hashimoto, Kenji; Ohashi, Yutaka; Takei, Nori; Mori, Norio

    2001-01-01

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

  7. Identification of a cKit+ Colonic Crypt Base Secretory Cell That Supports Lgr5+ Stem Cells in Mice

    PubMed Central

    Rothenberg, Michael E.; Nusse, Ysbrand; Kalisky, Tomer; Lee, John J.; Dalerba, Piero; Scheeren, Ferenc; Lobo, Neethan; Kulkarni, Subhash; Sim, Sopheak; Qian, Dalong; Beachy, Philip A.; Pasricha, Pankaj J.; Quake, Stephen R.; Clarke, Michael F.

    2013-01-01

    Background & Aims Paneth cells contribute to the small intestinal niche of Lgr5+ stem cells. Although the colon also contains Lgr5+ stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5+ stem cells. Methods We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. Results Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117+ crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit+ goblet cells were interdigitated with Lgr5+ stem cells. In vivo, this colonic cKit+ population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit+ cells. When isolated from mouse colon, cKit+ cells promoted formation of organoids from Lgr5+ stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit+ cells using a toxin-conjugated antibody, organoid formation decreased. Conclusions cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5+stem cells. PMID:22333952

  8. Granule Associated Serine Proteases of Hematopoietic Cells – An Analysis of Their Appearance and Diversification during Vertebrate Evolution

    PubMed Central

    Akula, Srinivas; Thorpe, Michael; Boinapally, Vamsi; Hellman, Lars

    2015-01-01

    Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish. PMID:26569620

  9. Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

    PubMed

    Park, Soonhong; Ahuja, Malini; Kim, Min Seuk; Brailoiu, G Cristina; Jha, Archana; Zeng, Mei; Baydyuk, Maryna; Wu, Ling-Gang; Wassif, Christopher A; Porter, Forbes D; Zerfas, Patricia M; Eckhaus, Michael A; Brailoiu, Eugen; Shin, Dong Min; Muallem, Shmuel

    2016-02-01

    Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV. PMID:26682800

  10. Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells

    PubMed Central

    Vomund, Anthony N.; Zinselmeyer, Bernd H.; Hughes, Jing; Calderon, Boris; Valderrama, Carolina; Ferris, Stephen T.; Wan, Xiaoxiao; Kanekura, Kohsuke; Carrero, Javier A.; Urano, Fumihiko; Unanue, Emil R.

    2015-01-01

    Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell–phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system. PMID:26324934

  11. Synaptic excitation of individual rat cerebellar granule cells in situ: evidence for the role of NMDA receptors.

    PubMed

    D'Angelo, E; De Filippi, G; Rossi, P; Taglietti, V

    1995-04-15

    1. Current-clamp recordings were made in whole-cell patch-clamp configuration from ninety-one granule cells in parasagittal cerebellar slices obtained from 21- to 31-day-old rats. Recordings were performed at 30 degrees C. 2. Resting membrane potential was -58 +/- 6 mV (n = 43). The membrane voltage response to step current injection showed inward rectification consistent with increasing input resistance during membrane depolarization. Over -35 +/- 7 mV (n = 14) repetitive firing with little or no adaptation was activated. Spike frequency increased nearly linearly with injected current. 3. Unitary EPSPs obtained by stimulating the mossy fibre bundle had an amplitude of 11.4 +/- 2.1 mV (n = 22, holding potential = -75 mV). Synchronous activation of greater than one to two mossy fibres was needed to elicit action potentials. Antidromic stimulation elicited antidromic spikes and also EPSPs, presumably through a mossy fibre 'axon reflex'. 4. EPSPs were brought about by NMDA and non-NMDA receptor activation, accounting for about 70 and 30%, respectively, of peak amplitude at the holding potential of -70 mV. The EPSP decay conformed to passive membrane discharge after blocking the NMDA receptors. 5. No appreciable correlation was found between the time-to-peak and decay time constant of the EPSPs, consistent with the compact electrotonic structure of these neurons. 6. During membrane depolarization EPSP amplitude increased transiently, due to both a voltage-dependent increase of the NMDA component and inward rectification. In addition, EPSPs slowed down due to a slowdown of the NMDA component. 7. Temporal summation during high-frequency stimulation was sustained by NMDA receptors, whose contribution to depolarization tended to prevail over that of non-NMDA receptors during the trains. A block of the NMDA receptors resulted in reduced depolarization and output spike frequency. 8. This study, as well as extending previous knowledge to the intracellular level in vivo, provides evidence for a primary role of NMDA receptors in determining mossy fibre excitation of granule cells. It is suggested that the marked voltage dependence of the EPSP time course, which was mainly caused by voltage dependence in NMDA conductance, promotes the NMDA receptor-dependent enhancement of granule cell coding observed during repetitive mossy fibre activity. PMID:7602534

  12. Enduring changes in tonic GABAA receptor signaling in dentate granule cells after controlled cortical impact brain injury in mice.

    PubMed

    Boychuk, Jeffery A; Butler, Corwin R; Halmos, Katalin Cs; Smith, Bret N

    2016-03-01

    Changes in functional GABAAR signaling in hippocampus have previously been evaluated using pre-clinical animal models of either diffuse brain injury or extreme focal brain injury that precludes measurement of cells located ipsilateral to injury. As a result, there is little information about the status of functional GABAAR signaling in dentate granule cells (DGCs) located ipsilateral to focal brain injury, where significant cellular changes have been documented. We used whole-cell patch-clamp recordings from hippocampal slices to measure changes in GABAARs in dentate granule cells (DGCs) at 1-2, 3-5, and 8-13weeks after controlled cortical impact (CCI) brain injury. Synaptic and tonic GABAAR currents (ITonicGABA) were measured in DGCs at baseline conditions and during application of the GABAAR agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride (THIP) to assess in the function of ? subunit-containing GABAARs. DGCs ipsilateral to CCI exhibited no changes in the amplitude of resting ITonicGABA relative to DGCs after sham-injury or contralateral to CCI. In contrast, there was a significant reduction in the THIP-evoked ITonicGABA in DGCs ipsilateral to CCI at both time-points. Tonic GABAergic inhibition of DGCs ipsilateral to injury also exhibited reduced responsiveness to the neurosteroid THDOC. ITonicGABA in DGCs ipsilateral to CCI did not exhibit a change in sensitivity to L655,708, an inverse agonist with selectivity for ?5 subunit-containing GABAARs, suggesting a lack of functional change in GABAARs containing this subunit. At the 8-13week time-point, gene expression of GABAAR subunits expected to contribute to ITonicGABA (i.e., ?4, ?5 and ?) was not significantly altered by CCI injury in isolated dentate gyrus. Collectively, these results demonstrate enduring functional changes in ITonicGABA in DGCs ipsilateral to focal brain injury that occur independent of altered gene expression. PMID:26772635

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

    PubMed

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

    2014-09-01

    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

  14. PTBP1 is required for glucose-stimulated cap-independent translation of insulin granule proteins and Coxsackieviruses in beta cells

    PubMed Central

    Knoch, Klaus-Peter; Nath-Sain, Suchita; Petzold, Antje; Schneider, Hendryk; Beck, Mike; Wegbrod, Carolin; Snmez, Anke; Mnster, Carla; Friedrich, Anne; Roivainen, Merja; Solimena, Michele

    2014-01-01

    Glucose and GLP-1 stimulate not only insulin secretion, but also the post-transcriptional induction of insulin granule biogenesis. This process involves the nucleocytoplasmic translocation of the RNA binding protein PTBP1. Binding of PTBP1 to the 3?-UTRs of mRNAs for insulin and other cargoes of beta cell granules increases their stability. Here we show that glucose enhances also the binding of PTBP1 to the 5?-UTRs of these transcripts, which display IRES activity, and their translation exclusively in a cap-independent fashion. Accordingly, glucose-induced biosynthesis of granule cargoes was unaffected by pharmacological, genetic or Coxsackievirus-mediated inhibition of cap-dependent translation. Infection with Coxsackieviruses, which also depend on PTBP1 for their own cap-independent translation, reduced instead granule stores and insulin release. These findings provide insight into the mechanism for glucose-induction of insulin granule production and on how Coxsackieviruses, which have been implicated in the pathogenesis of type 1 diabetes, can foster beta cell failure. PMID:25061557

  15. Secretory diarrhoea: mechanisms and emerging therapies

    PubMed Central

    Thiagarajah, Jay R.; Donowitz, Mark; Verkman, Alan S.

    2016-01-01

    Diarrhoeal disease remains a major health burden worldwide. Secretory diarrhoeas are caused by certain bacterial and viral infections, inflammatory processes, drugs and genetic disorders. Fluid secretion across the intestinal epithelium in secretory diarrhoeas involves multiple ion and solute transporters, as well as activation of cyclic nucleotide and Ca2+ signalling pathways. In many secretory diarrhoeas, activation of Cl− channels in the apical membrane of enterocytes, including the cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl− channels, increases fluid secretion, while inhibition of Na+ transport reduces fluid absorption. Current treatment of diarrhoea includes replacement of fluid and electrolyte losses using oral rehydration solutions, and drugs targeting intestinal motility or fluid secretion. Therapeutics in the development pipeline target intestinal ion channels and transporters, regulatory proteins and cell surface receptors. This Review describes pathogenic mechanisms of secretory diarrhoea, current and emerging therapeutics, and the challenges in developing antidiarrhoeal therapeutics. PMID:26122478

  16. Transcription factor Foxq1 controls mucin gene expression and granule content in mouse stomach surface mucous cells

    PubMed Central

    Verzi, Michael P.; Khan, Abdul H.; Ito, Susumu; Shivdasani, Ramesh A.

    2010-01-01

    Background and Aims The gastric mucosa provides a stringent epithelial barrier and produces acid and enzymes that initiate digestion. In this regenerating tissue, progenitors differentiate continually into 4 principal specialized cell types, yet underlying mechanisms of differentiation are poorly understood. We identified stomach-restricted expression of the forkhead transcription factor FOXQ1. Methods We used a combination of genetic, histochemical, ultrastructural and molecular analysis to study gastric cell lineages with respect to FOXQ1. Results Within the developing and adult gastrointestinal tract, Foxq1 mRNA is restricted to the stomach and expressed predominantly in foveolar (pit) cells, the abundant mucin-producing cells that line the mucosal surface. Mice carrying Foxq1 coding mutations show virtual absence of mRNA and protein for the backbone of the major stomach mucin, MUC5AC. These observations correspond to a paucity of foveolar-cell secretory vesicles and notable loss of stomach but not intestinal mucus. Transcriptional profiling identified a surprisingly restricted set of genes with altered expression in Foxq1 mutant stomachs. MUC5AC is a highly tissue-restricted product that similarly depends on FOXQ1 in its other major site of expression, conjunctival goblet cells. Conclusions Taken together, these observations imply that promotion of gastric MUC5AC synthesis is a primary, cell-autonomous function of FOXQ1. This study is the first to implicate a transcription factor in terminal differentiation of foveolar cells and begins to define the requirements to assemble highly specialized organelles and cells in the gastric mucosa. PMID:18558092

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

    PubMed Central

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

    2014-01-01

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

  18. Vesicle-mediated secretion of human eosinophil granule-derived major basic protein.

    PubMed

    Melo, Rossana C N; Spencer, Lisa A; Perez, Sandra A C; Neves, Josiane S; Bafford, Staci P; Morgan, Ellen S; Dvorak, Ann M; Weller, Peter F

    2009-07-01

    Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases. PMID:19398958

  19. Identification of Wnt/?-catenin signaling pathway in dermal papilla cells of human scalp hair follicles: TCF4 regulates the proliferation and secretory activity of dermal papilla cell.

    PubMed

    Xiong, Ya; Liu, Yi; Song, Zhiqiang; Hao, Fei; Yang, Xichuan

    2014-01-01

    It is clear that the dermal papilla cell (DPC), which is located at the bottom of the hair follicle, is a special mesenchymal component, and it plays a leading role in regulating hair follicle development and periodic regeneration. Recent studies showed that the Wnt signaling pathway through ?-catenin (canonical Wnt signaling pathway) is an essential component in maintaining the hair-inducing activity of the dermal papilla and growth of hair papilla cells. However, the intrinsic pathways and regulating mechanism are largely unknown. In the previous work, we constructed a cDNA subtractive library of DPC and first found that the TCF4 gene, as a key factor of Wnt signaling pathway, was expressed as the upregulated gene of the hair follicle in low-passage DPC. This study was to explore the role of TCF4 in regulating the proliferation and secretory activity of DPC. We constructed a pcDNA3.0-TCF4 expression vector and transfected it into DPC to achieve stable expression by bangosome 2000. Furthermore, we used the method of chemosynthesis to synthesize three pairs of TCF4 siRNA and transfected them into DPC. Meanwhile, we compared the transfection group and non-transfection group. We first proposed that there was expression difference in TCF4 in DPC under different biological condition. This study may have a high impact on the molecular mechanism of follicular lesions and provide a new vision for the treatment of clinic diseases. PMID:24354472

  20. Selective disruption of Tcf7l2 in the pancreatic ? cell impairs secretory function and lowers ? cell mass.

    PubMed

    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

    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