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Sample records for lytic granule exocytosis

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

    DEFF Research Database (Denmark)

    Boding, Lasse; Hansen, Ann K; Meroni, Germana;

    2015-01-01

    We have recently shown that the E3 ubiquitin ligase midline 1 (MID1) is upregulated in murine cytotoxic lymphocytes (CTL), where it controls exocytosis of lytic granules and the killing capacity. Accordingly, CTL from MID1 knock-out (MID1(-/-)) mice have a 25-30% reduction in exocytosis of lytic...... granules and cytotoxicity compared to CTL from wild-type (WT) mice. We wondered why the MID1 gene knock-out did not affect exocytosis and cytotoxicity more severely and speculated whether MID2, a close homologue of MID1, might partially compensate for the loss of MID1 in MID1(-/-) CTL. Here, we showed...

  2. Platelet granule exocytosis: A comparison with chromaffin cells

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    Jennifer eFitch-Tewfik

    2013-06-01

    Full Text Available 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.

  3. Granule exocytosis is required for platelet spreading: differential sorting of α-granules expressing VAMP-7.

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    Peters, Christian G; Michelson, Alan D; Flaumenhaft, Robert

    2012-07-01

    There has been recent controversy as to whether platelet α-granules represent a single granule population or are composed of different subpopulations that serve discrete functions. To address this question, we evaluated the localization of vesicle-associated membrane proteins (VAMPs) in spread platelets to determine whether platelets actively sort a specific subpopulation of α-granules to the periphery during spreading. Immunofluorescence microscopy demonstrated that granules expressing VAMP-3 and VAMP-8 localized to the central granulomere of spread platelets along with the granule cargos von Willebrand factor and serotonin. In contrast, α-granules expressing VAMP-7 translocated to the periphery of spread platelets along with the granule cargos TIMP2 and VEFG. Time-lapse microscopy demonstrated that α-granules expressing VAMP-7 actively moved from the granulomere to the periphery during spreading. Platelets from a patient with gray platelet syndrome lacked α-granules and demonstrated only minimal spreading. Similarly, spreading was impaired in platelets obtained from Unc13d(Jinx) mice, which are deficient in Munc13-4 and have an exocytosis defect. These studies identify a new α-granule subtype expressing VAMP-7 that moves to the periphery during spreading, supporting the premise that α-granules are heterogeneous and demonstrating that granule exocytosis is required for platelet spreading.

  4. Partitioning and Exocytosis of Secretory Granules during Division of PC12 Cells

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    Nickolay Vassilev Bukoreshtliev

    2012-01-01

    Full Text Available The biogenesis, maturation, and exocytosis of secretory granules in interphase cells have been well documented, whereas the distribution and exocytosis of these hormone-storing organelles during cell division have received little attention. By combining ultrastructural analyses and time-lapse microscopy, we here show that, in dividing PC12 cells, the prominent peripheral localization of secretory granules is retained during prophase but clearly reduced during prometaphase, ending up with only few peripherally localized secretory granules in metaphase cells. During anaphase and telophase, secretory granules exhibited a pronounced movement towards the cell midzone and, evidently, their tracks colocalized with spindle microtubules. During cytokinesis, secretory granules were excluded from the midbody and accumulated at the bases of the intercellular bridge. Furthermore, by measuring exocytosis at the single granule level, we showed, that during all stages of cell division, secretory granules were competent for regulated exocytosis. In conclusion, our data shed new light on the complex molecular machinery of secretory granule redistribution during cell division, which facilitates their release from the F-actin-rich cortex and active transport along spindle microtubules.

  5. VAMP-7 links granule exocytosis to actin reorganization during platelet activation.

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    Koseoglu, Secil; Peters, Christian G; Fitch-Tewfik, Jennifer L; Aisiku, Omozuanvbo; Danglot, Lydia; Galli, Thierry; Flaumenhaft, Robert

    2015-07-30

    Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization.

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

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

    2013-01-01

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

  7. Mathematical modeling and statistical analysis of calcium-regulated insulin granule exocytosis in ß-cells from mice and humans

    DEFF Research Database (Denmark)

    Pedersen, Morten Gram; Cortese, Giuliana; Eliasson, Lena

    2011-01-01

    on depolarization-evoked Ca2+-currents and corresponding capacitance measurements. Using a statistical mixed-effects model, we show that the data indicate that pool depletion is negligible in response to short depolarizations in mouse ß-cells. We then review mathematical models of granule dynamics and exocytosis......Insulin is released from pancreatic ß-cells as a result of Ca2+-evoked exocytosis of dense-core granules. Secretion is biphasic, which has been suggested to correspond to the release of different granule pools. Here we review and carefully reanalyze previously published patch-clamp data...... in rodent ß-cells and present a mathematical description of Ca2+-evoked exocytosis in human ß-cells, which show clear differences to their rodent counterparts. The model suggests that L- and P/Q-type Ca2+-channels are involved to a similar degree in exocytosis during electrical activity in human ß-cells....

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

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    Nikhil R Gandasi

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

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

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    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.

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

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    Galvanovskis, Juris; Braun, Matthias; Rorsman, Patrik

    2011-02-06

    Pancreatic β-cells use Ca(2+)-dependent exocytosis of large dense core vesicles to release insulin. Exocytosis in β-cells has been studied biochemically, biophysically and optically. We have previously developed a biophysical method to monitor release of endogenous intragranular constituents that are co-released with insulin. This technique involves the expression of ionotropic membrane receptors in the β-cell plasma membrane and enables measurements of exocytosis of individual vesicles with sub-millisecond resolution. Like carbon fibre amperometry, this method allows fine details of the release process, like the expansion of the fusion pore (the narrow connection between the granule lumen and the extracellular space), to be monitored. Here, we discuss experimental data obtained with this method within the framework of a simple mathematical model that describes the release of low-molecular constituents during exocytosis of the insulin granules. Our findings suggest that the fusion pore functions as a molecular sieve, allowing differential release of low- and high-molecular-weight granule constituents.

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

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

    2009-12-04

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

  12. New Roles of Syntaxin-1A in Insulin Granule Exocytosis and Replenishment.

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    Liang, Tao; Qin, Tairan; Xie, Li; Dolai, Subhankar; Zhu, Dan; Prentice, Kacey J; Wheeler, Michael; Kang, Youhou; Osborne, Lucy; Gaisano, Herbert Y

    2017-02-10

    In type-2 diabetes (T2D), severely reduced islet syntaxin-1A (Syn-1A) levels contribute to insulin secretory deficiency. We generated β-cell-specific Syn-1A-KO (Syn-1A-βKO) mice to mimic β-cell Syn-1A deficiency in T2D. Glucose tolerance tests showed that Syn-1A-βKO mice exhibited blood glucose elevation corresponding to reduced blood insulin levels. Perifusion of Syn-1A-βKO islets showed impaired first- and second-phase glucose-stimulated insulin secretion (GSIS) resulting from reduction in readily releasable pool and granule pool refilling. To unequivocally determine the β-cell exocytotic defects caused by Syn-1A deletion, EM and total internal reflection fluorescence microscopy showed that Syn-1A-KO β-cells had a severe reduction in the number of secretory granules (SGs) docked onto the plasma membrane (PM) at rest and reduced SG recruitment to the PM after glucose stimulation, the latter indicating defects in replenishment of releasable pools required to sustain second-phase GSIS. Whereas reduced predocked SG fusion accounted for reduced first-phase GSIS, selective reduction of exocytosis of short-dock (but not no-dock) newcomer SGs accounted for the reduced second-phase GSIS. These Syn-1A actions on newcomer SGs were partly mediated by Syn-1A interactions with newcomer SG VAMP8.

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

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    Boding, Lasse; Hansen, Ann K; Meroni, Germana

    2014-01-01

    Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2A activity. Loss-of-function mutations in MID1 lead to the X-linked Opitz G/BBB syndrome characterized by defective midline development during embryogenesis. Here we show that MID1 is strongly...

  14. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

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    Alice C N Brown

    2011-09-01

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

  15. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

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

    2011-09-01

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

  16. Hydrogen sulfide induces hyperpolarization and decreases the exocytosis of secretory granules of rat GH3 pituitary tumor cells.

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    Mustafina, Alsu N; Yakovlev, Aleksey V; Gaifullina, Aisylu Sh; Weiger, Thomas M; Hermann, Anton; Sitdikova, Guzel F

    2015-10-02

    The aim of the present study was to evaluate the effects of hydrogen sulfide (H2S) on the membrane potential, action potential discharge and exocytosis of secretory granules in neurosecretory pituitary tumor cells (GH3). The H2S donor - sodium hydrosulfide (NaHS) induced membrane hyperpolarization, followed by truncation of spontaneous electrical activity and decrease of the membrane resistance. The NaHS effect was dose-dependent with an EC50 of 152 μM (equals effective H2S of 16-19 μM). NaHS effects were not altered after inhibition of maxi conductance calcium-activated potassium (BK) channels by tetraethylammonium or paxilline, but were significantly reduced after inhibition or activation of ATP-dependent potassium channels (KATP) by glibenclamide or by diazoxide, respectively. In whole-cell recordings NaHS increased the amplitude of KATP currents, induced by hyperpolarizing pulses and subsequent application of glibenclamide decreased currents to control levels. Using the fluorescent dye FM 1-43 exocytosis of secretory granules was analyzed in basal and stimulated conditions (high K(+) external solution). Prior application of NaHS decreased the fluorescence of the cell membrane in both conditions which links with activation of KATP currents (basal secretion) and activation of KATP currents and BK-currents (stimulated exocytosis). We suggest that H2S induces hyperpolarization of GH3 cells by activation of KATP channels which results in a truncation of spontaneous action potentials and a decrease of hormone release.

  17. Syntaxin 8 is required for efficient lytic granule trafficking in cytotoxic T lymphocytes.

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    Bhat, Shruthi S; Friedmann, Kim S; Knörck, Arne; Hoxha, Cora; Leidinger, Petra; Backes, Christina; Meese, Eckart; Keller, Andreas; Rettig, Jens; Hoth, Markus; Qu, Bin; Schwarz, Eva C

    2016-07-01

    Cytotoxic T lymphocytes (CTL) eliminate pathogen-infected and cancerous cells mainly by polarized secretion of lytic granules (LG, containing cytotoxic molecules like perforin and granzymes) at the immunological synapse (IS). Members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family are involved in trafficking (generation, transport and fusion) of vesicles at the IS. Syntaxin 8 (Stx8) is expressed in LG and colocalizes with the T cell receptor (TCR) upon IS formation. Here, we report the significance of Stx8 for human CTL cytotoxicity. We found that Stx8 mostly localized in late, recycling endosomal and lysosomal compartments with little expression in early endosomal compartments. Down-regulation of Stx8 by siRNA resulted in reduced cytotoxicity. We found that following perforin release of the pre-existing pool upon target cell contact, Stx8 down-regulated CTL regenerate perforin pools less efficiently and thus release less perforin compared to control CTL. CD107a degranulation, real-time and end-point population cytotoxicity assays, and high resolution microscopy support our conclusion that Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules to functional LG through the endosomal pathway in human CTL.

  18. Translocation of classical PKC and cortical granule exocytosis of human oocyte in germinal vesicle and metaphase Ⅱ stage

    Institute of Scientific and Technical Information of China (English)

    Xue-qing WU; Xiao ZHANG; Xiao-hong LI; Hui-hong CHENG; Yan-rong KUAI; Song WANG; Ying-lu GUO

    2006-01-01

    Aim: Protein kinase C (PKC) is as a family of serine/threonine kinases that can be activated by Ca2+, phospholipid and diacylglycerol. PKC plays an important role in oocyte maturation and activation. This study was undertaken to investigate classical PKC (cPKC) in human oocyte maturation and activation. Methods: Germinal vesicle (GV) and metaphase Ⅱ (MII) stage oocytes were collected from healthy women. The expression and distribution of cPKC were investigated by immunoflourescence. MII oocytes were treated with PKC activator or inhibitor and imaged using a laser confocal scanning microscope (LCSM). Results: In GV oocytes, PKC α,β1 and γ were localized to the germinal vesicles, with a weak expression in ooplasm. In MII oocytes, PKCα, β1 and γ were distributed evenly in ooplasm. After treatment with PKC activator, phorbol 12-myristate 13-acetate (PMA), cPKC translocated to the periphery of oocyte, and cortical granules (CG) exocytosis was found. When the oocytes were treated with PKC inhibitor, staurosporine, no translocation of cPKC and CG exocytosis were found. Conclusion: PKCα, β1 and γ exist in human oocytes and activation of these subunits could induce CG exocytosis in MII stage.

  19. Complexin 2 modulates vesicle-associated membrane protein (VAMP) 2-regulated zymogen granule exocytosis in pancreatic acini.

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    Falkowski, Michelle A; Thomas, Diana D H; Groblewski, Guy E

    2010-11-12

    Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca(2+)-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca(2+)-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca(2+)-sensitive regulatory pathway for zymogen granule exocytosis.

  20. Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini*

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    Falkowski, Michelle A.; Thomas, Diana D. H.; Groblewski, Guy E.

    2010-01-01

    Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca2+-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca2+-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca2+-sensitive regulatory pathway for zymogen granule exocytosis. PMID:20829354

  1. Post-ovulatory aging of mouse oocytes in vivo and in vitro: Effects of caffeine on exocytosis and translocation of cortical granules.

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    Zheng, Jie; Yin, Xun-Qiang; Ge, Wei; He, Gui-Fang; Qian, Wei-Ping; Ma, Jun-Yu; Shen, Wei; Yin, Shen; Sun, Qing-Yuan

    2016-11-01

    The developmental potential of post-ovulatory oocytes decreases with aging in vivo and in vitro. In this study, we aimed to investigate the effects of a potent antioxidant caffeine on cortical granules (CGs) distribution in mouse oocytes aging in vivo and in vitro. We found that in vivo administration of 150 mg/kg caffeine caused ovulation of some morphologically abnormal oocytes showing premature exocytosis or congregation of CGs, but significantly decreased abnormal distribution of CGs in oocytes aging for 6 h, 12 h and 18 h in vivo compared to those without caffeine treatment. Unexpectedly, supplementation of oocyte culture medium with 10 mmol/L caffeine accelerated CGs release of oocytes and the normal CG distribution rate dramatically decreased from 6 h in oocytes aging in vitro. It appeared that oocytes showed a high degree of abnormal CG distribution by aging for 18 h, and caffeine might delay oocyte CG exocytosis in vivo, but accelerates CG exocytosis in vitro. Our findings may have implications for improving assisted reproduction technologies.

  2. The F-actin modifier villin regulates insulin granule dynamics and exocytosis downstream of islet cell autoantigen 512

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    Hassan Mziaut

    2016-08-01

    Conclusion: Our findings show that villin controls the size of the F-actin cages restricting SGs and, thus, regulates their dynamics and availability for exocytosis. Evidence that villin acts downstream of Ica512 also indicates that SGs directly influence the remodeling properties of the cortical actin cytoskeleton for tight control of insulin secretion.

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

    Science.gov (United States)

    1989-01-01

    cascade during exocytosis. Keywords: Cyclo-oxygenase; Prostaglandin E2; Secretory granules; Mast cell ; Exocytosis; Lipid mediators; Inflammation; Arachidonic acid; Eicosanoids; Immunocytochemistry; Reprints. (KT)

  4. Exocytosis and endocytosis in juxtaglomerular cells

    DEFF Research Database (Denmark)

    Friis, U G; Jensen, B L; Hansen, Pernille B. Lærkegaard;

    2000-01-01

    that the afferent arterioles lose a large number of renin granules after acute stimulation without changing the average granular volume. Current electrophysiological techniques have now permitted direct measurements of cell membrane capacitance in juxtaglomerular (JG) cells as a measure of net addition (exocytosis......The cellular events related to secretion of renin are not well understood. Here we review some of the evidence that has led to the current understanding of renin secretion as a process that involves exocytosis as the predominant mode of secretion. This is based on the observation of occasional......) or removal (endocytosis) of membrane material. With this technique we have shown that cAMP, which is a vasodilator and stimulates renin secretion, enhances net exocytosis at low concentrations, while at higher concentrations membrane retrieval processes are also stimulated. We suggest that both exocytosis...

  5. How intravesicular composition affects exocytosis.

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    Mark Wightman, R; Domínguez, Natalia; Borges, Ricardo

    2017-08-04

    Large dense core vesicles and chromaffin granules accumulate solutes at large concentrations (for instance, catecholamines, 0.5-1 M; ATP, 120-300 mM; or Ca(2+), 40 mM (12)). Solutes seem to aggregate to a condensed protein matrix, which is mainly composed of chromogranins, to elude osmotic lysis. This association is also responsible for the delayed release of catecholamines during exocytosis. Here, we compile experimental evidence, obtained since the inception of single-cell amperometry, demonstrating how the alteration of intravesicular composition promotes changes in the quantum characteristics of exocytosis. As chromaffin cells are large and their vesicles contain a high concentration of electrochemically detectable species, most experimental data comes from this cell model.

  6. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules.

    Science.gov (United States)

    Heijnen, H F; Schiel, A E; Fijnheer, R; Geuze, H J; Sixma, J J

    1999-12-01

    Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and alpha-granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or alpha-thrombin: microvesicles of 100 nm to 1 microm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and alpha-granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains alpha(IIb)-beta(3) and beta(1), GPIbalpha, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63(+) internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

  7. Ceramide triggers Weibel-Palade body exocytosis.

    Science.gov (United States)

    Bhatia, Rinky; Matsushita, Kenji; Yamakuchi, Munekazu; Morrell, Craig N; Cao, Wangsen; Lowenstein, Charles J

    2004-08-06

    The sphingolipid ceramide mediates a variety of stress responses, including vascular inflammation and thrombosis. Activated endothelial cells release Weibel-Palade bodies, granules containing von Willebrand factor (vWF) and P-selectin, which induce leukocyte rolling and platelet adhesion and aggregation. We hypothesized that ceramide induces vascular inflammation and thrombosis in part by triggering Weibel-Palade body exocytosis. We added ceramide to human aortic endothelial cells and assayed Weibel-Palade body exocytosis by measuring the concentration of vWF released into the media. Exogenous ceramide induces vWF release from endothelial cells in a dose-dependent manner. Activators of endogenous ceramide production, neutral sphingomyelinase, or tumor necrosis factor-alpha also induce Weibel-Palade body exocytosis. We next studied NO effects on ceramide-induced Weibel-Palade body exocytosis because NO can inhibit vascular inflammation. The NO donor S-nitroso-N-acetylpenicillamine decreases ceramide-induced vWF release in a dose-dependent manner, whereas the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester increases ceramide-induced vWF release. In summary, our findings show that endogenous ceramide triggers Weibel-Palade body exocytosis, and that endogenous NO inhibits ceramide-induced exocytosis. These data suggest a novel mechanism by which ceramide induces vascular inflammation and thrombosis.

  8. Endocytosis is required for exocytosis and priming of respiratory burst activity in human neutrophils.

    Science.gov (United States)

    Creed, T Michael; Tandon, Shweta; Ward, Richard A; McLeish, Kenneth R

    2017-06-21

    Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47(phox) phosphorylation-dependent translocation of cytosolic NADPH oxidase components. Clathrin-mediated endocytosis was recently reported to be necessary for priming, but the mechanism linking endocytosis to priming was not identified. The present study examined the hypothesis that endocytosis regulates neutrophil priming by controlling granule exocytosis. Clathrin-mediated endocytosis by isolated human neutrophils was inhibited by chlorpromazine, monodansylcadaverine, and sucrose. Exocytosis of granule subsets was measured as release of granule components by ELISA or chemiluminescence. ROS generation was measured as extracellular release of superoxide as reduction of ferrocytochrome c. p38 MAPK activation and p47(phox) phosphorylation were measured by immunoblot analysis. Statistical analysis was performed using a one-way ANOVA with the Tukey-Kramer multiple-comparison test. Inhibition of endocytosis prevented priming of superoxide release by TNFα and inhibited TNFα stimulation and priming of exocytosis of all four granule subsets. Inhibition of endocytosis did not reduce TNFα-stimulated p38 MAPK activation or p47(phox) phosphorylation. Inhibition of NADPH oxidase activity blocked TNFα stimulation of secretory vesicle and gelatinase granule exocytosis. Endocytosis is linked to priming of respiratory burst activity through ROS-mediated control of granule exocytosis.

  9. Exocytosis through the lens

    Directory of Open Access Journals (Sweden)

    Alicja eGraczyk

    2013-10-01

    Full Text Available Exocytosis, the process in which material is transported from the cell interior to the extracellular space, proceeds through a complex mechanism. Defects in this process are linked to a number of serious illnesses including diabetes, cancer and a range of neuropathologies. In neuroendocrine cells, exocytosis involves the fusion of secretory vesicles, carrying signalling molecules, with the plasma membrane through the coordinated interplay of proteins, lipids and small molecules. This process is highly regulated and occurs in a complex three-dimensional environment within the cell precisely coupled to the stimulus. The study of exocytosis poses significant challenges, involving rapidly changing, nano-scale, protein-protein and protein-lipid interactions, at specialised sites in the cell. Over the last decade our understanding of neuroendocrine exocytosis has been greatly enhanced by developments in fluorescence microscopy. Modern microscopy encompasses a toolbox of advanced techniques, pushing the limits of sensitivity and resolution, to probe different properties of exocytosis. In more recent years, the development of super-resolution microscopy techniques, side-stepping the limits of optical resolution imposed by the physical properties of light, have started to provide an unparalleled view of exocytosis. In this review we will discuss how advances in fluorescence microscopy are shedding light on the spatial and temporal organisation of the exocytotic machinery.

  10. Exocytosis and endocytosis in neurodocrine cells: inseparable membranes !

    Directory of Open Access Journals (Sweden)

    Sébastien eHouy

    2013-10-01

    Full Text Available Although much has been learned concerning the mechanisms of secretory vesicle formation and fusion at donor and acceptor membrane compartments, relatively little attention has been paid towards understanding how cells maintain a homeostatic membrane balance through vesicular trafficking. In neurons and neuroendocrine cells, release of neurotransmitters, neuropeptides and hormones occurs through calcium-regulated exocytosis at the plasma membrane. To allow recycling of secretory vesicle components and to preserve organelles integrity, cells must initiate and regulate compensatory membrane uptake. This review relates the fate of secretory granule membranes after full fusion exocytosis in neuroendocrine cells. In particular, we focus on the potential role of lipids in preserving and sorting secretory granule membranes after exocytosis and we discuss the potential mechanisms of membrane retrieval.

  11. Calcium sensing in exocytosis

    DEFF Research Database (Denmark)

    Gustavsson, Natalia; Wu, Bingbing; Han, Weiping

    2012-01-01

    an increase in intracellular calcium levels. Besides the triggering role, calcium signaling modulates the precise amount and kinetics of vesicle release. Thus, it is a central question to understand the molecular machineries responsible for calcium sensing in exocytosis. Here we provide an overview of our...... current understanding of calcium sensing in neurotransmitter release and hormone secretion....

  12. Kinetics of human sperm acrosomal exocytosis.

    Science.gov (United States)

    Sosa, C M; Pavarotti, M A; Zanetti, M N; Zoppino, F C M; De Blas, G A; Mayorga, L S

    2015-03-01

    The acrosome reaction is a unique event in the lifespan of sperm characterized by the exocytosis of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal membrane and the plasma membrane. This unique regulated exocytosis is mediated by essentially the same membrane fusion machinery present in neuroendocrine cells. However, whereas secretion in neuroendocrine cells occurs in less than a second, the acrosome reaction is normally assessed after several minutes of incubation with inducers. In this report, we measured the kinetics of human sperm exocytosis triggered by two stimuli (calcium ionophore and progesterone) by using electron microscopy and three different approaches based on the incorporation of fluorescent Pisum sativum agglutinin into the acrosome upon opening of fusion pores connecting the extracellular medium with the acrosomal lumen. The results with the different methods are consistent with a slow kinetics (t½ = 14 min). We also manipulated the system to measure different steps of the process. We observed that cytosolic calcium increased with a relatively fast kinetics (t½ = 0.1 min). In contrast, the swelling of the acrosomal granule that precedes exocytosis was a slow process (t½ = 13 min). When swelling was completed, the fusion pore opening was fast (t½ = 0.2 min). The results indicate that acrosomal swelling is the slowest step and it determines the kinetics of the acrosome reaction. After the swelling is completed, the efflux of calcium from intracellular stores triggers fusion pores opening and the release of hybrid vesicles in seconds.

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

    OpenAIRE

    Johnson, Joseph M.; Betz, William J.

    2007-01-01

    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 concen...

  14. Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

    Science.gov (United States)

    Barg, S; Knowles, M K; Chen, X; Midorikawa, M; Almers, Wolfhard

    2010-11-30

    Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

  15. Epigallocatechin gallate inhibits endothelial exocytosis.

    Science.gov (United States)

    Yamakuchi, Munekazu; Bao, Clare; Ferlito, Marcella; Lowenstein, Charles J

    2008-07-01

    Consumption of green tea is associated with a decrease in cardiovascular mortality. The beneficial health effects of green tea are attributed in part to polyphenols, organic compounds found in tea that lower blood pressure, reduce body fat, decrease LDL cholesterol, and inhibit inflammation. We hypothesized that epigallocatechin gallate (EGCG), the most abundant polyphenol in tea, inhibits endothelial exocytosis, the initial step in leukocyte trafficking and vascular inflammation. To test this hypothesis, we treated human umbilical-vein endothelial cells with EGCG and other polyphenols, and then measured endothelial exocytosis. We found that EGCG decreases endothelial exocytosis in a concentration-dependent manner, with the effects most prominent after 4 h of treatment. Other catechin polyphenols had no effect on endothelial cells. By inhibiting endothelial exocytosis, EGCG decreases leukocyte adherence to endothelial cells. In searching for the mechanism by which EGCG affects endothelial cells, we found that EGCG increases Akt phosphorylation, eNOS phosphorylation, and nitric oxide (NO) production. NOS inhibition revealed that NO mediates the anti-inflammatory effects of EGCG. Our data suggest that polyphenols can decrease vascular inflammation by increasing the synthesis of NO, which blocks endothelial exocytosis.

  16. DPP-4 inhibitor des-F-sitagliptin treatment increased insulin exocytosis from db/db mice {beta} cells

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-09

    Highlights: {yields} Anti-diabetic new drug, DPP-4 inhibitor, can affect the insulin exocytosis. {yields} DPP-4 inhibitor treatment altered syntaxin 1 expression. {yields} Treatment of db/db mice with DPP-4 inhibitor increased insulin release. -- Abstract: Incretin promotes insulin secretion acutely. Recently, orally-administered DPP-4 inhibitors represent a new class of anti-hyperglycemic agents. Indeed, inhibitors of dipeptidyl peptidase-IV (DPP-4), sitagliptin, has just begun to be widely used as therapeutics for type 2 diabetes. However, the effects of sitagliptin-treatment on insulin exocytosis from single {beta}-cells are yet unknown. We therefore investigated how sitagliptin-treatment in db/db mice affects insulin exocytosis by treating db/db mice with des-F-sitagliptin for 2 weeks. Perfusion studies showed that 2 weeks-sitagliptin treatment potentiated insulin secretion. We then analyzed insulin granule motion and SNARE protein, syntaxin 1, by TIRF imaging system. TIRF imaging of insulin exocytosis showed the increased number of docked insulin granules and increased fusion events from them during first-phase release. In accord with insulin exocytosis data, des-F-sitagliptin-treatment increased the number of syntaxin 1 clusters on the plasma membrane. Thus, our data demonstrated that 2-weeks des-F-sitagliptin-treatment increased the fusion events of insulin granules, probably via increased number of docked insulin granules and that of syntaxin 1 clusters.

  17. RNA granules

    OpenAIRE

    Anderson, Paul; Kedersha, Nancy

    2006-01-01

    Cytoplasmic RNA granules in germ cells (polar and germinal granules), somatic cells (stress granules and processing bodies), and neurons (neuronal granules) have emerged as important players in the posttranscriptional regulation of gene expression. RNA granules contain various ribosomal subunits, translation factors, decay enzymes, helicases, scaffold proteins, and RNA-binding proteins, and they control the localization, stability, and translation of their RNA cargo. We review the relationshi...

  18. Observation of Insulin Exocytosis by a Pancreatic (3 Cell Line with Total Internal Reflection Fluorescence Microscopy

    Institute of Scientific and Technical Information of China (English)

    Zhao-ying Fu; Ya-ping Wang; Yu Chen

    2011-01-01

    @@ INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radio-immunoassay.However,these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis.In recent years,an imaging technique known as total internal reflection fluorescence (TIRF) microscopy has been employed to study insulin secretion.1-4 This imaging technique can explore events taking place near or on live cell membrane,such as secretory granule movement,exocytosis,vesicle content release,and membrane fusion.5-10 In the present paper,we applied TIRF microscopy to the observation of insulin exocytosis by the pancreatic β cell line Ins-1.

  19. Phage lytic enzymes: a history

    Institute of Scientific and Technical Information of China (English)

    David; Trudil

    2015-01-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of ‘bacteria-eaters’ or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well(Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specifi c disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay(Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes–from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  20. Mutational analysis of VAMP domains implicated in Ca2+-induced insulin exocytosis.

    Science.gov (United States)

    Regazzi, R; Sadoul, K; Meda, P; Kelly, R B; Halban, P A; Wollheim, C B

    1996-01-01

    Vesicle-associated membrane protein-2 (VAMP-2) and cellubrevin are associated with the membrane of insulin-containing secretory granules and of gamma-aminobutyric acid (GABA)-containing synaptic-like vesicles of pancreatic beta-cells. We found that a point mutation in VAMP-2 preventing targeting to synaptic vesicles also impairs the localization on insulin-containing secretory granules, suggesting a similar requirement for vesicular targeting. Tetanus toxin (TeTx) treatment of permeabilized HIT-T15 cells leads to the proteolytic cleavage of VAMP-2 and cellubrevin and causes the inhibition of Ca2+-triggered insulin exocytosis. Transient transfection of HIT-T15 cells with VAMP-1, VAMP-2 or cellubrevin made resistant to the proteolytic action of TeTx by amino acid replacements in the cleavage site restored Ca2+-stimulated secretion. Wild-type VAMP-2, wild-type cellubrevin or a mutant of VAMP-2 resistant to TeTx but not targeted to secretory granules were unable to rescue Ca2+-evoked insulin release. The transmembrane domain and the N-terminal region of VAMP-2 were not essential for the recovery of stimulated exocytosis, but deletions preventing the binding to SNAP-25 and/or to syntaxin I rendered the protein inactive in the reconstitution assay. Mutations of putative phosphorylation sites or of negatively charged amino acids in the SNARE motif recognized by clostridial toxins had no effect on the ability of VAMP-2 to mediate Ca2+-triggered secretion. We conclude that: (i) both VAMP-2 and cellubrevin can participate in the exocytosis of insulin; (ii) the interaction of VAMP-2 with syntaxin and SNAP-25 is required for docking and/or fusion of secretory granules with the plasma membrane; and (iii) the phosphorylation of VAMP-2 is not essential for Ca2+-stimulated insulin exocytosis. Images PMID:9003771

  1. Actomyosin II contractility expels von Willebrand factor from Weibel-Palade bodies during exocytosis.

    Science.gov (United States)

    Nightingale, Thomas D; White, Ian J; Doyle, Emily L; Turmaine, Mark; Harrison-Lavoie, Kimberly J; Webb, Kathleen F; Cramer, Louise P; Cutler, Daniel F

    2011-08-22

    The study of actin in regulated exocytosis has a long history with many different results in numerous systems. A major limitation on identifying precise mechanisms has been the paucity of experimental systems in which actin function has been directly assessed alongside granule content release at distinct steps of exocytosis of a single secretory organelle with sufficient spatiotemporal resolution. Using dual-color confocal microscopy and correlative electron microscopy in human endothelial cells, we visually distinguished two sequential steps of secretagogue-stimulated exocytosis: fusion of individual secretory granules (Weibel-Palade bodies [WPBs]) and subsequent expulsion of von Willebrand factor (VWF) content. Based on our observations, we conclude that for fusion, WPBs are released from cellular sites of actin anchorage. However, once fused, a dynamic ring of actin filaments and myosin II forms around the granule, and actomyosin II contractility squeezes VWF content out into the extracellular environment. This study therefore demonstrates how discrete actin cytoskeleton functions within a single cellular system explain actin filament-based prevention and promotion of specific exocytic steps during regulated secretion.

  2. Characterisation of Weibel-Palade body fusion by amperometry in endothelial cells reveals fusion pore dynamics and the effect of cholesterol on exocytosis.

    Science.gov (United States)

    Cookson, Emma A; Conte, Ianina L; Dempster, John; Hannah, Matthew J; Carter, Tom

    2013-12-01

    Regulated secretion from endothelial cells is mediated by Weibel-Palade body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.

  3. Brief exposure to copper activates lysosomal exocytosis.

    Science.gov (United States)

    Peña, Karina; Coblenz, Jessica; Kiselyov, Kirill

    2015-04-01

    Copper (Cu) is essential mineral, but its toxicity necessitates existence of powerful machinery responsible for the extraction of excess Cu from the cell. Cu exposure was recently shown to induce the translocation of Cu pump ATP7B to the lysosomes followed by lysosomal exocytosis. Here we sought to investigate the mechanisms underlying the effect of Cu on lysosomal exocytosis. We found that brief exposure to Cu activates lysosomal exocytosis, which was measured as a release of the lysosomal digestive enzyme β-hexosaminidase (β-hex) into the extracellular medium and by the presence lysosomal protein LAMP1 at the plasma membrane. Such release depends on calcium (Ca) and on the lysosomal SNARE VAMP7. ATP7B knockdown using RNAi suppressed the basal lysosomal exocytosis, but did not affect the ability of Cu to activate it. ATP7B knockdown was associated with sustained oxidative stress. The removal of Ca from the extracellular medium suppressed the Cu-dependent component of the lysosomal exocytosis. We propose that Cu promotes lysosomal exocytosis by facilitating a Ca-dependent step of the lysosomal exocytosis.

  4. Munc18b is a major mediator of insulin exocytosis in rat pancreatic β-cells.

    Science.gov (United States)

    Lam, Patrick P L; Ohno, Mitsuyo; Dolai, Subhankar; He, Yu; Qin, Tairan; Liang, Tao; Zhu, Dan; Kang, Youhou; Liu, Yunfeng; Kauppi, Maria; Xie, Li; Wan, Wilson C Y; Bin, Na-Rhum; Sugita, Shuzo; Olkkonen, Vesa M; Takahashi, Noriko; Kasai, Haruo; Gaisano, Herbert Y

    2013-07-01

    Sec1/Munc18 proteins facilitate the formation of trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes that mediate fusion of secretory granule (SG) with plasma membrane (PM). The capacity of pancreatic β-cells to exocytose insulin becomes compromised in diabetes. β-Cells express three Munc18 isoforms of which the role of Munc18b is unknown. We found that Munc18b depletion in rat islets disabled SNARE complex formation formed by syntaxin (Syn)-2 and Syn-3. Two-photon imaging analysis revealed in Munc18b-depleted β-cells a 40% reduction in primary exocytosis (SG-PM fusion) and abrogation of almost all sequential SG-SG fusion, together accounting for a 50% reduction in glucose-stimulated insulin secretion (GSIS). In contrast, gain-of-function expression of Munc18b wild-type and, more so, dominant-positive K314L/R315L mutant promoted the assembly of cognate SNARE complexes, which caused potentiation of biphasic GSIS. We found that this was attributed to a more than threefold enhancement of both primary exocytosis and sequential SG-SG fusion, including long-chain fusion (6-8 SGs) not normally (2-3 SG fusion) observed. Thus, Munc18b-mediated exocytosis may be deployed to increase secretory efficiency of SGs in deeper cytosolic layers of β-cells as well as additional primary exocytosis, which may open new avenues of therapy development for diabetes.

  5. VAMP-8 segregates mast cell-preformed mediator exocytosis from cytokine trafficking pathways.

    Science.gov (United States)

    Tiwari, Neeraj; Wang, Cheng-Chun; Brochetta, Cristiana; Ke, Gou; Vita, Francesca; Qi, Zeng; Rivera, Juan; Soranzo, Maria Rosa; Zabucchi, Giuliano; Hong, Wanjin; Blank, Ulrich

    2008-04-01

    Inflammatory responses by mast cells are characterized by massive exocytosis of prestored granular mediators followed by cytokine/chemokine release. The vesicular trafficking mechanisms involved remain poorly understood. Vesicular-associated membrane protein-8 (VAMP-8), a member of the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) family of fusion proteins initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. Here we show that in bone marrow-derived mast cells (BMMCs) VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. This was associated with increased SNARE complex formation with the target t-SNAREs, SNAP-23 and syntaxin-4. VAMP-8-deficient BMMCs exhibited a markedly reduced degranulation response after IgE+ antigen-, thapsigargin-, or ionomycin-induced stimulation. VAMP-8-deficient mice also showed reduced plasma histamine levels in passive systemic anaphylaxis experiments, while cytokine/chemokine release was not affected. Unprocessed TNF accumulated at the plasma membrane where it colocalized with a VAMP-3-positive vesicular compartment but not with VAMP-8. The findings demonstrate that VAMP-8 segregates secretory lysosomal granule exocytosis in mast cells from cytokine/chemokine molecular trafficking pathways.

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

    Directory of Open Access Journals (Sweden)

    Ekkapongpisit M

    2012-04-01

    Full Text Available Maneerat Ekkapongpisit1,*, Antonino Giovia1,*, Giuseppina Nicotra1, Matteo Ozzano1, Giuseppe Caputo2,3, Ciro Isidoro1 1Laboratory of Molecular Pathology and Nanobioimaging, Department of Health Sciences, Università del Piemonte Orientale "A. Avogadro", Novara, Italy; 2Department of Chemistry, University of Turin, Turin, 3Cyanine Technology SpA, Torino, Italy *These authors contributed equally to this workBackground: 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.Keywords: secretory lysosomes, inflammation, nanoparticles, vesicular traffic

  7. Kinetics of MTT-formazan exocytosis in phagocytic and non-phagocytic cells.

    Science.gov (United States)

    Molinari, Beatriz L; Tasat, Deborah R; Palmieri, Mónica A; Cabrini, Rómulo L

    2005-01-01

    MTT is taken up by cells by endocytosis and reduced to formazan in the endosomal/lysosomal compartment. Formazan is deposited intracellularly as blue granules and is later exocytosed as needle-like formazan crystals. The present study involves an analysis of the pattern of exocytosis of MTT in different cell types showing clearcut differences in the response that can be associated to their ability to phagocytose. To further assess the characteristics of the exocytic mechanism of MTT/formazan, different experimental conditions were assayed. When culture medium with decreasing serum concentration was used as a metabolic modulator no variations were observed in the proportion of cells with formazan crystals. Conversely, the markedly sensitivity of phagocytic cells to increasing concentrations of genistein constituted a remarkable difference with non-phagocytic cells. These results must be considered when the modulation of MTT exocytosis is used as a signal of the progress of human diseases.

  8. Phentolamine inhibits exocytosis of glucagon by Gi2 protein-dependent activation of calcineurin in rat pancreatic alpha -cells.

    Science.gov (United States)

    Høy, M; Bokvist, K; Xiao-Gang, W; Hansen, J; Juhl, K; Berggren, P O; Buschard, K; Gromada, J

    2001-01-12

    Capacitance measurements were used to investigate the molecular mechanisms by which imidazoline compounds inhibit glucagon release in rat pancreatic alpha-cells. The imidazoline compound phentolamine reversibly decreased depolarization-evoked exocytosis >80% without affecting the whole-cell Ca(2+) current. During intracellular application through the recording pipette, phentolamine produced a concentration-dependent decrease in the rate of exocytosis (IC(50) = 9.7 microm). Another imidazoline compound, RX871024, exhibited similar effects on exocytosis (IC(50) = 13 microm). These actions were dependent on activation of pertussis toxin-sensitive G(i2) proteins but were not associated with stimulation of ATP-sensitive K(+) channels or adenylate cyclase activity. The inhibitory effect of phentolamine on exocytosis resulted from activation of the protein phosphatase calcineurin and was abolished by cyclosporin A and deltamethrin. Exocytosis was not affected by intracellular application of specific alpha(2), I(1), and I(2) ligands. Phentolamine reduced glucagon release (IC(50) = 1.2 microm) from intact islets by 40%, an effect abolished by pertussis toxin, cyclosporin A, and deltamethrin. These data suggest that imidazoline compounds inhibit glucagon secretion via G(i2)-dependent activation of calcineurin in the pancreatic alpha-cell. The imidazoline binding site is likely to be localized intracellularly and probably closely associated with the secretory granules.

  9. Pathogen recognition by Toll-like receptor 2 activates Weibel-Palade body exocytosis in human aortic endothelial cells.

    Science.gov (United States)

    Into, Takeshi; Kanno, Yosuke; Dohkan, Jun-ichi; Nakashima, Misako; Inomata, Megumi; Shibata, Ken-ichiro; Lowenstein, Charles J; Matsushita, Kenji

    2007-03-16

    The endothelial cell-specific granule Weibel-Palade body releases vasoactive substances capable of modulating vascular inflammation. Although innate recognition of pathogens by Toll-like receptors (TLRs) is thought to play a crucial role in promotion of inflammatory responses, the molecular basis for early-phase responses of endothelial cells to bacterial pathogens has not fully been understood. We here report that human aortic endothelial cells respond to bacterial lipoteichoic acid (LTA) and synthetic bacterial lipopeptides, but not lipopolysaccharide or peptidoglycan, to induce Weibel-Palade body exocytosis, accompanied by release or externalization of the storage components von Willebrand factor and P-selectin. LTA could activate rapid Weibel-Palade body exocytosis through a TLR2- and MyD88-dependent mechanism without de novo protein synthesis. This process was at least mediated through MyD88-dependent phosphorylation and activation of phospholipase Cgamma. Moreover, LTA activated interleukin-1 receptor-associated kinase-1-dependent delayed exocytosis with de novo protein synthesis and phospholipase Cgamma-dependent activation of the NF-kappaB pathway. Increased TLR2 expression by transfection or interferon-gamma treatment increased TLR2-mediated Weibel-Palade body exocytosis, whereas reduced TLR2 expression under laminar flow decreased the response. Thus, we propose a novel role for TLR2 in induction of a primary proinflammatory event in aortic endothelial cells through Weibel-Palade body exocytosis, which may be an important step for linking innate recognition of bacterial pathogens to vascular inflammation.

  10. Distinct actions of Rab3 and Rab27 GTPases on late stages of exocytosis of insulin.

    Science.gov (United States)

    Cazares, Victor A; Subramani, Arasakumar; Saldate, Johnny J; Hoerauf, Widmann; Stuenkel, Edward L

    2014-09-01

    Rab GTPases associated with insulin-containing secretory granules (SGs) are key in targeting, docking and assembly of molecular complexes governing pancreatic β-cell exocytosis. Four Rab3 isoforms along with Rab27A are associated with insulin granules, yet elucidation of the distinct roles of these Rab families on exocytosis remains unclear. To define specific actions of these Rab families we employ Rab3GAP and/or EPI64A GTPase-activating protein overexpression in β-cells from wild-type or Ashen mice to selectively transit the entire Rab3 family or Rab27A to a GDP-bound state. Ashen mice carry a spontaneous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Rab27A is essential for generation of the functionally defined immediately releasable pool (IRP) and central to regulating the size of the readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 GTPases, but not of Rab27A, is essential for a kinetically rapid filling of the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A GTPases demonstrate considerable functional overlap in building the readily releasable granule pool. Hence, while Rab3 and Rab27A cooperate to generate release-ready SGs in β-cells, they also direct unique kinetic and functional properties of the exocytotic pathway.

  11. The GTPase Rab37 Participates in the Control of Insulin Exocytosis.

    Directory of Open Access Journals (Sweden)

    Sanda Ljubicic

    Full Text Available Rab37 belongs to a subclass of Rab GTPases regulating exocytosis, including also Rab3a and Rab27a. Proteomic studies indicate that Rab37 is associated with insulin-containing large dense core granules of pancreatic β-cells. In agreement with these observations, we detected Rab37 in extracts of β-cell lines and human pancreatic islets and confirmed by confocal microscopy the localization of the GTPase on insulin-containing secretory granules. We found that, as is the case for Rab3a and Rab27a, reduction of Rab37 levels by RNA interference leads to impairment in glucose-induced insulin secretion and to a decrease in the number of granules in close apposition to the plasma membrane. Pull-down experiments revealed that, despite similar functional effects, Rab37 does not interact with known Rab3a or Rab27a effectors and is likely to operate through a different mechanism. Exposure of insulin-secreting cells to proinflammatory cytokines, fatty acids or oxidized low-density lipoproteins, mimicking physiopathological conditions that favor the development of diabetes, resulted in a decrease in Rab37 expression. Our data identify Rab37 as an additional component of the machinery governing exocytosis of β-cells and suggest that impaired expression of this GTPase may contribute to defective insulin release in pre-diabetic and diabetic conditions.

  12. Localization of MTT formazan in lipid droplets. An alternative hypothesis about the nature of formazan granules and aggregates

    OpenAIRE

    G. Diaz; Melis, M; Musinu, A; M Piludu; Piras, M; AM Falchi

    2009-01-01

    MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dihphenyltetrazolium bromide) assay is a widely used method to assess cell viability and proliferation. MTT is readily taken up by cells and enzymatically reduced to formazan, a dark compound which accumulates in cytoplasmic granules. Formazan is later eliminated by the cell by a mechanisms often indicated as exocytosis, that produces characteristic needle-like aggregates on the cell surface. The shape of formazan aggregates and the rate of exocytosis c...

  13. Polyamines are present in mast cell secretory granules and are important for granule homeostasis.

    Directory of Open Access Journals (Sweden)

    Gianni García-Faroldi

    Full Text Available BACKGROUND: Mast cell secretory granules accommodate a large number of components, many of which interact with highly sulfated serglycin proteoglycan (PG present within the granules. Polyamines (putrescine, spermidine and spermine are absolutely required for the survival of the vast majority of living cells. Given the reported ability of polyamines to interact with PGs, we investigated the possibility that polyamines may be components of mast cell secretory granules. METHODOLOGY/PRINCIPAL FINDINGS: Spermidine was released by mouse bone marrow derived mast cells (BMMCs after degranulation induced by IgE/anti-IgE or calcium ionophore A23187. Additionally, both spermidine and spermine were detected in isolated mouse mast cell granules. Further, depletion of polyamines by culturing BMMCs with α-difluoromethylornithine (DFMO caused aberrant secretory granule ultrastructure, impaired histamine storage, reduced serotonin levels and increased β-hexosaminidase content. A proteomic approach revealed that DFMO-induced polyamine depletion caused an alteration in the levels of a number of proteins, many of which are connected either with the regulated exocytosis or with the endocytic system. CONCLUSIONS/SIGNIFICANCE: Taken together, our results show evidence that polyamines are present in mast cell secretory granules and, furthermore, indicate an essential role of these polycations during the biogenesis and homeostasis of these organelles.

  14. SNARE Requirements En Route to Exocytosis

    DEFF Research Database (Denmark)

    Mohrmann, Ralf; Sørensen, Jakob Balslev

    2012-01-01

    and their complexes before and during vesicle exocytosis is still limited. Here, we review recent progress in this expanding field with emphasis on the question of fusion complex stoichiometry, i.e., how many SNARE proteins and complexes are needed for the fusion of a vesicle with the plasma membrane....

  15. Eosinophil secretion of granule-derived cytokines

    Directory of Open Access Journals (Sweden)

    Lisa A Spencer

    2014-10-01

    Full Text Available 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.

  16. Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

    Science.gov (United States)

    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.

  17. Twin screw granulation: steps in granule growth.

    Science.gov (United States)

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

    2012-11-15

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

  18. Lytic to temperate switching of viral communities

    Science.gov (United States)

    Knowles, B.; Silveira, C. B.; Bailey, B. A.; Barott, K.; Cantu, V. A.; Cobián-Güemes, A. G.; Coutinho, F. H.; Dinsdale, E. A.; Felts, B.; Furby, K. A.; George, E. E.; Green, K. T.; Gregoracci, G. B.; Haas, A. F.; Haggerty, J. M.; Hester, E. R.; Hisakawa, N.; Kelly, L. W.; Lim, Y. W.; Little, M.; Luque, A.; McDole-Somera, T.; McNair, K.; de Oliveira, L. S.; Quistad, S. D.; Robinett, N. L.; Sala, E.; Salamon, P.; Sanchez, S. E.; Sandin, S.; Silva, G. G. Z.; Smith, J.; Sullivan, C.; Thompson, C.; Vermeij, M. J. A.; Youle, M.; Young, C.; Zgliczynski, B.; Brainard, R.; Edwards, R. A.; Nulton, J.; Thompson, F.; Rohwer, F.

    2016-03-01

    Microbial viruses can control host abundances via density-dependent lytic predator-prey dynamics. Less clear is how temperate viruses, which coexist and replicate with their host, influence microbial communities. Here we show that virus-like particles are relatively less abundant at high host densities. This suggests suppressed lysis where established models predict lytic dynamics are favoured. Meta-analysis of published viral and microbial densities showed that this trend was widespread in diverse ecosystems ranging from soil to freshwater to human lungs. Experimental manipulations showed viral densities more consistent with temperate than lytic life cycles at increasing microbial abundance. An analysis of 24 coral reef viromes showed a relative increase in the abundance of hallmark genes encoded by temperate viruses with increased microbial abundance. Based on these four lines of evidence, we propose the Piggyback-the-Winner model wherein temperate dynamics become increasingly important in ecosystems with high microbial densities; thus ‘more microbes, fewer viruses’.

  19. Optical control of calcium-regulated exocytosis.

    Science.gov (United States)

    Izquierdo-Serra, Mercè; Trauner, Dirk; Llobet, Artur; Gorostiza, Pau

    2013-03-01

    Neurons signal to each other and to non-neuronal cells as those in muscle or glands, by means of the secretion of neurotransmitters at chemical synapses. In order to dissect the molecular mechanisms of neurotransmission, new methods for directly and reversibly triggering neurosecretion at the presynaptic terminal are necessary. Here we exploit the calcium permeability of the light-gated channel LiGluR in order to reversibly manipulate cytosolic calcium concentration, thus controlling calcium-regulated exocytosis. Bovine chromaffin cells expressing LiGluR were stimulated with light. Exocytic events were detected by amperometry or by whole-cell patch-clamp to quantify membrane capacitance and calcium influx. Amperometry reveals that optical stimulation consistently triggers exocytosis in chromaffin cells. Secretion of catecholamines can be adjusted between zero and several Hz by changing the wavelength of illumination. Differences in secretion efficacy are found between the activation of LiGluR and native voltage-gated calcium channels (VGCCs). Our results show that the distance between sites of calcium influx and vesicles ready to be released is longer when calcium influx is triggered by LiGluR instead of native VGCCs. LiGluR activation directly and reversibly increases the intracellular calcium concentration. Light-gated calcium influx allows for the first time to control calcium-regulated exocytosis without the need of applying depolarizing solutions or voltage clamping in chromaffin cells. LiGluR is a useful tool to study the secretory mechanisms and their spatiotemporal patterns in neurotransmission, and opens a window to study other calcium-dependent processes such as muscular contraction or cell migration.

  20. Biphasic regulation of lysosomal exocytosis by oxidative stress.

    Science.gov (United States)

    Ravi, Sreeram; Peña, Karina A; Chu, Charleen T; Kiselyov, Kirill

    2016-11-01

    Oxidative stress drives cell death in a number of diseases including ischemic stroke and neurodegenerative diseases. A better understanding of how cells recover from oxidative stress is likely to lead to better treatments for stroke and other diseases. The recent evidence obtained in several models ties the process of lysosomal exocytosis to the clearance of protein aggregates and toxic metals. The mechanisms that regulate lysosomal exocytosis, under normal or pathological conditions, are only beginning to emerge. Here we provide evidence for the biphasic effect of oxidative stress on lysosomal exocytosis. Lysosomal exocytosis was measured using the extracellular levels of the lysosomal enzyme beta-hexosaminidase (ß-hex). Low levels or oxidative stress stimulated lysosomal exocytosis, but inhibited it at high levels. Deletion of the lysosomal ion channel TRPML1 eliminated the stimulatory effect of low levels of oxidative stress. The inhibitory effects of oxidative stress appear to target the component of lysosomal exocytosis that is driven by extracellular Ca(2+). We propose that while moderate oxidative stress promotes cellular repair by stimulating lysosomal exocytosis, at high levels oxidative stress has a dual pathological effect: it directly causes cell damage and impairs damage repair by inhibiting lysosomal exocytosis. Harnessing these adaptive mechanisms may point to pharmacological interventions for diseases involving oxidative proteotoxicity or metal toxicity.

  1. Exocytosis and endocytosis: modes, functions, and coupling mechanisms.

    Science.gov (United States)

    Wu, Ling-Gang; Hamid, Edaeni; Shin, Wonchul; Chiang, Hsueh-Cheng

    2014-01-01

    Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis.

  2. Lytic clavicular lesions in fibromatosis colli

    Energy Technology Data Exchange (ETDEWEB)

    Sartoris, D.J.; Parker, B.R.; Mochizuki, R.M.

    1983-06-01

    Two patients with fibromatosis colli (congenital torticollis) presented with lytic lesions in the clavicle at the insertion of the fibrosed clavicular head of the sternocleidomastoid muscle. Biopsy of one lesion showed intraosseous fibrosis. These lesions are probably not uncommon but radiographs are rarely performed in uncomplicated cases.

  3. Epac, Rap and Rab3 act in concert to mobilize calcium from sperm's acrosome during exocytosis.

    Science.gov (United States)

    Ruete, María C; Lucchesi, Ornella; Bustos, Matías A; Tomes, Claudia N

    2014-08-27

    Exocytosis of sperm's single secretory granule or acrosome (acrosome reaction, AR) is a highly regulated event essential for fertilization. The AR begins with an influx of calcium from the extracellular milieu and continues with the synthesis of cAMP and the activation of its target Epac. The cascade bifurcates into a Rab3-GTP-driven limb that assembles the fusion machinery and a Rap-GTP-driven limb that mobilizes internal calcium. To understand the crosstalk between the two signaling cascades, we applied known AR inhibitors in three experimental approaches: reversible, stage-specific blockers in a functional assay, a far-immunofluorescence protocol to detect active Rab3 and Rap, and single cell-confocal microscopy to visualize fluctuations in internal calcium stores. Our model system was human sperm with their plasma membrane permeabilized with streptolysin O and stimulated with external calcium. The inhibition caused by reagents that prevented the activation of Rap was reversed by mobilizing intracellular calcium pharmacologically, whereas that caused by AR inhibitors that impeded Rab3's binding to GTP was not. Both limbs of the exocytotic cascade joined at or near the stage catalyzed by Rab3 in a unidirectional, hierarchical connection in which the intra-acrosomal calcium mobilization arm was subordinated to the fusion protein arm; somewhere after Rab3, the pathways became independent. We delineated the sequence of events that connect an external calcium signal to internal calcium mobilization during exocytosis. We have taken advantage of the versatility of the sperm model to investigate how cAMP, calcium, and the proteinaceous fusion machinery coordinate to accomplish secretion. Because the requirement of calcium from two different sources is not unique to sperm and fusion proteins are highly conserved, our findings might contribute to elucidate mechanisms that operate in regulated exocytosis in other secretory cell types.

  4. Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

    Science.gov (United States)

    Chang, Hsin-Fang; Bzeih, Hawraa; Schirra, Claudia; Chitirala, Praneeth; Halimani, Mahantappa; Cordat, Emmanuelle; Krause, Elmar; Rettig, Jens; Pattu, Varsha

    2016-09-15

    CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

  5. The voltage-gated proton channel Hv1/VSOP inhibits neutrophil granule release.

    Science.gov (United States)

    Okochi, Yoshifumi; Aratani, Yasuaki; Adissu, Hibret A; Miyawaki, Nana; Sasaki, Mari; Suzuki, Kazuo; Okamura, Yasushi

    2016-01-01

    Neutrophil granule exocytosis is crucial for host defense and inflammation. Neutrophils contain 4 types of granules, the exocytotic release of which is differentially regulated. This exocytosis is known to be driven by diverse mediators, including calcium and nucleotides, but the precise molecular mechanism remains largely unknown. We show in the present study that voltage-gated proton (Hv) channels are necessary for the proper release of azurophilic granules in neutrophils. On activation of NADPH oxidase by PMA and IgG, neutrophils derived from Hvcn1 gene knockout mouse exhibited greater secretion of MPO and elastase than WT cells. In contrast, release of LTF enriched in specific granules was not enhanced in these cells. The excess release of azurophilic granules in Hv1/VSOP-deficient neutrophils was suppressed by inhibiting NADPH oxidase activity and, in part, by valinomycin, a potassium ionophore. In addition, Hv1/VSOP-deficient mice exhibited more severe lung inflammation after intranasal Candida albicans infection than WT mice. These findings suggest that the Hv channel acts to specifically dampen the release of azurophilic granules through, in part, the suppression of increased positive charges at the plasma membrane accompanied by the activation of NADPH oxidase in neutrophils. © Society for Leukocyte Biology.

  6. Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis.

    Science.gov (United States)

    Lee, Tae-Sun; Lee, Joo-Young; Kyung, Jae Won; Yang, Yoosoo; Park, Seung Ju; Lee, Seulgi; Pavlovic, Igor; Kong, Byoungjae; Jho, Yong Seok; Jessen, Henning J; Kweon, Dae-Hyuk; Shin, Yeon-Kyun; Kim, Sung Hyun; Yoon, Tae-Young; Kim, Seyun

    2016-07-19

    Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6 Synaptotagmin 1 (Syt1), a Ca(2+) sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7 Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6 In addition, 5-IP7-dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca(2+) levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca(2+) These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.

  7. Granulation of fine powder

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ching-Fong

    2016-08-09

    A mixture of fine powder including thorium oxide was converted to granulated powder by forming a first-green-body and heat treating the first-green-body at a high temperature to strengthen the first-green-body followed by granulation by crushing or milling the heat-treated first-green-body. The granulated powder was achieved by screening through a combination of sieves to achieve the desired granule size distribution. The granulated powder relies on the thermal bonding to maintain its shape and structure. The granulated powder contains no organic binder and can be stored in a radioactive or other extreme environment. The granulated powder was pressed and sintered to form a dense compact with a higher density and more uniform pore size distribution.

  8. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Strobel, Claudia, E-mail: Claudia.Strobel@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany); Oehring, Hartmut [Jena University Hospital – Friedrich Schiller University Jena, Institute of Anatomy II (Germany); Herrmann, Rudolf [University of Augsburg, Department of Physics (Germany); Förster, Martin [Jena University Hospital – Friedrich Schiller University Jena, Department of Internal Medicine I, Division of Pulmonary Medicine and Allergy/Immunology (Germany); Reller, Armin [University of Augsburg, Department of Physics (Germany); Hilger, Ingrid, E-mail: ingrid.hilger@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany)

    2015-05-15

    Although cytotoxicity and endocytosis of nanoparticles have been the subject of numerous studies, investigations regarding exocytosis as an important mechanism to reduce intracellular nanoparticle accumulation are rather rare and there is a distinct lack of knowledge. The current study investigated the behavior of human microvascular endothelial cells to exocytose cerium dioxide (CeO{sub 2}) nanoparticles (18.8 nm) by utilization of specific inhibitors [brefeldin A; nocodazole; methyl-β-cyclodextrin (MβcD)] and different analytical methods (flow cytometry, transmission electron microscopy, inductively coupled plasma mass spectrometry). Overall, it was found that endothelial cells were able to release CeO{sub 2} nanoparticles via exocytosis after the migration of nanoparticle containing endosomes toward the plasma membrane. The exocytosis process occurred mainly by fusion of vesicular membranes with plasma membrane resulting in the discharge of vesicular content to extracellular environment. Nevertheless, it seems to be likely that nanoparticles present in the cytosol could leave the cells in a direct manner. MβcD treatment led to the strongest inhibition of the nanoparticle exocytosis indicating a significant role of the plasma membrane cholesterol content in the exocytosis process. Brefeldin A (inhibitor of Golgi-to-cell-surface-transport) caused a higher inhibitory effect on exocytosis than nocodazole (inhibitor of microtubules). Thus, the transfer from distal Golgi compartments to the cell surface influenced the exocytosis process of the CeO{sub 2} nanoparticles more than the microtubule-associated transport. In conclusion, endothelial cells, which came in contact with nanoparticles, e.g., after intravenously applied nano-based drugs, can regulate their intracellular nanoparticle amount, which is necessary to avoid adverse nanoparticle effects on cells.

  9. Lipids implicated in the journey of a secretory granule: from biogenesis to fusion.

    Science.gov (United States)

    Tanguy, Emeline; Carmon, Ophélie; Wang, Qili; Jeandel, Lydie; Chasserot-Golaz, Sylvette; Montero-Hadjadje, Maité; Vitale, Nicolas

    2016-06-01

    The regulated secretory pathway begins with the formation of secretory granules by budding from the Golgi apparatus and ends by their fusion with the plasma membrane leading to the release of their content into the extracellular space, generally following a rise in cytosolic calcium. Generation of these membrane-bound transport carriers can be classified into three steps: (i) cargo sorting that segregates the cargo from resident proteins of the Golgi apparatus, (ii) membrane budding that encloses the cargo and depends on the creation of appropriate membrane curvature, and (iii) membrane fission events allowing the nascent carrier to separate from the donor membrane. These secretory vesicles then mature as they are actively transported along microtubules toward the cortical actin network at the cell periphery. The final stage known as regulated exocytosis involves the docking and the priming of the mature granules, necessary for merging of vesicular and plasma membranes, and the subsequent partial or total release of the secretory vesicle content. Here, we review the latest evidence detailing the functional roles played by lipids during secretory granule biogenesis, recruitment, and exocytosis steps. In this review, we highlight evidence supporting the notion that lipids play important functions in secretory vesicle biogenesis, maturation, recruitment, and membrane fusion steps. These effects include regulating various protein distribution and activity, but also directly modulating membrane topology. The challenges ahead to understand the pleiotropic functions of lipids in a secretory granule's journey are also discussed. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

  10. Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules

    Science.gov (United States)

    Han, Xiaofan; Li, Pin; Yang, Zhenghao; Huang, Xiaoshuai; Wei, Guoqin; Sun, Yujie; Kang, Xuya; Hu, Xueting; Deng, Qiuping; Chen, Liangyi; He, Aibin; Huo, Yingqing; Li, Dong; Betzig, Eric; Luo, Jincai

    2017-01-01

    Endothelial exocytosis of Weibel–Palade body (WPB) is one of the first lines of defence against vascular injury. However, the mechanisms that control WPB exocytosis in the final stages (including the docking, priming and fusion of granules) are poorly understood. Here we show that the focal adhesion protein zyxin is crucial in this process. Zyxin downregulation inhibits the secretion of von Willebrand factor (VWF), the most abundant cargo in WPBs, from human primary endothelial cells (ECs) induced by cAMP agonists. Zyxin-deficient mice exhibit impaired epinephrine-stimulated VWF release, prolonged bleeding time and thrombosis, largely due to defective endothelial secretion of VWF. Using live-cell super-resolution microscopy, we visualize previously unappreciated reorganization of pre-existing actin filaments around WPBs before fusion, dependent on zyxin and an interaction with the actin crosslinker α-actinin. Our findings identify zyxin as a physiological regulator of endothelial exocytosis through reorganizing local actin network in the final stage of exocytosis. PMID:28256511

  11. Mining the granule proteome

    DEFF Research Database (Denmark)

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

    2015-01-01

    Proteomics of secretory granules is an emerging strategy for identifying secreted proteins, including potentially novel candidate biomarkers and peptide hormones. In addition, proteomics can provide information about the abundance, localization and structure (post-translational modification) of g...

  12. Localization of MTT formazan in lipid droplets. An alternative hypothesis about the nature of formazan granules and aggregates

    Directory of Open Access Journals (Sweden)

    G Diaz

    2009-08-01

    Full Text Available MTT (3-(4, 5-dimethyl-2-thiazolyl-2, 5-dihphenyltetrazolium bromide assay is a widely used method to assess cell viability and proliferation. MTT is readily taken up by cells and enzymatically reduced to formazan, a dark compound which accumulates in cytoplasmic granules. Formazan is later eliminated by the cell by a mechanisms often indicated as exocytosis, that produces characteristic needle-like aggregates on the cell surface. The shape of formazan aggregates and the rate of exocytosis change in the presence of bioactive amyloid b peptides (Ab and cholesterol. Though the cellular mechanisms involved in MTT reduction have been extensively investigated, the exact nature of formazan granules and the process of exocytosis are still obscure. Using Nile Red, which stains differentially neutral and polar lipids, and a fluorescent analog of cholesterol (NBD-cholesterol, we found that formazan localized in lipid droplets, consistent with the lipophilic nature of formazan. However, formazan granules and aggregates were also found to form after killing cells with paraformaldehyde fixation. Moreover, formazan aggregates were also obtained in cell-free media, using ascorbic acid to reduce MTT. The density and shape of formazan aggregates obtained in cell-free media was sensitive to cholesterol and Ab. In cells, electron microscopy failed to detect the presence of secretory vesicles, but revealed unusual fibers of 50 nm of diameter extending throughout the cytoplasm. Taken together, these findings suggest that formazan efflux is driven by physico-chemical interactions at molecular level without involving higher cytological mechanisms.

  13. Localization of MTT formazan in lipid droplets. An alternative hypothesis about the nature of formazan granules and aggregates.

    Science.gov (United States)

    Diaz, Giacomo; Melis, M; Musin, A; Piludu, M; Piras, M; Falchi, A M

    2007-01-01

    MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dihphenyltetrazolium bromide) assay is a widely used method to assess cell viability and proliferation. MTT is readily taken up by cells and enzymatically reduced to formazan, a dark compound which accumulates in cytoplasmic granules. Formazan is later eliminated by the cell by a mechanisms often indicated as exocytosis, that produces characteristic needle-like aggregates on the cell surface. The shape of formazan aggregates and the rate of exocytosis change in the presence of bioactive amyloid beta peptides (Abeta) and cholesterol. Though the cellular mechanisms involved in MTT reduction have been extensively investigated, the exact nature of formazan granules and the process of exocytosis are still obscure. Using Nile Red, which stains differentially neutral and polar lipids, and a fluorescent analog of cholesterol (NBD-cholesterol), we found that formazan localized in lipid droplets, consistent with the lipophilic nature of formazan. However, formazan granules and aggregates were also found to form after killing cells with paraformaldehyde fixation. Moreover, formazan aggregates were also obtained in cell-free media, using ascorbic acid to reduce MTT. The density and shape of formazan aggregates obtained in cell-free media was sensitive to cholesterol and Abeta. In cells, electron microscopy failed to detect the presence of secretory vesicles, but revealed unusual fibers of 50 nm of diameter extending throughout the cytoplasm. Taken together, these findings suggest that formazan efflux is driven by physico-chemical interactions at molecular level without involving higher cytological mechanisms.

  14. The nature of exocytosis in the yolk trophoblastic layer of silver arowana (Osteoglossum bicirrhosum) juvenile, the representative of ancient teleost fishes.

    Science.gov (United States)

    Jaroszewska, Marta; Dabrowski, Konrad

    2009-11-01

    We have chosen the silver arowana (Osteoglossum bicirrhosum), a representative of the most ancient teleost family Osteoglossidae, to address the question of yolk nutrients utilization. Silver arowana have particularly large eggs (1-1.5 cm of diameter) and a unique morphology of the yolk. We present evidence that the yolk cytoplasmic zone (ycz) in the "yolksac juveniles" is a very complex structure involved in sequential processes of yolk hydrolysis, lipoprotein particles synthesis, their transport, and exocytosis. Vacuoles filled with yolk granules in different stages of digestion move from the vitellolysis zone through the ycz to be emptied into the microvillar interspace in the process of exocytosis. The area of the ycz with the abundance of the mitochondria must play an important role in providing energy for both the transport of vacuoles and the release of their contents. Therefore, we postulate that the function of yolk syncytial layer (ysl) as the "early embryonic patterning center" transforms in fish larvae or yolksac juveniles into a predominantly specialized role as the yolk trophoblastic layer (ytl) involved in yolk nutrients utilization. In addition to discovering the mechanism of transformation of the ysl function into ytl function, we suggest that the machinery involved in nutrient mobilization and exocytosis in yolk of arowana yolksac juveniles can be very attractive system for studies of regulatory processes in almost all secretory pathways in animal cells.

  15. The relation between granule size, granule stickiness, and torque in the high-shear granulation process

    NARCIS (Netherlands)

    Bouwman, A.M.; Henstra, M.J.; Hegge, J.J.M.E.; Zhang, Z.; Ingram, A.; Seville, J.P.K.; Frijlink, H.W.

    2005-01-01

    Purpose. To investigate the background of the observed relationship between measured torque and granule size in high-shear granulation processes. Methods. Torque was measured during the granulation process; the behavior of individual wet granules during compaction was investigated using micromanipul

  16. Viral reproductive strategies: How can lytic viruses be evolutionarily competitive?

    Science.gov (United States)

    Komarova, Natalia L

    2007-12-21

    Viral release strategies can be roughly classified as lytic (the ones that accumulate inside the host cell and exit in a burst, killing the cell), and budding (the ones that are produced and released from the host cell gradually). Here we study the evolutionary competition between the two strategies. If all the parameters, such as the rate of viral production, cell life-span and the neutralizing capacity of the antibodies, were the same for lytic and budding viruses, the budding life-strategy would have a large evolutionary advantage. The question arises what makes lytic viruses evolutionarily competitive. We propose that it is the different removal capacity of the antibodies against budding and lytic virions. The latter exit the cell in a large burst such that the antibodies are "flooded" and a larger proportion of virions can escape the immune system and spread to new cells. We create two spatial models of virus-antibody interaction and show that for realistic parameter values, the effect of antibody flooding can indeed take place. We also argue that the lytic life cycle, including a relatively large burst-size, has evolved to promote survival in the face of antibody attack. According to the calculations, in the absence of efficient antibodies, the optimal burst size of lytic viruses would be only a few virus particles, as opposed to the observed 10(2)-10(5) viral particles. Similarly, there is an evolutionary pressure to extend the life-span as a response to antibody action.

  17. ATP storage and uptake by isolated pancreatic zymogen granules

    DEFF Research Database (Denmark)

    Haanes, Kristian Agmund; Novak, Ivana

    2010-01-01

    ATP is released from pancreatic acini in response to cholinergic and hormonal stimulation. The same stimuli cause exocytosis of ZG (zymogen granules) and release of digestive enzymes. The aim of the present study was to determine whether ZG stored ATP and to characterize the uptake mechanism...... for ATP transport into the ZG. ZG were isolated and the ATP content was measured using luciferin/luciferase assays and was related to protein in the sample. The estimate of ATP concentration in freshly isolated granules was 40-120 µM. The ATP uptake had an apparent Km value of 4.9±2.1 mM when granules...... were incubated without Mg2+ and a Km value of 0.47±0.05 mM in the presence of Mg2+, both in pH 6.0 buffers. The uptake of ATP was significantly higher at pH 7.2 compared with pH 6.0 solutions. The anion transport blockers DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate) and Evans Blue inhibited ATP...

  18. In vitro secretion of zymogens by bovine pancreatic acini and ultra-structural analysis of exocytosis

    Directory of Open Access Journals (Sweden)

    Sivalingam Jayaveni

    2016-03-01

    Full Text Available The aim of this study is to establish a bovine pancreatic acinar cell culture model with longer viability and functionality. The cells could be maintained in a functional state for upto 20 days with normal morphology. Cells were positive for amylase as observed by immunofluorescence staining. Acinar cells are spherical and range about 2–3 µm in diameter. The porosome formed by exocytosis and heterogenous enzyme granules of size ranging 100–300 nm were seen on the surface of cells by electron microscopy. The activity of the enzymes was high on day 15 and the activity profile of the enzymes is in the order: protease>lipase>amylase and the enzymes were identified by SDS-PAGE. Long-term culture of bovine pancreatic acini could be useful in studying the pathogenesis of pancreatitis. Since the bovine genome shares about 80% identity with the human genome, the cells derived from bovine pancreas can be engineered and used as a potential xenotransplant to treat conditions like pancreatitis as the tissue source is abundantly available.

  19. The intravesicular cocktail and its role in the regulation of exocytosis.

    Science.gov (United States)

    Estévez-Herrera, Judith; González-Santana, Ayoze; Baz-Dávila, Rebeca; Machado, José D; Borges, Ricardo

    2016-06-01

    The accumulation of neurotransmitters within secretory vesicles (SVs) far exceeds the theoretical tonic concentrations in the cytosol, a phenomenon that has captivated the attention of scientists for decades. For instance, chromaffin granules can accumulate close to molar concentrations of catecholamines, along with many other products like ATP, calcium, peptides, chromogranins, ascorbate, and other nucleotides. In this short review, we will summarize the interactions that are currently believed to occur between the elements that make up the vesicular cocktail in the acidic environment of SVs, and how they permit the accumulation of such high concentrations of certain components. In addition, we will examine how the vesicular cocktail regulates the exocytosis of neurotransmitters. In this review, we have highlighted the mechanisms that permit the storage of neurotransmitters and hormones inside secretory vesicles. We also have proposed a novel model based in the intravesicular interactions of the main components of this inner cocktail - catecholamines, ATP, and chromogranins - to allow the accumulation of near molar concentrations of transmitters in secretory vesicles. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

  20. Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

    Energy Technology Data Exchange (ETDEWEB)

    Somanath, Sangeeta [Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT (United Kingdom); Partridge, Christopher J. [Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and Churchill Hospital, University of Oxford, Oxford, OX3 7LJ (United Kingdom); Marshall, Catriona [Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT (United Kingdom); Rowe, Tony [CSL Limited, 45 Poplar Road, Parkville, Victoria 3052 (Australia); Turner, Mark D., E-mail: mark.turner@ntu.ac.uk [Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, NG11 8NS (United Kingdom)

    2016-04-29

    Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation. - Highlights: • Snapin mediates granule docking. • Snapin binds SNAP-25. • SNARE complex forms downstream.

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

    Science.gov (United States)

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

    2014-10-01

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

  2. Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy

    Science.gov (United States)

    Zhang, Jinxie; Zhang, Xudong; Liu, Gan; Chang, Danfeng; Liang, Xin; Zhu, Xianbing; Tao, Wei; Mei, Lin

    2016-01-01

    The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine. PMID:27698943

  3. Dynamics of SNARE assembly and disassembly during sperm acrosomal exocytosis.

    Directory of Open Access Journals (Sweden)

    Gerardo A De Blas

    2005-10-01

    Full Text Available The dynamics of SNARE assembly and disassembly during membrane recognition and fusion is a central issue in intracellular trafficking and regulated secretion. Exocytosis of sperm's single vesicle--the acrosome--is a synchronized, all-or-nothing process that happens only once in the life of the cell and depends on activation of both the GTP-binding protein Rab3 and of neurotoxin-sensitive SNAREs. These characteristics make acrosomal exocytosis a unique mammalian model for the study of the different phases of the membrane fusion cascade. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca2+ chelator we show that, in unactivated sperm, SNAREs are locked in heterotrimeric cis complexes. Upon Ca2+ entry into the cytoplasm, Rab3 is activated and triggers NSF/alpha-SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose trans complexes that are resistant to NSF/alpha-SNAP and differentially sensitive to cleavage by two vesicle-associated membrane protein (VAMP-specific neurotoxins. Ca2+ must be released from inside the acrosome to trigger the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis.

  4. Granule-Dependent Natural Killer Cell Cytotoxicity to Fungal Pathogens

    Science.gov (United States)

    Ogbomo, Henry; Mody, Christopher H.

    2017-01-01

    Natural killer (NK) cells kill or inhibit the growth of a number of fungi including Cryptococcus, Candida, Aspergillus, Rhizopus, and Paracoccidioides. Although many fungi are not dangerous, invasive fungal pathogens, such as Cryptococcus neoformans, cause life-threatening disease in individuals with impaired cell-mediated immunity. While there are similarities to cell-mediated killing of tumor cells, there are also important differences. Similar to tumor killing, NK cells directly kill fungi in a receptor-mediated and cytotoxic granule-dependent manner. Unlike tumor cell killing where multiple NK cell-activating receptors cooperate and signal events that mediate cytotoxicity, only the NKp30 receptor has been described to mediate signaling events that trigger the NK cell to mobilize its cytolytic payload to the site of interaction with C. neoformans and Candida albicans, subsequently leading to granule exocytosis and fungal killing. More recently, the NKp46 receptor was reported to bind Candida glabrata adhesins Epa1, 6, and 7 and directly mediate fungal clearance. A number of unanswered questions remain. For example, is only one NK cell-activating receptor sufficient for signaling leading to fungal killing? Are the signaling pathways activated by fungi similar to those activated by tumor cells during NK cell killing? How do the cytolytic granules traffic to the site of interaction with fungi, and how does this process compare with tumor killing? Recent insights into receptor use, intracellular signaling and cytolytic granule trafficking during NK cell-mediated fungal killing will be compared to tumor killing, and the implications for therapeutic approaches will be discussed. PMID:28123389

  5. Lytic and non-lytic permeabilization of cardiolipin-containing lipid bilayers induced by cytochrome C.

    Directory of Open Access Journals (Sweden)

    Jian Xu

    Full Text Available The release of cytochrome c (cyt c from mitochondria is an important early step during cellular apoptosis, however the precise mechanism by which the outer mitochondrial membrane becomes permeable to these proteins is as yet unclear. Inspired by our previous observation of cyt c crossing the membrane barrier of giant unilamellar vesicle model systems, we investigate the interaction of cyt c with cardiolipin (CL-containing membranes using the innovative droplet bilayer system that permits electrochemical measurements with simultaneous microscopy observation. We find that cyt c can permeabilize CL-containing membranes by induction of lipid pores in a dose-dependent manner, with membrane lysis eventually observed at relatively high (µM cyt c concentrations due to widespread pore formation in the membrane destabilizing its bilayer structure. Surprisingly, as cyt c concentration is further increased, we find a regime with exceptionally high permeability where a stable membrane barrier is still maintained between droplet compartments. This unusual non-lytic state has a long lifetime (>20 h and can be reversibly formed by mechanically separating the droplets before reforming the contact area between them. The transitions between behavioural regimes are electrostatically driven, demonstrated by their suppression with increasing ionic concentrations and their dependence on CL composition. While membrane permeability could also be induced by cationic PAMAM dendrimers, the non-lytic, highly permeable membrane state could not be reproduced using these synthetic polymers, indicating that details in the structure of cyt c beyond simply possessing a cationic net charge are important for the emergence of this unconventional membrane state. These unexpected findings may hold significance for the mechanism by which cyt c escapes into the cytosol of cells during apoptosis.

  6. Painful Lytic Lesions of the Foot : A Case Report

    Directory of Open Access Journals (Sweden)

    R Vaishya

    2015-03-01

    Full Text Available The presence of lytic lesions in the bones of foot raises a number of diagnostic possibilities ranging from infection, inflammatory pathology to neoplastic conditions. Although the radiological picture is not pathognomonic of any pathology, clinical history and histopathological examination can help to clinch the diagnosis. We present a case of multiple lytic lesions of the foot and discuss possible differential diagnoses. The patient was diagnosed as a case of madura foot and the lesions responded to surgical debridement and anti-fungal treatment with a good functional outcome. Madura foot is an uncommon, chronic granulomatous fungal or bacterial infection with a predilection in people who walk barefoot. Although known for a specific geographical distribution, madura foot should be kept as a possible diagnosis in patients presenting with lytic lesions of the foot due to population emigration across the world.

  7. Exocytosis sensitivity to growth hormone-releasing hormone in subsets of GH cells in rats under different corticosterone conditions. Ultrastructural study using microwave irradiation for fixation and immunocytochemistry.

    Science.gov (United States)

    Ozawa, Hitoshi; Han, Fang; Kawata, Mitsuhiro

    2004-12-01

    Growth hormone (GH) cells in the rat anterior pituitary have been morphologically classified into three subtypes: type I (mature) containing large secretory granules about 350 nm in diameter, type II (intermediate) containing a mixture of large and small granules, and type III (immature) containing small granules about 150 nm in diameter. However, the functional implications of morphological heterogeneity, especially the different sensitivities to growth hormone-releasing hormone (GRH) under different corticosteroid conditions have not been elucidated to date. In the present study, by application of microwave irradiation (MWI) for fixation and immunocytochemistry, new findings of the exocytotic response have been revealed among the subsets of GH cells following adrenalectomy (ADX), corticosterone treatment and/or GRH treatment. The MWI gave effective results for fixation, especially for the permeability of the fixative, and showed good results for immunoelectron microscopy using the protein-A gold method. Moreover, the use of MWI greatly shortened the fixation, processing and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The number of exocytotic figures was low in all subtypes of GH cells in the sham-operated control rats. GRH treatment induced a significant increase in exocytosis in each subtype of GH cells, particularly in type I (mature) and type II (intermediate) GH cells in the control rats. GRH injection to rats for 4 days after ADX also showed an increase in exocytosis, but the degree was significantly less in comparison with the GRH injection in the control group. Corticosterone replacement given to ADX rats induced a clear recovery of the exocytotic response to GRH to the control level. Serum GH content measured by radioimmunoassay correlated with these morphological results. These results suggest that the secretion of GH stimulated by GRH is closely related to corticosteroids, and

  8. Naegleria fowleri: contact-dependent secretion of electrondense granules (EDG).

    Science.gov (United States)

    Chávez-Munguía, Bibiana; Villatoro, Lizbeth Salazar; Omaña-Molina, Maritza; Rodríguez-Monroy, Marco Aurelio; Segovia-Gamboa, Norma; Martínez-Palomo, Adolfo

    2014-07-01

    The free living amoeba Naegleria fowleri is pathogenic to humans but also to other mammalians. These amoebae may invade the nasal mucosa and migrate into the brain causing cerebral hemorrhagic necrosis, a rapidly fatal infection. Knowledge of the cytolytic mechanism involved in the destruction of brain tissues by Naegleria trophozoites is limited. In other amoebic species, such as Entamoeba histolytica, we have previously reported the possible lytic role of small cytoplasmic components endowed with proteolytic activities, known as electrondense granules (EDG). Using transmission electron microscopy we now report that EDG, seldom found in long term cultured N. fowleri, are present in abundance in trophozoites recovered from experimental mice brain lesions. Numerous EDG were also observed in amoebae incubated with collagen substrates or cultured epithelial cells. SDS-PAGE assays of concentrated supernatants of these trophozoites, containing EDG, revealed proteolytic activities. These results suggest that EDG may have a clear role in the cytopathic mechanisms of this pathogenic amoeba.

  9. Synaptobrevin transmembrane domain influences exocytosis by perturbing vesicle membrane curvature.

    Science.gov (United States)

    Chang, Che-Wei; Jackson, Meyer B

    2015-07-07

    Membrane fusion requires that nearly flat lipid bilayers deform into shapes with very high curvature. This makes membrane bending a critical force in determining fusion mechanisms. A lipid bilayer will bend spontaneously when material is distributed asymmetrically between its two monolayers, and its spontaneous curvature (C0) will influence the stability of curved fusion intermediates. Prior work on Ca(2+)-triggered exocytosis revealed that fusion pore lifetime (τ) varies with vesicle content (Q), and showed that this relation reflects membrane bending energetics. Lipids that alter C0 change the dependence of τ on Q. These results suggested that the greater stability of an initial exocytotic fusion pore associated with larger vesicles reflects the need to bend more membrane during fusion pore dilation. In this study, we explored the possibility of manipulating C0 by mutating the transmembrane domain (TMD) of the vesicle membrane protein synaptobrevin 2 (syb2). Amperometric measurements of exocytosis in mouse chromaffin cells revealed that syb2 TMD mutations altered the relation between τ and Q. The effects of these mutations showed a striking periodicity, changing sign as the structural perturbation moved through the inner and outer leaflets. Some glycine and charge mutations also influenced the dependence of τ on Q in a manner consistent with expected changes in C0. These results suggest that side chains in the syb2 TMD influence the kinetics of exocytosis by perturbing the packing of the surrounding lipids. The present results support the view that membrane bending occurs during fusion pore expansion rather than during fusion pore formation. This supports the view of an initial fusion pore through two relatively flat membranes formed by protein. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Granulation of increasingly hydrophobic formulations using a twin screw granulator.

    Science.gov (United States)

    Yu, Shen; Reynolds, Gavin K; Huang, Zhenyu; de Matas, Marcel; Salman, Agba D

    2014-11-20

    The application of twin screw granulation in the pharmaceutical industry has generated increasing interest due to its suitability for continuous processing. However, an understanding of the impact of formulation properties such as hydrophobicity on intermediate and finished product quality has not yet been established. Hence, the current work investigated the granulation behaviour of three formulations containing increasing amounts of hydrophobic components using a Consigma™-1 twin screw granulator. Process conditions including powder feed rate, liquid to solid ratio, granulation liquid composition and screw configuration were also evaluated. The size of the wet granules was measured in order to enable exploration of granulation behaviour in isolation without confounding effects from downstream processes such as drying. The experimental observations indicated that the granulation process was not sensitive to the powder feed rate. The hydrophobicity led to heterogeneous liquid distribution and hence a relatively large proportion of un-wetted particles. Increasing numbers of kneading elements led to high shear and prolonged residence time, which acted to enhance the distribution of liquid and feeding materials. The bimodal size distributions considered to be characteristic of twin screw granulation were primarily ascribed to the breakage of relatively large granules by the kneading elements.

  11. Exocytosis and polarity in plant cells: insights by studying cellulose synthase complexes and the exocyst

    NARCIS (Netherlands)

    Ying Zhang, Ying

    2012-01-01

    The work presented in this thesis covers aspects of exocytosis, plant cell growth and cell wall formation. These processes are strongly linked as cell growth and cell wall formation occur simultaneously and exocytosis is the process that delivers cell wall components to the existing cell wall and in

  12. Stretch-regulated Exocytosis/Endocytosis in Bladder Umbrella Cells

    Science.gov (United States)

    Truschel, Steven T.; Wang, Edward; Ruiz, Wily G.; Leung, Som-Ming; Rojas, Raul; Lavelle, John; Zeidel, Mark; Stoffer, David; Apodaca, Gerard

    2002-01-01

    The epithelium of the urinary bladder must maintain a highly impermeable barrier despite large variations in urine volume during bladder filling and voiding. To study how the epithelium accommodates these volume changes, we mounted bladder tissue in modified Ussing chambers and subjected the tissue to mechanical stretch. Stretching the tissue for 5 h resulted in a 50% increase in lumenal surface area (from ∼2900 to 4300 μm2), exocytosis of a population of discoidal vesicles located in the apical cytoplasm of the superficial umbrella cells, and release of secretory proteins. Surprisingly, stretch also induced endocytosis of apical membrane and 100% of biotin-labeled membrane was internalized within 5 min after stretch. The endocytosed membrane was delivered to lysosomes and degraded by a leupeptin-sensitive pathway. Last, we show that the exocytic events were mediated, in part, by a cyclic adenosine monophosphate, protein kinase A-dependent process. Our results indicate that stretch modulates mucosal surface area by coordinating both exocytosis and endocytosis at the apical membrane of umbrella cells and provide insight into the mechanism of how mechanical forces regulate membrane traffic in nonexcitable cells. PMID:11907265

  13. Granule stores from cellubrevin/VAMP-3 null mouse platelets exhibit normal stimulus-induced release.

    Science.gov (United States)

    Schraw, Todd D; Rutledge, Tara W; Crawford, Garland L; Bernstein, Audrey M; Kalen, Amanda L; Pessin, Jeffery E; Whiteheart, Sidney W

    2003-09-01

    It is widely accepted that the platelet release reaction is mediated by heterotrimeric complexes of integral membrane proteins known as SNAREs (SNAP receptors). In an effort to define the precise molecular machinery required for platelet exocytosis, we have analyzed platelets from cellubrevin/VAMP-3 knockout mice. Cellubrevin/VAMP-3 has been proposed to be a critical v-SNARE for human platelet exocytosis; however, data reported here suggest that it is not required for platelet function. Upon stimulation with increasing concentrations of thrombin, collagen, or with thrombin for increasing time there were no differences in secretion of [3H]-5HT (dense core granules), platelet factor IV (alpha granules), or hexosaminidase (lysosomes) between null and wild-type platelets. There were no gross differences in bleeding times nor in agonist-induced aggregation measured in platelet-rich plasma or with washed platelets. Western blotting of wild-type, heterozygous, and null platelets confirmed the lack of cellubrevin/VAMP-3 in nulls and showed that most elements of the secretion machinery are expressed at similar levels. While the secretory machinery in mice was similar to humans, mice did express apparently higher levels of synaptobrevin/VAMP-2. These data show that the v-SNARE, cellubrevin/VAMP-3 is not a requirement for the platelet release reaction in mice.

  14. Extrasynaptic exocytosis and its mechanisms: a source of molecules mediating volume transmission in the nervous system

    Directory of Open Access Journals (Sweden)

    Citlali eTrueta

    2012-09-01

    Full Text Available We review the evidence of exocytosis from extrasynaptic sites in the soma, dendrites and axonal varicosities of central and peripheral neurons of vertebrates and invertebrates, and how it may contribute to signaling in the nervous system. The finding of secretory vesicles in extrasynaptic sites of neurons, the presence of transmitters in the extracellular space outside synaptic clefts, and the mismatch between exocytosis sites and the location of receptors for these molecules in neurons and glial cells, have long suggested that in addition to synaptic communication, transmitters are released and act extrasynaptically. The catalog of these molecules includes low molecular weight transmitters such as monoamines, acetylcholine, glutamate, GABA, ATP, and a list of peptides including substance P, BDNF, and oxytocin. By comparing the mechanisms of extrasynaptic exocytosis of different molecules in various neuron types we show that extrasynaptic exocytosis is a widespread mechanism for communication in the nervous system that uses certain common mechanisms, which are different from those of synaptic exocytosis but similar to those of exocytosis from excitable endocrine cells. Somatic exocytosis, which has been measured directly in different neuron types, starts after high-frequency electrical activity or long experimental depolarizations and may continue for several minutes after the end of stimulation. Activation of L-type calcium channels, calcium release from intracellular stores and vesicle transport couples excitation and exocytosis from small clear or large dense core vesicles in release sites lacking postsynaptic counterparts. The presence of synaptic and extrasynaptic exocytosis endows individual neurons with a wide variety of time- and space-dependent communication possibilities. Extrasynaptic exocytosis may be the major source of signaling molecules producing volume transmission and by doing so may be part of a long duration signaling mode in

  15. Isolation of RNP granules

    DEFF Research Database (Denmark)

    Jønson, Lars; Nielsen, Finn Cilius; Christiansen, Jan

    2011-01-01

    be regarded as a supramolecular assembly of RNA and protein, probably representing several overlapping post-transcriptional operons. The present protocol describes how RNP granules may be isolated by the transgenic expression of a 3X FLAG version of an RNA-binding protein under tetracycline control via...... the tetracycline receptor/operator complex. In this way, inclusion of an appropriate tetracycline concentration ensures expression of the tagged version at the endogenous level, and the 3X FLAG tag is a convenient "handle" for the subsequent immunoprecipitation by immobilized anti-FLAG antibody....

  16. Granulation techniques and technologies: recent progresses.

    Science.gov (United States)

    Shanmugam, Srinivasan

    2015-01-01

    Granulation, the process of particle enlargement by agglomeration technique, is one of the most significant unit operations in the production of pharmaceutical dosage forms, mostly tablets and capsules. Granulation process transforms fine powders into free-flowing, dust-free granules that are easy to compress. Nevertheless, granulation poses numerous challenges due to high quality requirement of the formed granules in terms of content uniformity and physicochemical properties such as granule size, bulk density, porosity, hardness, moisture, compressibility, etc. together with physical and chemical stability of the drug. Granulation process can be divided into two types: wet granulation that utilize a liquid in the process and dry granulation that requires no liquid. The type of process selection requires thorough knowledge of physicochemical properties of the drug, excipients, required flow and release properties, to name a few. Among currently available technologies, spray drying, roller compaction, high shear mixing, and fluid bed granulation are worth of note. Like any other scientific field, pharmaceutical granulation technology also continues to change, and arrival of novel and innovative technologies are inevitable. This review focuses on the recent progress in the granulation techniques and technologies such as pneumatic dry granulation, reverse wet granulation, steam granulation, moisture-activated dry granulation, thermal adhesion granulation, freeze granulation, and foamed binder or foam granulation. This review gives an overview of these with a short description about each development along with its significance and limitations.

  17. Granule Size Distribution and Porosity of Granule Packing

    Institute of Scientific and Technical Information of China (English)

    DAI Shu-hua; SHEN Feng-man; YU Ai-bing

    2008-01-01

    The granule size distribution and the porosity of the granule packing process were researched.For realizing the optimizing control of the whole sintering production process,researchers must know the factors influencing the granule size distribution and the porosity.Therefore,tests were carried out in the laboratory with regard to the influences of the size and size distribution of raw materials and the total moisture content on the size and size distribution of granule.Moreover,tests for finding out the influences of the moisture content and the granule volume fraction on the porosity were also carried out.The results show that (1) the raw material has little influence on granulation when its size is in the range of 0.51 mm to 1.0 mm;(2) the influence of the material size on granule size plays a dominant role,and in contrast,the moisture content creates a minor effect on granule size;(3) in binary packing system,with the increase in the constituent volume fraction,the porosity initially increases and then decreases,and there is a minimum value on the porosity curve of the binary mixture system;(4) the minimum value of the porosity in binary packing system occurs at different locations for different moisture contents,and this value shifts from right to left on the porosity curve with increasing the moisture content;(5) the addition of small granules to the same size component cannot create a significant influence on the porosity,whereas the addition of large granules to the same system can greatly change the porosity.

  18. Multidimensional modelling of anaerobic granules

    DEFF Research Database (Denmark)

    Picioreanu, C.; Batstone, Damien J.; van Loosdrecht, M.C.M.

    2005-01-01

    . The model uses individual-based representation of biomass particles within the granule (biofilm), and describes spreading by an iterative pushing technique. The overall computational domain consists of one granule, and is divided into a grid with Cartesian coordinates. The number of grid elements does...

  19. Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion

    Energy Technology Data Exchange (ETDEWEB)

    Petrov, Alexey M., E-mail: fysio@rambler.ru; Zakyrjanova, Guzalija F., E-mail: guzik121192@mail.ru; Yakovleva, Anastasia A., E-mail: nastya1234qwer@mail.ru; Zefirov, Andrei L., E-mail: zefiroval@rambler.ru

    2015-01-02

    Highlights: • We examine the involvement of PKC in MCD induced synaptic vesicle exocytosis. • PKC inhibitor does not decrease the effect MCD on MEPP frequency. • PKC inhibitor prevents MCD induced FM1-43 unloading. • PKC activation may switch MCD induced exocytosis from kiss-and-run to a full mode. • Inhibition of phospholipase C does not lead to similar change in exocytosis. - Abstract: Previous studies demonstrated that depletion of membrane cholesterol by 10 mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis.

  20. Effects of Perfluorocarbons on surfactant exocytosis and membrane properties in isolated alveolar type II cells

    Directory of Open Access Journals (Sweden)

    Ravasio Andrea

    2010-05-01

    Full Text Available Abstract Background Perfluorocarbons (PFC are used to improve gas exchange in diseased lungs. PFC have been shown to affect various cell types. Thus, effects on alveolar type II (ATII cells and surfactant metabolism can be expected, data, however, are controversial. Objective The study was performed to test two hypotheses: (I the effects of PFC on surfactant exocytosis depend on their respective vapor pressures; (II different pathways of surfactant exocytosis are affected differently by PFC. Methods Isolated ATII cells were exposed to two PFC with different vapor pressures and spontaneous surfactant exocytosis was measured. Furthermore, surfactant exocytosis was stimulated by either ATP, PMA or Ionomycin. The effects of PFC on cell morphology, cellular viability, endocytosis, membrane permeability and fluidity were determined. Results The spontaneous exocytosis was reduced by PFC, however, the ATP and PMA stimulated exocytosis was slightly increased by PFC with high vapor pressure. In contrast, Ionomycin-induced exocytosis was decreased by PFC with low vapor pressure. Cellular uptake of FM 1-43 - a marker of membrane integrity - was increased. However, membrane fluidity, endocytosis and viability were not affected by PFC incubation. Conclusions We conclude that PFC effects can be explained by modest, unspecific interactions with the plasma membrane rather than by specific interactions with intracellular targets.

  1. Granulated zeolite plant "Alusil", Zvornik

    Directory of Open Access Journals (Sweden)

    Stanković Mirjana S.

    2003-01-01

    Full Text Available The IGPC's Engineering Department designed basic technological and machine projects for a granulated zeolite production plant, on the basis of which a pilot plant with an initial capacity of 5,000 t/y was constructed in 1984, within Birač - Zvornik production complex. The technology in these projects was developed in the laboratories of the IGPC.Several goals were realized by designing a granulated zeolite production plant. This technology is one of the newest state of the art high tech technologies. The product meets all quality demands, as well as environmental regulations, by which granulated zeolite production for various uses was developed. The granulated zeolite production process is fully automatized, and the product has uniform quality. There is no waste material in granulated zeolite production, because all products with unsatisfactory quality are returned to the process. The production process can be controlled manually, which is necessary during start - up, and repairs.

  2. Cortex Peptidoglycan Lytic Activity in Germinating Bacillus anthracis Spores▿

    OpenAIRE

    2008-01-01

    Bacterial endospore dormancy and resistance properties depend on the relative dehydration of the spore core, which is maintained by the spore membrane and its surrounding cortex peptidoglycan wall. During spore germination, the cortex peptidoglycan is rapidly hydrolyzed by lytic enzymes packaged into the dormant spore. The peptidoglycan structures in both dormant and germinating Bacillus anthracis Sterne spores were analyzed. The B. anthracis dormant spore peptidoglycan was similar to that fo...

  3. Moluo Yangping Granule (

    Institute of Scientific and Technical Information of China (English)

    LI; En-fu

    2001-01-01

    Peptic ulcer is a common and frequently encountered disease. H2 receptor blocker and several other drugs have been used in treating peptic ulcer (PU) since many years ago, but there still remain a rather large number of intractable ulcer and recurrent ulcer. Recently the discovery of Helicobacter pylori (HP), as well as the relationship between HP and PU relapse was confirmed, but there still lacks of perfect therapeutic program for anti-HP infection(1). From June 1993 to August 1996, we used a pure TCM preparation Moluo Yangping granule (摩罗疡平冲剂, MYG) in treating 64 PU patients, and satisfactory results have been obtained. The report is as follows.

  4. A green-light inducible lytic system for cyanobacterial cells.

    Science.gov (United States)

    Miyake, Kotone; Abe, Koichi; Ferri, Stefano; Nakajima, Mitsuharu; Nakamura, Mayumi; Yoshida, Wataru; Kojima, Katsuhiro; Ikebukuro, Kazunori; Sode, Koji

    2014-01-01

    Cyanobacteria are an attractive candidate for the production of biofuel because of their ability to capture carbon dioxide by photosynthesis and grow on non-arable land. However, because huge quantities of water are required for cultivation, strict water management is one of the greatest issues in algae- and cyanobacteria-based biofuel production. In this study, we aim to construct a lytic cyanobacterium that can be regulated by a physical signal (green-light illumination) for future use in the recovery of biofuel related compounds. We introduced T4 bacteriophage-derived lysis genes encoding holin and endolysin under the control of the green-light regulated cpcG2 promoter in Synechocystis sp. PCC 6803. When cells harboring the lysis genes were illuminated with both red and green light, we observed a considerable decrease in growth rate, a significant increase in cellular phycocyanin released in the medium, and a considerable fraction of dead cells. These effects were not observed when these cells were illuminated with only red light, or when cells not containing the lysis genes were grown under either red light or red and green light. These results indicate that our constructed green-light inducible lytic system was clearly induced by green-light illumination, resulting in lytic cells that released intracellular phycocyanin into the culture supernatant. This property suggests a future possibility to construct photosynthetic genetically modified organisms that are unable to survive under sunlight exposure. Expression of the self-lysis system with green-light illumination was also found to greatly increase the fragility of the cell membrane, as determined by subjecting the induced cells to detergent, osmotic-shock, and freeze-thaw treatments. A green-light inducible lytic system was constructed in Synechocystis sp. PCC 6803. The engineered lytic cyanobacterial cells should be beneficial for the recovery of biofuels and related compounds from cells with minimal effort

  5. Neuropeptide exocytosis involving synaptotagmin-4 and oxytocin in hypothalamic programming of body weight and energy balance.

    Science.gov (United States)

    Zhang, Guo; Bai, Hua; Zhang, Hai; Dean, Camin; Wu, Qiang; Li, Juxue; Guariglia, Sara; Meng, Qingyuan; Cai, Dongsheng

    2011-02-10

    Hypothalamic neuropeptides play essential roles in regulating energy and body weight balance. Energy imbalance and obesity have been linked to hypothalamic signaling defects in regulating neuropeptide genes; however, it is unknown whether dysregulation of neuropeptide exocytosis could be critically involved. This study discovered that synaptotagmin-4, an atypical modulator of synaptic exocytosis, is expressed most abundantly in oxytocin neurons of the hypothalamus. Synaptotagmin-4 negatively regulates oxytocin exocytosis, and dietary obesity is associated with increased vesicle binding of synaptotagmin-4 and thus enhanced negative regulation of oxytocin release. Overexpressing synaptotagmin-4 in hypothalamic oxytocin neurons and centrally antagonizing oxytocin in mice are similarly obesogenic. Synaptotagmin-4 inhibition prevents against dietary obesity by normalizing oxytocin release and energy balance under chronic nutritional excess. In conclusion, the negative regulation of synaptotagmin-4 on oxytocin release represents a hypothalamic basis of neuropeptide exocytosis in controlling obesity and related diseases. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Involvement of conventional kinesin in glucose-stimulated secretory granule movements and exocytosis in clonal pancreatic beta-cells.

    Science.gov (United States)

    Varadi, Aniko; Ainscow, Edward K; Allan, Victoria J; Rutter, Guy A

    2002-11-01

    Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal beta-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein (phogrin.EGFP), and employed immunoadsorption to obtain highly purified insulin-containing vesicles. Whereas several kinesin family members were expressed in this cell type, only conventional kinesin heavy chain (KHC) was detected in vesicle preparations. Expression of a dominant-negative KHC motor domain (KHC(mut)) blocked all vesicular movements with velocity >0.4 micro m second(-1), which demonstrates that kinesin activity was essential for vesicle motility in live beta-cells. Moreover, expression of KHC(mut) strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Finally, vesicle movement was stimulated by ATP dose-dependently in permeabilized cells, which suggests that glucose-induced increases in cytosolic [ATP] mediate the effects of the sugar in vivo, by enhancing kinesin activity. These data therefore provide evidence for a novel mechanism whereby glucose may enhance insulin release.

  7. Alpha-1-antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis

    DEFF Research Database (Denmark)

    Clemmensen, Stine N; Jacobsen, Lars C; Rørvig, Sara

    2011-01-01

    1AT is produced at all stages of myeloid maturation in the bone marrow. The production increases as neutrophils enter circulation and increases further upon migration to tissues as observed in skin windows and when blood neutrophils are incubated with granulocyte colony-stimulating factor...

  8. Genetic deletion of mouse platelet glycoprotein Ibbeta produces a Bernard-Soulier phenotype with increased alpha-granule size.

    Science.gov (United States)

    Kato, Kazunobu; Martinez, Constantino; Russell, Susan; Nurden, Paquita; Nurden, Alan; Fiering, Steven; Ware, Jerry

    2004-10-15

    Here we report the characterization of a mouse model of the Bernard-Soulier syndrome generated by a targeted disruption of the gene encoding the glycoprotein (GP) Ibbeta subunit of the GP Ib-IX complex. Similar to a Bernard-Soulier model generated by disruption of the mouse GP Ibalpha subunit, GP Ibbeta(Null) mice display macrothrombocytopenia and a severe bleeding phenotype. When examined by transmission electron microscopy, the large platelets produced by a GP Ibbeta(Null) genotype revealed alpha-granules with increased size as compared with the alpha-granules from control mouse platelets. Data are presented linking the overexpression of a septin protein, SEPT5, to the presence of larger alpha-granules in the GP Ibbeta(Null) platelet. The SEPT5 gene resides approximately 250 nucleotides 5' to the GP Ibbeta gene and has been associated with modulating exocytosis from neurons and platelets as part of a presynaptic protein complex. Fusion mRNA transcripts present in megakaryocytes can contain both the SEPT5 and GP Ibbeta coding sequences as a result in an imperfect polyadenylation signal within the 3' end of both the human and mouse SEPT5 genes. We observed a 2- to 3-fold increase in SEPT5 protein levels in platelets from GP Ibbeta(Null) mice. These results implicate SEPT5 levels in the maintenance of normal alpha-granule size and may explain the variant granules associated with human GP Ibbeta mutations and the Bernard-Soulier syndrome.

  9. A TRP Channel in the Lysosome Regulates Large Particle Phagocytosis via Focal Exocytosis

    OpenAIRE

    2013-01-01

    Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here we identified Mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers, and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML...

  10. Genipin inhibits endothelial exocytosis via nitric oxide in cultured human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Guang-fa WANG; Shao-yu WU; Jin-jun RAO; Lin L(U); Wei XU; Jian-xin PANG; Zhong-qiu LIU; Shu-guang WU; Jia-jie ZHANG

    2009-01-01

    Aim: Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor (VWF), P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin,an aglycon of geniposide, inhibits endothelial exocytosis.Methods: Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase (eNOS) and phospho-eNOS present. Nitric oxide (NO) was measured in the cell supernatants as nitrite using an NO Colorimetric Assay.Results: Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time-dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis.Conclusion: Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel antiinflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.

  11. Complexin II plays a positive role in Ca2+-triggered exocytosis by facilitating vesicle priming

    DEFF Research Database (Denmark)

    Cai, Haijiang; Reim, Kerstin; Varoqueaux, Frederique

    2008-01-01

    the complex and inhibiting membrane fusion. However, several other studies also suggest that CPXs are positive regulators of neurotransmitter release. Thus, whether CPXs are positive or negative regulators of exocytosis is not known, much less the stage in the vesicle life cycle at which they function. Here...... regulate the size of the primed vesicle pools and have a positive role in Ca(2+)-triggered exocytosis....

  12. Linking granulation performance with residence time and granulation liquid distributions in twin-screw granulation: An experimental investigation.

    Science.gov (United States)

    Kumar, Ashish; Alakarjula, Maija; Vanhoorne, Valérie; Toiviainen, Maunu; De Leersnyder, Fien; Vercruysse, Jurgen; Juuti, Mikko; Ketolainen, Jarkko; Vervaet, Chris; Remon, Jean Paul; Gernaey, Krist V; De Beer, Thomas; Nopens, Ingmar

    2016-07-30

    Twin-screw granulation is a promising wet granulation technique for the continuous manufacturing of pharmaceutical solid dosage forms. A twin screw granulator displays a short residence time. Thus, the solid-liquid mixing must be achieved quickly by appropriate arrangement of transport and kneading elements in the granulator screw allowing the production of granules with a size distribution appropriate for tableting. The distribution of residence time and granulation liquid is governed by the field conditions (such as location and length of mixing zones) in the twin-screw granulator, thus contain interesting information on granulation time, mixing and resulting sub-processes such as wetting, aggregation and breakage. In this study, the impact of process (feed rate, screw speed and liquid-to-solid ratio) and equipment parameters (number of kneading discs and stagger angle) on the residence time (distribution), the granulation liquid-powder mixing and the resulting granule size distributions during twin-screw granulation were investigated. Residence time and axial mixing data was extracted from tracer maps and the solid-liquid mixing was quantified from moisture maps, obtained by monitoring the granules at the granulator outlet using near infra-red chemical imaging (NIR-CI). The granule size distribution was measured using the sieving method. An increasing screw speed dominantly reduced the mean residence time. Interaction of material throughput with the screw speed and with the number of kneading discs led to most variation in the studied responses including residence time and mixing capacity. At a high screw speed, granulation yield improved due to high axial mixing. However, increasing material throughput quickly lowers the yield due to insufficient mixing of liquid and powder. Moreover, increasing liquid-to-solid ratio resulted in more oversized granules, and the fraction of oversized granules further increased at higher throughput. Although an increasing number

  13. Granulation techniques and technologies: recent progresses

    OpenAIRE

    Srinivasan Shanmugam

    2015-01-01

    Granulation, the process of particle enlargement by agglomeration technique, is one of the most significant unit operations in the production of pharmaceutical dosage forms, mostly tablets and capsules. Granulation process transforms fine powders into free-flowing, dust-free granules that are easy to compress. Nevertheless, granulation poses numerous challenges due to high quality requirement of the formed granules in terms of content uniformity and physicochemical proper...

  14. New Electrochemical Methods for Studying Nanoparticle Electrocatalysis and Neuronal Exocytosis

    Science.gov (United States)

    Cox, Jonathan T.

    This dissertation presents the construction and application of micro and nanoscale electrodes for electroanalytical analysis. The studies presented herein encompass two main areas: electrochemical catalysis, and studies of the dynamics of single cell exocytosis. The first portion of this dissertation engages the use of Pt nanoelectrodes to study the stability and electrocatalytic properties of materials. A single nanoparticle electrode (SNPE) was fabricated by immobilizing a single Au nanoparticle on a Pt disk nanoelectrode via an amine-terminated silane cross linker. In this manner we were able to effectively study the electrochemistry and electrocatalytic activity of single Au nanoparticles and found that the electrocatalytic activity is dependent on nanoparticle size. This study can further the understanding of the structure-function relationship in nanoparticle based electrocatalysis. Further work was conducted to probe the stability of Pt nanoelectrodes under conditions of potential cycling. Pt based catalysts are known to deteriorate under such conditions due to losses in electrochemical surface area and Pt dissolution. By using Pt disk nanoelectrodes we were able to study Pt dissolution via steady-state voltammetry. We observed an enhanced dissolution rate and higher charge density on nanoelectrodes than that previously found on macro scale electrodes. The goal of the second portion of this dissertation is to develop new analytical methods to study the dynamics of exocytosis from single cells. The secretion of neurotransmitters plays a key role in neuronal communication, and our studies highlight how bipolar electrochemistry can be employed to enhance detection of neurotransmitters from single cells. First, we developed a theory to quantitatively characterize the voltammetric behavior of bipolar carbon fiber microelectrodes and secondly applied those principles to single cell detection. We showed that by simply adding an additional redox mediator to the back

  15. Natural killer lytic-associated molecule plays a role in controlling tumor dissemination and metastasis

    Directory of Open Access Journals (Sweden)

    Richard Glenn Hoover

    2012-12-01

    Full Text Available Natural killer lytic-associated molecule (NKLAM is an E3 ubiquitin ligase that plays a major role in the cytolytic activity of NK cells. NKLAM is rapidly synthesized and then targeted to the granule membranes of NK cells upon NK activation. Previous studies have shown an essential role for NKLAM in NK killing activity in vitro. These findings were extended to an in vivo model of NK-mediated tumor killing in which NKLAM-deficient knockout (KO mice injected with B16 melanoma cells were found to have significantly higher numbers of pulmonary tumor nodules than wild type (WT mice. To further investigate the role of NKLAM and NK function in tumor immunity in vivo, we utilized additional tumor models to compare tumor development and progression in NKLAM KO and WT mice. Primary tumor growth, dissemination, and metastasis of RMA-S lymphoma cells and E0771 breast cancer cells were evaluated. Both tumor cell lines were stably transfected with constructs that allow expression of green fluorescent protein (GFP, which serves as a tumor-specific marker. Intravenous injection of NK-sensitive RMA-S lymphoma cells resulted in greater dissemination of lymphoma cells in NKLAM KO mice than in WT mice. Lymphoma cells were found in the lymph nodes and bone marrow of NKLAM KO mice two weeks after injection; few detectable tumor cells remained in WT mice. E0771 syngeneic breast cancer cells were injected into the mammary pads of NKLAM KO and WT mice. Primary tumor growth was greater in NKLAM KO than in WT mice. More significantly, there were four to five fold more tumor cells in the blood and lungs of NKLAM KO than in WT mice two weeks after injection of tumor cells into the mammary pad. These results indicate that NKLAM plays a role in tumor development in vivo, especially in controlling tumor dissemination and metastasis to distant sites.

  16. Structural characterization of Lytic Polysaccharide MonoOxygenases

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of copper-containingmetalloenzymes that have been found to oxidatively degrade polysaccharides (and recently alsooligosaccharides). They dependent on redox partners to provide them with electrons and they utilizemolecular oxygen to cleave......) and their interaction with substratehave been structurally characterized. A number of structures of LsAA9A have been obtained in complexwith a range of cellulosic- and hemicellulosic substrates and with the active site Cu in different redox state.Two of the LsAA9A structures with the active site Cu in essentially a Cu...

  17. Pore opening dynamics in the exocytosis of serotonin

    Science.gov (United States)

    Ramirez-Santiago, Guillermo; Cercos, Montserrat G.; Martinez-Valencia, Alejandro; Salinas Hernandez, Israel; Rodríguez-Sosa, Leonardo; de-Miguel, Francisco F.

    2015-03-01

    The current view of the exocytosis of transmitter molecules is that it starts with the formation of a fusion pore that connects the intravesicular and the extracellular spaces, and is completed by the release of the rest of the transmitter contained in the vesicle upon the full fusion and collapse of the vesicle with the plasma membrane. However, under certain circumstances, a rapid closure of the pore before the full vesicle fusion produces only a partial release of the transmitter. Here we show that whole release of the transmitter occurs through fusion pores that remain opened for tens of milliseconds without vesicle collapse. This was demonstrated through amperometric measurements of serotonin release from electrodense vesicles in the axon of leech Retzius neurons and mathematical modelling. By modeling transmitter release with a diffusion equation subjected to boundary conditions that are defined by the experiment, we showed that those pores with a fast half rise time constant remained opened and allowed the full quantum release without vesicle collapse, whereas pores with a slow rise time constant closed rapidly, thus producing partial release. We conclude that a full transmitter release may occur through the fusion pore in the absence of vesicle collapse. This work was founded by a DGAPA-UNAM grants IN200914 and IN118410 CONACYT GRANT 130031, and CONACyT doctoral fellowships.

  18. LIPIDS IN REGULATED EXOCYTOSIS: WHAT ARE THEY DOING?

    Directory of Open Access Journals (Sweden)

    Mohamed Raafet Ammar

    2013-09-01

    Full Text Available TThe regulated secretory pathway in neuroendocrine cells ends with the release of hormones and neurotransmitters following a rise in cytosolic calcium. This process known as regulated exocytosis involves the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE proteins, the synaptic vesicle VAMP (synaptobrevin and the plasma membrane proteins syntaxin and SNAP-25. Although there is much evidence suggesting that SNARE proteins play a key role in the fusion machinery, other cellular elements regulating the kinetics, the extent of fusion, and the preparation of vesicle for release have received less attention. Among those factors, lipids have also been proposed to play important functions both at the level of secretory vesicle recruitment and late membrane fusion steps. Here, we will review the latest evidence supporting the concept of the fusogenic activity of lipids, and also discuss how this may be achieved. These possibilities include the recruitment and sequestration of the components of the exocytotic machinery, regulation of protein function and direct effects on membrane topology.

  19. Exocytosis of MTT formazan could exacerbate cell injury.

    Science.gov (United States)

    Lü, Lanhai; Zhang, Lihong; Wai, Maria Sen Mun; Yew, David Tai Wai; Xu, Jie

    2012-06-01

    MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method is one of the most widely used methods to analyze cell proliferation and viability. It is taken up through endocytosis and is reduced by mitochondrial enzymes as well as endosomal/lysosomal compartments, then is transported to cell surfaces to form needle-like MTT formazans; however the effect of MTT itself still remains elusive. Our objective was to investigate the direct effects of MTT on in vitro SH-SY5Y cells. Results showed that the endocytosis of MTT did not cause obvious lesion and induce cell death, but the metabolism and exocytosis of MTT could dramatically damage cells. Our results also indicated that MTT could activate apoptosis related factors such as caspase-8, caspase-3 or accelerate the leakage of cell contents after the appearance of MTT formazan crystals. The present data suggest MTT method should be carefully chosen; otherwise the cell viability would be underestimated and incomparable.

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

    Science.gov (United States)

    Tsutusumi, T; Kurabuchi, S; Aiyama, S

    1989-06-01

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

  1. Granulopoiesis and granules of human neutrophils

    DEFF Research Database (Denmark)

    Cowland, Jack B; Borregaard, Niels

    2016-01-01

    Granules are essential for the ability of neutrophils to fulfill their role in innate immunity. Granule membranes contain proteins that react to environmental cues directing neutrophils to sites of infection and initiate generation of bactericidal oxygen species. Granules are densely packed...... with proteins that contribute to microbial killing when liberated to the phagosome or extracellularly. Granules are, however, highly heterogeneous and are traditionally subdivided into azurophil granules, specific granules, and gelatinase granules in addition to secretory vesicles. This review will address...... issues pertinent to formation of granules, which is a process intimately connected to maturation of neutrophils from their precursors in the bone marrow. We further discuss possible mechanisms by which decisions are made regarding sorting of proteins to constitutive secretion or storage in granules...

  2. An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis.

    Directory of Open Access Journals (Sweden)

    Wei Xiong

    Full Text Available AIM: Neurotransmitter release is elicited by an elevation of intracellular Ca(2+ concentration ([Ca(2+](i. The action potential triggers Ca(2+ influx through Ca(2+ channels which causes local changes of [Ca(2+](i for vesicle release. However, any direct role of extracellular Ca(2+ (besides Ca(2+ influx on Ca(2+-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG neurons and chromaffin cells, widely used models for studying vesicle exocytosis. RESULTS: Using photolysis of caged Ca(2+ and caffeine-induced release of stored Ca(2+, we found that extracellular Ca(2+ inhibited exocytosis following moderate [Ca(2+](i rises (2-3 µM. The IC(50 for extracellular Ca(2+ inhibition of exocytosis (ECIE was 1.38 mM and a physiological reduction (∼30% of extracellular Ca(2+ concentration ([Ca(2+](o significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca(2+](o. The calcimimetics Mg(2+, Cd(2+, G418, and neomycin all inhibited exocytosis. The extracellular Ca(2+-sensing receptor (CaSR was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE. CONCLUSION/SIGNIFICANCE: As an extension of the classic Ca(2+ hypothesis of synaptic release, physiological levels of extracellular Ca(2+ play dual roles in evoked exocytosis by providing a source of Ca(2+ influx, and by directly regulating quantal size and release probability in neuronal cells.

  3. Zona Pellucida Induces Activation of Phospholipase A2 During Acrosomal Exocytosis in Guinea PigSpermatozoa

    Institute of Scientific and Technical Information of China (English)

    袁玉英; 陈文颖; 石其贤; 毛丽珍; 俞淑清; 方昕; E.R.S.Roldan

    2005-01-01

    Objective To investigate whether PLA2 is involved in ZP-stimulated acrosomal exocytosis,if Ca2+ is required for activation of PLA2,and sigal transduction pathways modulating PLA2. Methods Guinea-pig spermatozoa were capacitated and labeled in low Ca2+ medium with [14C]choline chloride or [14C] arachidonic acid,and were then exposed to millimolar Ca2+ and various reagents and stimulated with ZP. Results Precapacitated spermatozoa exposed to millimolar Ca2+ and stimulated with ZP experienced increases in arachidonic acid(AA)and lysophosphatidylcholine (lysoPC) and a parallel decrease in phosphatidylcholine (PC);these chages are indicative of PLA2 activation.Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid,a PLA2 inhibitor, before treatment with ZP.Stimulation with ZP in medium without added Ca2+,or in medium with millimolar Ca2+ and EGTA or La3+ resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin,a G1 protein inhibitor, before stimulation with ZP,blocked the release of AA and lysoPC and acrosomal exocytosis.Exposure of spermatozoa to the DAG kinase inhibitor R59022 before ZP stimulation led to a significant increase in the generation of lysoPC and exocytosis.Conclusion These results indicate very strongly that PLA2 plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA2 activation requires Ca2+ internalization,and that it is regulated by signal transduction pathways involving G proteins and DAG.

  4. Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosis.

    Directory of Open Access Journals (Sweden)

    Ian B Hogue

    2014-12-01

    Full Text Available Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.

  5. Vesicle-associated membrane protein 3 (VAMP-3) and VAMP-8 are present in human platelets and are required for granule secretion.

    Science.gov (United States)

    Polgár, János; Chung, Sul-Hee; Reed, Guy L

    2002-08-01

    Secretion of platelet granules is necessary for normal hemostasis. Platelet secretion requires soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex formation between different members of the syntaxin, SNAP-25, and vesicle-associated membrane protein (VAMP) gene families. Using microcapillary reverse-phase high-performance liquid chromatography-nano-electrospray tandem mass spectrometry, we identified VAMP-3 and VAMP-8 as VAMP isoforms coimmunoprecipitated from platelets with syntaxin 4. Immunoblotting experiments confirmed the presence of VAMP-3 and VAMP-8 but not VAMP-1 or VAMP-2 in platelets. To examine the effect of VAMP proteins on platelet secretion, soluble recombinant (r) VAMP-2, rVAMP-3, and rVAMP-8 were incubated with streptolysin O-permeabilized platelets. Secretion of alpha granules (monitored by flow cytometric measurement of P-selectin) was blocked, and dense-granule secretion (assessed by release of carbon 14-serotonin) was almost completely inhibited by rVAMP-3, whereas rVAMP-8 inhibited secretion of dense granules but not alpha granules. In contrast, rVAMP-2, which formed SNARE complexes in vitro, had no effect on platelet exocytosis. We conclude that VAMP-3 and VAMP-8 form SNARE complexes with platelet syntaxin 4 and are required for platelet granule secretion.

  6. Linking granulation performance with residence time and granulation liquid distributions in twin-screw granulation: An experimental investigation

    DEFF Research Database (Denmark)

    Kumar, Ashish; Alakarjula, Maija; Vanhoorne, Valérie

    2016-01-01

    Twin-screw granulation is a promising wet granulation technique for the continuous manufacturing of pharmaceutical solid dosage forms. A twin screw granulator displays a short residence time. Thus, the solid-liquid mixing must be achieved quickly by appropriate arrangement of transport and kneading...... elements in the granulator screw allowing the production of granules with a size distribution appropriate for tableting. The distribution of residence time and granulation liquid is governed by the field conditions (such as location and length of mixing zones) in the twin-screw granulator, thus contain...... interesting information on granulation time, mixing and resulting sub-processes such as wetting, aggregation and breakage. In this study, the impact of process (feed rate, screw speed and liquid-to-solid ratio) and equipment parameters (number of kneading discs and stagger angle) on the residence time...

  7. A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis

    Science.gov (United States)

    Guatimosim, C; Romano-Silva, M A; Cruz, J S; Beirão, P S L; Kalapothakis, E; Moraes-Santos, T; Cordeiro, M N; Diniz, C R; Gomez, M V; Prado, M A M

    1997-01-01

    The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. Using ω-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by ω-agatoxin IVA, an antagonist of P/Q calcium channels. ω-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. It is concluded that Tx3-3 potently inhibits ω-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes. PMID:9351520

  8. A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis.

    Science.gov (United States)

    Samie, Mohammad; Wang, Xiang; Zhang, Xiaoli; Goschka, Andrew; Li, Xinran; Cheng, Xiping; Gregg, Evan; Azar, Marlene; Zhuo, Yue; Garrity, Abigail G; Gao, Qiong; Slaugenhaupt, Susan; Pickel, Jim; Zolov, Sergey N; Weisman, Lois S; Lenk, Guy M; Titus, Steve; Bryant-Genevier, Marthe; Southall, Noel; Juan, Marugan; Ferrer, Marc; Xu, Haoxing

    2013-09-16

    Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.

  9. Ischemia-induced exocytosis of Weibel-Palade bodies mobilizes stem cells.

    Science.gov (United States)

    Kuo, Mei-Chuan; Patschan, Daniel; Patschan, Susann; Cohen-Gould, Leona; Park, Hyeong-Cheon; Ni, Jei; Addabbo, Francesco; Goligorsky, Michael S

    2008-12-01

    Recruitment of various stem and progenitor cells is crucial for the regeneration of an injured organ. Levels of uric acid, one of the prototypical "alarm signals," surge after ischemia-reperfusion injury. Exogenous uric acid rapidly mobilizes endothelial progenitor cells and hematopoietic stem cells and protects the kidney from ischemia. The relatively fast responses to uric acid suggest that preformed second messengers may be released from a storage pool. Here, it is reported that monosodium urate (MSU) results in exocytosis of Weibel-Palade bodies in vitro and in vivo, leading to the release of IL-8, von Willebrand factor, and angiopoietin 2 in the culture medium or circulation. Confocal and immunoelectron microscopy confirmed depletion of von Willebrand factor in MSU-treated aortic endothelial cells. Angiopoietin 2 alone induced exocytosis of Weibel-Palade bodies, mobilized hematopoietic stem cells and depleted splenic endothelial progenitor cells, partially reproducing the actions of MSU. In addition, pretreatment with angiopoietin 2 protected the kidneys from an ischemic insult, suggesting that the previously reported renoprotection conferred by MSU likely results from exocytosis of Weibel-Palade bodies. Furthermore, experiments with toll-like receptor 4 (TLR-4)-and TLR-2-deficient mice demonstrated that uric acid-induced exocytosis of Weibel-Palade bodies is mediated by TLR-4 and that uric acid-induced release of IL-8 requires both TLR-2 and TLR-4. In summary, these results suggest that exocytosis of Weibel-Palade bodies links postischemic repair with inflammation and mobilization of stem cells.

  10. Exocytosis of serotonin from the neuronal soma is sustained by a serotonin and calcium-dependent feedback loop

    Directory of Open Access Journals (Sweden)

    Carolina eLeon-Pinzon

    2014-06-01

    Full Text Available The soma of many neurons releases large amounts of transmitter molecules through an exocytosis process that continues for hundreds of seconds after the end of the triggering stimulus. Transmitters released in this way modulate the activity of neurons, glia and blood vessels over vast volumes of the nervous system. Here we studied how somatic exocytosis is maintained for such long periods in the absence of electrical stimulation and transmembrane Ca2+ entry. Somatic exocytosis of serotonin from dense core vesicles could be triggered by a train of 10 action potentials at 20 Hz in Retzius neurons of the leech. However, the same number of action potentials produced at 1 Hz failed to evoke any exocytosis. The 20-Hz train evoked exocytosis through a sequence of intracellular Ca2+ transients, with each transient having a different origin, timing and intracellular distribution. Upon electrical stimulation, transmembrane Ca2+ entry through L-type channels activated Ca2+-induced Ca2+ release. A resulting fast Ca2+ transient evoked an early exocytosis of serotonin from sparse vesicles resting close to the plasma membrane. This Ca2+ transient also triggered the transport of distant clusters of vesicles towards the plasma membrane. Upon exocytosis, the released serotonin activated autoreceptors coupled to phospholipase C, which in turn produced an intracellular Ca2+ increase in the submembrane shell. This localized Ca2+ increase evoked new exocytosis as the vesicles in the clusters arrived gradually at the plasma membrane. In this way, the extracellular serotonin elevated the intracellular Ca2+ and this Ca2+ evoked more exocytosis. The resulting positive feedback loop maintained exocytosis for the following hundreds of seconds until the last vesicles in the clusters fused. Since somatic exocytosis displays similar kinetics in neurons releasing different types of transmitters, the data presented here contributes to understand the cellular basis of paracrine

  11. Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii.

    Science.gov (United States)

    Merabishvili, Maia; Vandenheuvel, Dieter; Kropinski, Andrew M; Mast, Jan; De Vos, Daniel; Verbeken, Gilbert; Noben, Jean-Paul; Lavigne, Rob; Vaneechoutte, Mario; Pirnay, Jean-Paul

    2014-01-01

    Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively), high burst size (125 and 145, respectively), stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections.

  12. Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii.

    Directory of Open Access Journals (Sweden)

    Maia Merabishvili

    Full Text Available Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively, high burst size (125 and 145, respectively, stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections.

  13. Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure

    Science.gov (United States)

    Villares, Ana; Moreau, Céline; Bennati-Granier, Chloé; Garajova, Sona; Foucat, Loïc; Falourd, Xavier; Saake, Bodo; Berrin, Jean-Guy; Cathala, Bernard

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers. PMID:28071716

  14. Increased Lytic Efficiency of Bovine Macrophages Trained with Killed Mycobacteria

    Science.gov (United States)

    Juste, Ramon A.; Alonso-Hearn, Marta; Garrido, Joseba M.; Abendaño, Naiara; Sevilla, Iker A.; Gortazar, Christian; de la Fuente, José; Dominguez, Lucas

    2016-01-01

    Innate immunity is evolutionarily conserved in multicellular organisms and was considered to lack memory until very recently. One of its more characteristic mechanisms is phagocytosis, the ability of cells to engulf, process and eventually destroy any injuring agent. We report the results of an ex vivo experiment in bovine macrophages in which improved clearance of Mycobacterium bovis (M. bovis) was induced by pre-exposure to a heat killed M. bovis preparation. The effects were independent of humoral and cellular adaptive immune responses and lasted up to six months. Specifically, our results demonstrate the existence of a training effect in the lytic phase of phagocytosis that can be activated by killed mycobacteria, thus suggesting a new mechanism of vaccine protection. These findings are compatible with the recently proposed concept of trained immunity, which was developed to explain the observation that innate immune responses provide unspecific protection against pathogens including other than those that originally triggered the immune response. PMID:27820836

  15. Structural characterization of Lytic Polysaccharide MonoOxygenases

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of copper-containingmetalloenzymes that have been found to oxidatively degrade polysaccharides (and recently alsooligosaccharides). They dependent on redox partners to provide them with electrons and they utilizemolecular oxygen to cleave......) and their interaction with substratehave been structurally characterized. A number of structures of LsAA9A have been obtained in complexwith a range of cellulosic- and hemicellulosic substrates and with the active site Cu in different redox state.Two of the LsAA9A structures with the active site Cu in essentially a Cu......(II) state show differences in thenature of the Cu-ligand with and without cellulosic substrate bound and provide structural insight into themechanistic action of LPMOs. Interestingly, for an LsAA9A complex structure with a hemicellulosicsubstrate (xylooligosaccharide) a corresponding difference...

  16. The role of VAMP7/TI-VAMP in cell polarity and lysosomal exocytosis in vivo.

    Science.gov (United States)

    Sato, Mahito; Yoshimura, Shinichiro; Hirai, Rika; Goto, Ayako; Kunii, Masataka; Atik, Nur; Sato, Takashi; Sato, Ken; Harada, Reiko; Shimada, Junko; Hatabu, Toshimitsu; Yorifuji, Hiroshi; Harada, Akihiro

    2011-10-01

    VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis.

  17. Asynchronous transmitter release: control of exocytosis and endocytosis at the salamander rod synapse.

    Science.gov (United States)

    Rieke, F; Schwartz, E A

    1996-05-15

    1. We have studied exocytosis and endocytosis in the synaptic terminal of salamander rods using a combination of Ca2+ imaging, capacitance measurement and the photolysis of Ca2+ buffers. 2. The average cytoplasmic Ca2+ concentration at the dark resting potential was 2-4 microM. 3. An average cytoplasmic Ca2+ concentration of 2-4 microM maintained a high rate of continuous exocytosis and endocytosis. 4. Changes in the rate of exocytosis were followed in less than 0.7 s by compensatory changes in the rate of endocytosis. 5. Vesicle cycling in the rod synapse is specialized for graded transmission and differs from that previously described for synapses that release synchronized bursts of transmitter.

  18. A quantitative study of exocytosis of titanium dioxide nanoparticles from neural stem cells

    Science.gov (United States)

    Wang, Yanli; Wu, Qiuxia; Sui, Keke; Chen, Xin-Xin; Fang, Jie; Hu, Xuefeng; Wu, Minghong; Liu, Yuanfang

    2013-05-01

    Nanoparticles (NPs) have been widely studied and applied in biomedicine and other fields. It is important to know the basic process of interaction between NPs and cells in terms of cellular endocytosis and exocytosis. However, little attention has been paid to the cellular exocytosis of NPs. Herein, using a multi-step cellular subculture method, we ascertain quantitatively the endocytosis and exocytosis of widely used TiO2 NPs using the neural stem cells (NSC) as a cellular model and ICP-AES as an analytic measure. Irrespective of the type and dose of TiO2 NPs, approximately 30% of the total TiO2 NPs entered NSCs after 48 h incubation. In the first 24 h after removing TiO2NPs, from the culture medium, about 35.0%, 34.6% and 41.7% of NP1 (50 nm), NP2 (30 nm) and NTs (nanotubes, 100 nm × 4-6 nm) were released (exocytosed) from cells, respectively. The release decreased over time, and became negligible at 72 h. Exocytosis did not happen during cell division. In addition, our results suggested that both endocytosis and exocytosis of TiO2NPs were energy-dependent processes, and NPs uptake by cells was influenced by serum proteins. Furthermore, we achieved primary dynamic confocal imaging of the exocytosis, allowing tracking of TiO2 NPs from NSCs. These findings may benefit studies on nanotoxicology and nanomedicine of TiO2 NPs.Nanoparticles (NPs) have been widely studied and applied in biomedicine and other fields. It is important to know the basic process of interaction between NPs and cells in terms of cellular endocytosis and exocytosis. However, little attention has been paid to the cellular exocytosis of NPs. Herein, using a multi-step cellular subculture method, we ascertain quantitatively the endocytosis and exocytosis of widely used TiO2 NPs using the neural stem cells (NSC) as a cellular model and ICP-AES as an analytic measure. Irrespective of the type and dose of TiO2 NPs, approximately 30% of the total TiO2 NPs entered NSCs after 48 h incubation. In the

  19. Variation of granule mass fraction with coordination number in wet granulation process

    Institute of Scientific and Technical Information of China (English)

    Papiya Roy; Manish Vashishtha; Rajesh Khanna; Duwuri Subbarao

    2009-01-01

    In granulation, fine particles combine to form a coarse granule in the form of a particle matrix partially or fully saturated with a binder liquid. The final product of granulation possesses a wide variety of granule size distributions with surface mean diameters which differ with operating conditions. The final granule size depends on the operating conditions, e.g. operating gas velocity, inlet air temperature, initial feed particle size, and viscosity of the binder. The objective of this paper is to find out the uniformity in the relation between the granule mass fraction in the final granule size distribution and the number of feed particles present in the granules. The total number of granules obtained depends on the experimental conditions but the granule mass fraction and the number of feed particles forming a single granule are independent of operating variables, feed material and method of granulation. The paper purports further to compare the uniform nature of mass fraction of the granules in final granule size distribution and the primary particles required to form that particular granule size irrespective of experimental conditions of granulation.

  20. Statistical analysis and comparison of a continuous high shear granulator with a twin screw granulator: Effect of process parameters on critical granule attributes and granulation mechanisms.

    Science.gov (United States)

    Meng, Wei; Kotamarthy, Lalith; Panikar, Savitha; Sen, Maitraye; Pradhan, Shankali; Marc, Michaelis; Litster, James D; Muzzio, Fernando J; Ramachandran, Rohit

    2016-11-20

    This study is concerned with identifying the design space of two different continuous granulators and their respective granulation mechanisms. Performance of a continuous high shear granulator and a twin screw granulator with paracetamol formulations were examined by face-centered cubic design, which focused on investigating key performance metrics, namely, granule size, porosity, flowability and particle morphology of granules as a function of essential input process parameters (liquid content, throughput and rotation speed). Liquid and residence time distribution tests were also performed to gain insights into the liquid-powder mixing and flow behavior. The results indicated that continuous high shear granulation was more sensitive to process variation and produced spherical granules with monomodal size distribution and distinct internal structure and strength variation. Twin screw granulation with such a particular screw configuration showed narrower design space and granules were featured with multimodal size distribution, irregular shape, less detectible porosity difference and tighter range of strength. Granulation mechanisms explored on the basis of nucleation and growth regime maps revealed that for most cases liquid binder was uniformly distributed with fast droplet penetration into the powder bed and that granule consolidation and coalescence mainly took place in the nucleation, steady growth and rapid growth regimes.

  1. Steering neuronal growth cones by shifting the imbalance between exocytosis and endocytosis.

    Science.gov (United States)

    Tojima, Takuro; Itofusa, Rurika; Kamiguchi, Hiroyuki

    2014-05-21

    Extracellular molecular cues guide migrating growth cones along specific routes during development of axon tracts. Such processes rely on asymmetric elevation of cytosolic Ca(2+) concentrations across the growth cone that mediates its attractive or repulsive turning toward or away from the side with Ca(2+) elevation, respectively. Downstream of these Ca(2+) signals, localized activation of membrane trafficking steers the growth cone bidirectionally, with endocytosis driving repulsion and exocytosis causing attraction. However, it remains unclear how Ca(2+) can differentially regulate these opposite membrane-trafficking events. Here, we show that growth cone turning depends on localized imbalance between exocytosis and endocytosis and identify Ca(2+)-dependent signaling pathways mediating such imbalance. In embryonic chicken dorsal root ganglion neurons, repulsive Ca(2+) signals promote clathrin-mediated endocytosis through a 90 kDa splice variant of phosphatidylinositol-4-phosphate 5-kinase type-1γ (PIPKIγ90). In contrast, attractive Ca(2+) signals facilitate exocytosis but suppress endocytosis via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (Cdk5) that can inactivate PIPKIγ90. Blocking CaMKII or Cdk5 leads to balanced activation of both exocytosis and endocytosis that causes straight growth cone migration even in the presence of guidance signals, whereas experimentally perturbing the balance restores the growth cone's turning response. Remarkably, the direction of this resumed turning depends on relative activities of exocytosis and endocytosis, but not on the type of guidance signals. Our results suggest that navigating growth cones can be redirected by shifting the imbalance between exocytosis and endocytosis, highlighting the importance of membrane-trafficking imbalance for axon guidance and, possibly, for polarized cell migration in general.

  2. RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Bello, Oscar D.; Zanetti, M. Natalia [Laboratorio de Biologia Celular y Molecular, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Laboratorio de Biologia Reproductiva, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Mayorga, Luis S. [Laboratorio de Biologia Celular y Molecular, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Michaut, Marcela A., E-mail: mmichaut@fcm.uncu.edu.ar [Laboratorio de Biologia Reproductiva, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Instituto de Ciencias Basicas, Universidad Nacional de Cuyo, Mendoza (5500) (Argentina)

    2012-03-10

    Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: Black-Right-Pointing-Pointer RIM and Munc13 are present in human sperm and localize to the acrosomal region. Black-Right-Pointing-Pointer RIM and Munc13 are necessary for acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Munc13 participate before the acrosomal calcium efflux. Black

  3. 5-hydroxymethylation of the EBV genome regulates the latent to lytic switch.

    Science.gov (United States)

    Wille, Coral K; Nawandar, Dhananjay M; Henning, Amanda N; Ma, Shidong; Oetting, Kayla M; Lee, Dennis; Lambert, Paul; Johannsen, Eric C; Kenney, Shannon C

    2015-12-29

    Latent Epstein-Barr virus (EBV) infection and cellular hypermethylation are hallmarks of undifferentiated nasopharyngeal carcinoma (NPC). However, EBV infection of normal oral epithelial cells is confined to differentiated cells and is lytic. Here we demonstrate that the EBV genome can become 5-hydroxymethylated and that this DNA modification affects EBV lytic reactivation. We show that global 5-hydroxymethylcytosine (5hmC)-modified DNA accumulates during normal epithelial-cell differentiation, whereas EBV+ NPCs have little if any 5hmC-modified DNA. Furthermore, we find that increasing cellular ten-eleven translocation (TET) activity [which converts methylated cytosine (5mC) to 5hmC] decreases methylation, and increases 5hmC modification, of lytic EBV promoters in EBV-infected cell lines containing highly methylated viral genomes. Conversely, inhibition of endogenous TET activity increases lytic EBV promoter methylation in an EBV-infected telomerase-immortalized normal oral keratinocyte (NOKs) cell line where lytic viral promoters are largely unmethylated. We demonstrate that these cytosine modifications differentially affect the ability of the two EBV immediate-early proteins, BZLF1 (Z) and BRLF1 (R), to induce the lytic form of viral infection. Although methylation of lytic EBV promoters increases Z-mediated and inhibits R-mediated lytic reactivation, 5hmC modification of lytic EBV promoters has the opposite effect. We also identify a specific CpG-containing Z-binding site on the BRLF1 promoter that must be methylated for Z-mediated viral reactivation and show that TET-mediated 5hmC modification of this site in NOKs prevents Z-mediated viral reactivation. Decreased 5-hydroxymethylation of cellular and viral genes may contribute to NPC formation.

  4. Midline 1 controls polarization and migration of murine cytotoxic T cells

    DEFF Research Database (Denmark)

    Boding, Lasse; Hansen, Ann K; Nielsen, Morten M;

    2014-01-01

    is strongly up-regulated in murine cytotoxic T lymphocytes (CTLs), and that it has a significant impact on exocytosis of lytic granules and the killing capacity of CTLs. The aims of the present study were to determine the localization of MID1 in migrating CTLs, and to investigate whether MID1 affects CTL...

  5. Complete Genome Sequences of Lytic Bacteriophages of Xanthomonas arboricola pv. juglandis.

    Science.gov (United States)

    Retamales, Julio; Vasquez, Ignacio; Santos, Leonardo; Segovia, Cristopher; Ayala, Manuel; Alvarado, Romina; Nuñez, Pablo; Santander, Javier

    2016-06-02

    Three bacteriophages, f20-Xaj, f29-Xaj, and f30-Xaj, with lytic activity against Xanthomonas arboricola pv. juglandis were isolated from walnut trees (VIII Bío Bío Region, Chile). These lytic bacteriophages have double-stranded DNA (dsDNA) genomes of 43,851 bp, 41,865 bp, and 44,262 bp, respectively. These are the first described bacteriophages with lytic activity against X. arboricola pv. juglandis that can be utilized as biocontrol agents.

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

    Science.gov (United States)

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

    2010-07-01

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

  7. Selective sorting of alpha-granule proteins.

    Science.gov (United States)

    Italiano, J E; Battinelli, E M

    2009-07-01

    One of the main functions of blood platelets is to secrete a variety of substances that can modify a developing thrombus, regulate the growth of the vasculature, promote wound repair, and contribute to cell-adhesive events. A majority of this vast array of secreted proteins are stored in alpha-granules. Until recently, it was assumed that platelets contained one homogeneous population of alpha-granules that undergo complete de-granulation during platelet activation. This review focuses on the mechanisms of alpha-granule biogenesis and secretion, with a particular emphasis on recent findings that clearly demonstrate that platelets contain distinct subpopulations of alpha-granules that undergo differential release during activation. We consider the implications of this new paradigm of platelet secretion, discuss mechanisms of alpha-granule biogenesis, and review the molecular basis of transport and delivery of alpha-granules to assembling platelets.

  8. Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells.

    Science.gov (United States)

    Oishi, Yohei; Arakawa, Toshiya; Tanimura, Akihiko; Itakura, Makoto; Takahashi, Masami; Tajima, Yoshifumi; Mizoguchi, Itaru; Takuma, Taishin

    2006-03-01

    We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH-GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20 degrees C for 2 h and then released by warming up to 37 degrees C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH-GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.

  9. Munc18/SNARE proteins' regulation of exocytosis in guinea pig duodenal Brunner's gland acini

    Institute of Scientific and Technical Information of China (English)

    Laura I Cosen-Binker; Gerry P Morris; Stephen Vanner; Herbert Y Gaisano

    2008-01-01

    AIM: To examine the molecular mechanism of exocytosis in the Brunner's gland acinar cell.METHODS: We used a submucosal preparation of guinea pig duodenal Brunner's gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy.RESULTS: One and 6 μmol/L carbachol evoked a dosedependent dilation of Brunner's gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact.CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner's gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.

  10. Hypertensive stretch regulates endothelial exocytosis of Weibel-Palade bodies through VEGF receptor 2 signaling pathways

    Institute of Scientific and Technical Information of China (English)

    Yan Xiong; Zhenqian Hu; Xiaofan Han; Beibei Jiang; Rongli Zhang; Xiaoyu Zhang; Yao Lu

    2013-01-01

    Regulated endothelial exocytosis of Weibel-Palade bodies (WPBs),the first stage in leukocyte trafficking,plays a pivotal role in inflammation and injury.Acute mechanical stretch has been closely associated with vascular inflammation,although the precise mechanism is unknown.Here,we show that hypertensive stretch regulates the exocytosis of WPBs of endothelial ceils (ECs) through VEGF receptor 2 (VEGFR2) signaling pathways.Stretch triggers a rapid release (within minutes) of von Willebrand factor and interleukin-8 from WPBs in cultured human ECs,promoting the interaction between leukocytes and ECs through the translocation of P-selectin to the cell membrane.We further show that hypertensive stretch significantly induces P-selectin translocation of intact ECs and enhances leukocyte adhesion both ex vivo and in vivo.Stretch-induced endothelial exocytosis is mediated via a VEGFR2/PLCy1/calcium pathway.Interestingly,stretch also induces a negative feedback via a VEGFR2/Akt/nitric oxide pathway.Such dual effects are confirmed using pharmacological and genetic approaches in carotid artery segments,as well as in acute hypertensive mouse models.These studies reveal mechanical stretch as a potent agonist for endothelial exocytosis,which is modulated by VEGFR2 signaling.Thus,VEGFR2 signaling pathways may represent novel therapeutic targets in limiting hypertensive stretch-related inflammation.

  11. Characterization of endocytosis and exocytosis of cationic nanoparticles in airway epithelium cells

    Science.gov (United States)

    Youta Dombu, Christophe; Kroubi, Maya; Zibouche, Rima; Matran, Regis; Betbeder, Didier

    2010-09-01

    A major challenge of drug delivery using colloids via the airway is to understand the mechanism implied in their interactions with epithelial cells. The purpose of this work was to characterize the process of endocytosis and exocytosis of cationic nanoparticles (NPs) made of maltodextrin which were developed as a delivery system for antigens in vaccine applications. Confocal microscopy demonstrated that these NP are rapidly endocytosed after as little as 3 min incubation, and that the endocytosis was also faster than NP binding since most of the NPs were found in the middle of the cells around the nuclei. A saturation limit was observed after a 40 min incubation, probably due to an equilibrium becoming established between endocytosis and exocytosis. Endocytosis was dramatically reduced at 4 °C compared with 37 °C, or by NaN3 treatment, both results suggesting an energy dependent process. Protamine pretreatment of the cells inhibited NPs uptake and we found that clathrin pathway is implied in their endocytosis. Cholesterol depletion increased NP uptake by 300% and this phenomenon was explained by the fact that cholesterol depletion totally blocked NP exocytosis. These results suggest that these cationic NPs interact with anionic sites, are quickly endocytosed via the clathrin pathway and that their exocytosis is cholesterol dependent, and are similar to those obtained in other studies with viruses such as influenza.

  12. Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+ -induced glucagon exocytosis in pancreas

    DEFF Research Database (Denmark)

    Gustavsson, Natalia; Wei, Shun-Hui; Hoang, Dong Nhut

    2009-01-01

    abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus...

  13. Characterization of endocytosis and exocytosis of cationic nanoparticles in airway epithelium cells

    Energy Technology Data Exchange (ETDEWEB)

    Dombu, Christophe Youta; Kroubi, Maya; Zibouche, Rima; Matran, Regis; Betbeder, Didier, E-mail: dbetbeder@aol.com [EA 4483, IFR 114, Laboratoire de Physiologie, Faculte de Medecine Pole Recherche, Universite de Lille 2, 1 place de Verdun, 59045 Lille Cedex (France)

    2010-09-03

    A major challenge of drug delivery using colloids via the airway is to understand the mechanism implied in their interactions with epithelial cells. The purpose of this work was to characterize the process of endocytosis and exocytosis of cationic nanoparticles (NPs) made of maltodextrin which were developed as a delivery system for antigens in vaccine applications. Confocal microscopy demonstrated that these NP are rapidly endocytosed after as little as 3 min incubation, and that the endocytosis was also faster than NP binding since most of the NPs were found in the middle of the cells around the nuclei. A saturation limit was observed after a 40 min incubation, probably due to an equilibrium becoming established between endocytosis and exocytosis. Endocytosis was dramatically reduced at 4 deg. C compared with 37 deg. C, or by NaN{sub 3} treatment, both results suggesting an energy dependent process. Protamine pretreatment of the cells inhibited NPs uptake and we found that clathrin pathway is implied in their endocytosis. Cholesterol depletion increased NP uptake by 300% and this phenomenon was explained by the fact that cholesterol depletion totally blocked NP exocytosis. These results suggest that these cationic NPs interact with anionic sites, are quickly endocytosed via the clathrin pathway and that their exocytosis is cholesterol dependent, and are similar to those obtained in other studies with viruses such as influenza.

  14. Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes

    NARCIS (Netherlands)

    Zonia, L.; Munnik, T.

    2008-01-01

    Pollen tubes are one of the fastest growing eukaryotic cells. Rapid anisotropic growth is supported by highly active exocytosis and endocytosis at the plasma membrane, but the subcellular localization of these sites is unknown. To understand molecular processes involved in pollen tube growth, it is

  15. Effect of intravenous anesthetic propofol on synaptic vesicle exocytosis at the frog neuromuscular junction

    Institute of Scientific and Technical Information of China (English)

    Luciana Ferreira LEITE; Renato Santiago GOMEZ; Matheus de Castro FONSECA; Marcus Vinicius GOMEZ; Cristina GUATIMOSIM

    2011-01-01

    Aim: To investigate the presynaptic effects of propofol, a short-acting intravenous anesthetic, in the frog neuromuscular junction. Methods: Frog cutaneous pectoris nerve muscle preparations were prepared. A fluorescent tool (FM1-43) was used to visualize the effect of propofol on synaptic vesicle exocytosos in the frog neuromuscular junction. Results: Low concentrations of propofol, ranging from 10 to 25 μmol/L, enhanced spontaneous vesicle exocytosis monitored by FM1-43 in a Ca2+-dependent and Na+-independent fashion. Higher concentrations of propofol (50, 100, and 200 μmol/L) had no effect on spontaneous exocytosis. By contrast, higher concentrations of propofol inhibited the Na+-dependent exocytosis evoked by 4-aminopyri-dine but did not affect the Na+-independent exocytosis evoked by KCI. This action was similar and non-additive with that observed by tetrodotoxin, a Na+ channel blocker.Conclusion: Our data suggest that propofol has a dose-dependent presynaptic effect at the neuromuscular transmission which mayhelp to understand some of the clinical effects of this agent on neuromuscular function.

  16. Hypertensive stretch regulates endothelial exocytosis of Weibel-Palade bodies through VEGF receptor 2 signaling pathways.

    Science.gov (United States)

    Xiong, Yan; Hu, Zhenqian; Han, Xiaofan; Jiang, Beibei; Zhang, Rongli; Zhang, Xiaoyu; Lu, Yao; Geng, Chenyang; Li, Wei; He, Yulong; Huo, Yingqing; Shibuya, Masabumi; Luo, Jincai

    2013-06-01

    Regulated endothelial exocytosis of Weibel-Palade bodies (WPBs), the first stage in leukocyte trafficking, plays a pivotal role in inflammation and injury. Acute mechanical stretch has been closely associated with vascular inflammation, although the precise mechanism is unknown. Here, we show that hypertensive stretch regulates the exocytosis of WPBs of endothelial cells (ECs) through VEGF receptor 2 (VEGFR2) signaling pathways. Stretch triggers a rapid release (within minutes) of von Willebrand factor and interleukin-8 from WPBs in cultured human ECs, promoting the interaction between leukocytes and ECs through the translocation of P-selectin to the cell membrane. We further show that hypertensive stretch significantly induces P-selectin translocation of intact ECs and enhances leukocyte adhesion both ex vivo and in vivo. Stretch-induced endothelial exocytosis is mediated via a VEGFR2/PLCγ1/calcium pathway. Interestingly, stretch also induces a negative feedback via a VEGFR2/Akt/nitric oxide pathway. Such dual effects are confirmed using pharmacological and genetic approaches in carotid artery segments, as well as in acute hypertensive mouse models. These studies reveal mechanical stretch as a potent agonist for endothelial exocytosis, which is modulated by VEGFR2 signaling. Thus, VEGFR2 signaling pathways may represent novel therapeutic targets in limiting hypertensive stretch-related inflammation.

  17. Histones link inflammation and thrombosis through the induction of Weibel-Palade body exocytosis.

    Science.gov (United States)

    Michels, A; Albánez, S; Mewburn, J; Nesbitt, K; Gould, T J; Liaw, P C; James, P D; Swystun, L L; Lillicrap, D

    2016-11-01

    Essentials Dysregulated DNA and histone release can promote pathological immunothrombosis. Weibel-Palade bodies (WPBs) are sentinel-like organelles that respond to proinflammatory stimuli. Histones induce WPB exocytosis in a caspase, calcium and charge-dependent mechanism. A targetable axis may exist between DNA/histones and WPBs in inflammation and immunothrombosis.

  18. Properties of Brucella-phages lytic for non-smooth Brucella strains.

    Science.gov (United States)

    Corbel, M J

    1984-01-01

    A series of host-range mutants has been selected for brucella-phage R. Two of these mutants designated R/O and R/C have been used for typing purposes. Phage R/O is lytic for non-smooth strains of Brucella abortus and for B. ovis. It is genetically unstable however and produces mutants lytic for smooth B. obortus and B. suis. Phage R/C is lytic for non-smooth B. abortus and for B. ovis and B. canis. It is much more stable than phages R or R/O and shows little or no lytic activity on smooth Brucella strains. It has been effective in differentiating B. canis from B. suis in tests on a limited number of strains. In their properties, all of the brucella-phages of the R series resemble their parent phage.

  19. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Dhananjay M Nawandar

    2015-10-01

    Full Text Available Epstein-Barr virus (EBV is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL in immunosuppressed patients. However, the cellular mechanism(s that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1 promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

  20. Induction of lytic pathways in T cell clones derived from wild-type or protein tyrosine kinase Fyn mutant mice.

    Science.gov (United States)

    Lancki, D W; Fields, P; Qian, D; Fitch, F W

    1995-08-01

    The OVA-reactive CD4+ Th1 clones and alloreactive CD8+ clones derived from wild-type or fyn-/- mice serve as model systems which have allowed us to investigate several aspects of the molecular events associated with T cell-mediated cytotoxicity, including 1) the differential utilization of two distinct cytolytic pathways by CD4+ Th1 clones and CD8+ CTL, 2) a comparison of the pathways of lysis induced by stimulation of the TCR or by alternative stimuli, 3) the requirement of Fyn for derivation of antigen-specific T-cell clones having properties of CD4+ Th1 and CD8+ CTL cells 4) the differential requirement of Fyn in the induction of responses by TCR and the alternative stimuli. Stimulation through the TCR, either by APC bearing relevant antigen or by immobilized anti-CD3 mAb, resulted in comparable levels of target cell lysis by clones from both wild-type and fyn-/- mice. These clones also utilize the Fas pathway to lyse target cells. Thus, Fyn does not appear to be required for expression of the Fas pathway when triggered through the TCR. In contrast, lysis of target cells by T-cell clones lacking Fyn was deficient when stimulated through Thy-1 or Ly-6C (using mAb) or with Con A or phorbol ester as compared to clones derived from wild-type mice. The basis for the defect in response to stimulation through the GPI-linked molecules appears to be a signaling defect which affects all of the functional responses we measured, while the defect in response to Con A stimulation appears to affect lysis but not lymphokine production. Thus, Fyn expression is selectively required for efficient activation of the Fas pathway of lysis through Thy-1, Ly-6C, and by Con A or phorbol ester in these T-cell clones. CD8+ clones derived from fyn-/- mutant mice, like clones derived from wild-type mice, display antigen-specific lysis, and appear to express perforin message and perforin protein. A Ca(++)-dependent (presumably perforin/exocytosis) component and Fas component of lysis was

  1. Enhancement of Lytic Activity by Leptin Is Independent From Lipid Rafts in Murine Primary Splenocytes.

    Science.gov (United States)

    Collin, Aurore; Noacco, Audrey; Talvas, Jérémie; Caldefie-Chézet, Florence; Vasson, Marie-Paule; Farges, Marie-Chantal

    2017-01-01

    Leptin, a pleiotropic adipokine, is known as a regulator of food intake, but it is also involved in inflammation, immunity, cell proliferation, and survival. Leptin receptor is integrated inside cholesterol-rich microdomains called lipid rafts, which, if disrupted or destroyed, could lead to a perturbation of lytic mechanism. Previous studies also reported that leptin could induce membrane remodeling. In this context, we studied the effect of membrane remodeling in lytic activity modulation induced by leptin. Thus, primary mouse splenocytes were incubated with methyl-β-cyclodextrin (β-MCD), a lipid rafts disrupting agent, cholesterol, a major component of cell membranes, or ursodeoxycholic acid (UDCA), a membrane stabilizer agent for 1 h. These treatments were followed by splenocyte incubation with leptin (absence, 10 and 100 ng/ml). Unlike β-MCD or cholesterol, UDCA was able to block leptin lytic induction. This result suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling. J. Cell. Physiol. 232: 101-109, 2017. © 2016 Wiley Periodicals, Inc.

  2. Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide.

    Directory of Open Access Journals (Sweden)

    Lindsay Hewlett

    Full Text Available BACKGROUND: Weibel-Palade bodies (WPB are endothelial cell (EC specific secretory organelles containing Von Willebrand factor (VWF. The temperature-dependence of Ca(2+-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca(2+-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs. PRINCIPAL FINDINGS: Ca(2+-driven WPB exocytosis occurred at all temperatures studied (7-37°C. The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s(-1 (37°C to 0.80±0.14 s(-1 (7°C and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C to 3.6±1.3% (7°C. A discrepancy was found between the reduction in Ca(2+-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75-80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion dispersal from WPBs, although slowed, was complete within 60-120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself. CONCLUSIONS: We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased.

  3. Twin screw granulation - review of current progress.

    Science.gov (United States)

    Thompson, M R

    2015-01-01

    Twin screw granulation (TSG) is a new process of interest to the pharmaceutical community that can continuously wet granulate powders, doing so at lower liquid concentrations and with better product consistency than found by a high shear batch mixer. A considerable body of research has evolved over the short time since this process was introduced but generally with little comparison of results. A certain degree of confidence has been developed through these studies related to how process variables and many attributes of machinery configuration will affect granulation but some major challenges still lay ahead related to scalability, variations in the processing regimes related to degree of channel fill and the impact of wetting and granulation of complex powder formulations. This review examines the current literature for wet granulation processes studied in twin screw extrusion machinery, summarizing the influences of operational and system parameters affecting granule properties as well as strives to provide some practical observations to newly interested users of the technique.

  4. Selective sorting of alpha-granule proteins

    OpenAIRE

    Italiano, J.E.; Battinelli, E. M.

    2009-01-01

    One of the main functions of blood platelets is to secrete a variety of substances that can modify a developing thrombus, regulate the growth of the vasculature, promote wound repair, and contribute to cell-adhesive events. The majority of this vast array of secreted proteins is stored in alpha-granules. Until recently, it was assumed that platelets contained one homogeneous population of alpha-granules that undergo complete de-granulation during platelet activation. This review focuses on th...

  5. The extrusion properties of potato granules

    OpenAIRE

    Kooi, Eng Teong

    1982-01-01

    Potato granules from different sources were found, on extrusion, to produce potato snacks of variable quality. In some instances strip formation was unsatisfactory, in other instances blistering of the snack occurred on frying. In total, about 20-25 batches of potato granules were examined and classified in relation to these two phenomena. The amylose/amylopectin ratios of these samples of potato granules were determined by the semi-micro potentiometric iodine titration technique, but it was ...

  6. Actin filaments and microtubule dual-granule transport in human adhered platelets: the role of alpha-dystrobrevins.

    Science.gov (United States)

    Cerecedo, Doris; Cisneros, Bulmaro; Mondragón, Ricardo; González, Sirenia; Galván, Iván J

    2010-04-01

    Upon activation with physiological stimuli, human platelets undergo morphological changes, centralizing their organelles and secreting effector molecules at the site of vascular injury. Previous studies have indicated that the actin filaments and microtubules of suspension-activated platelets play a critical role in granule movement and exocytosis; however, the participation of these cytoskeleton elements in adhered platelets remains unexplored. alpha- and beta-dystrobrevin members of the dystrophin-associated protein complex in muscle and non-muscle cells have been described as motor protein receptors that might participate in the transport of cellular components in neurons. Recently, we characterized the expression of dystrobrevins in platelets; however, their functional diversity within this cellular model had not been elucidated. The present study examined the contribution of actin filaments and microtubules in granule trafficking during the platelet adhesion process using cytoskeleton-disrupting drugs, quantification of soluble P-selectin, fluorescence resonance transfer energy analysis and immunoprecipitation assays. Likewise, we assessed the interaction of alpha-dystrobrevins with the ubiquitous kinesin heavy chain. Our results strongly suggest that microtubules and actin filaments participate in the transport of alpha and dense granules in the platelet adhesion process, during which alpha-dystrobrevins play the role of regulatory and adaptor proteins that govern trafficking events.

  7. Medical image of the week: granulation tissue

    Directory of Open Access Journals (Sweden)

    Ganesh A

    2014-03-01

    Full Text Available A 57 year old woman presented with a tickling sensation in the back of throat and intermittent bleeding from the healing stoma one month after decannulation of her tracheostomy tube. On bronchoscopy a granuloma with surrounding granulation tissue was present in the subglottic space (Figure 1. Argon plasma coagulation (APC was performed to cauterize the granulation tissue (Figure 2. Formation of granulation tissue after tracheostomy is a common complication which can result in tracheal stenosis. APC and electrocautery using flexible bronchoscopy has been shown to safely and effectively remove the granulation tissue.

  8. Mechanism of film formation during granules capsulation in fluidized bed

    OpenAIRE

    Ostroha, Ruslan; Yukhymenko, Mykola

    2013-01-01

    It is proposed to perform granules capsulation process in the device of fluidized bed. Analysis of different approaches to mathematical description of granules growth kinetics was made. Equation of size determination of received granules in the device is proposed including granules growth rate and changes of density of granules distribution according to sizes in film forming process.

  9. Granulation of coal fly ash by using different types of granule agents

    Science.gov (United States)

    Agusta, H.; Nisya, F. N.; Iman, R. N.; Bilad, D. B. C.

    2017-05-01

    The use of coal produces about 5% solid pollutant in the form of ash (fly ash and bottom ash). Of the total ash produced, about 10-20% is bottom ash and 80-90% is fly ash. This study was aimed at obtaining a type of adhesive which could be used as a fly granulation material for soil conditioner. The study was conducted at the pilot plant of Surfactant and Bioenergy Research Center (SBRC) LPPM IPB from April to August 2016. The fly ash used in this study was obtained from Kalimantan. A pan granulator was used in fly ash granule making process. Granule agent materials were diluted in the concentration of 5, 10, and 15%. Different types of granule agents, namely SBRC-M, SBRC-T, and SBRC-SC were used. The formed fly ash granules were then analyzed for their physical properties including particle density, fly ash granule pH, fly ash granule durability, and fly ash granule water holding capacity. Results showed that fly ash granules made from 15% of SBRC-M had the highest particle density (0.75 g/cm3). Fly ash granules made with SBRC-M had higher pH (10) than those made by using SBRC-SC adhesive (9.3) and SBRC-T (9). SBRC-T was found as the granule agent material which produced fly ash granules with the highest durability levels on average. In this study, the use of SBRC-M granule agent resulted in higher water holding capacity (WHC) (40.62%) than did SBRC-SC (38.79%) and SBRC-T (36.85%). As a granule agent, compared to SBRC-SC and SBRC-T, SBRC-M could produce fly ash granules with highest particle density, highest pH, good durability, and best water holding capacity.

  10. Discovery and industrial applications of lytic polysaccharide mono-oxygenases.

    Science.gov (United States)

    Johansen, Katja S

    2016-02-01

    The recent discovery of copper-dependent lytic polysaccharide mono-oxygenases (LPMOs) has opened up a vast area of research covering several fields of application. The biotech company Novozymes A/S holds patents on the use of these enzymes for the conversion of steam-pre-treated plant residues such as straw to free sugars. These patents predate the correct classification of LPMOs and the striking synergistic effect of fungal LPMOs when combined with canonical cellulases was discovered when fractions of fungal secretomes were evaluated in industrially relevant enzyme performance assays. Today, LPMOs are a central component in the Cellic CTec enzyme products which are used in several large-scale plants for the industrial production of lignocellulosic ethanol. LPMOs are characterized by an N-terminal histidine residue which, together with an internal histidine and a tyrosine residue, co-ordinates a single copper atom in a so-called histidine brace. The mechanism by which oxygen binds to the reduced copper atom has been reported and the general mechanism of copper-oxygen-mediated activation of carbon is being investigated in the light of these discoveries. LPMOs are widespread in both the fungal and the bacterial kingdoms, although the range of action of these enzymes remains to be elucidated. However, based on the high abundance of LPMOs expressed by microbes involved in the decomposition of organic matter, the importance of LPMOs in the natural carbon-cycle is predicted to be significant. In addition, it has been suggested that LPMOs play a role in the pathology of infectious diseases such as cholera and to thus be relevant in the field of medicine. © 2016 Authors; published by Portland Press Limited.

  11. The paradox of high shear granulation : the formation of non-homogeneous granules

    NARCIS (Netherlands)

    Dries, Kaspar van den

    2004-01-01

    Wet granulation is a process used for the particle size enlargement of primary powders. The mixing of a liquid with the powder glues the primary particles together, which results in the formation of the granules. The mixing action can be performed in many ways, like tumbling (drum granulation), flui

  12. Granule fraction inhomogeneity of calcium carbonate/sorbitol in roller compacted granules

    DEFF Research Database (Denmark)

    Bacher, Charlotte; Olsen, P.M.; Bertelsen, P.;

    2008-01-01

    The granule fraction inhomogeneity of roller compacted granules was examined on mixtures of three different morphologic forms of calcium carbonate and three particle sizes of sorbitol. The granule fraction inhomogeneity was determined by the distribution of the calcium carbonate in each of the 10...

  13. Different NK cell-activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity

    DEFF Research Database (Denmark)

    Wood, Stephanie M; Meeths, Marie; Chiang, Samuel C C

    2009-01-01

    of perforin-containing lytic granules induced by signals for natural and antibody-dependent cellular cytotoxicity. We demonstrate here that these signals fail to induce degranulation in resting NK cells from Rab27a-deficient patients. In resting NK cells from healthy subjects, endogenous Rab27a and Munc13...... functional antigen-1, NKG2D, or 2B4 induced colocalization of Rab27a, but not Munc13-4, with perforin. Conversely, engagement of antibody-dependent cellular cytotoxicity receptor CD16 induced colocalization of Munc13-4, but not Rab27a, with perforin. Furthermore, colocalization of Munc13-4 with perforin...

  14. Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

    Science.gov (United States)

    MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

    2015-04-24

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis.

  15. Regulated protein aggregation: stress granules and neurodegeneration

    Directory of Open Access Journals (Sweden)

    Wolozin Benjamin

    2012-11-01

    Full Text Available Abstract The protein aggregation that occurs in neurodegenerative diseases is classically thought to occur as an undesirable, nonfunctional byproduct of protein misfolding. This model contrasts with the biology of RNA binding proteins, many of which are linked to neurodegenerative diseases. RNA binding proteins use protein aggregation as part of a normal regulated, physiological mechanism controlling protein synthesis. The process of regulated protein aggregation is most evident in formation of stress granules. Stress granules assemble when RNA binding proteins aggregate through their glycine rich domains. Stress granules function to sequester, silence and/or degrade RNA transcripts as part of a mechanism that adapts patterns of local RNA translation to facilitate the stress response. Aggregation of RNA binding proteins is reversible and is tightly regulated through pathways, such as phosphorylation of elongation initiation factor 2α. Microtubule associated protein tau also appears to regulate stress granule formation. Conversely, stress granule formation stimulates pathological changes associated with tau. In this review, I propose that the aggregation of many pathological, intracellular proteins, including TDP-43, FUS or tau, proceeds through the stress granule pathway. Mutations in genes coding for stress granule associated proteins or prolonged physiological stress, lead to enhanced stress granule formation, which accelerates the pathophysiology of protein aggregation in neurodegenerative diseases. Over-active stress granule formation could act to sequester functional RNA binding proteins and/or interfere with mRNA transport and translation, each of which might potentiate neurodegeneration. The reversibility of the stress granule pathway also offers novel opportunities to stimulate endogenous biochemical pathways to disaggregate these pathological stress granules, and perhaps delay the progression of disease.

  16. Deconstructing complexin function in activating and clamping Ca2+-triggered exocytosis by comparing knockout and knockdown phenotypes.

    Science.gov (United States)

    Yang, Xiaofei; Cao, Peng; Südhof, Thomas C

    2013-12-17

    Complexin, a presynaptic protein that avidly binds to assembled SNARE complexes, is widely acknowledged to activate Ca(2+)-triggered exocytosis. In addition, studies of invertebrate complexin mutants and of mouse neurons with a double knockdown (DKD) of complexin-1 and -2 suggested that complexin maintains the readily releasable pool (RRP) of vesicles and clamps spontaneous exocytosis. In contrast, studies of mouse neurons with a double knockout (DKO) of complexin-1 and -2, largely carried out in hippocampal autapses, did not detect changes in the RRP size or in spontaneous exocytosis. To clarify complexin function, we here directly compared in two different preparations, cultured cortical and olfactory bulb neurons, the phenotypes of complexin DKD and DKO neurons. We find that complexin-deficient DKD and DKO neurons invariably exhibit a ~50% decrease in vesicle priming. Moreover, the DKD consistently increased spontaneous exocytosis, but the DKO did so in cortical but not olfactory bulb neurons. Furthermore, the complexin DKD but not the complexin DKO caused a compensatory increase in complexin-3 and -4 mRNA levels; overexpression of complexin-3 but not complexin-1 increased spontaneous exocytosis. Complexin-3 but not complexin-1 contains a C-terminal lipid anchor attaching it to the plasma membrane; addition of a similar lipid anchor to complexin-1 converted complexin-1 from a clamp into an activator of spontaneous exocytosis. Viewed together, our data suggest that complexin generally functions in priming and Ca(2+) triggering of exocytosis, and additionally contributes to the control of spontaneous exocytosis dependent on the developmental history of a neuron and on the subcellular localization of the complexin.

  17. The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Mai [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan); Kitaguchi, Tetsuya [Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABOIS), Waseda University, 11 Biopolis Way, 05-01/02 Helios, Singapore 138667 (Singapore); Numano, Rika [The Electronics-Inspired Interdisciplinary Research Institute (EIIRIS), Toyohashi University of Technology, 1-1 Hibarigaoka, Tennpaku-cho, Toyohashi, Aichi 441-8580 (Japan); Ikematsu, Kazuya [Forensic Pathology and Science, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523 (Japan); Kakeyama, Masaki [Laboratory of Environmental Health Sciences, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Murata, Masayuki; Sato, Ken [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan); Tsuboi, Takashi, E-mail: takatsuboi@bio.c.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Regulation of exocytosis by Rho GTPase Cdc42. Black-Right-Pointing-Pointer Cdc42 increases the number of fusion events from newly recruited vesicles. Black-Right-Pointing-Pointer Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

  18. Evidence for lysosomal exocytosis and release of aggrecan-degrading hydrolases from hypertrophic chondrocytes, in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Edward R. Bastow

    2012-02-01

    The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca2+-dependent lysosomal exocytosis. In this study we confirm that hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development. We show that lysosome-associated membrane protein 1 (LAMP1 is detected at the cell surface following in vitro treatment of epiphyseal chondrocytes with the calcium ionophore, ionomycin. Furthermore, we show that in addition to the lysosomal exocytosis markers, cathepsin D and β-hexosaminidase, ionomycin induces release of aggrecan- and hyaluronan-degrading activity from cultured epiphyseal chondrocytes. We identify VAMP-8 and VAMP7 as v-SNARE proteins with potential roles in lysosomal exocytosis in hypertrophic chondrocytes, based on their colocalisation with LAMP1 at the cell surface in secondary ossification centers in mouse tibiae. We propose that resorbing growth plate cartilage involves release of destructive hydrolases from hypertrophic chondrocytes, via lysosomal exocytosis.

  19. Inhibition of calcineurin phosphatase promotes exocytosis of renin from juxtaglomerular cells

    DEFF Research Database (Denmark)

    Madsen, Kirsten; Friis, Ulla Glenert; Gooch, Jennifer L;

    2010-01-01

    To examine the role of the calcium/calmodulin-dependent phosphatase calcineurin in regulation of renin release, we assayed exocytosis using whole-cell patch clamp of single juxtaglomerular cells in culture. The calcineurin inhibitor, cyclosporine A (CsA), significantly increased juxtaglomerular......A, but not tacrolimus, significantly stimulated renin release from cultured juxtaglomerular cells. Juxtaglomerular cells expressed the calcineurin isoforms A-beta and A-gamma but not A-alpha. Plasma renin concentrations (PRCs) were not different in wild-type, calcineurin A-alpha, or A-beta knockout mice but increased...... cell membrane capacitance, an index of cell surface area and an established measure of exocytosis in single-cell assays. This effect was mimicked by intracellular delivery of a calcineurin inhibitory peptide, the calcium chelator ethylene glycol tetraacetic acid (EGTA), or the calmodulin inhibitor W-13...

  20. Imaging Exocytosis of Single Synaptic Vesicles at a Fast CNS Presynaptic Terminal.

    Science.gov (United States)

    Midorikawa, Mitsuharu; Sakaba, Takeshi

    2015-11-01

    Synaptic vesicles are tethered to the active zone where they are docked/primed so that they can fuse rapidly upon Ca(2+) influx. To directly study these steps at a CNS presynaptic terminal, we used total internal reflection fluorescence (TIRF) microscopy at the live isolated calyx of Held terminal and measured the movements of single synaptic vesicle just beneath the plasma membrane. Only a subset of vesicles within the TIRF field underwent exocytosis. Following exocytosis, new vesicles (newcomers) approached the membrane and refilled the release sites slowly with a time constant of several seconds. Uniform elevation of the intracellular Ca(2+) using flash photolysis elicited an exocytotic burst followed by the sustained component, representing release of the readily releasable vesicles and vesicle replenishment, respectively. Surprisingly, newcomers were not released within a second of high Ca(2+). Instead, already-tethered vesicles became release-ready and mediated the replenishment. Our results reveal an important feature of conventional synapses.

  1. Linking cortical microtubule attachment and exocytosis [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Ivar Noordstra

    2017-04-01

    Full Text Available Exocytosis is a fundamental cellular process whereby secreted molecules are packaged into vesicles that move along cytoskeletal filaments and fuse with the plasma membrane. To function optimally, cells are strongly dependent on precisely controlled delivery of exocytotic cargo. In mammalian cells, microtubules serve as major tracks for vesicle transport by motor proteins, and thus microtubule organization is important for targeted delivery of secretory carriers. Over the years, multiple microtubule-associated and cortical proteins have been discovered that facilitate the interaction between the microtubule plus ends and the cell cortex. In this review, we focus on mammalian protein complexes that have been shown to participate in both cortical microtubule capture and exocytosis, thereby regulating the spatial organization of secretion. These complexes include microtubule plus-end tracking proteins, scaffolding factors, actin-binding proteins, and components of vesicle docking machinery, which together allow efficient coordination of cargo transport and release.

  2. Mesoporous silica nanoparticles enhance MTT formazan exocytosis in HeLa cells and astrocytes.

    Science.gov (United States)

    Fisichella, Matthieu; Dabboue, Hinda; Bhattacharyya, Sanjib; Saboungi, Marie-Louise; Salvetat, Jean-Paul; Hevor, Tobias; Guerin, Martine

    2009-06-01

    We report on the observation that mesoporous silica nanoparticles (MSNs), after being endocytosed, interfere with the MTT test in HeLa cells and astrocytes by accelerating the exocytosis of formazan crystals. The stimulation of MTT formazan exocytosis is probably related to perturbation of intracellular vesicle trafficking by MSN uptake as revealed by experiments in presence of chloroquine and genistein. Similar effect has been previously observed with a number of chemicals, especially with neurotoxic beta amyloid peptides, but not with nanoparticles. We showed also that MTT reduction test gives an overestimation of the cytotoxicity of mesoporous silica nanoparticles compared to other tests such as LDH activity, WST-1 test and flow cytometry. These findings show that MTT assay should not be used for the study of MSN toxicity, and that perturbation of intracellular trafficking has to be taken into account in evaluating biocompatibility of MSNs.

  3. Actin- and Myosin-Dependent Vesicle Loading of Presynaptic Docking Sites Prior to Exocytosis.

    Science.gov (United States)

    Miki, Takafumi; Malagon, Gerardo; Pulido, Camila; Llano, Isabel; Neher, Erwin; Marty, Alain

    2016-08-17

    Variance analysis of postsynaptic current amplitudes suggests the presence of distinct docking sites (also called release sites) where vesicles pause before exocytosis. Docked vesicles participate in the readily releasable pool (RRP), but the relation between docking site number and RRP size remains unclear. It is also unclear whether all vesicles of the RRP are equally release competent, and what cellular mechanisms underlie RRP renewal. We address here these questions at single glutamatergic synapses, counting released vesicles using deconvolution. We find a remarkably low variance of cumulative vesicle counts during action potential trains. This, combined with Monte Carlo simulations, indicates that vesicles transit through two successive states before exocytosis, so that the RRP is up to 2-fold higher than the docking site number. The transition to the second state has a very rapid rate constant, and is specifically inhibited by latrunculin B and blebbistatin, suggesting the involvement of actin and myosin. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Twin screw wet granulation: Binder delivery.

    Science.gov (United States)

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

    2015-06-20

    The effects of three ways of binder delivery into the twin screw granulator (TSG) on the residence time, torque, properties of granules (size, shape, strength) and binder distribution were studied. The binder distribution was visualised through the transparent barrel using high speed imaging as well as quantified using offline technique. Furthermore, the effect of binder delivery and the change of screw configuration (conveying elements only and conveying elements with kneading elements) on the surface velocity of granules across the screw channel were investigated using particle image velocimetry (PIV). The binder was delivered in three ways; all solid binder incorporated with powder mixture, 50% of solid binder mixed with powder mixture and 50% mixed with water, all the solid binder dissolved in water. Incorporation of all solid binder with powder mixture resulted in the relatively longer residence time and higher torque, narrower granule size distribution, more spherical granules, weaker big-sized granules, stronger small-sized granules and better binder distribution compared to that in other two ways. The surface velocity of granules showed variation from one screw to another as a result of uneven liquid distribution as well as shown a reduction while introducing the kneading elements into the screw configuration.

  5. Autophagy meets FUS-positive Stress Granules

    Science.gov (United States)

    Matus, Soledad; Bosco, Daryl A.; Hetz, Claudio

    2015-01-01

    Mutations in fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) are linked to familial cases of amyotrophic lateral sclerosis (ALS). Mutant FUS selectively accumulates into discrete cytosolic structures known as stress granules under various stress conditions. In addition, mutant FUS expression can alter the dynamics and morphology of stress granules. Although the link between mutant-FUS and stress granules is well established, the mechanisms modulating stress granule formation and disassembly in the context of ALS are poorly understood. In this issue of Neurobiology of Aging, Ryu and coworkers uncover the impact of autophagy on the potential toxicity of mutant-FUS positive stress granules. The authors provide evidence indicating that enhanced autophagy activity reduces the number of stress granules, which in the case of cells containing mutant-FUS positive stress granules, is neuroprotective. Overall, this study identifies an intersection between the proteostasis network and alterations in RNA metabolism in ALS through the dynamic assembly and disassembly of stress granules. PMID:25444610

  6. Efficacy of lytic Staphylococcus aureus bacteriophage against multidrug-resistant Staphylococcus aureus in mice.

    Science.gov (United States)

    Oduor, Joseph Michael Ochieng'; Onkoba, Nyamongo; Maloba, Fredrick; Arodi, Washingtone Ouma; Nyachieo, Atunga

    2016-11-24

    The use of bacteriophages as an alternative treatment method against multidrug-resistant bacteria has not been explored in Kenya. This study sought to determine the efficacy of environmentally obtained lytic bacteriophage against multidrug-resistant Staphylococcus aureus (MDRSA) bacterium in mice. Staphylococcus aureus bacterium and S. aureus-specific lytic phage were isolated from sewage and wastewater collected within Nairobi County, Kenya. Thirty mice were randomly assigned into three groups: MDRSA infection group (n = 20), phage-infection group (n = 5), and non-infection group (n = 5). The MDRSA infection group was further subdivided into three groups: clindamycin treatment (8 mg/kg; n = 5), lytic phage treatment (108 PFU/mL (n = 5), and a combination treatment of clindamycin and lytic phage (n = 5). Treatments were done at either 24 or 72 hours post-infection (p.i), and data on efficacy, bacterial load, and animal physical health were collected. Treatment with phage was more effective (100%) than with clindamycin (62.25% at 24 hours p.i and 87.5% at 72 hours p.i.) or combination treatment (75% at 24 hours p.i. and 90% at 72 hours p.i.) (p aureus lytic bacteriophage has therapeutic potential against MDRSA bacterium in mice.

  7. TRIM5α Promotes Ubiquitination of Rta from Epstein–Barr Virus to Attenuate Lytic Progression

    Science.gov (United States)

    Huang, Hsiang-Hung; Chen, Chien-Sin; Wang, Wen-Hung; Hsu, Shih-Wei; Tsai, Hsiao-Han; Liu, Shih-Tung; Chang, Li-Kwan

    2017-01-01

    Replication and transcription activator (Rta), a key protein expressed by Epstein–Barr virus (EBV) during the immediate-early stage of the lytic cycle, is responsible for the activation of viral lytic genes. In this study, GST-pulldown and coimmunoprecipitation assays showed that Rta interacts in vitro and in vivo with TRIM5α, a host factor known to be involved in the restriction of retroviral infections. Confocal microscopy results revealed that Rta colocalizes with TRIM5α in the nucleus during lytic progression. The interaction involves 190 amino acids in the N-terminal of Rta and the RING domain in TRIM5α, and it was further found that TRIM5α acts as an E3 ubiquitin ligase to promote Rta ubiquitination. Overexpression of TRIM5α reduced the transactivating capabilities of Rta, while reducing TRIM5α expression enhanced EBV lytic protein expression and DNA replication. Taken together, these results point to a critical role for TRIM5α in attenuating EBV lytic progression through the targeting of Rta for ubiquitination, and suggest that the restrictive capabilities of TRIM5α may go beyond retroviral infections. PMID:28105027

  8. A Herpesviral Lytic Protein Regulates the Structure of Latent Viral Chromatin

    Directory of Open Access Journals (Sweden)

    Priya Raja

    2016-05-01

    Full Text Available Latent infections by viruses usually involve minimizing viral protein expression so that the host immune system cannot recognize the infected cell through the viral peptides presented on its cell surface. Herpes simplex virus (HSV, for example, is thought to express noncoding RNAs such as latency-associated transcripts (LATs and microRNAs (miRNAs as the only abundant viral gene products during latent infection. Here we describe analysis of HSV-1 mutant viruses, providing strong genetic evidence that HSV-infected cell protein 0 (ICP0 is expressed during establishment and/or maintenance of latent infection in murine sensory neurons in vivo. Studies of an ICP0 nonsense mutant virus showed that ICP0 promotes heterochromatin and latent and lytic transcription, arguing that ICP0 is expressed and functional. We propose that ICP0 promotes transcription of LATs during establishment or maintenance of HSV latent infection, much as it promotes lytic gene transcription. This report introduces the new concept that a lytic viral protein can be expressed during latent infection and can serve dual roles to regulate viral chromatin to optimize latent infection in addition to its role in epigenetic regulation during lytic infection. An additional implication of the results is that ICP0 might serve as a target for an antiviral therapeutic acting on lytic and latent infections.

  9. Inhibition of the Epstein-Barr virus lytic cycle by moronic acid.

    Science.gov (United States)

    Chang, Fang-Rong; Hsieh, Yi-Chung; Chang, Yung-Fu; Lee, Kuo-Hsiung; Wu, Yang-Chang; Chang, Li-Kwan

    2010-03-01

    Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, during the immediate-early stage of the lytic cycle to activate the transcription of viral lytic genes. Our immunoblotting and flow cytometry analyses find that moronic acid, found in galls of Rhus chinensis and Brazilian propolis, at 10microM inhibits the expression of Rta, Zta, and an EBV early protein, EA-D, after lytic induction with sodium butyrate. This study also finds that moronic acids inhibits the capacity of Rta to activate a promoter that contains an Rta-response element, indicating that moronic acid interferes with the function of Rta. On the other hand, moronic acid does not appear to influence with the transactivation function of Zta. Therefore, the lack of expression of Zta and EA-D after moronic acid treatment is attributable to the inhibition of the transactivation functions of Rta. Because the expression of Zta, EA-D and many EBV lytic genes depends on Rta, the treatment of P3HR1 cells with moronic acid substantially reduces the numbers of EBV particles produced by the cells after lytic induction. This study suggests that moronic acid is a new structural lead for anti-EBV drug development.

  10. FcɛRI-mediated mast cell degranulation requires calcium-independent microtubule-dependent translocation of granules to the plasma membrane

    Science.gov (United States)

    Nishida, Keigo; Yamasaki, Satoru; Ito, Yukitaka; Kabu, Koki; Hattori, Kotaro; Tezuka, Tohru; Nishizumi, Hirofumi; Kitamura, Daisuke; Goitsuka, Ryo; Geha, Raif S.; Yamamoto, Tadashi; Yagi, Takeshi; Hirano, Toshio

    2005-01-01

    The aggregation of high affinity IgE receptors (Fcɛ receptor I [FcɛRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcɛRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcɛRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcɛRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule–plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway. PMID:15998803

  11. Amperometric post spike feet reveal most exocytosis is via extended kiss-and-run fusion

    OpenAIRE

    Lisa J Mellander; Raphaël Trouillon; Svensson, Maria I.; Ewing, Andrew G.

    2012-01-01

    The basis for communication between nerve cells lies in the process of exocytosis, the fusion of neurotransmitter filled vesicles with the cell membrane resulting in release of the signaling molecules. Even though much is known about this process, the extent that the vesicles are emptied upon fusion is a topic that is being debated. We have analyzed amperometric peaks corresponding to release at PC12 cells and find stable plateau currents during the decay of peaks, indicating closing of the v...

  12. Continuous twin screw granulation: influence of process variables on granule and tablet quality.

    Science.gov (United States)

    Vercruysse, J; Córdoba Díaz, D; Peeters, E; Fonteyne, M; Delaet, U; Van Assche, I; De Beer, T; Remon, J P; Vervaet, C

    2012-09-01

    The aim of the current study was to screen theophylline (125 mg) tablets manufactured via twin screw granulation in order to improve process understanding and knowledge of process variables that determine granule and tablet quality. A premix of theophylline anhydrate, α-lactose monohydrate and PVP (ratio: 30/67.5/2.5,w/w) was granulated with demineralized water. Experiments were done using the high-shear wet granulation module (based on twin screw granulation) of the ConsiGma™-25 unit (a continuous tablet manufacturing system) for particle size enlargement. After drying, granules were compressed using a MODUL™ P tablet press (compression force: 10 kN, tablet diameter: 12 mm). Using a D-optimal experimental design, the effect of several process variables (throughput (10-25 kg/h), screw speed (600-950 rpm), screw configuration (number (2, 4, 6 and 12) and angle (30°, 60° and 90°) of kneading elements), barrel temperature (25-40°C) and method of binder addition (dry versus wet)) on the granulation process (torque and temperature increase in barrel wall), granule (particle size distribution, friability and flowability) and tablet (tensile strength, porosity, friability, disintegration time and dissolution) quality was evaluated. The results showed that the quality of granules and tablets can be optimized by adjusting specific process variables (number of kneading elements, barrel temperature and binder addition method) during a granulation process using a continuous twin screw granulator.

  13. Development of granules from phyllanthus niruri spray-dried extract

    OpenAIRE

    Tatiane Pereira de Souza; José Luiz Gómez-Amoza; Ramón Martínez Pacheco; Pedro Ros Petrovick

    2009-01-01

    The aim of this study was to develop granules from Phyllanthus niruri spray-dried extract using dry and wet granulation and to assess techniques to enable the production of granules with improved technological characteristics and yields. Granules were characterized by granulometry, reological parameters, compression and hygroscopic behavior. Independent of the granulation technique, technologically developed granules presented particle diameter, bulk and tapped densities and compressibility i...

  14. A computational analysis framework for molecular cell dynamics: case-study of exocytosis.

    Science.gov (United States)

    Chen, Wenhai; Zhou, Wen; Xia, Tian; Gu, Xun

    2012-01-01

    One difficulty in conducting biologically meaningful dynamic analysis at the systems biology level is that in vivo system regulation is complex. Meanwhile, many kinetic rates are unknown, making global system analysis intractable in practice. In this article, we demonstrate a computational pipeline to help solve this problem, using the exocytotic process as an example. Exocytosis is an essential process in all eukaryotic cells that allows communication in cells through vesicles that contain a wide range of intracellular molecules. During this process a set of proteins called SNAREs acts as an engine in this vesicle-membrane fusion, by forming four-helical bundle complex between (membrane) target-specific and vesicle-specific SNAREs. As expected, the regulatory network for exocytosis is very complex. Based on the current understanding of the protein-protein interaction network related to exocytosis, we mathematically formulated the whole system, by the ordinary differential equations (ODE). We then applied a mathematical approach (called inverse problem) to estimating the kinetic parameters in the fundamental subsystem (without regulation) from limited in vitro experimental data, which fit well with the reports by the conventional assay. These estimates allowed us to conduct an efficient stability analysis under a specified parameter space for the exocytotic process with or without regulation. Finally, we discuss the potential of this approach to explain experimental observations and to make testable hypotheses for further experimentation.

  15. Alpha-latrotoxin Triggers Extracellular Ca2+-dependent Exocytosis and Sensitizes Fusion Machinery in Endocrine Cells

    Institute of Scientific and Technical Information of China (English)

    Zhi-Tao HU; Ping ZHAO; Jie LIU; Zheng-Xing WU; Tao XU

    2006-01-01

    α-Latrotoxin from the venom of black widow spider induces and augments neurotransmitter and hormone release by way of extracellular Ca2+ influx and cellular signal transduction pathways. By using whole cell current and capacitance recording, the photolysis of caged Ca2+, and Ca2+ microfluorometry and amperometry, we investigated the stimulating effect and mechanism of o-latrotoxin on exocytosis in rat pancreatic β cells, LβT2 cells and latrophilin plasmid-transfected INS-1 cells. Our data indicated that: (1) α-latrotoxin increased cytosolic Ca2+ concentration through the formation of cation-permitting pores and subsequent Ca2+ influx with the presence of extracellular Ca2+; (2) α-latrotoxin stimulated exocytosis in normal bath solution and its stimulating effect on secretion was eradicated in Ca2+-free bath solution; and (3) α-latrotoxin sensitized the molecular machinery of fusion through activation of protein kinase C and increased the response of cells to Ca2+ photolysed by a flash of ultraviolet light. In summary, α-latrotoxin induced exocytosis by way of Ca2+ influx and accelerated vesicle fusion by the sensitization of fusion machinery.

  16. Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line.

    Science.gov (United States)

    Bertrand, C A; Danahay, H; Poll, C T; Laboisse, C; Hopfer, U; Bridges, R J

    2004-02-01

    ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.

  17. Osmotic Stress Modulates the Balance between Exocytosis and Clathrin-Mediated Endocytosis in Arabidopsis thaliana.

    Science.gov (United States)

    Zwiewka, Marta; Nodzyński, Tomasz; Robert, Stéphanie; Vanneste, Steffen; Friml, Jiří

    2015-08-01

    The sessile life style of plants creates the need to deal with an often adverse environment, in which water availability can change on a daily basis, challenging the cellular physiology and integrity. Changes in osmotic conditions disrupt the equilibrium of the plasma membrane: hypoosmotic conditions increase and hyperosmotic environment decrease the cell volume. Here, we show that short-term extracellular osmotic treatments are closely followed by a shift in the balance between endocytosis and exocytosis in root meristem cells. Acute hyperosmotic treatments (ionic and nonionic) enhance clathrin-mediated endocytosis simultaneously attenuating exocytosis, whereas hypoosmotic treatments have the opposite effects. In addition to clathrin recruitment to the plasma membrane, components of early endocytic trafficking are essential during hyperosmotic stress responses. Consequently, growth of seedlings defective in elements of clathrin or early endocytic machinery is more sensitive to hyperosmotic treatments. We also found that the endocytotic response to a change of osmotic status in the environment is dominant over the presumably evolutionary more recent regulatory effect of plant hormones, such as auxin. These results imply that osmotic perturbation influences the balance between endocytosis and exocytosis acting through clathrin-mediated endocytosis. We propose that tension on the plasma membrane determines the addition or removal of membranes at the cell surface, thus preserving cell integrity.

  18. Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis

    Science.gov (United States)

    Najafinobar, Neda; Mellander, Lisa J.; Kurczy, Michael E.; Dunevall, Johan; Angerer, Tina B.; Fletcher, John S.; Cans, Ann-Sofie

    2016-09-01

    Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release.

  19. A Ca sup 2+ influx associated with exocytosis is specifically abolished in a Paramecium exocytotic mutant

    Energy Technology Data Exchange (ETDEWEB)

    Kerboeuf, D.; Cohen, J. (Centre National de la Recherche Scientifique, Gif-sur-Yvette (France))

    1990-12-01

    A Paramecium possesses secretory organelles called trichocysts which are docked beneath the plasma membrane awaiting an external stimulus that triggers their exocytosis. Membrane fusion is the sole event provoked by the stimulation and can therefore be studied per se. Using 3 microM aminoethyl dextran as a vital secretagogue, we analyzed the movements of calcium (Ca{sup 2+}) during the discharge of trichocysts. We showed that (a) external Ca{sup 2+}, at least at 3 X 10(-7) M, is necessary for AED to induce exocytosis; (b) a dramatic and transient influx of Ca{sup 2+} as measured from {sup 45}Ca uptake is induced by AED; (c) this influx is independent of the well-characterized voltage-operated Ca{sup 2+} channels of the ciliary membranes since it persists in a mutant devoid of these channels; and (d) this influx is specifically abolished in one of the mutants unable to undergo exocytosis, nd12. We propose that the Ca{sup 2+} influx induced by AED reflects an increase in membrane permeability through the opening of novel Ca{sup 2+} channel or the activation of other Ca{sup 2+} transport mechanism in the plasma membrane. The resulting rise in cytosolic Ca{sup 2+} concentration would in turn induce membrane fusion. The mutation nd12 would affect a gene product involved in the control of plasma membrane permeability to Ca{sup 2+}, specifically related to membrane fusion.

  20. A complete Rab screening reveals novel insights in Weibel-Palade body exocytosis.

    Science.gov (United States)

    Zografou, Sofia; Basagiannis, Dimitris; Papafotika, Alexandra; Shirakawa, Ryutaro; Horiuchi, Hisanori; Auerbach, Daniel; Fukuda, Mitsunori; Christoforidis, Savvas

    2012-10-15

    Weibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are Rab27a and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with Rab27a in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis.

  1. Minocycline recovers MTT-formazan exocytosis impaired by amyloid beta peptide.

    Science.gov (United States)

    Kreutzmann, Peter; Wolf, Gerald; Kupsch, Kathleen

    2010-10-01

    Minocycline, a tetracycline antibiotic, has been reported to exert beneficial effects in models of Alzheimer's disease (AD). To characterize the mechanisms underlying the putative minocycline-related neuroprotection, we studied its effect in an in vitro model of AD. Primary hippocampal cultures were treated with β-amyloid peptide (Aβ) and cell viability was assessed by standard MTT-assay. Incubation with 10 μM Aβ for 24 h significantly inhibits cellular MTT-reduction without inducing morphological signs of enhanced cell death or increase in release of lactate dehydrogenase. This indicates that cell viability was not affected. The inhibition of MTT-reduction by Aβ was due to an acceleration of MTT-formazan exocytosis. Intriguingly, the Aβ-triggered increase in MTT-formazan exocytosis was abolished by co-treatment with minocycline. In vehicle-treated cells minocycline had no effect on formazan exocytosis. This hitherto unrecognized property of minocycline has to be noticed in the elucidation of the underlying mechanism of this promising neuroprotectant.

  2. Evidence that nitric oxide synthase is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa.

    Science.gov (United States)

    Herrero, M B; Viggiano, J M; Pérez Martínez, S; de Gimeno, M F

    1997-01-01

    In a recent work, we detected nitric oxide synthase (NO synthase) in the acrosome and tail of mouse and human spermatozoa by an immunofluorescence technique. Also, NO-synthase inhibitors added during sperm capacitation in vitro reduced the percentage of oocytes fertilized in vitro, suggesting a role for NO synthase in sperm function. Therefore, in the present study the effect of three NO-synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-D-arginine methyl ester (D-NAME) and L-NG-nitro-arginine (NO2-arg), and of a nitric oxide donor, spermine-NONOate, on the progesterone-induced acrosome reaction of mouse sperm was examined. NO-synthase inhibitors were added at 0, 60 or 90 min during capacitation; at 120 min, mouse epididymal spermatozoa were exposed to 15 microM progesterone for another 15 min. In another set of experiments, different concentrations of spermine-NONOate were added to capacitated spermatozoa for 15 min; in these experiments, progesterone was not included. NO2-arg and L-NAME blocked progesterone-induced exocytosis regardless of the time at which these inhibitors were added. Moreover, D-NAME did not inhibit exocytosis. In contrast, spermine-NONOate stimulated the acrosomal exocytosis in vitro directly. These results provide evidence that mouse sperm NO synthase participates in the progesterone-induced acrosome reaction in vitro and that nitric oxide induces this event.

  3. Preparation and evaluation of granules with pH-dependent release by melt granulation.

    Science.gov (United States)

    Shiino, Kai; Iwao, Yasunori; Fujinami, Yukari; Itai, Shigeru

    2012-07-15

    This study had two objectives: (1) to prepare, by melt granulation in a high-shear mixer, granules containing acetaminophen (APAP) as a model drug and aminoalkyl methacrylate copolymer E (AMCE) as a pH-sensitive polymer that readily dissolves at pH values lower than 5, and (2) to investigate the effects of AMCE loading (5-15%) on granule properties and the in vitro release profile of drug from the granules. Compared with polymer-free granules, the granules containing 5% and 10% AMCE were found to have higher median diameters and wider particle size distributions. For the formulation containing 15% AMCE, on the other hand, the diameters and distribution were similar to those for polymer-free granules. From compression testing, load-displacement curves revealed that AMCE enhanced particle strength at ambient temperature and induced plastic strain, while suppressing fragmentation of the granules. In addition, from dissolution testing using media with pH 4.0 and pH 6.5, granules containing AMCE, except 15% AMCE loading, exhibited drug release with significant pH dependence. When the pH 4.0 and pH 6.5 dissolution profiles were further compared by calculating the difference factor (f(1)), the 5% AMCE granules showed the strongest pH dependence of drug release among all formulations in this study. Large cracks and breakage were observed on the surface of 10% AMCE granules after they were used in dissolution testing. The obtained results are attributed to the plastic strain properties of AMCE above its glass transition temperature, and to the irregular distribution of AMCE within granules. Hence, this study has demonstrated for the first time that the combination of melt granulation and AMCE incorporation enables the formulation of novel functional granules that exhibit pH-dependent release of the active ingredient. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Cytotoxic amyloid peptides inhibit cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction by enhancing MTT formazan exocytosis.

    Science.gov (United States)

    Liu, Y; Schubert, D

    1997-12-01

    Amyloid beta peptide (A beta) neurotoxicity is believed to play a central role in the pathogenesis of Alzheimer's disease. An early indicator of A beta toxicity is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to MTT formazan, a widely used assay for measuring cell viability. In this report we show that A beta and other cytotoxic amyloid peptides such as human amylin dramatically enhance MTT formazan exocytosis, resulting in the inhibition of cellular MTT reduction. Only the amyloid peptides that are known to be cytotoxic enhanced MTT formazan exocytosis. Basal MTT formazan exocytosis and amyloid peptide-enhanced MTT formazan exocytosis are blocked by several drugs with diverse known effects. These and other data suggest that MTT formazan exocytosis is a multistep process and that cytotoxic amyloid peptides enhance MTT formazan exocytosis through an intracellular signal transduction pathway.

  5. THE STUDY OF THE KINETIC OF NATURAL ZEOLITE GRANULES GROWTH AT DIFFERENT WAYS OF GRANULATION

    Directory of Open Access Journals (Sweden)

    Rybachuk VD

    2016-12-01

    Full Text Available Introduction. Active substances and excipients used in the manufacture of medicines in tablet form, in most cases, have poor technological properties. This fact determines the need for prior granulation of mass before compression. Granulators of various sizes and designs, running on different modes, made the formation, growth and consolidation of the powder particles that lead to obtain pellets of different shapes and sizes. From the literature it is known that granulation leads to two forms of granules: isodiametric and nonisodiametric. The first group of particles forms has globular shape with a smooth surface and the proportion in which the length, thickness and height are about the same. They are usually made by fluidized bed granulation, spray drying, pelletizing and granulation in dragee pan. Granules of nonisodiametric form in which length is several times the width and height are made mostly by extrusion and compacting. The geometrical parameters of obtained granules are affected by the properties of raw materials, the granulation modes, type and amount of added humidifier and so on. The shape and size of granules, from a technological point of view, are the key factors that contribute, except organoleptic characteristics of the product, its technological properties such as particle size distribution, bulk volume, the ability of the material to shrinkage, porosity, fluidity, mechanical strength and so on. Properly selected for specific conditions granulation method is able to provide the finished product with the specified technological parameters depending on the needs. The aim of this work was to study the effect of granulation method and its conditions on the kinetics of growth of the natural zeolite granules and some quality characteristics of obtained granules. Material & methods. As objects of study served the natural zeolite pellets produced using 3%, 5%, 7% and 10% potato starch paste and solution of polyvinylpyrrolidone (PVP

  6. Correlative microscopy of detergent granules.

    Science.gov (United States)

    van Dalen, G; Nootenboom, P; Heussen, P C M

    2011-03-01

    The microstructure of detergent products for textile cleaning determines to a large extent the physical properties of these products. Correlative microscopy was used to reveal the microstructure by reconciling images obtained by scanning electron microscopy with energy dispersive X-ray analysis, X-ray microtomography and Fourier transform infrared microscopy. These techniques were applied on the same location of a subsample of a spray-dried detergent base powder embedded in polyacrylate. In this way, the three-dimensional internal and external structure of detergent granules could be investigated from milli to nano scale with detailed spatial information about the components present. This will generate knowledge how to design optimal microstructures for laundry products to obtain product properties demanded by the market. This method is also very useful for other powder systems used in a large variety of industries (e.g. for pharmaceutical, food, ceramic and metal industries).

  7. The Lytic SA Phage Demonstrate Bactericidal Activity against Mastitis Causing Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Hamza Ameer

    2016-01-01

    Full Text Available Staphylococcus aureus is the major causative agent of mastitis among dairy animals as it causes intramammary gland infection. Due to antibiotic resistance and contamination of antibiotics in the milk of diseased animals; alternative therapeutic agents are required to cure mastitis. Lytic bacteriophages and their gene products can be potential therapeutic agents against bacteria as they are host specific and less harmful than antibiotics. In this study, Staphylococcus aureus were isolated from milk samples of the infected animals and identified biochemically. SA phage was isolated from sewage water showing lytic activity against Staphylococcus aureus isolates. The highest lytic activity of bacteriophages was observed at 37°C and pH 7, and the most suitable storage condition was at 4°C. SA phage efficiently reduced bacterial growth in the bacterial reduction assay. The characterization and bacterial growth reduction activity of the bacteriophages against Staphylococcus aureus signifies their underlying potential of phage therapy against mastitis.

  8. In vitro cytocidal effect of lytic peptides on several transformed mammalian cell lines.

    Science.gov (United States)

    Jaynes, J M; Julian, G R; Jeffers, G W; White, K L; Enright, F M

    1989-01-01

    Several types of transformed mammalian cells, derived from established cell lines, were found to be lysed in vitro by three novel lytic peptides (SB-37, SB-37*, and Shiva-1). This is in contrast with the behavior of normal cells, where the observed lytic activity of the peptides is greatly reduced. Based on experiments utilizing compounds which disrupt the cytoskeleton (colchicine and cytochalasin-D), it is surmised that alterations in the cytoskeleton of transformed cells increase their sensitivity to the cytolytic activity exerted by the peptides, primarily by causing a loss of osmotic integrity. Thus, a stable and regenerative cytoskeletal system, as that possessed by normal cells, would seem requisite to withstanding the lytic effects of the peptides.

  9. Salvinorin A exerts opposite presynaptic controls on neurotransmitter exocytosis from mouse brain nerve terminals.

    Science.gov (United States)

    Grilli, Massimo; Neri, Elisa; Zappettini, Stefania; Massa, Francesca; Bisio, Angela; Romussi, Giovanni; Marchi, Mario; Pittaluga, Anna

    2009-01-01

    We investigated the effects of salvinorin A on the basal and the 12 mM K(+)-evoked release of preloaded [(3)H]noradenaline ([(3)H]NA) and [(3)H]serotonin ([(3)H]5-HT) from mouse hippocampal nerve terminals (synaptosomes), as well as on the basal and 12mM K(+)-evoked release of preloaded [(3)H]dopamine ([(3)H]DA) from mouse striatal and prefrontal cortex (PFc) synaptosomes. Salvinorin A (0.1-1000 nM) failed to affect the basal release of amines, but inhibited the 12 mM K(+)-evoked, Ca(2+)-dependent, exocytotic-like release of [(3)H]5-HT and [(3)H]DA. At the same concentration, salvinorin A facilitated the 12 mM K(+)-evoked, Ca(2+)-dependent, exocytotic-like release of [(3)H]NA. These effects could not be observed in pertussis toxin (PTx) entrapped synaptosomes. The broad spectrum kappa-opioid receptor (KOR) antagonist norbinaltorphimine (norBNI, 1-100 nM) antagonized the inhibition of [(3)H]5-HT and [(3)H]DA exocytosis as well as the facilitation of [(3)H]NA overflow induced by 100 nM salvinorin A. The KOR agonist U69593 (1-100 nM) mimicked salvinorin A in inhibiting [(3)H]5-HT and of [(3)H]DA exocytosis, its effect being prevented by norBNI, but leaving unchanged the K(+)-evoked release of [(3)H]NA. The effects of Salvinorin A on neurotransmitter exocytosis were not prevented by the selective mu opioid (MOR) receptor antagonist CTAP (10-100 nM), whereas facilitation of [(3)H]NA exocytosis, but not inhibition of [(3)H]5-HT and [(3)H]DA K(+)-evoked release, was counteracted by the delta opioid receptor (DOR) antagonist naltrindole (1-100 nM). We conclude that salvinorin A presynaptically modulates central NA, 5-HT, and DA exocytosis evoked by a mild depolarizing stimulus by acting at presynaptic opioid receptors having different pharmacological profiles.

  10. In vitro cytocidal effect of novel lytic peptides on Plasmodium falciparum and Trypanosoma cruzi.

    Science.gov (United States)

    Jaynes, J M; Burton, C A; Barr, S B; Jeffers, G W; Julian, G R; White, K L; Enright, F M; Klei, T R; Laine, R A

    1988-10-01

    Plasmodium falciparum and Trypanosoma cruzi were killed by two novel lytic peptides (SB-37 and Shiva-1) in vitro. Human erythrocytes infected with P. falciparum, and Vero cells infected with T. cruzi, were exposed to these peptides. The result, in both cases, was a significant decrease in the level of parasite infection. Furthermore, the peptides had a marked cytocidal effect on trypomastigote stages of T. cruzi in media, whereas host eukaryotic cells were unaffected by the treatments. In view of the worldwide prevalence of these protozoan diseases and the lack of completely suitable treatments, lytic peptides may provide new and unique chemotherapeutic agents for the treatment of these infections.

  11. Listeria monocytogenes has a functional chitinolytic system and an active lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Paspaliari, Dafni Katerina; Loose, Jennifer S. M.; Larsen, Marianne Halberg

    2015-01-01

    B) and a multi-modular lytic polysaccharide monooxygenase (LmLPMO10). These enzymes have been related to virulence and their role in chitin metabolism is poorly understood. It is thus of interest to functionally characterize the individual enzymes in order to shed light on their roles in vivo. Our results......Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and Chi...

  12. Impact of screw configuration on the particle size distribution of granules produced by twin screw granulation.

    Science.gov (United States)

    Vercruysse, J; Burggraeve, A; Fonteyne, M; Cappuyns, P; Delaet, U; Van Assche, I; De Beer, T; Remon, J P; Vervaet, C

    2015-02-01

    Twin screw granulation (TSG) has been reported by different research groups as an attractive technology for continuous wet granulation. However, in contrast to fluidized bed granulation, granules produced via this technique typically have a wide and multimodal particle size distribution (PSD), resulting in suboptimal flow properties. The aim of the current study was to evaluate the impact of granulator screw configuration on the PSD of granules produced by TSG. Experiments were performed using a 25 mm co-rotating twin screw granulator, being part of the ConsiGma™-25 system (a fully continuous from-powder-to-tablet manufacturing line from GEA Pharma Systems). Besides the screw elements conventionally used for TSG (conveying and kneading elements), alternative designs of screw elements (tooth-mixing-elements (TME), screw mixing elements (SME) and cutters) were investigated using an α-lactose monohydrate formulation granulated with distilled water. Granulation with only conveying elements resulted in wide and multimodal PSD. Using kneading elements, the width of the PSD could be partially narrowed and the liquid distribution was more homogeneous. However, still a significant fraction of oversized agglomerates was obtained. Implementing additional kneading elements or cutters in the final section of the screw configuration was not beneficial. Furthermore, granulation with only TME or SME had limited impact on the width of the PSD. Promising results were obtained by combining kneading elements with SME, as for these configurations the PSD was narrower and shifted to the size fractions suitable for tableting.

  13. Asymmetric distribution in twin screw granulation.

    Science.gov (United States)

    Chan Seem, Tim; Rowson, Neil A; Gabbott, Ian; de Matas, Marcel; Reynolds, Gavin K; Ingram, Andy

    2016-09-01

    Positron Emission Particle Tracking (PEPT) was successfully employed to validate measured transverse asymmetry in material distribution in the conveying zones of a Twin Screw Granulator (TSG). Flow asymmetry was established to be a property of the granulator geometry and dependent on fill level. The liquid distribution of granules as a function of fill level was determined. High flow asymmetry at low fill level negatively affects granule nucleation leading to high variance in final uniformity. Wetting of material during nucleation was identified as a critical parameter in determining final granule uniformity and fill level is highlighted as a crucial control factor in achieving this. Flow asymmetry of dry material in conveying zones upstream of binder fluid injection leads to poor non-uniform wetting at nucleation and results in heterogeneous final product. The granule formation mechanism of 60°F kneading blocks is suggested to be primarily breakage of agglomerates formed during nucleation. Optimisation of screw configuration would be required to provide secondary growth. This work shows how fill dependent flow regimes affect granulation mechanisms.

  14. Undetectable bacterial resistance to phage lytic proteins from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

    Science.gov (United States)

    The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we tested for the emergence of resistant Staphylococcus aureus to any of three phage lytic proteins constructs. The investigated cell wall lytic enzymes w...

  15. Regional Variation in Lytic and Lysogenic Viral Infection in the Southern Ocean and Its Contribution to Biogeochemical Cycling

    NARCIS (Netherlands)

    Evans, C.; Brussaard, C.P.D.

    2012-01-01

    Lytic and lysogenic viral infection was investigated throughout the Southern Ocean at sites spanning the sub-Antarctic zone, the Antarctic Circumpolar Current, and an Antarctic continental sea. Higher lytic virus activity was recorded in the more productive sub-Antarctic zone than in the iron-limite

  16. Influence of heavy metals on biosynthesis, activity of lytic enzymes and growthstimulating factor of Streptomyces recifensis var. lyticus P-29

    Directory of Open Access Journals (Sweden)

    Т. P. Kilochok

    2005-02-01

    Full Text Available Influence of heavy metals on growth, biosynthesis, lytic action and growthstimulating activity enzymes complex of Streptomyces recifensis var. lyticus was studied. It was showed that salt of plumbum' has positive influence as on biosynthesis hydrolases (lytic endopeptidases, proteinases, amylases as well increase growthstimulating activity of preparation relatively the yeast

  17. Glinide, but Not Sulfonylurea, Can Evoke Insulin Exocytosis by Repetitive Stimulation: Imaging Analysis of Insulin Exocytosis by Secretagogue-Induced Repetitive Stimulations

    Directory of Open Access Journals (Sweden)

    Kyota Aoyagi

    2009-01-01

    Full Text Available To investigate the different effects between sulfonylurea (SU and glinide drugs in insulin secretion, pancreatic β-cells were repeatedly stimulated with SU (glimepiride or glinide (mitiglinide. Total internal reflection fluorescent (TIRF microscopy revealed that secondary stimulation with glimepiride, but not glucose and mitiglinide, failed to evoke fusions of insulin granules although primary stimulation with glucose, glimepiride, and mitiglinide induced equivalent numbers of exocytotic responses. Glimepiride, but not glucose and mitiglinide, induced abnormally sustained [Ca2+]i elevations and reductions of docked insulin granules on the plasma membrane. Our data suggest that the effect of glinide on insulin secretory mechanisms is similar to that of glucose.

  18. Compressibility and compactibility of granules produced by wet and dry granulation.

    Science.gov (United States)

    Bacher, C; Olsen, P M; Bertelsen, P; Sonnergaard, J M

    2008-06-24

    The bulk properties, compactibility and compressibility of granules produced by wet and dry granulation were compared applying a rotary tablet press, three different morphological forms of calcium carbonate and two particle sizes of sorbitol. Granules from both granulation methods possessed acceptable flow properties; however, the ground (Mikhart) and cubic (Scoralite) calcium carbonate demonstrated better die-filling abilities in the tablet press than the scalenhedral calcium carbonate (Sturcal). The wet processed granules showed in general larger compression properties. This was explained as these granules were mechanical stronger and had a higher initial porosity. In some cases, a large particle surface area of calcium carbonate and sorbitol resulted in a small, insignificant improvement of the consolidation characteristics. A correlation between the compression and compaction characteristics was demonstrated.

  19. A TECHNICAL NOTE ON GRANULATION TECHNOLOGY: A WAY TO OPTIMISE GRANULES

    Directory of Open Access Journals (Sweden)

    Mahammed Athar A. Saikh

    2013-01-01

    Full Text Available To provide an updated technical note on granulation technology (GT, mostly on novel GT, that will help researcher working/engaged in designing an efficient GT for getting granules with desired features. Granules were most widely used in the production of pharmaceutical oral dosage forms. Advancement in GT had revolutionized the sphere and resulted in development of several processes. Each and every process had advantages and disadvantages, and limitations. Depth knowledge in GT was a prerequisite to process product for obtaining targeted granulation with desired product parameters. In this regards updated literatures were collected from data bases, studied and was presented for easy reference of scientists engaged in granule production, so that they can adopt appropriate and suitable GT. Presented handy note will help researchers in designing a robust GT for getting optimised granule.

  20. Distribution of binder in granules produced by means of twin screw granulation.

    Science.gov (United States)

    Fonteyne, Margot; Fussell, Andrew Luke; Vercruysse, Jurgen; Vervaet, Chris; Remon, Jean Paul; Strachan, Clare; Rades, Thomas; De Beer, Thomas

    2014-02-28

    According to the quality by design principle processes may not remain black-boxes and full process understanding is required. The granule size distribution of granules produced via twin screw granulation is often found to be bimodal. The aim of this study was to gain a better understanding of binder distribution within granules produced via twin screw granulation in order to investigate if an inhomogeneous spread of binder is causing this bimodal size distribution. Theophylline-lactose-polyvinylpyrrolidone K30 (PVP) (30-67.5-2.5%, w/w) was used as a model formulation. The intra-granular distribution of PVP was evaluated by means of hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy. For the evaluated formulation, no PVP rich zones were detected when applying a lateral spatial resolution of 0.5 μm, indicating that PVP is homogenously distributed within the granules.

  1. THE STUDY OF THE KINETIC OF NATURAL ZEOLITE GRANULES GROWTH AT DIFFERENT WAYS OF GRANULATION

    OpenAIRE

    Rybachuk VD

    2016-01-01

    Introduction. Active substances and excipients used in the manufacture of medicines in tablet form, in most cases, have poor technological properties. This fact determines the need for prior granulation of mass before compression. Granulators of various sizes and designs, running on different modes, made the formation, growth and consolidation of the powder particles that lead to obtain pellets of different shapes and sizes. From the literature it is known that granulation leads to two forms ...

  2. Carbon Fiber Ultramicrodic Electrode Electrodeposited with Over-Oxidized Polypyrrole for Amperometric Detection of Vesicular Exocytosis from Pheochromocytoma Cell

    Science.gov (United States)

    Wang, Li; Xu, Huiren; Song, Yilin; Luo, Jinping; Xu, Shengwei; Zhang, Song; Liu, Juntao; Cai, Xinxia

    2015-01-01

    Vesicular exocytosis is ubiquitous, but it is difficult to detect within the cells' communication mechanism. For this purpose, a 2 μm ultramicrodic carbon fiber electrode was fabricated in this work based on electrodeposition with over-oxidized polypyrrole nanoparticle (PPyox-CFE), which was applied successfully for real-time monitoring of quantal exocytosis from individual pheochromocytoma (PC12) cells. PPyox-CFE was evaluated by dopamine (DA) solutions through cyclic voltammetry and amperometry electrochemical methods, and results revealed that PPyox-CFE improved the detection limit of DA. In particular, the sensitivity of DA was improved to 24.55 μA·μM−1·μm−2 using the PPyox-CFE. The ultramicrodic electrode combined with the patch-clamp system was used to detect vesicular exocytosis of DA from individual PC12 cells with 60 mM K+ stimulation. A total of 287 spikes released from 7 PC12 cells were statistically analyzed. The current amplitude (Imax) and the released charge (Q) of the amperometric spikes from the DA release by a stimulated PC12 cell is 45.1 ± 12.5 pA and 0.18 ± 0.04 pC, respectively. Furthermore, on average ∼562,000 molecules were released in each vesicular exocytosis. PPyox-CFE, with its capability of detecting vesicular exocytosis, has potential application in neuron communication research. PMID:25569759

  3. Age-dependent preferential dense-core vesicle exocytosis in neuroendocrine cells revealed by newly developed monomeric fluorescent timer protein.

    Science.gov (United States)

    Tsuboi, Takashi; Kitaguchi, Tetsuya; Karasawa, Satoshi; Fukuda, Mitsunori; Miyawaki, Atsushi

    2010-01-01

    Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO-tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K(+)] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.

  4. Carbon Fiber Ultramicrodic Electrode Electrodeposited with Over-Oxidized Polypyrrole for Amperometric Detection of Vesicular Exocytosis from Pheochromocytoma Cell

    Directory of Open Access Journals (Sweden)

    Li Wang

    2015-01-01

    Full Text Available Vesicular exocytosis is ubiquitous, but it is difficult to detect within the cells’ communication mechanism. For this purpose, a 2 µm ultramicrodic carbon fiber electrode was fabricated in this work based on electrodeposition with over-oxidized polypyrrole nanoparticle (PPyox-CFE, which was applied successfully for real-time monitoring of quantal exocytosis from individual pheochromocytoma (PC12 cells. PPyox-CFE was evaluated by dopamine (DA solutions through cyclic voltammetry and amperometry electrochemical methods, and results revealed that PPyox-CFE improved the detection limit of DA. In particular, the sensitivity of DA was improved to 24.55 µA·µM−1·µm−2 using the PPyox-CFE. The ultramicrodic electrode combined with the patch-clamp system was used to detect vesicular exocytosis of DA from individual PC12 cells with 60 mM K+ stimulation. A total of 287 spikes released from 7 PC12 cells were statistically analyzed. The current amplitude (Imax and the released charge (Q of the amperometric spikes from the DA release by a stimulated PC12 cell is 45.1 ± 12.5 pA and 0.18 ± 0.04 pC, respectively. Furthermore, on average ~562,000 molecules were released in each vesicular exocytosis. PPyox-CFE, with its capability of detecting vesicular exocytosis, has potential application in neuron communication research.

  5. Effectiveness of lytic bacteriophages in reducing E. coli O157:H7 populations introduced through cross-contamination on fresh cut lettuce

    Science.gov (United States)

    Previous research has shown that lytic bacteriophages (phages) can kill E. coli O157:H7 on produce surfaces. The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield) at 10^8 PFU/m...

  6. Cohesive strength of iron ore granules

    Directory of Open Access Journals (Sweden)

    Contreras Rafael Jaimes

    2017-01-01

    Full Text Available We present an experimental and numerical investigation of the mechanical strength of crude iron ore (Hematite granules in which capillary bonds between primary particles are the source of internal cohesion. The strength is measured by subjecting the granules to vertical compression between two plates. We show that the behavior of the granules is ductile with a well-defined plastic threshold which increases with the amount of water. It is found that the compressive strength scales with capillary cohesion with a pre-factor that is nearly independent of size polydispersity for the investigated range of parameters but increases with friction coefficient between primary particles. This weak dependence may be attributed to the class of fine particles which, due to their large number, behaves as a cohesive matrix that controls the strength of the granule.

  7. Cohesive strength of iron ore granules

    Science.gov (United States)

    Contreras, Rafael Jaimes; Berger, Nicolas; Izard, Edouard; Douce, Jean-François; Koltsov, Alexey; Delenne, Jean-Yves; Azema, Emilien; Nezamabadi, Saeid; van Loo, Frédéric; Pellenq, Roland; Radjai, Farhang

    2017-06-01

    We present an experimental and numerical investigation of the mechanical strength of crude iron ore (Hematite) granules in which capillary bonds between primary particles are the source of internal cohesion. The strength is measured by subjecting the granules to vertical compression between two plates. We show that the behavior of the granules is ductile with a well-defined plastic threshold which increases with the amount of water. It is found that the compressive strength scales with capillary cohesion with a pre-factor that is nearly independent of size polydispersity for the investigated range of parameters but increases with friction coefficient between primary particles. This weak dependence may be attributed to the class of fine particles which, due to their large number, behaves as a cohesive matrix that controls the strength of the granule.

  8. Atrial natriuretic peptide (ANP)-granules: ultrastructure ...

    African Journals Online (AJOL)

    AJB SERVER

    2006-12-29

    Dec 29, 2006 ... physiological conditions: temperature, dehydration and nutritional condition. The main ... Granule content is released into the extra-cellular space (extrusion). The ANP causes .... Studies carried out in humans (Tanaka et al.,.

  9. Measuring stellar granulation during planet transits

    CERN Document Server

    Chiavassa, A; Selsis, F; Leconte, J; Von Paris, P; Bordé, P; Magic, Z; Collet, R; Asplund, M

    2016-01-01

    Stellar activity and convection-related surface structures might cause bias in planet detection and characterization that use these transits. Surface convection simulations help to quantify the granulation signal. We used realistic three-dimensional radiative hydrodynamical simulations from the Stagger grid and synthetic images computed with the radiative transfer code Optim3D to model the transits of three prototype planets: a hot Jupiter, a hot Neptune, and a terrestrial planet. We computed intensity maps from RHD simulations of the Sun and a K-dwarf star at different wavelength bands from optical to far-infrared. We modeled the transit using synthetic stellar-disk images and emulated the temporal variation of the granulation intensity. We identified two types of granulation noise that act simultaneously during the planet transit: (i) the intrinsic change in the granulation pattern with timescales smaller than the usual planet transit, and (ii) the fact that the transiting planet occults isolated regions of...

  10. Complete Genome Sequence of a Lytic Siphoviridae Bacteriophage Infecting Several Serovars of Salmonella enterica

    Science.gov (United States)

    Paradiso, Rubina; Lombardi, Serena; Iodice, Maria Grazia; Riccardi, Marita Georgia; Orsini, Massimiliano; Bolletti Censi, Sergio; Galiero, Giorgio

    2016-01-01

    The bacteriophage 100268_sal2 was isolated from water buffalo feces in southern Italy, exhibiting lytic activity against several subspecies of Salmonella enterica. This bacteriophage belongs to the Siphoviridae family and has a 125,114-bp double-stranded DNA (ds-DNA) genome containing 188 coding sequences (CDSs). PMID:27688334

  11. Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication.

    Directory of Open Access Journals (Sweden)

    Giuseppe Balistreri

    2016-02-01

    Full Text Available Kaposi's sarcoma herpesvirus (KSHV causes Kaposi's sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication.

  12. The novel Shewanella putrefaciens-infecting bacteriophage Spp001: genome sequence and lytic enzymes.

    Science.gov (United States)

    Han, Feng; Li, Meng; Lin, Hong; Wang, Jingxue; Cao, Limin; Khan, Muhammad Naseem

    2014-06-01

    Shewanella putrefaciens has been identified as a specific spoilage organism commonly found in chilled fresh fish, which contributes to the spoilage of fish products. Limiting S. putrefaciens growth can extend the shelf-life of chilled fish. Endolysins, which are lytic enzymes produced by bacteriophages, have been considered an alternative to control bacterial growth, and have been useful in various applications, including food preservation. We report here, for the first time, the complete genome sequence of a novel phage Spp001, which lyses S. putrefaciens Sp225. The Spp001 genome comprises a 54,789-bp DNA molecule with 67 open reading frames and an average total G + C content of 49.42 %. In silico analysis revealed that the Spp001 open reading frames encode various putative functional proteins, including an endolysin (ORF 62); however, no sequence for genes encoding the holin polypeptides, which work in concert with endolysins, was identified. To examine further the lytic activity of Spp001, we analyzed the lytic enzyme-containing fraction from phages released at the end of the phage lytic cycle in S. putrefaciens, using diffusion and turbidimetric assays. The results show that the partially purified extract contained endolysin, as indicated by a high hydrolytic activity towards bacterial peptidoglycan decrease in the OD590 value by 0.160 in 15 min. The results will allow further investigation of the purification of natural Spp001 endolysin, the extension of Spp001 host range, and the applications of the phage-encoded enzymes.

  13. Crystal structure and mechanism of the lytic transglycosylase MltA from Escherichia coli

    NARCIS (Netherlands)

    van Straaten, Karin

    2006-01-01

    This thesis describes the determination and analysis of the 3D-structure of the lytic transglycosylase MltA from Escherichia coli by X-ray crystallography. This work aims to further increase our knowledge of the molecular details of the cleaving mechanism and the typical 1,6- anhydromuropeptide prod

  14. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Lo Leggio, Leila; Simmons, Thomas J.; Poulsen, Jens-Christian Navarro

    2015-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here...... substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes....

  15. Pain relief with percutaneous trochanteroplasty in a patient with bilateral trochanteric myelomatous lytic lesions.

    Science.gov (United States)

    Wahezi, Sayed E; Silva, Kyle; Najafi, Shervin

    2015-01-01

    Multiple myeloma is a hematologic malignancy associated with destructive bone loss. Lytic lesions, a hallmark of this cancer, can result in significant morbidity because of associated pain and structural osseous compromise. Osteoplasty has demonstrated efficacy in the treatment of myelomatous pain within the axial skeleton; however, there is limited evidence supporting the utility of osteoplasty to treat extra-spinal lesions. We describe a 67 year-old woman with stable IgA lambda multiple myeloma with sentinel bilateral greater trochanteric lytic lesions that was referred to our interventional pain management clinic for evaluation of bilateral lateral hip pain. Conservative treatment options including physical therapy, non-steroidal anti-inflammatory drugs (NSAIDs), oral opiates, and local corticosteroid injections to bilateral trochanteric bursae failed to offer pain relief. The patient underwent minimally invasive percutaneous trochanteroplasty with concomitant core biopsy of her bilateral trochanteric lytic lesions. The intended goals of this novel procedure were to determine the cause of the suspected lytic lesions, provide pain relief, and offer structural stability by safely implanting bone cement as part of a fracture prevention strategy. At 12 month follow-up, the patient's pain improved by 70% and she no longer required the use of pain medication. The patient also displayed a significant improvement in her day-to-day functioning and quality of life.

  16. The importance of lytic and nonlytic immune responses in viral infections

    DEFF Research Database (Denmark)

    Wodarz, Dominik; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup

    2002-01-01

    Antiviral immune effector mechanisms can be divided broadly into lytic and nonlytic components. We use mathematical models to investigate the fundamental question of which type of response is required to combat different types of viral infection. According to our model, the relative roles...

  17. Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication.

    Science.gov (United States)

    Balistreri, Giuseppe; Viiliäinen, Johanna; Turunen, Mikko; Diaz, Raquel; Lyly, Lauri; Pekkonen, Pirita; Rantala, Juha; Ojala, Krista; Sarek, Grzegorz; Teesalu, Mari; Denisova, Oxana; Peltonen, Karita; Julkunen, Ilkka; Varjosalo, Markku; Kainov, Denis; Kallioniemi, Olli; Laiho, Marikki; Taipale, Jussi; Hautaniemi, Sampsa; Ojala, Päivi M

    2016-02-01

    Kaposi's sarcoma herpesvirus (KSHV) causes Kaposi's sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication.

  18. Crystal structure and mechanism of the lytic transglycosylase MltA from Escherichia coli

    NARCIS (Netherlands)

    van Straaten, Karin

    2006-01-01

    This thesis describes the determination and analysis of the 3D-structure of the lytic transglycosylase MltA from Escherichia coli by X-ray crystallography. This work aims to further increase our knowledge of the molecular details of the cleaving mechanism and the typical 1,6- anhydromuropeptide prod

  19. Characterization of the lytic-lysogenic switch of the lactococcal bacteriophage Tuc2009

    NARCIS (Netherlands)

    Kenny, JG; Leach, S; de la Hoz, AB; Venema, G; Kok, J; Fitzgerald, GF; Nauta, A; Alonso, JC; van Sinderen, D; Kenny, John G.; Hoz, Ana B. de la; Fitzgerald, Gerald F.; Alonso, Juan C.

    2006-01-01

    Tuc2009 is a temperate bacteriophage of Lactococcus lactis subsp. cremoris UC509 which encodes a CI- and Cro-type lysogenic-lytic switch region. A helix-swap of the 0 helices of the closely related Cl-type proteins from the lactococcal phages r1t and Tuc2009 revealed the crucial elements involved in

  20. STUDIES ON THE BACTERIOPHAGE OF D'HERELLE : I. IS THE LYTIC PRINCIPLE VOLATILE?

    Science.gov (United States)

    Bronfenbrenner, J J; Korb, C

    1925-01-01

    The lytic principle concerned in the phenomenon of transmissible lysis is not volatile. The results which have been taken to indicate volatility are, in our opinion, to be attributed to the transfer to the distillate of minute droplets of the original active filtrate.

  1. Acid resistance of starch granules

    Energy Technology Data Exchange (ETDEWEB)

    Nara, S.; Sakakura, M.; Komiya, T.

    1983-08-01

    When potato starch was hydrolyzed to form Naegeli amylodextrin by 16% sulfuric acid at 30/sup 0/C, only the amorphous portion of the starch granules was deteriorated. The crystallinity of Naegeli amylodextrin showing the hydrolysis ratio of 0.22 was 1.28 times as large as that of original starch. The hydrolysis process at above 45/sup 0/C was given by two exponential equations. The value of acid resistance portion (C/sub 0/) at 30 and 38/sup 0/C was 100%, while the values at 45, 50 and 55/sup 0/C were 67, 38 and 18%, respectively. The high value of C/sub 0/ generally showed the high acid resistance in the various starches. Sweet potato and waxy rice starches were more easily hydrolysed than other starches, although they gave the relatively high value of C/sub 0/. Thus, it was slightly more difficult for low acid resistance portion of potato starch to be hydrolyzed than for that of other starches. Moreover, that of waxy rice was easily hydrolyzed.

  2. Hippocampal granule cells opt for early retirement.

    Science.gov (United States)

    Alme, C B; Buzzetti, R A; Marrone, D F; Leutgeb, J K; Chawla, M K; Schaner, M J; Bohanick, J D; Khoboko, T; Leutgeb, S; Moser, E I; Moser, M-B; McNaughton, B L; Barnes, C A

    2010-10-01

    Increased excitability and plasticity of adult-generated hippocampal granule cells during a critical period suggests that they may "orthogonalize" memories according to time. One version of this "temporal tag" hypothesis suggests that young granule cells are particularly responsive during a specific time period after their genesis, allowing them to play a significant role in sculpting CA3 representations, after which they become much less responsive to any input. An alternative possibility is that the granule cells active during their window of increased plasticity, and excitability become selectively tuned to events that occurred during that time and participate in later reinstatement of those experiences, to the exclusion of other cells. To discriminate between these possibilities, rats were exposed to different environments at different times over many weeks, and cell activation was subsequently assessed during a single session in which all environments were revisited. Dispersing the initial experiences in time did not lead to the increase in total recruitment at reinstatement time predicted by the selective tuning hypothesis. The data indicate that, during a given time frame, only a very small number of granule cells participate in many experiences, with most not participating significantly in any. Based on these and previous data, the small excitable population of granule cells probably correspond to the most recently generated cells. It appears that, rather than contributing to the recollection of long past events, most granule cells, possibly 90-95%, are effectively "retired." If granule cells indeed sculpt CA3 representations (which remains to be shown), then a possible consequence of having a new set of granule cells participate when old memories are reinstated is that new representations of these experiences might be generated in CA3. Whatever the case, the present data may be interpreted to undermine the standard "orthogonalizer" theory of the role of

  3. Intracranial localization of arachnoid granulations in rats

    Institute of Scientific and Technical Information of China (English)

    Yanan Dong; Min Yu; Lei Meng; Yong Jiang; Jun Gao; Honghai Peng; Jianguo Shi

    2008-01-01

    BACKGROUND: This paper describes histomorphologic studies on arachnoid granulations in rats, which have not been investigated in China to our knowledge.OBJECTIVE: To observe the distribution of intracranial arachnoid granulations in rats. DESIGN, TIME AND SETTING: The observational experiment was performed in the Academy of Life Sciences of Shandong Taishan Medical College from May to August 2004.MATERIALS: Thirty healthy adult Wistar rats (3-4 months old) of SPF grade, equal numbers of each sex, were selected for this study. Methylene blue parenteral solution was provided by Jiangsu Jichuan Pharmaceutical Company (China), and an optical microscope (Type: CH20; Olympus Co. Ltd., Japan) was used for observation of the histomorphology of the arachnoid granulations.METHOD: Injection of methylene blue parenteral solution into the cerebellomedullary cistern of rats.MAIN OUTCOME MEASURE: The blue stained parts of the lateral sinus were sectioned, stained by hematoxylin and eosin, and then observed under the microscope.RESULTS: The cavitas subarachnoidealis had extensive blue staining after methylene blue injection, while the arachnoid and dura were without dye accumulation. The blue dye indicated the location of the arachnoid granulations. The location of these granulations was fixed, mainly in the lateral sinus at both sides of confluence within 4 mm of the internal jugular vein.CONCLUSION: The arachnoid granulations of the rat were located mainly in the lateral sinuses of the dura mater.

  4. Pituitary adenylate cyclase activating polypeptide modulates catecholamine storage and exocytosis in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Yan Dong

    Full Text Available A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD. Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM. PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising

  5. Mesenchymal Stem Cells Exhibit Regulated Exocytosis in Response to Chemerin and IGF.

    Directory of Open Access Journals (Sweden)

    J Dinesh Kumar

    Full Text Available Mesenchymal stem cells (MSCs play important roles in tissue repair and cancer progression. Our recent work suggests that some mesenchymal cells, notably myofibroblasts exhibit regulated exocytosis resembling that seen in neuroendocrine cells. We now report that MSCs also exhibit regulated exocytosis. Both a G-protein coupled receptor agonist, chemerin, and a receptor tyrosine kinase stimulant, IGF-II, evoked rapid increases in secretion of a marker protein, TGFβig-h3. The calcium ionophore, ionomycin, also rapidly increased secretion of TGFβig-h3 while inhibitors of translation (cycloheximide or secretory protein transport (brefeldin A had no effect, indicating secretion from preformed secretory vesicles. Inhibitors of the chemerin and IGF receptors specifically reduced the secretory response. Confocal microscopy of MSCs loaded with Fluo-4 revealed chemerin and IGF-II triggered intracellular Ca2+ oscillations requiring extracellular calcium. Immunocytochemistry showed co-localisation of TGFβig-h3 and MMP-2 to secretory vesicles, and transmission electron-microscopy showed dense-core secretory vesicles in proximity to the Golgi apparatus. Proteomic studies on the MSC secretome identified 64 proteins including TGFβig-h3 and MMP-2 that exhibited increased secretion in response to IGF-II treatment for 30min and western blot of selected proteins confirmed these data. Gene ontology analysis of proteins exhibiting regulated secretion indicated functions primarily associated with cell adhesion and in bioassays chemerin increased adhesion of MSCs and adhesion, proliferation and migration of myofibroblasts. Thus, MSCs exhibit regulated exocytosis that is compatible with an early role in tissue remodelling.

  6. Cycling of dense core vesicles involved in somatic exocytosis of serotonin by leech neurons

    Directory of Open Access Journals (Sweden)

    Citlali eTrueta

    2012-06-01

    Full Text Available We studied the cycling of dense core vesicles producing somatic exocytosis of serotonin. Our experiments were made using electron microscopy and vesicle staining with fluorescent dye FM1-43 in Retzius neurons of the leech, which secrete serotonin from clusters of dense core vesicles in a frequency-dependent manner. Electron micrographs of neurons at rest or after 1 Hz stimulation showed two pools of dense core vesicles. A perinuclear pool near Golgi apparatuses, from which vesicles apparently form, and a peripheral pool with vesicle clusters at a distance from the plasma membrane. By contrast, after 20 Hz electrical stimulation 47% of the vesicle clusters were apposed to the plasma membrane, with some omega exocytosis structures. Dense core and small clear vesicles apparently originating from endocytosis were incorporated in multivesicular bodies. In another series of experiments, neurons were stimulated at 20 Hz while bathed in a solution containing peroxidase. Electron micrographs of these neurons contained gold particles coupled to anti-peroxidase antibodies in dense core vesicles and multivesicular bodies located near the plasma membrane. Cultured neurons depolarized with high potassium in the presence of FM1-43 displayed superficial fluorescent spots, each reflecting a vesicle cluster. A partial bleaching of the spots followed by another depolarization in the presence of FM1-43 produced restaining of some spots, other spots disappeared, some remained without restaining and new spots were formed. Several hours after electrical stimulation the FM1-43 spots accumulated at the center of the somata. This correlated with electron micrographs of multivesicular bodies releasing their contents near Golgi apparatuses. Our results suggest that dense core vesicle cycling related to somatic serotonin release involves two steps: the production of clear vesicles and multivesicular bodies after exocytosis, and the formation of new dense core vesicles in

  7. β-Hydroxybutyrate supports synaptic vesicle cycling but reduces endocytosis and exocytosis in rat brain synaptosomes.

    Science.gov (United States)

    Hrynevich, Sviatlana V; Waseem, Tatyana V; Hébert, Audrey; Pellerin, Luc; Fedorovich, Sergei V

    2016-02-01

    The ketogenic diet is used as a prophylactic treatment for different types of brain diseases, such as epilepsy or Alzheimer's disease. In such a diet, carbohydrates are replaced by fats in everyday food, resulting in an elevation of blood-borne ketone bodies levels. Despite clinical applications of this treatment, the molecular mechanisms by which the ketogenic diet exerts its beneficial effects are still uncertain. In this study, we investigated the effect of replacing glucose by the ketone body β-hydroxybutyrate as the main energy substrate on synaptic vesicle recycling in rat brain synaptosomes. First, we observed that exposing presynaptic terminals to nonglycolytic energy substrates instead of glucose did not alter the plasma membrane potential. Next, we found that synaptosomes were able to maintain the synaptic vesicle cycle monitored with the fluorescent dye acridine orange when glucose was replaced by β-hydroxybutyrate. However, in presence of β-hydroxybutyrate, synaptic vesicle recycling was modified with reduced endocytosis. Replacing glucose by pyruvate also led to a reduced endocytosis. Addition of β-hydroxybutyrate to glucose-containing incubation medium was without effect. Reduced endocytosis in presence of β-hydroxybutyrate as sole energy substrate was confirmed using the fluorescent dye FM2-10. Also we found that replacement of glucose by ketone bodies leads to inhibition of exocytosis, monitored by FM2-10. However this reduction was smaller than the effect on endocytosis under the same conditions. Using both acridine orange in synaptosomes and the genetically encoded sensor synaptopHluorin in cortical neurons, we observed that replacing glucose by β-hydroxybutyrate did not modify the pH gradient of synaptic vesicles. In conclusion, the nonglycolytic energy substrates β-hydroxybutyrate and pyruvate are able to support synaptic vesicle recycling. However, they both reduce endocytosis. Reduction of both endocytosis and exocytosis together with

  8. P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells.

    Science.gov (United States)

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

    2011-04-01

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

  9. Hypotonicity-induced Renin exocytosis from juxtaglomerular cells requires aquaporin-1 and cyclooxygenase-2

    DEFF Research Database (Denmark)

    Friis, Ulla G; Madsen, Kirsten; Svenningsen, Per

    2009-01-01

    The mechanism by which extracellular hypotonicity stimulates release of renin from juxtaglomerular (JG) cells is unknown. We hypothesized that osmotically induced renin release depends on water movement through aquaporin-1 (AQP1) water channels and subsequent prostanoid formation. We recorded...... membrane capacitance (C(m)) by whole-cell patch clamp in single JG cells as an index of exocytosis. Hypotonicity increased C(m) significantly and enhanced outward current. Indomethacin, PLA(2) inhibition, and an antagonist of prostaglandin transport impaired the C(m) and current responses to hypotonicity...

  10. Study of light scattering by a granulated coated sphere - a model of granulated blood cells

    NARCIS (Netherlands)

    M.A. Yurkin; D. de Kanter; A.G. Hoekstra

    2008-01-01

    We performed extensive simulations of light scattering by granulated coated sphere model using the discrete dipole approximation and varying model parameters in the ranges of sizes and refractive indices of granulated blood cells. We compared these results with predictions of Maxwell-Garnett effecti

  11. Granule size control and targeting in pulsed spray fluid bed granulation.

    Science.gov (United States)

    Ehlers, Henrik; Liu, Anchang; Räikkönen, Heikki; Hatara, Juha; Antikainen, Osmo; Airaksinen, Sari; Heinämäki, Jyrki; Lou, Honxiang; Yliruusi, Jouko

    2009-07-30

    The primary aim of the study was to investigate the effects of pulsed liquid feed on granule size. The secondary aim was to increase knowledge of this technique in granule size targeting. Pulsed liquid feed refers to the pump changing between on- and off-positions in sequences, called duty cycles. One duty cycle consists of one on- and off-period. The study was performed with a laboratory-scale top-spray fluid bed granulator with duty cycle length and atomization pressure as studied variables. The liquid feed rate, amount and inlet air temperature were constant. The granules were small, indicating that the powder has only undergone ordered mixing, nucleation and early growth. The effect of atomizing pressure on granule size depends on inlet air relative humidity, with premature binder evaporation as a reason. The duty cycle length was of critical importance to the end product attributes, by defining the extent of intermittent drying and rewetting. By varying only the duty cycle length, it was possible to control granule nucleation and growth, with a wider granule size target range in increased relative humidity. The present study confirms that pulsed liquid feed in fluid bed granulation is a useful tool in end product particle size targeting.

  12. A tetanus toxin sensitive protein other than VAMP 2 is required for exocytosis in the pancreatic acinar cell.

    Science.gov (United States)

    Padfield, P J

    2000-11-01

    The neurotoxin sensitivity of regulated exocytosis in the pancreatic acinar cell was investigated using streptolysin-O permeabilized pancreatic acini. Treatment of permeabilized acini with botulinum toxin B (BoNT/B) or botulinum toxin D (BoNT/D) had no detectable effect on Ca(2+)-dependent amylase secretion but did result in the complete cleavage of VAMP 2. In comparison, tetanus toxin (TeTx) treatment both significantly inhibited Ca(2+)-dependent amylase secretion and cleaved VAMP 2. These results indicate that regulated exocytosis in the pancreatic acinar cell requires a tetanus toxin sensitive protein(s) other than VAMP 2.

  13. Investigation of Physicochemical Drug Properties to Prepare Fine Globular Granules Composed of Only Drug Substance in Fluidized Bed Rotor Granulation.

    Science.gov (United States)

    Mise, Ryohei; Iwao, Yasunori; Kimura, Shin-Ichiro; Osugi, Yukiko; Noguchi, Shuji; Itai, Shigeru

    2015-01-01

    The effect of some drug properties (wettability and particle size distribution) on granule properties (mean particle size, particle size distribution, sphericity, and granule strength) were investigated in a high (>97%) drug-loading formulation using fluidized bed rotor granulation. Three drugs: acetaminophen (APAP); ibuprofen (IBU); and ethenzamide (ETZ) were used as model drugs based on their differences in wettability and particle size distribution. Granules with mean particle sizes of 100-200 µm and a narrow particle size distribution (PSD) could be prepared regardless of the drug used. IBU and ETZ granules showed a higher sphericity than APAP granules, while APAP and ETZ granules exhibited higher granule strength than IBU. The relationship between drug and granule properties suggested that the wettability and the PSD of the drugs were critical parameters affecting sphericity and granule strength, respectively. Furthermore, the dissolution profiles of granules prepared with poorly water-soluble drugs (IBU and ETZ) showed a rapid release (80% release in 20 min) because of the improved wettability with granulation. The present study demonstrated for the first time that fluidized bed rotor granulation can prepare high drug-loaded (>97%) globular granules with a mean particle size of less than 200 µm and the relationship between physicochemical drug properties and the properties of the granules obtained could be readily determined, indicating the potential for further application of this methodology to various drugs.

  14. Aerobic granulation of protein-rich granules from nitrogen-lean wastewaters.

    Science.gov (United States)

    Chen, Yu-You; Ju, Sheau-Pyng; Lee, Duu-Jong

    2016-10-01

    Proteins (PN)-rich granules are stable in structure in long-term reactor operations. This study proposed to cultivate PN-rich granules with PN/polysaccharides (PS) >20 from nitrogen lean wastewater, with ammonia-nitrogen as sole nitrogen source at chemical oxygen demand (COD)/N of 153.8. The yielded granules can sustain their structural stability in sequencing batch reactor mode for sufficient treatment of wastewaters up to 7000mg/L COD and with COD/Nnitrogen fed in the nitrogen-lean wastewater is converted to the PN in the granules. The replacement of ammonia by nitrate as sole nitrogen source led to granules enriched with γ-proteobacteria which are easily deteriorated at low OLR.

  15. Cloning and sequencing of parafusin, a calcium-dependent exocytosis-related phosphoglycoprotein.

    Science.gov (United States)

    Subramanian, S V; Wyroba, E; Andersen, A P; Satir, B H

    1994-10-11

    A cDNA for parafusin, an evolutionarily conserved phosphoglycoprotein involved in exocytosis, has been cloned and sequenced from a unicellular eukaryote, Paramecium tetraurelia. A Paramecium cDNA library was screened with an oligonucleotide probe synthesized to an internal amino acid sequence of isolated parafusin. The insert was 3 kb long with an open reading frame of 1.75 kb. Data base searches of the deduced amino acid sequence showed that Paramecium parafusin had a 50.7% sequence identity to rabbit muscle phosphoglucomutase, although no detectable phosphoglucomutase activity has been detected in isolated parafusin. The deduced parafusin amino acid sequence had four inserts and two deletions, which might confer on the protein specific functions in signal transduction events related to exocytosis. Furthermore, searches for potential phosphorylation sites showed the presence of a protein kinase C site (KDFSFR) specific to parafusin. Southern blot analysis with probes specific for parafusin and phosphoglucomutase suggested that these proteins were products of different genes. We propose that parafusin and phosphoglucomutase are members of a superfamily that conserve homologies important for the tertiary structure of the molecules.

  16. The effects of hypokalaemia on the hormone exocytosis in adenohypophysis and prolactinoma cell culture model systems.

    Science.gov (United States)

    Molnár, Z; Pálföldi, R; László, A; Radács, M; László, M; Hausinger, P; Tiszlavicz, L; Rázga, Z; Valkusz, Z; Gálfi, M

    2014-11-01

    The extracellular ion milieu determines the exocytosis mechanism that is coupled to spontaneous electrical activity. The K(+) ion plays crucial role in this mechanism: as the potassium current is associated with membrane hyperpolarization and hormone release through protein cascade activation. The primary aim of this study was to investigate the response mechanisms of normal adenohypophysis and adenohypophyseal prolactinoma cell populations at different extracellular K(+) levels with an otherwise isoionic milieu of all other essential ions. We focused on prolactin (PRL) and adrenocorticotrophic hormone (ACTH) release.In our experimental study, female Wistar rats (n=20) were treated with estrone-acetate (150 μg/kg b.w./week) for 6 months to induce prolactinomas in the adenohypophysis. Primary, monolayer cell cultures were prepared by enzymatic and mechanical digestion. PRL and ACTH hormone presence was measured by radioimmunoassay or immuno-chemiluminescence assay. Immunocytochemistry was used to assess the apoptotic cells.Differences between the effects of hypokalaemia on normal adenohypophysis cultures and prolactinoma cell populations were investigated. Significant alteration (pprolactinoma cell cultures compared to untreated groups. Immunocyto-chemistry showed that Bcl-2 expression was reduced under hypokalaemic conditions.The decrease in hormone exocytosis was tightly correlated to the extracellular K(+) in both cell types, leading to the conclusion that external K(+) may be the major factor for the inhibition of hormone release. The significant increase in hormone content in supernatant media suggests that hypokalaemia may play important role in apoptosis.

  17. Redox-filled Carbon-Fiber Microelectrodes for Single-Cell Exocytosis

    Science.gov (United States)

    Cox, Jonathan T.; Gunderson, Christopher G.; Zhang, Bo

    2014-01-01

    Carbon-fiber microelectrodes (CFEs) are the primary electroanalytical tool in single-cell exocytosis and in-vivo studies. Here we report a new study on the kinetic properties of electrolyte-filled CFEs in single-cell measurements and demonstrate that the addition of outer sphere redox species, such as Fe(CN)63− and Ru(NH3)63+, in the backfill electrolyte solution can greatly enhance the kinetic response of CFEs. We show that at 750 mV, a voltage normally applied for detection of dopamine, the presence of fast outer sphere redox species in the backfilling solution significantly enhances the kinetic response of CFEs toward fast dopamine detection at single PC12 cells. Moreover, we also demonstrate that the use of Fe(CN)63− in the backfilling solution has enabled direct measurement of dopamine at applied voltages as low as 200 mV. This kinetic enhancement is believed to be due to faster electron-transfer kinetics on the coupling pole as compared to the sluggish reduction of oxygen. We anticipate that such redox-filled CFE ultramicroelectrodes will find many useful applications in single cell exocytosis and in-vivo sensing. PMID:24833889

  18. Formation of volutin granules in Corynebacterium glutamicum.

    Science.gov (United States)

    Pallerla, Srinivas Reddy; Knebel, Sandra; Polen, Tino; Klauth, Peter; Hollender, Juliane; Wendisch, Volker F; Schoberth, Siegfried M

    2005-02-01

    Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted.

  19. Amylase activity in substrate deficiency aerobic granules.

    Science.gov (United States)

    Lee, Chuen-Chi; Lee, Duu-Jong; Lai, Juin-Yih

    2009-01-01

    Immunohistochemical staining was applied together with the multicolor fluorescent scheme to demonstrate the amylase activity for polysaccharide hydrolysis in stored or starved aerobic granules that are in substrate deficiency. If sufficient nutrients were present, alpha-amylase and beta-amylase were found close to the surface layer of the original granules. Following storage or starvation during which most external nutrients were depleted, the alpha-amylase and beta-amylase were distributed over the entire granule interior, suggesting endogenous respiration at the core of the granule. In particular, the fluorescent intensities of alpha-amylase and beta-amylase were enriched 5-20 microm from the edge of the internal cavity, suggesting the strong correlation between polysaccharide hydrolysis and the formation of interior cavities. The secreted amylase was located near the living cells, suggesting that the polysaccharide hydrolysis is restricted to local environment that occurs near the functional strains. Internal hydrolysis within the core, for the case of both proteins and polysaccharides should correspond in principle to the loss of granule stability.

  20. Measuring stellar granulation during planet transits

    Science.gov (United States)

    Chiavassa, A.; Caldas, A.; Selsis, F.; Leconte, J.; Von Paris, P.; Bordé, P.; Magic, Z.; Collet, R.; Asplund, M.

    2017-01-01

    Context. Stellar activity and convection-related surface structures might cause bias in planet detection and characterization that use these transits. Surface convection simulations help to quantify the granulation signal. Aims: We used realistic three-dimensional (3D) radiative hydrodynamical (RHD) simulations from the Stagger grid and synthetic images computed with the radiative transfer code Optim3D to model the transits of three prototype planets: a hot Jupiter, a hot Neptune, and a terrestrial planet. Methods: We computed intensity maps from RHD simulations of the Sun and a K-dwarf star at different wavelength bands from optical to far-infrared that cover the range of several ground- and space-based telescopes which observe exoplanet transits. We modeled the transit using synthetic stellar-disk images obtained with a spherical-tile imaging method and emulated the temporal variation of the granulation intensity generating random images covering a granulation time-series of 13.3 h. We measured the contribution of the stellar granulation on the light curves during the planet transit. Results: We identified two types of granulation noise that act simultaneously during the planet transit: (i) the intrinsic change in the granulation pattern with timescale (e.g., 10 min for solar-type stars assumed in this work) is smaller than the usual planet transit ( hours as in our prototype cases); and (ii) the fact that the transiting planet occults isolated regions of the photosphere that differ in local surface brightness as a result of convective-related surface structures. First, we showed that our modeling approach returns granulation timescale fluctuations that are comparable with what has been observed for the Sun. Then, our statistical approach shows that the granulation pattern of solar and K-dwarf-type stars have a non-negligible effect of the light curve depth during the transit, and, consequentially on the determination of the planet transit parameters such as the

  1. Antimicrobial-Coated Granules for Disinfecting Water

    Science.gov (United States)

    Akse, James R.; Holtsnider, John T.; Kliestik, Helen

    2011-01-01

    Methods of preparing antimicrobialcoated granules for disinfecting flowing potable water have been developed. Like the methods reported in the immediately preceding article, these methods involve chemical preparation of substrate surfaces (in this case, the surfaces of granules) to enable attachment of antimicrobial molecules to the surfaces via covalent bonds. A variety of granular materials have been coated with a variety of antimicrobial agents that include antibiotics, bacteriocins, enzymes, bactericides, and fungicides. When employed in packed beds in flowing water, these antimicrobial-coated granules have been proven effective against gram-positive bacteria, gram-negative bacteria, fungi, and viruses. Composite beds, consisting of multiple layers containing different granular antimicrobial media, have proven particularly effective against a broad spectrum of microorganisms. These media have also proven effective in enhancing or potentiating the biocidal effects of in-line iodinated resins and of very low levels of dissolved elemental iodine.

  2. MEMBRANE-COATING GRANULES OF KERATINIZING EPITHELIA.

    Science.gov (United States)

    MATOLTSY, A G; PARAKKAL, P F

    1965-02-01

    The purpose of this study has been to obtain information on the development of the envelop of horny cells that resists the action of keratinolytic agents. Toward this end the epidermis, oral mucosa, and tongue epithelium of various vertebrates, as well as the isolated envelopes of horny cells, were examined by electron microscopy. It was found that small cytoplasmic granules (1,000 to 5,000 A) that develop within differentiating epithelial cells move toward the cell periphery, and after fusion with the plasma membrane, empty their contents into the intercellular spaces. The content of the granules spreads over the cell surfaces, and subsequently a thickened and coated cell envelope is formed that resists the action of keratinolytic agent. The membrane-coating granule is regarded as a specific differentiation product of the keratinizing epithelium. It contains numerous inner membranes and is assumed to engage in synthetic activities such as, perhaps, the formation of polysaccharides.

  3. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes

    DEFF Research Database (Denmark)

    Pierce, Brian; Wittrup Agger, Jane; Zhang, Zhenghong

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-...

  4. Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage.

    Science.gov (United States)

    James, C E; Stanley, K N; Allison, H E; Flint, H J; Stewart, C S; Sharp, R J; Saunders, J R; McCarthy, A J

    2001-09-01

    A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.

  5. Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase.

    Science.gov (United States)

    O'Dell, William B; Agarwal, Pratul K; Meilleur, Flora

    2017-01-16

    Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. We have determined high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed "pre-bound" molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygen activation. These results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme-substrate complex. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Involvement of Complexin 2 in Docking, Locking and Unlocking of Different SNARE Complexes during Sperm Capacitation and Induced Acrosomal Exocytosis.

    NARCIS (Netherlands)

    Tsai, P.S.; Brewis, I.A.; van Maaren, J.; Gadella, B.M.

    2012-01-01

    Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of O

  7. Estradiol inhibits depolarization-evoked exocytosis in PC12 cells via N-type voltage-gated calcium channels.

    NARCIS (Netherlands)

    Adams, K.L.; Maxson, M.M.; Mellander, L.; Westerink, R.H.S.; Ewing, A.G.

    2010-01-01

    Fast neuromodulatory effects of 17-β-estradiol (E2) on cytosolic calcium concentration ([Ca(2+)](i)) have been reported in many cell types, but little is known about its direct effects on vesicular neurotransmitter secretion (exocytosis). We examined the effects of E2 on depolarization-evoked [Ca(2+

  8. Arachnoid granulation affected by subarachnoid hemorrhage

    Directory of Open Access Journals (Sweden)

    R.P. Chopard

    1993-11-01

    Full Text Available The purpose of this study was to investigate using light microscopy the fibro-cellular components of arachnoid granulations affected by mild and severe subarachnoid hemorrage. The erythrocytes were in the channels delimitated by collagenous and elastic bundles and arachnoid cells, showing their tortuous and intercommunicating row from the pedicle to the fibrous capsule. The core portion of the pedicle and the center represented a principal route to the bulk outflow of cerebrospinal fluid and erythrocytes. In the severe hemorrhage, the fibrocellular components are desorganized, increasing the extracellular channels. We could see arachnoid granulations without erythrocytes, which cells showed big round nucleous suggesting their transformation into phagocytic cells.

  9. Molecular composition of IMP1 ribonucleoprotein granules

    DEFF Research Database (Denmark)

    Jønson, Lars; Vikesaa, Jonas; Krogh, Anders

    2007-01-01

    Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in mo......Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization...

  10. Neutrophil granules in health and disease

    DEFF Research Database (Denmark)

    Häger, M; Cowland, J B; Borregaard, N

    2010-01-01

    Neutrophil granules store proteins that are critically important for the neutrophil to move from the vascular bed to tissues and to kill microorganisms. This is illustrated in nature when individual proteins are deleted due to inherited mutations of their cognate genes, and such deficiencies result...... in the conditions leucocyte adhesion deficiency and chronic granulomatous disease. The granules of the neutrophil have traditionally been divided into two or three major types but are instead a continuum where several subtypes can be identified with differences in protein content and propensity for mobilization...

  11. Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

    OpenAIRE

    1989-01-01

    P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomp...

  12. Involvement of Noxa in mediating cellular ER stress responses to lytic virus infection

    OpenAIRE

    2011-01-01

    Noxa is a Bcl-2 homology domain-containing pro-apoptotic mitochondrial protein. Noxa mRNA and protein expression are upregulated by dsRNA or virus, and ectopic Noxa expression enhances cellular sensitivity to virus or dsRNA-induced apoptosis. Here we demonstrate that Noxa null baby mouse kidney (BMK) cells are deficient in normal cytopathic response to lytic viruses, and that reconstitution of the knockout cells with wild type Noxa restored normal cytopathic responses. Noxa regulation by viru...

  13. How Cancer Cells Become Resistant to Cationic Lytic Peptides: It's the Sugar!

    Science.gov (United States)

    Pierce, Joshua G

    2017-02-16

    In this issue of Cell Chemical Biology, Ishikawa et al. (2017) demonstrate that the loss of cell-surface anionic saccharides can impart resistance toward anticancer peptides. This study provides the first insight into potential resistance mechanisms toward cationic lytic peptides and highlights the important, yet previously unappreciated, role cell-surface glycans can play in cellular resistance mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Regulation of the Spore Cortex Lytic Enzyme SleB in Bacillus anthracis

    OpenAIRE

    2014-01-01

    Bacillus anthracis is the causative agent of the disease anthrax and poses a threat due to its potential to be used as a biological weapon. The spore form of this bacterium is an extremely resistant structure, making spore decontamination exceptionally challenging. During spore germination, nutrient germinants interact with Ger receptors, triggering a cascade of events. A crucial event in this process is degradation of the cortex peptidoglycan by germination-specific lytic enzymes (GSLEs),...

  15. Continuous melt granulation: Influence of process and formulation parameters upon granule and tablet properties.

    Science.gov (United States)

    Monteyne, Tinne; Vancoillie, Jochem; Remon, Jean-Paul; Vervaet, Chris; De Beer, Thomas

    2016-10-01

    The pharmaceutical industry has a growing interest in alternative manufacturing models allowing automation and continuous production in order to improve process efficiency and reduce costs. Implementing a switch from batch to continuous processing requires fundamental process understanding and the implementation of quality-by-design (QbD) principles. The aim of this study was to examine the relationship between formulation-parameters (type binder, binder concentration, drug-binder miscibility), process-parameters (screw speed, powder feed rate and granulation temperature), granule properties (size, size distribution, shape, friability, true density, flowability) and tablet properties (tensile strength, friability, dissolution rate) of four different drug-binder formulations using Design of experiments (DOE). Two binders (polyethylene glycol (PEG) and Soluplus®) with a different solid state, semi-crystalline vs amorphous respectively, were combined with two model-drugs, metoprolol tartrate (MPT) and caffeine anhydrous (CAF), both having a contrasting miscibility with the binders. This research revealed that the granule properties of miscible drug-binder systems depended on the powder feed rate and barrel filling degree of the granulator whereas the granule properties of immiscible systems were mainly influenced by binder concentration. Using an amorphous binder, the tablet tensile strength depended on the granule size. In contrast, granule friability was more important for tablet quality using a brittle binder. However, this was not the case for caffeine-containing blends, since these phenomena were dominated by the enhanced compression properties of caffeine Form I, which was formed during granulation. Hence, it is important to gain knowledge about formulation behavior during processing since this influences the effect of process parameters onto the granule and tablet properties.

  16. Granule size distributions after twin-screw granulation - Do not forget the feeding systems.

    Science.gov (United States)

    Meier, R; Thommes, M; Rasenack, N; Moll, K-P; Krumme, M; Kleinebudde, P

    2016-09-01

    The aim of this study was to investigate the influence of qualitatively different powder feeder performances on resulting granule size distributions after twin-screw granulation of a highly drug loaded, hydrophobic mixture and a mannitol powder. It was shown that powder feeder related problems usually cannot be identified by trusting in the values given by the feeder. Therefore, a newly developed model for the evaluation of the performance of powder feeders was introduced and it was tried to connect this model to residence time distributions in twin-screw granulation processes. The influence of feeder performances on resulting granule size distributions varied, depending on the applied screw configuration and the used powder. Regarding the hydrophobic and highly drug loaded formulation, which was granulated at an L/S-ratio of 0.5, a pure conveying screw and a medium kneading configuration, consisting of 60° kneading blocks were negatively influenced by poor feeder settings. For optimal settings more narrow distributions could be obtained. For an extensive kneading configuration good and poor settings resulted in mono-modal granule size distributions but were differing in the overall size. Mannitol, a model substance for a liquid sensitive formulation was granulated at an L/S-ratio of 0.075. It was even more important to maintain optimal feeding as mannitol was highly affected by poor feeder performances. Even an extensive kneading configuration could not level the errors in powder feeder performance, resulting in qualitatively different granule size distributions. The results of this study demonstrate the importance of detailed knowledge about applied feeding systems to gain optimal performance in twin-screw granulation.

  17. Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.

    Directory of Open Access Journals (Sweden)

    Xiaonan Dong

    2010-07-01

    Full Text Available Upon viral infection, the mitochondrial antiviral signaling (MAVS-IKKbeta pathway is activated to restrict viral replication. Manipulation of immune signaling events by pathogens has been an outstanding theme of host-pathogen interaction. Here we report that the loss of MAVS or IKKbeta impaired the lytic replication of gamma-herpesvirus 68 (gammaHV68, a model herpesvirus for human Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. gammaHV68 infection activated IKKbeta in a MAVS-dependent manner; however, IKKbeta phosphorylated and promoted the transcriptional activation of the gammaHV68 replication and transcription activator (RTA. Mutational analyses identified IKKbeta phosphorylation sites, through which RTA-mediated transcription was increased by IKKbeta, within the transactivation domain of RTA. Moreover, the lytic replication of recombinant gammaHV68 carrying mutations within the IKKbeta phosphorylation sites was greatly impaired. These findings support the conclusion that gammaHV68 hijacks the antiviral MAVS-IKKbeta pathway to promote viral transcription and lytic infection, representing an example whereby viral replication is coupled to host immune activation.

  18. Diversity of phage infection types and associated terminology: the problem with 'Lytic or lysogenic'.

    Science.gov (United States)

    Hobbs, Zack; Abedon, Stephen T

    2016-04-01

    Bacteriophages, or phages, are viruses of members of domain Bacteria. These viruses play numerous roles in shaping the diversity of microbial communities, with impact differing depending on what infection strategies specific phages employ. From an applied perspective, these especially are communities containing undesired or pathogenic bacteria that can be modified through phage-mediated bacterial biocontrol, that is, through phage therapy. Here we seek to categorize phages in terms of their infection strategies as well as review or suggest more descriptive, accurate or distinguishing terminology. Categories can be differentiated in terms of (1) whether or not virion release occurs (productive infections versus lysogeny, pseudolysogeny and/or the phage carrier state), (2) the means of virion release (lytic versus chronic release) and (3) the degree to which phages are genetically equipped to display lysogenic cycles (temperate versus non-temperate phages). We address in particular the use or overuse of what can be a somewhat equivocal phrase, 'Lytic or lysogenic', especially when employed as a means of distinguishing among phages types. We suggest that the implied dichotomy is inconsistent with both modern as well as historical understanding of phage biology. We consider, therefore, less ambiguous terminology for distinguishing between 'Lytic' versus 'Lysogenic' phage types.

  19. KSHV Targeted Therapy: An Update on Inhibitors of Viral Lytic Replication

    Directory of Open Access Journals (Sweden)

    Natacha Coen

    2014-11-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. Since the discovery of KSHV 20 years ago, there is still no standard treatment and the management of virus-associated malignancies remains toxic and incompletely efficacious. As the majority of tumor cells are latently infected with KSHV, currently marketed antivirals that target the virus lytic cycle have shown inconsistent results in clinic. Nevertheless, lytic replication plays a major role in disease progression and virus dissemination. Case reports and retrospective studies have pointed out the benefit of antiviral therapy in the treatment and prevention of KSHV-associated diseases. As a consequence, potent and selective antivirals are needed. This review focuses on the anti-KSHV activity, mode of action and current status of antiviral drugs targeting KSHV lytic cycle. Among these drugs, different subclasses of viral DNA polymerase inhibitors and compounds that do not target the viral DNA polymerase are being discussed. We also cover molecules that target cellular kinases, as well as the potential of new drug targets and animal models for antiviral testing.

  20. Host transcript accumulation during lytic KSHV infection reveals several classes of host responses.

    Directory of Open Access Journals (Sweden)

    Sanjay Chandriani

    Full Text Available Lytic infection by Kaposi's sarcoma-associated herpesvirus (KSHV is associated with an extensive shutoff of host gene expression, mediated chiefly by accelerated mRNA turnover due to expression of the viral SOX protein. We have previously identified a small number of host mRNAs that can escape SOX-mediated degradation. Here we present a detailed, transcriptome-wide analysis of host shutoff, with careful microarray normalization to allow rigorous determination of the magnitude and extent of transcript loss. We find that the extent of transcript reduction represents a continuum of susceptibilities of transcripts to virus-mediated shutoff. Our results affirm that the levels of over 75% of host transcripts are substantially reduced during lytic infection, but also show that another approximately 20% of cellular mRNAs declines only slightly (less than 2-fold during the course of infection. Approximately 2% of examined cellular genes are strongly upregulated during lytic infection, most likely due to transcriptional induction of mRNAs that display intrinsic SOX-resistance.

  1. Isolation and characterization of lytic phages TSE1-3 against Enterobacter cloacae

    Directory of Open Access Journals (Sweden)

    Khawaja Komal Ameer

    2016-01-01

    Full Text Available The emergence of antibiotic resistant bacterial pathogens is becoming a major challenge for patient care. The utilization of alternative therapies for infectious diseases other than antibiotics is an urgent need of today medical practice. The utilization of lytic bacteriophages and their gene products as therapeutic agents against antibiotic resistant bacteria is one of the convincing alternative approaches. Here we present the isolation and characterization of three lytic bacteriophages TSE1-3 against Enterobacter cloacae from sewage effluent. The isolates maintained antibacterial activity for 10 hours of incubation followed by the development of phage resistance. Their stability at different temperatures and pH, established their possible application in phage therapy. The highest activity of the phages was observed at 37°C and pH 7.0, while they gave lytic activity up to 60°C. The latent period of all the TSE phages was 20 minutes, while the burst size was 360 for TSE1, 270 for TSE2 and 311 for TSE3. The phages were harboring double-stranded DNA larger than 12kb in size. Further research into the phages genome and proteins, animal experiments, delivery parameters and clinical trials may lead to their utilization in phage therapy.

  2. Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances.

    Science.gov (United States)

    Södergren, Anna L; Svensson Holm, Ann-Charlotte B; Ramström, Sofia; Lindström, Eva G; Grenegård, Magnus; Öllinger, Karin

    2016-01-01

    Exocytosis of lysosomal contents from platelets has been speculated to participate in clearance of thrombi and vessel wall remodelling. The mechanisms that regulate lysosomal exocytosis in platelets are, however, still unclear. The aim of this study was to identify the pathways underlying platelet lysosomal secretion and elucidate how this process is controlled by platelet inhibitors. We found that high concentrations of thrombin induced partial lysosomal exocytosis as assessed by analysis of the activity of released N-acetyl-β-glucosaminidase (NAG) and by identifying the fraction of platelets exposing the lysosomal-associated membrane protein (LAMP)-1 on the cell surface by flow cytometry. Stimulation of thrombin receptors PAR1 or PAR4 with specific peptides was equally effective in inducing LAMP-1 surface expression. Notably, lysosomal exocytosis in response to thrombin was significantly reduced if the secondary activation by ADP was inhibited by the P2Y12 antagonist cangrelor, while inhibition of thromboxane A2 formation by treatment with acetylsalicylic acid was of minor importance in this regard. Moreover, the NO-releasing drug S-nitroso-N-acetyl penicillamine (SNAP) or the cyclic AMP-elevating eicosanoid prostaglandin I2 (PGI2) significantly suppressed lysosomal exocytosis. We conclude that platelet inhibitors that mimic functional endothelium such as PGI2 or NO efficiently counteract lysosomal exocytosis. Furthermore, we suggest that secondary release of ADP and concomitant signaling via PAR1/4- and P2Y12 receptors is important for efficient platelet lysosomal exocytosis by thrombin.

  3. Steroid hormones block amyloid fibril-induced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formazan exocytosis: relationship to neurotoxicity.

    Science.gov (United States)

    Liu, Y; Schubert, D

    1998-12-01

    Perhaps the most reproducible early event induced by the interaction of amyloid beta peptide (A beta) with the cell is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. We recently demonstrated that cytotoxic amyloid peptides such as A beta and human amylin inhibit cellular MTT reduction by dramatically enhancing MTT formazan exocytosis. We now show the following: (a) Insulin and glucagon, when converted to fibrils with beta-pleated sheet structure, induce MTT formazan exocytosis that is indistinguishable from that induced by A beta. NAC35, an amyloidogenic fragment of alpha-synuclein (or NACP), also induces MTT formazan exocytosis. (b) All protein fibrils with the beta-pleated sheet structure examined are toxic to rat hippocampal neurons. (c) Many sterol sex hormones (e.g., estradiol and progesterone) block amyloid fibril-enhanced MTT formazan exocytosis as well as MTT formazan exocytosis in control cells by acting at a common late step in the exocytic pathway. Steroids fail, however, to protect hippocampal neurons from acute amyloid fibril toxicity. These findings suggest that the ability to enhance MTT formazan exocytosis and to induce neurotoxicity are common biological activities of protein fibrils with beta-pleated sheet structure but that enhanced MTT formazan exocytosis is not sufficient for acute A beta neurotoxicity.

  4. Rock mechanics modeling based on soft granulation theory

    CERN Document Server

    Owladeghaffari, H

    2008-01-01

    This paper describes application of information granulation theory, on the design of rock engineering flowcharts. Firstly, an overall flowchart, based on information granulation theory has been highlighted. Information granulation theory, in crisp (non-fuzzy) or fuzzy format, can take into account engineering experiences (especially in fuzzy shape-incomplete information or superfluous), or engineering judgments, in each step of designing procedure, while the suitable instruments modeling are employed. In this manner and to extension of soft modeling instruments, using three combinations of Self Organizing Map (SOM), Neuro-Fuzzy Inference System (NFIS), and Rough Set Theory (RST) crisp and fuzzy granules, from monitored data sets are obtained. The main underlined core of our algorithms are balancing of crisp(rough or non-fuzzy) granules and sub fuzzy granules, within non fuzzy information (initial granulation) upon the open-close iterations. Using different criteria on balancing best granules (information pock...

  5. Ca²⁺ signals promote GLUT4 exocytosis and reduce its endocytosis in muscle cells.

    Science.gov (United States)

    Li, Q; Zhu, X; Ishikura, S; Zhang, D; Gao, J; Sun, Y; Contreras-Ferrat, A; Foley, K P; Lavandero, S; Yao, Z; Bilan, P J; Klip, A; Niu, W

    2014-07-15

    Elevating cytosolic Ca(2+) stimulates glucose uptake in skeletal muscle, but how Ca(2+) affects intracellular traffic of GLUT4 is unknown. In tissue, changes in Ca(2+) leading to contraction preclude analysis of the impact of individual, Ca(2+)-derived signals. In L6 muscle cells stably expressing GLUT4myc, the Ca(2+) ionophore ionomycin raised cytosolic Ca(2+) and caused a gain in cell surface GLUT4myc. Extra- and intracellular Ca(2+) chelators (EGTA, BAPTA-AM) reversed this response. Ionomycin activated calcium calmodulin kinase II (CaMKII), AMPK, and PKCs, but not Akt. Silencing CaMKIIδ or AMPKα1/α2 partly reduced the ionomycin-induced gain in surface GLUT4myc, as did peptidic or small molecule inhibitors of CaMKII (CN21) and AMPK (Compound C). Compared with the conventional isoenzyme PKC inhibitor Gö6976, the conventional plus novel PKC inhibitor Gö6983 lowered the ionomycin-induced gain in cell surface GLUT4myc. Ionomycin stimulated GLUT4myc exocytosis and inhibited its endocytosis in live cells. siRNA-mediated knockdown of CaMKIIδ or AMPKα1/α2 partly reversed ionomycin-induced GLUT4myc exocytosis but did not prevent its reduced endocytosis. Compared with Gö6976, Gö6983 markedly reversed the slowing of GLUT4myc endocytosis triggered by ionomycin. In summary, rapid Ca(2+) influx into muscle cells accelerates GLUT4myc exocytosis while slowing GLUT4myc endocytosis. CaMKIIδ and AMPK stimulate GLUT4myc exocytosis, whereas novel PKCs reduce endocytosis. These results identify how Ca(2+)-activated signals selectively regulate GLUT4 exocytosis and endocytosis in muscle cells.

  6. `Full fusion' is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells

    Science.gov (United States)

    Oleinick, Alexander; Svir, Irina; Amatore, Christian

    2017-01-01

    Vesicular exocytosis is an essential and ubiquitous process in neurons and endocrine cells by which neurotransmitters are released in synaptic clefts or extracellular fluids. It involves the fusion of a vesicle loaded with chemical messengers with the cell membrane through a nanometric fusion pore. In endocrine cells, unless it closes after some flickering (`Kiss-and-Run' events), this initial pore is supposed to expand exponentially, leading to a full integration of the vesicle membrane into the cell membrane-a stage called `full fusion'. We report here a compact analytical formulation that allows precise measurements of the fusion pore expansion extent and rate to be extracted from individual amperometric spike time courses. These data definitively establish that, during release of catecholamines, fusion pores enlarge at most to approximately one-fifth of the radius of their parent vesicle, hence ruling out the ineluctability of `full fusion'.

  7. Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission.

    Science.gov (United States)

    Alabi, AbdulRasheed A; Tsien, Richard W

    2013-01-01

    Regulated exocytosis and endocytosis are critical to the function of many intercellular networks, particularly the complex neural circuits underlying mammalian behavior. Kiss-and-run (KR) is an unconventional fusion between secretory vesicles and a target membrane that releases intravesicular content through a transient, nanometer-sized fusion pore. The fusing vesicle retains its gross shape, precluding full integration into the planar membrane, and enough molecular components for rapid retrieval, reacidification, and reuse. KR makes judicious use of finite presynaptic resources, and mounting evidence suggests that it influences synaptic information transfer. Here we detail emerging perspectives on KR and its role in neurotransmission. We additionally formulate a restraining force hypothesis as a plausible mechanistic basis for KR and its physiological modulation in small nerve terminals. Clarification of the mechanism and function of KR has bearing on understanding the kinetic transitions underlying SNARE-mediated fusion, interactions between vesicles and their local environment, and the influence of release dynamics on neural information processing.

  8. 'Full fusion' is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells.

    Science.gov (United States)

    Oleinick, Alexander; Svir, Irina; Amatore, Christian

    2017-01-01

    Vesicular exocytosis is an essential and ubiquitous process in neurons and endocrine cells by which neurotransmitters are released in synaptic clefts or extracellular fluids. It involves the fusion of a vesicle loaded with chemical messengers with the cell membrane through a nanometric fusion pore. In endocrine cells, unless it closes after some flickering ('Kiss-and-Run' events), this initial pore is supposed to expand exponentially, leading to a full integration of the vesicle membrane into the cell membrane-a stage called 'full fusion'. We report here a compact analytical formulation that allows precise measurements of the fusion pore expansion extent and rate to be extracted from individual amperometric spike time courses. These data definitively establish that, during release of catecholamines, fusion pores enlarge at most to approximately one-fifth of the radius of their parent vesicle, hence ruling out the ineluctability of 'full fusion'.

  9. Granulate of stainless steel as compensator material

    NARCIS (Netherlands)

    J.P.C. van Santvoort (J. P C)

    1995-01-01

    textabstractCompensators produced with computer controlled milling devices usually consist of a styrofoam mould, filled with an appropriate material. We investigated granulate of stainless steel as filling material. This cheap, easy to use, clean and re-usable material can be obtained with an averag

  10. Structural studies on methylated starch granules

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Burgt, Y.E.M. van der; Bergsma, J.; Bleeker, I.P.; Mijland, P.J.H.C.; Kamerling, J.P.

    2000-01-01

    Granules of potato starch and amylopectin potato starch were methylated in aqueous alkaline suspension. Here, an overview is given of all structural aspects that have been studied with respect to the determination of the location of the substituents. Methods, which are generally applicable, were

  11. Starch Granule Variability in Wild Solanum Species

    Science.gov (United States)

    Because most of the dry matter of potato tubers is starch, an understanding of starch properties is important in potato improvement programs. Starch granule size is considered to influence tuber processing quality parameters such as gelatinization temperature, viscosity, and water holding capacity. ...

  12. Neutrophil granules in health and disease

    DEFF Research Database (Denmark)

    Häger, M; Cowland, J B; Borregaard, N

    2010-01-01

    Neutrophil granules store proteins that are critically important for the neutrophil to move from the vascular bed to tissues and to kill microorganisms. This is illustrated in nature when individual proteins are deleted due to inherited mutations of their cognate genes, and such deficiencies resu...

  13. Creep of granulated loose-fill insulation

    DEFF Research Database (Denmark)

    Rasmussen, Torben Valdbjørn

    with SP-Building Physics in Sweden and VTT Building Technology in Finland. For the round robin test a cellulosic fibre insulation material was used. The proposed standardised method for creep tests and theories are limited to cases when the granulated loose-fill material is exposed to a constant...

  14. High-shear granulation as a manufacturing method for cocrystal granules

    DEFF Research Database (Denmark)

    Rehder, Sönke; Christensen, Niels Peter Aae; Rantanen, Jukka

    2013-01-01

    influenced by the excipients, since in presence of calcium hydrogenphosphate, the poorly water-soluble salt calcium tartrate monohydrate was formed at high relative humidity. Interestingly, compactability was increased by cocrystal formation compared to that of the reference granules (piracetam...

  15. Vesicular uptake and exocytosis of l-aspartate is independent of sialin

    Science.gov (United States)

    Morland, Cecilie; Nordengen, Kaja; Larsson, Max; Prolo, Laura M.; Farzampour, Zoya; Reimer, Richard J.; Gundersen, Vidar

    2013-01-01

    The mechanism of release and the role of l-aspartate as a central neurotransmitter are controversial. A vesicular release mechanism for l-aspartate has been difficult to prove, as no vesicular l-aspartate transporter was identified until it was found that sialin could transport l-aspartate and l-glutamate when reconstituted into liposomes. We sought to clarify the release mechanism of l-aspartate and the role of sialin in this process by combining l-aspartate uptake studies in isolated synaptic vesicles with immunocyotchemical investigations of hippocampal slices. We found that radiolabeled l-aspartate was taken up into synaptic vesicles. The vesicular l-aspartate uptake, relative to the l-glutamate uptake, was twice as high in the hippocampus as in the whole brain, the striatum, and the entorhinal and frontal cortices and was not inhibited by l-glutamate. We further show that sialin is not essential for exocytosis of l-aspartate, as there was no difference in ATP-dependent l-aspartate uptake in synaptic vesicles from sialin-knockout and wild-type mice. In addition, expression of sialin in PC12 cells did not result in significant vesicle uptake of l-aspartate, and depolarization-induced depletion of l-aspartate from hippocampal nerve terminals was similar in hippocampal slices from sialin-knockout and wild-type mice. Further, there was no evidence for nonvesicular release of l-aspartate via volume-regulated anion channels or plasma membrane excitatory amino acid transporters. This suggests that l-aspartate is exocytotically released from nerve terminals after vesicular accumulation by a transporter other than sialin.—Morland, C., Nordengen, K., Larsson, M., Prolo, L. M., Farzampour, Z., Reimer, R. J., Gundersen, V. Vesicular uptake and exocytosis of l-aspartate is independent of sialin. PMID:23221336

  16. Chronic use of pravastatin reduces insulin exocytosis and increases β-cell death in hypercholesterolemic mice.

    Science.gov (United States)

    Lorza-Gil, Estela; Salerno, Alessandro G; Wanschel, Amarylis C B A; Vettorazzi, Jean F; Ferreira, Mônica S; Rentz, Thiago; Catharino, Rodrigo R; Oliveira, Helena C F

    2016-02-17

    We have previously demonstrated that hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice secrete less insulin than wild-type mice. Removing cholesterol from isolated islets using methyl-beta-cyclodextrin reversed this defect. In this study, we hypothesized that in vivo treatment of LDLr(-/-) mice with the HMGCoA reductase inhibitor pravastatin would improve glucose-stimulated insulin secretion. Female LDLr(-/-) mice were treated with pravastatin (400mg/L) for 1-3 months. Isolated pancreatic islets were assayed for insulin secretion rates, intracellular calcium oscillations, cholesterol levels, NAD(P)H and SNARE protein levels, apoptosis indicators and lipidomic profile. Two months pravastatin treatment reduced cholesterol levels in plasma, liver and islets by 35%, 25% and 50%, respectively. Contrary to our hypothesis, pravastatin treatment increased fasting and fed plasma levels of glucose and decreased markedly (40%) fed plasma levels of insulin. In addition, ex vivo glucose stimulated insulin secretion was significantly reduced after two and three months (36-48%, pinsulin secretion and insulinemia, two months pravastatin treatment did not affect glucose tolerance because it improved global insulin sensitivity. Pravastatin induced islet dysfunction was associated with marked reductions of exocytosis-related SNARE proteins (SNAP25, Syntaxin 1A, VAMP2) and increased apoptosis markers (Bax/Bcl2 protein ratio, cleaved caspase-3 and lower NAD(P)H production rates) observed in pancreatic islets from treated mice. In addition, several oxidized phospholipids, tri- and diacylglycerols and the proapoptotic lipid molecule ceramide were identified as markers of pravastatin-treated islets. Cell death and oxidative stress (H2O2 production) were confirmed in insulin secreting INS-1E cells treated with pravastatin. These results indicate that chronic treatment with pravastatin impairs the insulin exocytosis machinery and increases β-cell death. These findings suggest

  17. Apatite accumulation enhances the mechanical property of anammox granules

    NARCIS (Netherlands)

    Lin, Y. M.; Lotti, T.; Sharma, P. K.; van Loosdrecht, M. C. M.

    2013-01-01

    The strength of granular sludge is essential for the mechanical stability of the granules. Inorganic precipitants form a major factor influencing the strength of the granules. To check the possibility of apatite accumulation in anammox granules, and study its contribution to the mechanical strength

  18. Experimental investigation of granule size and shape dynamics in twin-screw granulation.

    Science.gov (United States)

    Kumar, Ashish; Vercruysse, Jurgen; Bellandi, Giacomo; Gernaey, Krist V; Vervaet, Chris; Remon, Jean Paul; De Beer, Thomas; Nopens, Ingmar

    2014-11-20

    A twin-screw granulator (TSG), a promising equipment for continuous high shear wet granulation (HSWG), achieves the desired level of mixing by a combination of the appropriate screw configuration and a suitable set of process settings (e.g. feed rate, screw speed, etc.), thus producing a certain granule size and shape distribution (GSSD). However, the primary sizing and shaping mechanism behind the resulting distribution is not well understood due to the opacity of the multiphase system in the granulator. This study experimentally characterised the GSSD dynamics along the TSG barrel length in order to understand the function of individual screw modules and process settings, as well as their interaction. Particle size analysis of granules collected at the outlet of the TSG suggested significant interaction between the process and screw configuration parameters influencing the heterogeneity in the GSSD. By characterising the samples collected along the screw length, a variable influence of the screw modules at different process conditions was observed. At low liquid-to-solid ratio (L/S), the first kneading module seemed to play a significant role in mixing, whereas the second kneading module was found to be more involved in reshaping the granules. At high L/S and high throughput, aggregation mainly took place in the second kneading module changing the GSSD. The results obtained from this study will be further used for the calibration and validation of a mechanistic model and, hence, support future development of a more detailed understanding of the HSWG process in a TSG.

  19. Microbial starch binding domains are superior to granule bound starch synthase 1 for anchoring luciferase to potato starch granules

    NARCIS (Netherlands)

    Ji, Q.; Vincken, J.P.; Suurs, L.C.J.M.; Visser, R.G.F.

    2006-01-01

    Microbial starch-binding domains (SBD) and granule-bound starch synthase I (GBSSI) are proteins which are accumulated in potato starch granules. The efficiency of SBD and GBSSI for targeting active luciferase reporter proteins to granules during starch biosynthesis was compared. GBSSI or SBD sequenc

  20. Form, formation, and deformation : the influence of material properties and process conditions on the shape of granules produced by high shear granulation

    NARCIS (Netherlands)

    Bouwman, Anneke Margriet

    2005-01-01

    High shear granulation The introduction of this thesis describes the high shear granulation process. High shear granulation is a commonly used unit operation to produce larger granules of primary particles. The granulation process has been described to consist of different stages. The initial growth

  1. Protein Methylation and Stress Granules: Posttranslational Remodeler or Innocent Bystander?

    Directory of Open Access Journals (Sweden)

    Wen Xie

    2011-01-01

    Full Text Available Stress granules contain a large number of post-translationally modified proteins, and studies have shown that these modifications serve as recruitment tags for specific proteins and even control the assembly and disassembly of the granules themselves. Work originating from our laboratory has focused on the role protein methylation plays in stress granule composition and function. We have demonstrated that both asymmetrically and symmetrically dimethylated proteins are core constituents of stress granules, and we have endeavored to understand when and how this occurs. Here we seek to integrate this data into a framework consisting of the currently known post-translational modifications affecting stress granules to produce a model of stress granule dynamics that, in turn, may serve as a benchmark for understanding and predicting how post-translational modifications regulate other granule types.

  2. Enhanced aerobic nitrifying granulation by static magnetic field.

    Science.gov (United States)

    Wang, Xin-Hua; Diao, Mu-He; Yang, Ying; Shi, Yi-Jing; Gao, Ming-Ming; Wang, Shu-Guang

    2012-04-01

    One of the main challenging issues for aerobic nitrifying granules in treating high strength ammonia wastewater is the long granulation time required for activated sludge to transform into aerobic granules. The present study provides a novel strategy for enhancing aerobic nitrifying granulation by applying an intensity of 48.0mT static magnetic field. The element analysis showed that the applied magnetic field could promote the accumulation of iron compounds in the sludge. And then the aggregation of iron decreased the full granulation time from 41 to 25days by enhancing the setting properties of granules and stimulating the secretion of extracellular polymeric substances (EPS). Long-term, cycle experiments and fluorescence in-situ hybridization (FISH) analysis proved that an intensity of 48.0mT magnetic field could enhance the activities and growth of nitrite-oxidizing bacteria (NOB). These findings suggest that magnetic field is helpful and reliable for accelerating the aerobic nitrifying granulation.

  3. Noncanonical microRNAs and endogenous siRNAs in lytic infection of murine gammaherpesvirus.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available MicroRNA (miRNA and endogenous small interfering RNA (endo-siRNA are two essential classes of small noncoding RNAs (sncRNAs in eukaryotes. The class of miRNA is diverse and there exist noncanonical miRNAs that bypass the canonical miRNA biogenesis pathway. In order to identify noncanonical miRNAs and endo-siRNAs responding to virus infection and study their potential function, we sequenced small-RNA species from cells lytically infected with murine gammaherpesvirus 68 (MHV68. In addition to three novel canonical miRNAs in mouse, two antisense miRNAs in virus and 25 novel noncanonical miRNAs, including miRNAs derived from transfer RNAs, small nucleolar RNAs and introns, in the host were identified. These noncanonical miRNAs exhibited features distinct from that of canonical miRNAs in lengths of hairpins, base pairings and first nucleotide preference. Many of the novel miRNAs are conserved in mammals. Besides several known murine endo-siRNAs detected by the sequencing profiling, a novel locus in the mouse genome was identified to produce endo-siRNAs. This novel endo-siRNA locus is comprised of two tandem inverted B4 short interspersed nuclear elements (SINEs. Unexpectedly, the SINE-derived endo-siRNAs were found in a variety of sequencing data and virus-infected cells. Moreover, a murine miRNA was up-regulated more than 35 fold in infected than in mock-treated cells. The putative targets of the viral and the up-regulated murine miRNAs were potentially involved in processes of gene transcription and protein phosphorylation, and localized to membranes, suggesting their potential role in manipulating the host basal immune system during lytic infection. Our results extended the number of noncanonical miRNAs in mammals and shed new light on their potential functions of lytic infection of MHV68.

  4. Induction of Epstein-Barr Virus Lytic Replication by Recombinant Adenoviruses Expressing the Zebra Gene with EBV Specific Promoters

    Institute of Scientific and Technical Information of China (English)

    Lu CHEN; Juan YIN; Yi CHEN; Jiang ZHONG

    2005-01-01

    The latent Epstein-Barr virus (EBV) is found in the cells of many tumors. For example, EBV is detectable in almost all cases, and in almost all tumor cells, of non-keratinizing nasopharyngeal carcinoma.Activating the latent virus, which will result in its lytic replication and the death of tumor cells, is a potential approach for the treatment of EBV-associated cancers. In this study, three recombinant adenoviruses were constructed to express the Zebra gene, an EBV gene responsible for switching from the latent state to lytic replication. EBV-specific promoters were used in order to limit Zebra expression in EBV-positive cells, and reduce the potential side effects. The EBV promoters used were Cp, Zp and a dual promoter combining both promoters, CpZp. The Zebra protein was detected in HEK293 cells as well as the EBV-positive D98-HR1 cells infected with recombinant viruses. An EBV lytic replication early antigen, EA-D, was also detected in infected D98-HR1, implying the initiation of lytic replication. In the cell viability assay, Zebra-expressing adenoviruses had little effect on EBV-negative HeLa cells, while significantly reducing the cell viability and proliferation of D98-HR1 cells. The results indicate that EBV virus promoters can be used in adenovirus vectors to express the Zebra gene and induce EBV lytic replication in D98-HR1 cells.

  5. Hemoglobin expression in rat experimental granulation tissue

    Institute of Scientific and Technical Information of China (English)

    Miretta Tommila; Christoffer Stark; Anne Jokilammi; Ville Peltonen; Risto Penttinen; Erika Ekholm

    2011-01-01

    The general opinion that hemoglobin is only a carrier protein for oxygen and carbon dioxide has been challenged by several recent studies showing hemoglobin expression in other cells than those of the erythroid series, for example, in macrophages. We discovered β-globin expression in rat experimental granulation tissue induced by subcutaneously implanted cellulose sponges. Closer investigation revealed also α-globin expression. The first peak of the biphasic globin expression noticed during granulation tissue formation correlated with the invasion of monocytes/macrophages, whereas the second one seemed to be connected to the appearance of hematopoietic progenitors. Data presented in this study indicate globin expression both in macrophages and in immature erythroid cells as validated by erythroid-specific markers.

  6. The morphological details of globular keratohyalin granules.

    Science.gov (United States)

    Westerhof, W; Dingemans, K P

    1986-10-01

    In quickly dividing epithelia such as that of the tongue, keratohyalin formation takes place in globular keratohyalin granules (KHG). This is in contrast with the irregular KHG as seen in normal, slowly dividing epidermis. The morphogenesis of the globular KHG is explained in this study. In small KHG, dense aggregates of ribosomes can be seen at the site of blebs. It is suggested that these blebs framed with ribosomes are internalized giving rise to "dense homogeneous deposits" or "single granules". Lipid droplets occur in the upper spinous and horny layer. Globular KHG also contain variable amounts of lipids, and the lipid content seems to be inversely related to the protein content, dependent on the degree of cell differentiation or on the rate of cell turnover. It is suggested that in epithelia with a high cell turnover few rigid keratohyalin components are dispersed in lipids, which maintain a globular shape due to the surface tension.

  7. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

    Science.gov (United States)

    Lo Leggio, Leila; Simmons, Thomas J; Poulsen, Jens-Christian N; Frandsen, Kristian E H; Hemsworth, Glyn R; Stringer, Mary A; von Freiesleben, Pernille; Tovborg, Morten; Johansen, Katja S; De Maria, Leonardo; Harris, Paul V; Soong, Chee-Leong; Dupree, Paul; Tryfona, Theodora; Lenfant, Nicolas; Henrissat, Bernard; Davies, Gideon J; Walton, Paul H

    2015-01-22

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.

  8. Regulation of latency to lytic life cycle:multiple tricks by KSHV RTA

    Institute of Scientific and Technical Information of China (English)

    Jiemin Wong

    2010-01-01

    @@ Higher Education Press and Springer-Verlag Berlin Heidelberg 2010The herpesviruses are large enveloped DNA viruses that infect a wide spectrum hosts including human being. A key characteristic of all herpesviruses is their ability to establish life-time latency within the infected host and to periodically reactivate and enter the iytic replication to produce infectious virus progeny. During latency the 120-300 kb double-stranded DNA genomes of these viruses are maintained as multiple copies of circular episomes within the nuclei of the host cells. Lytic replication is marked by an increase in viral gene expression and the production of infectious virus progeny.

  9. Percutaneous aspiration biopsy in cervical spine lytic lesions. Indications and technique

    Energy Technology Data Exchange (ETDEWEB)

    Tampieri, D.; Weill, A.; Melanson, D.; Ethier, R. (Montreal Neurological Inst. and Hospital, PQ (Canada). Dept. of Neuroradiology)

    1991-02-01

    We describe the technique and the results of the percutaneous aspiration biopsy (PAB) in a series of 9 patients presenting with neck pain and different degrees of myelopathy, in whom the cervical spine X-ray demonstrated lytic lesions of unknown origin. PAB is a useful, relatively safe technique, and leads to histological diagnosis between metastatic and inflammatory processes. Furthermore, in inflammatory lesions with negative hemoculture, PAB may help in detecting the micro-organism responsible and therefore allow a better antibiotic treatment. (orig.).

  10. Reduced insulin exocytosis in human pancreatic β-cells with gene variants linked to type 2 diabetes

    DEFF Research Database (Denmark)

    Rosengren, Anders H; Braun, Matthias; Mahdi, Taman;

    2012-01-01

    The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features...... in relation to genetic risk profiles in diabetic and nondiabetic donors. Islets from donors with T2D exhibited impaired insulin secretion, which was more pronounced in lean than obese diabetic donors. We assessed the impact of 14 disease susceptibility variants on measures of glucose sensing, exocytosis......, and structure. Variants near TCF7L2 and ADRA2A were associated with reduced glucose-induced insulin secretion, whereas susceptibility variants near ADRA2A, KCNJ11, KCNQ1, and TCF7L2 were associated with reduced depolarization-evoked insulin exocytosis. KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2 variants...

  11. 钙离子与植物胞吐作用%Roles of Calcium Ion in Plant Cell Exocytosis

    Institute of Scientific and Technical Information of China (English)

    宋华东

    2012-01-01

    本文总结了Ca2+在花粉管、胚芽鞘原生质体、糊粉层原生质体、根冠原生质体等植物细胞胞吐过程中的调节作用,Rop GTPases与Ca2+、annexirs与Ca2+在胞吐中的相互协调作用,以及植物细胞中存在的两条分泌途径.%The regulation roles of Ca2+ on exocytosis in pollen tubes, coleoptile protoplasts, aleurone layer protoplasts and root cap protoplasts, and the concerted regulation of Rop GTPases and annexing with Ca respectively in exocytosis were reviewed in this paper. Two secretory pathways present in plant cells were also introduced.

  12. Zymophagy: Selective Autophagy of Secretory Granules

    Directory of Open Access Journals (Sweden)

    Maria I. Vaccaro

    2012-01-01

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

  13. Influence of binder droplet dimension on granulation rate during fluidized bed granulation.

    Science.gov (United States)

    Fujiwara, Maya; Dohi, Masafumi; Otsuka, Tomoko; Yamashita, Kazunari; Sako, Kazuhiro

    2013-01-01

    Here, we statistically identified the critical factor of the granulation rate during the fluidized bed granulation process. Lactose was selected as the excipient and was granulated with several binders, including hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and polyvinylpyrrolidone. The viscosity, density, and surface tension of the binder solution, contact angle, and the work done during adhesion and cohesion between the binder and lactose, mist diameter, Stokes number, and the dimension of the droplet were considered. The Stokes number was defined as the ratio of the inertial force to the viscous-damping force of a particle. We confirmed that droplet diameter after adhesion had the highest correlation coefficient with the granulation rate constant in our investigated parameters. Partial least squares regression revealed two critical principal components of the granulation rate: one relating to the droplet dimension, which is composed of mist diameter and diameter and thickness of the droplet after adhesion of the binder to the lactose surface; and the other relating to wettability, which involves the work done during adhesion and cohesion, surface tension, and the thickness of the droplet after adhesion of the binder to the lactose surface.

  14. Solar Radiation Induces Non-Nuclear Perturbations and a False Start to Regulated Exocytosis in Cryptosporidium parvum

    Science.gov (United States)

    King, Brendon J.; Hoefel, Daniel; Ee Wong, Pao; Monis, Paul T.

    2010-01-01

    Stratospheric ozone depletion, climate warming and acidification of aquatic ecosystems have resulted in elevated levels of solar radiation reaching many aquatic environments with an increased deleterious impact on a wide range of living organisms. While detrimental effects on living organisms are thought to occur primarily through DNA damage, solar UV can also damage cellular proteins, lipids and signalling pathways. Cryptosporidium, a member of the eukaryotic phylum Apicomplexa, contain numerous vesicular secretory organelles and their discharge via regulated exocytosis is essential for the successful establishment of infection. Using flow cytometric techniques we demonstrate that solar UV rapidly induces sporozoite exocytosis resulting in a significant reduction in the ability of sporozoites to attach and invade host cells. We found that solar UV induced sporozoite membrane depolarization, resulting in reduced cellular ATP and increased cytosolic calcium. These changes were accompanied by a reduction in the internal granularity of sporozoites, indicative of apical organelle discharge, which was confirmed by analysis of sporozoites with an exocytosis-sensitive dye. The precise timing of apical organelle discharge in the presence of a compatible host cell is critical for sporozoite attachment and invasion. Our results demonstrate for the first time how solar UV radiation can interfere with exocytosis, a fundamental cellular process in all eukaryotic cells. We contend that not only may the forecast increases in solar radiation in both aquatic and terrestrial environments significantly affect members of the Apicomplexa, solar UV-induced membrane depolarizations resulting in cytosolic calcium perturbation may affect a wider range of eukaryotic organisms through antagonistic effects on a myriad of calcium dependant cellular functions. PMID:20668710

  15. Inhibition by cromoglycate and some flavonoids of nucleoside diphosphate kinase and of exocytosis from permeabilized mast cells.

    Science.gov (United States)

    Martin, M. W.; O'Sullivan, A. J.; Gomperts, B. D.

    1995-01-01

    1. The anti-allergic compound, cromoglycate, is reported to possess affinity for, and to suppress the autophosphorylation of a 72kDa protein having the sequence of nucleoside diphosphate kinase (NDPK). 2. We have tested the ability of cromoglycate, and a panel of ten structurally related flavonoids of plant origin, to inhibit the NDPK reaction and the exocytotic process of permeabilized mast cells. The conditions of permeabilization (use of an isotonic medium based on sodium glutamate) were selected to ensure that NDPK activity would be an essential component in the induction of Ca(2+)-induced exocytosis in which ATP is required for generation of GTP. For comparison, we also measured the inhibition of exocytosis induced by GTP-gamma-S; this proceeds in the absence of ATP and bypasses the need for NDPK activity. 3. We found that cromoglycate does not discriminate between Ca2+ and GTP-gamma-S-induced exocytosis and is a poor inhibitor of NDPK activity. Concentrations in the millimolar range are required for inhibition of all these functions. By comparison, many of the flavonoids are effective at concentrations in the micromolar range. 4. While we were unable to discern any systematic relationships between their ability to inhibit the three functions, two compounds, quercetin and genistein, inhibit Ca(2+)-induced, but not GTP-gamma-S-induced exocytosis. Inhibition of the late stages of the stimulus-response pathway in mast cells by these compounds is therefore likely to be due to inhibition of NDPK and the consequent failure to generate GTP. PMID:7582506

  16. Solar radiation induces non-nuclear perturbations and a false start to regulated exocytosis in Cryptosporidium parvum.

    Science.gov (United States)

    King, Brendon J; Hoefel, Daniel; Wong, Pao Ee; Monis, Paul T

    2010-07-23

    Stratospheric ozone depletion, climate warming and acidification of aquatic ecosystems have resulted in elevated levels of solar radiation reaching many aquatic environments with an increased deleterious impact on a wide range of living organisms. While detrimental effects on living organisms are thought to occur primarily through DNA damage, solar UV can also damage cellular proteins, lipids and signalling pathways. Cryptosporidium, a member of the eukaryotic phylum Apicomplexa, contain numerous vesicular secretory organelles and their discharge via regulated exocytosis is essential for the successful establishment of infection. Using flow cytometric techniques we demonstrate that solar UV rapidly induces sporozoite exocytosis resulting in a significant reduction in the ability of sporozoites to attach and invade host cells. We found that solar UV induced sporozoite membrane depolarization, resulting in reduced cellular ATP and increased cytosolic calcium. These changes were accompanied by a reduction in the internal granularity of sporozoites, indicative of apical organelle discharge, which was confirmed by analysis of sporozoites with an exocytosis-sensitive dye. The precise timing of apical organelle discharge in the presence of a compatible host cell is critical for sporozoite attachment and invasion. Our results demonstrate for the first time how solar UV radiation can interfere with exocytosis, a fundamental cellular process in all eukaryotic cells. We contend that not only may the forecast increases in solar radiation in both aquatic and terrestrial environments significantly affect members of the Apicomplexa, solar UV-induced membrane depolarizations resulting in cytosolic calcium perturbation may affect a wider range of eukaryotic organisms through antagonistic effects on a myriad of calcium dependant cellular functions.

  17. Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways

    Science.gov (United States)

    Buckingham, Erin M.; Jarosinski, Keith W.; Jackson, Wallen; Carpenter, John E.

    2016-01-01

    ABSTRACT Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome. IMPORTANCE VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an

  18. Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

    Science.gov (United States)

    Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E

    2015-01-01

    Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that

  19. Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

    Directory of Open Access Journals (Sweden)

    Daungruthai Jarukanont

    Full Text Available Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We

  20. A novel Netrin-1–sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

    Science.gov (United States)

    Winkle, Cortney C.; McClain, Leslie M.; Valtschanoff, Juli G.; Park, Charles S.; Maglione, Christopher

    2014-01-01

    Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion. PMID:24778312

  1. Interaction between MyRIP and the actin cytoskeleton regulates Weibel-Palade body trafficking and exocytosis.

    Science.gov (United States)

    Conte, Ianina L; Hellen, Nicola; Bierings, Ruben; Mashanov, Gregory I; Manneville, Jean-Baptiste; Kiskin, Nikolai I; Hannah, Matthew J; Molloy, Justin E; Carter, Tom

    2016-02-01

    Weibel-Palade body (WPB)-actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A-MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP-actin interactions can also occur. To evaluate the specific contribution of MyRIP-actin and MyRIP-MyoVa binding in WPB trafficking and Ca(2+)-driven exocytosis, we used EGFP-MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca(2+)-driven exocytosis requires both MyoVa- and actin-binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments, we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa.

  2. Identification of Novel Small Organic Compounds with Diverse Structures for the Induction of Epstein-Barr Virus (EBV) Lytic Cycle in EBV-Positive Epithelial Malignancies.

    Science.gov (United States)

    Choi, Chung King; Ho, Dona N; Hui, Kwai Fung; Kao, Richard Y; Chiang, Alan K S

    2015-01-01

    Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) lytic cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV lytic cycle through alternative cellular pathways may aid in defining the mechanisms leading to lytic cycle reactivation and improve cells' responsiveness towards lytic induction. We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV lytic cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. All five compounds are structurally diverse from each other and distinct from phorbol esters or HDAC inhibitors. They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers. Two of the five compounds with rapid lytic-inducing action were further studied for their mechanisms of induction of EBV lytic cycle. Unlike HDAC inhibitors, lytic induction by both compounds was not inhibited by rottlerin, a specific inhibitor of PKCδ. Interestingly, both compounds could cooperate with HDAC inhibitors to enhance EBV lytic cycle induction in EBV-positive epithelial cancer cells, paving way for the development of strategies to increase cells' responsiveness towards lytic reactivation. One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways. These structurally diverse novel organic compounds may represent potential new classes of chemicals that can be used to investigate any alternative mechanism(s) leading to EBV

  3. Protozoacidal Trojan-Horse: use of a ligand-lytic peptide for selective destruction of symbiotic protozoa within termite guts.

    Science.gov (United States)

    Sethi, Amit; Delatte, Jennifer; Foil, Lane; Husseneder, Claudia

    2014-01-01

    For novel biotechnology-based termite control, we developed a cellulose bait containing freeze-dried genetically engineered yeast which expresses a protozoacidal lytic peptide attached to a protozoa-recognizing ligand. The yeast acts as a 'Trojan-Horse' that kills the cellulose-digesting protozoa in the termite gut, which leads to the death of termites, presumably due to inefficient cellulose digestion. The ligand targets the lytic peptide specifically to protozoa, thereby increasing its protozoacidal efficiency while protecting non-target organisms. After ingestion of the bait, the yeast propagates in the termite's gut and is spread throughout the termite colony via social interactions. This novel paratransgenesis-based strategy could be a good supplement for current termite control using fortified biological control agents in addition to chemical insecticides. Moreover, this ligand-lytic peptide system could be used for drug development to selectively target disease-causing protozoa in humans or other vertebrates.

  4. Genomic sequence and evolution of marine cyanophage P60: a new insight on lytic and lysogenic phages.

    Science.gov (United States)

    Chen, Feng; Lu, Jingrang

    2002-05-01

    The genome of cyanophage P60, a lytic virus which infects marine Synechococcus WH7803, was completely sequenced. The P60 genome contained 47,872 bp with 80 potential open reading frames that were mostly similar to the genes found in lytic phages like T7, phi-YeO3-12, and SIO1. The DNA replication system, consisting of primase-helicase and DNA polymerase, appeared to be more conserved in podoviruses than in siphoviruses and myoviruses, suggesting that DNA replication genes could be the critical elements for lytic phages. Strikingly high sequence similarities in the regions coding for nucleotide metabolism were found between cyanophage P60 and marine unicellular cyanobacteria.

  5. Epstein-Barr virus (EBV Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells.

    Directory of Open Access Journals (Sweden)

    Yen-Ju Chen

    Full Text Available Epstein-Barr virus (EBV Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-β-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV, to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1 an ideal environment for virus reactivation if EBV or KSHV coexists and (2 a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.

  6. Epstein-Barr virus (EBV) Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells.

    Science.gov (United States)

    Chen, Yen-Ju; Tsai, Wan-Hua; Chen, Yu-Lian; Ko, Ying-Chieh; Chou, Sheng-Ping; Chen, Jen-Yang; Lin, Su-Fang

    2011-03-10

    Epstein-Barr virus (EBV) Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox)-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-β-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1) an ideal environment for virus reactivation if EBV or KSHV coexists and (2) a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.

  7. Simian virus 40 late proteins possess lytic properties that render them capable of permeabilizing cellular membranes.

    Science.gov (United States)

    Daniels, Robert; Rusan, Nasser M; Wilbuer, Anne-Kathrin; Norkin, Leonard C; Wadsworth, Patricia; Hebert, Daniel N

    2006-07-01

    Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumulated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correlated with late protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the late proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the late proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.

  8. Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS.

    Science.gov (United States)

    Borges-Pereira, Lucas; Budu, Alexandre; McKnight, Ciara A; Moore, Christina A; Vella, Stephen A; Hortua Triana, Miryam A; Liu, Jing; Garcia, Celia R S; Pace, Douglas A; Moreno, Silvia N J

    2015-11-01

    Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca(2+) oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca(2+) enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca(2+) changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca(2+) oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca(2+) influx. This is the first study showing, in real time, Ca(2+) signals preceding egress and their direct link with motility, an essential virulence trait.

  9. CTCF interacts with the lytic HSV-1 genome to promote viral transcription

    Science.gov (United States)

    Lang, Fengchao; Li, Xin; Vladimirova, Olga; Hu, Benxia; Chen, Guijun; Xiao, Yu; Singh, Vikrant; Lu, Danfeng; Li, Lihong; Han, Hongbo; Wickramasinghe, J. M. A. S. P.; Smith, Sheryl T.; Zheng, Chunfu; Li, Qihan; Lieberman, Paul M.; Fraser, Nigel W.; Zhou, Jumin

    2017-01-01

    CTCF is an essential chromatin regulator implicated in important nuclear processes including in nuclear organization and transcription. Herpes Simplex Virus-1 (HSV-1) is a ubiquitous human pathogen, which enters productive infection in human epithelial and many other cell types. CTCF is known to bind several sites in the HSV-1 genome during latency and reactivation, but its function has not been defined. Here, we report that CTCF interacts extensively with the HSV-1 DNA during lytic infection by ChIP-seq, and its knockdown results in the reduction of viral transcription, viral genome copy number and virus yield. CTCF knockdown led to increased H3K9me3 and H3K27me3, and a reduction of RNA pol II occupancy on viral genes. Importantly, ChIP-seq analysis revealed that there is a higher level of CTD Ser2P modified RNA Pol II near CTCF peaks relative to the Ser5P form in the viral genome. Consistent with this, CTCF knockdown reduced the Ser2P but increased Ser5P modified forms of RNA Pol II on viral genes. These results suggest that CTCF promotes HSV-1 lytic transcription by facilitating the elongation of RNA Pol II and preventing silenced chromatin on the viral genome. PMID:28045091

  10. Viroporin potential of the lentivirus lytic peptide (LLP domains of the HIV-1 gp41 protein

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2007-11-01

    Full Text Available Abstract Background Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development. Virally induced changes in membrane ionic permeability induced by lytic viruses of many families contribute to cytopathogenesis. HIV-1 induces disturbances in plasma membrane ion transport. The carboxyl terminus of TM (gp41 contains potential amphipathic α-helical motifs identified through their structural similarities to naturally occurring cytolytic peptides. These sequences have been dubbed lentiviral lytic peptides (LLP -1, -2, and -3. Results Peptides corresponding to the LLP domains (from a clade B virus partition into lipid membranes, fold into α-helices and disrupt model membrane permeability. A peptide corresponding to the LLP-1 domain of a clade D HIV-1 virus, LLP-1D displayed similar activity to the LLP-1 domain of the clade B virus in all assays, despite a lack of amino acid sequence identity. Conclusion These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin. Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs.

  11. Parosteal osteosarcoma dedifferentiating into telangiectatic osteosarcoma: importance of lytic changes and fluid cavities at imaging

    Energy Technology Data Exchange (ETDEWEB)

    Azura, M. [Istituto Ortopedico Rizzoli, Musculoskeletal Oncological Surgery Department, Bologna (Italy); University of Malaya, Department of Orthopaedic Surgery, Kuala Lumpur (Malaysia); Vanel, D. [Radiology, Istituto Ortopedico Rizzoli, Bologna (Italy); Istituti Rizzoli, Anatomia Patologica, Bologna (Italy); Alberghini, M. [Pathology, Istituto Ortopedico Rizzoli, Bologna (Italy); Picci, P.; Staals, E.; Mercuri, M. [Istituto Ortopedico Rizzoli, Musculoskeletal Oncological Surgery Department, Bologna (Italy)

    2009-07-15

    This study was performed to assess the imaging findings in cases of parosteal osteosarcoma dedifferentiated into telangiectatic osteosarcoma. Parosteal osteosarcoma is a low-grade well-differentiated malignant tumor. Dedifferentiation into a more aggressive lesion is frequent and usually visible on imaging as a central lytic area in a sclerotic mass. Only one case of differentiation into a telangiectatic osteosarcoma has been reported. As it has practical consequences, with a need for aggressive chemotherapy, we looked for this rather typical imaging pattern. Review of 199 cases of surface osteosarcomas (including 86 parosteal, of which 23 were dedifferentiated) revealed lesions suggesting a possible telangiectatic osteosarcoma on imaging examinations in five cases (cavities with fluid). Histology confirmed three cases (the two other only had hematoma inside a dedifferentiated tumor). There were three males, aged 24, 28, and 32. They had radiographs and CT, and two an MR examination. Lesions involved the distal femur, proximal tibia, and proximal humerus. The parosteal osteosarcoma was a sclerotic, regular mass, attached to the cortex. A purely lytic mass, partially composed of fluid cavities was easily detected on CT and MR. It involved the medullary cavity twice, and remained outside the bone once. Histology confirmed the two components in each case. Two patients died of pulmonary metastases and one is alive. Knowledge of this highly suggestive pattern should help guide the initial biopsy to diagnose the two components of the tumor, and guide aggressive treatment. (orig.)

  12. Overexpression of antimicrobial lytic peptides protects grapevine from Pierce's disease under greenhouse but not field conditions.

    Science.gov (United States)

    Li, Zhijian T; Hopkins, Donald L; Gray, Dennis J

    2015-10-01

    Pierce's disease (PD) caused by Xylella fastidiosa prevents cultivation of grapevine (Vitis vinifera) and susceptible hybrids in the southeastern United States and poses a major threat to the grape industry of California and Texas. Genetic resistance is the only proven control of X. fastidiosa. Genetic engineering offers an alternative to heretofore ineffective conventional breeding in order to transfer only PD resistance traits into elite cultivars. A synthetic gene encoding lytic peptide LIMA-A was introduced into V. vinifera and a Vitis hybrid to assess in planta inhibition of X. fastidiosa. Over 1050 independent transgenic plant lines were evaluated in the greenhouse, among which nine lines were selected and tested under naturally-inoculated field conditions. These selected plant lines in the greenhouse remain disease-free for 10 years, to date, even with multiple manual pathogen inoculations. However, all these lines in the field, including a grafted transgenic rootstock, succumbed to PD within 7 years. We conclude that in planta production of antimicrobial lytic peptides does not provide durable PD resistance to grapevine under field conditions.

  13. Preliminary survey of local bacteriophages with lytic activity against multi-drug resistant bacteria.

    Science.gov (United States)

    Latz, Simone; Wahida, Adam; Arif, Assuda; Häfner, Helga; Hoß, Mareike; Ritter, Klaus; Horz, Hans-Peter

    2016-10-01

    Bacteriophages (phages) represent a potential alternative for combating multi-drug resistant bacteria. Because of their narrow host range and the ever emergence of novel pathogen variants the continued search for phages is a prerequisite for optimal treatment of bacterial infections. Here we performed an ad hoc survey in the surroundings of a University hospital for the presence of phages with therapeutic potential. To this end, 16 aquatic samples of different origins and locations were tested simultaneously for the presence of phages with lytic activity against five current, but distinct strains each from the ESKAPE-group (i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae). Phages could be isolated for 70% of strains, covering all bacterial species except S. aureus. Apart from samples from two lakes, freshwater samples were largely devoid of phages. By contrast, one liter of hospital effluent collected at a single time point already contained phages active against two-thirds of tested strains. In conclusion, phages with lytic activity against nosocomial pathogens are unevenly distributed across environments with the prime source being the immediate hospital vicinity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Global mRNA degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency.

    Directory of Open Access Journals (Sweden)

    Justin M Richner

    2011-07-01

    Full Text Available During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68 SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment.

  15. Investigating the lytic activity and structural properties of Staphylococcus aureus phenol soluble modulin (PSM) peptide toxins.

    Science.gov (United States)

    Laabei, Maisem; Jamieson, W David; Yang, Yi; van den Elsen, Jean; Jenkins, A Toby A

    2014-12-01

    The ubiquitous bacterial pathogen, Staphylococcus aureus, expresses a large arsenal of virulence factors essential for pathogenesis. The phenol-soluble modulins (PSMs) are a family of cytolytic peptide toxins which have multiple roles in staphylococcal virulence. To gain an insight into which specific factors are important in PSM-mediated cell membrane disruption, the lytic activity of individual PSM peptides against phospholipid vesicles and T cells was investigated. Vesicles were most susceptible to lysis by the PSMα subclass of peptides (α1-3 in particular), when containing between 10 and 30mol% cholesterol, which for these vesicles is the mixed solid ordered (so)-liquid ordered (lo) phase. Our results show that the PSMβ class of peptides has little effect on vesicles at concentrations comparable to that of the PSMα class and exhibited no cytotoxicity. Furthermore, within the PSMα class, differences emerged with PSMα4 showing decreased vesicle and cytotoxic activity in comparison to its counterparts, in contrast to previous studies. In order to understand this, peptides were studied using helical wheel projections and circular dichroism measurements. The degree of amphipathicity, alpha-helicity and properties such as charge and hydrophobicity were calculated, allowing a structure-function relationship to be inferred. The degree of alpha-helicity of the peptides was the single most important property of the seven peptides studied in predicting their lytic activity. These results help to redefine this class of peptide toxins and also highlight certain membrane parameters required for efficient lysis.

  16. Effect of metals on the lytic cycle of the coccolithovirus, EhV86.

    Directory of Open Access Journals (Sweden)

    Martha eGledhill

    2012-04-01

    Full Text Available In this study we show that metals, and in particular copper (Cu, can disrupt the lytic cycle in the Emiliania huxleyi - EhV86 host-virus system. Numbers of virus particles produced per E. huxleyi cell and E. huxleyi lysis rates were reduced by Cu at total metal concentrations over 500 nM in the presence of EDTA (ethylenediaminetetraacetic acid, and 250 nM in the absence of EDTA in acute short term exposure experiments. Zinc (Zn, cadmium (Cd and cobalt (Co were not observed to affect the lysis rate of EhV86 in these experiments. The cellular glutathione (GSH content increased in virus infected cells, but not as a result of metal exposure. In contrast, the cellular content of phytochelatins (PCs increased only in response to metal exposure. The increase in gluthatione content is consistent with increases in the production of reactive oxygen species (ROS on viral infection, while increases in PC content are likely linked to metal homeostasis and indicate that metal toxicity to the host was not affected by viral infection. We propose that Cu prevents lytic production of EhV86 by interfering with virus DNA (deoxyribonucleic acid synthesis through a transcriptional block, which ultimately suppresses the formation of ROS, a biochemical response required for successful virus infection.

  17. PARTIAL CHARACTERIZATION OF A LYTIC METHICILLIN RESISTANT-STAPHYLOCOCCUS AUREUS BACTERIOPHAGE

    Directory of Open Access Journals (Sweden)

    Sulaiman Al-Yousef

    2014-12-01

    Full Text Available A marked increase in the infection incidence caused by methicillin-resistant Staphylococcus aureus (MRSA strains has been noted in medical practice in recent years. This study was conducted to study the biological and characterize of MRSA-phage. Methicillin resistance of Staphylococcus aureus was detected and confirmed by determining of the MIC of oxacillin by the standard agar dilution method. Phage was biologically purified using single plaque technique, then phage characterization were studied using host range, adsorption time, particle morphology and its structural protein. MRSA phage showing lytic nature was purified by repeated plating after picking of single isolated plaques. This phage is active against all 11 isolates either of S. aureus or MRSA tested as hosts. Phage produced clear plaques indicating their lytic nature. This phage was concentrated employing polyethylene glycol (PEG-NaCl precipitation method. Morphologically, MRSA Phage has a hexagonal head having a long non-contractile tail, indicating his icosahedral nature. Adsorption studies showed 100% adsorption of MRSA-Phage after 35 minutes of exposure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE experimentation indicated that the phage particles contain one major structural protein (about 30 Kda.

  18. Preparation and characterization of polyclonal antibody against Kaposi's sarcoma-associated herpesvirus lytic gene encoding RTA.

    Science.gov (United States)

    Fan, Weifei; Tang, Qiao; Shen, Chenyou; Qin, Di; Lu, Chun; Yan, Qin

    2015-11-01

    Replication and transcription activator (RTA) is a critical lytic protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). To prepare rabbit polyclonal antibody against RTA, three antigenic polypeptides of KSHV RTA were initially synthesized. The fragment of RTA was cloned into p3FlagBsd to construct the recombinant plasmid, pRTA-Flag. 293 T and EA.hy926 cells were transfected with pRTA-Flag to obtain RTA-Flag fusion protein, which was detected using anti-Flag antibody. Next, New Zealand white rabbits were immunized with keyhole limpet hemocyanin-conjugated peptides to generate polyclonal antibodies against RTA. Enzyme-linked immunosorbent assays were performed to characterize the polyclonal antibodies, and the titers of the polyclonal antibodies against RTA were greater than 1:11,000. Western blotting and immunofluorescence assay revealed that the prepared antibody reacted specifically with the RTA-Flag fusion protein as well as the native viral protein in KSHV-infected primary effusion lymphoma cells. Collectively, our work successfully constructed the recombinant expression vector, pRTA-Flag, and prepared the polyclonal antibody against RTA, which was valuable for investigating the biochemical and biological functions of the critical KSHV lytic gene.

  19. WET-GRANULATION RESEARCH WITH APPLICATION TO SCALE-UP

    Institute of Scientific and Technical Information of China (English)

    Gabriel I.Tardos

    2005-01-01

    Granulation is a unit operation by which larger granules are produced from fine, powdery particles to improve appearance, flow properties and mixedness, reduce dustiness and, in general, produce engineered particles with superior attributes. Agglomeration in wet granulation is achieved by introducing a "binder" fluid onto a shearing mass of fine powders. This paper gives a general overview of the process with emphasis on a simplified granulation model based on a dimensionless parameter containing inertia and viscous dissipation energies between colliding particles: the so-called Stokes number. The model incorporates most common features of all granulation devices (mixers) used in the pharmaceutical industry.Also described in the paper is a computer simulation that captures the movement of flowing powder in an ideal mixer-granulator with constant shear rate. A fraction of the total number of particles is wet (covered by binder and therefore "sticky") while the rest of the particles are dry. The numerical simulation depicts two distinct regimes of agglomeration found in a typical granulator: granule growth and subsequent breakup. During granule growth-simulations, final granule size and shape distributions are obtained by analyzing the size and shape of formed granules using a pattern-recognition technique. A second kind of simulation, also using rapid granular flow modeling, follows the rotation and deformation of an "agglomerate" held together by a liquid binder. Results from these simulations yield critical values of the Stokes number. Below the critical value, the agglomerates are stable and only rotate in response to shear while above the critical value they break into two or more pieces. At the critical value, they attain a steady elongated shape. Using values of the critical Stokes number, the model predicts the size of formed granules.The existence of the critical state in which granules attain a characteristic elongated shape is used to measure shear forces

  20. Formation of artificial granules for proving gelation as the main mechanism of aerobic granulation in biological wastewater treatment.

    Science.gov (United States)

    Li, Yun; Yang, Shu-Fang; Zhang, Jian-Jun; Li, Xiao-Yan

    2014-01-01

    In this study, gelation-facilitated biofilm formation as a new mechanism is proposed for the phenomenon of aerobic granulation in biological wastewater treatment. To obtain an experimental proof for the gelation-based theory, the granulation process was simulated in a chemical system using latex particles for bacterial cells and organic polymers (alginate and peptone) for extracellular polymeric substances (EPS) in a solution with the addition of cations (Ca²⁺, Mg²⁺ and Fe³⁺). The results showed that at a low alginate content (70 mg g⁻¹ mixed liquid suspended solids (MLSS)) flocculation was observed in the suspension with loose flocs. At a higher alginate content (180 mg g⁻¹ MLSS), together with discharge of small flocs, formation of artificial gel granules was successfully achieved leading to granulation. The artificial granules show a morphological property similar to that of actual microbial granules. However, if the protein content increased, granulation became difficult with little gel formation. The experimental work demonstrates the importance of the bonding interactions between EPS functional groups and cations in gel formation and granulation. The laboratory results on the formation of artificial granules provide a sound proof for the theory of gelation-facilitated biofilm formation as the main mechanism for aerobic granulation in sludge suspensions.

  1. Bronchogenic adenocarcinoma presenting as a synchronous solitary lytic skull lesion with ischaemic stroke--case report and literature review.

    LENUS (Irish Health Repository)

    O'Connell, David

    2011-01-01

    The authors describe a rare case of metastatic bronchogenic adenocarcinoma in a 55-year-old man presenting with concomittant solitary lytic skull lesion and ischaemic stroke. Metastatic bronchogenic carcinoma is known to present as lytic skull lesions. Primary brain tumours are also known to cause ischaemic brain injury. An underlying stroke risk may be exagerated by cranial tumour surgery. Patients with brain tumours are well known to be predisposed to an increased risk of developing thromboembolic disease. It is unusual to see metastatic bronchogenic adenocarcinoma presenting as ischaemic stroke with a background of concomittant cerebral metastasis. The aetio-pathogenesis of this rare occurrence is discussed with a review of literature.

  2. Visualization and understanding of the granulation liquid mixing and distribution during continuous twin screw granulation using NIR chemical imaging.

    Science.gov (United States)

    Vercruysse, Jurgen; Toiviainen, Maunu; Fonteyne, Margot; Helkimo, Niko; Ketolainen, Jarkko; Juuti, Mikko; Delaet, Urbain; Van Assche, Ivo; Remon, Jean Paul; Vervaet, Chris; De Beer, Thomas

    2014-04-01

    Over the last decade, there has been increased interest in the application of twin screw granulation as a continuous wet granulation technique for pharmaceutical drug formulations. However, the mixing of granulation liquid and powder material during the short residence time inside the screw chamber and the atypical particle size distribution (PSD) of granules produced by twin screw granulation is not yet fully understood. Therefore, this study aims at visualizing the granulation liquid mixing and distribution during continuous twin screw granulation using NIR chemical imaging. In first instance, the residence time of material inside the barrel was investigated as function of screw speed and moisture content followed by the visualization of the granulation liquid distribution as function of different formulation and process parameters (liquid feed rate, liquid addition method, screw configuration, moisture content and barrel filling degree). The link between moisture uniformity and granule size distributions was also studied. For residence time analysis, increased screw speed and lower moisture content resulted to a shorter mean residence time and narrower residence time distribution. Besides, the distribution of granulation liquid was more homogenous at higher moisture content and with more kneading zones on the granulator screws. After optimization of the screw configuration, a two-level full factorial experimental design was performed to evaluate the influence of moisture content, screw speed and powder feed rate on the mixing efficiency of the powder and liquid phase. From these results, it was concluded that only increasing the moisture content significantly improved the granulation liquid distribution. This study demonstrates that NIR chemical imaging is a fast and adequate measurement tool for allowing process visualization and hence for providing better process understanding of a continuous twin screw granulation system.

  3. PKCε ACTIVATION PROMOTES FGF-2 EXOCYTOSIS AND INDUCES ENDOTHELIAL CELL PROLIFERATION AND SPROUTING

    Science.gov (United States)

    Monti, Martina; Donnini, Sandra; Morbidelli, Lucia; Giachetti, Antonio; Mochly-Rosen, Daria; Mignatti, Paolo; Ziche, Marina

    2013-01-01

    Protein kinase C epsilon (PKCε) activation controls fibroblast growth factor-2 (FGF-2) angiogenic signaling. Here, we examined the effect of activating PKCε on FGF-2 dependent vascular growth and endothelial activation. ψεRACK, a selective PKCε agonist induces pro-angiogenic responses in endothelial cells, including formation of capillary like structures and cell growth. These effects are mediated by FGF-2 export to the cell membrane, as documented by biotinylation and immunofluorescence, and FGF-2/FGFR1 signaling activation, as attested by ERK1/2-STAT-3 phosphorylation and de novo FGF-2 synthesis. Similarly, vascular endothelial growth factor (VEGF) activates PKCε in endothelial cells, and promotes FGF-2 export and FGF-2/FGFR1 signaling activation. ψεRACK fails to elicit responses in FGF-2−/− endothelial cells, and in cells pretreated with methylamine (MeNH2), an exocytosis inhibitor, indicating that both intracellular FGF-2 and its export toward the membrane are required for the ψεRACK activity. In vivo ψεRACK does not induce angiogenesis in the rabbit cornea. However, ψεRACK promotes VEGF angiogenic responses, an effect sustained by endothelial FGF-2 release and synthesis, since anti-FGF-2 antibody strongly attenuates VEGF responses. The results demonstrate that PKCε stimulation promotes angiogenesis and modulates VEGF activity, by inducing FGF-2 release and autocrine signaling. PMID:23880610

  4. Kir6.2△C26 Channel Traffics to Plasma Membrane by Constitutive Exocytosis

    Institute of Scientific and Technical Information of China (English)

    Ping ZHAO; Wei LI; Yong-Ming DONG; Dan ZHU; An-Lian QU; Tao XU; Zheng-Xing WU

    2006-01-01

    Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels regulate many cellular functions by coupling the metabolic state of the cell to the changes in membrane potential. Truncation of C-terminal 26 amino acid residues of Kir6.2 protein (Kir6.2△C26) deletes its endoplasmic reticulum retention signal, allowing functional expression of Kir6.2 in the absence of sulfonylurea receptor subunit. pEGFP-Kir6.2△C26 and pKir6.2△C26-IRES2-EGFP expression plasmids were constructed and transfected into HEK293 cells. We identified that Kir6.2△C26 was localized on the plasma membrane and trafficked to the plasmalemma by means of constitutive exocytosis of Kir6.2△C26 transport vesicles, using epi-fluorescence and total internal reflection fluorescence microscopy. Our electrophysiological data showed that Kir6.2△C26 alone expressed KATP currents, whereas EGFP-Kir6.2△C26 fusion protein displayed no KATP channel activity.

  5. HIV-1 intersection with CD4 T cell vesicle exocytosis: intercellular communication goes viral

    Directory of Open Access Journals (Sweden)

    Helena eSoares

    2014-09-01

    Full Text Available In cells of the immune system the secretion of extracellular vesicles is modulated through cellular activation. In particular, T cell activation is achieved through cell-cell contacts with antigen presenting cells and the consequent formation of a specialized signaling junction called the immunological synapse. Recent works on CD4 T cells have elucidated that cognate antigen recognition by the T cell receptor (TCR engages two distinct exocytic events. The first, involves the exocytic targeting of signaling molecules at the synaptic membrane and drives the functional architecture of the immunological synapse. The second, enlists the extracellular secretion of the TCR itself, once the functional architecture of the immunological synapse is accomplished. HIV-1, a human lymphotropic virus, has evolved sophisticated mechanisms to co-opt CD4 T cell physiology. Notably, it has become apparent that HIV-1 intersects the regulated secretory system of CD4 T cells in order to bud from the plasma membrane of the infected cell and to promote bystander cell death. Here, I review the relevance of CD4 vesicle exocytosis to immune regulation and to HIV-1 pathogenesis and discuss their potential therapeutic applications.

  6. BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis.

    Science.gov (United States)

    Béguin, Pascal; Nagashima, Kazuaki; Mahalakshmi, Ramasubbu N; Vigot, Réjan; Matsunaga, Atsuko; Miki, Takafumi; Ng, Mei Yong; Ng, Yu Jin Alvin; Lim, Chiaw Hwee; Tay, Hock Soon; Hwang, Le-Ann; Firsov, Dmitri; Tang, Bor Luen; Inagaki, Nobuya; Mori, Yasuo; Seino, Susumu; Launey, Thomas; Hunziker, Walter

    2014-04-28

    Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca(2+)-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca(2+) overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain-binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca(2+) channel activity at the plasma membrane, resulting in the inhibition of Ca(2+)-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.

  7. Peritoneal fluid modulates the sperm acrosomal exocytosis induced by N-acetylglucosaminyl neoglycoprotein

    Directory of Open Access Journals (Sweden)

    E.P. Passos

    1999-01-01

    Full Text Available The effect of peritoneal fluid (PF on the human sperm acrosome reaction (AR was tested. Sperm was pre-incubated with PF and the AR was induced by calcium ionophore A23187 and a neoglycoprotein bearing N-acetylglycosamine residues (NGP. The AR induced by calcium ionophore was inhibited 40% by PF from controls (PFc and 50% by PF from the endometriosis (PFe group, but not by PF from infertile patients without endometriosis (PFi. No significant differences were found in the spontaneous AR. When the AR was induced by NGP, pre-incubation with PFc reduced (60% the percentage of AR, while PFe and PFi caused no significant differences. The average rates of acrosome reactions obtained in control, NGP- and ionophore-treated sperm showed that NGP-induced exocytosis differed significantly between the PFc (11% and PFe/PFi groups (17%, and the ionophore-induced AR was higher for PFi (33% than PFc/PFe (25%. The incidence of the NGP-induced AR was reduced in the first hour of pre-incubation with PFc and remained nearly constant throughout 4 h of incubation. The present data indicate that PF possesses a protective factor which prevents premature AR.

  8. Bone regeneration in rabbit calvaria with novel monetite granules.

    Science.gov (United States)

    Tamimi, Faleh; Torres, Jesús; Kathan, Carlos; Baca, Rafael; Clemente, Celia; Blanco, Luis; Lopez Cabarcos, Enrique

    2008-12-15

    The aim of this study was to evaluate whether local application of monetite granules would induce bone regeneration in critical size defects on rabbits calvaria. Novel monetite granules were synthesized by thermal conversion of preset brushite cement. Twelve female New Zealand rabbits were used for this study. Two identical 10-mm-diameter bicortical cranial defects were created in each animal. One of the defects was grafted with monetite granules while the contralateral was left unfilled as negative control. Animals were sacrificed at 4 and 8 weeks after surgery, and biopsies were taken for histological and histomorphometrical evaluation under light microscopy. Wilcoxon test was used for statistical analysis. The histological observations showed signs of graft resorption as newly formed bone tissue grew surrounding and penetrating the monetite granules. Histomorphometric evaluation showed that the augmented bone volume as well as the augmented mineral tissue was higher in the defects treated with monetite granules (p monetite granules in bone defects enhances bone healing significantly.

  9. Flow of formulation granules through a conical hopper

    Directory of Open Access Journals (Sweden)

    Mazumder B

    2008-01-01

    Full Text Available Gravity flow characteristics of various pharmaceutical granules through static conical hoppers of different cone angles were studied. Mass flow rate depends on properties of granules and cone angles when environmental conditions such as temperature and relative humidity are kept within a fixed range. The granules were made with active pharmaceutical ingredients as per Indian pharmacopoeia with other additives like binders and diluents. Lubricants were added with the granules to observe their effects on mass flow rate. Magnesium stearate and colloidal silicon dioxide of different proportions were used as lubricants after granulation. A new dimensionally analyzed equation was developed to predict flow rate of the granules. The developed equation agreed well with the experimental data with a percentage deviation of ±10%.

  10. Information granules and entropy theory in information systems

    Institute of Scientific and Technical Information of China (English)

    LIANG JiYe; QIAN YuHua

    2008-01-01

    Information granulation and entropy theory are two main approaches to research uncertainty of an information system,which have been widely applied in many practical issues.In this paper,the characterizations and representations of information granules under various binary relations are investigated in information systems,an axiom definition of information granulation is presented,and some existing definitions of information granulation become its special forms.Entropy theory in information systems is further developed and the granulation monotonicity of each of them is proved.Moreover,the complement relationship between information granulation and entropy is established.This investigation unifies the results of measures for uncertainties in complete information systems and incomplete information systems.

  11. The use of lytic bacteriophages to reduce E. coli O157:H7 on fresh cut lettuce introduced through cross-contamination

    Science.gov (United States)

    The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield) at 108 PFU/ml or a control (phosphate buffered saline, PBS) was applied to lettuce by either 1) spraying on to lettuce piec...

  12. Phage lysin LysK can be truncated to its CHAP domain and retain lytic activity against live antibiotic-resistant staphylococci.

    Science.gov (United States)

    Horgan, Marianne; O'Flynn, Gary; Garry, Jennifer; Cooney, Jakki; Coffey, Aidan; Fitzgerald, Gerald F; Ross, R Paul; McAuliffe, Olivia

    2009-02-01

    A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus. This is the first known report of a truncated phage lysin which retains high lytic activity against live staphylococcal cells.

  13. Giant arachnoid granulation in a patient with benign intracranial hypertension

    Energy Technology Data Exchange (ETDEWEB)

    Kiroglu, Yilmaz; Yaqci, Baki; Cirak, Bayram; Karabulut, Nevzat [Pamukkale University, Department of Radiology, School of Medicine, Denizli (Turkey)

    2008-10-15

    We report magnetic resonance (MR), computed tomography (CT) and angiographic imaging of an unusual giant arachnoid granulation in the superior sagittal sinus in a man with headache and vertigo. Intrasinus pressure measurements revealed a significant pressure gradient across the lesion. MR imaging is useful to identify giant arachnoid granulation and dural sinus thrombosis, whereas dural sinus pressure measurement in certain cases of giant arachnoid granulations can be used to evaluate the lesion as the cause of the patient's symptoms. (orig.)

  14. Comparison of low shear, high shear, and fluid bed granulation during low dose tablet process development.

    Science.gov (United States)

    Hausman, Debra S

    2004-03-01

    Three processing methods were compared to develop a low dose (0.1%) immediate release tablet. Similar formulations were used to evaluate low shear, high shear, and fluid bed granulation methods. For each granulation process, the drug was dissolved or suspended in the granulating fluid and sprayed into the granulator. Both water and methanol were evaluated as granulating fluids. The low shear granulation was performed in a Patterson-Kelley V-Blender with I-bar. The high shear granulation was performed in a GRAL (top entry impeller) and a Diosna (bottom mounted impeller). Fluid bed granulation was also performed using top-spray. Acceptable content uniformity was obtained using each technology. The type of granulator and granulating solvent affected the granulation particle size distributions and bulk/tap densities. However, the addition of extragranular microcrystalline cellulose minimized the effect of variable granulation properties and allowed similar tablets to be produced from each granulation process.

  15. Freeze granulation for the processing of silicon nitride ceramics

    Energy Technology Data Exchange (ETDEWEB)

    Lyckfeldt, O. [Swedish Ceramic Inst., Goeteborg (Sweden); Rundgren, K. [Permascand AB, Ljungaverk (Sweden); Sjoestedt, M. [PowderPro HB, Hisings Backa (Sweden)

    2004-07-01

    Freeze granulation (LS-2, PowderPro HB, Sweden) has been demonstrated as a favourable alternative to conventional granulation methods (spray drying, sieve granulation etc) in the production of granules for the pressing of high-performance ceramic powders. Freeze granulation/freeze drying prevents the migration of pressing aids or particle fines to the granule surface, as is the case in spray drying. This ensures granule homogeneity and an easy breakdown of granules during pressing. This, in turn, results in defect minimisation and optimal conditions for the sintering and the development of the desired material properties. In this study silicon nitride materials have been produced using freeze granulation, pressing and sintering to validate the performance. Materials with competitive properties were manufactured based on medium-cost, direct-nitrided powders (SicoNide P95, Permascand AB, Sweden), various pressing and sintering aid compositions as well as various pressing and sintering schedules. MgO vs Fe{sub 2}O{sub 3} as sintering aid, PEG vs PVA as binder and higher pressure at the initial uniaxial pressing were found to promote the sintering performance. (orig.)

  16. Application of the Molecular Combing Technique to Starch Granules

    Directory of Open Access Journals (Sweden)

    Zhong-Dong Liu

    2009-10-01

    Full Text Available The molecular combing technique was used to dissociate the nanostructural units of starch granules from the starch fragments after a gelatinization process. With the help of atomic force microscopy (AFM, we observed that some nanostructural chains were just flowing out of the granules. It proves that there are substantive nanostructural units in the starch granules, a phenomenon not previously observed, so these nanostructural units were defined as suspected intermediates. Furthermore, we conclude that blocklets of starch granules are formed through twisting or distortion of nanochains.

  17. CHARACTERIZATION AND ADSORPTION PROPERTIES OF POROUS CARBON NANOFIBER GRANULES

    Institute of Scientific and Technical Information of China (English)

    Jiuling Chen; Qinghai Chen; Yongdan Li

    2006-01-01

    The properties of the porous granules produced by agglomeration of catalytically grown carbon nanofibers were investigated in this work. The single pellet crushing strength of the granules is high, e.g., 1.6-2.5 MPa. They have adsorption at 298 K of benzene or phenol on the granules is much lower than that on activated carbon and depends not only on the specific surface area of the carbon material but also on the sewing structure of the granules and the morphology of the carbon nanofibers. Treatment in dilute nitric acid appreciably reduces such adsorption.

  18. Aerobic granulation of pure bacterial strain Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    Sunil S ADAV; Duu-Jong LEE

    2008-01-01

    The objective of this study is to cultivate aer-obic granules by pure bacterial strain, Bacillus thuringien-sis, in a sequencing batch reactor. Stable granules sized 2.0-2.2 mm were formed in the reactor after a five-week cultivation. These granules exhibited excellent settling attributes, and degraded phenol at rates of 1.49 and concentration, respectively. Confocal laser scanning microscopic test results show that Bacillus thuringiensis was distributed over the initial small aggregates, and the outer edge of the granule was away from the core regime in the following stage.

  19. Osseointegration of alumina bioceramic granules: A comparative experimental study

    Science.gov (United States)

    Rerikh, V. V.; Avetisyan, A. R.; Zaydman, A. M.; Anikin, K. A.; Bataev, V. A.; Nikulina, A. A.; Sadovoy, M. A.; Aronov, A. M.; Semantsova, E. S.

    2016-08-01

    To perform a comparative analysis of osseointegration of bioceramic alumina-based granules, hydroxyapatite-based granules, and deproteinized bone granules. The experiment was conducted on 52 adult male Kyoto-Wistar rats weighing 350 to 520 g. The animals were divided into five matched groups that differed only in the type of an implanted material. The granules were implanted in the lumbar vertebral bodies and in the distal right femur of each laboratory animal. Two months after surgery, the animals were euthanized, followed by tissue sampling for morphological studies. An examination of specimens from the groups with implanted alumina granules revealed the newly formed trabecular bone with remodeling signs. The bone tissue filled the intragranular space, tightly adhering to the granule surface. There was no connective tissue capsule on the border between bone tissue and alumina granules. Cylindrical bioceramic alumina-based granules with an open internal channel have a higher strength surpassing than that of analogs and the osseointegration ability close to that of hydroxyapatite and deproteinized bone granules.

  20. Microbial granulation for lactic acid production.

    Science.gov (United States)

    Kim, Dong-Hoon; Lee, Mo-Kwon; Hwang, Yuhoon; Im, Wan-Taek; Yun, Yeo-Myeong; Park, Chul; Kim, Mi-Sun

    2016-01-01

    This work investigated the formation of microbial granules to boost the productivity of lactic acid (LA). The flocculated form of LA-producing microbial consortium, dominated by Lactobacillus sp. (91.5% of total sequence), was initially obtained in a continuous stirred-tank reactor (CSTR), which was fed with 2% glucose and operated at a hydraulic retention time (HRT) of 12 h and pH 5.0 ± 0.1 under a thermophilic condition (50°C). The mixed liquor in the CSTR was then transferred to an up-flow anaerobic sludge blanket reactor (UASB). The fermentation performance and granulation process were monitored with a gradual decrease of HRT from 8.0 to 0.17 h, corresponding to an increase in the substrate loading from 60 to 2,880 g glucose L(-1) d(-1) . As the operation continued, the accumulation of biomass in the UASB was clearly observed, which changed from flocculent to granular form with decrease in HRT. Up to the HRT decrease to 0.5 h, the LA concentration was maintained at 19-20 g L(-1) with over 90% of substrate removal efficiency. However, further decrease of HRT resulted in a decrease of LA concentration with increase in residual glucose. Nevertheless, the volumetric LA productivity continuously increased, reaching 67 g L-fermenter (-1) h(-1) at HRT 0.17 h. The size of LA-producing granules and hydrophobicity gradually increased with decrease in HRT, reaching 6.0 mm and 60%, respectively. These biogranules were also found to have high settling velocities and low porosities, ranging 2.69-4.73 cm s(-1) and 0.39-0.92, respectively. © 2015 Wiley Periodicals, Inc.

  1. Disrupted dynamics of F-actin and insulin granule fusion in INS-1 832/13 beta-cells exposed to glucotoxicity: partial restoration by glucagon-like peptide 1.

    Science.gov (United States)

    Quinault, Aurore; Gausseres, Blandine; Bailbe, Danielle; Chebbah, Nella; Portha, Bernard; Movassat, Jamileh; Tourrel-Cuzin, Cecile

    2016-08-01

    Actin dynamics in pancreatic β-cells is involved in insulin exocytosis but the molecular mechanisms of this dynamics and its role in biphasic insulin secretion in pancreatic β-cells is largely unknown. Moreover, the impact of a glucotoxic environment on the sub-cortical actin network dynamics is poorly studied. In this study, we investigate the behavior of insulin granules and the subcortical actin network dynamics in INS-1 832/13 β-cells submitted to a normal or glucotoxic environment. Our results show that glucose stimulation leads to a reorganization of the subcortical actin network with a rupture of its interactions with t-SNARE proteins (Syntaxin 1A and SNAP-25), promoting insulin secretion in INS-1 832/13 β-cells. Prolonged exposure of INS-1 832/13 β-cells to high-glucose levels (glucotoxicity) leads to the densification of the cortical actin network, which prevents its reorganization under acute glucose, and diminishes the glucose-stimulated insulin secretion, as shown by the decreased number of fusion events. The most interesting in our results is the partial restoration by GLP-1 of the insulin secretion ability from high-glucose treated INS-1 832/13 cells. This improved insulin exocytosis is associated with partial restored actin dynamics and fusion events during the two phases of the secretion, with a preferential involvement of Epac2 signaling in the first phase and a rather involvement of PKA signaling in the second phase of insulin exocytosis. All these data provide some new insights into the mechanism by which current therapeutics may be improving insulin secretion.

  2. Observation of polyphosphate granules in cable bacteria

    Science.gov (United States)

    Yang, T.; Nielsen, L. P.; Risgaard-Petersen, N.

    2015-12-01

    Cable bacteria are long filamentous bacteria that capable for long distance electron transport: transporting electrons derived from oxidizing sulfide in anoxic layers, to oxygen at the sediment surface, over a distance of centimeters. Cable bacteria are found in many types of freshwater and marine sediment all over the world, with density of approximately thousands of kilometers per square meter. These long filaments are composed by individual cells closely related to Desulfobulbaceae, connected with a shared outer membrane inside which the strings structure are presumed to be highly conductive. The observed doubling time of cells within the filament is about 20 min, which is among the shortest compare to other bacteria. In these cable cells, we constantly observed polyphosphate granules (poly-P), regardless of cell dimension and shape. This is very interesting since it has long been recognized that the microbial polyP content is low during rapid growth and increases under unfavorable conditions, for example, increasing sulfide concentration and anoxia resulted in a decomposition of poly-P in Beggiatoa. Here, we investigated marine cable bacteria from Netherland and Aarhus Bay, focusing on the poly-P dynamics under various redox conditions. In poly-P stained cells, typically there are two big poly-P granules locate at each polar. In dividing cells, however, the morphology of poly-P changed to six small granules precisely arranged to two row. Moreover, the cells seem be able to continuously divide more than one time without elongation step. These varied poly-P morphologies demonstrate that poly-P is closely related to the cell growth and cell division, by an unknown mechanism. Individual cable filaments were picked up and were exposed to different redox conditions; our primary data indicated the cable cells could suffer anoxic condition better than oxic condition. We also detected decomposition of poly-P under anoxia. These results call for an in-depth examination

  3. Distribution of binder in granules produced by means of twin screw granulation

    DEFF Research Database (Denmark)

    Fonteyne, Margot; Fussell, Andrew Luke; Vercruysse, Jurgen

    2014-01-01

    -granular distribution of PVP was evaluated by means of hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy. For the evaluated formulation, no PVP rich zones were detected when applying a lateral spatial resolution of 0.5 μm, indicating that PVP is homogenously distributed within the granules....

  4. In planta modification of potato starch granule biogenesis by different granule-bound fusion proteins

    NARCIS (Netherlands)

    Nazarian, F.

    2007-01-01

    Starch is composed of amylose and amylopectin and it is deposited in amyloplasts/choloroplasts as semi-crystalline granules. Many biosynthetic enzymes are involved in starch degradation and biosynthesis. Some microbial starch degrading enzymes have a Starch Binding Domain (SBD) which has affinity fo

  5. Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains.

    Directory of Open Access Journals (Sweden)

    Renata Urban-Chmiel

    Full Text Available The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves.The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC® BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR.Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904 and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101.The results obtained indicate the need for further research aimed at isolating and characterizing

  6. Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains.

    Science.gov (United States)

    Urban-Chmiel, Renata; Wernicki, Andrzej; Stęgierska, Diana; Dec, Marta; Dudzic, Anna; Puchalski, Andrzej

    2015-01-01

    The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves. The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR). Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101. The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages

  7. Evaluation of granulated lactose as a carrier for DPI formulations 1: effect of granule size.

    Science.gov (United States)

    Du, Ping; Du, Ju; Smyth, Hugh D C

    2014-12-01

    The objective of this study was to investigate the effect of large granulated lactose carrier particle systems on aerosol performance of dry powder inhaler formulations. Granulated lactose carriers with average sizes ranging from 200 to 1,000 μm were prepared and subsequently fractionated into separate narrow size powders. The fractionated granulated lactose (GL) samples were characterized in terms of size, specific surface area, surface roughness, morphology, density, flowability, and solid-state. The in vitro aerosolization performance was performed on the different size fractions of GL samples from a commercial inhaler device (Aerolizer®) with a model formulation (2% w/w salbutamol sulfate). The cascade impaction parameters employed were 60 or 90 L/min with standard (aperture size, 0.6 mm) or modified piercing holes (aperture size, 1.2 mm) of the inhaler loaded capsules. It was shown that the largest size fraction formulation (850-1000 μm) had a slight improvement in the fine particle fraction (FPF) compared to immediately preceding size fractions, explained by a smaller adhesive force between drug and carrier. Compared to commercial piercing holes, enlarged piercing holes generated a slight decreasing trend of FPF as the lactose powder sizes increased from 200-250 μm to 600-850 μm, perhaps due to the reduced detachment force by flow forces. The size, surface roughness, density, and flowability of lactose carrier as well as device design all contributed to the aerosol dispersion performance of granulated lactose-based adhesive mixtures. It was concluded that poorer or enhanced redispersion performance is not an inherent property to the significantly large size of granulated lactose carriers as previously contended.

  8. Influence of process variable and physicochemical properties on the granulation mechanism of mannitol in a fluid bed top spray granulator.

    Science.gov (United States)

    Bouffard, Jonathan; Kaster, Meagan; Dumont, Hubert

    2005-10-01

    This study investigated the influence of specific process variables, including the hydroxypropyl cellulose (HPC) binder solution atomization, on the fluidized bed top spray granulation of mannitol. Special attention was given to the relationship between wetting and the granule growth profile. The atomization of the HPC binder solution using a binary nozzle arrangement produced droplets of decreasing size as the atomization pressure was increased, while changes in the spray rate had little effect on the mean droplet size. Increasing the HPC binder concentration from 2 to 8% w/w increased the binder droplet size and was most likely attributed to higher solution viscosity. The top spray granulation of mannitol showed induction type growth behavior. Process conditions like high spray rate, low fluidizing air velocity and binder solution concentration that promote the availability of HPC binder solution at the surface of the particles appeared to be key in enhancing nucleation and growth of the granules. Increasing the bed moisture level, up to a certain value, reduced the contribution of attrition to the overall growth profile of the granule and, more significantly, produced less granule breakage on drying. It was observed that the mean granule size could be reduced as much as 40% between the end of granulation and the end of drying for lower initial bed moisture level despite a shorter drying phase. High atomization pressure, especially when maintained during the drying phase, contributed substantially to granule breakage.

  9. Conditional knockout of tumor overexpressed gene in mouse neurons affects RNA granule assembly, granule translation, LTP and short term habituation.

    Directory of Open Access Journals (Sweden)

    Elisa Barbarese

    Full Text Available In neurons, specific RNAs are assembled into granules, which are translated in dendrites, however the functional consequences of granule assembly are not known. Tumor overexpressed gene (TOG is a granule-associated protein containing multiple binding sites for heterogeneous nuclear ribonucleoprotein (hnRNP A2, another granule component that recognizes cis-acting sequences called hnRNP A2 response elements (A2REs present in several granule RNAs. Translation in granules is sporadic, which is believed to reflect monosomal translation, with occasional bursts, which are believed to reflect polysomal translation. In this study, TOG expression was conditionally knocked out (TOG cKO in mouse hippocampal neurons using cre/lox technology. In TOG cKO cultured neurons granule assembly and bursty translation of activity-regulated cytoskeletal associated (ARC mRNA, an A2RE RNA, are disrupted. In TOG cKO brain slices synaptic sensitivity and long term potentiation (LTP are reduced. TOG cKO mice exhibit hyperactivity, perseveration and impaired short term habituation. These results suggest that in hippocampal neurons TOG is required for granule assembly, granule translation and synaptic plasticity, and affects behavior.

  10. Hepatocyte growth factor pathway upregulation in the bone marrow microenvironment in multiple myeloma is associated with lytic bone disease

    DEFF Research Database (Denmark)

    Kristensen, Ida B; Christensen, Jacob H; Lyng, Maria Bibi

    2013-01-01

    Lytic bone disease (LBD) in multiple myeloma (MM) is caused by osteoclast hyperactivation and osteoblast inhibition. Based on in vitro studies, the hepatocyte growth factor (HGF) pathway is thought to be central in osteoblast inhibition. We evaluated the gene expression of the HGF pathway in vivo...

  11. Lytic infection of Lactococcus lactis by bacteriophages Tuc2009 and c2 triggers alternative transcriptional host responses.

    Science.gov (United States)

    Ainsworth, Stuart; Zomer, Aldert; Mahony, Jennifer; van Sinderen, Douwe

    2013-08-01

    Here we present an entire temporal transcriptional profile of Lactococcus lactis subsp. cremoris UC509.9 undergoing lytic infection with two distinct bacteriophages, Tuc2009 and c2. Furthermore, corresponding high-resolution whole-phage genome tiling arrays of both bacteriophages were performed throughout lytic infection. Whole-genome microarrays performed at various time points postinfection demonstrated a rather modest impact on host transcription. The majority of changes in the host transcriptome occur during late infection stages; few changes in host gene transcription occur during the immediate and early infection stages. Alterations in the L. lactis UC509.9 transcriptome during lytic infection appear to be phage specific, with relatively few differentially transcribed genes shared between cells infected with Tuc2009 and those infected with c2. Despite the apparent lack of a coordinated general phage response, three themes common to both infections were noted: alternative transcription of genes involved in catabolic flux and energy production, differential transcription of genes involved in cell wall modification, and differential transcription of genes involved in the conversion of ribonucleotides to deoxyribonucleotides. The transcriptional profiles of both bacteriophages during lytic infection generally correlated with the findings of previous studies and allowed the confirmation of previously predicted promoter sequences. In addition, the host transcriptional response to lysogenization with Tuc2009 was monitored along with tiling array analysis of Tuc2009 in the lysogenic state. Analysis identified 44 host genes with altered transcription during lysogeny, 36 of which displayed levels of transcription significantly reduced from those for uninfected cells.

  12. Probing the structure of glucan lyases – the lytic members of GH31 - by sequence analysis, circular dichroism and proteolysis

    DEFF Research Database (Denmark)

    Ernst, Heidi; Lo Leggio, Leila; Yu, Shukun

    2005-01-01

    Glucan lyase (GL) is a polysaccharide lyase with unique characteristics. It is involved in an alternative pathway for the degradation of alpha-glucans, the anhydrofructose pathway. Sequence similarity suggests that this lytic enzyme belongs to glycoside hydrolase family 31, for which until very r...

  13. Lytic Infection of Lactococcus lactis by Bacteriophages Tuc2009 and c2 Triggers Alternative Transcriptional Host Responses

    NARCIS (Netherlands)

    Ainsworth, S.; Zomer, A.L.; Mahony, J.; Sinderen, D. van

    2013-01-01

    Here we present an entire temporal transcriptional profile of Lactococcus lactis subsp. cremoris UC509.9 undergoing lytic infection with two distinct bacteriophages, Tuc2009 and c2. Furthermore, corresponding high-resolution whole-phage genome tiling arrays of both bacteriophages were performed thro

  14. In vivo dynamics of EBNA1-oriP interaction during latent and lytic replication of Epstein-Barr virus.

    Science.gov (United States)

    Daikoku, Tohru; Kudoh, Ayumi; Fujita, Masatoshi; Sugaya, Yutaka; Isomura, Hiroki; Tsurumi, Tatsuya

    2004-12-24

    The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is required for maintenance of the viral genome DNA during the latent phase of EBV replication but continues to be synthesized after the induction of viral productive replication. An EBV genome-wide chromatin immunoprecipitation assay revealed that EBNA1 constantly binds to oriP of the EBV genome during not only latent but also lytic infection. Although the total levels of EBNA1 proved constant throughout the latter, the levels of the oriP-bound form were increased as lytic infection proceeded. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins are clustered. Confocal laser microscopic analyses revealed that whereas EBNA1 was distributed broadly in nuclei as fine punctate dots during the latent phase of infection, the protein became redistributed to the viral replication compartments and localized as distinct spots within and/or nearby the compartments after the induction of lytic replication. Taking these findings into consideration, oriP regions of the EBV genome might be organized by EBNA1 into replication domains that may set up scaffolding for lytic replication and transcription.

  15. Epiphyseal involvement in Erdheim-Chester disease: radiographic and scintigraphic findings in a case with lytic lesions

    Energy Technology Data Exchange (ETDEWEB)

    Ruiz-Hernandez, G.; Tajahuerce-Romera, G.M.; Latorre-Ibanez, M.D.; Lara-Pomares, A. [Servicio de Medicina Nuclear, Hospital Provincial de Castellon (Spain); Vila-Fayos, V. [Servicio de Reumatologia, Hospital Comarcal de Vinaroz (Spain)

    2000-08-01

    We reported a symmetric increase of activity in lower links secondary to Erdheim-Chester disease and demonstrated by bone scans and radiographs. An inusual scintigraphic and radiographic appearance with epiphyseal involvement and lytic lesions is described. Differential diagnosis of bone scan and radiographic findings is discussed. (orig.)

  16. Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Pierce, Brian; Wittrup Agger, Jane; Wichmann, Jesper

    2017-01-01

    The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety...

  17. Lytic Infection of Lactococcus lactis by Bacteriophages Tuc2009 and c2 Triggers Alternative Transcriptional Host Responses

    NARCIS (Netherlands)

    Ainsworth, S.; Zomer, A.L.; Mahony, J.; Sinderen, D. van

    2013-01-01

    Here we present an entire temporal transcriptional profile of Lactococcus lactis subsp. cremoris UC509.9 undergoing lytic infection with two distinct bacteriophages, Tuc2009 and c2. Furthermore, corresponding high-resolution whole-phage genome tiling arrays of both bacteriophages were performed

  18. High resolution crystal structures of the Escherichia coli lytic transglycosylase Slt70 and its complex with a peptidoglycan fragment

    NARCIS (Netherlands)

    Asselt, Erik J. van; Thunnissen, Andy-Mark W.H.; Dijkstra, Bauke W.

    1999-01-01

    The 70 kDa soluble lytic transglycosylase (Slt70) from Escherichia coli is an exo-muramidase, that catalyses the cleavage of the glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues in peptidoglycan, the main structural component of the bacterial cell wall. This cleavage is

  19. A subset of replication proteins enhances origin recognition and lytic replication by the Epstein-Barr virus ZEBRA protein.

    Directory of Open Access Journals (Sweden)

    Ayman El-Guindy

    Full Text Available ZEBRA is a site-specific DNA binding protein that functions as a transcriptional activator and as an origin binding protein. Both activities require that ZEBRA recognizes DNA motifs that are scattered along the viral genome. The mechanism by which ZEBRA discriminates between the origin of lytic replication and promoters of EBV early genes is not well understood. We explored the hypothesis that activation of replication requires stronger association between ZEBRA and DNA than does transcription. A ZEBRA mutant, Z(S173A, at a phosphorylation site and three point mutants in the DNA recognition domain of ZEBRA, namely Z(Y180E, Z(R187K and Z(K188A, were similarly deficient at activating lytic DNA replication and expression of late gene expression but were competent to activate transcription of viral early lytic genes. These mutants all exhibited reduced capacity to interact with DNA as assessed by EMSA, ChIP and an in vivo biotinylated DNA pull-down assay. Over-expression of three virally encoded replication proteins, namely the primase (BSLF1, the single-stranded DNA-binding protein (BALF2 and the DNA polymerase processivity factor (BMRF1, partially rescued the replication defect in these mutants and enhanced ZEBRA's interaction with oriLyt. The findings demonstrate a functional role of replication proteins in stabilizing the association of ZEBRA with viral DNA. Enhanced binding of ZEBRA to oriLyt is crucial for lytic viral DNA replication.

  20. Probing the structure of glucan lyases – the lytic members of GH31 - by sequence analysis, circular dichroism and proteolysis

    DEFF Research Database (Denmark)

    Ernst, Heidi; Lo Leggio, Leila; Yu, Shukun

    2005-01-01

    Glucan lyase (GL) is a polysaccharide lyase with unique characteristics. It is involved in an alternative pathway for the degradation of alpha-glucans, the anhydrofructose pathway. Sequence similarity suggests that this lytic enzyme belongs to glycoside hydrolase family 31, for which until very r...

  1. Isolation and characterization of lytic vibriophage against Vibrio cholerae O1 from environmental water samples in Kelantan, Malaysia.

    Science.gov (United States)

    Al-Fendi, Ali; Shueb, Rafidah Hanim; Ravichandran, Manickam; Yean, Chan Yean

    2014-10-01

    Water samples from a variety of sources in Kelantan, Malaysia (lakes, ponds, rivers, ditches, fish farms, and sewage) were screened for the presence of bacteriophages infecting Vibrio cholerae. Ten strains of V. cholerae that appeared to be free of inducible prophages were used as the host strains. Eleven bacteriophage isolates were obtained by plaque assay, three of which were lytic and further characterized. The morphologies of the three lytic phages were similar with each having an icosahedral head (ca. 50-60 nm in diameter), a neck, and a sheathed tail (ca. 90-100 nm in length) characteristic of the family Myoviridae. The genomes of the lytic phages were indistinguishable in length (ca. 33.5 kb), nuclease sensitivity (digestible with DNase I, but not RNase A or S1 nuclease), and restriction enzyme sensitivity (identical banding patterns with HindIII, no digestion with seven other enzymes). Testing for infection against 46 strains of V. cholerae and 16 other species of enteric bacteria revealed that all three isolates had a narrow host range and were only capable of infecting V. cholerae O1 El Tor Inaba. The similar morphologies, indistinguishable genome characteristics, and identical host ranges of these lytic isolates suggests that they represent one phage, or several very closely related phages, present in different water sources. These isolates are good candidates for further bio-phage-control studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Drug distribution in wet granulation: foam versus spray.

    Science.gov (United States)

    Tan, Melvin X L; Nguyen, Thanh H; Hapgood, Karen P

    2013-09-01

    Foam granulation technology is a new wet granulation approach for pharmaceutical formulations. This study evaluates the performance of foam and spray granulation in achieving uniform drug distribution using a model formulation. To observe wetting and nuclei formation, single drop/foam penetration experiments were performed on a static powder bed comprised of varying compositions of hydrophilic/hydrophobic glass ballotini, and hydrophilic lactose/hydrophobic salicylic acid respectively. High shear granulation experiments were performed in a 5L mixer using varying compositions of hydrophilic lactose and hydrophobic salicylic acid. Four percent hydroxylpropyl methylcellulose (HPMC) solution was delivered at 90 g/min as either a foam (92% FQ) or an atomized spray whilst recording impeller power consumption. After drying, the granule size distribution was measured and the granule composition was estimated using gravimetric filtration in methanol. Foam penetration was less dependent on the powder hydrophobicity compared to drop penetration. For glass ballotini powder mixtures, foam induced nucleation created nuclei with relatively uniform structure and size regardless of the powder hydrophobicity. For salicylic acid and lactose mixtures, increasing the proportion of salicylic acid reduced the nuclei granule size for both foam and drop binder addition. The granule drug distribution was not significantly affected by the binder addition method. Processing conditions, including liquid binder amount, impeller speed, wet massing, and the wettability properties of the formulation were the dominant factors for delivering homogeneous granules. The study reveals that foam and spray granulation involve different nucleation mechanisms - spray tends to incur early liquid penetration whereas foam granulation operates well in mechanical dispersion.

  3. Rheological and fractal hydrodynamics of aerobic granules.

    Science.gov (United States)

    Tijani, H I; Abdullah, N; Yuzir, A; Ujang, Zaini

    2015-06-01

    The structural and hydrodynamic features for granules were characterized using settling experiments, predefined mathematical simulations and ImageJ-particle analyses. This study describes the rheological characterization of these biologically immobilized aggregates under non-Newtonian flows. The second order dimensional analysis defined as D2=1.795 for native clusters and D2=1.099 for dewatered clusters and a characteristic three-dimensional fractal dimension of 2.46 depicts that these relatively porous and differentially permeable fractals had a structural configuration in close proximity with that described for a compact sphere formed via cluster-cluster aggregation. The three-dimensional fractal dimension calculated via settling-fractal correlation, U∝l(D) to characterize immobilized granules validates the quantitative measurements used for describing its structural integrity and aggregate complexity. These results suggest that scaling relationships based on fractal geometry are vital for quantifying the effects of different laminar conditions on the aggregates' morphology and characteristics such as density, porosity, and projected surface area.

  4. Microbial granulation for lactic acid production

    DEFF Research Database (Denmark)

    Kim, Dong-Hoon; Lee, Mo-Kwon; Hwang, Yuhoon

    2016-01-01

    was fed with 2% glucose and operated at a hydraulic retention time (HRT) of 12 h and pH 5.0 ± 0.1 under a thermophilic condition (50°C). The mixed liquor in the CSTR was then transferred to an up-flow anaerobic sludge blanket reactor (UASB). The fermentation performance and granulation process were......This work investigated the formation of microbial granules to boost the productivity of lactic acid (LA). The flocculated form of LA-producing microbial consortium, dominated by Lactobacillus sp. (91.5% of total sequence), was initially obtained in a continuous stirred-tank reactor (CSTR), which...... monitored with a gradual decrease of HRT from 8.0 to 0.17 h, corresponding to an increase in the substrate loading from 60 to 2,880 g glucose L−1d−1. As the operation continued, the accumulation of biomass in the UASB was clearly observed, which changed from flocculent to granular form with decrease in HRT...

  5. "Lytic" lesions in autologous bone grafts: demonstration of medullary air pockets on post mortem computed tomography.

    Science.gov (United States)

    Rotman, A; Hamilton, K; O'Donnell, C

    2007-12-01

    Donor bone grafts are an important aspect of orthopaedic surgery. The use of plain film as a pathological screening tool before donor bone dispatch has revealed "lytic" lesions in proximal humeri. Donor demographics did not support the diagnosis of myeloma and subsequent computed tomography (CT) scans of these bones identified the lesions as air, not pathology. In total, 27 long bones were scanned and 100% (27/27 cases) exhibited air within the trabecular bone. Three distinct patterns were found: ovoid, linear/branching, and broad channel. A longitudinal course of CT scans was performed to identify at which stage air appeared within the bone. Pre-retrieval, preprocessing, and postprocessing scans revealed that air originated between the retrieval and preprocessing stages of donor bone preparation. There may be multiple aetiology of this phenomenon, including bone retrieval and natural decomposition.

  6. Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Christine Sattler

    2016-03-01

    Full Text Available An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt. Using murine gammaherpesvirus 68 (MHV-68 as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues.

  7. Lytic polysaccharide monooxygenases: a crystallographer's view on a new class of biomass-degrading enzymes

    Directory of Open Access Journals (Sweden)

    Kristian E. H. Frandsen

    2016-11-01

    Full Text Available Lytic polysaccharide monooxygenases (LPMOs are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future.

  8. Isolation and characterization of a T7-like lytic phage for Pseudomonas fluorescens

    Directory of Open Access Journals (Sweden)

    Neubauer Peter

    2008-10-01

    Full Text Available Abstract Background Despite the proven relevance of Pseudomonas fluorescens as a spoilage microorganism in milk, fresh meats and refrigerated food products and the recognized potential of bacteriophages as sanitation agents, so far no phages specific for P. fluorescens isolates from dairy industry have been closely characterized in view of their lytic efficiency. Here we describe the isolation and characterization of a lytic phage capable to infect a variety of P. fluorescens strains isolated from Portuguese and United States dairy industries. Results Several phages were isolated which showed a different host spectrum and efficiency of lysis. One of the phages, phage ϕIBB-PF7A, was studied in detail due to its efficient lysis of a wide spectrum of P. fluorescens strains and ribotypes. Phage ϕIBB-PF7A with a head diameter of about 63 nm and a tail size of about 13 × 8 nm belongs morphologically to the Podoviridae family and resembles a typical T7-like phage, as analyzed by transmission electron microscopy (TEM. The phage growth cycle with a detected latent period of 15 min, an eclipse period of 10 min, a burst size of 153 plaque forming units per infected cell, its genome size of approximately 42 kbp, and the size and N-terminal sequence of one of the protein bands, which gave similarity to the major capsid protein 10A, are consistent with this classification. Conclusion The isolated T7-like phage, phage ϕIBB-PF7A, is fast and efficient in lysing different P. fluorescens strains and may be a good candidate to be used as a sanitation agent to control the prevalence of spoilage causing P. fluorescens strains in dairy and food related environments.

  9. In vitro management of hospital Pseudomonas aeruginosa biofilm using indigenous T7-like lytic phage.

    Science.gov (United States)

    Ahiwale, Sangeeta; Tamboli, Nilofer; Thorat, Kiran; Kulkarni, Rajendra; Ackermann, Hans; Kapadnis, Balasaheb

    2011-02-01

    Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1-HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.

  10. The FIKK kinase of Toxoplasma gondii is not essential for the parasite's lytic cycle.

    Science.gov (United States)

    Skariah, S; Walwyn, O; Engelberg, K; Gubbels, M-J; Gaylets, C; Kim, N; Lynch, B; Sultan, A; Mordue, D G

    2016-05-01

    FIKK kinases are a novel family of kinases unique to the Apicomplexa. While most apicomplexans encode a single FIKK kinase, Plasmodium falciparum expresses 21 and piroplasms do not encode a FIKK kinase. FIKK kinases share a conserved C-terminal catalytic domain, but the N-terminal region is highly variable and contains no known functional domains. To date, FIKK kinases have been primarily studied in P. falciparum and Plasmodium berghei. Those that have been studied are exported from the parasite and associate with diverse locations in the infected erythrocyte cytosol or membrane. Deletion of individual P. falciparum FIKK kinases indicates that they may play a role in modification of the infected erythrocyte. The current study characterises the single FIKK gene in Toxoplasma gondii to evaluate the importance of the FIKK kinase in an apicomplexan that has a single FIKK kinase. The TgFIKK gene encoded a protein of approximately 280kDa. Endogenous tagging of the FIKK protein with Yellow Fluorescent Protein showed that the FIKK protein exclusively localised to the posterior end of tachyzoites. A Yellow Fluorescent Protein-tagged FIKK and a Ty-tagged FIKK both co-localised with T. gondii membrane occupation and recognition nexus protein to the basal complex and were localised apical to inner membrane complex protein-5 and Centrin2. Deletion of TgFIKK, surprisingly, had no detectable effect on the parasite's lytic cycle in vitro in human fibroblast cells or in acute virulence in vivo. Thus, our results clearly show that while the FIKK kinase is expressed in tachyzoites, it is not essential for the lytic cycle of T. gondii. Copyright © 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  11. Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.

    Directory of Open Access Journals (Sweden)

    Marie Samanovic

    2009-01-01

    Full Text Available Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.

  12. Broad-range lytic bacteriophages that kill Staphylococcus aureus local field strains.

    Science.gov (United States)

    Abatángelo, Virginia; Peressutti Bacci, Natalia; Boncompain, Carina A; Amadio, Ariel A; Carrasco, Soledad; Suárez, Cristian A; Morbidoni, Héctor R

    2017-01-01

    Staphylococcus aureus is a very successful opportunistic pathogen capable of causing a variety of diseases ranging from mild skin infections to life-threatening sepsis, meningitis and pneumonia. Its ability to display numerous virulence mechanisms matches its skill to display resistance to several antibiotics, including β-lactams, underscoring the fact that new anti-S. aureus drugs are urgently required. In this scenario, the utilization of lytic bacteriophages that kill bacteria in a genus -or even species- specific way, has become an attractive field of study. In this report, we describe the isolation, characterization and sequencing of phages capable of killing S. aureus including methicillin resistant (MRSA) and multi-drug resistant S. aureus local strains from environmental, animal and human origin. Genome sequencing and bio-informatics analysis showed the absence of genes encoding virulence factors, toxins or antibiotic resistance determinants. Of note, there was a high similarity between our set of phages to others described in the literature such as phage K. Considering that reported phages were obtained in different continents, it seems plausible that there is a commonality of genetic features that are needed for optimum, broad host range anti-staphylococcal activity of these related phages. Importantly, the high activity and broad host range of one of our phages underscores its promising value to control the presence of S. aureus in fomites, industry and hospital environments and eventually on animal and human skin. The development of a cocktail of the reported lytic phages active against S. aureus-currently under way- is thus, a sensible strategy against this pathogen.

  13. Revisiting the Cellulosimicrobium cellulans yeast-lytic β-1,3-glucanases toolbox: A review

    Directory of Open Access Journals (Sweden)

    Ferrer Pau

    2006-03-01

    Full Text Available Abstract Cellulosimicrobium cellulans (also known with the synonyms Cellulomonas cellulans, Oerskovia xanthineolytica, and Arthrobacter luteus is an actinomycete that excretes yeast cell wall lytic enzyme complexes containing endo-β-1,3-glucanases [EC 3.2.1.39 and 3.2.1.6] as key constituents. Three genes encoding endo-β-1,3-glucanases from two C. cellulans strains have been cloned and characterised over the past years. The βglII and βglIIA genes from strain DSM 10297 (also known as O. xanthineolytica LL G109 encoded proteins of 40.8 and 28.6 kDa, respectively, whereas the β-1,3-glucanase gene from strain ATCC 21606 (also known as A. luteus 73–14 encoded a 54.5 kDa protein. Alignment of their deduced amino acid sequences reveal that βglII and βglIIA have catalytic domains assigned to family 16 of glycosyl hydrolases, whereas the catalytic domain from the 54.5 kDa glucanase belongs to family 64. Notably, both βglII and the 54.5 kDa β-1,3-glucanase are multidomain proteins, having a lectin-like C-terminal domain that has been assigned to family 13 of carbohydrate binding modules, and that confers to β-1,3-glucanases the ability to lyse viable yeast cells. Furthermore, βglII may also undergo posttranslational proteolytic processing of its C-terminal domain, resulting in a truncated enzyme retaining its glucanase activity but with very low yeast-lytic activity. In this review, the diversity in terms of structural and functional characteristics of the C. cellulans β-1,3-glucanases has been compiled and compared.

  14. Lytic Characteristics and Identification of Two Alga-lysing Bacterial Strains

    Institute of Scientific and Technical Information of China (English)

    PEI Haiyan; HU Wenrong

    2006-01-01

    All previously reported bacterial species which are capable of lysing harmful algae have been isolated from coastal environments in which harmful algae blooms have occurred. Due to the low concentration of alga-lysing bacteria in an algal bloom, it is difficult to isolate the alga-lysing bacteria by existing methods. In this paper, two algae-lysing bacterial strains,P01 and P03, have been isolated from a biosystem immobilized on a sponge that was highly effective in removing algae and microcystins. Their lysing modes and effects on Microcystis aeruginosa have been studied. The results show that the degradation processes of these two strains for M. aeruginosa accorded with a first-order reaction model when the chlorophylla concentration was in the range from 0 to 1000 μg L-1. The degradation rate constants were 0.106 7, 0.127 4 and 0.279 2 for P01and0.0683, 0.0744 and 0.02897 for P03, when the bacterial densities were 8.6 × 105, 8.6 × 106 and 8.6 × 107cells mL 1, respectively. Moreover, the two bacterial strains had favourable lytic effects not only on M. aeruginosa, but also on Chlorella and Scene-desmus. Their lytic effect on M. aeruginosa did not require physical cell to cell contact, but proceeded by the production of an extracellular product. The bacterial strains were identified as Bacillus species by PCR amplification of the 16S rRNA gene, BLAST analysis, and comparison with sequences in the GenBank nucleotide database.

  15. Structure of the Bacteriophage [phi]KZ Lytic Transglycosylase gp144

    Energy Technology Data Exchange (ETDEWEB)

    Fokine, Andrei; Miroshnikov, Konstantin A.; Shneider, Mikhail M.; Mesyanzhinov, Vadim V.; Rossmann, Michael G. (SOIBC); (Purdue)

    2008-04-02

    Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage {phi}KZ has been determined to 2.5-{angstrom} resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. {phi}KZ gp144 is a 260-residue {alpha}-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G d-Ala-d-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu{sup 115} in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the {phi}KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine){sub 4} has been determined to 2.6-{angstrom} resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

  16. Delta-9 tetrahydrocannabinol (THC inhibits lytic replication of gamma oncogenic herpesviruses in vitro

    Directory of Open Access Journals (Sweden)

    Friedman Herman

    2004-09-01

    Full Text Available Abstract Background The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC, has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication. Methods Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV and Epstein-Barr virus (EBV replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS of monkeys, murine gamma herpesvirus 68 (MHV 68, and herpes simplex type 1 (HSV-1 was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method. Results Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68. Conclusions THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC

  17. 21 CFR 520.970a - Flunixin meglumine granules.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Flunixin meglumine granules. 520.970a Section 520...) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS ORAL DOSAGE FORM NEW ANIMAL DRUGS § 520.970a Flunixin meglumine granules. (a) Specifications. Each 10-gram packet contains flunixin meglumine equivalent to 250...

  18. Application of granulating of tires; Aplicaciones de granulado de NFUs

    Energy Technology Data Exchange (ETDEWEB)

    Jimenez Justicia, A.

    2001-07-01

    At present, used tires are mainly used as fuel, retreading and granulating of tires. In this article. I will focus on the last option, talking about some of the multiple applications and going into detail of the making process of the granulation of used tires in a recycling plant. (Author)

  19. Cerebellar Granule Cells: Dense, Rich and Evolving Representations

    NARCIS (Netherlands)

    Badura, Aleksandra; De Zeeuw, Chris I

    2017-01-01

    For half a century it was assumed that granule cells use ultra-sparse encoding, but now in vivo calcium-imaging studies have shown that large ensembles of granule cells provide dense signals, which themselves evolve and adapt during training.

  20. Osseous drill holes to promote granulation tissue: Radiologic appearance

    Energy Technology Data Exchange (ETDEWEB)

    Resnik, C.S.; Reiner, B.I.; Diaconis, J.N. (Maryland Univ. Medical System/Hospital, Baltimore, MD (USA). Dept. of Radiology); Goldberg, N.H. (Maryland Univ. Medical System/Hospital, Baltimore, MD (USA). Div. of Plastic Surgery)

    1991-04-01

    Skin grafting following extensive soft-tissue loss is often delayed until adequate granulation tissue can be generated. Surgical drill holes into the marrow cavity promote development of granulation tissue. This article illustrates the radiology appearance of these drill holes in four patients. (orig.).

  1. Investigation of Compost Fertilizer Granulation Parameters Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Y Ghasemi

    2015-03-01

    Full Text Available Nowadays compost fertilizers are suitable alternative to chemical fertilizers, due to the threats for human health and agriculture products. The most important problems for applying the compost fertilizer in the farm are: transportation (high volume, high moisture content, spreading problem, impurity such as dust and storage. To solve the problems mentioned, pressing process such as converting the compost to pellets and granules are suggested. In this research the effects of some granulation parameters on the percent of useful granules in a laboratory scale rotating drum was evaluated. The percentage of useful granules decreased by increasing the granulation time and increased by increasing the percentage of drum filling. The optimal conditions for granules production was achieved at drum rotational speed of 40.38 rpm, granulation time of 15 min, drum filling of 10% and molasse percentage of 40.97. According to these conditions, the response for useful granule was estimated as 81.6% with R2 of 0.924.

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

    Science.gov (United States)

    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.

  3. Monetite granules versus particulate autologous bone in bone regeneration.

    Science.gov (United States)

    Torres, Jesús; Tamimi, Iskandar; Cabrejos-Azama, Jatsue; Tresguerres, Isabel; Alkhraisat, Mohammad; López-Cabarcos, Enrique; Hernández, Gonzalo; Tamimi, Faleh

    2015-07-01

    The aim of this study was to test bone tissue response to monetite granules in comparison with intramembranous autologous bone graft in a rabbit calvaria critical size defect model. Novel monetite granules were synthesized by thermal conversion of set brushite cement. Eight female New Zealand rabbits were used for this study. Two identical 10mm diameter bicortical cranial defects were created in each animal. One of the defects was grafted with monetite granules while the contralateral was grafted with granules of intramembranous autologous bone as control. Animals were sacrificed 8 weeks after surgery and biopsies were taken for histological and histomorphometrical evaluation under light microscopy. Wilcoxon test was used for statistical analysis. The bone defects treated with either autologous bone or monetite granules were able to heal within the study period. Upon histological observation the defects treated with autologous bone granules resembled the adjacent intact calvaria, whereas the defects treated with monetite showed a high infiltration of new bone and only 13.4±8.4% of remaining granules. The percentage of bone volume in the defects of the control group (71±9%) was 16% higher than in the study group (55±10%) (p0.05). The amount of augmented mineralized tissue in the bone defects obtained with monetite granules was not significantly different from that obtained with autologous bone. This study confirms the potential of monetite based biomaterials as an alternative to autologous bone graft. Copyright © 2015 Elsevier GmbH. All rights reserved.

  4. Understanding and predicting bed humidity in fluidized bed granulation.

    Science.gov (United States)

    Hu, Xinhui; Cunningham, John; Winstead, Denita

    2008-04-01

    Bed humidity is a critical parameter that needs to be controlled in a fluidized bed granulation to ensure reliability. To predict and control the bed humidity during the fluidized bed granulation process, a simple model based on the mass conservation of moisture was developed. The moisture mass balance model quantitatively simulates the effects of spray rate, binder solution concentration, airflow rate, inlet air temperature, and dew point on the bed humidity. The model was validated by a series of granulations performed in different scale granulators including Glatt GPCG-1, GPCG-15, and GPCG-60. Good agreement was observed between the theoretical prediction and the measured loss on drying (LOD). The model developed in the current work enables us to choose the appropriate parameters for the fluidized bed granulation and can be used as a valuable tool in process scaling-up.

  5. A Calculus on Granules from Rough Inclusions in Information Systems

    Institute of Scientific and Technical Information of China (English)

    Lech Polkowski

    2006-01-01

    Indiscernibility relations induced in information systems in the sense of Pawlak,define granules of knowledge as unions of their equivalence classes,leading to regular structures like Boolean algebras. Apart from Boolean operations on granules, under afixed set of attributes,that induce the partition topology on their collection, one may search for more flexible schemes that would induce in collections of granules some non-trivial topologies and in particular quasi-metrics on granules, allowing for richer structures. A way of realization of this program has been proposed as the paradigm of rough mereology; although abstract, stemming from an alternative set theory, yet it offers a calculus based on kind of quasi-metrics that allows for discussing granules from the point of view of their closeness. In thiswork, we would liketopresent theideas of this approach and basic results with short discussion of applications.

  6. Biophysics of active vesicle transport, an intermediate step that couples excitation and exocytosis of serotonin in the neuronal soma.

    Directory of Open Access Journals (Sweden)

    Francisco F De-Miguel

    Full Text Available Transmitter exocytosis from the neuronal soma is evoked by brief trains of high frequency electrical activity and continues for several minutes. Here we studied how active vesicle transport towards the plasma membrane contributes to this slow phenomenon in serotonergic leech Retzius neurons, by combining electron microscopy, the kinetics of exocytosis obtained from FM1-43 dye fluorescence as vesicles fuse with the plasma membrane, and a diffusion equation incorporating the forces of local confinement and molecular motors. Electron micrographs of neurons at rest or after stimulation with 1 Hz trains showed cytoplasmic clusters of dense core vesicles at 1.5±0.2 and 3.7±0.3 µm distances from the plasma membrane, to which they were bound through microtubule bundles. By contrast, after 20 Hz stimulation vesicle clusters were apposed to the plasma membrane, suggesting that transport was induced by electrical stimulation. Consistently, 20 Hz stimulation of cultured neurons induced spotted FM1-43 fluorescence increases with one or two slow sigmoidal kinetics, suggesting exocytosis from an equal number of vesicle clusters. These fluorescence increases were prevented by colchicine, which suggested microtubule-dependent vesicle transport. Model fitting to the fluorescence kinetics predicted that 52-951 vesicles/cluster were transported along 0.60-6.18 µm distances at average 11-95 nms(-1 velocities. The ATP cost per vesicle fused (0.4-72.0, calculated from the ratio of the ΔG(process/ΔG(ATP, depended on the ratio of the traveling velocity and the number of vesicles in the cluster. Interestingly, the distance-dependence of the ATP cost per vesicle was bistable, with low energy values at 1.4 and 3.3 µm, similar to the average resting distances of the vesicle clusters, and a high energy barrier at 1.6-2.0 µm. Our study confirms that active vesicle transport is an intermediate step for somatic serotonin exocytosis by Retzius neurons and provides a

  7. Biophysics of active vesicle transport, an intermediate step that couples excitation and exocytosis of serotonin in the neuronal soma.

    Science.gov (United States)

    De-Miguel, Francisco F; Santamaría-Holek, Iván; Noguez, Paula; Bustos, Carlos; Hernández-Lemus, Enrique; Rubí, J Miguel

    2012-01-01

    Transmitter exocytosis from the neuronal soma is evoked by brief trains of high frequency electrical activity and continues for several minutes. Here we studied how active vesicle transport towards the plasma membrane contributes to this slow phenomenon in serotonergic leech Retzius neurons, by combining electron microscopy, the kinetics of exocytosis obtained from FM1-43 dye fluorescence as vesicles fuse with the plasma membrane, and a diffusion equation incorporating the forces of local confinement and molecular motors. Electron micrographs of neurons at rest or after stimulation with 1 Hz trains showed cytoplasmic clusters of dense core vesicles at 1.5±0.2 and 3.7±0.3 µm distances from the plasma membrane, to which they were bound through microtubule bundles. By contrast, after 20 Hz stimulation vesicle clusters were apposed to the plasma membrane, suggesting that transport was induced by electrical stimulation. Consistently, 20 Hz stimulation of cultured neurons induced spotted FM1-43 fluorescence increases with one or two slow sigmoidal kinetics, suggesting exocytosis from an equal number of vesicle clusters. These fluorescence increases were prevented by colchicine, which suggested microtubule-dependent vesicle transport. Model fitting to the fluorescence kinetics predicted that 52-951 vesicles/cluster were transported along 0.60-6.18 µm distances at average 11-95 nms(-1) velocities. The ATP cost per vesicle fused (0.4-72.0), calculated from the ratio of the ΔG(process)/ΔG(ATP), depended on the ratio of the traveling velocity and the number of vesicles in the cluster. Interestingly, the distance-dependence of the ATP cost per vesicle was bistable, with low energy values at 1.4 and 3.3 µm, similar to the average resting distances of the vesicle clusters, and a high energy barrier at 1.6-2.0 µm. Our study confirms that active vesicle transport is an intermediate step for somatic serotonin exocytosis by Retzius neurons and provides a quantitative method

  8. Hilar mossy cell circuitry controlling dentate granule cell excitability

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    Seiichiro eJinde

    2013-02-01

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

  9. A highly sustainable and versatile granulation method of nanodrugs via their electrostatic adsorption onto chitosan microparticles as the granulation substrates.

    Science.gov (United States)

    Yang, Yue; Hadinoto, Kunn

    2013-08-16

    Nanodrugs play important roles in enhancing the sustainability of pharmaceutical manufacturing via their ability to enhance the bioavailability of poorly soluble drugs, resulting in less drug wastage and less mass/energy consumed in their manufacturing. Despite their sustainability enhancement capability, solid dosage form manufacturing of nanodrugs remains lacking from the sustainability perspective. One example is the granulation of nanodrugs prior to tablet preparation, where existing methods (e.g. wet granulation, spray granulation, spray drying) require high energy and time expenses, or are highly intricate often leading to product inconsistencies. Herein we present an alternative nanodrug granulation method via electrostatic adsorption of the nanodrugs onto chitosan microparticles acting as granulation substrates. The method is sustainable involving only mixing of aqueous suspensions of the nanodrugs and substrates under ambient conditions, followed by washing and drying. We investigate the effects of substrate's physical characteristics and nanodrug to substrate ratio on the nanodrug loading in the granules, content uniformity, nanodrug recovery, and granule flowability. Ciprofloxacin and curcumin nanoplexes prepared by drug-polyelectrolyte complexation are used as the model nanodrugs with neutrally, positively, and negatively charged chitosan microparticles as the substrates. Granules having 25% (w/w) nanodrug loading at 50% (w/w) recovery with good flowability have been successfully prepared. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. The temporal characteristics of Ca2+ entry through L-type and T-type Ca2+ channels shape exocytosis efficiency in chick auditory hair cells during development.

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    Levic, Snezana; Dulon, Didier

    2012-12-01

    During development, synaptic exocytosis by cochlear hair cells is first initiated by patterned spontaneous Ca(2+) spikes and, at the onset of hearing, by sound-driven graded depolarizing potentials. The molecular reorganization occurring in the hair cell synaptic machinery during this developmental transition still remains elusive. We characterized the changes in biophysical properties of voltage-gated Ca(2+) currents and exocytosis in developing auditory hair cells of a precocial animal, the domestic chick. We found that immature chick hair cells (embryonic days 10-12) use two types of Ca(2+) currents to control exocytosis: low-voltage-activating, rapidly inactivating (mibefradil sensitive) T-type Ca(2+) currents and high-voltage-activating, noninactivating (nifedipine sensitive) L-type currents. Exocytosis evoked by T-type Ca(2+) current displayed a fast release component (RRP) but lacked the slow sustained release component (SRP), suggesting an inefficient recruitment of distant synaptic vesicles by this transient Ca(2+) current. With maturation, the participation of L-type Ca(2+) currents to exocytosis largely increased, inducing a highly Ca(2+) efficient recruitment of an RRP and an SRP component. Notably, L-type-driven exocytosis in immature hair cells displayed higher Ca(2+) efficiency when triggered by prerecorded native action potentials than by voltage steps, whereas similar efficiency for both protocols was found in mature hair cells. This difference likely reflects a tighter coupling between release sites and Ca(2+) channels in mature hair cells. Overall, our results suggest that the temporal characteristics of Ca(2+) entry through T-type and L-type Ca(2+) channels greatly influence synaptic release by hair cells during cochlear development.

  11. Fluidization of Wet Granulates under Hydrodynamic Shear

    CERN Document Server

    Battiato, I

    2012-01-01

    We investigate the fluidization threshold of three-dimensional cohesive granulates under hydrodynamic shear forces exerted by a creeping ow. A continuum model of flow through porous media provides an analytical expression for the average drag force on a single grain. The balance equation for the forces and a force propagation model are then used to investigate the effects of porosity and packing structure on the stability of the pile. We obtain a closed-form expression for the fluidization threshold of a regular packing of mono-disperse frictionless cohesive spherical grains in a planar fracture. The compound effect of structural (packing orientation and porosity) and dynamical properties of the system on its stability is quantified.

  12. Testing protozoacidal activity of ligand-lytic peptides against termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into termite hindgut).

    Science.gov (United States)

    Husseneder, Claudia; Sethi, Amit; Foil, Lane; Delatte, Jennifer

    2010-12-29

    We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004). In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni,Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic

  13. DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

    Science.gov (United States)

    Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

    2015-01-01

    Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

  14. Activation and Repression of Epstein-Barr Virus and Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycles by Short- and Medium-Chain Fatty Acids

    Science.gov (United States)

    Gorres, Kelly L.; Daigle, Derek; Mohanram, Sudharshan

    2014-01-01

    ABSTRACT The lytic cycles of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are induced in cell culture by sodium butyrate (NaB), a short-chain fatty acid (SCFA) histone deacetylase (HDAC) inhibitor. Valproic acid (VPA), another SCFA and an HDAC inhibitor, induces the lytic cycle of KSHV but blocks EBV lytic reactivation. To explore the hypothesis that structural differences between NaB and VPA account for their functional effects on the two related viruses, we investigated the capacity of 16 structurally related short- and medium-chain fatty acids to promote or prevent lytic cycle reactivation. SCFAs differentially affected EBV and KSHV reactivation. KSHV was reactivated by all SCFAs that are HDAC inhibitors, including phenylbutyrate. However, several fatty acid HDAC inhibitors, such as isobutyrate and phenylbutyrate, did not reactivate EBV. Reactivation of KSHV lytic transcripts could not be blocked completely by any fatty acid tested. In contrast, several medium-chain fatty acids inhibited lytic activation of EBV. Fatty acids that blocked EBV reactivation were more lipophilic than those that activated EBV. VPA blocked activation of the BZLF1 promoter by NaB but did not block the transcriptional function of ZEBRA. VPA also blocked activation of the DNA damage response that accompanies EBV lytic cycle activation. Properties of SCFAs in addition to their effects on chromatin are likely to explain activation or repression of EBV. We concluded that fatty acids stimulate the two related human gammaherpesviruses to enter the lytic cycle through different pathways. IMPORTANCE Lytic reactivation of EBV and KSHV is needed for persistence of these viruses and plays a role in carcinogenesis. Our direct comparison highlights the mechanistic differences in lytic reactivation between related human oncogenic gammaherpesviruses. Our findings have therapeutic implications, as fatty acids are found in the diet and produced by the human microbiota

  15. Cerebellar granule cells are predominantly generated by terminal symmetric divisions of granule cell precursors.

    Science.gov (United States)

    Nakashima, Kie; Umeshima, Hiroki; Kengaku, Mineko

    2015-06-01

    Neurons in the central nervous system (CNS) are generated by symmetric and asymmetric cell division of neural stem cells and their derivative progenitor cells. Cerebellar granule cells are the most abundant neurons in the CNS, and are generated by intensive cell division of granule cell precursors (GCPs) during postnatal development. Dysregulation of GCP cell cycle is causal for some subtypes of medulloblastoma. However, the details and mechanisms underlying neurogenesis from GCPs are not well understood. Using long-term live-cell imaging of proliferating GCPs transfected with a fluorescent newborn-granule cell marker, we found that GCPs underwent predominantly symmetric divisions, generating two GCPs or two neurons, while asymmetric divisions generating a GCP and a neuron were only occasionally observed, in both dissociated culture and within tissues of isolated cerebellar lobules. We found no significant difference in cell cycle length between proliferative and neurogenic divisions, or any consistent changes in cell cycle length during repeated proliferative division. Unlike neural stem cells in the cerebral cortex and spinal cord, which generate many neurons by repeated asymmetric division, cerebellar GCPs produce neurons predominantly by terminal symmetric division. These results indicate diverse mechanisms of neurogenesis in the mammalian brain. © 2015 Wiley Periodicals, Inc.

  16. Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Daniela F Angelini

    Full Text Available It has long been known that multiple sclerosis (MS is associated with an increased Epstein-Barr virus (EBV seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A and lytic (BZLF-1, BMLF-1 antigens in relapsing-remitting MS patients (n = 113 and healthy donors (HD (n = 43 and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.

  17. Involvement of VAMP-2 in exocytosis of IL-1 beta in turbot (Scophthalmus maximus) leukocytes after Vibrio anguillarum infection.

    Science.gov (United States)

    Chai, Yingmei; Huang, Xiaohang; Cong, Bailin; Liu, Shenghao; Chen, Kui; Li, Guangyou; Gaisano, Herbert Y

    2006-04-01

    Vibrio anguillarum is a major pathogen threatening the fish aquaculture in China. Infection of cultivated turbot (Scophthalmus maximus) with V. anguillarum induced rapid synthesis and secretion of IL-1beta, which initiates the innate immune response. SNARE proteins are known to regulate vesicular trafficking and fusion in all eukaryotes. Here, we determined whether SNARE proteins, specifically vesicle-associated membrane protein-2 (VAMP-2), are involved in regulated exocytosis of IL-1beta of leukocytes in marine fish. We show that VAMP-2 is present in turbot blood leukocytes, with nucleotide sequence identity of 88.2% and 93.0% to those of zebra fish and sea bass, respectively. After Vibrio infection, turbot leukocyte VAMP-2 was increased at the levels of transcription and translation in a temporal pattern coinciding with leukocyte IL-1beta secretion. Confocal microscopy localized VAMP-2 to vesicle structures in leukocytes. Taken together, our results suggest that VAMP-2 is involved in regulated exocytosis of cytokines in immunocytes in fish.

  18. Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo.

    Science.gov (United States)

    Sorvina, Alexandra; Shandala, Tetyana; Brooks, Douglas A

    2016-01-01

    The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. Here, we report that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10), appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. We propose that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens.

  19. Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo

    Directory of Open Access Journals (Sweden)

    Alexandra Sorvina

    2016-06-01

    Full Text Available The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. Here, we report that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10, appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. We propose that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens.

  20. Group IIA secretory phospholipase A2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons.

    Science.gov (United States)

    Wei, S; Ong, W Y; Thwin, M M; Fong, C W; Farooqui, A A; Gopalakrishnakone, P; Hong, W

    2003-01-01

    Recent evidence shows that secretory phospholipase A2 (sPLA2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA2 on vesicle exocytosis. External application of group IIA sPLA2 (purified crotoxin subunit B or purified human synovial sPLA2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA2 was dependent on calcium and sPLA2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.

  1. STXBP1 promotes Weibel-Palade body exocytosis through its interaction with the Rab27A effector Slp4-a.

    Science.gov (United States)

    van Breevoort, Dorothee; Snijders, Ambrosius P; Hellen, Nicola; Weckhuysen, Sarah; van Hooren, Kathinka W E M; Eikenboom, Jeroen; Valentijn, Karine; Fernandez-Borja, Mar; Ceulemans, Berten; De Jonghe, Peter; Voorberg, Jan; Hannah, Matthew; Carter, Tom; Bierings, Ruben

    2014-05-15

    Vascular endothelial cells contain unique rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs), which contain the hemostatic protein von Willebrand factor (VWF) and a cocktail of angiogenic and inflammatory mediators. We have shown that the Rab27A effector synaptotagmin-like protein 4-a (Slp4-a) plays a critical role in regulating hormone-evoked WPB exocytosis. Using a nonbiased proteomic screen for targets for Slp4-a, we now identify syntaxin-binding protein 1 (STXBP1) and syntaxin-2 and -3 as endogenous Slp4-a binding partners in endothelial cells. Coimmunoprecipitations showed that STXBP1 interacts with syntaxin-2 and -3, but not with syntaxin-4. Small interfering RNA-mediated silencing of STXBP1 expression impaired histamine- and forskolin-induced VWF secretion. To further substantiate the role of STXBP1, we isolated blood outgrowth endothelial cells (BOECs) from an early infantile epileptic encephalopathy type 4 (EIEE4) patient carrying a de novo mutation in STXBP1. STXBP1-haploinsufficient EIEE4 BOECs contained similar numbers of morphologically normal WPBs compared with control BOECs of healthy donors; however, EIEE4 BOECs displayed significantly impaired histamine- and forskolin-stimulated VWF secretion. Based on these findings, we propose that the Rab27A-Slp4-a complex on WPB promotes exocytosis through an interaction with STXBP1, thereby controlling the release of vaso-active substances in the vasculature.

  2. VAMP3 is associated with endothelial weibel-palade bodies and participates in their Ca(2+)-dependent exocytosis.

    Science.gov (United States)

    Pulido, Inés Rojo; Jahn, Reinhard; Gerke, Volker

    2011-05-01

    Weibel-Palade bodies (WPBs) are secretory organelles of endothelial cells that store the thrombogenic glycoprotein von Willebrand factor (vWF). Endothelial activation, e.g. by histamine and thrombin, triggers the Ca(2+)-dependent exocytosis of WPB that releases vWF into the vasculature and thereby initiates platelet capture and thrombus formation. Towards understanding the molecular mechanisms underlying this regulated WPB exocytosis, we here identify components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery associated with WPB. We show that vesicle-associated membrane protein (VAMP) 3 and VAMP8 are present on WPB and that VAMP3, but not VAMP8 forms a stable complex with syntaxin 4 and SNAP23, two plasma membrane-associated SNAREs in endothelial cells. By introducing mutant SNARE proteins into permeabilized endothelial cells we also show that soluble VAMP3 but not VAMP8 mutants comprising the cytoplasmic domain interfere with efficient vWF secretion. This indicates that endothelial cells specifically select VAMP 3 over VAMP8 to cooperate with syntaxin 4 and SNAP23 in the Ca(2+)-triggered fusion of WPB with the plasma membrane. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

  3. A Granule Model for Evaluating Adhesion of Pharmaceutical Binders

    Directory of Open Access Journals (Sweden)

    Hossein Orafai

    2003-10-01

    Full Text Available Granule capability is defined in terms of the strength of individual granule and friability of granulation batch to withstand breaking, abrasion and compactibility. Binder(s are added to perform the above properties .The common methods to asses their capability are to test crushing strength of the granules directly and to make statistical analysis and /or testing the friability of bulk granulation. In this work four substrate models including polymethylmetacrylate beads(PMMA,glass powder, acetaminophen, and para-aminobebzoic acid were chosen. The binder models were corn starch, gelatin, methylcellulose (MC and hydroxypropylmethylcellulose (HPMC. After massing the substrates with the binder solutions, discs were produced by the mean of the mold technique. The discs were dried and conditioned and then tested for tensile strength while the failed areas were scanned by SEM. Various granulations were made and the results of friability and crush strength were compared with the discs strength .The bond areas in the SEM showed the trend with the binder concentration .A comparison of the standard deviation shows that discs have much lower level of the strength than granules. The resulting discs showed a higher performance which is related to the stems for the discs shape .In conclusion, this method is a simple and is applicable to differentiate efficacy of binder under studies.

  4. Optimization for blast furnace slag dry cooling granulation device

    Science.gov (United States)

    Dazhan, Sheng; Yali, Wang; Ruiyun, Wang; Suping, Cui; Xiaoyu, Ma

    2017-03-01

    Since the large accumulation amount of blast furnace slag (BFS) with recycling value, it has become a hot topic for recovery utilization. Compared with the existing various BFS granulation process, the dry granulation process can promote the use of blast furnace granulated slag as cement substitute and concrete admixtures. Our research group developed a novel dry cooling granulation experiment device to treat BFS. However, there are still some problems to be solved. The purpose of this research is to improve the cooling and granulation efficiency of the existing dry type cooling equipment. This topic uses the FLUENT simulation software to study the impact of the number of air inlet on the cooling effect of the device. The simulation result is that the device possessing eight air inlets can increase the number of hot and cold gas exchanged, resulting in a better cooling effect. According to the power consumption, LCA analysis was carried out on the cooling granulation process. The results show that the device equipped eight air inlets not only improved the original equipment cooling granulation effect, but also increased resource utilization ratio, realized energy-saving and emission reduction.

  5. UNCERTAINTY HANDLING IN DISASTER MANAGEMENT USING HIERARCHICAL ROUGH SET GRANULATION

    Directory of Open Access Journals (Sweden)

    H. Sheikhian

    2015-08-01

    Full Text Available Uncertainty is one of the main concerns in geospatial data analysis. It affects different parts of decision making based on such data. In this paper, a new methodology to handle uncertainty for multi-criteria decision making problems is proposed. It integrates hierarchical rough granulation and rule extraction to build an accurate classifier. Rough granulation provides information granules with a detailed quality assessment. The granules are the basis for the rule extraction in granular computing, which applies quality measures on the rules to obtain the best set of classification rules. The proposed methodology is applied to assess seismic physical vulnerability in Tehran. Six effective criteria reflecting building age, height and material, topographic slope and earthquake intensity of the North Tehran fault have been tested. The criteria were discretized and the data set was granulated using a hierarchical rough method, where the best describing granules are determined according to the quality measures. The granules are fed into the granular computing algorithm resulting in classification rules that provide the highest prediction quality. This detailed uncertainty management resulted in 84% accuracy in prediction in a training data set. It was applied next to the whole study area to obtain the seismic vulnerability map of Tehran. A sensitivity analysis proved that earthquake intensity is the most effective criterion in the seismic vulnerability assessment of Tehran.

  6. The Leader Protein of Cardioviruses Inhibits Stress Granule Assembly ▿

    Science.gov (United States)

    Borghese, Fabian; Michiels, Thomas

    2011-01-01

    Stress granules (SG) are cytoplasmic aggregates of stalled translation preinitiation complexes that form in cells exposed to various environmental stresses. Here, we show that stress granules assemble in cells infected with Theiler's murine encephalomyelitis virus (TMEV) mutants carrying alterations in the leader (L) protein, but not in cells infected with wild-type TMEV. Stress granules also formed in STAT1-deficient cells, suggesting that SG formation was not a consequence of increased type I interferon (IFN) production when cells were infected with the mutant virus. Ectopic expression of the wild-type L protein was sufficient to inhibit stress granule formation induced by sodium arsenite or thapsigargin treatment. In conclusion, TMEV infection induces stress granule assembly, but this process is inhibited by the L protein. Unlike poliovirus-induced stress granules, TMEV-induced stress granules did not contain the nuclear protein Sam68 but contained polypyrimidine tract binding protein (PTB), an internal ribosome entry site (IRES)-interacting protein. Moreover, G3BP was not degraded and was found in SG after TMEV infection, suggesting that SG content could be virus specific. Despite the colocalization of PTB with SG and the known interaction of PTB with viral RNA, in situ hybridization and immunofluorescence assays failed to detect viral RNA trapped in infection-induced SG. Recombinant Theiler's viruses expressing the L protein of Saffold virus 2 (SAFV-2), a closely related human theilovirus, or the L protein of mengovirus, an encephalomyocarditis virus (EMCV) strain, also inhibited infection-induced stress granule assembly, suggesting that stress granule antagonism is a common feature of cardiovirus L proteins. PMID:21752908

  7. The leader protein of cardioviruses inhibits stress granule assembly.

    Science.gov (United States)

    Borghese, Fabian; Michiels, Thomas

    2011-09-01

    Stress granules (SG) are cytoplasmic aggregates of stalled translation preinitiation complexes that form in cells exposed to various environmental stresses. Here, we show that stress granules assemble in cells infected with Theiler's murine encephalomyelitis virus (TMEV) mutants carrying alterations in the leader (L) protein, but not in cells infected with wild-type TMEV. Stress granules also formed in STAT1-deficient cells, suggesting that SG formation was not a consequence of increased type I interferon (IFN) production when cells were infected with the mutant virus. Ectopic expression of the wild-type L protein was sufficient to inhibit stress granule formation induced by sodium arsenite or thapsigargin treatment. In conclusion, TMEV infection induces stress granule assembly, but this process is inhibited by the L protein. Unlike poliovirus-induced stress granules, TMEV-induced stress granules did not contain the nuclear protein Sam68 but contained polypyrimidine tract binding protein (PTB), an internal ribosome entry site (IRES)-interacting protein. Moreover, G3BP was not degraded and was found in SG after TMEV infection, suggesting that SG content could be virus specific. Despite the colocalization of PTB with SG and the known interaction of PTB with viral RNA, in situ hybridization and immunofluorescence assays failed to detect viral RNA trapped in infection-induced SG. Recombinant Theiler's viruses expressing the L protein of Saffold virus 2 (SAFV-2), a closely related human theilovirus, or the L protein of mengovirus, an encephalomyocarditis virus (EMCV) strain, also inhibited infection-induced stress granule assembly, suggesting that stress granule antagonism is a common feature of cardiovirus L proteins.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-04-01

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

  9. Utility of lytic bacteriophage in the treatment of multidrug-resistant Pseudomonas aeruginosa septicemia in mice

    Directory of Open Access Journals (Sweden)

    Vinodkumar C

    2008-07-01

    Full Text Available Drug resistance is the major cause of increase in morbidity and mortality in neonates. One thousand six hundred forty-seven suspected septicemic neonates were subjected for microbiological analysis over a period of 5 years. Forty-two P. aeruginosa were isolated and the antibiogram revealed that 28 P. aeruginosa were resistant to almost all the common drugs used (multidrug-resistant. The emergence of antibiotic-resistant bacterial strains is one of the most critical problems of modern medicine. As a result, a novel and most effective approaches for treating infection caused by multidrug-resistant bacteria are urgently required. In this context, one intriguing approach is to use bacteriophages (viruses that kill bacteria in the treatment of infection caused by drug-resistant bacteria. In the present study, the utility of lytic bacteriophages to rescue septicemic mice with multidrug-resistant (MDR P. aeruginosa infection was evaluated. MDR P. aeruginosa was used to induce septicemia in mice by intraperitoneal (i.p. injection of 10 7 CFU. The resulting bacteremia was fatal within 48 hrs. The phage strain used in this study had lytic activity against a wide range of clinical isolates of MDR P. aeruginosa. A single i.p. injection of 3 x 10 9 PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue septicemic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of septicemic mice could be affected only by phage strains able to grow in vitro on the bacterial host used to infect the animals and when such strains are heat-inactivated, they lose their ability to rescue the infected mice. Multidrug-resistant bacteria have

  10. Survival of Salmonella Newport on Whole and Fresh-Cut Cucumbers Treated with Lytic Bacteriophages.

    Science.gov (United States)

    Sharma, Manan; Dashiell, Gwendolyn; Handy, Eric T; East, Cheryl; Reynnells, Russell; White, Chanelle; Nyarko, Esmond; Micallef, Shirley; Hashem, Fawzy; Millner, Patricia D

    2017-04-01

    Salmonella enterica associated with consumption of cucumbers ( Cucumis sativus ) has led to foodborne outbreaks in the United States. Whole and fresh-cut cucumbers are susceptible to S. enterica contamination during growing, harvesting, and postharvest handling. The application of lytic bacteriophages specific for S. enterica was evaluated to reduce Salmonella populations on cucumbers. Unwaxed cucumbers ('Lisboa' variety, or mini-cucumbers purchased at retail) were inoculated with Salmonella Newport (5 log CFU per cucumber) and were sprayed with 3.2 mL of phosphate-buffered saline (control) or 10 log PFU/ml of SalmoFresh, a Salmonella-specific bacteriophage preparation (phage), to deliver 4.76 × 10(7) PFU/cm(2). Cucumbers were stored at 10 or 22°C for 7 days. Inoculated mini-cucumbers were sliced with a sterile knife to investigate Salmonella transfer to mesocarp, and cut pieces were stored at 4°C for 2 days. Populations (log CFU per cucumber) of Salmonella Newport on phage-treated whole cucumbers were significantly (P cucumbers (4.27 ± 0.37) on day 0. Populations on phage-treated cucumbers stored at 10°C were 1.72 ± 0.77 and 1.56 ± 0.46, which were significantly lower than those on control-treated cucumbers (3.20 ± 0.48 and 2.33 ± 0.25) on days 1 and 4, respectively. Between days 0 and 1, populations on control-treated cucumbers stored at 10 and 22°C declined by 1.07 and 2.47 log CFU per cucumber, respectively. At 22°C, Salmonella Newport populations declined by 2.37 log CFU per cucumber between days 0 and 1. Phage application to whole cucumbers before slicing did not reduce the transfer of Salmonella Newport to fresh-cut slices. Lytic phage application may be a potential intervention to reduce Salmonella populations on whole cucumbers.

  11. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

    NARCIS (Netherlands)

    Loose, Jennifer S. M.; Forsberg, Zarah; Fraaije, Marco W.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2014-01-01

    The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass

  12. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

    NARCIS (Netherlands)

    Loose, Jennifer S. M.; Forsberg, Zarah; Fraaije, Marco W.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2014-01-01

    The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass

  13. Inhibition of Epstein-Barr Virus Lytic Cycle by an Ethyl Acetate Subfraction Separated from Polygonum cuspidatum Root and Its Major Component, Emodin

    Directory of Open Access Journals (Sweden)

    Ching-Yi Yiu

    2014-01-01

    Full Text Available Polygonum cuspidatum is widely used as a medicinal herb in Asia. In this study, we examined the ethyl acetate subfraction F3 obtained from P. cuspidatum root and its major component, emodin, for their capacity to inhibit the Epstein-Barr virus (EBV lytic cycle. The cell viability was determined by the MTT [3-(4,5-dimethyldiazol-2-yl-2,5-diphenyltetrazolium bromide] method. The expression of EBV lytic proteins was analyzed by immunoblot, indirect immunofluorescence and flow cytometric assays. Real-time quantitative PCR was used to assess the EBV DNA replication and the transcription of lytic genes, including BRLF1 and BZLF1. Results showed that the F3 and its major component emodin inhibit the transcription of EBV immediate early genes, the expression of EBV lytic proteins, including Rta, Zta, and EA-D and reduces EBV DNA replication, showing that F3 and emodin are potentially useful as an anti-EBV drug.

  14. A novel role of IL-17–producing lymphocytes in mediating lytic bone disease in multiple myeloma

    Science.gov (United States)

    Noonan, Kimberly; Marchionni, Luigi; Anderson, Judy; Pardoll, Drew; Roodman, G. David

    2010-01-01

    Osteoclast (OC)–mediated lytic bone disease remains a cause of major morbidity in multiple myeloma. Here we demonstrate the critical role of interleukin-17–producing marrow infiltrating lymphocytes (MILs) in OC activation and development of bone lesions in myeloma patients. Unlike MILs from normal bone marrow, myeloma MILs possess few regulatory T cells (Tregs) and demonstrate an interleukin-17 phenotype that enhances OC activation. In univariate analyses of factors mediating bone destruction, levels of cytokines that selectively induce and maintain the Th17 phenotype tightly correlated with the extent of bone disease in myeloma. In contrast, MILs activated under conditions that skew toward a Th1 phenotype significantly reduced formation of mature OC. These findings demonstrate that interleukin-17 T cells are critical to the genesis of myeloma bone disease and that immunologic manipulations shifting MILs from a Th17 to a Th1 phenotype may profoundly diminish lytic bone lesions in multiple myeloma. PMID:20664052

  15. The algae-lytic ability of bacterium DC10 and the influence of environmental factors on the ability

    Institute of Scientific and Technical Information of China (English)

    SHI Shunyu; LIU Yongding; SHEN Yinwu; LI Genbao

    2005-01-01

    A lysing-bacterium DC10, isolated from Dianchi Lake of Yunnan Province, was characterized to be Pseudomonas sp. It was able to lyse some algae well, such as Microcystis viridis, Selenastrum capricornutum, and so on. In this study, it was shown that the bacterium lysed the algae by releasing a substance; the best lytic effects were achieved at Iow temperatures and in the dark. Different concentrations of CaCI2 and NaNO3 influenced the lytic effects;the ability to lyse algae decreased in the following order: pH 4 > pH 9 > pH 7 > pH 5.5. It was significant to develop a special technology with this kind of bacterium for controlling the bloomforming planktonic microalgae.

  16. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

    Directory of Open Access Journals (Sweden)

    Denis Avey

    2015-07-01

    Full Text Available Kaposi's Sarcoma-Associated Herpesvirus (KSHV is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK mitogen-activated protein kinase (MAPK pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45

  17. Lytic enzyme production optimization using low-cost substrates and its application in the clarification of xanthan gum culture broth

    Science.gov (United States)

    da Silva, Cíntia Reis; Silva, Marilia Lordelo Cardoso; Kamida, Helio Mitoshi; Goes-Neto, Aristoteles; Koblitz, Maria Gabriela Bello

    2014-01-01

    Lytic enzymes are widely used in industrial biotechnology as they are able to hydrolyze the bacterial cell wall. One application of these enzymes is the clarification of the culture broth for the production of xanthan gum, because of its viability in viscous media and high specificity. The screening process for filamentous fungi producing lytic enzymes, the optimization of production of these enzymes by the selected microorganism, and the optimization of the application of the enzymes produced in the clarification of culture broth are presented in this article. Eleven fungal isolates were tested for their ability to produce enzymes able to increase the transmittance of the culture broth containing cells of Xanthomonas campestris. To optimize the secretion of lytic enzymes by the selected microorganism the following variables were tested: solid substrate, initial pH, incubation temperature, and addition of inducer (gelatin). Thereafter, secretion of the enzymes over time of incubation was assessed. To optimize the clarification process a central composite rotational design was applied in which the pH of the reaction medium, the dilution of the broth, and the reaction temperature were evaluated. The isolate identified as Aspergillus tamarii was selected for increasing the transmittance of the broth from 2.1% to 54.8%. The best conditions for cultivation of this microorganism were: use of coconut husk as solid substrate, with 90% moisture, at 30°C for 20 days. The lytic enzymes produced thereby were able to increase the transmittance of the culture broth from 2.1% to 70.6% at 65°C, without dilution and without pH adjustment. PMID:25473487

  18. Morphological diversity of cultured cold-active lytic bacteriophages isolated from the Napahai plateau wetland in China

    Institute of Scientific and Technical Information of China (English)

    Xiuling Ji; Chunjing Zhang; Anxiu Kuang; Jiankai Li; Yinshan Cui; Kunhao Qin; Lianbing Lin; Benxu Cheng; Qi Zhang; Yunlin Wei

    2015-01-01

    Dear Editor,Viruses are the most abundant,diverse,and ubiquitous entities(approximately 1031)on Earth.They play major roles in horizontal gene transfer,the regulation of bacterial community structures,as well as nutrient and energy cycles of marine ecosystems(Danovaro et al.,2008).In particular,lytic bacteriophages(phages)can infect and kill bacteria without harming human or animal

  19. In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL

    OpenAIRE

    2012-01-01

    The bacterial endospore is the most resilient biological structure known. Multiple protective integument layers shield the spore core and promote spore dehydration and dormancy. Dormancy is broken when a spore germinates and becomes a metabolically active vegetative cell. Germination requires the breakdown of a modified layer of peptidoglycan (PG) known as the spore cortex. This study reports in vitro and in vivo analyses of the Bacillus anthracis SleL protein. SleL is a spore cortex lytic en...

  20. Correlation between microbial community and granule conductivity in anaerobic bioreactors for brewery wastewater treatment

    DEFF Research Database (Denmark)

    Shrestha, Pravin; Malvankar, Nikhil S.; Werner, Jeffrey

    2014-01-01

    granules from an aerobic bioreactor designed for phosphate removal were not. There was a moderate correlation (r = 0.67) between the abundance of Geobacter species in the UASB granules and granule conductivity, suggesting that Geobacter contributed to granule conductivity. These results, coupled...

  1. Potential antiviral lignans from the roots of Saururus chinensis with activity against Epstein-Barr virus lytic replication.

    Science.gov (United States)

    Cui, Hui; Xu, Bo; Wu, Taizong; Xu, Jun; Yuan, Yan; Gu, Qiong

    2014-01-24

    Epstein-Barr virus (EBV) is a member of the γ-herpes virus subfamily and has been implicated in the pathogenesis of several human malignancies. Bioassay-guided fractionation was conducted on an EtOAc-soluble extract of the roots of Saururus chinensis and monitored using an EBV lytic replication assay. This led to the isolation of 19 new (1-19) and nine known (20-28) lignans. The absolute configurations of the new lignans were established by Mosher's ester, ECD, and computational methods. Eight lignans, including three sesquineolignans (19, 23, and 24) and five dineolignans (3, 4, 26, 27, and 28), exhibited inhibitory effects toward EBV lytic replication with EC50 values from 1.09 to 7.55 μM and SI values from 3.3 to 116.4. In particular, manassantin B (27) exhibited the most promising inhibition, with an EC50 of 1.72 μM, low cytotoxicity, CC50 > 200 μM, and SI > 116.4. This is the first study demonstrating that lignans possess anti-EBV lytic replication activity.

  2. Advanced lytic lesion is a poor mobilization factor in peripheral blood stem cell collection in patients with multiple myeloma.

    Science.gov (United States)

    Jung, Sung-Hoon; Yang, Deok-Hwan; Ahn, Jae-Sook; Kim, Yeo-Kyeoung; Kim, Hyeoung-Joon; Lee, Je-Jung

    2014-12-01

    This study examined the incidence and predictors of peripheral blood stem cell (PBSC) mobilization failure in patients with multiple myeloma (MM). Retrospective data for 104 patients who received granulocyte colony-stimulating factor (G-CSF) alone or with cyclophosphamide as mobilization regimens were analyzed. The rates of mobilization failure using two definitions of failure (mobilization failure were evaluated using logistic regression analysis which included age, advanced osteolytic lesions, bone marrow cellularity before mobilization, platelet count, body mass index before mobilization, and mobilization method. Lytic bone lesions were assessed using a conventional skeletal survey, and advanced osteolytic lesions were defined as lytic lesions in more than three skeletal sites regardless of the number of lytic lesions. On multivariate analysis, advanced osteolytic lesions [hazard ratio (HR) = 10.95, P = 0.001] and age ≥60 years (HR = 5.45, P = 0.016) were associated with a PBSC yield mobilization (HR = 4.72, P = 0.005), and G-CSF only mobilization (HR 10.52, P mobilization failure in MM patients.

  3. Biomimetic aqueous-core lipid nanoballoons integrating a multiple emulsion formulation: a suitable housing system for viable lytic bacteriophages.

    Science.gov (United States)

    Balcão, Victor M; Glasser, Cássia A; Chaud, Marco V; del Fiol, Fernando S; Tubino, Matthieu; Vila, Marta M D C

    2014-11-01

    The emergence of antibiotic-resistant bacterial strains and the weak penetration of antibiotics into bacterial biofilms put an emphasis in the need for safe and effective alternatives for antimicrobial treatments. The application of strictly lytic bacteriophages (or phages) has been proposed as an alternative (or complement) to conventional antibiotics, allowing release of the natural predators of bacteria directly to the site of infection. In the present research effort, production of bacteriophage derivatives (starting from lytic phage particle isolates), encompassing full stabilization of their three-dimensional structure, has been attempted via housing said bacteriophage particles within lipid nanovesicles integrating a multiple water-in-oil-in-water (W/O/W) emulsion. As a proof-of-concept for the aforementioned strategy, bacteriophage particles with broad lytic spectrum were entrapped within the aqueous core of lipid nanoballoons integrating a W/O/W multiple emulsion. Long-term storage of the multiple emulsions produced did not lead to leaching of phage particles, thus proving the effectiveness of the encapsulation procedure.

  4. Isolation and characterization of five lytic bacteriophages infecting a Vibrio strain closely related to Vibrio owensii.

    Science.gov (United States)

    Yu, Yan-Ping; Gong, Ting; Jost, Günter; Liu, Wen-Hua; Ye, De-Zan; Luo, Zhu-Hua

    2013-11-01

    Vibrio owensii is a potential bacterial pathogen in marine aquaculture system. In this study, five lytic phages specific against Vibrio strain B8D, closely related to V. owensii, were isolated from seawater of an abalone farm. The phages were characterized with respect to morphology, genome size, growth phenotype, as well as thermal, and pH stability. All phages were found to belong to the family Siphoviridae with long noncontractile tails and terminal fibers. Restriction analysis indicated that the five phages were dsDNA viruses with molecular weights ranging from c. 30 to 48 kb. One-step growth experiments revealed that the phages were heterogeneous in latent periods (10-70 min), rise periods (40-70 min), and burst sizes [23-331 plaque-forming units (PFU) per infected cell] at the same host strain. All phages were thermal stable and were tolerant to a wide range of pH. The results indicated that these phages could be potential candidates of a phage cocktail for biological control of V. owensii in aquaculture systems.

  5. Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan.

    Science.gov (United States)

    Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz Ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

    2015-01-01

    This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

  6. Lytic phages obscure the cost of antibiotic resistance in Escherichia coli

    Science.gov (United States)

    Tazzyman, Samuel J; Hall, Alex R

    2015-01-01

    The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods. PMID:25268496

  7. Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species.

    Science.gov (United States)

    Hikima, Sonomi; Hikima, Jun ichi; Rojtinnakorn, Jiraporn; Hirono, Ikuo; Aoki, Takashi

    2003-10-16

    Lysozyme cDNA was isolated from a kuruma shrimp, Marsupenaeus japonicus, hemocyte cDNA library. The cDNA consists of 1055 base pairs (bp) and encodes a chicken-type (c-type) lysozyme with a deduced amino acid sequence of 156 residues. The kuruma shrimp lysozyme has a high identity (79.7%) with pacific white shrimp lysozyme, and low to moderate identities (33.3-43.0%) with lysozymes of insects and vertebrates. Comparisons with other c-type lysozymes from invertebrates and vertebrates showed that the two catalytic residues (Glu58 and Asp75) and the eight cysteine residue motif were completely conserved. Two novel insertion sequences were also observed in the kuruma and pacific white shrimp lysozyme amino acid sequences. Interestingly, phylogenetic analysis revealed that the kuruma shrimp lysozyme was more closely related to vertebrate c-type lysozymes. Expression of the cDNA in insect cells, using a baculovirus expression system, yielded a recombinant lysozyme with optimum activity at pH 7.5 and 50 degrees C, as evaluated by a lysoplate assay. The kuruma shrimp lysozyme displayed lytic activities against several Vibrio species and fish pathogens, including Vibrio penaeicida (a pathogenic bacteria to the kuruma shrimp) and suggested that shrimp lysozyme affects a greater variety of pathogens.

  8. Hemoglobin is a co-factor of human trypanosome lytic factor.

    Directory of Open Access Journals (Sweden)

    Justin Widener

    2007-09-01

    Full Text Available Trypanosome lytic factor (TLF is a high-density lipoprotein (HDL subclass providing innate protection to humans against infection by the protozoan parasite Trypanosoma brucei brucei. Two primate-specific plasma proteins, haptoglobin-related protein (Hpr and apolipoprotein L-1 (ApoL-1, have been proposed to kill T. b. brucei both singularly or when co-assembled into the same HDL. To better understand the mechanism of T. b. brucei killing by TLF, the protein composition of TLF was investigated using a gentle immunoaffinity purification technique that avoids the loss of weakly associated proteins. HDL particles recovered by immunoaffinity absorption, with either anti-Hpr or anti-ApoL-1, were identical in protein composition and specific activity for T. b. brucei killing. Here, we show that TLF-bound Hpr strongly binds Hb and that addition of Hb stimulates TLF killing of T. b. brucei by increasing the affinity of TLF for its receptor, and by inducing Fenton chemistry within the trypanosome lysosome. These findings suggest that TLF in uninfected humans may be inactive against T. b. brucei prior to initiation of infection. We propose that infection of humans by T. b. brucei causes hemolysis that triggers the activation of TLF by the formation of Hpr-Hb complexes, leading to enhanced binding, trypanolytic activity, and clearance of parasites.

  9. Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells.

    Science.gov (United States)

    Estes, Kathleen A; Szumowski, Suzannah C; Troemel, Emily R

    2011-09-01

    The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo.

  10. Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay.

    Science.gov (United States)

    Kittl, Roman; Kracher, Daniel; Burgstaller, Daniel; Haltrich, Dietmar; Ludwig, Roland

    2012-10-26

    Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification. Four pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH. P. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components.

  11. Involvement of Noxa in mediating cellular ER stress responses to lytic virus infection.

    Science.gov (United States)

    Rosebeck, Shaun; Sudini, Kuladeep; Chen, Tiannan; Leaman, Douglas W

    2011-09-01

    Noxa is a Bcl-2 homology domain-containing pro-apoptotic mitochondrial protein. Noxa mRNA and protein expression are upregulated by dsRNA or virus, and ectopic Noxa expression enhances cellular sensitivity to virus or dsRNA-induced apoptosis. Here we demonstrate that Noxa null baby mouse kidney (BMK) cells are deficient in normal cytopathic response to lytic viruses, and that reconstitution of the knockout cells with wild-type Noxa restored normal cytopathic responses. Noxa regulation by virus mirrored its regulation by proteasome inhibitors or ER stress inducers and the ER stress response inhibitor salubrinal protected cells against viral cytopathic effects. Noxa mRNA and protein were synergistically upregulated by IFN or dsRNA when combined with ER stress inducers, leading to Noxa/Mcl-1 interaction, activation of Bax and pro-apoptotic caspases, degradation of Mcl-1, loss of mitochondrial membrane potential and initiation of apoptosis. These data highlight the importance of ER stress in augmenting the expression of Noxa following viral infection.

  12. Isolation and characterisation of lytic bacteriophages of Klebsiella pneumoniae and Klebsiella oxytoca.

    Science.gov (United States)

    Karumidze, Natia; Kusradze, Ia; Rigvava, Sophio; Goderdzishvili, Marine; Rajakumar, Kumar; Alavidze, Zemphira

    2013-03-01

    Klebsiella bacteria have emerged as an increasingly important cause of community-acquired nosocomial infections. Extensive use of broad-spectrum antibiotics in hospitalised patients has led to both increased carriage of Klebsiella and the development of multidrug-resistant strains that frequently produce extended-spectrum β-lactamases and/or other defences against antibiotics. Many of these strains are highly virulent and exhibit a strong propensity to spread. In this study, six lytic Klebsiella bacteriophages were isolated from sewage-contaminated river water in Georgia and characterised as phage therapy candidates. Two of the phages were investigated in greater detail. Biological properties, including phage morphology, nucleic acid composition, host range, growth phenotype, and thermal and pH stability were studied for all six phages. Limited sample sequencing was performed to define the phylogeny of the K. pneumoniae- and K. oxytoca-specific bacteriophages vB_Klp_5 and vB_Klox_2, respectively. Both of the latter phages had large burst sizes, efficient rates of adsorption and were stable under different adverse conditions. Phages reported in this study are double-stranded DNA bacterial viruses belonging to the families Podoviridae and Siphoviridae. One or more of the six phages was capable of efficiently lysing ~63 % of Klebsiella strains comprising a collection of 123 clinical isolates from Georgia and the United Kingdom. These phages exhibit a number of properties indicative of potential utility in phage therapy cocktails.

  13. Characterisation and genome sequence of the lytic Acinetobacter baumannii bacteriophage vB_AbaS_Loki

    Science.gov (United States)

    Wand, Matthew E.; Briers, Yves; Lavigne, Rob; Sutton, J. Mark; Reynolds, Darren M.

    2017-01-01

    Acinetobacter baumannii has emerged as an important nosocomial pathogen in healthcare and community settings. While over 100 of Acinetobacter phages have been described in the literature, relatively few have been sequenced. This work describes the characterisation and genome annotation of a new lytic Acinetobacter siphovirus, vB_AbaS_Loki, isolated from activated sewage sludge. Sequencing revealed that Loki encapsulates a 41,308 bp genome, encoding 51 predicted open reading frames. Loki is most closely related to Acinetobacter phage IME_AB3 and more distantly related to Burkholderia phage KL1, Paracoccus phage vB_PmaS_IMEP1 and Pseudomonas phages vB_Pae_Kakheti25, vB_PaeS_SCH_Ab26 and PA73. Loki is characterised by a narrow host range, among the 40 Acinetobacter isolates tested, productive infection was only observed for the propagating host, A. baumannii ATCC 17978. Plaque formation was found to be dependent upon the presence of Ca2+ ions and adsorption to host cells was abolished upon incubation with a mutant of ATCC 17978 encoding a premature stop codon in lpxA. The complete genome sequence of vB_AbaS_Loki was deposited in the European Nucleotide Archive (ENA) under the accession number LN890663. PMID:28207864

  14. Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

    Science.gov (United States)

    Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

    2015-01-01

    Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract. PMID:26691463

  15. Parachlamydia acanthamoeba is endosymbiotic or lytic for Acanthamoeba polyphaga depending on the incubation temperature.

    Science.gov (United States)

    Greub, Gilbert; La Scola, Bernard; Raoult, Didier

    2003-06-01

    Parachlamydiaceae are potential emerging pathogens that naturally infect free-living amoebae. We investigated the affects of incubation temperature on the growth and cytopathic effect of P. acanthamoeba in Acanthamoeba polyphaga. A. polyphaga were infected with P. acanthamoeba and incubated at different temperatures for ten days. Bacterial growth was quantified by real-time PCR. Cytopathic effects were determined by counting the number of cysts and viable amoebae (unstained with trypan blue) in Nageotte counting chambers. Uninfected amoebae cultures were used as negative control. At 32, 35, and 37 degrees C, we observed a significant decrease in the number of viable A. polyphaga that contrasted with the delayed and smaller decrease in the number of living A. polyphaga observed at 25, 28, and 30 degrees C. Higher incubation temperature, which is associated with amoebal lysis, surprisingly was not associated with increased growth rate. P. acanthamoeba is lytic for A. polyphaga at 32-37 degrees C but endosymbiotic at 25-30 degrees C. This suggests that A. polyphaga may be a reservoir of endosymbionts at the lower temperature of the nasal mucosa, which may be liberated by lysis at higher temperature, for instance, when the amoeba is inhaled and reaches the lower respiratory tract.

  16. Identification of a membrane-bound prepore species clarifies the lytic mechanism of actinoporins

    CERN Document Server

    Morante, Koldo; Gil-Cartón, David; Redondo-Morata, Lorena; Sot, Jesús; Scheuring, Simon; Valle, Mikel; González-Mañas, Juan Manuel; Tsumoto, Kouhei; Caaveiro, Jose M M

    2016-01-01

    Pore-forming toxins (PFT) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of $\\beta$-PFT. However, in the class of $\\alpha$-PFT like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C (FraC) bound to lipid vesicles. The size of the prepore coincides that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N-terminus is not inserte...

  17. Characterization of potential lytic bacteriophage against Vibrio alginolyticus and its therapeutic implications on biofilm dispersal.

    Science.gov (United States)

    Sasikala, Dakshinamurthy; Srinivasan, Pappu

    2016-12-01

    Vibrio alginolyticus is a leading cause of vibriosis, presenting opportunistic infections to humans associated with raw seafood contamination. At present, phage therapy that acts as an alternative sanitizing agent is explored for targeting V. alginolyticus. The study outcome revealed that the phage VP01 with its extreme lytic effect showed a high potential impact on the growth of V. alginolyticus as well as biofilm formation. Electron microscopy revealed the phage resemblance to Myoviridae, based on its morphology. Further study clarified that the phage VP01 possesses a broad host spectrum and amazing phage sensitivity at different pH, high thermal stability, and high burst size of 415 PFU/cell. In addition, the investigation of phage co-culturing against this pathogen resulted in a significant growth reduction even at less MOIs 0.1 and 1. These results suggest that the phage could be a promising candidate for the control of V. alginolyticus infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Use of lytic bacteriophages to reduce Salmonella Enteritidis in experimentally contaminated chicken cuts

    Directory of Open Access Journals (Sweden)

    L Fiorentin

    2005-12-01

    Full Text Available Reducing Salmonella contamination in poultry is of major importance to prevent the introduction of this microorganism into the food chain. Salmonellae may spread during storage time (shelf life whenever pre-harvest control fails or post-harvest contamination occurs. Therefore, preventive measures should also be used in the post-harvest level of poultry production in order to control salmonellae. Chicken skin samples were experimentally contaminated by immersing whole legs (thighs and drumsticks in a suspension containing 10(6 colony forming units per milliliter (CFU/mL of Salmonella Enteritidis phage type 4 (SE PT4 at the slaughter day. One day later, samples from one group were immersed in a suspension pool containing 10(9 CFU/mL of each of three wild salmonella-lytic bacteriophages previously isolated from feces of free-range chickens. Salmonella counting was performed at three-day intervals in the chicken legs stored at 5°C and showed a significant reduction (P<0.05 of SE PT4 in bacteriophage-treated cuts on days 3, 6 and 9 post-treatment. These findings suggest that the use of bacteriophages may reduce SE PT4 in chicken skin. Further studies are encouraged and might demonstrate the potential of this approach as an efficient and safe technique to be routinelly used for Salmonella control in chicken products.

  19. Chromatin Modulation of Herpesvirus Lytic Gene Expression: Managing Nucleosome Density and Heterochromatic Histone Modifications

    Directory of Open Access Journals (Sweden)

    Thomas M. Kristie

    2016-03-01

    Full Text Available Like their cellular hosts, herpesviruses are subject to the regulatory impacts of chromatin assembled on their genomes. Upon infection, these viruses are assembled into domains of chromatin with heterochromatic signatures that suppress viral gene expression or euchromatic characteristics that promote gene expression. The organization and modulation of these chromatin domains appear to be intimately linked to the coordinated expression of the different classes of viral genes and thus ultimately play an important role in the progression of productive infection or the establishment and maintenance of viral latency. A recent report from the Knipe laboratory (J. S. Lee, P. Raja, and D. M. Knipe, mBio 7:e02007-15, 2016 contributes to the understanding of the dynamic modulation of chromatin assembled on the herpes simplex virus genome by monitoring the levels of characteristic heterochromatic histone modifications (histone H3 lysine 9 and 27 methylation associated with a model viral early gene during the progression of lytic infection. Additionally, this study builds upon previous observations that the viral immediate-early protein ICP0 plays a role in reducing the levels of heterochromatin associated with the early genes.

  20. Lytic phages obscure the cost of antibiotic resistance in Escherichia coli.

    Science.gov (United States)

    Tazzyman, Samuel J; Hall, Alex R

    2015-03-17

    The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods.

  1. Characterisation and genome sequence of the lytic Acinetobacter baumannii bacteriophage vB_AbaS_Loki.

    Science.gov (United States)

    Turner, Dann; Wand, Matthew E; Briers, Yves; Lavigne, Rob; Sutton, J Mark; Reynolds, Darren M

    2017-01-01

    Acinetobacter baumannii has emerged as an important nosocomial pathogen in healthcare and community settings. While over 100 of Acinetobacter phages have been described in the literature, relatively few have been sequenced. This work describes the characterisation and genome annotation of a new lytic Acinetobacter siphovirus, vB_AbaS_Loki, isolated from activated sewage sludge. Sequencing revealed that Loki encapsulates a 41,308 bp genome, encoding 51 predicted open reading frames. Loki is most closely related to Acinetobacter phage IME_AB3 and more distantly related to Burkholderia phage KL1, Paracoccus phage vB_PmaS_IMEP1 and Pseudomonas phages vB_Pae_Kakheti25, vB_PaeS_SCH_Ab26 and PA73. Loki is characterised by a narrow host range, among the 40 Acinetobacter isolates tested, productive infection was only observed for the propagating host, A. baumannii ATCC 17978. Plaque formation was found to be dependent upon the presence of Ca2+ ions and adsorption to host cells was abolished upon incubation with a mutant of ATCC 17978 encoding a premature stop codon in lpxA. The complete genome sequence of vB_AbaS_Loki was deposited in the European Nucleotide Archive (ENA) under the accession number LN890663.

  2. Phase transition time delays in irradiated superheated superconducting granules

    CERN Document Server

    Abplanalp, M; Czapek, G; Diggelmann, U; Furlan, M; Gabutti, A; Janos, S; Moser, U; Pozzi, R; Pretzl, Klaus P; Schmiemann, K; Perret-Gallix, D; Van den Brandt, B; Konter, J A; Mango, S

    1994-01-01

    The time difference between a particle interaction in a Superheated Superconducting Granule (SSG) and the resulting phase transition signal has been explored. Detectors containing Zn and Sn SSG were irradiated with neutrons and protons to study the heating mechanism taking place in nuclear recoil and ionizing events. Scattered neutrons have been detected by a scintillator hodoscope behind the SSG with a recoil energy measurement resolution of 10\\% and an interaction time resolution of 1ns. The fast transition of the metastable granules allowed to determine the elapsed time between an energy deposition and the phase transition signal. In the case of Sn granules, the results show that the time distributions are narrow and independent of the deposited energy in nuclear recoil and ionizing events. In Zn, however, the time distributions are much broader and depend on the energy deposition in the granule.

  3. Treatment of hyper-granulated limb wounds in horses

    Directory of Open Access Journals (Sweden)

    O. A. Bader

    2011-01-01

    Full Text Available This study was performed to investigate the different methods of treating hyper granulation tissue on experimentally induced wounds in equine limbs. Wounds were induced by removal of a skin patch and subcutaneous tissue for about 5-7 cm width and 6-8 cm in length from the dorsal and lateral aspect of the fore and hind limbs below the carpal and tarsal joints. The wounds were left open without treatment and the animals were trained 2-2.5 hours every day for about 3-5 weeks until hyper granulation tissue was developed. The schedule for the treatment of hyper granulation was divided into five groups each contained eight wounds of hyper granulation tissue; each main group was divided into two subgroups. The subgroups of first, second, third, fourth and fifth groups were treated by the following schedules: bandage alone; copper sulphate ointment 10%; silver nitrate ointment 2%; red mercury ointment 11%; and laser therapy (at a total dose of 9.72 Joule / cm2 respectively. While the second subgroups were treated by surgical resection of the hyper granulation tissue, followed by the same treatments applied on the first subgroup. The bandage for all experimental groups was changed every 48 hours until healing was occurred. The clinical and histological observation of the first group revealed that the healing take long period comparing with other groups. The mean of wound healing were 65 days in non surgical removal of hyper granulation tissue subgroup, while 57 days in surgical removed of hyper granulation tissue subgroup. The results of the second, third, fourth groups revealed that the caustic material especially red mercury has a role in healing processes through depressing the hyper granulation tissue. The mean of wound healing of the second group was 42.25 days in non surgical removal of hyper granulation tissue subgroup while 37.25 days in surgically removed hyper granulation tissue subgroup. In the third group the mean of wound healing was 45

  4. Heterogeneity of secretory granules of silent pituitary adenomas

    DEFF Research Database (Denmark)

    Holck, S; Wewer, U M; Albrechtsen, R

    1988-01-01

    Silent pituitary adenomas were compared with hormonally active tumors taking into account the size, number, and ultrastructural characteristics of secretory granules (SG). The study group (a total of 79 primary pituitary adenomas) comprised 27 silent, 21 growth hormone (GH)-producing-, 16 prolactin...... (PRL)-producing-, 5 GH-PRL-producing- and 10 adrenocorticotropic hormone (ACTH)-producing adenomas. The SG of silent adenomas were significantly smaller than SG in endocrine active adenomas. All hormonally inactive tumors also contained small (mean, 94 nm) specific cytoplasmic granules, designated...... approximately 10 to 50% of the granules in each cell. These granules were not seen in hormonally active tumors and considered therefore diagnostic of silent pituitary adenomas....

  5. In vitro binding of puroindolines to wheat starch granules

    DEFF Research Database (Denmark)

    Sørensen, Helle Aagaard; Darlington, H.F.; Shewry, P.R.

    2001-01-01

    Puroindoline (pin) preparations made from flours of hard and soft wheats contained a mixture of pin-a, 0.19/0.53 alpha -amylase inhibitor, and purothionins. Starch granule preparations from the same cultivars were treated with proteinase to remove surface proteins and incubated with solutions...... of the pin preparations. Binding of pin-a and purothionins but not the 0.19/0.53 inhibitor was observed with no apparent differences between the behavior of the pin preparations or starch granule preparations from hard or soft types. No binding was observed when several other proteins (bovine serum albumin......, total albumins, a commercial preparation of wheat alpha -amylase inhibitors, and barley beta -amylase) were incubated with the starch granules under the same conditions, indicating that in vitro binding can be used to study specific starch granule and protein interactions....

  6. The Distribution of Synaptotagmin Ⅱ in RBL-2H3 and Its Regulation on Exocytosis of Lysosomes in RBL-2H3

    Institute of Scientific and Technical Information of China (English)

    Jicheng Zhang; Jianmin Wu; Shixiu Pan; Wenli Lv

    2005-01-01

    Synaptotagmin (Syt) constitutes a family of membrane-trafficking proteins, so far nearly 20 Syts have been discovered. Extensive work showed that synatotagmins were a potential Ca2+ sensor for regulated exocytosis. This study was to investigate the expression and location of synaptotagmin Ⅱ (Syt2) in RBL-2H3 (RBL) and its role in regulating exocytosis of RBL. The expression of Syt2 in RBL was confirmed by Western blot. The recombinant expression vector pEGFP-N1-Syt2 was constructed and transfected into RBL by electroporation, the stable transfectant RBL-Syt2-S expressing fusion protein Syt2-EGFP were obtained and Syt2 was highly concentrated at plasma membrane with little detected in cytoplasm. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. Compared with control, the release of cathepsin D by RBL-Syt2-S was markedly decreased. The results indicated that Syt2 played a negative regulation in exocytosis of lysosomes in RBL.

  7. Development of granules from Phyllanthus niruri spray-dried extract

    Directory of Open Access Journals (Sweden)

    Tatiane Pereira de Souza

    2009-12-01

    Full Text Available The aim of this study was to develop granules from Phyllanthus niruri spray-dried extract using dry and wet granulation and to assess techniques to enable the production of granules with improved technological characteristics and yields. Granules were characterized by granulometry, reological parameters, compression and hygroscopic behavior. Independent of the granulation technique, technologically developed granules presented particle diameter, bulk and tapped densities and compressibility indexes suitable for a solid dosage form. The compression behavior showed plastic and fragmentary deformation for granules produced by the dry granulation technique and predominantly plastic deformation for wet granulation. Concerning the humidity sorption, the study showed that granules absorb less humidity than the spray-dried extract. However, granules with Eudragit® E 100 were the least hygroscopic.O objetivo deste estudo foi desenvolver grânulos de extrato Phyllantus niruri seco por aspersão e por granulação úmida e avaliar técnicas que possibilitem a produção de grânulos com características tecnológicas e rendimentos aperfeiçoados. Os grânulos foram caracterizados por granulometria, parâmetros reológicos, compressão e comportamento higroscópico. Independentemente da técnica de granulação, os grânulos tecnologicamente desenvolvidos apresentaram diâmetro de partículas, densidades aparente e compactada e índices de compressibilidade adequados para a formulação sólida. O comportamento de compressão mostrou deformação plástica e elástica para os grânulos produzidos por técnicas de granulação seca e, predominantemente, deformação plástica para a granulação úmida. Com relação à absorção da umidade, o estudo mostrou que os grânulos absorvem menos umidade do que o extrato seco por aspersão. Entretanto, os grânulos com Eudragit E 100 foram os menos higroscópicos.

  8. Fluidization of wet granulates under shear.

    Science.gov (United States)

    Rahbari, S H Ebrahimnazhad; Vollmer, J; Herminghaus, S; Brinkmann, M

    2010-12-01

    Small amounts of a wetting liquid render sand a stiff and moldable material. The cohesive forces between the sand grains are caused by capillary bridges at the points of contact. Due to the finite strength of these bridges wet sand undergoes a transition from an arrested (i.e., solidified) to a fluidized state under an externally applied shear force. The transition between these two dynamic states is studied in a MD-type simulation of a two-dimensional assembly of bidisperse frictionless disks under the action of a cosine force profile. In addition to soft core repulsion the disks interact through a hysteretic and short ranged attractive force modeling the effect of the capillary bridges. In this model the transition between the fluidized and the arrested state is discontinuous and hysteretic. The parameter dependence of the critical force for solidification is modeled by combining theoretical arguments with a detailed numerical exploration of the transition. We address a range of densities from slightly below close packing until slightly above densities where the system approaches a shear-banded state. Differences and similarities of the transition in wet granulates to the jamming transition are also addressed.

  9. Giant arachnoid granulation mimicking dural sinus thrombosis.

    Science.gov (United States)

    Ayaz, Ercan; Atalay, Basak; Baysal, Begumhan; Senturk, Senem; Aslan, Ahmet

    2017-01-01

    Arachnoid granulations (AG) are composed of dense, collagenous connective tissue that includes clusters of arachnoid cells. They tend to invaginate into the dural sinuses, through which cerebrospinal fluid enters the venous system. AG are most commonly seen at the junction between the middle and lateral thirds of the transverse sinuses near the entry sites of the superficial veins. Presently described is the case of a 21-year-old female who presented at the clinic with recurrent headaches. Magnetic resonance (MR) imaging revealed a 3.5-cm lesion, which extended from confluens sinuum through the superior sagittal sinus. The lesion had created a scallop-shaped area of erosion in the neighboring occipital bone. To exclude sinus thrombosis, MR venography was performed, which displayed a maintained venous flow around the lesion. Headaches were treated symptomatically with medical therapy. Giant AG can be misdiagnosed as dural sinus thrombosis. MR imaging combined with MR venography is the most useful diagnostic tool to differentiate giant AG from dural sinus thrombosis.

  10. Brain Herniation into Giant Arachnoid Granulation: An Unusual Case

    Science.gov (United States)

    Santos, Gonçalo Roque

    2017-01-01

    Arachnoid granulations are structures filled with cerebrospinal fluid (CSF) that extend into the venous sinuses through openings in the dura mater and allow the drainage of CSF from subarachnoid space into venous system. Usually they are asymptomatic but can be symptomatic when large enough to cause sinus occlusion. We report a rare case of a brain herniation into a giant arachnoid granulation in an asymptomatic elderly male patient, which was discovered incidentally. PMID:28392955

  11. Preparation of Glass Ceramic Based on Granulated Slag and Cullet

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The glass-ceramic was prepared on the basis of materials of granulated slag containing high-calcium oxide and cullet.The content of granulated slag ranges from 50%-60%wt in the glass compositions. The samples were analyzed by DTA, SEM and XRD.The results show that the main crystal phase of the glass-ceramic is β-CaSiO3,Which is in scattering fiber or column form.The applying properties have also been measured.

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

    OpenAIRE

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

    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 β-Catenin in anaphase cells was analysed, which showed that there was a significant asymmetry in the distribution of β-Catenin in dividing GNPs. Further, inhibition of S...

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

    Science.gov (United States)

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

    2015-05-15

    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 β-Catenin in anaphase cells was analysed, which showed that there was a significant asymmetry in the distribution of β-Catenin in dividing GNPs. Further, inhibition of Sonic Hedgehog (Shh) signalling had an effect on plane of cell division. Asymmetric distribution of β-Catenin was shown to occur towards the source of a localized extracellular cue.

  14. Quantitative analysis of synaptic vesicle pool replenishment in cultured cerebellar granule neurons using FM dyes.

    Science.gov (United States)

    Cheung, Giselle; Cousin, Michael A

    2011-11-11

    After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis(1,2). The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation(1,2). It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli. FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals(3-8). They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane(9). Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal(10), the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified. Here, we present a protocol that has been modified to

  15. Preventive effect of Ganfujian granule on experimental hepatocarcinoma in rats

    Institute of Scientific and Technical Information of China (English)

    Yan Qian; Chang-Quan Ling

    2004-01-01

    AIM: To investigate the inhibitory effect of dietary and medicinal formula Ganfujian granule on diethylnitrosamine (DEN)-induced hepatocarcinoma in rats.METHODS: Male SD rats had free access to water containing 0.1 g/L DEN for 16 weeks, during which the rats fed with standard diet or administration of Ganfujian granule (30.4 g/Kg in diet). At weeks 4, 8, 12 and 16 of hepatocarcinogenesis 5 rats of each group were sacrificed, and at week 20 another 30 rats were sacrificed from each group. The end point for survival observation was at week 28.Immunochemistry methods were used to examine the effect of Ganfujian granule on the process of hepatocarcinogenesis including proliferation of hepatocytes and cell cycle modulation.RESULTS: Ganfujian granule could reduce and delay the incidence of hepatocarcinoma in rats and prolong the survival of animals. In addition, Ganfujian granule had a marked inhibitory effect on high expression of cyclin dependent kinase (CDK4) during the whole process of hepatocarcinogenesis and cyclin D1 at week 16 and the number of proliferating cell nuclear antigen (PCNA) positive cells in different stages of hepatocarcinogenesis.CONCLUSION: Ganfujian granule can reduce and delay the incidence of hepatocarcinoma in rats by exerting direct or indirect effects on cell cycle and inhibiting uncontrolled proliferation of hepatocytes.

  16. Monitoring exocytosis and release from individual mast cells by capillary electrophoresis and UV imaging microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Yeung, E.S. [Ames Lab., IA (United States)]|[Iowa State Univ., Ames, IA (United States); Lillard, S.J. [Stanford Univ., CA (United States); McCloskey, M.A. [Iowa State Univ., Ames, IA (United States)

    1997-12-31

    The complex temporal evolution of on-column exocytotic release of serotonin from individual peritoneal mast cells (RPMCs) was monitored by using capillary electrophoresis and UV imaging microscopy. Laser-induced native fluorescence detection with 275-nm excitation was used, and a detection limit of 1.7 amol (S/N = 3; rms) was obtained for serotonin. A physiological running buffer was used to ensure that the cell remained viable throughout. The secretagogue was polymyxin B sulfate (Pmx). Following the injection of a single mast cell into the capillary, electromigration of Pmx toward and past the cell induced degranulation and release of serotonin. The time course of release was registered in the electropherograms with subsecond resolution. Subsequent introduction of SDS caused the cell to lyse completely and allowed the residual serotonin to be quantified. The average amount of serotonin observed per RPMC was 1.6 {+-} 0.6 fmol; the average percentage of serotonin released was 28 {+-} 14%. Events that are consistent with released serontonin from single submicron granules (250 aL each) were evident, each of which contained an average amount of 5.9 {+-} 3 amol. Alternatively, UV movies can be taken of the entire event to provide temporal and spatial information.

  17. The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis.

    Science.gov (United States)

    Bierings, Ruben; Hellen, Nicola; Kiskin, Nikolai; Knipe, Laura; Fonseca, Ana-Violeta; Patel, Bijal; Meli, Athina; Rose, Marlene; Hannah, Matthew J; Carter, Tom

    2012-09-27

    Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis.

  18. Investigation of Physicochemical Drug Properties to Prepare Fine Globular Granules Composed of Only Drug Substance in Fluidized Bed Rotor Granulation

    National Research Council Canada - National Science Library

    Mise, Ryohei; Iwao, Yasunori; Kimura, Shin-ichiro; Osugi, Yukiko; Noguchi, Shuji; Itai, Shigeru

    2015-01-01

    ...%) drug-loading formulation using fluidized bed rotor granulation. Three drugs: acetaminophen (APAP); ibuprofen (IBU); and ethenzamide (ETZ) were used as model drugs based on their differences in wettability and particle size distribution...

  19. Twin screw wet granulation: the study of a continuous twin screw granulator using Positron Emission Particle Tracking (PEPT) technique.

    Science.gov (United States)

    Lee, Kai T; Ingram, Andy; Rowson, Neil A

    2012-08-01

    In this paper, Positron Emission Particle Tracking (PEPT) techniques are utilised to track the trajectory of single particles through the mixing and conveying zones of a Twin Screw Granulator (TSG). A TSG consisting of conveying zones and mixing zones is used in this study. The mixing zones are arranged with kneading discs at an angle of 30°, 60° or 90°. Experiments were carried out using different mixing configurations with various screw speed and total mass flow rate. The PEPT data obtained were then utilised to obtain the residence time distribution (RTD) and the Peclet number in an attempt to gain some insight into the mixing of the process. The fill level of the granulator was also estimated to study the mechanism of granulation. As might be expected, it was shown that the residence time of the granulation process increases with decreasing screw speed. It also increases with increasing angle of the arrangement of kneading blocks in the mixing zones, but will decreases when powder feed rate is increased. The fill level of the mixing zone in particular increases when the screw speed decreases or when powder feed rate increases. Furthermore, the fill level of the granulator will increase when the mixing zone configuration changes from 30° to 90°. It is shown that the granulator is never fully filled, even using 90° mixer elements implying limited compaction which may explain why the granules produced are porous compared with those from a high shear mixer. Interestingly, the RTD analysis reveals that the extent of axial mixing in the mixing zone of the granulator does not change significantly for different configurations and process conditions. There is evidence of a tail in the RTD which implies some material hold up and channelling.

  20. Impact of screw elements on continuous granulation with a twin-screw extruder.

    Science.gov (United States)

    Djuric, Dejan; Kleinebudde, Peter

    2008-11-01

    The influence of different screw element types on wet granulation process with a twin-screw extruder was investigated. Lactose granules were prepared with different screw configurations such as conveying, combing mixer and kneading elements. The use of kneading blocks led to an almost complete agglomeration of lactose, whereas kneading and combing mixer elements resulted in smaller granules in comparison. Granule porosity varied between 17.4% and 50.6%. Granule friability values ranged from 1.2% to 38.5%. Conveying elements led to the most porous and friable granules, whereas kneading blocks produced the densest and least friable granules. Combing mixer elements produced granules with median properties. A linear correlation between granule porosity and the natural logarithm of granule friability was detected. Flowability of granules was also influenced by the element type. Compressed granules with higher granule porosities resulted in tablets with higher tensile strength values and vice versa. Twin-screw extruders proved to be a versatile tool for wet granulation. By the choice of a suitable screw element granule and tablet characteristics were influenced.

  1. Synaptobrevin N-terminally bound to syntaxin-SNAP-25 defines the primed vesicle state in regulated exocytosis

    DEFF Research Database (Denmark)

    Walter, Alexander M; Wiederhold, Katrin; Bruns, Dieter;

    2010-01-01

    ) to the syntaxin-SNAP-25 (target membrane SNAREs) acceptor complex or whether the reaction is arrested upstream of that step. In this study, by a combination of in vitro biophysical measurements and time-resolved exocytosis measurements in adrenal chromaffin cells, we find that mutations of the N......-terminal interaction layers of the SNARE bundle inhibit assembly in vitro and vesicle priming in vivo without detectable changes in triggering speed or fusion pore properties. In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration. Between the two domains, we identify...... a region exquisitely sensitive to mutation, possibly constituting a switch. Our data are consistent with a model in which the N terminus of the SNARE complex assembles during vesicle priming, followed by Ca(2+)-triggered C-terminal assembly and membrane fusion....

  2. Involvement of complexin 2 in docking, locking and unlocking of different SNARE complexes during sperm capacitation and induced acrosomal exocytosis.

    Directory of Open Access Journals (Sweden)

    Pei-Shiue J Tsai

    Full Text Available Acrosomal exocytosis (AE is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM with the outer acrosomal membrane (OAM. This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3. By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1 recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2 the assembly of a fusion-driving SNARE complex which executes Ca(2+-dependent AE, (3 the disassembly of the earlier reported docking SNARE complex, (4 the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca(2+ which are all involved in AE make sperm an ideal model for studying exocytosis.

  3. Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity-dependent bulk endocytosis.

    Science.gov (United States)

    Morton, Andrew; Marland, Jamie R K; Cousin, Michael A

    2015-08-01

    Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine-dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium-dependent events such as activity-dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity-dependent dynamin I dephosphorylation was also arrested in EGTA-treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. To determine the minimal requirements for ADBE triggering, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. We found that SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient to trigger ADBE.

  4. DCP-LA stimulates AMPA receptor exocytosis through CaMKII activation due to PP-1 inhibition.

    Science.gov (United States)

    Kanno, Takeshi; Yaguchi, Takahiro; Nagata, Tetsu; Tanaka, Akito; Nishizaki, Tomoyuki

    2009-10-01

    The linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) by inhibiting protein phosphatase-1 (PP-1). DCP-LA induced a transient huge facilitation of synaptic transmission monitored from the CA1 region of rat hippocampal slices, which was largely inhibited by the CaMKII inhibitor KN-93. DCP-LA potentiated kainate-evoked whole-cell membrane currents for Xenopus oocytes expressing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors composed of the GluR1, GluR3, GluR1/GluR2, GluR1/GluR3, and GluR1/GluR2/GluR3 subunits, and the potentiation was significantly inhibited by KN-93. A similar potentiation was still found with mutant GluR1 (S831A) receptor lacking CaMKII phosphorylation site. The GluR1 and GluR2 subunits formed AMPA receptors in the rat hippocampus, and DCP-LA increased expression of both the subunits on the plasma membrane. The DCP-LA action was blocked by KN-93 and the exocytosis inhibitor botulinum toxin type A, but not by the endocytosis inhibitor phenylarsine oxide. DCP-LA, thus, appears to activate CaMKII through PP-1 inhibition, that stimulates AMPA receptor exocytosis to increase expression of the receptors on the plasma membrane, responsible for potentiate AMPA receptor responses and facilitation of hippocampal synaptic transmission.

  5. Molecular characterization of Trypanosoma cruzi SAP proteins with host-cell lysosome exocytosis-inducing activity required for parasite invasion.

    Science.gov (United States)

    Zanforlin, Tamiris; Bayer-Santos, Ethel; Cortez, Cristian; Almeida, Igor C; Yoshida, Nobuko; da Silveira, José Franco

    2013-01-01

    To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms. Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP

  6. Using lytic bacteriophages to eliminate or significantly reduce contamination of food by foodborne bacterial pathogens.

    Science.gov (United States)

    Sulakvelidze, Alexander

    2013-10-01

    Bacteriophages (also called 'phages') are viruses that kill bacteria. They are arguably the oldest (3 billion years old, by some estimates) and most ubiquitous (total number estimated to be 10(30) -10(32) ) known organisms on Earth. Phages play a key role in maintaining microbial balance in every ecosystem where bacteria exist, and they are part of the normal microflora of all fresh, unprocessed foods. Interest in various practical applications of bacteriophages has been gaining momentum recently, with perhaps the most attention focused on using them to improve food safety. That approach, called 'phage biocontrol', typically includes three main types of applications: (i) using phages to treat domesticated livestock in order to reduce their intestinal colonization with, and shedding of, specific bacterial pathogens; (ii) treatments for decontaminating inanimate surfaces in food-processing facilities and other food establishments, so that foods processed on those surfaces are not cross-contaminated with the targeted pathogens; and (iii) post-harvest treatments involving direct applications of phages onto the harvested foods. This mini-review primarily focuses on the last type of intervention, which has been gaining the most momentum recently. Indeed, the results of recent studies dealing with improving food safety, and several recent regulatory approvals of various commercial phage preparations developed for post-harvest food safety applications, strongly support the idea that lytic phages may provide a safe, environmentally-friendly, and effective approach for significantly reducing contamination of various foods with foodborne bacterial pathogens. However, some important technical and nontechnical problems may need to be addressed before phage biocontrol protocols can become an integral part of routine food safety intervention strategies implemented by food industries in the USA.

  7. Characterization of four lytic transducing bacteriophages of luminescent Vibrio harveyi isolated from shrimp (Penaeus monodon) hatcheries.

    Science.gov (United States)

    Thiyagarajan, Sanjeevi; Chrisolite, Bagthasingh; Alavandi, Shankar V; Poornima, Modem; Kalaimani, Natarajan; Santiago, T Chinnappan

    2011-12-01

    Four lytic bacteriophages designated as φVh1, φVh2, φVh3, and φVh4 were isolated from commercial shrimp hatcheries, possessing broad spectrum of infectivity against luminescent Vibrio harveyi isolates, considering their potential as biocontrol agent of luminescent bacterial disease in shrimp hatcheries, and were characterized by electron microscopy, genomic analysis, restriction enzyme analysis (REA), and pulsed-field gel electrophoresis (PFGE). Three phages φVh1, φVh2, and φVh4 had an icosahedral head of 60-115 nm size with a long, noncontractile tail of 130-329 × 1-17 nm, belonged to the family Siphoviridae. φVh3 had an icosahedral head (72 ± 5 nm) with a short tail (27 × 12 nm) and belonged to Podoviridae. REA with DraI and PFGE of genomic DNA digested with ScaI and XbaI and cluster analysis of their banding patterns indicated that φVh3 was distinct from the other three siphophages. PFGE-based genome mean size of the four bacteriophages φVh1, φVh2, φVh3, and φVh4 was estimated to be about 85, 58, 64, and 107 kb, respectively. These phages had the property of generalized transduction as demonstrated by transduction with plasmid pHSG 396 with frequencies ranging from 4.1 × 10(-7) to 2 × 10(-9) per plaque-forming unit, suggesting a potential ecological role in gene transfer among aquatic vibrios.

  8. killerFLIP: a novel lytic peptide specifically inducing cancer cell death.

    Science.gov (United States)

    Pennarun, B; Gaidos, G; Bucur, O; Tinari, A; Rupasinghe, C; Jin, T; Dewar, R; Song, K; Santos, M T; Malorni, W; Mierke, D; Khosravi-Far, R

    2013-10-31

    One of the objectives in the development of effective cancer therapy is induction of tumor-selective cell death. Toward this end, we have identified a small peptide that, when introduced into cells via a TAT cell-delivery system, shows a remarkably potent cytoxicity in a variety of cancer cell lines and inhibits tumor growth in vivo, whereas sparing normal cells and tissues. This fusion peptide was named killerFLIP as its sequence was derived from the C-terminal domain of c-FLIP, an anti-apoptotic protein. Using structure activity analysis, we determined the minimal bioactive core of killerFLIP, namely killerFLIP-E. Structural analysis of cells using electron microscopy demonstrated that killerFLIP-E triggers cell death accompanied by rapid (within minutes) plasma membrane permeabilization. Studies of the structure of the active core of killerFLIP (-E) indicated that it possesses amphiphilic properties and self-assembles into micellar structures in aqueous solution. The biochemical properties of killerFLIP are comparable to those of cationic lytic peptides, which participate in defense against pathogens and have also demonstrated anticancer properties. We show that the pro-cell death effects of killerFLIP are independent of its sequence similarity with c-FLIPL as killerFLIP-induced cell death was largely apoptosis and necroptosis independent. A killerFLIP-E variant containing a scrambled c-FLIPL motif indeed induced similar cell death, suggesting the importance of the c-FLIPL residues but not of their sequence. Thus, we report the discovery of a promising synthetic peptide with novel anticancer activity in vitro and in vivo.

  9. Chemical modification of methionines in a cobra venom cytotoxin differentiates between lytic and binding domains.

    Science.gov (United States)

    Stevens-Truss, R; Hinman, C L

    1996-08-01

    Cytotoxin-III from Naja naja atra (CTX) was chemically modified at either or both of its two methionine residues: Over 50% oxidation of methionine-26 occurred with a 1:1 molar ratio of chloramine-T:methionine; at a 5:1 molar ratio, methionine-26 was almost completely oxidized, while methionine-24 was modified only 26%; at a 10:1 molar ratio, both methionines were completely oxidized. Each oxidized derivative demonstrated a lower toxicity toward T-cells than toward heart cells. Conversely, binding to heart cells was affected more than binding to T-cells. Cyanogen bromide cleaved native CTX at both methionines, excising phenyl-alanine-25 and methionine-26 and converting methionine-24 to homoserine lactone. This treatment of CTX eliminated cytotoxicity toward both heart and T-cells, but had only a modest effect upon T-cell binding, as had 50% oxidation of methionine-26, suggesting that CTX lytic and binding regions may be distinct. A selective loss in heart cell binding following oxidation of methionine-24 further suggests that different parts of CTX may interact with the two types of target cells. Perturbation of the relatively flat hydrophobic surface of the CTX' triple-stranded beta-sheet could result from the introduction of negative charge due to methionine-24 oxidation. Alternatively, amino acid side chain participation in a CTX binding domain may be altered by the potential formation of a new hydrogen bond between tyrosine-51 and methionine-24 sulfoxide, as revealed by computer modeling of the completely oxidized CTX derivative.

  10. Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry.

    Science.gov (United States)

    Westereng, Bjørge; Arntzen, Magnus Ø; Agger, Jane Wittrup; Vaaje-Kolstad, Gustav; Eijsink, Vincent G H

    2017-01-01

    Lytic polysaccharide monooxygenases perform oxidative cleavage of glycosidic bonds in various polysaccharides. The majority of LMPOs studied so far possess activity on either cellulose or chitin and analysis of these activities is therefore the main focus of this review. Notably, however, the number of LPMOs that are active on other polysaccharides is increasing. The products generated by LPMOs from cellulose are either oxidized in the downstream end (at C1) or upstream end (at C4), or at both ends. These modifications only result in small structural changes, which makes both chromatographic separation and product identification by mass spectrometry challenging. The changes in physicochemical properties that are associated with oxidation need to be considered when choosing analytical approaches. C1 oxidation leads to a sugar that is no longer reducing but instead has an acidic functionality, whereas C4 oxidation leads to products that are inherently labile at high and low pH and that exist in a keto-gemdiol equilibrium that is strongly shifted toward the gemdiol in aqueous solutions. Partial degradation of C4-oxidized products leads to the formation of native products, which could explain why some authors claim to have observed glycoside hydrolase activity for LPMOs. Notably, apparent glycoside hydrolase activity may also be due to small amounts of contaminating glycoside hydrolases since these normally have much higher catalytic rates than LPMOs. The low catalytic turnover rates of LPMOs necessitate the use of sensitive product detection methods, which limits the analytical possibilities considerably. Modern liquid chromatography and mass spectrometry have become essential tools for evaluating LPMO activity, and this chapter provides an overview of available methods together with a few novel tools. The methods described constitute a suite of techniques for analyzing oxidized carbohydrate products, which can be applied to LPMOs as well as other carbohydrate

  11. Antibacterial efficacy of lytic bacteriophages against antibiotic-resistant Klebsiella species.

    Science.gov (United States)

    Karamoddini, M Khajeh; Fazli-Bazzaz, B S; Emamipour, F; Ghannad, M Sabouri; Jahanshahi, A R; Saed, N; Sahebkar, A

    2011-07-07

    Bacterial resistance to antibiotics is a leading and highly prevalent problem in the treatment of infectious diseases. Bacteriophages (phages) appear to be effective and safe alternatives for the treatment of resistant infections because of their specificity for bacterial species and lack of infectivity in eukaryotic cells. The present study aimed to isolate bacteriophages against Klebsiella spp. and evaluate their efficacy against antibiotic-resistant species. Seventy-two antibiotic-resistant Klebsiella spp. were isolated from samples of patients who referred to the Ghaem Hospital (Mashhad, Iran). Lytic bacteriophages against Klebsiella spp. were isolated from wastewater of the septic tank of the same hospital. Bactericidal activity of phages against resistant Klebsiella spp. was tested in both liquid (tube method; after 1 and 24 h of incubation) and solid (double-layer agar plate method; after 24 h of incubation) phases. In each method, three different concentrations of bacteriophages (low: 10(7) PFU/mL) were used. Bacteriophages showed promising bactericidal activity at all assessed concentrations, regardless of the test method and duration of incubation. Overall, bactericidal effects were augmented at higher concentrations. In the tube method, higher activity was observed after 24 h of incubation compared to the 1-h incubation. The bactericidal effects were also higher in the tube method compared to the double-layer agar plate method after 24 h of incubation. The findings of the present study suggest that bacteriophages possess effective bactericidal activity against resistant Klebsiella spp. These bactericidal activities are influenced by phage concentration, duration of incubation, and test method.

  12. In vitro model for lytic replication, latency, and transformation of an oncogenic alphaherpesvirus.

    Science.gov (United States)

    Schermuly, Julia; Greco, Annachiara; Härtle, Sonja; Osterrieder, Nikolaus; Kaufer, Benedikt B; Kaspers, Bernd

    2015-06-09

    Marek's disease virus (MDV) is an alphaherpesvirus that causes deadly T-cell lymphomas in chickens and serves as a natural small animal model for virus-induced tumor formation. In vivo, MDV lytically replicates in B cells that transfer the virus to T cells in which the virus establishes latency. MDV also malignantly transforms CD4+ T cells with a T(reg) signature, ultimately resulting in deadly lymphomas. No in vitro infection system for primary target cells of MDV has been available due to the short-lived nature of these cells in culture. Recently, we characterized cytokines and monoclonal antibodies that promote survival of cultured chicken B and T cells. We used these survival stimuli to establish a culture system that allows efficient infection of B and T cells with MDV. We were able to productively infect with MDV B cells isolated from spleen, bursa or blood cultured in the presence of soluble CD40L. Virus was readily transferred from infected B to T cells stimulated with an anti-TCRαVβ1 antibody, thus recapitulating the in vivo situation in the culture dish. Infected T cells could then be maintained in culture for at least 90 d in the absence of TCR stimulation, which allowed the establishment of MDV-transformed lymphoblastoid cell lines (LCL). The immortalized cells had a signature comparable to MDV-transformed CD4+ α/β T cells present in tumors. In summary, we have developed a novel in vitro system that precisely reflects the life cycle of an oncogenic herpesivrus in vivo and will allow us to investigate the interaction between virus and target cells in an easily accessible system.

  13. Antibacterial Efficacy of Lytic Bacteriophages against Antibiotic-Resistant Klebsiella Species

    Directory of Open Access Journals (Sweden)

    M. Khajeh Karamoddini

    2011-01-01

    Full Text Available Bacterial resistance to antibiotics is a leading and highly prevalent problem in the treatment of infectious diseases. Bacteriophages (phages appear to be effective and safe alternatives for the treatment of resistant infections because of their specificity for bacterial species and lack of infectivity in eukaryotic cells. The present study aimed to isolate bacteriophages against Klebsiella spp. and evaluate their efficacy against antibiotic-resistant species. Seventy-two antibiotic-resistant Klebsiella spp. were isolated from samples of patients who referred to the Ghaem Hospital (Mashhad, Iran. Lytic bacteriophages against Klebsiella spp. were isolated from wastewater of the septic tank of the same hospital. Bactericidal activity of phages against resistant Klebsiella spp. was tested in both liquid (tube method; after 1 and 24 h of incubation and solid (double-layer agar plate method; after 24 h of incubation phases. In each method, three different concentrations of bacteriophages (low: 107 PFU/mL were used. Bacteriophages showed promising bactericidal activity at all assessed concentrations, regardless of the test method and duration of incubation. Overall, bactericidal effects were augmented at higher concentrations. In the tube method, higher activity was observed after 24 h of incubation compared to the 1-h incubation. The bactericidal effects were also higher in the tube method compared to the double-layer agar plate method after 24 h of incubation. The findings of the present study suggest that bacteriophages possess effective bactericidal activity against resistant Klebsiella spp. These bactericidal activities are influenced by phage concentration, duration of incubation, and test method.

  14. Cyclin-dependent kinase activity controls the onset of the HCMV lytic cycle.

    Directory of Open Access Journals (Sweden)

    Martin Zydek

    Full Text Available The onset of human cytomegalovirus (HCMV lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2 cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state.

  15. Lytic effects of normal serum on isolated postonchospheral and metacestode stages of Taenia taeniaeformis.

    Science.gov (United States)

    Conder, G A; Picone, J; Geary, A M; deHoog, J; Williams, J F

    1983-06-01

    Postonchospheral stages of Taenia taeniaeformis liberated from rat livers by enzymatic digestion at 1 to 10 days postinfection (DPI) and metacestodes dissected from infected livers at 22, 34, and 69 DPI were exposed in vitro to immune rat serum (IRS) and to normal serum from rats (NRS), human beings (NHS), or guinea pigs (NGS). The onset of rapid and destructive tegumental changes in all organisms exposed to any of the sera was demonstrated to be complement-dependent because the reaction was: (a) inhibited by treatment of serum at 56 C for 30 min; (b) inhibited by prior incubation of serum with zymosan or with complement-fixing, soluble products derived from larvae of T. taeniaeformis maintained in vitro (IVP); and (c) abolished by the addition of EDTA. Lytic effects occurred on exposure to agammaglobulinemic sheep serum, and lysis in the presence of IRS and NRS was shown to result in consumption of available hemolytic complement. Surface changes consisted of vesiculation in the microvillar or microthrix layers followed by sloughing of the tegument, eventually leading to collapse of the cystic bladder and cessation of flame cell activity, or, in the case of early postonchospheral forms, complete disintegration of the organism. When IVP was added to NHS, reduction of hemolytic complement activity was associated with the electrophoretic conversion of C3, and Factor B, but there was little or no consumption of C1. The observations support the hypothesis that complement-mediated effector mechanisms must be counteracted to ensure survival of parasites in vivo, and that the capacity for release of soluble nonspecific complement-fixing factors by taeniid larvae may have an important role to play in this process.

  16. EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization.

    Science.gov (United States)

    Pérez-Quintero, Luis-Alberto; Roncagalli, Romain; Guo, Huaijian; Latour, Sylvain; Davidson, Dominique; Veillette, André

    2014-04-07

    Ewing's sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase Cγ, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation.

  17. Identification of a major QTL controlling the content of B-type starch granules in Aegilops

    Science.gov (United States)

    Howard, Thomas; Rejab, Nur Ardiyana; Griffiths, Simon; Leigh, Fiona; Leverington-Waite, Michelle; Simmonds, James; Uauy, Cristobal; Trafford, Kay

    2011-01-01

    Starch within the endosperm of most species of the Triticeae has a unique bimodal granule morphology comprising large lenticular A-type granules and smaller near-spherical B-type granules. However, a few wild wheat species (Aegilops) are known to lack B-granules. Ae. peregrina and a synthetic tetraploid Aegilops with the same genome composition (SU) were found to differ in B-granule number. The synthetic tetraploid had normal A- and B-type starch granules whilst Ae. peregrina had only A-granules because the B-granules failed to initiate. A population segregating for B-granule number was generated by crossing these two accessions and was used to study the genetic basis of B-granule initiation. A combination of Bulked Segregant Analysis and QTL mapping identified a major QTL located on the short arm of chromosome 4S that accounted for 44.4% of the phenotypic variation. The lack of B-granules in polyploid Aegilops with diverse genomes suggests that the B-granule locus has been lost several times independently during the evolution of the Triticeae. It is proposed that the B-granule locus is susceptible to silencing during polyploidization and a model is presented to explain the observed data based on the assumption that the initiation of B-granules is controlled by a single major locus per haploid genome. PMID:21227932

  18. Microbial community variation in cryoconite granules on Qaanaaq Glacier, NW Greenland.

    Science.gov (United States)

    Uetake, Jun; Tanaka, Sota; Segawa, Takahiro; Takeuchi, Nozomu; Nagatsuka, Naoko; Motoyama, Hideaki; Aoki, Teruo

    2016-09-01

    Cryoconite granules are aggregations of microorganisms with mineral particles that form on glacier surfaces. To understand the processes by which the granules develop, this study focused on the altitudinal distribution of the granules and photosynthetic microorganisms on the glacier, bacterial community variation with granules size and environmental factors affecting the growth of the granules. Size-sorted cryoconite granules collected from five different sites on Qaanaaq Glacier were analyzed. C and N contents were significantly higher in large (diameter greater than 250 μm) granules than in smaller (diameter 30-249 μm) granules. Bacterial community structures, based on 16S rRNA gene amplicon sequencing, were different between the smaller and larger granules. The filamentous cyanobacterium Phormidesmis priestleyi was the dominant bacterial species in larger granules. Multivariate analysis suggests that the abundance of mineral particles on the glacier surface is the main factor controlling growth of these cyanobacteria. These results show that the supply of mineral particles on the glacier enhances granule development, that P. priestleyi is likely the key species for primary production and the formation of the granules and that the bacterial community in the granules changes over the course of the