WorldWideScience

Sample records for human gmx33 golgi

  1. Characterization of the human GARP (Golgi associated retrograde protein) complex

    International Nuclear Information System (INIS)

    Liewen, Heike; Meinhold-Heerlein, Ivo; Oliveira, Vasco; Schwarzenbacher, Robert; Luo Guorong; Wadle, Andreas; Jung, Martin; Pfreundschuh, Michael; Stenner-Liewen, Frank

    2005-01-01

    The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells

  2. Golgi-type I and Golgi-type II neurons in the ventral anterior thalamic nucleus of the adult human: morphological features and quantitative analysis.

    Science.gov (United States)

    Al-Hussain Bani Hani, Saleh M; El-Dwairi, Qasim A; Bataineh, Ziad M; Al-Haidari, Mohammad S; Al-Alami, Jamil

    2008-05-01

    The morphological and quantitative features of neurons in the adult human ventral anterior thalamic nucleus were studied in Golgi preparations. Two neuronal types were found and their quantitative features were studied. Golgi-type I neurons were medium to large cells with dense dendritic trees and dendritic protrusions and short hair-like appendages. They have somatic mean diameter of 30.8 microm (+/-9.4, n = 85). They have an average 100.3 dendritic branches, 48.97 dendritic branching points, and 58.85 dendritic tips. The mean diameters of their primary, secondary, and tertiary dendrites were 3.1 microm (+/-1, n = 80), 1.85 microm (+/-0.8, n = 145), and 1.5 microm (+/-0.4, n = 160), respectively. Golgi-type II neurons were small to medium cells with few sparsely branching dendrites and dendritic stalked appendages with or without terminal swellings. They have somatic mean diameters of 22.2 microm (+/-5.8, n = 120). They have an average 33.76 dendritic branches, 16.49 dendritic branching points, and 21.97 dendritic tips. The mean diameters of their primary, secondary, and tertiary dendrites were 1.6 microm (+/-0.86, n = 70), 1.15 microm (+/-0.55, n = 118), and 1 microm (+/-0.70, n = 95), respectively. These quantitative data may form the basis for further quantitative studies involving aging or some degenerative diseases that may affect cell bodies and/or dendritic trees of the Golgi-type I and/or Golgi-type II thalamic neurons.

  3. Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Soonthornsit, Jeerawat [Laboratory for Cell and Developmental Biology, Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita, Kyoto 603-8555 (Japan); Yamaguchi, Yoko; Tamura, Daisuke [Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Ishida, Ryuichi; Nakakoji, Yoko; Osako, Shiho [Laboratory for Cell and Developmental Biology, Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita, Kyoto 603-8555 (Japan); Yamamoto, Akitsugu [Department of Animal Bioscience, Nagahama Institute of Bio-Science and Technology, 266 Tamura, Nagahama, Shiga, 526‐0829 (Japan); Nakamura, Nobuhiro, E-mail: osaru3@cc.kyoto-su.ac.jp [Laboratory for Cell and Developmental Biology, Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita, Kyoto 603-8555 (Japan); Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)

    2014-11-01

    The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1–2 h after the addition of a control medium. The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A{sub 2} inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A{sub 2} was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus. - Highlights: • The Golgi apparatus reversibly disassembles by low pH treatment. • The cis-Golgi disassembles quickly generating tubular structures. • Both anterograde and retrograde transport between the ER and the Golgi apparatus are reduced. • Phospholipase A{sub 2} inhibitors (ONO

  4. IntraGolgi distribution of the Conserved Oligomeric Golgi (COG) complex

    International Nuclear Information System (INIS)

    Vasile, Eliza; Oka, Toshihiko; Ericsson, Maria; Nakamura, Nobuhiro; Krieger, Monty

    2006-01-01

    The Conserved Oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in membrane trafficking and glycoconjugate synthesis. COG appears to participate in retrograde vesicular transport and is required to maintain normal Golgi structure and function. COG mutations interfere with normal transport, distribution, and/or stability of Golgi proteins associated with glycoconjugate synthesis and trafficking, and lead to failure of spermatogenesis in Drosophila melanogaster, misdirected migration of gonadal distal tip cells in Caenorhabditis elegans, and type II congenital disorders of glycosylation in humans. The mechanism by which COG influences Golgi structure and function is unclear. Immunogold electron microscopy was used to visualize the intraGolgi distribution of a functional, hemagglutinin epitope-labeled COG subunit, Cog1-HA, that complements the Cog1-deficiency in Cog1-null Chinese hamster ovary cells. COG was found to be localized primarily on or in close proximity to the tips and rims of the Golgi's cisternae and their associated vesicles and on vesicles and vesiculo-tubular structures seen on both the cis and trans-Golgi Network faces of the cisternal stacks, in some cases on COPI containing vesicles. These findings support the proposal that COG is directly involved in controlling vesicular retrograde transport of Golgi resident proteins throughout the Golgi apparatus

  5. Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus.

    Science.gov (United States)

    Soonthornsit, Jeerawat; Yamaguchi, Yoko; Tamura, Daisuke; Ishida, Ryuichi; Nakakoji, Yoko; Osako, Shiho; Yamamoto, Akitsugu; Nakamura, Nobuhiro

    2014-11-01

    The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1-2h after the addition of a control medium. The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A2 inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A2 was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Vibration sensitivity of human muscle spindles and Golgi tendon organs.

    Science.gov (United States)

    Fallon, James B; Macefield, Vaughan G

    2007-07-01

    The responses of the various muscle receptors to vibration are more complicated than a naïve categorization into stretch (muscle spindle primary ending), length (muscle spindle secondary endings), and tension (Golgi tendon organs) receptors. To emphasize the similarity of responses to small length changes, we recorded from 58 individual muscle afferents subserving receptors in the ankle or toe dorsiflexors of awake human subjects (32 primary endings, 20 secondary endings, and six Golgi tendon organs). Transverse sinusoidal vibration was applied to the distal tendon of the receptor-bearing muscle, while subjects either remained completely relaxed or maintained a weak isometric contraction of the appropriate muscle. In relaxed muscle, few units responded in a 1:1 manner to vibration, and there was no evidence of a preferred frequency of activation. In active muscle the response profiles of all three receptor types overlapped, with no significant difference in threshold between receptor types. These results emphasize that when intramuscular tension increases during a voluntary contraction, Golgi tendon organs and muscle spindle secondary endings, not just muscle spindle primary endings, can effectively encode small imposed length changes.

  7. Depletion of the human N-terminal acetyltransferase hNaa30 disrupts Golgi integrity and ARFRP1 localization.

    Science.gov (United States)

    Starheim, Kristian K; Kalvik, Thomas V; Bjørkøy, Geir; Arnesen, Thomas

    2017-04-30

    The organization of the Golgi apparatus (GA) is tightly regulated. Golgi stack scattering is observed in cellular processes such as apoptosis and mitosis, and has also been associated with disruption of cellular lipid metabolism and neurodegenerative diseases. Our studies show that depletion of the human N-α-acetyltransferase 30 (hNaa30) induces fragmentation of the Golgi stack in HeLa and CAL-62 cell lines. The GA associated GTPase ADP ribosylation factor related protein 1 (ARFRP1) was previously shown to require N-terminal acetylation for membrane association and based on its N-terminal sequence, it is likely to be a substrate of hNaa30. ARFRP1 is involved in endosome-to- trans -Golgi network (TGN) traffic. We observed that ARFRP1 shifted from a predominantly cis -Golgi and TGN localization to localizing both Golgi and non-Golgi vesicular structures in hNaa30-depleted cells. However, we did not observe loss of membrane association of ARFRP1. We conclude that hNaa30 depletion induces Golgi scattering and induces aberrant ARFRP1 Golgi localization. © 2017 The Author(s).

  8. Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

    Science.gov (United States)

    Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

    2015-02-06

    Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

  9. Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus

    DEFF Research Database (Denmark)

    Röttger, S; White, J; Wandall, H H

    1998-01-01

    O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation......, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation...... of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we...

  10. The small G protein Arl5 contributes to endosome-to-Golgi traffic by aiding the recruitment of the GARP complex to the Golgi

    Directory of Open Access Journals (Sweden)

    Cláudia Rosa-Ferreira

    2015-03-01

    Full Text Available The small G proteins of the Arf family play critical roles in membrane trafficking and cytoskeleton organization. However, the function of some members of the family remains poorly understood including Arl5 which is widely conserved in eukaryotes. Humans have two closely related Arl5 paralogues (Arl5a and Arl5b, and both Arl5a and Arl5b localize to the trans-Golgi with Arl5b being involved in retrograde traffic from endosomes to the Golgi apparatus. To investigate the function of Arl5, we have used Drosophila melanogaster as a model system. We find that the single Arl5 orthologue in Drosophila also localizes to the trans-Golgi, but flies lacking the Arl5 gene are viable and fertile. By using both liposome and column based affinity chromatography methods we find that Arl5 interacts with the Golgi-associated retrograde protein (GARP complex that acts in the tethering of vesicles moving from endosomes to the trans-Golgi network (TGN. In Drosophila tissues the GARP complex is partially displaced from the Golgi when Arl5 is absent, and the late endosomal compartment is enlarged. In addition, in HeLa cells GARP also becomes cytosolic upon depletion of Arl5b. These phenotypes are consistent with a role in endosome-to-Golgi traffic, but are less severe than loss of GARP itself. Thus it appears that Arl5 is one of the factors that directs the recruitment of the GARP complex to the trans-Golgi, and this function is conserved in both flies and humans.

  11. A novel Golgi retention signal RPWS for tumor suppressor UBIAD1.

    Directory of Open Access Journals (Sweden)

    Xian Wang

    Full Text Available UBIAD1 plays critical roles in physiology including vitamin K and CoQ10 biosynthesis as well as pathophysiology including dyslipimedia-induced SCD (Schnyder's corneal dystrophy, Parkinson's disease, cardiovascular disease and bladder carcinoma. Since the subcellular localization of UBIAD1 varies in different cell types, characterization of the exact subcellular localization of UBIAD1 in specific human disease is vital for understanding its molecular mechanism. As UBIAD1 suppresses bladder carcinoma, we studied its subcellular localization in human bladder carcinoma cell line T24. Since fluorescent images of UBIAD1-EGFP in T24, human prostate cancer cell line PC-3, human embryonic kidney cell line HEK293 and human hepatocyte cell line L02 are similar, these four cell lines were used for present study. Using a combination of fluorescent microscopy and immunohistochemistry, it was found that UBIAD1 localized on the Golgi and endoplasmic reticulum (ER, but not on the plasma membrane, of T24 and HEK293 cells. Using scanning electron microscopy and western blot analysis, we found that UBIAD1 is enriched in the Golgi fraction extracted from the L02 cells, verifying the Golgi localization of UBAID1. Site-directed mutagenesis showed that the RPWS motif, which forms an Arginine finger on the UBIAD1 N terminus, serves as the Golgi retention signal. With both cycloheximide and brefeldin A inhibition assays, it was shown that UBIAD1 may be transported from the endoplasmic reticulum (ER to the Golgi by a COPII-mediated mechanism. Based upon flow cytometry analysis, it is shown that mutation of the RPWS motif reduced the UBIAD1-induced apoptosis of T24 cells, indicating that the proper Golgi localization of UBIAD1 influences its tumor suppressant activity. This study paves the way for further understanding the molecular mechanism of UBIAD1 in human diseases.

  12. Proteomic dissection of the Arabidopsis Golgi and trans-Golgi network

    DEFF Research Database (Denmark)

    Parsons, Harriet Tempé; Drakakaki, Georgia; Heazlewood, Joshua L.

    2013-01-01

    The plant Golgi apparatus and trans-Golgi network are major endomembrane trafficking hubs within the plant cell and are involved in a diverse and vital series of functions to maintain plant growth and development. Recently, a series of disparate technical approaches have been used to isolate...

  13. Phospholipase D is involved in the formation of Golgi associated clathrin coated vesicles in human parotid duct cells.

    Directory of Open Access Journals (Sweden)

    Lorena Brito de Souza

    Full Text Available Phospholipase D (PLD has been implicated in many cellular functions, such as vesicle trafficking, exocytosis, differentiation, and proliferation. The aim of this study was to characterize the role of PLD in HSY cells, a human cell line originating from the intercalated duct of the parotid gland. As the function and intracellular localization of PLD varies according to cell type, initially, the intracellular localization of PLD1 and PLD2 was determined. By immunofluorescence, PLD1 and PLD2 both showed a punctate cytoplasmic distribution with extensive co-localization with TGN-46. PLD1 was also found in the nucleus, while PLD2 was associated with the plasma membrane. Treatment of cells with the primary alcohol 1-butanol inhibits the hydrolysis of phosphatidylcoline by PLD thereby suppressing phosphatidic acid (PA production. In untreated HSY cells, there was only a slight co-localization of PLD with the clathrin coated vesicles. When HSY cells were incubated with 1-butanol the total number of clathrin coated vesicles increased, especially in the juxtanuclear region and the co-localization of PLD with the clathrin coated vesicles was augmented. Transmission electron microscopy confirmed that the number of Golgi-associated coated vesicles was greater. Treatment with 1-butanol also affected the Golgi apparatus, increasing the volume of the Golgi saccules. The decrease in PA levels after treatment with 1-butanol likewise resulted in an accumulation of enlarged lysosomes in the perinuclear region. Therefore, in HSY cells PLD appears to be involved in the formation of Golgi associated clathrin coated vesicles as well as in the structural maintenance of the Golgi apparatus.

  14. Trafficking of human ADAM 12-L: retention in the trans-Golgi network

    DEFF Research Database (Denmark)

    Hougaard, S; Loechel, F; Xu, X

    2000-01-01

    We have investigated the trafficking of the membrane-anchored form of human ADAM 12 (ADAM 12-L) fused to a green fluorescence protein tag. Subcellular localization of the protein in transiently transfected cells was determined by fluorescence microscopy and trypsin sensitivity. Full-length ADAM 12...... the cytoplasmic and transmembrane domains, but not the Src homology 3 domain (SH3) binding sites. These results raise the possibility that a trafficking checkpoint in the trans-Golgi network is one of the cellular mechanisms for regulation of ADAM 12-L function, by allowing a rapid release of ADAM 12-L...

  15. Human Diseases Associated with Form and Function of the Golgi Complex

    Directory of Open Access Journals (Sweden)

    Jeremy C. Simpson

    2013-09-01

    Full Text Available The Golgi complex lies at the heart of the secretory pathway and is responsible for modifying proteins and lipids, as well as sorting newly synthesized molecules to their correct destination. As a consequence of these important roles, any changes in its proteome can negatively affect its function and in turn lead to disease. Recently, a number of proteins have been identified, which when either depleted or mutated, result in diseases that affect various organ systems. Here we describe how these proteins have been linked to the Golgi complex, and specifically how they affect either the morphology, membrane traffic or glycosylation ability of this organelle.

  16. Defects in the COG complex and COG-related trafficking regulators affect neuronal Golgi function.

    Directory of Open Access Journals (Sweden)

    Leslie K Climer

    2015-10-01

    Full Text Available The Conserved Oligomeric Golgi (COG complex is an evolutionarily conserved hetero-octameric protein complex that has been proposed to organize vesicle tethering at the Golgi apparatus. Defects in seven of the eight COG subunits are linked to Congenital Disorders of Glycosylation (CDG-type II, a family of rare diseases involving misregulation of protein glycosylation, alterations in Golgi structure, variations in retrograde trafficking through the Golgi and system-wide clinical pathologies. A troublesome aspect of these diseases are the neurological pathologies such as low IQ, microcephaly and cerebellar atrophy. The essential function of the COG complex is dependent upon interactions with other components of trafficking machinery, such as Rab-GTPases and SNAREs. COG-interacting Rabs and SNAREs have been implicated in neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease. Defects in Golgi maintenance disrupts trafficking and processing of essential proteins, frequently associated with and contributing to compromised neuron function and human disease. Despite the recent advances in molecular neuroscience, the subcellular bases for most neurodegenerative diseases are poorly understood. This article gives an overview of the potential contributions of the COG complex and its Rab and SNARE partners in the pathogenesis of different neurodegenerative disorders.

  17. cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells.

    Science.gov (United States)

    Ito, Yoko; Uemura, Tomohiro; Shoda, Keiko; Fujimoto, Masaru; Ueda, Takashi; Nakano, Akihiko

    2012-08-01

    The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.

  18. Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

    Directory of Open Access Journals (Sweden)

    Danming Tang

    2012-09-01

    GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.

  19. Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

    Science.gov (United States)

    Tang, Danming; Zhang, Xiaoyan; Huang, Shijiao; Yuan, Hebao; Li, Jie; Wang, Yanzhuang

    2016-01-01

    In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking. PMID:26538023

  20. Structural Insights into Arl1-Mediated Targeting of the Arf-GEF BIG1 to the trans-Golgi

    Directory of Open Access Journals (Sweden)

    Antonio Galindo

    2016-07-01

    Full Text Available The GTPase Arf1 is the major regulator of vesicle traffic at both the cis- and trans-Golgi. Arf1 is activated at the cis-Golgi by the guanine nucleotide exchange factor (GEF GBF1 and at the trans-Golgi by the related GEF BIG1 or its paralog, BIG2. The trans-Golgi-specific targeting of BIG1 and BIG2 depends on the Arf-like GTPase Arl1. We find that Arl1 binds to the dimerization and cyclophilin binding (DCB domain in BIG1 and report a crystal structure of human Arl1 bound to this domain. Residues in the DCB domain that bind Arl1 are required for BIG1 to locate to the Golgi in vivo. DCB domain-binding residues in Arl1 have a distinct conformation from those in known Arl1-effector complexes, and this plasticity allows Arl1 to interact with different effectors of unrelated structure. The findings provide structural insight into how Arf1 GEFs, and hence active Arf1, achieve their correct subcellular distribution.

  1. Shifted Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65 results in formation of high mannose N-glycans in aggressive prostate cancer cells.

    Science.gov (United States)

    Bhat, Ganapati; Hothpet, Vishwanath-Reddy; Lin, Ming-Fong; Cheng, Pi-Wan

    2017-11-01

    There is a pressing need for biomarkers that can distinguish indolent from aggressive prostate cancer to prevent over-treatment of patients with indolent tumor. Golgi targeting of glycosyltransferases was characterized by confocal microscopy after knockdown of GM130, giantin, or both. N-glycans on a trans-Golgi enzyme β4galactosyltransferase-1 isolated by immunoprecipitation from androgen-sensitive and independent prostate cancer cells were determined by matrix-assisted laser desorption-time of flight-mass spectrometry. In situ proximity ligation assay was employed to determine co-localization of (a) α-mannosidase IA, an enzyme required for processing Man 8 GlcNAc 2 down to Man 5 GlcNAc 2 to enable synthesis of complex-type N-glycans, with giantin, GM130, and GRASP65, and (b) trans-Golgi glycosyltransferases with high mannose N-glycans terminated with α3-mannose. Defective giantin in androgen-independent prostate cancer cells results in a shift of Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65. Consequently, trans-Golgi enzymes and cell surface glycoproteins acquire high mannose N-glycans, which are absent in cells with functional giantin. In situ proximity ligation assays of co-localization of α-mannosidase IA with GM130 and GRASP65, and trans-Golgi glycosyltransferases with high mannose N-glycans are negative in androgen-sensitive LNCaP C-33 cells but positive in androgen-independent LNCaP C-81 and DU145 cells, and LNCaP C-33 cells devoid of giantin. In situ proximity ligation assays of Golgi localization of α-mannosidase IA at giantin versus GM130-GRASP65 site, and absence or presence of N-glycans terminated with α3-mannose on trans-Golgi glycosyltransferases may be useful for distinguishing indolent from aggressive prostate cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation.

    Science.gov (United States)

    Jansen, Jos C; Cirak, Sebahattin; van Scherpenzeel, Monique; Timal, Sharita; Reunert, Janine; Rust, Stephan; Pérez, Belén; Vicogne, Dorothée; Krawitz, Peter; Wada, Yoshinao; Ashikov, Angel; Pérez-Cerdá, Celia; Medrano, Celia; Arnoldy, Andrea; Hoischen, Alexander; Huijben, Karin; Steenbergen, Gerry; Quelhas, Dulce; Diogo, Luisa; Rymen, Daisy; Jaeken, Jaak; Guffon, Nathalie; Cheillan, David; van den Heuvel, Lambertus P; Maeda, Yusuke; Kaiser, Olaf; Schara, Ulrike; Gerner, Patrick; van den Boogert, Marjolein A W; Holleboom, Adriaan G; Nassogne, Marie-Cécile; Sokal, Etienne; Salomon, Jody; van den Bogaart, Geert; Drenth, Joost P H; Huynen, Martijn A; Veltman, Joris A; Wevers, Ron A; Morava, Eva; Matthijs, Gert; Foulquier, François; Marquardt, Thorsten; Lefeber, Dirk J

    2016-02-04

    Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal

  3. Golgi structure formation, function, and post-translational modifications in mammalian cells.

    Science.gov (United States)

    Huang, Shijiao; Wang, Yanzhuang

    2017-01-01

    The Golgi apparatus is a central membrane organelle for trafficking and post-translational modifications of proteins and lipids in cells. In mammalian cells, it is organized in the form of stacks of tightly aligned flattened cisternae, and dozens of stacks are often linked laterally into a ribbon-like structure located in the perinuclear region of the cell. Proper Golgi functionality requires an intact architecture, yet Golgi structure is dynamically regulated during the cell cycle and under disease conditions. In this review, we summarize our current understanding of the relationship between Golgi structure formation, function, and regulation, with focus on how post-translational modifications including phosphorylation and ubiquitination regulate Golgi structure and on how Golgi unstacking affects its functions, in particular, protein trafficking, glycosylation, and sorting in mammalian cells.

  4. Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

    Science.gov (United States)

    Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

    2015-01-01

    The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. PMID:26157166

  5. The role of Golgi reassembly and stacking protein 65 phosphorylation in H2O2-induced cell death and Golgi morphological changes.

    Science.gov (United States)

    Ji, Guang; Zhang, Weiwei; Quan, Moyuan; Chen, Yang; Qu, Hui; Hu, Zhiping

    2016-12-01

    This study aimed to investigate the effects of H 2 O 2 -induced oxidative stress on cell viability and survival, as well as changes in the distribution of Golgi apparatus and in the level of Golgi reassembly and stacking protein 65 (GRASP65). Cell viability of cultured N2a cells treated with H 2 O 2 was measured by the MTT assay. Apoptosis was measured by flow cytometry analyses. Cells labeled by indirect immunofluorescence were observed under confocal microscope to detect any Golgi morphological alterations; electron microscopy of Golgi apparatus was also done. Expression of GRASP65 and phospho-GRASP65 was examined by immunoblotting. H 2 O 2 treatment reduced the cell viability and raised the cell mortality of N2a cells in a time-dependent manner. Notable changes were only observed in the distribution and morphology of Golgi apparatus at 6 h after H 2 O 2 treatment. The expression of GRASP65 showed no significant changes at different time points; the phosphorylated GRASP65 level was significantly increased after H 2 O 2 treatment, peaked at 3 h, and finally dropped at 6 h. Taken together, GRASP65 phosphorylation may have a critical role in inducing cell death at the early stage after H 2 O 2 treatment, while its role in H 2 O 2 -induced Golgi morphological changes may be complex.

  6. Phosphorylation of p37 is important for Golgi disassembly at mitosis

    International Nuclear Information System (INIS)

    Kaneko, Yayoi; Tamura, Kaori; Totsukawa, Go; Kondo, Hisao

    2010-01-01

    Research highlights: → p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis. → Phosphorylated p37 does not bind to Golgi membranes. → p37 phosphorylation inhibits p97/p37-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled at early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 by Cdc2 results in mitotic inhibition of the p97/p47 pathway . In this study, we demonstrate that p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis, and this phosphorylated p37 does not bind to Golgi membranes. Using an in vitro Golgi reassembly assay, we show that mutated p37(S56D, T59D), which mimics mitotic phosphorylation, does not cause any cisternal regrowth, indicating that p37 phosphorylation inhibits the p97/p37 pathway. Our results demonstrate that p37 phosphorylation on Serine-56 and Threonine-59 is important for Golgi disassembly at mitosis.

  7. Mena-GRASP65 interaction couples actin polymerization to Golgi ribbon linking.

    Science.gov (United States)

    Tang, Danming; Zhang, Xiaoyan; Huang, Shijiao; Yuan, Hebao; Li, Jie; Wang, Yanzhuang

    2016-01-01

    In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking. © 2016 Tang et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  8. Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast.

    Science.gov (United States)

    Banerjee, Subhrajit; Kane, Patricia M

    2017-09-15

    Luminal pH and phosphoinositide content are fundamental features of organelle identity. Vacuolar H + -ATPases (V-ATPases) drive organelle acidification in all eukaryotes, and membrane-bound a-subunit isoforms of the V-ATPase are implicated in organelle-specific targeting and regulation. Earlier work demonstrated that the endolysosomal lipid PI(3,5)P 2 activates V-ATPases containing the vacuolar a-subunit isoform in Saccharomyces cerevisiae Here we demonstrate that PI(4)P, the predominant Golgi phosphatidylinositol (PI) species, directly interacts with the cytosolic amino terminal (NT) domain of the yeast Golgi V-ATPase a-isoform Stv1. Lysine-84 of Stv1NT is essential for interaction with PI(4)P in vitro and in vivo, and interaction with PI(4)P is required for efficient localization of Stv1-containing V-ATPases. The cytosolic NT domain of the human V-ATPase a2 isoform specifically interacts with PI(4)P in vitro, consistent with its Golgi localization and function. We propose that NT domains of V o a-subunit isoforms interact specifically with PI lipids in their organelles of residence. These interactions can transmit organelle-specific targeting or regulation information to V-ATPases. © 2017 Banerjee and Kane. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  9. The trans-Golgi Network and the Golgi Stacks Behave Independently During Regeneration After Brefeldin A Treatment in Tobacco BY-2 Cells.

    Science.gov (United States)

    Ito, Yoko; Toyooka, Kiminori; Fujimoto, Masaru; Ueda, Takashi; Uemura, Tomohiro; Nakano, Akihiko

    2017-04-01

    The trans-Golgi network (TGN) plays an essential role in intracellular membrane trafficking. In plant cells, recent live-cell imaging studies have revealed the dynamic behavior of the TGN independent from the Golgi apparatus. In order to better understand the relationships between the two organelles, we examined their dynamic responses to the reagent brefeldin A (BFA) and their recovery after BFA removal. Golgi markers responded to BFA similarly over a range of concentrations, whereas the behavior of the TGN was BFA concentration dependent. The TGN formed aggregates at high concentrations of BFA; however, TGN proteins relocalized to numerous small vesicular structures dispersed throughout the cytoplasm at lower BFA concentrations. During recovery from weak BFA treatment, the TGN started to regenerate earlier than the completion of the Golgi. The regeneration of the two organelles proceeded independently of each other for a while, and eventually was completed by their association. Our data suggest that there is some degree of autonomy for the regeneration of the TGN and the Golgi in tobacco BY-2 cells. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  10. The Cirque du Soleil of Golgi membrane dynamics.

    Science.gov (United States)

    Bankaitis, Vytas A

    2009-07-27

    The role of lipid metabolic enzymes in Golgi membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid-specific acyltransferase, LPAAT3, contributes to Golgi membrane dynamics by suppressing tubule formation.

  11. The Cirque du Soleil of Golgi membrane dynamics

    OpenAIRE

    Bankaitis, Vytas A.

    2009-01-01

    The role of lipid metabolic enzymes in Golgi membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid?specific acyltransferase, LPAAT3, contributes to Golgi membrane dynamics by suppressing tubule formation.

  12. G-rich, a Drosophila selenoprotein, is a Golgi-resident type III membrane protein

    International Nuclear Information System (INIS)

    Chen, Chang Lan; Shim, Myoung Sup; Chung, Jiyeol; Yoo, Hyun-Seung; Ha, Ji Min; Kim, Jin Young; Choi, Jinmi; Zang, Shu Liang; Hou, Xiao; Carlson, Bradley A.; Hatfield, Dolph L.; Lee, Byeong Jae

    2006-01-01

    G-rich is a Drosophila melanogaster selenoprotein, which is a homologue of human and mouse SelK. Subcellular localization analysis using GFP-tagged G-rich showed that G-rich was localized in the Golgi apparatus. The fusion protein was co-localized with the Golgi marker proteins but not with an endoplasmic reticulum (ER) marker protein in Drosophila SL2 cells. Bioinformatic analysis of G-rich suggests that this protein is either type II or type III transmembrane protein. To determine the type of transmembrane protein experimentally, GFP-G-rich in which GFP was tagged at the N-terminus of G-rich, or G-rich-GFP in which GFP was tagged at the C-terminus of G-rich, were expressed in SL2 cells. The tagged proteins were then digested with trypsin, and analyzed by Western blot analysis. The results showed that the C-terminus of the G-rich protein was exposed to the cytoplasm indicating it is a type III microsomal membrane protein. G-rich is First selenoprotein identified in the Golgi apparatus

  13. Regulation of ER-Golgi Transport Dynamics by GTPases in Budding Yeast

    Directory of Open Access Journals (Sweden)

    Yasuyuki Suda

    2018-01-01

    Full Text Available A large number of proteins are synthesized de novo in the endoplasmic reticulum (ER. They are transported through the Golgi apparatus and then delivered to their proper destinations. The ER and the Golgi play a central role in protein processing and sorting and show dynamic features in their forms. Ras super family small GTPases mediate the protein transport through and between these organelles. The ER-localized GTPase, Sar1, facilitates the formation of COPII transport carriers at the ER exit sites (ERES on the ER for the transport of cargo proteins from the ER to the Golgi. The Golgi-localized GTPase, Arf1, controls intra-Golgi, and Golgi-to-ER transport of cargo proteins by the formation of COPI carriers. Rab GTPases localized at the Golgi, which are responsible for fusion of membranes, are thought to establish the identities of compartments. Recent evidence suggests that these small GTPases regulate not only discrete sites for generation/fusion of transport carriers, but also membrane dynamics of the organelles where they locate to ensure the integrity of transport. Here we summarize the current understandings about the membrane traffic between these organelles and highlight the cutting-edge advances from super-resolution live imaging of budding yeast, Saccharomyces cerevisiae.

  14. Non-synaptic signaling from cerebellar climbing fibers modulates Golgi cell activity.

    Science.gov (United States)

    Nietz, Angela K; Vaden, Jada H; Coddington, Luke T; Overstreet-Wadiche, Linda; Wadiche, Jacques I

    2017-10-13

    Golgi cells are the principal inhibitory neurons at the input stage of the cerebellum, providing feedforward and feedback inhibition through mossy fiber and parallel fiber synapses. In vivo studies have shown that Golgi cell activity is regulated by climbing fiber stimulation, yet there is little functional or anatomical evidence for synapses between climbing fibers and Golgi cells. Here, we show that glutamate released from climbing fibers activates ionotropic and metabotropic receptors on Golgi cells through spillover-mediated transmission. The interplay of excitatory and inhibitory conductances provides flexible control over Golgi cell spiking, allowing either excitation or a biphasic sequence of excitation and inhibition following single climbing fiber stimulation. Together with prior studies of spillover transmission to molecular layer interneurons, these results reveal that climbing fibers exert control over inhibition at both the input and output layers of the cerebellar cortex.

  15. The Arf-GDP-regulated recruitment of GBF1 to Golgi membranes requires domains HDS1 and HDS2 and a Golgi-localized protein receptor.

    Science.gov (United States)

    Quilty, Douglas; Chan, Calvin J; Yurkiw, Katherine; Bain, Alexandra; Babolmorad, Ghazal; Melançon, Paul

    2018-04-19

    We previously proposed a novel mechanism by which the enzyme Golgi-specific Brefeldin A resistance factor 1 (GBF1) is recruited to the membranes of the cis -Golgi, based on in vivo experiments. Here, we extended our in vivo analysis on the production of regulatory Arf-GDP and observed that ArfGAP2 and ArfGAP3 do not play a role in GBF1 recruitment. We confirm that Arf-GDP localization is critical, as a TGN-localized Arf-GDP mutant protein fails to promote GBF1 recruitment. We also reported the establishment of an in vitro GBF1 recruitment assay that supports the regulation of GBF1 recruitment by Arf-GDP. This in vitro assay yielded further evidence for the requirement of a Golgi-localized protein because heat denaturation or protease treatment of Golgi membranes abrogated GBF1 recruitment. Finally, combined in vivo and in vitro measurements indicated that the recruitment to Golgi membranes via a putative receptor requires only the HDS1 and HDS2 domains in the C-terminal half of GBF1. © 2018. Published by The Company of Biologists Ltd.

  16. Journeys through the Golgi--taking stock in a new era.

    Science.gov (United States)

    Emr, Scott; Glick, Benjamin S; Linstedt, Adam D; Lippincott-Schwartz, Jennifer; Luini, Alberto; Malhotra, Vivek; Marsh, Brad J; Nakano, Akihiko; Pfeffer, Suzanne R; Rabouille, Catherine; Rothman, James E; Warren, Graham; Wieland, Felix T

    2009-11-16

    The Golgi apparatus is essential for protein sorting and transport. Many researchers have long been fascinated with the form and function of this organelle. Yet, despite decades of scrutiny, the mechanisms by which proteins are transported across the Golgi remain controversial. At a recent meeting, many prominent Golgi researchers assembled to critically evaluate the core issues in the field. This report presents the outcome of their discussions and highlights the key open questions that will help guide the field into a new era.

  17. OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface

    International Nuclear Information System (INIS)

    Zhou, You; Li, Shiqian; Maeyraenpaeae, Mikko I.; Zhong, Wenbin; Baeck, Nils; Yan, Daoguang; Olkkonen, Vesa M.

    2010-01-01

    We characterize here ORP11, a member of the oxysterol-binding protein family. ORP11 is present at highest levels in human ovary, testis, kidney, liver, stomach, brain, and adipose tissue. Immunohistochemistry demonstrates abundant ORP11 in the epithelial cells of kidney tubules, testicular tubules, caecum, and skin. ORP11 in HEK293 cells resides on Golgi complex and LE, co-localizing with GFP-Rab9, TGN46, GFP-Rab7, and a fluorescent medial-trans-Golgi marker. Under electron microscopic observation, cells overexpressing ORP11 displayed lamellar lipid bodies associated with vacuolar structures or the Golgi complex, indicating a disturbance of lipid trafficking. N-terminal fragment of ORP11 (aa 1-292) localized partially to Golgi, but displayed enhanced localization on Rab7- and Rab9-positive LE, while the C-terminal ligand-binding domain (aa 273-747) was cytosolic, demonstrating that the membrane targeting determinants are N-terminal. Yeast two-hybrid screen revealed interaction of ORP11 with the related ORP9. The interacting region was delineated within aa 98-372 of ORP9 and aa 154-292 of ORP11. Overexpressed ORP9 was able to recruit EGFP-ORP11 to membranes, and ORP9 silencing inhibited ORP11 Golgi association. The results identify ORP11 as an OSBP homologue distributing at the Golgi-LE interface and define the ORP9-ORP11 dimer as a functional unit that may act as an intracellular lipid sensor or transporter.

  18. OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, You [Minerva Foundation Institute for Medical Research, Biomedicum 2U, and National Institute for Health and Welfare/Public Health Genomics Unit, Biomedicum 1, FI-00290, Helsinki (Finland); Li, Shiqian [Department of Biology, Jinan University, Guangzhou 510632 (China); Maeyraenpaeae, Mikko I. [Wihuri Research Institute, FI-00140 Helsinki, and the Department of Forensic Medicine, FI-00014 University of Helsinki (Finland); Zhong, Wenbin [Department of Biology, Jinan University, Guangzhou 510632 (China); Baeck, Nils [Institute of Biomedicine/Anatomy, FI-00014 University of Helsinki, Helsinki (Finland); Yan, Daoguang [Department of Biology, Jinan University, Guangzhou 510632 (China); Olkkonen, Vesa M., E-mail: vesa.olkkonen@helsinki.fi [Minerva Foundation Institute for Medical Research, Biomedicum 2U, and National Institute for Health and Welfare/Public Health Genomics Unit, Biomedicum 1, FI-00290, Helsinki (Finland); Institute of Biomedicine/Anatomy, FI-00014 University of Helsinki, Helsinki (Finland)

    2010-11-15

    We characterize here ORP11, a member of the oxysterol-binding protein family. ORP11 is present at highest levels in human ovary, testis, kidney, liver, stomach, brain, and adipose tissue. Immunohistochemistry demonstrates abundant ORP11 in the epithelial cells of kidney tubules, testicular tubules, caecum, and skin. ORP11 in HEK293 cells resides on Golgi complex and LE, co-localizing with GFP-Rab9, TGN46, GFP-Rab7, and a fluorescent medial-trans-Golgi marker. Under electron microscopic observation, cells overexpressing ORP11 displayed lamellar lipid bodies associated with vacuolar structures or the Golgi complex, indicating a disturbance of lipid trafficking. N-terminal fragment of ORP11 (aa 1-292) localized partially to Golgi, but displayed enhanced localization on Rab7- and Rab9-positive LE, while the C-terminal ligand-binding domain (aa 273-747) was cytosolic, demonstrating that the membrane targeting determinants are N-terminal. Yeast two-hybrid screen revealed interaction of ORP11 with the related ORP9. The interacting region was delineated within aa 98-372 of ORP9 and aa 154-292 of ORP11. Overexpressed ORP9 was able to recruit EGFP-ORP11 to membranes, and ORP9 silencing inhibited ORP11 Golgi association. The results identify ORP11 as an OSBP homologue distributing at the Golgi-LE interface and define the ORP9-ORP11 dimer as a functional unit that may act as an intracellular lipid sensor or transporter.

  19. Induced oligomerization targets Golgi proteins for degradation in lysosomes.

    Science.gov (United States)

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

    2015-12-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. © 2015 Tewari et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  20. Golgi organization and the apical extension of fungal hyphae: an essential relationship.

    Science.gov (United States)

    Harris, Steven D

    2013-07-01

    The Golgi apparatus performs crucial functions in the sorting and processing of proteins destined for secretion from eukaryotic cells. In filamentous fungi, organization of the Golgi apparatus reflects the unique challenges brought about by the highly polarized nature of hyphal growth. Recent results show that Golgi compartments are spatially segregated within hyphal tip cells in a manner that depends upon the integrity of the cytoskeleton. Moreover, loss of normal Golgi organization stops polarized hyphal extension and triggers de-polarization of the hyphal tip. These results emphasize the point that a spatially organized and dynamic Golgi apparatus represents an adaptation that is as important for hyphal extension as is the presence of a Spitzenkörper. In addition, they also identify regulatory mechanisms that could enable controlled de-polarization of hyphae during development or infection-related morphogenesis. © 2013 John Wiley & Sons Ltd.

  1. Synthesis of cell wall xylans and glucans by golgi membranes

    International Nuclear Information System (INIS)

    Gibeaut, D.M.; Carpita, N.C.

    1989-01-01

    We investigated the biosynthesis of mixed-linkage β-D-glucan and glucuronoarabinoxylans which make up the hemicellulosic matrix of the primary cell walls of maize and other cereal grasses. The Golgi apparatus was enriched from plasma membrane and other organelles by flotation density gradient centrifugation. Glucan synthase I and II, which are established markers for Golgi and plasma membrane, respectively, displayed considerable overlap in conventional separations with sucrose density gradients. Flotation gradients improved separation of the membranes substantially, but the different synthases themselves also incorporated radioactivity from either 10 μM or 1 mM UDP-[ 14 C]-glucose into polymer. Relative incorporation of radioactivity into polymers from UDP-[ 14 C]-xylose by the various membrane fractions was nearly identical to relative IDPase activities, indicating that combined xylosyl transferase-xylan synthase represents a new, unequivocal marker for the Golgi apparatus. We also have developed techniques of gas-liquid chromatography and radiogas proportional counting to achieve capillary quality separation of partially methylated alditol acetates with simultaneous determination of radioactivity in the derivatives. Digestion of polymeric products by specific endo-glycanohydrolases to diagnostic oligosaccharides also reveal specific kinds of polysaccharides synthesized by the Golgi membranes. A combination of these techniques provides unequivocal determination of the linkage structure of specific polymers synthesized by the purified Golgi apparatus

  2. Proteomic characterization of golgi membranes enriched from Arabidopsis suspension cell cultures

    DEFF Research Database (Denmark)

    Hansen, Sara Fasmer; Ebert, Berit; Rautengarten, Carsten

    2016-01-01

    The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has...... historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes...... from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization...

  3. Golgi enrichment and proteomic analysis of developing Pinus radiata xylem by free-flow electrophoresis.

    Directory of Open Access Journals (Sweden)

    Harriet T Parsons

    Full Text Available Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557.

  4. The asymmetrical structure of Golgi apparatus membranes revealed by in situ atomic force microscope.

    Directory of Open Access Journals (Sweden)

    Haijiao Xu

    Full Text Available The Golgi apparatus has attracted intense attentions due to its fascinating morphology and vital role as the pivot of cellular secretory pathway since its discovery. However, its complex structure at the molecular level remains elusive due to limited approaches. In this study, the structure of Golgi apparatus, including the Golgi stack, cisternal structure, relevant tubules and vesicles, were directly visualized by high-resolution atomic force microscope. We imaged both sides of Golgi apparatus membranes and revealed that the outer leaflet of Golgi membranes is relatively smooth while the inner membrane leaflet is rough and covered by dense proteins. With the treatment of methyl-β-cyclodextrin and Triton X-100, we confirmed the existence of lipid rafts in Golgi apparatus membrane, which are mostly in the size of 20 nm -200 nm and appear irregular in shape. Our results may be of significance to reveal the structure-function relationship of the Golgi complex and pave the way for visualizing the endomembrane system in mammalian cells at the molecular level.

  5. 42 CFR 86.33 - Human subjects; animal welfare.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Human subjects; animal welfare. 86.33 Section 86.33... Occupational Safety and Health Direct Traineeships § 86.33 Human subjects; animal welfare. Where the...) Chapter 1-43 of the Department Grants Administration Manual 2 068 concerning animal welfare. 2 See...

  6. Disturbed neuronal ER-Golgi sorting of unassembled glycine receptors suggests altered subcellular processing is a cause of human hyperekplexia.

    Science.gov (United States)

    Schaefer, Natascha; Kluck, Christoph J; Price, Kerry L; Meiselbach, Heike; Vornberger, Nadine; Schwarzinger, Stephan; Hartmann, Stephanie; Langlhofer, Georg; Schulz, Solveig; Schlegel, Nadja; Brockmann, Knut; Lynch, Bryan; Becker, Cord-Michael; Lummis, Sarah C R; Villmann, Carmen

    2015-01-07

    Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) α1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR α1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment and cis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/β2-3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR α1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/β2-3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding. Copyright © 2015 the authors 0270-6474/15/350422-16$15.00/0.

  7. Physiological Roles of Plant Post-Golgi Transport Pathways in Membrane Trafficking.

    Science.gov (United States)

    Uemura, Tomohiro

    2016-10-01

    Membrane trafficking is the fundamental system through which proteins are sorted to their correct destinations in eukaryotic cells. Key regulators of this system include RAB GTPases and soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). Interestingly, the numbers of RAB GTPases and SNAREs involved in post-Golgi transport pathways in plant cells are larger than those in animal and yeast cells, suggesting that plants have evolved unique and complex post-Golgi transport pathways. The trans-Golgi network (TGN) is an important organelle that acts as a sorting station in the post-Golgi transport pathways of plant cells. The TGN also functions as the early endosome, which is the first compartment to receive endocytosed proteins. Several endocytosed proteins on the plasma membrane (PM) are initially targeted to the TGN/EE, then recycled back to the PM or transported to the vacuole for degradation. The recycling and degradation of the PM localized proteins is essential for the development and environmental responses in plant. The present review describes the post-Golgi transport pathways that show unique physiological functions in plants. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Inheritance of the Golgi Apparatus and Cytokinesis Are Controlled by Degradation of GBF1

    Directory of Open Access Journals (Sweden)

    Roberto Magliozzi

    2018-06-01

    Full Text Available Summary: Although much is known about how chromosome segregation is coupled to cell division, how intracellular organelles partition during mitotic division is poorly understood. We report that the phosphorylation-dependent degradation of the ARFGEF GBF1 regulates organelle trafficking during cell division. We show that, in mitosis, GBF1 is phosphorylated on Ser292 and Ser297 by casein kinase-2 allowing recognition by the F-box protein βTrCP. GBF1 interaction with βTrCP recruits GBF1 to the SCFβTrCP ubiquitin ligase complex, triggering its degradation. Phosphorylation and degradation of GBF1 occur along microtubules at the intercellular bridge of telophase cells and are required for Golgi membrane positioning and postmitotic Golgi reformation. Indeed, expression of a non-degradable GBF1 mutant inhibits the transport of the Golgi cluster adjacent to the midbody toward the Golgi twin positioned next to the centrosome and results in defective Golgi reassembly and cytokinesis failure. These findings define a mechanism that controls postmitotic Golgi reassembly and inheritance. : Magliozzi et al. demonstrate that, in mitosis, the ARFGEF GBF1 is targeted for ubiquitin-dependent degradation by casein kinase-2 and the SCFβTrCP ubiquitin ligase and show that GBF1 proteolysis is required for Golgi inheritance and accurate cell division. Keywords: cell division, cytokinesis, mitosis, Golgi apparatus, GBF1, ubiquitin-proteasome system, protein degradation, Cullin-RING ubiquitin ligase

  9. Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp

    2013-04-05

    Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

  10. Study of ethanol-induced Golgi disorganization reveals the potential mechanism of alcohol-impaired N-glycosylation

    Science.gov (United States)

    Casey, Carol A.; Bhat, Ganapati; Holzapfel, Melissa S.; Petrosyan, Armen

    2016-01-01

    Background It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi, however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation. Methods HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35 mM ethanol for 72 h. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Characterization of Golgi-associated mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), α-1,2-mannosidase (Man-I) and α-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by 3D Structured Illumination Microscopy (SIM). Results First, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor, however the high mannose-type N-glycans are increased. Further analysis by 3D SIM microscopy revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of Arf1 GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (a) EtOH specifically blocks activation of Arf1, and (b) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man

  11. A small-molecule switch for Golgi sulfotransferases.

    Science.gov (United States)

    de Graffenried, Christopher L; Laughlin, Scott T; Kohler, Jennifer J; Bertozzi, Carolyn R

    2004-11-30

    The study of glycan function is a major frontier in biology that could benefit from small molecules capable of perturbing carbohydrate structures on cells. The widespread role of sulfotransferases in modulating glycan function makes them prime targets for small-molecule modulators. Here, we report a system for conditional activation of Golgi-resident sulfotransferases using a chemical inducer of dimerization. Our approach capitalizes on two features shared by these enzymes: their requirement of Golgi localization for activity on cellular substrates and the modularity of their catalytic and localization domains. Fusion of these domains to the proteins FRB and FKBP enabled their induced assembly by the natural product rapamycin. We applied this strategy to the GlcNAc-6-sulfotransferases GlcNAc6ST-1 and GlcNAc6ST-2, which collaborate in the sulfation of L-selectin ligands. Both the activity and specificity of the inducible enzymes were indistinguishable from their WT counterparts. We further generated rapamycin-inducible chimeric enzymes comprising the localization domain of a sulfotransferase and the catalytic domain of a glycosyltransferase, demonstrating the generality of the system among other Golgi enzymes. The approach provides a means for studying sulfate-dependent processes in cellular systems and, potentially, in vivo.

  12. Nerve growth factor induced changes in the Golgi apparatus of PC-12 rat pheochromocytoma cells as studied by ligand endocytosis, cytochemical and morphometric methods.

    Science.gov (United States)

    Hickey, W F; Stieber, A; Hogue-Angeletti, R; Gonatas, J; GOnatas, N K

    1983-10-01

    Cells of the PC-12 rat pheochromocytoma cell line respond to nerve growth factor (NGF) by sprouting neurites and biochemically differentiating into sympathetic ganglion-like cells. NGF-stimulated ('differentiated') and unstimulated ('undifferentiated') cells were studied by cytochemical techniques for the localization of the enzymes acid phosphatase (ACPase) and thiamine pyrophosphatase (TPPase), and by a morphometric analysis of the distribution of endocytosed wheat-germ agglutinin labelled with horseradish peroxidase (WGA-HRP). Both cytochemical stains showed the enzymes to be distributed in lysosomes and certain cisternae of the Golgi apparatus in both NGF stimulated and unstimulated cells. ACPase was not confined to GERL (Golgi-endoplasmic reticulum-lysosome) as in certain other cells. The morphometric studies demonstrated that the reaction product of the internalized WGA-HRP occupied 4.7% of the cytoplasmic area in unstimulated cells and 4.5% in NGF-stimulated ones. Despite this similarity, the distribution of the WGA-HRP among the studied intracellular compartments in these two cell groups varied. In the NGF-stimulated cells 3.3% of the WGA-HRP reaction product was found in the innermost Golgi cisterna(e) while in unstimulated cells only 0.3% was seen in this compartment. Similarly, 4.3% of the WGA-HRP stain was found in small vesicles at the 'trans' aspect of the Golgi apparatus in stimulated cells, when only 0.3% of the stain occupied this compartment in 'undifferentiated' cells. The morphometric analysis also revealed that when the PC-12 cells were stimulated with NGF, the Golgi apparatus increased in area by approximately 70%. These findings are consistent with the hypothesis that NGF induced differentiation of PC-12 cells is coupled with enhanced endocytosis of WGA and probably of its 'receptor' to the innermost Golgi cisterna(e) and the closely associated vesicles.

  13. Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools*

    Science.gov (United States)

    Goto, Asako; Charman, Mark; Ridgway, Neale D.

    2016-01-01

    Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50–70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. PMID:26601944

  14. A role for Sar1 and ARF1 GTPases during Golgi biogenesis in the protozoan parasite Trypanosoma brucei

    Science.gov (United States)

    Yavuz, Sevil; Warren, Graham

    2017-01-01

    A single Golgi stack is duplicated and partitioned into two daughter cells during the cell cycle of the protozoan parasite Trypanosoma brucei. The source of components required to generate the new Golgi and the mechanism by which it forms are poorly understood. Using photoactivatable GFP, we show that the existing Golgi supplies components directly to the newly forming Golgi in both intact and semipermeabilized cells. The movement of a putative glycosyltransferase, GntB, requires the Sar1 and ARF1 GTPases in intact cells. In addition, we show that transfer of GntB from the existing Golgi to the new Golgi can be recapitulated in semipermeabilized cells and is sensitive to the GTP analogue GTPγS. We suggest that the existing Golgi is a key source of components required to form the new Golgi and that this process is regulated by small GTPases. PMID:28495798

  15. Nonrandom γ-TuNA-dependent spatial pattern of microtubule nucleation at the Golgi.

    Science.gov (United States)

    Sanders, Anna A W M; Chang, Kevin; Zhu, Xiaodong; Thoppil, Roslin J; Holmes, William R; Kaverina, Irina

    2017-11-07

    Noncentrosomal microtubule (MT) nucleation at the Golgi generates MT network asymmetry in motile vertebrate cells. Investigating the Golgi-derived MT (GDMT) distribution, we find that MT asymmetry arises from nonrandom nucleation sites at the Golgi (hotspots). Using computational simulations, we propose two plausible mechanistic models of GDMT nucleation leading to this phenotype. In the "cooperativity" model, formation of a single GDMT promotes further nucleation at the same site. In the "heterogeneous Golgi" model, MT nucleation is dramatically up-regulated at discrete and sparse locations within the Golgi. While MT clustering in hotspots is equally well described by both models, simulating MT length distributions within the cooperativity model fits the data better. Investigating the molecular mechanism underlying hotspot formation, we have found that hotspots are significantly smaller than a Golgi subdomain positive for scaffolding protein AKAP450, which is thought to recruit GDMT nucleation factors. We have further probed potential roles of known GDMT-promoting molecules, including γ-TuRC-mediated nucleation activator (γ-TuNA) domain-containing proteins and MT stabilizer CLASPs. While both γ-TuNA inhibition and lack of CLASPs resulted in drastically decreased GDMT nucleation, computational modeling revealed that only γ-TuNA inhibition suppressed hotspot formation. We conclude that hotspots require γ-TuNA activity, which facilitates clustered GDMT nucleation at distinct Golgi sites. © 2017 Sanders et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  16. The cerebellar Golgi cell and spatiotemporal organization of granular layer activity

    Directory of Open Access Journals (Sweden)

    Egidio eD‘Angelo

    2013-05-01

    Full Text Available The cerebellar granular layer has been suggested to perform a complex spatiotemporal reconfiguration of incoming mossy fiber signals. Central to this role is the inhibitory action exerted by Golgi cells over granule cells: Golgi cells inhibit granule cells through double feedforward and feedback inhibitory loops and generate a broad lateral inhibition that extends beyond the afferent synaptic field. This characteristic connectivity has recently been investigated in great detail and been correlated with specific functional properties of the neuron. These include theta-frequency pacemaking, network entrainment into coherent oscillations and phase resetting. Important advances have also been made in terms of determining the membrane and synaptic properties of the neuron, and clarifying the mechanisms of activation by input bursts. Moreover, voltage sensitive dye imaging and multi-electrode array recordings, combined with mathematical simulations based on realistic computational models, have improved our understanding of the impact of Golgi cell activity on granular layer circuit computations. These investigations have highlighted the critical role of Golgi cells in: generating dense clusters of granule cell activity organized in center-surround structures, implementing combinatorial operations on multiple mossy fiber inputs, regulating transmission gain and cut-off frequency, controlling spike timing and burst transmission, and determining the sign, intensity and extension of long-term synaptic plasticity at the mossy fiber-granule cell relay. This review considers recent advances in the field, highlighting the functional implications of Golgi cells for granular layer network computation and indicating new challenges for cerebellar research.

  17. Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools.

    Science.gov (United States)

    Goto, Asako; Charman, Mark; Ridgway, Neale D

    2016-01-15

    Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

    International Nuclear Information System (INIS)

    Wang, Ying-Nai; Wang, Hongmei; Yamaguchi, Hirohito; Lee, Hong-Jen; Lee, Heng-Huan; Hung, Mien-Chie

    2010-01-01

    Research highlights: → ARF1 activation is involved in the EGFR transport to the ER and the nucleus. → Assembly of γ-COP coatomer mediates EGFR transport to the ER and the nucleus. → Golgi-to-ER retrograde trafficking regulates nuclear transport of EGFR. -- Abstract: Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH 2 -terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with γ-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.

  19. CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation

    NARCIS (Netherlands)

    Jansen, Jos C.; Cirak, Sebahattin; van Scherpenzeel, Monique; Timal, Sharita; Reunert, Janine; Rust, Stephan; Pérez, Belén; Vicogne, Dorothée; Krawitz, Peter; Wada, Yoshinao; Ashikov, Angel; Pérez-Cerdá, Celia; Medrano, Celia; Arnoldy, Andrea; Hoischen, Alexander; Huijben, Karin; Steenbergen, Gerry; Quelhas, Dulce; Diogo, Luisa; Rymen, Daisy; Jaeken, Jaak; Guffon, Nathalie; Cheillan, David; van den Heuvel, Lambertus P.; Maeda, Yusuke; Kaiser, Olaf; Schara, Ulrike; Gerner, Patrick; van den Boogert, Marjolein A. W.; Holleboom, Adriaan G.; Nassogne, Marie-Cécile; Sokal, Etienne; Salomon, Jody; van den Bogaart, Geert; Drenth, Joost P. H.; Huynen, Martijn A.; Veltman, Joris A.; Wevers, Ron A.; Morava, Eva; Matthijs, Gert; Foulquier, François; Marquardt, Thorsten; Lefeber, Dirk J.

    2016-01-01

    Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are

  20. Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Hallée, Stéphanie; Thériault, Catherine; Gagnon, Dominic; Kehrer, Jessica; Frischknecht, Friedrich; Mair, Gunnar R; Richard, Dave

    2018-03-26

    Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages. © 2018 John Wiley & Sons Ltd.

  1. Quantifying Golgi structure using EM: combining volume-SEM and stereology for higher throughput.

    Science.gov (United States)

    Ferguson, Sophie; Steyer, Anna M; Mayhew, Terry M; Schwab, Yannick; Lucocq, John Milton

    2017-06-01

    Investigating organelles such as the Golgi complex depends increasingly on high-throughput quantitative morphological analyses from multiple experimental or genetic conditions. Light microscopy (LM) has been an effective tool for screening but fails to reveal fine details of Golgi structures such as vesicles, tubules and cisternae. Electron microscopy (EM) has sufficient resolution but traditional transmission EM (TEM) methods are slow and inefficient. Newer volume scanning EM (volume-SEM) methods now have the potential to speed up 3D analysis by automated sectioning and imaging. However, they produce large arrays of sections and/or images, which require labour-intensive 3D reconstruction for quantitation on limited cell numbers. Here, we show that the information storage, digital waste and workload involved in using volume-SEM can be reduced substantially using sampling-based stereology. Using the Golgi as an example, we describe how Golgi populations can be sensed quantitatively using single random slices and how accurate quantitative structural data on Golgi organelles of individual cells can be obtained using only 5-10 sections/images taken from a volume-SEM series (thereby sensing population parameters and cell-cell variability). The approach will be useful in techniques such as correlative LM and EM (CLEM) where small samples of cells are treated and where there may be variable responses. For Golgi study, we outline a series of stereological estimators that are suited to these analyses and suggest workflows, which have the potential to enhance the speed and relevance of data acquisition in volume-SEM.

  2. The Golgin GMAP210/TRIP11 anchors IFT20 to the Golgi complex.

    Directory of Open Access Journals (Sweden)

    John A Follit

    2008-12-01

    Full Text Available Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane.

  3. Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins

    NARCIS (Netherlands)

    Sinka, Rita; Gillingham, Alison K.; Kondylis, Vangelis; Munro, Sean

    2008-01-01

    Vesicles and other carriers destined for the Golgi apparatus must be guided to the correct cisternae. Golgins, long coiled-coil proteins that localize to particular Golgi subdomains via their C termini, are candidate regulators of vesicle sorting. In this study, we report that the GRIP domain

  4. Mutations in TRAPPC12 Manifest in Progressive Childhood Encephalopathy and Golgi Dysfunction.

    Science.gov (United States)

    Milev, Miroslav P; Grout, Megan E; Saint-Dic, Djenann; Cheng, Yong-Han Hank; Glass, Ian A; Hale, Christopher J; Hanna, David S; Dorschner, Michael O; Prematilake, Keshika; Shaag, Avraham; Elpeleg, Orly; Sacher, Michael; Doherty, Dan; Edvardson, Simon

    2017-08-03

    Progressive childhood encephalopathy is an etiologically heterogeneous condition characterized by progressive central nervous system dysfunction in association with a broad range of morbidity and mortality. The causes of encephalopathy can be either non-genetic or genetic. Identifying the genetic causes and dissecting the underlying mechanisms are critical to understanding brain development and improving treatments. Here, we report that variants in TRAPPC12 result in progressive childhood encephalopathy. Three individuals from two unrelated families have either a homozygous deleterious variant (c.145delG [p.Glu49Argfs ∗ 14]) or compound-heterozygous variants (c.360dupC [p.Glu121Argfs ∗ 7] and c.1880C>T [p. Ala627Val]). The clinical phenotypes of the three individuals are strikingly similar: severe disability, microcephaly, hearing loss, spasticity, and characteristic brain imaging findings. Fibroblasts derived from all three individuals showed a fragmented Golgi that could be rescued by expression of wild-type TRAPPC12. Protein transport from the endoplasmic reticulum to and through the Golgi was delayed. TRAPPC12 is a member of the TRAPP protein complex, which functions in membrane trafficking. Variants in several other genes encoding members of the TRAPP complex have been associated with overlapping clinical presentations, indicating shared and distinct functions for each complex member. Detailed understanding of the TRAPP-opathies will illuminate the role of membrane protein transport in human disease. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  5. Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae

    Science.gov (United States)

    Beznoussenko, Galina V; Parashuraman, Seetharaman; Rizzo, Riccardo; Polishchuk, Roman; Martella, Oliviano; Di Giandomenico, Daniele; Fusella, Aurora; Spaar, Alexander; Sallese, Michele; Capestrano, Maria Grazia; Pavelka, Margit; Vos, Matthijn R; Rikers, Yuri GM; Helms, Volkhard; Mironov, Alexandre A; Luini, Alberto

    2014-01-01

    The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression–maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes. DOI: http://dx.doi.org/10.7554/eLife.02009.001 PMID:24867214

  6. Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO.

    Directory of Open Access Journals (Sweden)

    Panjamaporn Sangwung

    Full Text Available Endothelial nitric oxide synthase (eNOS is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3 was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.

  7. PERMANGANATE FIXATION OF THE GOLGI COMPLEX AND OTHER CYTOPLASMIC STRUCTURES OF MAMMALIAN TESTES

    Science.gov (United States)

    Mollenhauer, Hilton H.; Zebrun, William

    1960-01-01

    Observations on the fine structure of KMnO4-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO4. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO4-fixed testes in view of what has already been reported for mammalian testes fixed in OsO4. PMID:13771855

  8. Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools*

    OpenAIRE

    Goto, Asako; Charman, Mark; Ridgway, Neale D.

    2015-01-01

    Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to de...

  9. Cholesterol depletion of enterocytes. Effect on the Golgi complex and apical membrane trafficking

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Niels-Christiansen, L L; Thorsen, Evy

    2000-01-01

    Intestinal brush border enzymes, including aminopeptidase N and sucrase-isomaltase, are associated with "rafts" (membrane microdomains rich in cholesterol and sphingoglycolipids). To assess the functional role of rafts in the present work, we studied the effect of cholesterol depletion on apical......, the rates of the Golgi-associated complex glycosylation and association with rafts of newly synthesized aminopeptidase N were reduced, and less of the enzyme had reached the brush border membrane after 2 h of labeling. In contrast, the basolateral Na(+)/K(+)-ATPase was neither missorted nor raft......-associated. Our results implicate the Golgi complex/trans-Golgi network in raft formation and suggest a close relationship between this event and apical membrane trafficking....

  10. La técnica de impregnación argéntica de Golgi. Conmemoración del centenario del premio nobel de Medicina (1906 compartido por Camillo Golgi y Santiago Ramón y Cajal

    Directory of Open Access Journals (Sweden)

    Orlando Torres-Fernández

    2006-12-01

    Full Text Available La técnica de Golgi es un sencillo procedimiento histológico que revela la morfología neuronal completa en tres dimensiones. Este método se fundamenta en la formación de depósitos opacos intracelulares de cromato argéntico, producto de la reacción entre el bicromato de potasio y el nitrato de plata (reacción negra. Camillo Golgi, su descubridor, y Santiago Ramón y Cajal, su principal exponente, recibieron el premio nobel de Medicina y Fisiología en 1906 por su contribución al conocimiento de la estructura del sistema nervioso. Gran parte de sus logros se obtuvieron a través de la aplicación del método de impregnación argéntica. Sin embargo, Golgi y Cajal tenían interpretaciones diferentes sobre la estructura del tejido nervioso. Golgi era defensor de la teoría reticular, la cual proponía que el sistema nervioso estaba conformado por una red de células fusionadas a través de los axones a manera de un sincitio. Por el contrario, la doctrina neuronal, defendida por Cajal, sostenía que las neuronas eran células independientes. También se debe a Golgi y su reazione nera el descubrimiento del organelo celular conocido como ‘aparato de Golgi'. La microscopía electrónica confirmó los postulados de la doctrina neuronal, así como la existencia del complejo de Golgi, y contribuyó al resurgimiento de la técnica de impregnación argéntica. Aunque existen métodos modernos de tinción intracelular que revelan imágenes excelentes de la morfología neuronal, la técnica de Golgi se mantiene vigente por ser un método más práctico y menos costoso para el estudio de la morfología normal y patológica de las neuronas.

  11. Foot-and-mouth disease virus-induced RNA polymerase is associated with Golgi apparatus.

    OpenAIRE

    Polatnick, J; Wool, S H

    1985-01-01

    Electrophoretic analysis of the Golgi apparatus isolated by differential centrifugation from radiolabeled cells infected with foot-and-mouth disease virus showed about 10 protein bands. The virus-induced RNA polymerase was identified by immunoprecipitation and electron microscope staining procedures. Pulse-chase experiments indicated that the polymerase passed through the Golgi apparatus in less than 1 h.

  12. Stathmin 1/2-triggered microtubule loss mediates Golgi fragmentation in mutant SOD1 motor neurons

    NARCIS (Netherlands)

    Bellouze, Sarah; Baillat, Gilbert; Buttigieg, Dorothée; de la Grange, Pierre; Rabouille, Catherine; Haase, Georg

    2016-01-01

    BACKGROUND: Pathological Golgi fragmentation represents a constant pre-clinical feature of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) but its molecular mechanisms remain hitherto unclear. RESULTS: Here, we show that the severe Golgi fragmentation in transgenic

  13. Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi*

    Science.gov (United States)

    Klayman, Lauren M.; Wedegaertner, Philip B.

    2017-01-01

    Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit βγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by βγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by βγ inhibition, demonstrating that βγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate βγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for βγ regulation of TGN to PM transport and a novel role for βγ in mediating Golgi fragmentation. PMID:27994056

  14. Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi.

    Science.gov (United States)

    Klayman, Lauren M; Wedegaertner, Philip B

    2017-02-03

    Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit βγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by βγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by βγ inhibition, demonstrating that βγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate βγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for βγ regulation of TGN to PM transport and a novel role for βγ in mediating Golgi fragmentation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. The Arabidopsis Golgi-localized GDP-L-fucose transporter is required for plant development

    DEFF Research Database (Denmark)

    Rautengarten, Carsten; Ebert, Berit; Liu, Lifeng

    2016-01-01

    assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures......Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport...... accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development....

  16. The Arabidopsis Golgi-localized GDP-L-fucose transporter is required for plant development.

    Science.gov (United States)

    Rautengarten, Carsten; Ebert, Berit; Liu, Lifeng; Stonebloom, Solomon; Smith-Moritz, Andreia M; Pauly, Markus; Orellana, Ariel; Scheller, Henrik Vibe; Heazlewood, Joshua L

    2016-07-06

    Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development.

  17. A novel, modernized Golgi-Cox stain optimized for CLARITY cleared tissue.

    Science.gov (United States)

    Kassem, Mustafa S; Fok, Sandra Y Y; Smith, Kristie L; Kuligowski, Michael; Balleine, Bernard W

    2018-01-15

    High resolution neuronal information is extraordinarily useful in understanding the brain's functionality. The development of the Golgi-Cox stain allowed observation of the neuron in its entirety with unrivalled detail. Tissue clearing techniques, e.g., CLARITY and CUBIC, provide the potential to observe entire neuronal circuits intact within tissue and without previous restrictions with regard to section thickness. Here we describe an improved Golgi-Cox stain method, optimised for use with CLARITY and CUBIC that can be used in both fresh and fixed tissue. Using this method, we were able to observe neurons in their entirety within a fraction of the time traditionally taken to clear tissue (48h). We were also able to show for the first-time that Golgi stained tissue is fluorescent when visualized using a multi-photon microscope, allowing us to image synaptic spines with a detail previously unachievable. These novel methods provide cheap and easy to use techniques to investigate the morphology of cellular processes in the brain at a new-found depth, speed, utility and detail, without previous restrictions of time, tissue type and section thickness. This is the first application of a Golgi-Cox stain to cleared brain tissue, it is investigated and discussed in detail, describing different methodologies that may be used, a comparison between the different clearing techniques and lastly the novel interaction of these techniques with this ultra-rapid stain. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import.

    Science.gov (United States)

    Peterson, Nathan A; Anderson, Tavis K; Wu, Xiao-Jun; Yoshino, Timothy P

    2013-07-09

    Carbohydrate structures of surface-expressed and secreted/excreted glycoconjugates of the human blood fluke Schistosoma mansoni are key determinants that mediate host-parasite interactions in both snail and mammalian hosts. Fucose is a major constituent of these immunologically important glycans, and recent studies have sought to characterize fucosylation-associated enzymes, including the Golgi-localized fucosyltransferases that catalyze the transfer of L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. Importantly, GDP-L-fucose is the only nucleotide-sugar donor used by fucosyltransferases and its availability represents a bottleneck in fucosyl-glycotope expression. A homology-based genome-wide bioinformatics approach was used to identify and molecularly characterize the enzymes that contribute to GDP-L-fucose synthesis and Golgi import in S. mansoni. Putative functions were further investigated through molecular phylogenetic and immunocytochemical analyses. We identified homologs of GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER), which constitute a de novo pathway for GDP-L-fucose synthesis, in addition to a GDP-L-fucose transporter (GFT) that putatively imports cytosolic GDP-L-fucose into the Golgi. In silico primary sequence analyses identified characteristic Rossman loop and short-chain dehydrogenase/reductase motifs in GMD and GMER as well as 10 transmembrane domains in GFT. All genes are alternatively spliced, generating variants of unknown function. Observed quantitative differences in steady-state transcript levels between miracidia and primary sporocysts may contribute to differential glycotope expression in early larval development. Additionally, analyses of protein expression suggest the occurrence of cytosolic GMD and GMER in the ciliated epidermal plates and tegument of miracidia and primary sporocysts, respectively, which is consistent with previous localization of highly

  19. The critical role of Golgi cells in regulating spatio-temporal integration and plasticity at the cerebellum input stage

    Directory of Open Access Journals (Sweden)

    2008-07-01

    Full Text Available After the discovery at the end of the 19th century (Golgi, 1883, the Golgi cell was precisely described by S.R. y Cajal (see Cajal, 1987, 1995 and functionally identified as an inhibitory interneuron 50 years later by J.C. Eccles and colleagues (Eccles e al., 1967. Then, its role has been casted by Marr (1969 within the Motor Learning Theory as a codon size regulator of granule cell activity. It was immediately clear that Golgi cells had to play a critical role, since they are the main inhibitory interneuron of the granular layer and control activity of as many as 100 millions granule cells. In vitro, Golgi cells show pacemaking, resonance, phase-reset and rebound-excitation in the theta-frequency band. These properties are likely to impact on their activity in vivo, which shows irregular spontaneous beating modulated by sensory inputs and burst responses to punctuate stimulation followed by a silent pause. Moreover, investigations have given insight into Golgi cells connectivity within the cerebellar network and on their impact on the spatio-temporal organization of activity. It turns out that Golgi cells can control both the temporal dynamics and the spatial distribution of information transmitted through the cerebellar network. Moreover, Golgi cells regulate the induction of long-term synaptic plasticity at the mossy fiber - granule cell synapse. Thus, the concept is emerging that Golgi cells are of critical importance for regulating granular layer network activity bearing important consequences for cerebellar computation as a whole.

  20. Human eosinophils constitutively express a unique serine protease, PRSS33.

    Science.gov (United States)

    Toyama, Sumika; Okada, Naoko; Matsuda, Akio; Morita, Hideaki; Saito, Hirohisa; Fujisawa, Takao; Nakae, Susumu; Karasuyama, Hajime; Matsumoto, Kenji

    2017-07-01

    Eosinophils play important roles in asthma, especially airway remodeling, by producing various granule proteins, chemical mediators, cytokines, chemokines and proteases. However, protease production by eosinophils is not fully understood. In the present study, we investigated the production of eosinophil-specific proteases/proteinases by transcriptome analysis. Human eosinophils and other cells were purified from peripheral blood by density gradient sedimentation and negative/positive selections using immunomagnetic beads. Protease/proteinase expression in eosinophils and release into the supernatant were evaluated by microarray analysis, qPCR, ELISA, flow cytometry and immunofluorescence staining before and after stimulation with eosinophil-activating cytokines and secretagogues. mRNAs for extracellular matrix proteins in human normal fibroblasts were measured by qPCR after exposure to recombinant protease serine 33 (PRSS33) protein (rPRSS33), created with a baculovirus system. Human eosinophils expressed relatively high levels of mRNA for metalloproteinase 25 (MMP25), a disintegrin and metalloprotease 8 (ADAM8), ADAM10, ADAM19 and PRSS33. Expression of PRSS33 was the highest and eosinophil-specific. PRSS33 mRNA expression was not affected by eosinophil-activating cytokines. Immunofluorescence staining showed that PRSS33 was co-localized with an eosinophil granule protein. PRSS33 was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but its cell surface expression was increased. rPRSS33 stimulation of human fibroblasts increased expression of collagen and fibronectin mRNAs, at least in part via protease-activated receptor-2 activation. Activated eosinophils may induce fibroblast extracellular matrix protein synthesis via cell surface expression of PRSS33, which would at least partly explain eosinophils' role(s) in airway remodeling. Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier

  1. Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells

    DEFF Research Database (Denmark)

    Hansen, Jesper S; Færgeman, Nils J; Kragelund, Birthe B

    2008-01-01

    showed that ACBP targeted to the ER (endoplasmic reticulum) and Golgi in a ligand-binding-dependent manner. A variant Y28F/K32A-FACI-50, which is unable to bind acyl-CoA, did no longer show association with the ER and became segregated from the Golgi, as analysed by intensity correlation calculations....... Depletion of fatty acids from cells by addition of FAFBSA (fatty-acid-free BSA) significantly decreased FACI-50 association with the Golgi, whereas fatty acid overloading increased Golgi association, strongly supporting that ACBP associates with the Golgi in a ligand-dependent manner. FRAP (fluorescence...... recovery after photobleaching) showed that the fatty-acid-induced targeting of FACI-50 to the Golgi resulted in a 5-fold reduction in FACI-50 mobility. We suggest that ACBP is targeted to the ER and Golgi in a ligand-binding-dependent manner in living cells and propose that ACBP may be involved...

  2. Autometallographic (AMG) technique used for enhancement of the Golgi-Cox staining gives good contrast andhigh resolution of dendrites and spines

    DEFF Research Database (Denmark)

    Orlowski, Dariusz

    Despite the existence of many newer staining methods, Golgi staining still remains the primary method forvisualization of the dendrites and spines. The black deposit in the Golgi-Cox impregnated cells is a Mercuricsulphide, therefore autometallographic (AMG) technique which is used for visualizat...... of dendrites and spines in the rat hippocampus. The describedmethod will be of value for future behavioural-anatomical studies, examining changes in dendrite branching andspine density caused by brain diseases and their subsequent treatment.......Despite the existence of many newer staining methods, Golgi staining still remains the primary method forvisualization of the dendrites and spines. The black deposit in the Golgi-Cox impregnated cells is a Mercuricsulphide, therefore autometallographic (AMG) technique which is used...... for visualization of the metals and metalsulphides/selenides in tissue may be used to enhance the Golgi-Cox staining. We demonstrated accordingly thatuse of AMG enhancement method on the Golgi-Cox staining gives good contrast and high resolution of dendritesand spines. Moreover, this method is cheaper and more...

  3. Somal and dendritic development of human CA3 pyramidal neurons from midgestation to middle childhood: a quantitative Golgi study.

    Science.gov (United States)

    Lu, Dahua; He, Lixin; Xiang, Wei; Ai, Wei-Min; Cao, Ye; Wang, Xiao-Sheng; Pan, Aihua; Luo, Xue-Gang; Li, Zhiyuan; Yan, Xiao-Xin

    2013-01-01

    The CA3 area serves a key relay on the tri-synaptic loop of the hippocampal formation which supports multiple forms of mnemonic processing, especially spatial learning and memory. To date, morphometric data about human CA3 pyramidal neurons are relatively rare, with little information available for their pre- and postnatal development. Herein, we report a set of developmental trajectory data, including somal growth, dendritic elongation and branching, and spine formation, of human CA3 pyramidal neurons from midgestation stage to middle childhood. Golgi-impregnated CA3 pyramidal neurons in fetuses at 19, 20, 26, 35, and 38 weeks of gestation (GW) and a child at 8 years of age (Y) were analyzed by Neurolucida morphometry. Somal size of the impregnated CA3 cells increased age-dependently among the cases. The length of the apical and basal dendrites of these neurons increased between 26 GW to 38 GW, and appeared to remain stable afterward until 8 Y. Dendritic branching points increased from 26 GW to 38 GW, with that on the apical dendrites slightly reduced at 8 Y. Spine density on the apical and basal dendrites increased progressively from 26 GW to 8 Y. These data suggest that somal growth and dendritic arborization of human CA3 pyramidal neurons occur largely during the second to third trimester. Spine development and likely synaptogenesis on CA3 pyramidal cells progress during the third prenatal trimester and may continue throughout childhood. Copyright © 2012 Wiley Periodicals, Inc.

  4. The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit from the Golgi Apparatus.

    Science.gov (United States)

    Lee, Myoung Hui; Yoo, Yun-Joo; Kim, Dae Heon; Hanh, Nguyen Hong; Kwon, Yun; Hwang, Inhwan

    2017-07-01

    Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis ( Arabidopsis thaliana ) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4 , atpra1.f4 , was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na + /K + -ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA : AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus. © 2017 American Society of Plant Biologists. All Rights Reserved.

  5. A model for the self-organization of vesicular flux and protein distributions in the Golgi apparatus.

    Directory of Open Access Journals (Sweden)

    Iaroslav Ispolatov

    Full Text Available The generation of two non-identical membrane compartments via exchange of vesicles is considered to require two types of vesicles specified by distinct cytosolic coats that selectively recruit cargo, and two membrane-bound SNARE pairs that specify fusion and differ in their affinities for each type of vesicles. The mammalian Golgi complex is composed of 6-8 non-identical cisternae that undergo gradual maturation and replacement yet features only two SNARE pairs. We present a model that explains how distinct composition of Golgi cisternae can be generated with two and even a single SNARE pair and one vesicle coat. A decay of active SNARE concentration in aging cisternae provides the seed for a cis[Formula: see text]trans SNARE gradient that generates the predominantly retrograde vesicle flux which further enhances the gradient. This flux in turn yields the observed inhomogeneous steady-state distribution of Golgi enzymes, which compete with each other and with the SNAREs for incorporation into transport vesicles. We show analytically that the steady state SNARE concentration decays exponentially with the cisterna number. Numerical solutions of rate equations reproduce the experimentally observed SNARE gradients, overlapping enzyme peaks in cis, medial and trans and the reported change in vesicle nature across the Golgi: Vesicles originating from younger cisternae mostly contain Golgi enzymes and SNAREs enriched in these cisternae and extensively recycle through the Endoplasmic Reticulum (ER, while the other subpopulation of vesicles contains Golgi proteins prevalent in older cisternae and hardly reaches the ER.

  6. Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

    DEFF Research Database (Denmark)

    Klemm, Robin W; Ejsing, Christer S.; Surma, Michal A

    2009-01-01

    The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane...... trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN...... than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery....

  7. Sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus

    International Nuclear Information System (INIS)

    Futerman, A.H.; Stieger, B.; Hubbard, A.L.; Pagano, R.E.

    1990-01-01

    The intracellular site of sphingomyelin (SM) synthesis was examined in subcellular fractions from rat liver using a radioactive ceramide analog N-([1-14C]hexanoyl)-D-erythro-sphingosine. This lipid readily transferred from a complex with bovine serum albumin to liver fractions without disrupting the membranes, and was metabolized to radioactive SM. To prevent degradation of the newly synthesized SM to ceramide, all experiments were performed in the presence of EDTA to minimize neutral sphingomyelinase activity and at neutral pH to minimize acid sphingomyelinase activity. An intact Golgi apparatus fraction gave an 85-98-fold enrichment of SM synthesis and a 58-83-fold enrichment of galactosyltransferase activity. Controlled trypsin digestion demonstrated that SM synthesis was localized to the lumen of intact Golgi apparatus vesicles. Although small amounts of SM synthesis were detected in plasma membrane and rough microsome fractions, after accounting for contamination by Golgi apparatus membranes, their combined activity contributed less than 13% of the total SM synthesis in rat liver. Subfractions of the Golgi apparatus were obtained and characterized by immunoblotting and biochemical assays using cis/medial (mannosidase II) and trans (sialyltransferase and galactosyltransferase) Golgi apparatus markers. The specific activity of SM synthesis was highest in enriched cis and medial fractions but far lower in a trans fraction. We conclude that SM synthesis in rat liver occurs predominantly in the cis and medial cisternae of the Golgi apparatus and not at the plasma membrane or endoplasmic reticulum as has been previously suggested

  8. A rapid method combining Golgi and Nissl staining to study neuronal morphology and cytoarchitecture.

    Science.gov (United States)

    Pilati, Nadia; Barker, Matthew; Panteleimonitis, Sofoklis; Donga, Revers; Hamann, Martine

    2008-06-01

    The Golgi silver impregnation technique gives detailed information on neuronal morphology of the few neurons it labels, whereas the majority remain unstained. In contrast, the Nissl staining technique allows for consistent labeling of the whole neuronal population but gives very limited information on neuronal morphology. Most studies characterizing neuronal cell types in the context of their distribution within the tissue slice tend to use the Golgi silver impregnation technique for neuronal morphology followed by deimpregnation as a prerequisite for showing that neuron's histological location by subsequent Nissl staining. Here, we describe a rapid method combining Golgi silver impregnation with cresyl violet staining that provides a useful and simple approach to combining cellular morphology with cytoarchitecture without the need for deimpregnating the tissue. Our method allowed us to identify neurons of the facial nucleus and the supratrigeminal nucleus, as well as assessing cellular distribution within layers of the dorsal cochlear nucleus. With this method, we also have been able to directly compare morphological characteristics of neuronal somata at the dorsal cochlear nucleus when labeled with cresyl violet with those obtained with the Golgi method, and we found that cresyl violet-labeled cell bodies appear smaller at high cellular densities. Our observation suggests that cresyl violet staining is inadequate to quantify differences in soma sizes.

  9. Golgi bypass: skirting around the heart of classical secretion

    NARCIS (Netherlands)

    Grieve, A.; Rabouille, C.

    2011-01-01

    Classical secretion consists of the delivery of transmembrane and soluble proteins to the plasma membrane and the extracellular medium, respectively, and is mediated by the organelles of the secretory pathway, the Endoplasmic Reticulum (ER), the ER exit sites, and the Golgi, as described by the

  10. Phase I study of anticolon cancer humanized antibody A33.

    Science.gov (United States)

    Welt, Sydney; Ritter, Gerd; Williams, Clarence; Cohen, Leonard S; John, Mary; Jungbluth, Achim; Richards, Elizabeth A; Old, Lloyd J; Kemeny, Nancy E

    2003-04-01

    Humanized A33 (huA33; IgG1) monoclonal antibody detects a determinant expressed by 95% of colorectal cancers and can activate immune cytolytic mechanisms. The present study was designed to (a) define the toxicities and maximum tolerated dose of huA33 and (b) determine huA33 immunogenicity. Patients (n = 11) with advanced chemotherapy-resistant colorectal cancer received 4-week cycles of huA33 at 10, 25, or 50 mg/m(2)/week. Serum samples were analyzed using biosensor technology for evidence of human antihuman antibody (HAHA) response. Eight of 11 patients developed a HAHA response. Significant toxicity was limited to four patients who developed high HAHA titers. In two of these cases, infusion-related reactions such as fevers, rigors, facial flushing, and changes in blood pressure were observed, whereas in the other two cases, toxicity consisted of skin rash, fever, or myalgia. Of three patients who remained HAHA negative, one achieved a radiographic partial response, with reduction of serum carcinoembryonic antigen from 80 to 3 ng/ml. Four patients had radiographic evidence of stable disease (2, 4, 6, and 12 months), with significant reductions (>25%) in serum carcinoembryonic antigen levels in two cases. The complementarity-determining region-grafted huA33 antibody is immunogenic in the majority of colon cancer patients (73%). HAHA activity can be measured reproducibly and quantitatively by BIACORE analysis. Whereas the huA33 construct tested here may be too immunogenic for further clinical development, the antitumor effects observed in the absence of antibody-mediated toxicity and in this heavily pretreated patient population warrant clinical testing of other IgG1 humanized versions of A33 antibody.

  11. Voltage-Dependent Intrinsic Bursting in Olfactory Bulb Golgi Cells

    Science.gov (United States)

    Pressler, R. Todd; Rozman, Peter A.; Strowbridge, Ben W.

    2013-01-01

    In the mammalian olfactory bulb (OB), local synaptic circuits modulate the evolving pattern of activity in mitral and tufted cells following olfactory sensory stimulation. GABAergic granule cells, the most numerous interneuron subtype in this brain region, have been extensively studied. However, classic studies using Golgi staining methods…

  12. A catechol oxidase AcPPO from cherimoya (Annona cherimola Mill.) is localized to the Golgi apparatus.

    Science.gov (United States)

    Olmedo, Patricio; Moreno, Adrián A; Sanhueza, Dayan; Balic, Iván; Silva-Sanzana, Christian; Zepeda, Baltasar; Verdonk, Julian C; Arriagada, César; Meneses, Claudio; Campos-Vargas, Reinaldo

    2018-01-01

    Cherimoya (Annona cherimola) is an exotic fruit with attractive organoleptic characteristics. However, it is highly perishable and susceptible to postharvest browning. In fresh fruit, browning is primarily caused by the polyphenol oxidase (PPO) enzyme catalyzing the oxidation of o-diphenols to quinones, which polymerize to form brown melanin pigment. There is no consensus in the literature regarding a specific role of PPO, and its subcellular localization in different plant species is mainly described within plastids. The present work determined the subcellular localization of a PPO protein from cherimoya (AcPPO). The obtained results revealed that the AcPPO- green fluorescent protein co-localized with a Golgi apparatus marker, and AcPPO activity was present in Golgi apparatus-enriched fractions. Likewise, transient expression assays revealed that AcPPO remained active in Golgi apparatus-enriched fractions obtained from tobacco leaves. These results suggest a putative function of AcPPO in the Golgi apparatus of cherimoya, providing new perspectives on PPO functionality in the secretory pathway, its effects on cherimoya physiology, and the evolution of this enzyme. Copyright © 2017. Published by Elsevier B.V.

  13. Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein (GARP) Complex with the t-SNARE Syntaxin 6

    Science.gov (United States)

    Abascal-Palacios, Guillermo; Schindler, Christina; Rojas, Adriana L; Bonifacino, Juan S.; Hierro, Aitor

    2016-01-01

    Summary The Golgi-Associated Retrograde Protein (GARP) is a tethering complex involved in the fusion of endosome-derived transport vesicles to the trans-Golgi network through interaction with components of the Syntaxin 6/Syntaxin 16/Vti1a/VAMP4 SNARE complex. The mechanisms by which GARP and other tethering factors engage the SNARE fusion machinery are poorly understood. Herein we report the structural basis for the interaction of the human Ang2 subunit of GARP with Syntaxin 6 and the closely related Syntaxin 10. The crystal structure of Syntaxin 6 Habc domain in complex with a peptide from the N terminus of Ang2 shows a novel binding mode in which a di-tyrosine motif of Ang2 interacts with a highly conserved groove in Syntaxin 6. Structure-based mutational analyses validate the crystal structure and support the phylogenetic conservation of this interaction. The same binding determinants are found in other tethering proteins and syntaxins, suggesting a general interaction mechanism. PMID:23932592

  14. Xyloglucan biosynthesis by Golgi membranes from suspension-cultured sycamore (Acer pseudoplatanus) cells

    International Nuclear Information System (INIS)

    White, A.R.; Xin, Yi

    1990-01-01

    Xyloglucan is a major hemicellulose polysaccharide in plant cell walls. Biosynthesis of such cell wall polysaccharides is closely linked to the process of plant cell growth and development. Xyloglucan polysaccharides consist of a β-1,4 glucan backbone synthesized by xyloglucan synthase and sidechains of xylose, galactose, and fucose added by other transferase enzymes. Most plant Golgi and plasma membranes also contain glucan synthases I ampersand II, which make β-1,4 and β-1,3 glucans, respectively. All of these enzymes have very similar activities. Cell walls on suspension-cultured cells from Acer pseudoplatanus (sycamore maple) were enzymatically softened prior to cell disruption by passing through a 30 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on top-loaded or flotation sucrose density gradients. Samples were collected by gradient fractionation and assayed for membrane markers and xyloglucan and glucan synthase activities. Standard marker assays (cyt. c reductase for eR, IDPase ampersand UDPase for Golgi, and eosin 5'-malelmide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of 14 C-labeled sugars from UDP-glucose and UDP-xylose was used to detect xyloglucan synthase, glucan synthases I ampersand II, and xylosyl transferase in Golgi membrane fractions. These activities overlapped, although distinct peaks of xyloglucan synthase and xylosyl transferase were found. Ca ++ had a stimulatory effect on glucan synthases I ampersand II, while Mn ++ had an inhibitory effect on glucan synthase I in the presence of Ca ++ . The similarity of these various synthase activities demonstrates the need for careful structural characterization of newly synthesized polysaccharides

  15. Retrograde transport of protein toxins through the Golgi apparatus

    DEFF Research Database (Denmark)

    Sandvig, Kirsten; Skotland, Tore; van Deurs, Bo

    2013-01-01

    at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER...

  16. Restoration of compact Golgi morphology in advanced prostate cancer enhances susceptibility to galectin-1-induced apoptosis by modifying mucin O-glycan synthesis.

    Science.gov (United States)

    Petrosyan, Armen; Holzapfel, Melissa S; Muirhead, David E; Cheng, Pi-Wan

    2014-12-01

    Prostate cancer progression is associated with upregulation of sialyl-T antigen produced by β-galactoside α-2,3-sialyltransferase-1 (ST3Gal1) but not with core 2-associated polylactosamine despite expression of core 2 N-acetylglucosaminyltransferase-L (C2GnT-L/GCNT1). This property allows androgen-refractory prostate cancer cells to evade galectin-1 (LGALS1)-induced apoptosis, but the mechanism is not known. We have recently reported that Golgi targeting of glycosyltransferases is mediated by golgins: giantin (GOLGB1) for C2GnT-M (GCNT3) and GM130 (GOLGA2)-GRASP65 (GORASP1) or GM130-giantin for core 1 synthase. Here, we show that for Golgi targeting, C2GnT-L also uses giantin exclusively whereas ST3Gal1 uses either giantin or GM130-GRASP65. In addition, the compact Golgi morphology is detected in both androgen-sensitive prostate cancer and normal prostate cells, but fragmented Golgi and mislocalization of C2GnT-L are found in androgen-refractory cells as well as primary prostate tumors (Gleason grade 2-4). Furthermore, failure of giantin monomers to be phosphorylated and dimerized prevents Golgi from forming compact morphology and C2GnT-L from targeting the Golgi. On the other hand, ST3Gal1 reaches the Golgi by an alternate site, GM130-GRASP65. Interestingly, inhibition or knockdown of non-muscle myosin IIA (MYH9) motor protein frees up Rab6a GTPase to promote phosphorylation of giantin by polo-like kinase 3 (PLK3), which is followed by dimerization of giantin assisted by protein disulfide isomerase A3 (PDIA3), and restoration of compact Golgi morphology and targeting of C2GnT-L. Finally, the Golgi relocation of C2GnT-L in androgen-refractory cells results in their increased susceptibility to galectin-1-induced apoptosis by replacing sialyl-T antigen with polylactosamine. This study demonstrates the importance of Golgi morphology and regulation of glycosylation and provides insight into how the Golgi influences cancer progression and metastasis. ©2014 American

  17. GOLGA2, Encoding A Master Regulator of Golgi Apparatus, Is Mutated in A Patient with A Neuromuscular Disorder

    OpenAIRE

    Shamseldin, Hanan E; Bennett, Alexis H; Alfadhel, Majid; Gupta, Vandana; Alkuraya, Fowzan S

    2016-01-01

    Golgi apparatus (GA) is a membrane-bound organelle that serves a multitude of critical cellular functions including protein secretion and sorting, and cellular polarity. Many Mendelian diseases are caused by mutations in genes encoding various components of GA. GOLGA2 encodes GM130, a necessary component for the assembly of GA as a single complex, and its deficiency has been found to result in severe cellular phenotypes. We describe the first human patient with a homozygous apparently loss of...

  18. Pathological changes of Golgi complex in hemocytoblasts of spleen of young axolotls after x-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Turska, R.

    1975-01-01

    The data available up to now concerning the structure of Golgi complex in different types of normal and pathologically changed cells are very divergent. Results are reported from detailed studies on changes occurring within the Golgi complex in the spleen of three-month old axolotls after x-ray irradiation with a single dose of 1,200 R and killed by decapitation 15, 30, 60 minutes and 3, 6, and 12 hours after irradiation.

  19. Galacturonomannan and Golgi-derived membrane linked to growth and shaping of biogenic calcite

    Science.gov (United States)

    Marsh, M. E.; Ridall, A. L.; Azadi, P.; Duke, P. J.

    2002-01-01

    The coccolithophores are valuable models for the design and synthesis of composite materials, because the cellular machinery controlling the nucleation, growth, and patterning of their calcitic scales (coccoliths) can be examined genetically. The coccoliths are formed within the Golgi complex and are the major CaCO(3) component in limestone sediments-particularly those of the Cretaceous period. In this study, we describe mutants lacking a sulfated galacturonomannan and show that this polysaccharide in conjunction with the Golgi-derived membrane is directly linked to the growth and shaping of coccolith calcite but not to the initial orientated nucleation of the mineral phase.

  20. Golgi apparatus-localized synaptotagmin 2 is required for unconventional secretion in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Haiyan Zhang

    Full Text Available BACKGROUND: Most secretory proteins contain signal peptides that direct their sorting to the ER and secreted via the conventional ER/Golgi transport pathway, while some signal-peptide-lacking proteins have been shown to export through ER/Golgi independent secretory pathways. Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus that is active against both prokaryotic and eukaryotic cells. The hygromycin phosphotransferase (HYG(R can phosphorylate and inactivate the hygromycin B, and has been widely used as a positive selective marker in the construction of transgenic plants. However, the localization and trafficking of HYG(R in plant cells remain unknown. Synaptotagmins (SYTs are involved in controlling vesicle endocytosis and exocytosis as calcium sensors in animal cells, while their functions in plant cells are largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: We found Arabidopsis synaptotagmin SYT2 was localized on the Golgi apparatus by immunofluorescence and immunogold labeling. Surprisingly, co-expression of SYT2 and HYG(R caused hypersensitivity of the transgenic Arabidopsis plants to hygromycin B. HYG(R, which lacks a signal sequence, was present in the cytoplasm as well as in the extracellular space in HYG(R-GFP transgenic Arabidopsis plants and its secretion is not sensitive to brefeldin A treatment, suggesting it is not secreted via the conventional secretory pathway. Furthermore, we found that HYG(R-GFP was truncated at carboxyl terminus of HYG(R shortly after its synthesis, and the cells deficient SYT2 failed to efficiently truncate HYG(R-GFP,resulting in HYG(R-GFP accumulated in prevacuoles/vacuoles, indicating that SYT2 was involved in HYG(R-GFP trafficking and secretion. CONCLUSION/SIGNIFICANCE: These findings reveal for the first time that SYT2 is localized on the Golgi apparatus and regulates HYG(R-GFP secretion via the unconventional protein transport from the cytosol to the extracelluar matrix in

  1. Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33.

    Science.gov (United States)

    Ritter, G; Cohen, L S; Williams, C; Richards, E C; Old, L J; Welt, S

    2001-09-15

    Mouse monoclonal antibody A33 (mAb A33) recognizes a M(r) 43,000 cell surface glycoprotein (designated A33) expressed in human colonic epithelium and colon cancer but absent from most other normal tissues. In patients, mAb A33 localizes with high specificity to colon cancer and is retained for up to 6 weeks in the cancer but cleared rapidly from normal colon (5-6 days). As a carrier of (125)I or (131)I, mAb A33 has shown antitumor activity. Induction of strong human anti-mouse antibody (immunoglobulin; HAMA) responses in patients, however, limits the use of the murine mAb A33 to very few injections. A humanized version of this antibody (huAb A33) has been prepared for Phase I and II clinical studies in patients with colon cancer. In those studies, immunogenicity of huAb A33 has been monitored using a novel, highly sensitive BIACORE method, which allows measurement of human anti-human antibodies (HAHAs) without the use of secondary reagents. We found that 63% (26 of 41) of the patients treated with repeated doses of huAb A33 developed HAHAs against a conformational antigenic determinant located in the V(L) and V(H) regions of huAb A33. Detailed serological analysis showed two distinct types of HAHAs. HAHA of type I (49% of patients) was characterized by an early onset with peak HAHA levels after 2 weeks of treatment, which declined with ongoing huAb A33 treatment. HAHA of type II (17% of patients) was characterized by a typically later onset of HAHA than in type I and by progressively increasing HAHA levels with each subsequent huAb A33 administration. Colon cancer patients with type I HAHAs did not develop infusion-related adverse events. In contrast, HAHA of type II was indicative of infusion-related adverse events. By using this new method, we were able to distinguish these two types of HAHAs in patients while on antibody treatment, allowing patients to be removed from study prior to the onset of severe infusion-related adverse events.

  2. A smart drug: a pH-responsive photothermal ablation agent for Golgi apparatus activated cancer therapy.

    Science.gov (United States)

    Xue, Fengfeng; Wen, Ying; Wei, Peng; Gao, Yilin; Zhou, Zhiguo; Xiao, Shuzhang; Yi, Tao

    2017-06-13

    We report a pH-responsive photothermal ablation agent (pH-PTT) based on cyanine dyes for photothermal therapy (PTT). The nanoparticles formed by BSA and pH-PTT preferentially accumulated in the Golgi apparatus of cancer cells compared to normal cells, and thus can be specifically activated by the acidic Golgi apparatus in cancer cells for effective PTT both ex vivo and in vivo.

  3. Golgi bypass for local delivery of axonal proteins, fact or fiction?

    Science.gov (United States)

    González, Carolina; Cornejo, Víctor Hugo; Couve, Andrés

    2018-04-06

    Although translation of cytosolic proteins is well described in axons, much less is known about the synthesis, processing and trafficking of transmembrane and secreted proteins. A canonical rough endoplasmic reticulum or a stacked Golgi apparatus has not been detected in axons, generating doubts about the functionality of a local route. However, axons contain mRNAs for membrane and secreted proteins, translation factors, ribosomal components, smooth endoplasmic reticulum and post-endoplasmic reticulum elements that may contribute to local biosynthesis and plasma membrane delivery. Here we consider the evidence supporting a local secretory system in axons. We discuss exocytic elements and examples of autonomous axonal trafficking that impact development and maintenance. We also examine whether unconventional post-endoplasmic reticulum pathways may replace the canonical Golgi apparatus. Copyright © 2018. Published by Elsevier Ltd.

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

    Science.gov (United States)

    Diederen, J H; Vullings, H G

    1995-03-01

    The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.

  5. Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei.

    Science.gov (United States)

    Ooi, Cher-Pheng; Smith, Terry K; Gluenz, Eva; Wand, Nadina Vasileva; Vaughan, Sue; Rudenko, Gloria

    2018-06-01

    The predominant secretory cargo of bloodstream form Trypanosoma brucei is variant surface glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre-cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post-mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post-mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol-anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans-face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi. © 2018 The Authors. Traffic published by John Wiley & Sons Ltd.

  6. Chlamydia trachomatis intercepts Golgi-derived sphingolipids through a Rab14-mediated transport required for bacterial development and replication.

    Directory of Open Access Journals (Sweden)

    Anahí Capmany

    2010-11-01

    Full Text Available Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation.

  7. Chlamydia trachomatis intercepts Golgi-derived sphingolipids through a Rab14-mediated transport required for bacterial development and replication.

    Science.gov (United States)

    Capmany, Anahí; Damiani, María Teresa

    2010-11-22

    Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation.

  8. Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells.

    Science.gov (United States)

    Liu, Jianping; Hayashi, Kyoko; Matsuoka, Ken

    2015-01-01

    S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.

  9. Vesicular transport of progeny parvovirus particles through ER and Golgi regulates maturation and cytolysis.

    Science.gov (United States)

    Bär, Séverine; Rommelaere, Jean; Nüesch, Jürg P F

    2013-09-01

    Progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. This process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. Here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. By colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny PV particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane. Besides known factors like sar1, sec24, rab1, the ERM family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing COPII-vesicles. These proteins also contribute to the transport through ER and Golgi of the well described analogue of cellular proteins, the secreted Gaussia luciferase in absence of virus infection. It is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. Finally, parvovirus egress via ER and Golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). While not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through ER and Golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway.

  10. Adiponectin release and insulin receptor targeting share trans-Golgi-dependent endosomal trafficking routes

    Directory of Open Access Journals (Sweden)

    Maria Rödiger

    2018-02-01

    Full Text Available Objective: Intracellular vesicle trafficking maintains cellular structures and functions. The assembly of cargo-laden vesicles at the trans-Golgi network is initiated by the ARF family of small GTPases. Here, we demonstrate the role of the trans-Golgi localized monomeric GTPase ARFRP1 in endosomal-mediated vesicle trafficking of mature adipocytes. Methods: Control (Arfrp1flox/flox and inducible fat-specific Arfrp1 knockout (Arfrp1iAT−/− mice were metabolically characterized. In vitro experiments on mature 3T3-L1 cells and primary mouse adipocytes were conducted to validate the impact of ARFRP1 on localization of adiponectin and the insulin receptor. Finally, secretion and transferrin-based uptake and recycling assays were performed with HeLa and HeLa M-C1 cells. Results: We identified the ARFRP1-based sorting machinery to be involved in vesicle trafficking relying on the endosomal compartment for cell surface delivery. Secretion of adiponectin from fat depots was selectively reduced in Arfrp1iAT−/− mice, and Arfrp1-depleted 3T3-L1 adipocytes revealed an accumulation of adiponectin in Rab11-positive endosomes. Plasma adiponectin deficiency of Arfrp1iAT−/− mice resulted in deteriorated hepatic insulin sensitivity, increased gluconeogenesis and elevated fasting blood glucose levels. Additionally, the insulin receptor, undergoing endocytic recycling after ligand binding, was less abundant at the plasma membrane of adipocytes lacking Arfrp1. This had detrimental effects on adipose insulin signaling, followed by insufficient suppression of basal lipolytic activity and impaired adipose tissue expansion. Conclusions: Our findings suggest that adiponectin secretion and insulin receptor surface targeting utilize the same post-Golgi trafficking pathways that are essential for an appropriate systemic insulin sensitivity and glucose homeostasis. Keywords: Adiponectin, ARFRP1, Exocytosis, Insulin receptor, trans-Golgi

  11. Role of myristoylation in membrane attachment and function of G alpha i-3 on Golgi membranes.

    Science.gov (United States)

    Brand, S H; Holtzman, E J; Scher, D A; Ausiello, D A; Stow, J L

    1996-05-01

    Heterotrimeric G protein alpha-subunits localized on the cytoplasmic face of Golgi membranes are involved in regulating vesicle trafficking and protein secretion. We investigated the role of myristoylation in attachment of the G alpha i-3 subunit to Golgi membranes. G alpha i-3 was epitope-tagged by insertion of a FLAG sequence at an NH2-terminal site predicted to interfere with myristoylation, and the resulting NT-alpha i-3 construct was stably transfected and expressed in polarized epithelial LLC-PK1 cells. Metabolic labeling confirmed that the translation product of NT-alpha i-3 was not myristoylated. In contrast to endogenous G alpha 1-3, which is tightly bound to Golgi membranes, the unmyristoylated FLAG-tagged NT-alpha i-3 did not attach to membranes; it was localized by immunofluorescence in the cytoplasm of LLC-PK1 cells and was detected only in the cytosol fraction of cell homogenates. Pertussis toxin-dependent ADP-ribosylation was used to test the ability of NT-alpha i-3 to interact with membrane-bound beta gamma-subunits. In both in vitro and in vivo assays, cytosolic NT-alpha i-3 alone was not ADP-ribosylated, although in the presence of membranes it could interact with G beta gamma-subunits to form heterotrimers. The expression of NT-alpha i-3 in LLC-PK1 cells altered the rate of basolateral secretion of sulfated proteoglycans, consistent with the demonstrated function of endogenous G alpha i-3. These data are consistent with a model in which G alpha i-3 utilizes NH2-terminal myristoylation to bind to Golgi membranes and to maximize its interaction with G beta gamma-subunits. Furthermore, our results show that stable attachment of G alpha i-3 to Golgi membranes is not required for it to participate as a regulatory element in vesicle trafficking in the secretory pathway.

  12. Transport According to GARP: Receiving Retrograde Cargo at the Trans-Golgi Network

    Science.gov (United States)

    Bonifacino, Juan S.; Hierro, Aitor

    2010-01-01

    Tethering factors are large protein complexes that capture transport vesicles and enable their fusion with acceptor organelles at different stages of the endomembrane system. Recent studies have shed new light on the structure and function of a heterotetrameric tethering factor named Golgi-associated retrograde protein (GARP), which promotes fusion of endosome-derived, retrograde transport carriers to the trans-Golgi network (TGN). X-ray crystallography of the Vps53 and Vps54 subunits of GARP has revealed that this complex is structurally related to other tethering factors such as the exocyst, COG and Dsl1, indicating that they all might work by a similar mechanism. Loss of GARP function compromises the growth, fertility and/or viability of the defective organisms, underscoring the essential nature of GARP-mediated retrograde transport. PMID:21183348

  13. Live-cell imaging of post-golgi transport vesicles in cultured hippocampal neurons

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Misonou, Hiroaki

    2015-01-01

    compartments of neurons. In the past two decades, the establishment and advancement of fluorescent protein technology have provided us with opportunities to study how proteins are trafficked in living cells. However, live imaging of trafficking processes in neurons necessitate imaging tools to distinguish...... the several different routes that neurons use for protein trafficking. Here we provide a novel protocol to selectively visualize post-Golgi transport vesicles carrying fluorescent-labeled ion channel proteins in living neurons. Further, we provide a number of analytical tools we developed to quantify...... mechanisms by which post-Golgi vesicles are trafficked in neurons. Our protocol uniquely combines the classic temperature-block with close monitoring of the transient expression of transfected protein tagged with fluorescent proteins, and provides a quick and easy way to study protein trafficking in living...

  14. GOLGA2, encoding a master regulator of golgi apparatus, is mutated in a patient with a neuromuscular disorder.

    Science.gov (United States)

    Shamseldin, Hanan E; Bennett, Alexis H; Alfadhel, Majid; Gupta, Vandana; Alkuraya, Fowzan S

    2016-02-01

    Golgi apparatus (GA) is a membrane-bound organelle that serves a multitude of critical cellular functions including protein secretion and sorting, and cellular polarity. Many Mendelian diseases are caused by mutations in genes encoding various components of GA. GOLGA2 encodes GM130, a necessary component for the assembly of GA as a single complex, and its deficiency has been found to result in severe cellular phenotypes. We describe the first human patient with a homozygous apparently loss of function mutation in GOLGA2. The phenotype is a neuromuscular disorder characterized by developmental delay, seizures, progressive microcephaly, and muscular dystrophy. Knockdown of golga2 in zebrafish resulted in severe skeletal muscle disorganization and microcephaly recapitulating loss of function human phenotype. Our data suggest an important developmental role of GM130 in humans and zebrafish.

  15. Monocrotaline pyrrole-induced megalocytosis of lung and breast epithelial cells: Disruption of plasma membrane and Golgi dynamics and an enhanced unfolded protein response

    International Nuclear Information System (INIS)

    Mukhopadhyay, Somshuvra; Shah, Mehul; Patel, Kirit; Sehgal, Pravin B.

    2006-01-01

    The pyrrolizidine alkaloid monocrotaline (MCT) initiates pulmonary hypertension by inducing a 'megalocytosis' phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of monocrotaline (MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the 'Golgi blockade' hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells. MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane caveolin-1 and E-cadherin, cortical actin together with a circumnuclear accumulation of clathrin heavy chain (CHC) and α-tubulin. Flotation analyses revealed losses/alterations in the association of caveolin-1, E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1α and glucose regulated protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of PERK kinase and protein disulfide isomerase (PDI). These data further support the hypothesis that an MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis

  16. The predominant cholecystokinin in human plasma and intestine is cholecystokinin-33

    DEFF Research Database (Denmark)

    Rehfeld, J F; Sun, G; Christensen, T

    2001-01-01

    Cholecystokinin (CCK) occurs in multiple molecular forms; the major ones are CCK-58, -33, -22, and -8. Their relative abundance in human plasma and intestine, however, is debated. To settle the issue, extracts of intestinal biopsies and plasma from 10 human subjects have been examined by chromato......Cholecystokinin (CCK) occurs in multiple molecular forms; the major ones are CCK-58, -33, -22, and -8. Their relative abundance in human plasma and intestine, however, is debated. To settle the issue, extracts of intestinal biopsies and plasma from 10 human subjects have been examined...... by chromatography, enzyme cleavages, and measurements using a library of sequence-specific RIAs. Plasma samples were drawn in the fasting state and at intervals after a meal. The abundance of the larger forms varied with the 8 C-terminal assays in the library, as 2 assays overestimated and 3 underestimated...... the amounts present. One assay, however, measured carboxyamidated and O:-sulfated CCKs with equimolar potency before and after tryptic cleavage. This assay showed that the predominant plasma form is CCK-33, both in the fasting state ( approximately 51%) and postprandially ( approximately 57%), whereas CCK-22...

  17. Evidence that proliferation of golgi apparatus depends on both de novo generation from the endoplasmic reticulum and formation from pre-existing stacks during the growth of tobacco BY-2 cells.

    Science.gov (United States)

    Abiodun, Moses Olabiyi; Matsuoka, Ken

    2013-04-01

    In higher plants, the numbers of cytoplasmic-distributed Golgi stacks differ based on function, age and cell type. It has not been clarified how the numbers are controlled, whether all the Golgi apparatus in a cell function equally and whether the increase in Golgi number is a result of the de novo formation from the endoplasmic reticulum (ER) or fission of pre-existing stacks. A tobacco prolyl 4-hydroxylase (NtP4H1.1), which is a cis-Golgi-localizing type II membrane protein, was tagged with a photoconvertible fluorescent protein, mKikGR (monomeric Kikume green red), and expressed in tobacco bright yellow 2 (BY-2) cells. Transformed cells were exposed to purple light to convert the fluorescence from green to red. A time-course analysis after the conversion revealed a progressive increase in green puncta and a decrease in the red puncta. From 3 to 6 h, we observed red, yellow and green fluorescent puncta corresponding to pre-existing Golgi; Golgi containing both pre-existing and newly synthesized protein; and newly synthesized Golgi. Analysis of the number and fluorescence of Golgi at different phases of the cell cycle suggested that an increase in Golgi number with both division and de novo synthesis occurred concomitantly with DNA replication. Investigation with different inhibitors suggested that the formation of new Golgi and the generation of Golgi containing both pre-existing and newly synthesized protein are mediated by different machineries. These results and modeling based on quantified results indicate that the Golgi apparatuses in tobacco BY-2 cells are not uniform and suggest that both de novo synthesis from the ER and Golgi division contribute almost equally to the increase in proliferating cells.

  18. Interleukin-33 induces mucin gene expression and goblet cell hyperplasia in human nasal epithelial cells.

    Science.gov (United States)

    Ishinaga, Hajime; Kitano, Masako; Toda, Masaaki; D'Alessandro-Gabazza, Corina N; Gabazza, Esteban C; Shah, Said Ahmad; Takeuchi, Kazuhiko

    2017-02-01

    We investigated whether IL-33 is involved in mucus overproduction and goblet cell hyperplasia in eosinophilic chronic rhinosinusitis (ECRS). IL-33 mRNA was significantly higher in the eosinophilic CRS group than in the non-eosinophilic CRS group from human nasal polyps. IL-33 induced MUC5AC mRNA and MUC5AC protein, and also goblet cell hyperplasia at air liquid interface culture in human nasal epithelial cells. In addition to that, IL-33 induced MUC5B and FOXA3, and reduces FOXJmRNA. In conclusion, our present study demonstrated that the direct evidence of IL-33 which lead to increase mucin gene and protein expression, as well as goblet cell hyperplasia. This study provides novel insights into the role of IL-33 on mucus overproduction in eosinophilic inflammation of human airways. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Live-cell imaging of dual-labeled Golgi stacks in tobacco BY-2 cells reveals similar behaviors for different cisternae during movement and brefeldin A treatment.

    Science.gov (United States)

    Madison, Stephanie L; Nebenführ, Andreas

    2011-09-01

    In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusual organelle displays a wide range of dynamic behaviors such as whole stack motility, constant membrane flux through the cisternae, and Golgi enzyme recycling through the ER. In order to further investigate various aspects of Golgi stack dynamics and integrity, we co-expressed pairs of established Golgi markers in tobacco BY-2 cells to distinguish sub-compartments of the Golgi during monensin treatments, movement, and brefeldin A (BFA)-induced disassembly. A combination of cis and trans markers revealed that Golgi stacks remain intact as they move through the cytoplasm. The Golgi stack orientation during these movements showed a slight preference for the cis side moving ahead, but trans cisternae were also found at the leading edge. During BFA treatments, the different sub-compartments of about half of the observed stacks fused with the ER sequentially; however, no consistent order could be detected. In contrast, the ionophore monensin resulted in swelling of trans cisternae while medial and particularly cis cisternae were mostly unaffected. Our results thus demonstrate a remarkable equivalence of the different cisternae with respect to movement and BFA-induced fusion with the ER. In addition, we propose that a combination of dual-label fluorescence microscopy and drug treatments can provide a simple alternative approach to the determination of protein localization to specific Golgi sub-compartments.

  20. Conditional deletion of Cadherin 13 perturbs Golgi cells and disrupts social and cognitive behaviors.

    Science.gov (United States)

    Tantra, M; Guo, L; Kim, J; Zainolabidin, N; Eulenburg, V; Augustine, G J; Chen, A I

    2018-02-15

    Inhibitory interneurons mediate the gating of synaptic transmission and modulate the activities of neural circuits. Disruption of the function of inhibitory networks in the forebrain is linked to impairment of social and cognitive behaviors, but the involvement of inhibitory interneurons in the cerebellum has not been assessed. We found that Cadherin 13 (Cdh13), a gene implicated in autism spectrum disorder and attention-deficit hyperactivity disorder, is specifically expressed in Golgi cells within the cerebellar cortex. To assess the function of Cdh13 and utilize the manipulation of Cdh13 expression in Golgi cells as an entry point to examine cerebellar-mediated function, we generated mice carrying Cdh13-floxed alleles and conditionally deleted Cdh13 with GlyT2::Cre mice. Loss of Cdh13 results in a decrease in the expression/localization of GAD67 and reduces spontaneous inhibitory postsynaptic current (IPSC) in cerebellar Golgi cells without disrupting spontaneous excitatory postsynaptic current (EPSC). At the behavioral level, loss of Cdh13 in the cerebellum, piriform cortex and endopiriform claustrum have no impact on gross motor coordination or general locomotor behaviors, but leads to deficits in cognitive and social abilities. Mice lacking Cdh13 exhibit reduced cognitive flexibility and loss of preference for contact region concomitant with increased reciprocal social interactions. Together, our findings show that Cdh13 is critical for inhibitory function of Golgi cells, and that GlyT2::Cre-mediated deletion of Cdh13 in non-executive centers of the brain, such as the cerebellum, may contribute to cognitive and social behavioral deficits linked to neurological disorders. © 2018 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.

  1. A Quantitative Golgi Study of Dendritic Morphology in the Mice Striatal Medium Spiny Neurons

    Directory of Open Access Journals (Sweden)

    Ana Hladnik

    2017-04-01

    Full Text Available In this study we have provided a detailed quantitative morphological analysis of medium spiny neurons (MSNs in the mice dorsal striatum and determined the consistency of values among three groups of animals obtained in different set of experiments. Dendritic trees of 162 Golgi Cox (FD Rapid GolgiStain Kit impregnated MSNs from 15 adult C57BL/6 mice were 3-dimensionally reconstructed using Neurolucida software, and parameters of dendritic morphology have been compared among experimental groups. The parameters of length and branching pattern did not show statistically significant difference and were highly consistent among groups. The average neuronal soma surface was between 160 μm2 and 180 μm2, and the cells had 5–6 primary dendrites with close to 40 segments per neuron. Sholl analysis confirmed regular pattern of dendritic branching. The total length of dendrites was around 2100 μm with the average length of individual branching (intermediate segment around 22 μm and for the terminal segment around 100 μm. Even though each experimental group underwent the same strictly defined protocol in tissue preparation and Golgi staining, we found inconsistency in dendritic volume and soma surface. These changes could be methodologically influenced during the Golgi procedure, although without affecting the dendritic length and tree complexity. Since the neuronal activity affects the dendritic thickness, it could not be excluded that observed volume inconsistency was related with functional states of neurons prior to animal sacrifice. Comprehensive analyses of tree complexity and dendritic length provided here could serve as an additional tool for understanding morphological variability in the most numerous neuronal population of the striatum. As reference values they could provide basic ground for comparisons with the results obtained in studies that use various models of genetically modified mice in explaining different pathological conditions that

  2. Research advances in association between Golgi protein 73 and liver diseases

    Directory of Open Access Journals (Sweden)

    WEI Fengxian

    2017-08-01

    Full Text Available Golgi protein 73 (GP73 has a very low expression level in normal people, while it has a significantly higher expression level in patients with liver diseases and hepatocellular carcinoma (HCC, and therefore, it may become a new marker for HCC. This article introduces the distribution of GP73 in human body and definitions of different subtypes of GP73 and elaborates on its association with benign/malignant liver diseases and surgical operation based on the subtypes of GP73, as well as the application of GP73 in the differentiation of benign/malignant liver diseases. Since GP73 is closely associated with the development, progression, and prognosis of liver diseases, this article summarizes the latest advances in basic research, introduces the structural basis of fucosylated GP73 and proliferation, migration, and invasion of hepatoma cells and known signaling pathways, and lists the factors which affect the expression of GP73.

  3. Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated

    NARCIS (Netherlands)

    Kok, JW; Babia, T; Klappe, K; Egea, G; Hoekstra, D

    1998-01-01

    Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degrees C, or in streptolysin O-permeabilized cells by

  4. Aberrant accumulation of the diabetes autoantigen GAD65 in Golgi membranes in conditions of ER stress and autoimmunity

    DEFF Research Database (Denmark)

    Phelps, Edward A; Cianciaruso, Chiara; Michael, Iacovos P

    2016-01-01

    Pancreatic islet beta cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in beta cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes GABA......, an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary beta cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes...... release from stressed and/or damaged beta cells, triggering autoimmunity....

  5. Proliferation of the Golgi apparatus in tobacco BY-2 cells during cell proliferation after release from the stationary phase of growth.

    Science.gov (United States)

    Abiodun, Moses; Matsuoka, Ken

    2013-08-01

    We have recently developed a new method aimed at mass photo-conversion of photo-convertible fluorescence protein (PFP) fluorescence in transformed tobacco BY-2 cells. Using this method we reported recently that the Golgi apparatus is generated by the de novo formation from ER and the division of pre-existing Golgi stacks with similar extents In this work we report that the proliferation of the Golgi apparatus in tobacco cells that enter the growing cycle from the non-dividing cycle is quite similar to that in rapidly growing cells and that de novo formation from the ER and division of pre-existing stacks seems to contribute almost equally to the proliferation.

  6. Golgi-associated Rab14, a new regulator for Chlamydia trachomatis infection outcome.

    Science.gov (United States)

    Capmany, Anahí; Leiva, Natalia; Damiani, María Teresa

    2011-09-01

    Chlamydia trachomatis is the causing agent of the most frequent bacterial sexually-transmitted diseases worldwide and is an underlying cause of chronic pelvic inflammatory diseases and cervical cancer. It is an obligate intracellular bacterium that establishes a close relationship with the Golgi complex and parasites the biosynthetic machinery of host cells. In a recent study, we have demonstrated that Rab14, a newly-described Golgi-associated Rab, is involved in the delivery of sphingolipids to the growing bacteria-containing vacuole. The interference with Rab14-controlled trafficking pathways delays chlamydial inclusion enlargement, decreases bacterial lipid uptake, negatively impact on bacterial differentiation, and reduces bacterial progeny and infectivity. C. trachomatis manipulation of host trafficking pathways for the acquisition of endogenously-biosynthesized nutrients arises as one of the characteristics of this highly evolved pathogen. The development of therapeutic strategies targeted to interfere with bacterium-host cell interaction is a new challenge for pharmacological approaches to control chlamydial infections.

  7. Hsp20 Protects against Oxygen-Glucose Deprivation/Reperfusion-Induced Golgi Fragmentation and Apoptosis through Fas/FasL Pathway

    Directory of Open Access Journals (Sweden)

    Bingwu Zhong

    2015-01-01

    Full Text Available Cerebral ischemia-reperfusion injury plays an important role in the development of tissue injury after acute ischemic stroke. Finding effective neuroprotective agents has become a priority in the treatment of ischemic stroke. The Golgi apparatus (GA is a pivotal organelle and its protection is an attractive target in the treatment of cerebral ischemia-reperfusion injury. Protective effects of Hsp20, a potential cytoprotective agent due to its chaperone-like activity and involvement in regulation of many vital processes, on GA were assessed in an ischemia-reperfusion injury model. Mouse neuroblastoma Neuro2a (N2a cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR insult. OGDR induces Golgi fragmentation, apoptosis, and p115 cleavage in N2a cells. However, transfection with Hsp20 significantly attenuates OGDR-induced Golgi fragmentation and apoptosis. Hsp20 interacts with Bax, decreases FasL and Bax expression, and inhibits caspases 3 and p115 cleavage in N2a cells exposed to OGDR. Our data demonstrate that increased Hsp20 expression protects against OGDR-induced Golgi fragmentation and apoptosis, likely through interaction with Bax and subsequent amelioration of the OGDR-induced elevation in p115 cleavage via the Fas/FasL signaling pathway. This neuroprotective potential of Hsp20 against OGDR insult and the underlying mechanism will pave the way for its potential clinical application for cerebral ischemia-reperfusion related disorders.

  8. Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition

    Science.gov (United States)

    Stoffel, Wilhelm; Hammels, Ina; Jenke, Bitta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Schauss, Astrid; Etich, Julia; Heilig, Juliane; Zaucke, Frank

    2016-01-01

    Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus–pituitary–somatotropic hypoplasia. The unbiased smpd3−/− mouse mutant and derived smpd3−/− primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3−/− mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition. PMID:27882938

  9. Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

    International Nuclear Information System (INIS)

    Mesa, Rosana; Magadan, Javier; Barbieri, Alejandro; Lopez, Cecilia; Stahl, Philip D.; Mayorga, Luis S.

    2005-01-01

    The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN)

  10. Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding.

    Science.gov (United States)

    Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H-J

    2011-04-08

    Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

  11. TCR¿ is transported to and retained in the Golgi apparatus independently of other TCR chains: implications for TCR assembly

    DEFF Research Database (Denmark)

    Dietrich, J; Kastrup, J; Lauritsen, Jens Peter Holst

    1999-01-01

    . This study focused on the intracellular localization and transport of partially assembled TCR complexes as determined by confocal microscopy analyses. We found that none of the TCR chains except for TCRzeta were allowed to exit the ER in T cell variants in which the hexameric CD3gammaepsilonTi alphabetaCD3...... deltaepsilon complex was not formed. Interestingly, TCRzeta was exported from the ER independently of other TCR chains and was predominantly located in a compartment identified as the Golgi apparatus. Furthermore, in the TCRzeta-negative cell line MA5.8, the hexameric CD3gammaepsilonTi alphabetaCD3...... the ER to the Golgi apparatus independently of each other and that these partial TCR complexes are unable to be efficiently expressed at the cell surface suggest that final TCR assembly occurs in the Golgi apparatus....

  12. Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

    International Nuclear Information System (INIS)

    Bonekamp, Nina A.; Vormund, Kerstin; Jacob, Ralf; Schrader, Michael

    2010-01-01

    The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.

  13. Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

    Science.gov (United States)

    de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

    2003-01-10

    The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

  14. Marine sponges (Porifera: Demospongiae) from the Gulf of México, new records and redescription of Erylus trisphaerus (de Laubenfels, 1953).

    Science.gov (United States)

    Ugalde, Diana; Gómez, Patricia; Simões, Nuno

    2015-01-19

    Marine sponges usually constitute the most diverse group of the benthic community in coral reefs. Although they are reasonably well studied at the northern Gulf of Mexico (GMx), the southern GMx is poorly known and lacks records from many major reef systems that lie off the Mexican coast. The present taxonomic study is the first sponge account from Alacranes reef, the largest coral reef system in the GMx, and from the shallow reef banks of Sisal, both in the northwest Yucatan Peninsula. The 19 species herein described represent the first sponge fauna records from these reefs. Among these, seven species represent new record for GMx: Erylus formosus, Cliona flavifodina, Spirastrella aff. mollis, Strongylacidon bermuda, Topsentia bahamensis, Agelas tubulata and Chelonaplysilla aff. erecta. Twelve species are new records for the Southern GMx: Erylus trisphaerus, Cliona amplicavata, Chondrilla caribensis, Halichondria lutea, Hymeniacidon caerulea, Axinella corrugata, Dragmacidon reticulatum, Chalinula molitba, Amphimedon caribica, A. complanata, Hyatella cavernosa and Dysidea variabilis. Additionally, a redescription of Erylus trisphaerus is presented which had not been reviewed since its original description in 1953 off Western Florida, except that it was listed for north La Habana, Cuba. 

  15. Short length transmembrane domains having voluminous exoplasmic halves determine retention of Type II membrane proteins in the Golgi complex

    OpenAIRE

    Quiroga, Rodrigo; Trenchi, Alejandra; Gonzalez Montoro, Ayelén; Valdez, Javier Esteban; Maccioni, Hugo Jose Fernando

    2017-01-01

    It is still unclear why some proteins that travel along the secretory pathway are retained in the Golgi complex whereas others make their way to the plasma membrane. Recent bioinformatic analyses on a large number of single-spanning membrane proteins support the hypothesis that specific features of the transmembrane domain (TMD) are relevant to the sorting of these proteins to particular organelles. Here we experimentally test this hypothesis for Golgi and plasma membrane proteins. Using the ...

  16. GABARAP activates ULK1 and traffics from the centrosome dependent on Golgi partners WAC and GOLGA2/GM130

    OpenAIRE

    Joachim, Justin; Tooze, Sharon A.

    2016-01-01

    ABSTRACT WAC and GOLGA2/GM130 are 2 Golgi proteins that affect autophagy; however, their mechanism of action was unknown. We have shown that WAC binding to GOLGA2 at the Golgi displaces GABARAP from GOLGA2 to allow the maintenance of a nonlipidated centrosomal GABARAP pool. Centrosomal GABARAP can traffic to autophagic structures during starvation. In addition GABARAP specifically promotes ULK1 activation and this is independent of GABARAP lipidation but likely requires a LIR-mediated GABARAP...

  17. Cholesterol Regulates Syntaxin 6 Trafficking at trans-Golgi Network Endosomal Boundaries

    Directory of Open Access Journals (Sweden)

    Meritxell Reverter

    2014-05-01

    Full Text Available Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN. Here, using Chinese hamster ovary (CHO Niemann-Pick type C1 (NPC1 mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6 accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs. This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.

  18. Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9.

    Directory of Open Access Journals (Sweden)

    Xinwei Liu

    Full Text Available Oxysterol binding protein (OSBP and OSBP-related proteins (ORPS have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE were poor ligands for OSBP. In contrast, both long (ORP9L and short (ORP9S variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.

  19. Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9).

    Science.gov (United States)

    Liu, Xinwei; Ridgway, Neale D

    2014-01-01

    Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.

  20. Hepatic trans-Golgi action coordinated by the GTPase ARFRP1 is crucial for lipoprotein lipidation and assembly[S

    Science.gov (United States)

    Hesse, Deike; Radloff, Katrin; Jaschke, Alexander; Lagerpusch, Merit; Chung, Bomee; Tailleux, Anne; Staels, Bart; Schürmann, Annette

    2014-01-01

    The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles. PMID:24186947

  1. Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval.

    Directory of Open Access Journals (Sweden)

    Jennifer Hirst

    2018-01-01

    Full Text Available The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR, GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.

  2. GABARAP activates ULK1 and traffics from the centrosome dependent on Golgi partners WAC and GOLGA2/GM130.

    Science.gov (United States)

    Joachim, Justin; Tooze, Sharon A

    2016-05-03

    WAC and GOLGA2/GM130 are 2 Golgi proteins that affect autophagy; however, their mechanism of action was unknown. We have shown that WAC binding to GOLGA2 at the Golgi displaces GABARAP from GOLGA2 to allow the maintenance of a nonlipidated centrosomal GABARAP pool. Centrosomal GABARAP can traffic to autophagic structures during starvation. In addition GABARAP specifically promotes ULK1 activation and this is independent of GABARAP lipidation but likely requires a LIR-mediated GABARAP-ULK1 interaction.

  3. PI3K class II α regulates δ-opioid receptor export from the trans-Golgi network.

    Science.gov (United States)

    Shiwarski, Daniel J; Darr, Marlena; Telmer, Cheryl A; Bruchez, Marcel P; Puthenveedu, Manojkumar A

    2017-08-01

    The interplay between signaling and trafficking by G protein-coupled receptors (GPCRs) has focused mainly on endocytic trafficking. Whether and how surface delivery of newly synthesized GPCRs is regulated by extracellular signals is less understood. Here we define a signaling-regulated checkpoint at the trans -Golgi network (TGN) that controls the surface delivery of the delta opioid receptor (δR). In PC12 cells, inhibition of phosphoinositide-3 kinase (PI3K) activity blocked export of newly synthesized δR from the Golgi and delivery to the cell surface, similar to treatment with nerve growth factor (NGF). Depletion of class II phosphoinositide-3 kinase α (PI3K C2A), but not inhibition of class I PI3K, blocked δR export to comparable levels and attenuated δR-mediated cAMP inhibition. NGF treatment displaced PI3K C2A from the Golgi and optogenetic recruitment of the PI3K C2A kinase domain to the TGN-induced δR export downstream of NGF. Of importance, PI3K C2A expression promotes export of endogenous δR in primary trigeminal ganglion neurons. Taken together, our results identify PI3K C2A as being required and sufficient for δR export and surface delivery in neuronal cells and suggest that it could be a key modulator of a novel Golgi export checkpoint that coordinates GPCR delivery to the surface. © 2017 Shiwarski et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  4. Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection [version 2; referees: 1 approved, 3 approved with reservations

    Directory of Open Access Journals (Sweden)

    Peter Wild

    2018-02-01

    Full Text Available Background: Herpesvirus capsids are assembled in the nucleus, translocated to the perinuclear space by budding, acquiring tegument and envelope, or released to the cytoplasm via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” is essential for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of alternative exit routes. Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1 or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1 by transmission and scanning electron microscopy, employing freezing technique protocols. Results:  The Golgi complex is a compact entity in a juxtanuclear position covered by a membrane on the cis face. Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced. Conclusions: The data suggest that virions derived by budding at nuclear membranes are intraluminally transported from the perinuclear space via Golgi -endoplasmic reticulum transitions into Golgi cisternae for packaging. Virions derived by budding at nuclear membranes are infective like Us3 deletion mutants, which  accumulate in the perinuclear space. Therefore, i de-envelopment followed by re-envelopment is not essential for production of infective progeny virus, ii the process taking place at the outer nuclear

  5. 3D Printing of Plant Golgi Stacks from Their Electron Tomographic Models.

    Science.gov (United States)

    Mai, Keith Ka Ki; Kang, Madison J; Kang, Byung-Ho

    2017-01-01

    Three-dimensional (3D) printing is an effective tool for preparing tangible 3D models from computer visualizations to assist in scientific research and education. With the recent popularization of 3D printing processes, it is now possible for individual laboratories to convert their scientific data into a physical form suitable for presentation or teaching purposes. Electron tomography is an electron microscopy method by which 3D structures of subcellular organelles or macromolecular complexes are determined at nanometer-level resolutions. Electron tomography analyses have revealed the convoluted membrane architectures of Golgi stacks, chloroplasts, and mitochondria. But the intricacy of their 3D organizations is difficult to grasp from tomographic models illustrated on computer screens. Despite the rapid development of 3D printing technologies, production of organelle models based on experimental data with 3D printing has rarely been documented. In this chapter, we present a simple guide to creating 3D prints of electron tomographic models of plant Golgi stacks using the two most accessible 3D printing technologies.

  6. FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus

    Science.gov (United States)

    Lee, Seongju; Chang, Jaerak; Blackstone, Craig

    2016-01-01

    The endosomal network maintains cellular homeostasis by sorting, recycling and degrading endocytosed cargoes. Retromer organizes the endosomal sorting pathway in conjunction with various sorting nexin (SNX) proteins. The SNX27–retromer complex has recently been identified as a major endosomal hub that regulates endosome-to-plasma membrane recycling by preventing lysosomal entry of cargoes. Here, we show that SNX27 directly interacts with FAM21, which also binds retromer, within the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex. This interaction is required for the precise localization of SNX27 at an endosomal subdomain as well as for recycling of SNX27-retromer cargoes. Furthermore, FAM21 prevents cargo transport to the Golgi apparatus by controlling levels of phosphatidylinositol 4-phosphate, which facilitates cargo dissociation at the Golgi. Together, our results demonstrate that the SNX27–retromer–WASH complex directs cargoes to the plasma membrane by blocking their transport to lysosomes and the Golgi. PMID:26956659

  7. The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis

    DEFF Research Database (Denmark)

    Rautengarten, Carsten; Ebert, Berit; Moreno, Ignacio

    2014-01-01

    Delivery of nucleotide sugar substrates into the Golgi apparatus and endoplasmic reticulum for processes such as cell wall biosynthesis and protein glycosylation is critical for plant growth and development. Plant genomes encode large families of uncharacterized nucleotide sugar transporters that...

  8. Characterization of p28, a novel ERGIC/"cis"-Golgi protein, required for Golgi ribbon formation. pH measurements in the early secretory pathway "in vivo"

    OpenAIRE

    Kögler, Eva Jutta

    2008-01-01

    The secretory pathway of mammalian cells consists of several compartments. Transport between these organelles is accomplished via vesicular carriers or maturation. For non abundant proteins it is thought that transport receptors help the proteins to exit the ER in an effective way. The best characterized mammalian cargo receptor is ERGIC-53, which transports blood coagulation factor V and VIII, cathespin C and Z as well as alpha1-antitrypsin. It localizes to the ER Golgi intermediate compartm...

  9. Selectivity of neuronal [3H]GABA accumulation in the visual cortex as revealed by Golgi staining of the labeled neurons

    International Nuclear Information System (INIS)

    Somogyi, P.; Freund, T.F.; Kisvarday, Z.F.; Halasz, N.

    1981-01-01

    [ 3 H]GABA was injected into the visual cortex of rats in vivo. The labeled amino acid was demonstrated by autoradiography using semithin sections of Golgi material. Selective accumulation was seen in the perikarya of Golgi-stained, gold-toned, aspinous stellate neurons. Spine-laden pyramidal-like cells did not show labeling. This method gives direct information about the dendritic arborization of a neuron, and its putative transmitter, and allows the identification of its synaptic connections. (Auth.)

  10. Human papillomavirus 33 worldwide genetic variation and associated risk of cervical cancer

    Science.gov (United States)

    Chen, Alyce A.; Heideman, Daniëlle A.M.; Boon, Debby; Chen, Zigui; Burk, Robert D.; De Vuyst, Hugo; Gheit, Tarik; Snijders, Peter J.F.; Tommasino, Massimo; Franceschi, Silvia; Clifford, Gary M.

    2014-01-01

    Human papillomavirus (HPV) 33, a member of the HPV16-related alpha-9 species group, is found in approximately 5% of cervical cancers worldwide. The current study aimed to characterize the genetic diversity of HPV33 and to explore the association of HPV33 variants with the risk for cervical cancer. Taking advantage of the International Agency for Research on Cancer biobank, we sequenced the entire E6 and E7 open reading frames of 213 HPV33-positive cervical samples from 30 countries. We identified 28 HPV33 variants that formed 5 phylogenetic groups: the previously identified A1, A2, and B (sub) lineages and the novel A3 and C (sub)lineages. The A1 sublineage was strongly over-represented in cervical cases compared to controls in both Africa and Europe. In conclusion, we provide a classification system for HPV33 variants based on the sequence of E6 and E7 and suggest that the association of HPV33 with cervical cancer may differ by variant (sub)lineage. PMID:24314666

  11. Knockdown of human serine/threonine kinase 33 suppresses human small cell lung carcinoma by blocking RPS6/BAD signaling transduction.

    Science.gov (United States)

    Sun, E L; Liu, C X; Ma, Z X; Mou, X Y; Mu, X A; Ni, Y H; Li, X L; Zhang, D; Ju, Y R

    2017-01-01

    Small cell lung cancer (SCLC) is characterized by rapid growth rate and a tendency to metastasize to distinct sites of patients' bodies. The human serine/threonine kinase 33 (STK33) gene has shown its potency as a therapeutic target for prevention of lung carcinomas including non-small cell lung cancer (NSCLC), but its function in the oncogenesis and development of SCLC remains unrevealed. In the current study, it was hypothesized that STK33 played a key role in the proliferation, survival, and invasion of SCLC cells. The expression of STK33 in human SCLC cell lines NCI-H466 and DMS153 was inhibited by specific shRNA. The cell proliferation, cell apoptosis, and cell invasion of the cells were assessed with a series of in vitro assays. To explore the mechanism through which STK33 gene exerted its function in the carcinogenesis of SCLC cells, the effect of STK33 knockdown on the activity of S6K1/RPS6/BAD signaling was detected. Then the results were further confirmed with STK33 inhibitor ML281 and in vivo assays. The results demonstrated that inhibition of STK33 in SCLC cells suppressed the cell proliferation and invasion while induced cell apoptosis. Associated with the change in the phenotypic features, knockdown of STK33 also decreased the phosphorylation of RPS6 and BAD while increased the expression of cleaved caspase 9, indicating that apoptosis induced by STK33 suppression was mediated via mitochondrial pathway. Similar to the results of STK33 knockdown, incubating NCI-H466 cells with STK33 inhibitor also reduced the cell viability by suppressing RPS6/BAD pathways. Additionally, STK33 knockdown also inhibited tumor growth and RPS6/BAD activity in mice models. Findings outlined in our study were different from that in NSCLC to some extent: knockdown of STK33 in SCLC cells induced the apoptosis through mitochondrial pathway but independent of S6K1 function, inferring that the function of STK33 might be cancer type specific.

  12. Role of the Small GTPase Rho3 in Golgi/Endosome trafficking through functional interaction with adaptin in Fission Yeast.

    Directory of Open Access Journals (Sweden)

    Ayako Kita

    Full Text Available BACKGROUND: We had previously identified the mutant allele of apm1(+ that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1 complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we isolated rho3(+, which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl(- sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl(-, and valproic acid. Green fluorescent protein (GFP-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner. CONCLUSIONS/SIGNIFICANCE: Taken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence

  13. STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells.

    Science.gov (United States)

    Erdmann, Roman S; Toomre, Derek; Schepartz, Alanna

    2017-01-01

    Long time-lapse super-resolution imaging in live cells requires a labeling strategy that combines a bright, photostable fluorophore with a high-density localization probe. Lipids are ideal high-density localization probes, as they are >100 times more abundant than most membrane-bound proteins and simultaneously demark the boundaries of cellular organelles. Here, we describe Cer-SiR, a two-component, high-density lipid probe that is exceptionally photostable. Cer-SiR is generated in cells via a bioorthogonal reaction of two components: a ceramide lipid tagged with trans-cyclooctene (Cer-TCO) and a reactive, photostable Si-rhodamine dye (SiR-Tz). These components assemble within the Golgi apparatus of live cells to form Cer-SiR. Cer-SiR is benign to cellular function, localizes within the Golgi at a high density, and is sufficiently photostable to enable visualization of Golgi structure and dynamics by 3D confocal or long time-lapse STED microscopy.

  14. Proton accumulation and ATPase activity in Golgi apparatus-enriched vesicles from rat liver

    International Nuclear Information System (INIS)

    Yeh, H.I.; van Rossum, G.D.

    1991-01-01

    We have studied the mechanism by which liver Golgi apparatus maintains the acidity of its contents, using a subcellular fraction from rat liver highly enriched in Golgi marker enzymes. Proton accumulation (measured by quenching of acridine-orange fluorescence) and anion-dependent ATPase were characterized and compared. Maximal ATPase and proton accumulation required ATP; GTP and other nucleotides gave 10% to 30% of maximal activity. Among anions, Cl- and Br- approximately doubled the activities; others were much less effective. Half-maximal increase of ATPase and H+ uptake required 55 mmol/L and 27 mmol/L Cl-, respectively. In predominantly chloride media, SCN- and NO3- markedly inhibited H+ uptake. Nitrate competitively inhibited both the chloride-dependent ATPase (apparent Ki 6 mmol/L) and proton uptake (apparent Ki 2 mmol/L). Nitrate and SCN- also inhibited uptake of 36Cl. Replacing K+ with Na+ had no effect on the initial rate of proton uptake but somewhat reduced the steady state attained. Replacement of K+ with NH4+ and choline reduced proton uptake without affecting ATPase. The ATPase and H+ uptake were supported equally well by Mg2+ or Mn2+. The ATPase was competitively inhibited by 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (apparent Ki 39 mumol/L). Other agents inhibiting both H+ uptake and ATPase were N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, chlorpromazine, diethylstilbestrol, Zn2+, Co2+ and Cu2+. In the Cl- medium, accumulated protons were released by ionophores at the relative rates, monensin = nigericin greater than valinomycin greater than carbonyl cyanide mchlorophenylhydrazone; the last of these also reduced ATPase activity. In the absence of Cl-, monensin and valinomycin both stimulated the ATPase. These results show a close association between ATPase activity and acidification of liver Golgi vesicles

  15. Morphometric golgi study of some cortical locations in wag/rij and aci rat strains

    NARCIS (Netherlands)

    Karpova, A.V.; Bikbaev, A.F.; Coenen, A.M.L.; Luijtelaar, E.L.J.M. van; Luijtelaar, E.L.J.M. van; Kuznetsova, G.D.; Coenen, A.M.L.; Chepurnov, S.A.

    2004-01-01

    The present study was aimed to investigate the neuronal organization of two neocortical frontal zones using a Golgi staining technique in genetic epileptic rats, WAG/Rij's. One cortical zone was a specific part of the somatosensory cortex, which was recently proposed to contain a cortical epileptic

  16. The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter*

    Science.gov (United States)

    Geisler, Christoph; Kotu, Varshika; Sharrow, Mary; Rendić, Dubravko; Pöltl, Gerald; Tiemeyer, Michael; Wilson, Iain B. H.; Jarvis, Donald L.

    2012-01-01

    Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac1 flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mistargeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac1 mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac1 Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac1 mutant. These results validate the Drosophila nac1 mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency. PMID:22745127

  17. The GTPase Rab43 Controls the Anterograde ER-Golgi Trafficking and Sorting of GPCRs

    Directory of Open Access Journals (Sweden)

    Chunman Li

    2017-10-01

    Full Text Available G-protein-coupled receptors (GPCRs constitute the largest superfamily of cell-surface signaling proteins. However, mechanisms underlying their surface targeting and sorting are poorly understood. Here, we screen the Rab family of small GTPases in the surface transport of multiple GPCRs. We find that manipulation of Rab43 function significantly alters the surface presentation and signaling of all GPCRs studied without affecting non-GPCR membrane proteins. Rab43 specifically regulates the transport of nascent GPCRs from the endoplasmic reticulum (ER to the Golgi. More interestingly, Rab43 directly interacts with GPCRs in an activation-dependent fashion. The Rab43-binding domain identified in the receptors effectively converts non-GPCR membrane protein transport into a Rab43-dependent pathway. These data reveal a crucial role for Rab43 in anterograde ER-Golgi transport of nascent GPCRs, as well as the ER sorting of GPCR members by virtue of its ability to interact directly.

  18. FMNL2 and -3 regulate Golgi architecture and anterograde transport downstream of Cdc42

    DEFF Research Database (Denmark)

    Kage, Frieda; Steffen, Anika; Ellinger, Adolf

    2017-01-01

    The Rho-family small GTPase Cdc42 localizes at plasma membrane and Golgi complex and aside from protrusion and migration operates in vesicle trafficking, endo- and exocytosis as well as establishment and/or maintenance of cell polarity. The formin family members FMNL2 and -3 are actin assembly fa...

  19. Retention in the Golgi apparatus and expression on the cell surface of Cfr/Esl-1/Glg-1/MG-160 are regulated by two distinct mechanisms.

    Science.gov (United States)

    Miyaoka, Yuichiro; Kato, Hidenori; Ebato, Kazuki; Saito, Shigeru; Miyata, Naoko; Imamura, Toru; Miyajima, Atsushi

    2011-11-15

    Cfr (cysteine-rich fibroblast growth factor receptor) is an Fgf (fibroblast growth factor)-binding protein without a tyrosine kinase. We have shown previously that Cfr is involved in Fgf18 signalling via Fgf receptor 3c. However, as Cfr is also known as Glg (Golgi apparatus protein)-1 or MG-160 and occurs in the Golgi apparatus, it remains unknown how the distribution of Cfr is regulated. In the present study, we performed a mutagenic analysis of Cfr to show that two distinct regions contribute to its distribution and stability. First, the C-terminal region retains Cfr in the Golgi apparatus. Secondly, the Cfr repeats in the extracellular juxtamembrane region destabilizes Cfr passed through the Golgi apparatus. This destabilization does not depend on the cleavage and secretion of the extracellular domain of Cfr. Furthermore, we found that Cfr with a GPI (glycosylphosphatidylinositol) anchor was predominantly expressed on the cell surface in Ba/F3 cells and affected Fgf18 signalling in a similar manner to the full-length Cfr, indicating that the interaction of Cfr with Fgfs on the cell surface is important for its function in Fgf signalling. These results suggest that the expression of Cfr in the Golgi apparatus and on the plasma membrane is finely tuned through two distinct mechanisms for exhibiting different functions.

  20. IL-33 stimulates expression of the GPR84 (EX33) fatty acid receptor gene and of cytokine and chemokine genes in human adipocytes.

    Science.gov (United States)

    Zaibi, Mohamed S; Kępczyńska, Małgorzata A; Harikumar, Parvathy; Alomar, Suliman Y; Trayhurn, Paul

    2018-05-15

    Expression of GPCR fatty acid sensor/receptor genes in adipocytes is modulated by inflammatory mediators, particularly IL-1β. In this study we examined whether the IL-1 gene superfamily member, IL-33, also regulates expression of the fatty acid receptor genes in adipocytes. Human fat cells, differentiated from preadipocytes, were incubated with IL-33 at three different dose levels for 3 or 24 h and mRNA measured by qPCR. Treatment with IL-33 induced a dose-dependent increase in GPR84 mRNA at 3 h, the level with the highest dose being 13.7-fold greater than in controls. Stimulation of GPR84 expression was transitory; the mRNA level was not elevated at 24 h. In contrast to GPR84, IL-33 had no effect on GPR120 expression. IL-33 markedly stimulated expression of the IL1B, CCL2, IL6, CXCL2 and CSF3 genes, but there was no effect on ADIPOQ expression. The largest effect was on CSF3, the mRNA level of which increased 183-fold over controls at 3 h with the highest dose of IL-33; there was a parallel increase in the secretion of G-CSF protein into the medium. It is concluded that in human adipocytes IL-33, which is synthesised in adipose tissue, has a strong stimulatory effect on the expression of cytokine and chemokine genes, particularly CSF3, and on the expression of GPR84, a pro-inflammatory fatty acid receptor. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

    Science.gov (United States)

    McKenzie, Jenna E.; Raisley, Brent; Zhou, Xin; Naslavsky, Naava; Taguchi, Tomohiko; Caplan, Steve; Sheff, David

    2012-01-01

    Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and ER. To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes, recycling endosomes, late endosomes and lysosomes. All cargos pass through early endosomes, but may take different routes to the Golgi. Retromer dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-Mannose-6-Phosphate Receptor, which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CD8-Mannose-6-Phosphate Receptor was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the early endosomes, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB. PMID:22540229

  2. The organization of the Golgi complex and microtubules in skeletal muscle is fiber type-dependent

    DEFF Research Database (Denmark)

    Ralston, E; Lu, Z; Ploug, Thorkil

    1999-01-01

    Skeletal muscle has a nonconventional Golgi complex (GC), the organization of which has been a subject of controversy in the past. We have now examined the distribution of the GC by immunofluorescence and immunogold electron microscopy in whole fibers from different rat muscles, both innervated a...

  3. Plasma Membrane Targeting of Protocadherin 15 Is Regulated by the Golgi-Associated Chaperone Protein PIST

    Directory of Open Access Journals (Sweden)

    Hongyun Nie

    2016-01-01

    Full Text Available Protocadherin 15 (PCDH15 is a core component of hair cell tip-links and crucial for proper function of inner ear hair cells. Mutations of PCDH15 gene cause syndromic and nonsyndromic hearing loss. At present, the regulatory mechanisms responsible for the intracellular transportation of PCDH15 largely remain unknown. Here we show that PIST, a Golgi-associated, PDZ domain-containing protein, interacts with PCDH15. The interaction is mediated by the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI of PCDH15. Through this interaction, PIST retains PCDH15 in the trans-Golgi network (TGN and reduces the membrane expression of PCDH15. We have previously showed that PIST regulates the membrane expression of another tip-link component, cadherin 23 (CDH23. Taken together, our finding suggests that PIST regulates the intracellular trafficking and membrane targeting of the tip-link proteins CDH23 and PCDH15.

  4. Human Papillomavirus 16 Infection Induces VAP-Dependent Endosomal Tubulation.

    Science.gov (United States)

    Siddiqa, Abida; Massimi, Paola; Pim, David; Broniarczyk, Justyna; Banks, Lawrence

    2018-03-15

    Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans -Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the trans -Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex. IMPORTANCE Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans -Golgi network. Indeed, the loss of

  5. Human endothelial progenitor cells internalize high-density lipoprotein.

    Directory of Open Access Journals (Sweden)

    Kaemisa Srisen

    Full Text Available Endothelial progenitor cells (EPCs originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL, and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate, cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal

  6. Evaluation of a magnetic particles-based chemiluminescence enzyme immunoassay for Golgi protein 73 in human serum.

    Science.gov (United States)

    Liu, Xiangyi; Wan, Xiaohua; Lu, Sheng; Zhang, Lijun; Yu, Shaohua; Lu, Xinxin

    2015-05-20

    Golgi protein 73 (GP73) is regarded as a potential serum biomarker for early diagnosis of hepatocellular carcinoma (HCC). We developed a rapid magnetic particles-based chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of serum GP73. Fluorescein isothiocyanate (FITC) and alkaline phosphatase (ALP) were used to label 2 different monoclonal antibodies to GP73. Serum GP73 was captured with labeled antibodies and formed a sandwiched immunoreaction. The magnetic particles (MPs) coated with anti-FITC antibody were used as a means of separation of the GP73 protein from other serum proteins. After adding the enzyme substrate solution, the relative light unit (RLU) was measured. A MPs-CLEIA for serum GP73 was established and evaluated. The RLU was directly proportional to the concentration of GP73. The method linearity was 5-600 μg/l. Limit of the blank was 2.19 μg/l. The intra- and inter-assay imprecision was 73-0.89), and the sensitivity and specificity, with cut-off value of 115.6 μg/l, were 75.4% and 92.1%, respectively. The proposed method demonstrates an acceptable performance for quantifying serum GP73. This assay could be appropriate for routine use in clinical laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Nucleocytoplasmic shuttling of STK16 (PKL12), a Golgi-resident serine/threonine kinase involved in VEGF expression regulation

    International Nuclear Information System (INIS)

    Guinea, Barbara; Ligos, Jose Manuel; Lain de Lera, Teresa; Martin-Caballero, Juan; Flores, Juana; Gonzalez de la Pena, Manuel; Garcia-Castro, Javier; Bernad, Antonio

    2006-01-01

    PKL12/STK16 protein is the first identified mammalian member of a ser/thr kinase subfamily that is conserved across several kingdoms, with a broad expression pattern in murine tissues and cell types. Endogenous STK16 subcellular localization was evaluated by indirect immunofluorescence in NIH/3T3 and NRK cells, demonstrating a Golgi-associated pattern that appears to be independent of signals provided by integrin pathways. When cells were treated with brefeldin A (BFA) or nocodazole, drugs that promote Golgi disorganization, we observed STK16 translocation to the nuclear compartment. Constitutive overexpression of this protein by retroviral vectors also promotes accumulation of STK16 in the nuclear compartment, as shown by subfractionation studies. A kinase-dead STK16 mutant (E202A) was used to demonstrate that both the Golgi association and the nuclear translocation capabilities seem to be independent of the STK16 kinase activity. In addition, we show that STK16 overexpression in several cell lines enhances their capacity to produce and secrete VEGF. To confirm these data in vivo, we injected tumor cells overexpressing STK16 into immunodeficient BALBc/SCID mice. HT1080-derived tumors overexpressing STK16 showed increased volume and number of blood vessels compared to controls. Altogether, these data concur with previous reports suggesting a potential role for STK16 as a transcriptional co-activator

  8. The human protooncogene product p33pim is expressed during fetal hematopoiesis and in diverse leukemias

    International Nuclear Information System (INIS)

    Amson, R.; Przedborski, S.; Telerman, A.; Sigaux, F.; Flandrin, G.; Givol, D.

    1989-01-01

    The authors measured the human pim-1 protooncogene (PIM) expression during fetal development and in hematopoietic malignancies. The data indicate that during human fetal hematopoiesis the 33-kDa pim product, p33pim, is highly expressed in the liver and the spleen. In contrast, a the adult stage it is only slightly expressed in circulating granulocytes. Out of 70 hematopoietic malignancies analyzed, 51 patients and 19 cell lines, p33pim was overexpressed in ∼ 30% of the samples, particularly in myeloid and lymphoid acute leukemias. This overexpression was unrelated to any stage of cellular differentiation and was not due to gene rearrangement or amplification. These results imply a physiological role of the pim-1 protooncogene during hematopoietic development and a deregulation in various leukemias

  9. Role of the Conserved Ologomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

    National Research Council Canada - National Science Library

    Zolov, Sergey

    2004-01-01

    .... We propose that the COG3 protein plays one of the main roles in these processes. We utilized RNA interference assay to knockdown COG3p in HeLa cells to determine the effect of its depletion on Golgi proteins localization...

  10. Ramón y Cajal erroneously identified as Camillo Golgi on a souvenir postage stamp.

    Science.gov (United States)

    Triarhou, Lazaros C; del Cerro, Manuel

    2012-01-01

    Focusing on a philatelic oddity that erringly identifies a picture of Santiago Ramón y Cajal as that of Camillo Golgi, this brief article examines official and unofficial stamp issues honoring the two great neuroanatomists, one from Spain and the other from Italy, who were early Nobel Prize winners in Physiology or Medicine.

  11. Analysis of thick brain sections by obverse-reverse computer microscopy: application of a new, high clarity Golgi-Nissl stain.

    Science.gov (United States)

    Glaser, E M; Van der Loos, H

    1981-08-01

    Exceptionally clear Golgi-Nissl sections of 300 micron thickness have been morphometrically studied by light microscopy using oil immersion objectives. The clarity results from a new variation of a staining procedure that combines Golgi and Nissl images in one section. A viewing technique has been developed that permits a histologic preparation to be examined from its obverse (or normally viewed) side and its reverse (or under) side. The technique was designed for use with a computer microscope but can be employed with any light microscope whose stage position can be measured within 100 micron. Sections thicker than 300 micron can be studied dependent on the working distance of the objective lens, provided that the clarity of the material permits it.

  12. Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

    Science.gov (United States)

    Hoang, Huy-Dung; Maruyama, Jun-ichi

    2014-01-01

    Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. PMID:25362068

  13. COPA mutations impair ER-Golgi transport causing hereditary autoimmune-mediated lung disease and arthritis

    Science.gov (United States)

    Watkin, Levi B.; Jessen, Birthe; Wiszniewski, Wojciech; Vece, Timothy; Jan, Max; Sha, Youbao; Thamsen, Maike; Santos-Cortez, Regie L. P.; Lee, Kwanghyuk; Gambin, Tomasz; Forbes, Lisa; Law, Christopher S.; Stray-Petersen, Asbjørg; Cheng, Mickie H.; Mace, Emily M.; Anderson, Mark S.; Liu, Dongfang; Tang, Ling Fung; Nicholas, Sarah K.; Nahmod, Karen; Makedonas, George; Canter, Debra; Kwok, Pui-Yan; Hicks, John; Jones, Kirk D.; Penney, Samantha; Jhangiani, Shalini N.; Rosenblum, Michael D.; Dell, Sharon D.; Waterfield, Michael R.; Papa, Feroz R.; Muzny, Donna M.; Zaitlen, Noah; Leal, Suzanne M.; Gonzaga-Jauregui, Claudia; Boerwinkle, Eric; Eissa, N. Tony; Gibbs, Richard A.; Lupski, James R.; Orange, Jordan S.; Shum, Anthony K.

    2015-01-01

    Advances in genomics have allowed unbiased genetic studies of human disease with unexpected insights into the molecular mechanisms of cellular immunity and autoimmunity1. We performed whole exome sequencing (WES) and targeted sequencing in patients with an apparent Mendelian syndrome of autoimmune disease characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease (ILD). In five families, we identified four unique deleterious variants in the Coatomer subunit alpha (COPA) gene all located within the same functional domain. We hypothesized that mutant COPA leads to a defect in intracellular transport mediated by coat protein complex I (COPI)2–4. We show that COPA variants impair binding of proteins targeted for retrograde Golgi to ER transport and demonstrate that expression of mutant COPA leads to ER stress and the upregulation of Th17 priming cytokines. Consistent with this pattern of cytokine expression, patients demonstrated a significant skewing of CD4+ T cells toward a T helper 17 (Th17) phenotype, an effector T cell population implicated in autoimmunity5,6. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease. These findings provide a unique opportunity to understand how alterations in cellular homeostasis caused by a defect in the intracellular trafficking pathway leads to the generation of human autoimmune disease. PMID:25894502

  14. α-Synuclein-induced lysosomal dysfunction occurs through disruptions in protein trafficking in human midbrain synucleinopathy models.

    Science.gov (United States)

    Mazzulli, Joseph R; Zunke, Friederike; Isacson, Ole; Studer, Lorenz; Krainc, Dimitri

    2016-02-16

    Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by the accumulation of protein aggregates comprised of α-synuclein (α-syn). A major barrier in treatment discovery for PD is the lack of identifiable therapeutic pathways capable of reducing aggregates in human neuronal model systems. Mutations in key components of protein trafficking and cellular degradation machinery represent important risk factors for PD; however, their precise role in disease progression and interaction with α-syn remains unclear. Here, we find that α-syn accumulation reduced lysosomal degradation capacity in human midbrain dopamine models of synucleinopathies through disrupting hydrolase trafficking. Accumulation of α-syn at the cell body resulted in aberrant association with cis-Golgi-tethering factor GM130 and disrupted the endoplasmic reticulum-Golgi localization of rab1a, a key mediator of vesicular transport. Overexpression of rab1a restored Golgi structure, improved hydrolase trafficking and activity, and reduced pathological α-syn in patient neurons. Our work suggests that enhancement of lysosomal hydrolase trafficking may prove beneficial in synucleinopathies and indicates that human midbrain disease models may be useful for identifying critical therapeutic pathways in PD and related disorders.

  15. Evidence of two genetic clusters of manatees with low genetic diversity in Mexico and implications for their conservation

    Science.gov (United States)

    Nourisson, C.; Morales-Vela, B.; Padilla-Saldivar, J.; Tucker, K.P.; Clark, A.; Olivera-Gomez, L. D.; Bonde, R.; McGuire, P.

    2011-01-01

    The Antillean manatee (Trichechus manatus manatus) occupies the tropical coastal waters of the Greater Antilles and Caribbean, extending from Mexico along Central and South America to Brazil. Historically, manatees were abundant in Mexico, but hunting during the pre-Columbian period, the Spanish colonization and throughout the history of Mexico, has resulted in the significantly reduced population occupying Mexico today. The genetic structure, using microsatellites, shows the presence of two populations in Mexico: the Gulf of Mexico (GMx) and Chetumal Bay (ChB) on the Caribbean coast, with a zone of admixture in between. Both populations show low genetic diversity (GMx: NA=2.69; HE=0.41 and ChB: NA=3.0; HE=0.46). The lower genetic diversity found in the GMx, the largest manatee population in Mexico, is probably due to a combination of a founder effect, as this is the northern range of the sub-species of T. m. manatus, and a bottleneck event. The greater genetic diversity observed along the Caribbean coast, which also has the smallest estimated number of individuals, is possibly due to manatees that come from the GMx and Belize. There is evidence to support limited or unidirectional gene flow between these two important areas. The analyses presented here also suggest minimal evidence of a handful of individual migrants possibly between Florida and Mexico. To address management issues we suggest considering two distinct genetic populations in Mexico, one along the Caribbean coast and one in the riverine systems connected to the GMx. ?? 2011 Springer Science+Business Media B.V.

  16. Evidence of two genetic clusters of manatees with low genetic diversity in Mexico and implications for their conservation.

    Science.gov (United States)

    Nourisson, Coralie; Morales-Vela, Benjamín; Padilla-Saldívar, Janneth; Tucker, Kimberly Pause; Clark, Annmarie; Olivera-Gómez, Leon David; Bonde, Robert; McGuire, Peter

    2011-07-01

    The Antillean manatee (Trichechus manatus manatus) occupies the tropical coastal waters of the Greater Antilles and Caribbean, extending from Mexico along Central and South America to Brazil. Historically, manatees were abundant in Mexico, but hunting during the pre-Columbian period, the Spanish colonization and throughout the history of Mexico, has resulted in the significantly reduced population occupying Mexico today. The genetic structure, using microsatellites, shows the presence of two populations in Mexico: the Gulf of Mexico (GMx) and Chetumal Bay (ChB) on the Caribbean coast, with a zone of admixture in between. Both populations show low genetic diversity (GMx: N(A) = 2.69; H(E) = 0.41 and ChB: N(A) = 3.0; H(E) = 0.46). The lower genetic diversity found in the GMx, the largest manatee population in Mexico, is probably due to a combination of a founder effect, as this is the northern range of the sub-species of T. m. manatus, and a bottleneck event. The greater genetic diversity observed along the Caribbean coast, which also has the smallest estimated number of individuals, is possibly due to manatees that come from the GMx and Belize. There is evidence to support limited or unidirectional gene flow between these two important areas. The analyses presented here also suggest minimal evidence of a handful of individual migrants possibly between Florida and Mexico. To address management issues we suggest considering two distinct genetic populations in Mexico, one along the Caribbean coast and one in the riverine systems connected to the GMx.

  17. Evidence of two genetic clusters of manatees with low genetic diversity in Mexico and implications for their conservation

    Science.gov (United States)

    Nourisson, Coralie; Morales-Vela, Benjamin; Padilla-Saldivar, Janneth; Tucker, Kimberly Pause; Clark, Ann Marie; Olivera-Gomez, Leon David; Bonde, Robert; McGuire, Peter

    2011-01-01

    The Antillean manatee (Trichechus manatus manatus) occupies the tropical coastal waters of the Greater Antilles and Caribbean, extending from Mexico along Central and South America to Brazil. Historically, manatees were abundant in Mexico, but hunting during the pre-Columbian period, the Spanish colonization and throughout the history of Mexico, has resulted in the significantly reduced population occupying Mexico today. The genetic structure, using microsatellites, shows the presence of two populations in Mexico: the Gulf of Mexico (GMx) and Chetumal Bay (ChB) on the Caribbean coast, with a zone of admixture in between. Both populations show low genetic diversity (GMx: NA = 2.69; HE = 0.41 and ChB: NA = 3.0; HE = 0.46). The lower genetic diversity found in the GMx, the largest manatee population in Mexico, is probably due to a combination of a founder effect, as this is the northern range of the sub-species of T. m. manatus, and a bottleneck event. The greater genetic diversity observed along the Caribbean coast, which also has the smallest estimated number of individuals, is possibly due to manatees that come from the GMx and Belize. There is evidence to support limited or unidirectional gene flow between these two important areas. The analyses presented here also suggest minimal evidence of a handful of individual migrants possibly between Florida and Mexico. To address management issues we suggest considering two distinct genetic populations in Mexico, one along the Caribbean coast and one in the riverine systems connected to the GMx.

  18. Phospho-eNOS Ser-1176 is associated with the nucleoli and the Golgi complex in C6 rat glioma cells.

    Science.gov (United States)

    Klinz, Franz-Josef; Herberg, Natalie; Arnhold, Stefan; Addicks, Klaus; Bloch, Wilhelm

    2007-06-29

    Enzymatic activity of endothelial nitric oxide synthase (eNOS) is controlled by posttranslational modifications, protein-protein interactions, and subcellular localization. For example, N-terminal fatty acid modifications target eNOS to the Golgi complex where it becomes phosphorylated. We show here by immunofluorescence analysis that phospho-eNOS Ser-1176 is enriched in the perinuclear region of interphase C6 rat glioma cells. Confocal double immunofluorescence microscopy with the Golgi marker protein 58K revealed that phospho-eNOS Ser-1176 is associated with the Golgi complex. Surprisingly, we observed several spots in the nucleus of C6 cells that were positive for phospho-eNOS Ser-1176. Confocal double immunofluorescence analysis with the nucleolus marker protein fibrillarin revealed that within the nucleus phospho-eNOS Ser-1176 is exclusively associated with the nucleoli. It is known that in mitotic cells nucleoli are lost during prophase and rebuild during telophase. In agreement with this, we find no nucleoli-like distribution of phospho-eNOS Ser-1176 in metaphase and anaphase C6 glioma cells. Our finding that phospho-eNOS Ser-1176 is selectively associated with the nucleoli points to a so far unknown role for eNOS in interphase glioma cells.

  19. Endogenous glycosphingolipid acceptor specificity of sialosyltransferase systems in intact golgi membranes, synaptosomes, and synaptic plasma membranes from rat brain

    International Nuclear Information System (INIS)

    Durrie, R.; Saito, M.; Rosenberg, A.

    1988-01-01

    Preparations highly enriched in Golgi complex membranes, synaptosomes, and synaptic plasma membranes (SPM) by marker enzyme analysis and electron microscopic morphology were made from the brains of 28-day-old rats. These were incubated with cytidine 5'-monophosphate-N-acetyl[ 14 C]neuraminic acid (CMP-NeuAc) in a physiologic buffer, without detergents. Glycolipid sialosyltransferase activities (SATs) were measured by analyzing incorporation of radiolabeled NeuAc into endogenous membrane gangliosides. Golgi SAT was diversified in producing all the various molecular species of labeled gangliosides. Synaptosomal SAT exhibited a lower activity, but it was highly specific in its labeling pattern, with a marked preference for labeling NeuAcα2 → 8NeuAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer (GD3 ganglioside). SPM prepared from the synaptosomes retained the GD3-related SAT (or SAT-2), and the total specific activity increased, which suggests that the location of the synaptosomal activity is in the SPM. These results indicate that SAT activity in Golgi membranes differs from that in synaptosomes with regard to endogenous acceptor substrate specificity and SAT activity of synaptosomes should be located in the synaptosomal plasma membrane. This SAT could function as an ectoenzyme in concert with ecto-sialidase to modulate the GD3 and other ganglioside population in situ at the SPM of the central nervous system

  20. Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy

    NARCIS (Netherlands)

    Gaietta, Guido M; Giepmans, Ben N G; Deerinck, Thomas J; Smith, W Bryan; Ngan, Lucy; Llopis, Juan; Adams, Stephen R; Tsien, Roger Y; Ellisman, Mark H

    2006-01-01

    Combinations of molecular tags visible in light and electron microscopes become particularly advantageous in the analysis of dynamic cellular components like the Golgi apparatus. This organelle disassembles at the onset of mitosis and, after a sequence of poorly understood events, reassembles after

  1. Golgi localized barley MTP8 proteins facilitate Mn transport

    DEFF Research Database (Denmark)

    Pedas, Pai Rosager; Schiller, Michaela; Hegelund, Josefine Nymark

    2014-01-01

    Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2 , which encode membrane...... in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts...

  2. Golgi protein 73 versus alpha-fetoprotein as a biomarker for hepatocellular carcinoma: a diagnostic meta- analysis

    International Nuclear Information System (INIS)

    Zhou, Ying; Yin, Xin; Ying, Jun; Zhang, BoHeng

    2012-01-01

    There have been conflicting reports about serum golgi protein 73 (GP73) as one of the most promising serum markers for the diagnosis of hepatocellular carcinoma (HCC). This study was to make a systematic review about the diagnostic accuracy of serum GP73 versus alpha-fetoprotein (AFP) for HCC. After a systematic review of related studies, sensitivity, specificity and other measures about the accuracy of serum GP73 and AFP in the diagnosis of HCC were pooled using random-effects models. Summary receiver operating characteristic curve analysis was used to summarize the overall test performance. Eight studies were included in our meta-analysis. The summary estimates for serum GP73 and AFP in diagnosing HCC in the studies included were as follows: sensitivity, 76% (95% confidence interval (CI) 51-91%) vs. 70% (47-86%); specificity, 86% (95%CI 65-95%) vs. 89% (69-96%); diagnostic odds ratio (DOR), 18.59 (95%CI 5.33-64.91) vs. 18.00(9.41-34.46); and area under sROC, 0.88 (95%CI 0.77-0.99) vs. 0.86 (95%CI 0.84-0.87). The current evidence indicates that serum GP73 has a comparable accuracy to AFP for the diagnosis of HCC, while the value of serum GP73 in combination with AFP for HCC detection deserves further investigation

  3. Function of the Golgi-located phosphate transporter PHT4;6 is critical for senescence-associated processes in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Hassler, S.; Jung, B.; Lemke, L.; Novák, Ondřej; Strnad, Miroslav; Martinoia, E.; Neuhaus, H.E.

    2016-01-01

    Roč. 67, č. 15 (2016), s. 4671-4684 ISSN 0022-0957 R&D Projects: GA ČR GA15-22322S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : ammonium * cytokinin * golgi Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.830, year: 2016

  4. Orf virus interferes with MHC class I surface expression by targeting vesicular transport and Golgi

    Directory of Open Access Journals (Sweden)

    Rohde Jörg

    2012-07-01

    Full Text Available Abstract Background The Orf virus (ORFV, a zoonotic Parapoxvirus, causes pustular skin lesions in small ruminants (goat and sheep. Intriguingly, ORFV can repeatedly infect its host, despite the induction of a specific immunity. These immune modulating and immune evading properties are still unexplained. Results Here, we describe that ORFV infection of permissive cells impairs the intracellular transport of MHC class I molecules (MHC I as a result of structural disruption and fragmentation of the Golgi apparatus. Depending on the duration of infection, we observed a pronounced co-localization of MHC I and COP-I vesicular structures as well as a reduction of MHC I surface expression of up to 50%. These subversion processes are associated with early ORFV gene expression and are accompanied by disturbed carbohydrate trimming of post-ER MHC I. The MHC I population remaining on the cell surface shows an extended half-life, an effect that might be partially controlled also by late ORFV genes. Conclusions The presented data demonstrate that ORFV down-regulates MHC I surface expression in infected cells by targeting the late vesicular export machinery and the structure and function of the Golgi apparatus, which might aid to escape cellular immune recognition.

  5. TMEM199 Deficiency Is a Disorder of Golgi Homeostasis Characterized by Elevated Aminotransferases, Alkaline Phosphatase, and Cholesterol and Abnormal Glycosylation

    NARCIS (Netherlands)

    Jansen, Jos C.; Timal, Sharita; van Scherpenzeel, Monique; Michelakakis, Helen; Vicogne, Dorothée; Ashikov, Angel; Moraitou, Marina; Hoischen, Alexander; Huijben, Karin; Steenbergen, Gerry; van den Boogert, Marjolein A. W.; Porta, Francesco; Calvo, Pier Luigi; Mavrikou, Mersyni; Cenacchi, Giovanna; van den Bogaart, Geert; Salomon, Jody; Holleboom, Adriaan G.; Rodenburg, Richard J.; Drenth, Joost P. H.; Huynen, Martijn A.; Wevers, Ron A.; Morava, Eva; Foulquier, François; Veltman, Joris A.; Lefeber, Dirk J.

    2016-01-01

    Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously

  6. ECA3, a Golgi-localized P2A-type-ATPase, plays a crucial role in manganese nutrition in Arabidopsis

    DEFF Research Database (Denmark)

    Mills, Rebecca F.; Doherty, Melissa Louise; Lopez Marques, Rosa Laura

    2008-01-01

    and development, and transport processes play a key role in regulating their cellular levels. Arabidopsis (Arabidopsis thaliana) contains four P(2A)-type ATPase genes, AtECA1 to AtECA4, which are expressed in all major organs of Arabidopsis. To elucidate the physiological role of AtECA2 and AtECA3 in Arabidopsis...... not so striking because in this case all plants were severely affected. ECA3 partially restored the growth defect on high Mn of the yeast (Saccharomyces cerevisiae) pmr1 mutant, which is defective in a Golgi Ca/Mn pump (PMR1), and the yeast K616 mutant (Deltapmc1 Deltapmr1 Deltacnb1), defective in Golgi...

  7. A late IL-33 response after exposure to Schistosoma haematobium antigen is associated with an up-regulation of IL-13 in human eosinophils

    DEFF Research Database (Denmark)

    Wilson, S.; Jones, F. M.; Fofana, H. K. M.

    2013-01-01

    IL-33, a proposed alarmin, stimulates innate immune cells and Th2 cells to produce IL-13 and is rapidly upregulated upon antigen exposure in murine helminth infection. The human IL-33 response to helminth antigen was analysed in Malians infected with Schistosoma haematobium by disrupting parasite...... integrity via chemotherapy. Plasma IL-33 was measured pretreatment, and 24 h and 9 weeks post-treatment. At 24 h post-treatment, IL-33 levels were low. Nine week post-treatment IL-33 levels were elevated and were associated with an increase in intracellular IL-13 in eosinophils. Up......-regulation of intracellular IL-13 in eosinophils was also associated with eosinophil expression of ST2L, the IL-33 receptor. IL-33 may play an important downstream role in the human response to schistosome adult worm antigen exposure....

  8. Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

    Directory of Open Access Journals (Sweden)

    Andreea Popa

    2015-02-01

    Full Text Available Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

  9. Association of the golgi UDP-galactose transporter with UDP-galactose: ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum

    NARCIS (Netherlands)

    Sprong, H.; Degroote, S.; Nilsson, T.; Kawakita, M.; Ishida, N.; van der Sluijs, P.; van Meer, G.

    2003-01-01

    UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose: ceramide galactosyltransferase. It is not known how

  10. Identification of rice cornichon as a possible cargo receptor for the Golgi-localized sodium transporter OsHKT1;3

    Czech Academy of Sciences Publication Activity Database

    Rosas-Santiago, P.; Lagunas-Goméz, D.; Barkla, B. J.; Vera-Estrella, R.; Lalonde, S.; Jones, A.; Frommer, W. B.; Zimmermannová, Olga; Sychrová, Hana; Pantoja, O.

    2015-01-01

    Roč. 66, č. 9 (2015), s. 2733-2748 ISSN 0022-0957 R&D Projects: GA MŠk(CZ) LD13037 Institutional support: RVO:67985823 Keywords : Cornichon * endoplasmic reticulum * Golgi * OsHKT1-3 * protein–protein interaction Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.677, year: 2015

  11. GOLGA2/GM130, cis-Golgi Matrix Protein, is a Novel Target of Anticancer Gene Therapy

    OpenAIRE

    Chang, Seung-Hee; Hong, Seong-Ho; Jiang, Hu-Lin; Minai-Tehrani, Arash; Yu, Kyeong-Nam; Lee, Jae-Ho; Kim, Ji-Eun; Shin, Ji-Young; Kang, Bitna; Park, Sungjin; Han, Kiwon; Chae, Chanhee; Cho, Myung-Haing

    2012-01-01

    Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an im...

  12. Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

    International Nuclear Information System (INIS)

    Kadohira, Ikuko; Abe, Yoichiro; Nuriya, Mutsuo; Sano, Kazumi; Tsuji, Shoji; Arimitsu, Takeshi; Yoshimura, Yasunori; Yasui, Masato

    2008-01-01

    Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [ 32 P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition.

  13. A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants.

    Science.gov (United States)

    Brummell, D A; Catala, C; Lashbrook, C C; Bennett, A B

    1997-04-29

    Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.

  14. Genetic, structural, and chemical insights into the dual function of GRASP55 in germ cell Golgi remodeling and JAM-C polarized localization during spermatogenesis.

    Directory of Open Access Journals (Sweden)

    Amandine Cartier-Michaud

    2017-06-01

    Full Text Available Spermatogenesis is a dynamic process that is regulated by adhesive interactions between germ and Sertoli cells. Germ cells express the Junctional Adhesion Molecule-C (JAM-C, encoded by Jam3, which localizes to germ/Sertoli cell contacts. JAM-C is involved in germ cell polarity and acrosome formation. Using a proteomic approach, we demonstrated that JAM-C interacted with the Golgi reassembly stacking protein of 55 kDa (GRASP55, encoded by Gorasp2 in developing germ cells. Generation and study of Gorasp2-/- mice revealed that knock-out mice suffered from spermatogenesis defects. Acrosome formation and polarized localization of JAM-C in spermatids were altered in Gorasp2-/- mice. In addition, Golgi morphology of spermatocytes was disturbed in Gorasp2-/- mice. Crystal structures of GRASP55 in complex with JAM-C or JAM-B revealed that GRASP55 interacted via PDZ-mediated interactions with JAMs and induced a conformational change in GRASP55 with respect of its free conformation. An in silico pharmacophore approach identified a chemical compound called Graspin that inhibited PDZ-mediated interactions of GRASP55 with JAMs. Treatment of mice with Graspin hampered the polarized localization of JAM-C in spermatids, induced the premature release of spermatids and affected the Golgi morphology of meiotic spermatocytes.

  15. Kidney-differentiated cells derived from Lowe Syndrome patient's iPSCs show ciliogenesis defects and Six2 retention at the Golgi complex.

    Directory of Open Access Journals (Sweden)

    Wen-Chieh Hsieh

    Full Text Available Lowe syndrome is an X-linked condition characterized by congenital cataracts, neurological abnormalities and kidney malfunction. This lethal disease is caused by mutations in the OCRL1 gene, which encodes for the phosphatidylinositol 5-phosphatase Ocrl1. While in the past decade we witnessed substantial progress in the identification and characterization of LS patient cellular phenotypes, many of these studies have been performed in knocked-down cell lines or patient's cells from accessible cell types such as skin fibroblasts, and not from the organs affected. This is partially due to the limited accessibility of patient cells from eyes, brain and kidneys. Here we report the preparation of induced pluripotent stem cells (iPSCs from patient skin fibroblasts and their reprogramming into kidney cells. These reprogrammed kidney cells displayed primary cilia assembly defects similar to those described previously in cell lines. Additionally, the transcription factor and cap mesenchyme marker Six2 was substantially retained in the Golgi complex and the functional nuclear-localized fraction was reduced. These results were confirmed using different batches of differentiated cells from different iPSC colonies and by the use of the human proximal tubule kidney cell line HK2. Indeed, OCRL1 KO led to both ciliogenesis defects and Six2 retention in the Golgi complex. In agreement with Six2's role in the suppression of ductal kidney lineages, cells from this pedigree were over-represented among patient kidney-reprogrammed cells. We speculate that this diminished efficacy to produce cap mesenchyme cells would cause LS patients to have difficulties in replenishing senescent or damaged cells derived from this lineage, particularly proximal tubule cells, leading to pathological scenarios such as tubular atrophy.

  16. Functional genomics indicates yeast requires Golgi/ER transport, chromatin remodeling, and DNA repair for low dose DMSO tolerance

    Directory of Open Access Journals (Sweden)

    Brandon David Gaytán

    2013-08-01

    Full Text Available Dimethyl sulfoxide (DMSO is frequently utilized as a solvent in toxicological and pharmaceutical investigations. It is therefore important to establish the cellular and molecular targets of DMSO in order to differentiate its intrinsic effects from those elicited by a compound of interest. We performed a genome-wide functional screen in Saccharomyces cerevisiae to identify deletion mutants exhibiting sensitivity to 1% DMSO, a concentration standard to yeast chemical profiling studies. We report that mutants defective in Golgi/ER transport are sensitive to DMSO, including those lacking components of the conserved oligomeric Golgi (COG complex. Moreover, strains deleted for members of the SWR1 histone exchange complex are hypersensitive to DMSO, with additional chromatin remodeling mutants displaying a range of growth defects. We also identify DNA repair genes important for DMSO tolerance. Finally, we demonstrate that overexpression of histone H2A.Z, which replaces chromatin-associated histone H2A in a SWR1-catalyzed reaction, confers resistance to DMSO. Many yeast genes described in this study have homologs in more complex organisms, and the data provided is applicable to future investigations into the cellular and molecular mechanisms of DMSO toxicity.

  17. Interaction of the SPG21 protein ACP33/maspardin with the aldehyde dehydrogenase ALDH16A1

    Science.gov (United States)

    2017-01-01

    Mast syndrome (SPG21) is an autosomal-recessive complicated form of hereditary spastic paraplegia characterized by dementia, thin corpus callosum, white matter abnormalities, and cerebellar and extrapyramidal signs in addition to spastic paraparesis. A nucleotide insertion resulting in premature truncation of the SPG21 gene product acidic cluster protein 33 (ACP33)/maspardin underlies this disorder, likely causing loss of protein function. However, little is known about the function of maspardin. Here, we report that maspardin localizes prominently to cytoplasm as well as to membranes, possibly at trans-Golgi network/late endosomal compartments. Immunoprecipitation of maspardin with identification of coprecipitating proteins by mass spectrometry revealed the aldehyde dehydrogenase ALDH16A1 as an interacting protein. This interaction was confirmed using overexpressed proteins as well as by fusion protein pull down experiments, and these proteins colocalized in cells. Further studies of the function of ALDH16A1 and the role of the maspardin–ALDH16A1 interaction in neuronal cells may clarify the cellular pathogenesis of Mast syndrome. PMID:19184135

  18. Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis.

    Science.gov (United States)

    Partridge, Emily A; Le Roy, Christine; Di Guglielmo, Gianni M; Pawling, Judy; Cheung, Pam; Granovsky, Maria; Nabi, Ivan R; Wrana, Jeffrey L; Dennis, James W

    2004-10-01

    The Golgi enzyme beta1,6 N-acetylglucosaminyltransferase V (Mgat5) is up-regulated in carcinomas and promotes the substitution of N-glycan with poly N-acetyllactosamine, the preferred ligand for galectin-3 (Gal-3). Here, we report that expression of Mgat5 sensitized mouse cells to multiple cytokines. Gal-3 cross-linked Mgat5-modified N-glycans on epidermal growth factor and transforming growth factor-beta receptors at the cell surface and delayed their removal by constitutive endocytosis. Mgat5 expression in mammary carcinoma was rate limiting for cytokine signaling and consequently for epithelial-mesenchymal transition, cell motility, and tumor metastasis. Mgat5 also promoted cytokine-mediated leukocyte signaling, phagocytosis, and extravasation in vivo. Thus, conditional regulation of N-glycan processing drives synchronous modification of cytokine receptors, which balances their surface retention against loss via endocytosis.

  19. Identification and functional analysis of two Golgi-localized UDP-galactofuranose transporters with overlapping functions in Aspergillus niger.

    Science.gov (United States)

    Park, Joohae; Tefsen, Boris; Heemskerk, Marc J; Lagendijk, Ellen L; van den Hondel, Cees A M J J; van Die, Irma; Ram, Arthur F J

    2015-11-02

    Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter. Based on homology with recently identified UDP-Galf-transporters in A. fumigatus and A. nidulans, two putative UDP-Galf-transporters in A. niger were found. Their function and localization was determined by gene deletions and GFP-tagging studies, respectively. The two putative UDP-Galf-transporters in A. niger are homologous to each other and are predicted to contain eleven transmembrane domains (UgtA) or ten transmembrane domains (UgtB) due to a reduced length of the C-terminal part of the UgtB protein. The presence of two putative UDP-Galf-transporters in the genome was not unique for A. niger. From the twenty Aspergillus species analysed, nine species contained two additional putative UDP-Galf-transporters. Three of the nine species were outside the Aspergillus section nigri, indication an early duplication of UDP-Galf-transporters and subsequent loss of the UgtB copy in several aspergilli. Deletion analysis of the single and double mutants in A. niger indicated that the two putative UDP-Galf-transporters (named UgtA and UgtB) have a redundant function in UDP-Galf-transport as only the double mutant displayed a Galf-negative phenotype. The Galf-negative phenotype of the double mutant could be complemented by expressing either CFP-UgtA or CFP-UgtB fusion proteins from their endogenous promoters, indicating that both CFP-tagged proteins are functional. Both Ugt proteins co-localize with each other as well as with the GDP

  20. Force estimation from ensembles of Golgi tendon organs

    Science.gov (United States)

    Mileusnic, M. P.; Loeb, G. E.

    2009-06-01

    Golgi tendon organs (GTOs) located in the skeletal muscles provide the central nervous system with information about muscle tension. The ensemble firing of all GTO receptors in the muscle has been hypothesized to represent a reliable measure of the whole muscle force but the precision and accuracy of that information are largely unknown because it is impossible to record activity simultaneously from all GTOs in a muscle. In this study, we combined a new mathematical model of force sampling and transduction in individual GTOs with various models of motor unit (MU) organization and recruitment simulating various normal, pathological and neural prosthetic conditions. Our study suggests that in the intact muscle the ensemble GTO activity accurately encodes force information according to a nonlinear, monotonic relationship that has its steepest slope for low force levels and tends to saturate at the highest force levels. The relationship between the aggregate GTO activity and whole muscle tension under some pathological conditions is similar to one seen in the intact muscle during rapidly modulated, phasic excitation of the motor pool (typical for many natural movements) but quite different when the muscle is activated slowly or held at a given force level. Substantial deviations were also observed during simulated functional electrical stimulation.

  1. Regulation of traffic and organelle architecture of the ER-Golgi interface by signal transduction.

    Science.gov (United States)

    Tillmann, Kerstin D; Millarte, Valentina; Farhan, Hesso

    2013-09-01

    The components that control trafficking between organelles of the secretory pathway as well as their architecture were uncovered to a reasonable extent in the past decades. However, only recently did we begin to explore the regulation of the secretory pathway by cellular signaling. In the current review, we focus on trafficking between the endoplasmic reticulum and the Golgi apparatus. We highlight recent advances that have been made toward a better understanding of how the secretory pathway is regulated by signaling and discuss how this knowledge is important to obtain an integrative view of secretion in the context of other homeostatic processes such as growth and proliferation.

  2. 21 CFR 25.33 - Animal drugs.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 1 2010-04-01 2010-04-01 false Animal drugs. 25.33 Section 25.33 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ENVIRONMENTAL IMPACT CONSIDERATIONS Categorical Exclusions § 25.33 Animal drugs. The classes of actions listed in this section are...

  3. Comprehensive two-dimensional gel protein databases offer a global approach to the analysis of human cells: the transformed amnion cells (AMA) master database and its link to genome DNA sequence data

    DEFF Research Database (Denmark)

    Celis, J E; Gesser, B; Rasmussen, H H

    1990-01-01

    , mitochondria, Golgi, ribosomes, intermediate filaments, microfilaments and microtubules), levels in fetal human tissues, partial protein sequences (containing information on 48 human proteins microsequenced so far), cell cycle-regulated proteins, proteins sensitive to interferons alpha, beta, and gamma, heat...

  4. The human brain. Prenatal development and structure

    International Nuclear Information System (INIS)

    Marin-Padilla, Miguel

    2011-01-01

    This book is unique among the current literature in that it systematically documents the prenatal structural development of the human brain. It is based on lifelong study using essentially a single staining procedure, the classic rapid Golgi procedure, which ensures an unusual and desirable uniformity in the observations. The book is amply illustrated with 81 large, high-quality color photomicrographs never previously reproduced. These photomicrographs, obtained at 6, 7, 11, 15, 18, 20, 25, 30, 35, and 40 weeks of gestation, offer a fascinating insight into the sequential prenatal development of neurons, blood vessels, and glia in the human brain. (orig.)

  5. The human brain. Prenatal development and structure

    Energy Technology Data Exchange (ETDEWEB)

    Marin-Padilla, Miguel

    2011-07-01

    This book is unique among the current literature in that it systematically documents the prenatal structural development of the human brain. It is based on lifelong study using essentially a single staining procedure, the classic rapid Golgi procedure, which ensures an unusual and desirable uniformity in the observations. The book is amply illustrated with 81 large, high-quality color photomicrographs never previously reproduced. These photomicrographs, obtained at 6, 7, 11, 15, 18, 20, 25, 30, 35, and 40 weeks of gestation, offer a fascinating insight into the sequential prenatal development of neurons, blood vessels, and glia in the human brain. (orig.)

  6. The trans-Golgi SNARE syntaxin 10 is required for optimal development of Chlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    Andrea L Lucas

    2015-09-01

    Full Text Available Chlamydia trachomatis, an obligate intracellular pathogen, grows inside of a vacuole, termed the inclusion. Within the inclusion, the organisms differentiate from the infectious elementary body (EB into the reticulate body (RB. The RB communicates with the host cell through the inclusion membrane to obtain the nutrients necessary to divide, thus expanding the chlamydial population. At late time points within the developmental cycle, the RBs respond to unknown molecular signals to redifferentiate into infectious EBs to perpetuate the infection cycle. One strategy for Chlamydia to obtain necessary nutrients and metabolites from the host is to intercept host vesicular trafficking pathways. In this study we demonstrate that a trans-Golgi soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE, syntaxin 10, and/or syntaxin10-associated Golgi elements colocalize with the chlamydial inclusion. We hypothesized that Chlamydia utilizes the molecular machinery of syntaxin 10 at the inclusion membrane to intercept specific vesicular trafficking pathways in order to create and maintain an optimal intra-inclusion environment. To test this hypothesis, we used siRNA knockdown of syntaxin 10 to examine the impact of the loss of syntaxin 10 on chlamydial growth and development. Our results demonstrate that loss of syntaxin 10 leads to defects in normal chlamydial maturation including: variable inclusion size with fewer chlamydial organisms per inclusion, fewer infectious progeny, and delayed or halted RB-EB differentiation. These defects in chlamydial development correlate with an overabundance of NBD-lipid retained by inclusions cultured in syntaxin 10 knockdown cells. Overall, loss of syntaxin 10 at the inclusion membrane negatively affects Chlamydia. Understanding host machinery involved in maintaining an optimal inclusion environment to support chlamydial growth and development is critical towards understanding the molecular signals involved in

  7. Hepatitis C virus replication and Golgi function in brefeldin a-resistant hepatoma-derived cells.

    Directory of Open Access Journals (Sweden)

    Rayan Farhat

    Full Text Available Recent reports indicate that the replication of hepatitis C virus (HCV depends on the GBF1-Arf1-COP-I pathway. We generated Huh-7-derived cell lines resistant to brefeldin A (BFA, which is an inhibitor of this pathway. The resistant cell lines could be sorted into two phenotypes regarding BFA-induced toxicity, inhibition of albumin secretion, and inhibition of HCV infection. Two cell lines were more than 100 times more resistant to BFA than the parental Huh-7 cells in these 3 assays. This resistant phenotype was correlated with the presence of a point mutation in the Sec7 domain of GBF1, which is known to impair the binding of BFA. Surprisingly, the morphology of the cis-Golgi of these cells remained sensitive to BFA at concentrations of the drug that allowed albumin secretion, indicating a dichotomy between the phenotypes of secretion and Golgi morphology. Cells of the second group were about 10 times more resistant than parental Huh-7 cells to the BFA-induced toxicity. The EC50 for albumin secretion was only 1.5-1.8 fold higher in these cells than in Huh-7 cells. However their level of secretion in the presence of inhibitory doses of BFA was 5 to 15 times higher. Despite this partially effective secretory pathway in the presence of BFA, the HCV infection was almost as sensitive to BFA as in Huh-7 cells. This suggests that the function of GBF1 in HCV replication does not simply reflect its role of regulator of the secretory pathway of the host cell. Thus, our results confirm the involvement of GBF1 in HCV replication, and suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication.

  8. The role of the active site Zn in the catalytic mechanism of the GH38 Golgi alpha-mannosidase II: Implications from noeuromycin inhibition

    DEFF Research Database (Denmark)

    Bols, Mikael; Kuntz, Douglas A.; Rose, David R.

    2006-01-01

    Golgi alpha-mannosidase II (GMII) is a Family 38 glycosyl hydrolase involved in the eukaryotic N-glycosylation pathway in protein synthesis. Understanding of its catalytic mechanism has been of interest for the development of specific inhibitors that could lead to novel anti-metastatic or anti-in...

  9. Influence of copper on the Golgi apparatus of the meristematic cells of the horse-bean - Vicia faba c. v. Povazsky

    Energy Technology Data Exchange (ETDEWEB)

    Kostal, L

    1974-01-01

    The influence of copper on the Golgi apparatus of beans was investigated. At 1 mg/l copper was toxic within 24 hours. After application of 1 mg/l Cu, a striking increase in the number of split vesicles was noted together with a significant decrease in the number of cisterns. The endoplasmic reticulum is often fragmented after application of Cu, showing separation of membrane.

  10. Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport

    Science.gov (United States)

    Helm, Jared R.; Bentley, Marvin; Thorsen, Kevin D.; Wang, Ting; Foltz, Lauren; Oorschot, Viola; Klumperman, Judith; Hay, Jesse C.

    2014-01-01

    Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery. PMID:25006245

  11. Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.

    Science.gov (United States)

    Barz, W P; Walter, P

    1999-04-01

    Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

  12. The regulation of ER export and Golgi retention of ST3Gal5 (GM3/GM4 synthase) and B4GalNAcT1 (GM2/GD2/GA2 synthase) by arginine/lysine-based motif adjacent to the transmembrane domain.

    Science.gov (United States)

    Uemura, Satoshi; Shishido, Fumi; Kashimura, Madoka; Inokuchi, Jin-ichi

    2015-12-01

    In the Golgi maturation model, the Golgi cisternae dynamically mature along a secretory pathway. In this dynamic process, glycosyltransferases are transported from the endoplasmic reticulum (ER) to the Golgi apparatus where they remain and function. The precise mechanism behind this maturation process remains unclear. We investigated two glycosyltransferases, ST3Gal5 (ST3G5) and B4GalNAcT1 (B4GN1), involved in ganglioside synthesis and examined their signal sequences for ER export and Golgi retention. Reports have suggested that the [R/K](X)[R/K] motif functions as an ER exporting signal; however, this signal sequence is insufficient in stably expressed, full-length ST3G5. Through further analysis, we have clarified that the (2)R(3)R(X)(5) (9)K(X)(3) (13)K sequence in ST3G5 is essential for ER export. We have named the sequence the R/K-based motif. On the other hand, for ER export of B4GN1, the homodimer formation in addition to the R/K-based motif is required for ER export suggesting the importance of unidentified lumenal side interaction. We found that ST3G5 R2A/R3A and K9A/K13A mutants localized not only in Golgi apparatus but also in endosomes. Furthermore, the amounts of mature type asparagine-linked (N)-glycans in ST3G5 R2A/R3A and K9A/K13A mutants were decreased compared with those in wild-type proteins, and the stability of the mutants was lower. These results suggest that the R/K-based motif is necessary for the Golgi retention of ST3G5 and that the retention is involved in the maturation of N-glycans and in stability. Thus, several basic amino acids located on the cytoplasmic tail of ST3G5 play important roles in both ER export and Golgi retention. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Detection of Cytotoxic Activity of Lectin on Human Colon Adenocarcinoma (Sw480 and Epithelial Cervical Carcinoma (C33-A

    Directory of Open Access Journals (Sweden)

    Mirandeli Bautista

    2011-03-01

    Full Text Available Lectins comprise a heterogeneous class of proteins that recognize the carbohydrate moieties of glycoconjugates with high specificity. Numerous studies have shown that lectins are capable of recognizing specific carbohydrate moieties displayed by malignant cells or tissues. The present work was performed to investigate the effects of tepary bean (Phaseolus acutifolius lectins on proliferation, colony formation, and alteration of DNA synthesis of human malignant cells. Tepary bean lectin showed dose dependent  effects on the inhibition of viability as well as on colony formation in two human malignant cells lines (C33-A, Sw480; By contrast, tepary bean lectin only showed significant effects on DNA synthesis on Sw480 cells. Our results provide evidence of the anti- proliferative and cytotoxic effects of the tepary bean lectins on C33-A and Sw480 cells lines.

  14. Prostaglandin E (dmPGE{sub 2}) action in vitro on the activity of rat liver Golgi apparatus galactosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Kordowiak, A.M.; Tomecki, J.; Procyk, K.; Kapusta, P. [Uniwersytet Jagiellonski, Cracow (Poland)

    1993-12-31

    In vitro addition of 16,16`-dimethyl prostaglandin E{sub 2} to Golgi-rich membrane fraction in final concentration of 0.1 {mu}g/1 mg of protein increased generally the activity of galactosyltransferase in comparison with control. The percentage of phospholipids in the whole fraction was similar in both investigated groups, only the sum of phosphatidylenoamine + phosphatidic acid was significantly lower after addition of dmPGE{sub 2} than in the control (0.001 < P < 0.01). (author). 25 refs, 2 figs, 1 tab.

  15. VvGONST-A and VvGONST-B are Golgi-localised GDP-sugar transporters in grapevine (Vitis vinifera L.).

    Science.gov (United States)

    Utz, Daniella; Handford, Michael

    2015-02-01

    Plant nucleotide-sugar transporters (NSTs) are responsible for the import of nucleotide-sugar substrates into the Golgi lumen, for subsequent use in glycosylation reactions. NSTs are specific for either GDP- or UDP-sugars, and almost all transporters studied to date have been isolated from Arabidopsis thaliana L. In order to determine the conservation of the import mechanism in other higher plant species, here we report the identification and characterisation of VvGONST-A and VvGONST-B from grapevine (Vitis vinifera L. cv. Thompson Seedless), which are the orthologues of the GDP-sugar transporters GONST3 and GONST4 in Arabidopsis. Both grapevine NSTs possess the molecular features characteristic of GDP-sugar transporters, including a GDP-binding domain (GXL/VNK) towards the C-terminal. VvGONST-A and VvGONST-B expression is highest at berry setting and decreases throughout berry development and ripening. Moreover, we show using green fluorescent protein (GFP) tagged versions and brefeldin A treatments, that both are localised in the Golgi apparatus. Additionally, in vitro transport assays after expression of both NSTs in tobacco leaves indicate that VvGONST-A and VvGONST-B are capable of transporting GDP-mannose and GDP-glucose, respectively, but not a range of other UDP- and GDP-sugars. The possible functions of these NSTs in glucomannan synthesis and/or glycosylation of sphingolipids are discussed. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. 45 CFR 33.10 - Procedures for salary offset.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Procedures for salary offset. 33.10 Section 33.10 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION SALARY OFFSET § 33.10 Procedures for salary offset. (a) Method and source of deductions. Unless the employee and the Secretary have...

  17. A comparison of models to assess the atmospheric dispersion of resuspended radionuclides on the Nevada Test Site

    International Nuclear Information System (INIS)

    Green, J.R.; Eckart, R.E.

    1994-11-01

    A study of computer codes was made to determine the suitability of their use for modeling radionuclide dispersion from attachment to fugitive dust at the GMX safety shot area of the Nevada Test site. Two codes, the Industrial Source Complex 2 Long Term Model (ISCLT2) and the Fugitive Dust Model (FDM), were subsequently chosen to model the GMX site. Dose calculations were performed using the output values generated by the computer codes. The concentration values produced by the two codes were within a factor of two of each other and were not significantly different. The FDM, however, was felt to be a more useful code for use in calculating doses caused by attachment to fugitive dust

  18. Violacein induces death of resistant leukaemia cells via kinome reprogramming, endoplasmic reticulum stress and Golgi apparatus collapse.

    Directory of Open Access Journals (Sweden)

    Karla C S Queiroz

    Full Text Available It is now generally recognised that different modes of programmed cell death (PCD are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34(+/c-Kit(+/P-glycoprotein(+/MRP1(+ TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells.

  19. Differences in incidence and co-occurrence of vaccine and nonvaccine human papillomavirus types in Finnish population before human papillomavirus mass vaccination suggest competitive advantage for HPV33.

    Science.gov (United States)

    Merikukka, Marko; Kaasila, Marjo; Namujju, Proscovia B; Palmroth, Johanna; Kirnbauer, Reinhard; Paavonen, Jorma; Surcel, Heljä-Marja; Lehtinen, Matti

    2011-03-01

    To understand likelihood of type replacement after vaccination against the high-risk human papillomavirus (HPV) types, we evaluated competition of the seven most common genital HPV types in a population sample of unvaccinated, fertile-aged Finnish women. First trimester sera from two consecutive pregnancies were retrieved from 3,183 Finnish women (mean age, 23.1 years) of whom 42.3% had antibodies to at least one HPV type (6/11/16/18/31/33/45) at the baseline. Antibody positivity to more than one HPV types by the second pregnancy was common among the baseline HPV seropositives. However, compared to baseline HPV-seronegative women, significantly increased incidence rate ratios (IRRs), indicating an increased risk to seroconvert for another HPV type, were consistently noted only for HPV33 among baseline HPV16 or HPV18 antibody (ab)-positive women: HPV(16ab only) (→) (16&33ab) IRR 2.9 [95% confidence interval (CI) 1.6-5.4] and HPV(18ab only) (→) (18&33ab) IRR 2.5 (95% CI 1.1-6.0), irrespectively of the presence of antibodies to other HPV types at baseline: HPV(16ab) (→) (16&33ab) IRR 3.2 (95% CI 2.0-5.2) and HPV(18ab) (→) (18&33ab) IRR 3.6 (95% CI 2.1-5.9). Our findings suggest a possible competitive advantage for HPV33 over other genital HPV types in the unvaccinated population. HPV33 should be monitored for type replacement after HPV mass vaccination. Copyright © 2010 UICC.

  20. Nuclear IL-33 regulates soluble ST2 receptor and IL-6 expression in primary human arterial endothelial cells and is decreased in idiopathic pulmonary arterial hypertension

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Dongmin [Section of Vascular Biology, National Heart and Lung Institute, Imperial College London, London (United Kingdom); Perros, Frédéric [Faculté de Médecine, Université Paris-Sud, Paris, Clamart (France); Caramori, Gaetano [Dipartimento di Scienze Mediche, Sezione di Medicina Interna e Cardiorespiratoria, Centro Interdipartimentale per lo Studio delle Malattie Infiammatorie delle Vie Aeree e Patologie Fumo-Correlate, University of Ferrara, Ferrara (Italy); Meng, Chao [Section of Vascular Biology, National Heart and Lung Institute, Imperial College London, London (United Kingdom); Department of Geriatrics, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China); Dormuller, Peter [Faculté de Médecine, Université Paris-Sud, Paris, Clamart (France); Chou, Pai-Chien [Airways Disease, National Heart and Lung Institute (United Kingdom); Church, Colin [Scottish Pulmonary Vascular Unit, University of Glasgow (United Kingdom); Papi, Alberto; Casolari, Paolo [Dipartimento di Scienze Mediche, Sezione di Medicina Interna e Cardiorespiratoria, Centro Interdipartimentale per lo Studio delle Malattie Infiammatorie delle Vie Aeree e Patologie Fumo-Correlate, University of Ferrara, Ferrara (Italy); Welsh, David; Peacock, Andrew [Scottish Pulmonary Vascular Unit, University of Glasgow (United Kingdom); Humbert, Marc [Faculté de Médecine, Université Paris-Sud, Paris, Clamart (France); Adcock, Ian M. [Airways Disease, National Heart and Lung Institute (United Kingdom); Wort, Stephen J., E-mail: s.wort@imperial.ac.uk [Section of Vascular Biology, National Heart and Lung Institute, Imperial College London, London (United Kingdom)

    2014-08-15

    Highlights: • Nuclear IL-33 expression is reduced in vascular endothelial cells from PAH patients. • Knockdown of IL-33 leads to increased IL-6 and sST2 mRNA expression. • IL-33 binds homeobox motifs in target gene promoters and recruits repressor proteins. - Abstract: Idiopathic pulmonary arterial hypertension (IPAH) is an incurable condition leading to right ventricular failure and death and inflammation is postulated to be associated with vascular remodelling. Interleukin (IL)-33, a member of the “alarmin” family can either act on the membrane ST2 receptor or as a nuclear repressor, to regulate inflammation. We show, using immunohistochemistry, that IL-33 expression is nuclear in the vessels of healthy subjects whereas nuclear IL-33 is markedly diminished in the vessels of IPAH patients. This correlates with reduced IL-33 mRNA expression in their lung. In contrast, serum levels of IL-33 are unchanged in IPAH. However, the expression of the soluble form of ST2, sST2, is enhanced in the serum of IPAH patients. Knock-down of IL-33 in human endothelial cells (ECs) using siRNA is associated with selective modulation of inflammatory genes involved in vascular remodelling including IL-6. Additionally, IL-33 knock-down significantly increased sST2 release from ECs. Chromatin immunoprecipitation demonstrated that IL-33 bound multiple putative homeodomain protein binding motifs in the proximal and distal promoters of ST2 genes. IL-33 formed a complex with the histone methyltransferase SUV39H1, a transcriptional repressor. In conclusion, IL-33 regulates the expression of IL-6 and sST2, an endogenous IL-33 inhibitor, in primary human ECs and may play an important role in the pathogenesis of PAH through recruitment of transcriptional repressor proteins.

  1. The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA Proteins Alter Amyloid-β Precursor Protein (APP Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1.

    Directory of Open Access Journals (Sweden)

    Bjoern von Einem

    Full Text Available Proteolytic processing of amyloid-β precursor protein (APP by beta-site APP cleaving enzyme 1 (BACE1 is the initial step in the production of amyloid beta (Aβ, which accumulates in senile plaques in Alzheimer's disease (AD. Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα, sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.

  2. IL-33 activates tumor stroma to promote intestinal polyposis.

    Science.gov (United States)

    Maywald, Rebecca L; Doerner, Stephanie K; Pastorelli, Luca; De Salvo, Carlo; Benton, Susan M; Dawson, Emily P; Lanza, Denise G; Berger, Nathan A; Markowitz, Sanford D; Lenz, Heinz-Josef; Nadeau, Joseph H; Pizarro, Theresa T; Heaney, Jason D

    2015-05-12

    Tumor epithelial cells develop within a microenvironment consisting of extracellular matrix, growth factors, and cytokines produced by nonepithelial stromal cells. In response to paracrine signals from tumor epithelia, stromal cells modify the microenvironment to promote tumor growth and metastasis. Here, we identify interleukin 33 (IL-33) as a regulator of tumor stromal cell activation and mediator of intestinal polyposis. In human colorectal cancer, IL-33 expression was induced in the tumor epithelium of adenomas and carcinomas, and expression of the IL-33 receptor, IL1RL1 (also referred to as IL1-R4 or ST2), localized predominantly to the stroma of adenoma and both the stroma and epithelium of carcinoma. Genetic and antibody abrogation of responsiveness to IL-33 in the Apc(Min/+) mouse model of intestinal tumorigenesis inhibited proliferation, induced apoptosis, and suppressed angiogenesis in adenomatous polyps, which reduced both tumor number and size. Similar to human adenomas, IL-33 expression localized to tumor epithelial cells and expression of IL1RL1 associated with two stromal cell types, subepithelial myofibroblasts and mast cells, in Apc(Min/+) polyps. In vitro, IL-33 stimulation of human subepithelial myofibroblasts induced the expression of extracellular matrix components and growth factors associated with intestinal tumor progression. IL-33 deficiency reduced mast cell accumulation in Apc(Min/+) polyps and suppressed the expression of mast cell-derived proteases and cytokines known to promote polyposis. Based on these findings, we propose that IL-33 derived from the tumor epithelium promotes polyposis through the coordinated activation of stromal cells and the formation of a protumorigenic microenvironment.

  3. In Vitro and In Vivo Antitumor Effect of Anti-CD33 Chimeric Receptor-Expressing EBV-CTL against CD33+ Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    A. Dutour

    2012-01-01

    Full Text Available Genetic engineering of T cells with chimeric T-cell receptors (CARs is an attractive strategy to treat malignancies. It extends the range of antigens for adoptive T-cell immunotherapy, and major mechanisms of tumor escape are bypassed. With this strategy we redirected immune responses towards the CD33 antigen to target acute myeloid leukemia. To improve in vivo T-cell persistence, we modified human Epstein Barr Virus-(EBV- specific cytotoxic T cells with an anti-CD33.CAR. Genetically modified T cells displayed EBV and HLA-unrestricted CD33 bispecificity in vitro. In addition, though showing a myeloablative activity, they did not irreversibly impair the clonogenic potential of normal CD34+ hematopoietic progenitors. Moreover, after intravenous administration into CD33+ human acute myeloid leukemia-bearing NOD-SCID mice, anti-CD33-EBV-specific T cells reached the tumor sites exerting antitumor activity in vivo. In conclusion, targeting CD33 by CAR-modified EBV-specific T cells may provide additional therapeutic benefit to AML patients as compared to conventional chemotherapy or transplantation regimens alone.

  4. STK33 kinase activity is nonessential in KRAS-dependent cancer cells.

    Science.gov (United States)

    Babij, Carol; Zhang, Yihong; Kurzeja, Robert J; Munzli, Anke; Shehabeldin, Amro; Fernando, Manory; Quon, Kim; Kassner, Paul D; Ruefli-Brasse, Astrid A; Watson, Vivienne J; Fajardo, Flordeliza; Jackson, Angela; Zondlo, James; Sun, Yu; Ellison, Aaron R; Plewa, Cherylene A; San, Miguel Tisha; Robinson, John; McCarter, John; Schwandner, Ralf; Judd, Ted; Carnahan, Josette; Dussault, Isabelle

    2011-09-01

    Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors. ©2011 AACR.

  5. 32 CFR 33.33 - Supplies.

    Science.gov (United States)

    2010-07-01

    ...) Disposition. If there is a residual inventory of unused supplies exceeding $5,000 in total aggregate fair... 32 National Defense 1 2010-07-01 2010-07-01 false Supplies. 33.33 Section 33.33 National Defense... Requirements Changes, Property, and Subawards § 33.33 Supplies. (a) Title. Title to supplies acquired under a...

  6. E6 and E7 Gene Polymorphisms in Human Papillomavirus Types-58 and 33 Identified in Southwest China.

    Directory of Open Access Journals (Sweden)

    Zuyi Chen

    Full Text Available Cancer of the cervix is associated with infection by certain types of human papillomavirus (HPV. The gene variants differ in immune responses and oncogenic potential. The E6 and E7 proteins encoded by high-risk HPV play a key role in cellular transformation. HPV-33 and HPV-58 types are highly prevalent among Chinese women. To study the gene intratypic variations, polymorphisms and positive selections of HPV-33 and HPV-58 E6/E7 in southwest China, HPV-33 (E6, E7: n = 216 and HPV-58 (E6, E7: n = 405 E6 and E7 genes were sequenced and compared to others submitted to GenBank. Phylogenetic trees were constructed by Maximum-likelihood and the Kimura 2-parameters methods by MEGA 6 (Molecular Evolutionary Genetics Analysis version 6.0. The diversity of secondary structure was analyzed by PSIPred software. The selection pressures acting on the E6/E7 genes were estimated by PAML 4.8 (Phylogenetic Analyses by Maximun Likelihood version4.8 software. The positive sites of HPV-33 and HPV-58 E6/E7 were contrasted by ClustalX 2.1. Among 216 HPV-33 E6 sequences, 8 single nucleotide mutations were observed with 6/8 non-synonymous and 2/8 synonymous mutations. The 216 HPV-33 E7 sequences showed 3 single nucleotide mutations that were non-synonymous. The 405 HPV-58 E6 sequences revealed 8 single nucleotide mutations with 4/8 non-synonymous and 4/8 synonymous mutations. Among 405 HPV-58 E7 sequences, 13 single nucleotide mutations were observed with 10/13 non-synonymous mutations and 3/13 synonymous mutations. The selective pressure analysis showed that all HPV-33 and 4/6 HPV-58 E6/E7 major non-synonymous mutations were sites of positive selection. All variations were observed in sites belonging to major histocompatibility complex and/or B-cell predicted epitopes. K93N and R145 (I/N were observed in both HPV-33 and HPV-58 E6.

  7. The Indolic Diet-Derivative, 3,3′-Diindolylmethane, Induced Apoptosis in Human Colon Cancer Cells through Upregulation of NDRG1

    Directory of Open Access Journals (Sweden)

    A. Lerner

    2012-01-01

    Full Text Available N-myc downstream regulated gene-1 participates in carcinogenesis, angiogenesis, metastases, and anticancer drug resistance. In the present study, we analyzed the expression pattern of N-myc downstream regulated gene-1 following treatment of human colonic cancer cell lines; HCT-116 (well differentiated with wild-type p53 gene and Colo-320 (poorly differentiated with mutant p53 gene, with 3,3′-diindolylmethane, a well-established proapoptotic agent product derived from indole-3-carbinol. Treatment of Colo-320 and HCT-116 with 3,3′-diindolylmethane disclosed inhibition of cell viability in a dose-dependent manner, mediated through apoptosis induction. The increased expression of N-myc downstream regulated gene-1 was detected only in poorly differentiated colon cancer cells, Colo-320 cell line. Our results suggest that N-myc downstream regulated gene-1 expression is enhanced by 3,3′-diindolylmethane in poorly differentiated cells and followed by induction of apoptosis. 3,3′-diindolylmethane induced apoptosis may represent a new regulator of N-myc downstream regulated gene-1 in poorly differentiated colonic cancer cells.

  8. The nuclear retention signal of HPV16 L2 protein is essential for incoming viral genome to transverse the trans-Golgi network

    International Nuclear Information System (INIS)

    DiGiuseppe, Stephen; Bienkowska-Haba, Malgorzata; Hilbig, Lydia; Sapp, Martin

    2014-01-01

    The Human papillomavirus (HPV) capsid is composed of the major and minor capsid proteins, L1 and L2, respectively. Infectious entry requires a complex series of conformational changes in both proteins that lead to uptake and allow uncoating to occur. During entry, the capsid is disassembled and host cyclophilins dissociate L1 protein from the L2/DNA complex. Herein, we describe a mutant HPV16 L2 protein (HPV16 L2-R302/5A) that traffics pseudogenome to the trans-Golgi network (TGN) but fails to egress. Our data provide further evidence that HPV16 traffics through the TGN and demonstrates that L2 is essential for TGN egress. Furthermore, we show that cyclophilin activity is required for the L2/DNA complex to be transported to the TGN which is accompanied by a reduced L1 protein levels. - Highlights: • mNLS mutant HPV16 L2 protein traffics pseudogenome to the TGN but fails to egress. • Cyclophilin activity is required for trafficking of the L2/DNA complex to the TGN. • Majority of L1 protein is shed from the L2/DNA complex prior to reaching the TGN

  9. The nuclear retention signal of HPV16 L2 protein is essential for incoming viral genome to transverse the trans-Golgi network

    Energy Technology Data Exchange (ETDEWEB)

    DiGiuseppe, Stephen; Bienkowska-Haba, Malgorzata; Hilbig, Lydia; Sapp, Martin, E-mail: msapp1@lsuhsc.edu

    2014-06-15

    The Human papillomavirus (HPV) capsid is composed of the major and minor capsid proteins, L1 and L2, respectively. Infectious entry requires a complex series of conformational changes in both proteins that lead to uptake and allow uncoating to occur. During entry, the capsid is disassembled and host cyclophilins dissociate L1 protein from the L2/DNA complex. Herein, we describe a mutant HPV16 L2 protein (HPV16 L2-R302/5A) that traffics pseudogenome to the trans-Golgi network (TGN) but fails to egress. Our data provide further evidence that HPV16 traffics through the TGN and demonstrates that L2 is essential for TGN egress. Furthermore, we show that cyclophilin activity is required for the L2/DNA complex to be transported to the TGN which is accompanied by a reduced L1 protein levels. - Highlights: • mNLS mutant HPV16 L2 protein traffics pseudogenome to the TGN but fails to egress. • Cyclophilin activity is required for trafficking of the L2/DNA complex to the TGN. • Majority of L1 protein is shed from the L2/DNA complex prior to reaching the TGN.

  10. Alteraciones de la morfología dendrítica neuronal en la corteza cerebral de ratones infectados con rabia: un estudio con la técnica de Golgi

    Directory of Open Access Journals (Sweden)

    Orlando Torres-Fernández

    2007-12-01

    Conclusiones. Estos resultados son evidencia de que el virus de la rabia sí puede inducir daño neuronal estructural. Además, esta infección aparentemente interfiere con los mecanismos de impregnación argéntica del método de Golgi.

  11. Interleukin-33 promoting Th1 lymphocyte differentiation dependents on IL-12.

    Science.gov (United States)

    Komai-Koma, Mousa; Wang, Eryi; Kurowska-Stolarska, Mariola; Li, Dong; McSharry, Charles; Xu, Damo

    2016-03-01

    The pro-Th2 cytokine IL-33 is now emerging as an important Th1 cytokine-IFN-γ inducer in murine CD4(+) T cells that is essential for protective cell-mediated immunity against viral infection in mice. However, whether IL-33 can promote human Th1 cell differentiation and how IL-33 polarizes Th1 cells is less understood. We assessed the ability of IL-33 to induce Th1 cell differentiation and IFN-γ production in vitro and in vivo. We report here that IL-33 alone had no ability in Th1 cell polarization. However it potentiated IL-12-mediated Th1 cell differentiation and IFN-γ production in TCR-stimulated murine and human CD4(+) T cells in vitro and in vivo. IL-33 promoted Th1 cell development via MyD88 and synergized with IL-12 to enhance St2 and IL-12R expression in CD4(+) T cells. These data therefore provide a novel mechanism for Th1 cell differentiation and optimal induction of a Type 1 response. Thus, IL-33 is capable of inducing IL-12-dependent Th1 cell differentiation in human and mouse CD4(+) T cells. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

  12. 45 CFR 99.33 - Effective date of Assistant Secretary's decision.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Effective date of Assistant Secretary's decision. 99.33 Section 99.33 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION PROCEDURE FOR HEARINGS FOR THE CHILD CARE AND DEVELOPMENT FUND Posthearing Procedures, Decisions § 99.33...

  13. IL-1 family members IL-18 and IL-33 upregulate the inflammatory potential of differentiated human Th1 and Th2 cultures

    DEFF Research Database (Denmark)

    Blom, Lars; Poulsen, Lars K.

    2012-01-01

    The IL-1 family members IL-1ß, IL-18, and IL-33 are potent cytokines in relationship to amplifying the CD4(+) T cell cytokine production. To evaluate their impact on in vitro-differentiated human Th1 and Th2 cultures, such cultures were established from naive T cells, purified from healthy blood...... donors, and reactivated in the presence of IL-1ß, IL-18, or IL-33. Interestingly, we observe modifying responses in Th1 and Th2 cultures induced by IL-18 or IL-33 but not by IL-1ß, both contributing to amplify production of IL-5, IL-13, and IFN-¿. IL-18 or IL-33 stimulation of Th1 cultures resulted...... in increased IFN-¿ and IL-13 production concurrent with reduced IL-10 gene transcription and secretion even though Th1 cultures, in contrast to IL-18Ra, had low ST2L expression. Furthermore, adding IL-18 to Th1 cultures promoted Tbet mRNA expression and production. Th2 cultures stimulated with IL-18 or IL-33...

  14. Cytoarchitectonic and quantitative Golgi study of the hedgehog supraoptic nucleus.

    Science.gov (United States)

    Caminero, A A; Machín, C; Sanchez-Toscano, F

    1992-02-01

    A cytoarchitectural study was made of the supraoptic nucleus (SON) of the hedgehog with special attention to the quantitative comparison of its main neuronal types. The main purposes were (1) to relate the characteristics of this nucleus in the hedgehog (a primitive mammalian insectivorous brain) with those in the SONs of more evolutionarily advanced species; (2) to identify quantitatively the dendritic fields of the main neuronal types in the hedgehog SON and to study their synaptic connectivity. From a descriptive standpoint, 3 neuronal types were found with respect to the number of dendritic stems arising from the neuronal soma: bipolar neurons (48%), multipolar neurons (45.5%) and monopolar neurons (6.5%). Within the multipolar type 2 subtypes could be distinguished, taking into account the number of dendritic spines: (a) with few spines (93%) and (b) very spiny (7%). These results indicate that the hedgehog SON is similar to that in other species except for the very spiny neurons, the significance of which is discussed. In order to characterise the main types more satisfactorily (bipolar and multipolars with few spines) we undertook a quantitative Golgi study of their dendritic fields. Although the patterns of the dendritic field are similar in both neuronal types, the differences in the location of their connectivity can reflect functional changes and alterations in relation to the synaptic afferences.

  15. Qualitative analysis neurons in the adult human dentate nucleus

    Directory of Open Access Journals (Sweden)

    Marić Dušica

    2012-01-01

    Full Text Available Although many relevant findings regarding to the morphology and cytoarchitectural development of the dentate nucleus have been presented so far, very little qualitative information has been collected on neuronal morphology in the adult human dentate nucleus. The neurons were labelled by Golgi staining from thirty human cerebella, obtained from medico-legal forensic autopsies of adult human bodies and free of significant brain pathology. The human dentate neurons were qualitatively analyzed and these cells were classified into two main classes: the small and the large multipolar neurons. Considering the shape of the cell body, number of the primary dendrites, shape of the dendritic tree and their position within the dentate nucleus, three subclasses of the large multipolar neurons have been recognized. The classification of neurons from the human dentate nucleus has been qualitatively confirmed in fetuses and premature infants. This study represents the first qualitative analysis and classification of the large multipolar neurons in the dentate nucleus of the adult human.

  16. IL33 Promotes Colon Cancer Cell Stemness via JNK Activation and Macrophage Recruitment

    Science.gov (United States)

    Fang, Min; Li, Yongkui; Huang, Kai; Qi, Shanshan; Zhang, Jian; Zgodzinski, Witold; Majewski, Marek; Wallner, Grzegorz; Gozdz, Stanislaw; Macek, Pawel; Kowalik, Artur; Pasiarski, Marcin; Grywalska, Ewelina; Vatan, Linda; Nagarsheth, Nisha; Li, Wei; Zhao, Lili; Kryczek, Ilona; Wang, Guobin; Wang, Zheng; Zou, Weiping; Wang, Lin

    2018-01-01

    The expression and biological role of IL33 in colon cancer is poorly understood. In this study, we show that IL33 is expressed by vascular endothelial cells and tumor cells in the human colon cancer microenvironment. Administration of human IL33 and overexpression of murine IL33 enhanced human and murine colon cancer cell growth in vivo, respectively. IL33 stimulated cell sphere formation and prevented chemotherapy-induced tumor apoptosis. Mechanistically, IL33 activated core stem cell genes NANOG, NOTCH3, and OCT3/4 via the ST2 signaling pathway, and induced phosphorylation of c-Jun N terminal kinase (JNK) activation and enhanced binding of c-Jun to the promoters of the core stem cell genes. Moreover, IL33 recruited macrophages into the cancer microenvironment and stimulated them to produce prostaglandin E2, which supported colon cancer stemness and tumor growth. Clinically, tumor IL33 expression associated with poor survival in patients with metastatic colon cancer. Thus, IL33 dually targets tumor cells and macrophages and endows stem-like qualities to colon cancer cells to promote carcinogenesis. Collectively, our work reveals an immune-associated mechanism that extrinsically confers cancer cell stemness properties. Targeting the IL33 signaling pathway may offer an opportunity to treat patients with metastatic cancer. PMID:28249897

  17. Identification of novel resistance mechanisms to NAMPT inhibition via the de novo NAD+ biosynthesis pathway and NAMPT mutation.

    Science.gov (United States)

    Guo, Jun; Lam, Lloyd T; Longenecker, Kenton L; Bui, Mai H; Idler, Kenneth B; Glaser, Keith B; Wilsbacher, Julie L; Tse, Chris; Pappano, William N; Huang, Tzu-Hsuan

    2017-09-23

    Cancer cells have an unusually high requirement for the central and intermediary metabolite nicotinamide adenine dinucleotide (NAD + ), and NAD + depletion ultimately results in cell death. The rate limiting step within the NAD + salvage pathway required for converting nicotinamide to NAD + is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). Targeting NAMPT has been investigated as an anti-cancer strategy, and several highly selective small molecule inhibitors have been found to potently inhibit NAMPT in cancer cells, resulting in NAD + depletion and cytotoxicity. To identify mechanisms that could cause resistance to NAMPT inhibitor treatment, we generated a human fibrosarcoma cell line refractory to the highly potent and selective NAMPT small molecule inhibitor, GMX1778. We uncovered novel and unexpected mechanisms of resistance including significantly increased expression of quinolinate phosphoribosyl transferase (QPRT), a key enzyme in the de novo NAD + synthesis pathway. Additionally, exome sequencing of the NAMPT gene in the resistant cells identified a single heterozygous point mutation that was not present in the parental cell line. The combination of upregulation of the NAD + de novo synthesis pathway through QPRT over-expression and NAMPT mutation confers resistance to GMX1778, but the cells are only partially resistant to next-generation NAMPT inhibitors. The resistance mechanisms uncovered herein provide a potential avenue to continue exploration of next generation NAMPT inhibitors to treat neoplasms in the clinic. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Analysis of the dead layer of a detector of germanium with code ultrapure Monte Carlo SWORD-GEANT; Analisis del dead layer de un detector de germanio ultrapuro con el codigo de Monte Carlo SWORDS-GEANT

    Energy Technology Data Exchange (ETDEWEB)

    Gallardo, S.; Querol, A.; Ortiz, J.; Rodenas, J.; Verdu, G.

    2014-07-01

    In this paper the use of Monte Carlo code SWORD-GEANT is proposed to simulate an ultra pure germanium detector High Purity Germanium detector (HPGe) detector ORTEC specifically GMX40P4, coaxial geometry. (Author)

  19. Restoration of Compact Golgi Morphology in Advanced Prostate Cancer Enhances Susceptibility to Galectin-1-induced Apoptosis by Modifying Mucin O-glycan Synthesis

    OpenAIRE

    Petrosyan, Armen; Holzapfel, Melissa S.; Muirhead, David E.; Cheng, Pi-Wan

    2014-01-01

    Prostate cancer progression is associated with up-regulation of sialyl-T antigen produced by β-galactoside α-2,3-sialyltransferase-1 (ST3Gal1) but not with core 2-associated polylactosamine despite expression of core 2 N-acetylglucosaminyltransferase-L (C2GnT-L/GCNT1). This property allows androgen-refractory prostate cancer cells to evade galectin-1 (LGALS1)-induced apoptosis, but the mechanism is not known. We have recently reported that Golgi targeting of glycosyltransferases is mediated b...

  20. Cytoarchitectonic and quantitative Golgi study of the hedgehog supraoptic nucleus.

    Science.gov (United States)

    Caminero, A A; Machín, C; Sanchez-Toscano, F

    1992-01-01

    A cytoarchitectural study was made of the supraoptic nucleus (SON) of the hedgehog with special attention to the quantitative comparison of its main neuronal types. The main purposes were (1) to relate the characteristics of this nucleus in the hedgehog (a primitive mammalian insectivorous brain) with those in the SONs of more evolutionarily advanced species; (2) to identify quantitatively the dendritic fields of the main neuronal types in the hedgehog SON and to study their synaptic connectivity. From a descriptive standpoint, 3 neuronal types were found with respect to the number of dendritic stems arising from the neuronal soma: bipolar neurons (48%), multipolar neurons (45.5%) and monopolar neurons (6.5%). Within the multipolar type 2 subtypes could be distinguished, taking into account the number of dendritic spines: (a) with few spines (93%) and (b) very spiny (7%). These results indicate that the hedgehog SON is similar to that in other species except for the very spiny neurons, the significance of which is discussed. In order to characterise the main types more satisfactorily (bipolar and multipolars with few spines) we undertook a quantitative Golgi study of their dendritic fields. Although the patterns of the dendritic field are similar in both neuronal types, the differences in the location of their connectivity can reflect functional changes and alterations in relation to the synaptic afferences. Images Fig. 2 Fig. 3 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:1452481

  1. Active Dendrites and Differential Distribution of Calcium Channels Enable Functional Compartmentalization of Golgi Cells.

    Science.gov (United States)

    Rudolph, Stephanie; Hull, Court; Regehr, Wade G

    2015-11-25

    Interneurons are essential to controlling excitability, timing, and synaptic integration in neuronal networks. Golgi cells (GoCs) serve these roles at the input layer of the cerebellar cortex by releasing GABA to inhibit granule cells (grcs). GoCs are excited by mossy fibers (MFs) and grcs and provide feedforward and feedback inhibition to grcs. Here we investigate two important aspects of GoC physiology: the properties of GoC dendrites and the role of calcium signaling in regulating GoC spontaneous activity. Although GoC dendrites are extensive, previous studies concluded they are devoid of voltage-gated ion channels. Hence, the current view holds that somatic voltage signals decay passively within GoC dendrites, and grc synapses onto distal dendrites are not amplified and are therefore ineffective at firing GoCs because of strong passive attenuation. Using whole-cell recording and calcium imaging in rat slices, we find that dendritic voltage-gated sodium channels allow somatic action potentials to activate voltage-gated calcium channels (VGCCs) along the entire dendritic length, with R-type and T-type VGCCs preferentially located distally. We show that R- and T-type VGCCs located in the dendrites can boost distal synaptic inputs and promote burst firing. Active dendrites are thus critical to the regulation of GoC activity, and consequently, to the processing of input to the cerebellar cortex. In contrast, we find that N-type channels are preferentially located near the soma, and control the frequency and pattern of spontaneous firing through their close association with calcium-activated potassium (KCa) channels. Thus, VGCC types are differentially distributed and serve specialized functions within GoCs. Interneurons are essential to neural processing because they modulate excitability, timing, and synaptic integration within circuits. At the input layer of the cerebellar cortex, a single type of interneuron, the Golgi cell (GoC), carries these functions. The

  2. GPR107, a G-protein-coupled receptor essential for intoxication by Pseudomonas aeruginosa exotoxin A, localizes to the Golgi and is cleaved by furin.

    Science.gov (United States)

    Tafesse, Fikadu G; Guimaraes, Carla P; Maruyama, Takeshi; Carette, Jan E; Lory, Stephen; Brummelkamp, Thijn R; Ploegh, Hidde L

    2014-08-29

    A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. 45 CFR 305.33 - Determination of applicable percentages based on performance levels.

    Science.gov (United States)

    2010-10-01

    ..., DEPARTMENT OF HEALTH AND HUMAN SERVICES PROGRAM PERFORMANCE MEASURES, STANDARDS, FINANCIAL INCENTIVES, AND PENALTIES § 305.33 Determination of applicable percentages based on performance levels. (a) A State's... performance levels. 305.33 Section 305.33 Public Welfare Regulations Relating to Public Welfare OFFICE OF...

  4. Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells

    International Nuclear Information System (INIS)

    Lee, Minyoung; Park, Jung-Jin; Ko, Young-Gyu; Lee, Yun-Sil

    2012-01-01

    Previously, we found that β-galactoside α2,6-sialyltransferase (ST6Gal I), an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins and is frequently overexpressed in cancer cells, is up-regulated by ionizing radiation (IR) and cleaved to a form possessing catalytic activity comparable to that of the Golgi-localized enzyme. Moreover, this soluble form is secreted into the culture media. Induction of ST6Gal I significantly increased the migration of colon cancer cells via sialylation of integrin β1. Here, we further investigated the mechanisms underlying ST6Gal I cleavage, solubilization and release from cells, and addressed its functions, focusing primarily on cancer cell migration. We performed immunoblotting and lectin affinity assay to analyze the expression of ST6 Gal I and level of sialylated integrin β1. After ionizing radiation, migration of cells was measured by in vitro migration assay. α2, 6 sialylation level of cell surface was analyzed by flow cytometry. Cell culture media were concentrated and then analyzed for soluble ST6Gal I levels using an α2, 6 sialyltransferase sandwich ELISA. We found that ST6Gal I was cleaved by BACE1 (β-site amyloid precursor protein-cleaving enzyme), which was specifically overexpressed in response to IR. The soluble form of ST6Gal I, which also has sialyltransferase enzymatic activity, was cleaved from the Golgi membrane and then released into the culture media. Both non-cleaved and cleaved forms of ST6Gal I significantly increased colon cancer cell migration in a sialylation-dependent manner. The pro-migratory effect of the non-cleaved form of ST6Gal I was dependent on integrin β1 sialylation, whereas that of the cleaved form of ST6Gal I was not, suggesting that other intracellular sialylated molecules apart from cell surface molecules such as integrin β1 might be involved in mediating the pro-migratory effects of the soluble form of ST6Gal I. Moreover, production of soluble form ST6Gal I by

  5. GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells

    Directory of Open Access Journals (Sweden)

    Dawid Krokowski

    2017-12-01

    Full Text Available Summary: GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome. : Here, Krokowski et al. show that GADD34/PP1 protects the microtubule network, prevents Golgi fragmentation, and preserves protein trafficking independent of its action in the integrated stress response (ISR. In osmoadaptation, GADD34 facilitates trans-Golgi-mediated processing of the endoplasmic reticulum (ER-synthesized amino acid transporter SNAT2, which in turn increases amino acid uptake. Keywords: SNAT2, GADD34, hyperosmotic stress, amino acid transport, Golgi fragmentation, ISR

  6. Epidermal Expression and Regulation of Interleukin-33 during Homeostasis and Inflammation: Strong Species Differences.

    Science.gov (United States)

    Sundnes, Olav; Pietka, Wojciech; Loos, Tamara; Sponheim, Jon; Rankin, Andrew L; Pflanz, Stefan; Bertelsen, Vibeke; Sitek, Jan C; Hol, Johanna; Haraldsen, Guttorm; Khnykin, Denis

    2015-07-01

    IL-33 is a novel IL-1 family member with a putative role in inflammatory skin disorders and a complex biology. Therefore, recent conflicting data regarding its function in experimental models justify a close assessment of its tissue expression and regulation. Indeed, we report here that there are strong species differences in the expression and regulation of epidermal IL-33. In murine epidermis, IL-33 behaved similar to an alarmin, being constitutively expressed in keratinocyte nuclei and rapidly lost during acute inflammation. By contrast, human and porcine IL-33 were weakly expressed or absent in keratinocytes of noninflamed skin but induced during acute inflammation. To this end, we observed that expression of IL-33 in human keratinocytes but not murine keratinocytes was strongly induced by IFN-γ, and this upregulation completely depended on the presence of EGFR ligands. Accordingly, IFN-γ increased the expression of IL-33 in the basal layers of the epidermis in human ex vivo skin cultures only, despite good evidence of IFN-γ activity in cultures from both species. Together these findings demonstrate that a full understanding of IL-33 function in clinical settings must take species-specific differences into account.

  7. Human Ubc9 is involved in intracellular HIV-1 Env stability after trafficking out of the trans-Golgi network in a Gag dependent manner.

    Directory of Open Access Journals (Sweden)

    Christopher R Bohl

    Full Text Available The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER and its trafficking to the trans-Golgi network (TGN were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions.

  8. Localization of HCMV UL33 and US27 in endocytic compartments and viral membranes

    DEFF Research Database (Denmark)

    Fraile-Ramos, Alberto; Pelchen-Matthews, Annegret; Kledal, Thomas N

    2002-01-01

    and undergoes constitutive endocytosis and recycling. Here we studied the cellular distributions and trafficking of two other human cytomegalovirus chemokine receptor-like proteins, UL33 and US27, in transfected and human cytomegalovirus-infected cells. Immunofluorescence staining indicated that UL33 and US27......The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine receptor-like proteins. Although important in viral pathogenesis, little is known about the properties or functions of these proteins. We previously reported that US28 is located in endocytic vesicles......27 undergoes endocytosis. By immunogold labeling of cryosections and electron microscopy, UL33 was seen to localize to multivesicular bodies (MVBs or multivesicular endosomes). Electron microscopy analysis of human cytomegalovirus-infected cells showed that most virus particles wrapped individually...

  9. From Performance to Decision Processes in 33 Years: A History of Organizational Behavior and Human Decision Processes under James C. Naylor.

    Science.gov (United States)

    Weber

    1998-12-01

    For the past 33 years, Organizational Behavior and Human Decision Processes has thrived under a single editor. That editor, James C. Naylor, is retiring from his long stewardship. This article chronicles the course of the journal under Jim's direction and marks some of the accomplishments and changes over the past three decades that go to his credit. Copyright 1998 Academic Press.

  10. Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

    International Nuclear Information System (INIS)

    Azzeh, Maysa; Honigman, Alik; Taraboulos, Albert; Rouvinski, Alexander; Wolf, Dana G.

    2006-01-01

    Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (ΔUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the ΔUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or ΔUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites

  11. Quantitative analysis of basal dendritic tree of layer III pyramidal neurons in different areas of adult human frontal cortex.

    Science.gov (United States)

    Zeba, Martina; Jovanov-Milosević, Natasa; Petanjek, Zdravko

    2008-01-01

    Large long projecting (cortico-cortical) layer IIIc pyramidal neurons were recently disclosed to be in the basis of cognitive processing in primates. Therefore, we quantitatively examined the basal dendritic morphology of these neurons by using rapid Golgi and Golgi Cox impregnation methods among three distinct Brodmann areas (BA) of an adult human frontal cortex: the primary motor BA4 and the associative magnopyramidal BA9 from left hemisphere and the Broca's speech BA45 from both hemispheres. There was no statistically significant difference in basal dendritic length or complexity, as dendritic spine number or their density between analyzed BA's. In addition, we analyzed each of these BA's immunocytochemically for distribution of SMI-32, a marker of largest long distance projecting neurons. Within layer IIIc, the highest density of SMI-32 immunopositive pyramidal neurons was observed in associative BA9, while in primary BA4 they were sparse. Taken together, these data suggest that an increase in the complexity of cortico-cortical network within human frontal areas of different functional order may be principally based on the increase in density of large, SMI-32 immunopositive layer IIIc neurons, rather than by further increase in complexity of their dendritic tree and synaptic network.

  12. 25 CFR 26.33 - How do I show I need job training?

    Science.gov (United States)

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false How do I show I need job training? 26.33 Section 26.33 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR HUMAN SERVICES JOB PLACEMENT AND TRAINING PROGRAM Training Services § 26.33 How do I show I need job training? The need for Job Placement and...

  13. 3D Structure and Interaction of p24β and p24δ Golgi Dynamics Domains: Implication for p24 Complex Formation and Cargo Transport.

    Science.gov (United States)

    Nagae, Masamichi; Hirata, Tetsuya; Morita-Matsumoto, Kana; Theiler, Romina; Fujita, Morihisa; Kinoshita, Taroh; Yamaguchi, Yoshiki

    2016-10-09

    The p24 family consists of four subfamilies (p24α, p24β, p24γ, and p24δ), and the proteins are thought to form hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. The proteins possess a conserved luminal Golgi dynamics (GOLD) domain, whose functions are largely unknown. Here, we present structural and biochemical studies of p24β1 and p24δ1 GOLD domains. Use of GOLD domain-deleted mutants revealed that the GOLD domain of p24δ1 is required for proper p24 hetero-oligomeric complex formation and efficient transport of GPI-anchored proteins. The p24β1 and p24δ1 GOLD domains share a common β-sandwich fold with a characteristic intrasheet disulfide bond. The GOLD domain of p24δ1 crystallized as dimers, allowing the analysis of a homophilic interaction site. Surface plasmon resonance and solution NMR analyses revealed that p24β1 and p24δ1 GOLD domains interact weakly (K d = ~10 -4 M). Bi-protein titration provided interaction site maps. We propose that the heterophilic interaction of p24 GOLD domains contributes to the formation of the p24 hetero-oligomeric complex and to efficient cargo transport. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. 33 CFR 142.33 - Foot protection.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Foot protection. 142.33 Section 142.33 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OUTER... protection. (a) Personnel working in areas or engaged in activities where there is a reasonable probability...

  15. 1647 comments.pmd

    African Journals Online (AJOL)

    driniev

    ask for fax line). Cell: +2772 40 59 561. E-mail: frank.winde@gmx.de. Comments on: “Gold tailings as a source of waterborne uranium contamination of streams – The Koekemoerspruit as a case study” published in Water SA 30 (2) 219-240.

  16. 3,3'-Diindolylmethane is a novel mitochondrial H(+)-ATP synthase inhibitor that can induce p21(Cip1/Waf1) expression by induction of oxidative stress in human breast cancer cells.

    Science.gov (United States)

    Gong, Yixuan; Sohn, Heesook; Xue, Ling; Firestone, Gary L; Bjeldanes, Leonard F

    2006-05-01

    Epidemiologic evidence suggests that high dietary intake of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis in multiple organs. 3,3'-Diindolylmethane, one of the active products derived from Brassica vegetables, is a promising antitumor agent. Previous studies in our laboratory showed that 3,3'-diindolylmethane induced a G(1) cell cycle arrest in human breast cancer MCF-7 cells by a mechanism that included increased expression of p21. In the present study, the upstream events leading to p21 overexpression were further investigated. We show for the first time that 3,3'-diindolylmethane is a strong mitochondrial H(+)-ATPase inhibitor (IC(50) approximately 20 micromol/L). 3,3'-Diindolylmethane treatment induced hyperpolarization of mitochondrial inner membrane, decreased cellular ATP level, and significantly stimulated mitochondrial reactive oxygen species (ROS) production. ROS production, in turn, led to the activation of stress-activated pathways involving p38 and c-Jun NH(2)-terminal kinase. Using specific kinase inhibitors (SB203580 and SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal kinase (JNK) pathways in 3,3'-diindolylmethane-induced p21 mRNA transcription. In addition, antioxidants significantly attenuated 3,3'-diindolylmethane-induced activation of p38 and JNK and induction of p21, indicating that oxidative stress is the major trigger of these events. To further support the role of ROS in 3,3'-diindolylmethane-induced p21 overexpression, we showed that 3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial respiratory chain deficient rho(0) MCF-7 cells, in which 3,3'-diindolylmethane did not stimulate ROS production. Thus, we have established the critical role of enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21 up-regulation in human breast cancer cells.

  17. Golgi Study of Medium Spiny Neurons from Dorsolateral Striatum of the Turtle Trachemys scripta elegans.

    Science.gov (United States)

    González, Carolina; Mendoza, Janeth; Avila-Costa, María Rosa; Arias, Juan M; Barral, Jaime

    2013-10-25

    Comparative anatomy has shown similarities between reptilian and mammalian basal ganglia. Here the morphological characteristics of the medium spiny neurons (MSN) in the dorsolateral striatum (DLS) of the turtle are described after staining them with the Golgi technique. The soma of MSN in DLS showed three main forms: spherical, ovoid, and fusiform. The number of primary dendritic branches (3-4 dendrites/cell) was less than observed in mammals. The MSN axon originates mainly from the soma, and randomly it emerges at the beginning of the primary dendrite. The main differences between turtle and mammalian MSN were detected on dendritic spines. Short, thin, bifurcated and fungiform types of dendritic spines were observed in the turtle's MSN, according to their shape. In most of the analyzed spines, it was found that its length considerably exceeded that reported in mammals, with dendritic spines up to 8μm in length. These differences could play an important role in the modulation of motor networks preserved along the vertebrate evolution. Copyright © 2013. Published by Elsevier Ireland Ltd.

  18. Golgi Outpost Synthesis Impaired by Toxic Polyglutamine Proteins Contributes to Dendritic Pathology in Neurons

    Directory of Open Access Journals (Sweden)

    Chang Geon Chung

    2017-07-01

    Full Text Available Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. Here, we show that nuclear polyglutamine (polyQ toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs and a decreased supply of plasma membrane (PM in Drosophila class IV dendritic arborization (da (C4 da neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP, which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, our results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway.

  19. The Counter-Regulation of Atherogenesis: a Role for Interleukin-33

    Directory of Open Access Journals (Sweden)

    Pavel Kuneš

    2010-01-01

    Full Text Available The recently recognized cytokine interleukin-33 and its receptor ST2 play a favorable role during atherogenesis by inducing a Th1→Th2 shift of the immune response. IL-33 also protects the failing human heart from harmful biomechanical forces which lead to cardiomyocyte hypertrophy and exaggerated interstitial fibrosis. IL-33 inevitably displays side effects common to other Th2 cytokines, the most grave of which is a predisposition to allergic reactions. IL-33 is a nuclear transcription factor of endothelial cells. As such, it is abundant in nonproliferating vessels. Its down-regulation is required for angiogenesis, which may be profitable in wound healing or deleterious in tumor growth.

  20. Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles

    DEFF Research Database (Denmark)

    Hummel, R; Nørgaard, P; Andreasen, P H

    1992-01-01

    The F-antigen is a prominent liver protein which has been extensively used in studies on natural and induced immunological tolerance. However, its intracellular localization and biological function have remained elusive. It has generally been assumed that the F-antigen is confined phylogenetically...... of the Golgi apparatus and transport vesicles pointing to a role of TF-ag in membrane trafficking. Transcription of the TF-ag gene, as determined by run-on analyses, was only detectable in growing cells, and following transfer to starvation condition pre-existing TF-ag mRNA was rapidly degraded. The abundance...

  1. Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication

    Science.gov (United States)

    Taylor, Kathryne E.

    2015-01-01

    ABSTRACT It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment. IMPORTANCE While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both

  2. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.

    Science.gov (United States)

    Zeng, Wei; Lampugnani, Edwin R; Picard, Kelsey L; Song, Lili; Wu, Ai-Min; Farion, Isabela M; Zhao, Jia; Ford, Kris; Doblin, Monika S; Bacic, Antony

    2016-05-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  3. Mitochondrial damage and cholesterol storage in human hepatocellular carcinoma cells with silencing of UBIAD1 gene expression

    Directory of Open Access Journals (Sweden)

    Carlos R. Morales

    2014-01-01

    Full Text Available Heterozygous mutations in the UBIAD1 gene cause Schnyder corneal dystrophy characterized by abnormal cholesterol and phospholipid deposits in the cornea. Ubiad1 protein was recently identified as Golgi prenyltransferase responsible for biosynthesis of vitamin K2 and CoQ10, a key protein in the mitochondrial electron transport chain. Our study shows that silencing UBIAD1 in cultured human hepatocellular carcinoma cells causes dramatic morphological changes and cholesterol storage in the mitochondria, emphasizing an important role of UBIAD1 in mitochondrial function.

  4. 21 CFR 1250.33 - Utensils and equipment.

    Science.gov (United States)

    2010-04-01

    ... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... SANITATION Food Service Sanitation on Land and Air Conveyances, and Vessels § 1250.33 Utensils and equipment... of food or beverages, and the cleaning of food utensils, shall be so constructed as to be easily...

  5. The tumor suppressors p33ING1 and p33ING2 interact with alien in vivo and enhance alien-mediated gene silencing.

    Science.gov (United States)

    Fegers, Inga; Kob, Robert; Eckey, Maren; Schmidt, Oliver; Goeman, Frauke; Papaioannou, Maria; Escher, Niko; von Eggeling, Ferdinand; Melle, Christian; Baniahmad, Aria

    2007-11-01

    The tumor suppressor p33ING1 is involved in DNA repair and cell cycle regulation. Furthermore, p33ING1 is a transcriptional silencer that recognizes the histone mark for trimethylated lysine 4 at histone H3. Interestingly, expression of p33ING1 and p33ING2 is able to induce premature senescence in primary human fibroblasts. The corepressor Alien is involved in gene silencing mediated by selected members of nuclear hormone receptors. In addition, Alien acts as a corepressor for E2F1, a member of the E2F cell cycle regulatory family. Furthermore, recent findings suggest that Alien is complexed with transcription factors participating in DNA repair and chromatin. Here, using a proteomic approach by surface-enhanced laser desorption ionization and mass spectrometry (SELDI-MS) combined with immunological techniques, we show that Alien interacts in vivo with the tumor suppressor p33ING1 as well as with the related tumor suppressor candidate p33ING2. The interaction of Alien with p33ING1 and p33ING2 was confirmed in vitro with GST-pull-down, suggesting a direct binding of Alien to these factors. The binding domain was mapped to a central region of Alien. Functionally, the expression of p33ING1 or p33ING2 enhances the Alien-mediated silencing, suggesting that the interaction plays a role in transcriptional regulation. Thus, the findings suggest that the identified interaction between Alien and the tumor suppressors p33ING1 and p33ING2 reveals a novel cellular protein network.

  6. The Prion-like Domain in the Exomer-Dependent Cargo Pin2 Serves as a trans-Golgi Retention Motif

    Directory of Open Access Journals (Sweden)

    Alicja M. Ritz

    2014-04-01

    Full Text Available Prion and prion-like domains (PLDs are found in many proteins throughout the animal kingdom. We found that the PLD in the S. cerevisiae exomer-dependent cargo protein Pin2 is involved in the regulation of protein transport and localization. The domain serves as a Pin2 retention signal in the trans-Golgi network (TGN. Pin2 is localized in a polarized fashion at the plasma membrane of the bud early in the cell cycle and the bud neck at cytokinesis. This polarized localization is dependent on both exo- and endocytosis. Upon environmental stress, Pin2 is rapidly endocytosed, and the PLD aggregates and causes sequestration of Pin2. The aggregation of Pin2 is reversible upon stress removal and Pin2 is rapidly re-exported to the plasma membrane. Altogether, these data uncover a role for PLDs as protein localization elements.

  7. A traffic signal for heterodimeric amino acid transporters to transfer from the ER to the Golgi.

    Science.gov (United States)

    Ganapathy, Vadivel

    2009-01-15

    Heterodimeric amino acid transporters represent a unique class of transport systems that consist of a light chain that serves as the 'transporter proper' and a heavy chain that is necessary for targeting the complex to the plasma membrane. The currently prevailing paradigm assigns no role for the light chains in the cellular processing of these transporters. In this issue of the Biochemical Journal, Sakamoto et al. provide evidence contrary to this paradigm. Their studies with the rBAT -b(0,+)AT (related to b(0,+) amino acid transporter-b(0,+)-type amino acid transporter) heterodimeric amino acid transporter show that the C-terminus of the light chain b(0,+)AT contains a sequence motif that serves as the traffic signal for the transfer of the heterodimeric complex from the endoplasmic reticulum to the Golgi. This is a novel function for the light chain in addition to its already established role as the subunit responsible for the transport activity. These new findings also seem to be applicable to other heterodimeric amino acid transporters as well.

  8. Assignment of electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) to human chromosome 4q33 by fluorescence in situ hybridization and somatic cell hybridization.

    Science.gov (United States)

    Spector, E B; Seltzer, W K; Goodman, S I

    1999-08-01

    Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a nuclear-encoded protein located in the inner mitochondrial membrane. Inherited defects of ETF-QO cause glutaric acidemia type II. We here describe the localization of the ETF-QO gene to human chromosome 4q33 by somatic cell hybridization and fluorescence in situ hybridization. Copyright 1999 Academic Press.

  9. 33 CFR 159.121 - Sewage processing test.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Sewage processing test. 159.121...) POLLUTION MARINE SANITATION DEVICES Design, Construction, and Testing § 159.121 Sewage processing test. (a) The device must process human sewage in the manner for which it is designed when tested in accordance...

  10. Transcriptional changes common to human cocaine, cannabis and phencyclidine abuse.

    Directory of Open Access Journals (Sweden)

    Elin Lehrmann

    2006-12-01

    Full Text Available A major goal of drug abuse research is to identify and understand drug-induced changes in brain function that are common to many or all drugs of abuse. As these may underlie drug dependence and addiction, the purpose of the present study was to examine if different drugs of abuse effect changes in gene expression that converge in common molecular pathways. Microarray analysis was employed to assay brain gene expression in postmortem anterior prefrontal cortex (aPFC from 42 human cocaine, cannabis and/or phencyclidine abuse cases and 30 control cases, which were characterized by toxicology and drug abuse history. Common transcriptional changes were demonstrated for a majority of drug abuse cases (N = 34, representing a number of consistently changed functional classes: Calmodulin-related transcripts (CALM1, CALM2, CAMK2B were decreased, while transcripts related to cholesterol biosynthesis and trafficking (FDFT1, APOL2, SCARB1, and Golgi/endoplasmic reticulum (ER functions (SEMA3B, GCC1 were all increased. Quantitative PCR validated decreases in calmodulin 2 (CALM2 mRNA and increases in apolipoprotein L, 2 (APOL2 and semaphorin 3B (SEMA3B mRNA for individual cases. A comparison between control cases with and without cardiovascular disease and elevated body mass index indicated that these changes were not due to general cellular and metabolic stress, but appeared specific to the use of drugs. Therefore, humans who abused cocaine, cannabis and/or phencyclidine share a decrease in transcription of calmodulin-related genes and increased transcription related to lipid/cholesterol and Golgi/ER function. These changes represent common molecular features of drug abuse, which may underlie changes in synaptic function and plasticity that could have important ramifications for decision-making capabilities in drug abusers.

  11. The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters*

    Science.gov (United States)

    Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

    2014-01-01

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. PMID:25320081

  12. Dsc E3 ligase localization to the Golgi requires the ATPase Cdc48 and cofactor Ufd1 for activation of sterol regulatory element-binding protein in fission yeast.

    Science.gov (United States)

    Burr, Risa; Ribbens, Diedre; Raychaudhuri, Sumana; Stewart, Emerson V; Ho, Jason; Espenshade, Peter J

    2017-09-29

    Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases associated with diverse cellular activities (AAA + ) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released into the cytosol to enter the nucleus and regulate gene expression. Previously, we reported that Cdc48 binding to Rbd2 is required for Rbd2-mediated SREBP cleavage. Here, using affinity chromatography and mass spectrometry experiments, we identified Cdc48-binding proteins in S. pombe , generating a list of many previously unknown potential Cdc48-binding partners. We show that the established Cdc48 cofactor Ufd1 is required for SREBP cleavage but does not interact with the Cdc48-Rbd2 complex. Cdc48-Ufd1 is instead required at a step prior to Rbd2 function, during Golgi localization of the Dsc E3 ligase complex. Together, these findings demonstrate that two distinct Cdc48 complexes, Cdc48-Ufd1 and Cdc48-Rbd2, are required for SREBP activation and low-oxygen adaptation in S. pombe . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML.

    Science.gov (United States)

    Kung Sutherland, May S; Walter, Roland B; Jeffrey, Scott C; Burke, Patrick J; Yu, Changpu; Kostner, Heather; Stone, Ivan; Ryan, Maureen C; Sussman, Django; Lyon, Robert P; Zeng, Weiping; Harrington, Kimberly H; Klussman, Kerry; Westendorf, Lori; Meyer, David; Bernstein, Irwin D; Senter, Peter D; Benjamin, Dennis R; Drachman, Jonathan G; McEarchern, Julie A

    2013-08-22

    Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.

  14. The human SLC25A33 and SLC25A36 genes of solute carrier family 25 encode two mitochondrial pyrimidine nucleotide transporters.

    Science.gov (United States)

    Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

    2014-11-28

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Golgi protein 73 as a biomarker for hepatocellular carcinoma: A diagnostic meta-analysis.

    Science.gov (United States)

    Yang, Jing; Li, Jingjing; Dai, Weiqi; Wang, Fan; Shen, Miao; Chen, Kan; Cheng, Ping; Zhang, Yan; Wang, Chengfen; Zhu, Rong; Zhang, Huawei; Zheng, Yuanyuan; Wang, Junshan; Xia, Yujing; Lu, Jie; Zhou, Yingqun; Guo, Chuanyong

    2015-04-01

    Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the third leading cause of cancer-related mortality worldwide. Conflicting results have been reported regarding the use of serum Golgi protein 73 (GP73) as a promising serum marker for the diagnosis of HCC; therefore, the aim of the present study was to provide a systematic review of the diagnostic performance of GP73 for HCC. Following a systematic review of the relevant studies, a number of indices associated with the accuracy of the diagnostic performance of GP73, including the sensitivity and specificity, were pooled using Meta Disc 1.4 software. Data were presented as forest plots, and summary receiver operating characteristic (SROC) curve analysis was used to summarize the overall test performance. Eleven studies were included in this meta-analysis. The summary estimates for serum GP73 in diagnosing HCC were as follows: Sensitivity, 77% [95% confidence interval (CI), 75-79%]; specificity, 91% (95% CI, 90-92%); positive likelihood ratio, 4.34 (95% CI, 2.19-8.59); negative likelihood ratio, 0.30 (95% CI, 0.26-0.36) and diagnostic odds ratio, 15.78 (95% CI, 6.95-35.83). The area under the SROC curve was 0.8638, and the Q index was 0.7944. Significant heterogeneity was found. This meta-analysis indicates a moderate diagnostic value of GP73 in HCC; however, further studies with rigorous design, large sample size and multiregional cooperation are required.

  16. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus1[OPEN

    Science.gov (United States)

    Zeng, Wei; Picard, Kelsey L.; Song, Lili; Wu, Ai-Min; Farion, Isabela M.; Zhao, Jia; Ford, Kris; Bacic, Antony

    2016-01-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana. We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. PMID:26951434

  17. Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum.

    Science.gov (United States)

    Smith, T; Ferreira, L R; Hebert, C; Norris, K; Sauk, J J

    1995-08-04

    Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for

  18. Investigation of the 5q33.3 longevity locus and age-related phenotypes

    DEFF Research Database (Denmark)

    Nygaard, Marianne; Thinggaard, Mikael; Christensen, Kaare

    2017-01-01

    A large meta-analysis recently found the 5q33.3 locus to be associated with survival to ≥ 90 years and lower all-cause mortality, thus suggesting it as a third human longevity locus alongside APOE and FOXO3A. The 5q33.3 locus has previously been associated with blood pressure regulation and cardi......A large meta-analysis recently found the 5q33.3 locus to be associated with survival to ≥ 90 years and lower all-cause mortality, thus suggesting it as a third human longevity locus alongside APOE and FOXO3A. The 5q33.3 locus has previously been associated with blood pressure regulation...... and cardiovascular diseases in middle-aged individuals. However, part of the influence on mortality appears to be independent of cardiovascular phenotypes, and the role of the 5q33.3 locus in longevity and survival is therefore still partly unknown. We investigated the association between the longevity......-associated variant rs2149954 on chromosome 5q33.3 and age-related phenotypes in two cohorts of 1,588 and 1,271 long-lived individuals (mean ages 93.1 and 95.9 years, respectively) as well as in 700 middle-aged and 677 elderly individuals (mean ages 52.5 and 78.7 years). Altogether, nominally significant associations...

  19. Expression of interleukin-33 and its receptor ST2 in periapical granulomas and radicular cysts.

    Science.gov (United States)

    Velickovic, Milena; Pejnovic, Nada; Petrovic, Renata; Mitrovic, Slobodanka; Jeftic, Ilija; Kanjevac, Tatjana; Lukic, Aleksandra

    2016-01-01

    Interleukin-33 (IL-33) is a recently identified cytokine belonging to the IL-1 family and ligand for the IL-1 receptor-related protein ST2. IL-33/ST2 signaling plays a critical role in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the pathogenesis of periapical lesions is unknown. We aimed to investigate the expression patterns of IL-33 and ST2 in human periapical lesions. Periapical lesions (n = 36) and healthy periapical tissues (n = 10) were evaluated by immunohistochemistry using antibodies specific for human IL-33 and ST2. Lesion samples were further analyzed by double immunofluorescence to assess IL-33/ST2 co-expression. The numbers of IL-33- and ST2-positive fibroblasts were significantly higher in periapical lesions compared to healthy periapical tissues (both P 0.05). There were no significant differences in the numbers of IL-33- and ST2-positive fibroblasts and endothelial cells between periapical granulomas and radicular cysts (all P > 0.05). Similarly, numbers of ST2-positive mononuclear cells did not differ between periapical granulomas and radicular cysts (P > 0.05). The majority of epithelial cells in radicular cysts were IL-33 positive, while the small proportion of epithelial cells was ST2 positive. Double immunofluorescence analysis revealed IL-33/ST2 co-expression in fibroblasts and endothelial cells. IL-33 and ST2 are expressed in periapical granulomas and radicular cysts. Increased numbers of IL-33- and ST2-positive fibroblasts in periapical lesions when compared to healthy periapical tissues suggest that IL-33/ST2 signaling may be involved in periapical inflammation and tissue fibrosis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33.

    Science.gov (United States)

    Osbourn, Megan; Soares, Dinesh C; Vacca, Francesco; Cohen, E Suzanne; Scott, Ian C; Gregory, William F; Smyth, Danielle J; Toivakka, Matilda; Kemter, Andrea M; le Bihan, Thierry; Wear, Martin; Hoving, Dennis; Filbey, Kara J; Hewitson, James P; Henderson, Holly; Gonzàlez-Cìscar, Andrea; Errington, Claire; Vermeren, Sonja; Astier, Anne L; Wallace, William A; Schwarze, Jürgen; Ivens, Alasdair C; Maizels, Rick M; McSorley, Henry J

    2017-10-17

    Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Ultrasonography as a tool to study afferent feedback from the muscle-tendon complex during human walking

    DEFF Research Database (Denmark)

    Cronin, Neil J.; Klint, Richard af; Grey, Michael James

    2011-01-01

    In humans, one of the most common tasks in everyday life is walking, and sensory afferent feedback from peripheral receptors, particularly the muscle spindles and Golgi tendon organs (GTO), makes an important contribution to the motor control of this task. One factor that can complicate the ability...... with an examination of muscle activation to give a broader insight to neuromuscular interaction during walking. Despite the advances in understanding that these techniques have brought, there is clearly still a need for more direct methods to study both neural and mechanical parameters during human walking in order...... of these receptors to act as length, velocity and force transducers is the complex pattern of interaction between muscle and tendinous tissues, as tendon length is often considerably greater than muscle fibre length in the human lower limb. In essence, changes in muscle-tendon mechanics can influence the firing...

  2. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    International Nuclear Information System (INIS)

    Boura, Evzen; Nencka, Radim

    2015-01-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine

  3. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Boura, Evzen, E-mail: boura@uochb.cas.cz; Nencka, Radim, E-mail: nencka@uochb.cas.cz

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  4. Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone.

    Science.gov (United States)

    O'Dell, Ryan S; Ustine, Candida J M; Cameron, David A; Lawless, Sean M; Williams, Rebecca M; Zipfel, Warren R; Olson, Eric C

    2012-07-07

    The secreted ligand Reelin is believed to regulate the translocation of prospective layer 6 (L6) neocortical neurons into the preplate, a loose layer of pioneer neurons that overlies the ventricular zone. Recent studies have also suggested that Reelin controls neuronal orientation and polarized dendritic growth during this period of early cortical development. To explicitly characterize and quantify how Reelin controls this critical aspect of neurite initiation and growth we used a new ex utero explant model of early cortical development to selectively label a subset of L6 cortical neurons for complete 3-D reconstruction. The total neurite arbor sizes of neurons in Reelin-deficient (reeler mutant) and Dab1-deficient (Reelin-non-responsive scrambler mutant) cortices were quantified and unexpectedly were not different than control arbor lengths (p = 0.51). For each mutant, however, arbor organization was markedly different: mutant neurons manifested more primary processes (neurites emitted directly from the soma) than wild type, and these neurites were longer and displayed less branching. Reeler and scrambler mutant neurites extended tangentially rather than radially, and the Golgi apparatus that normally invests the apical neurite was compact in both reeler and scrambler mutants. Mutant cortices also exhibited a neurite "exclusion zone" which was relatively devoid of L6 neuron neurites and extended at least 15 μm beneath the pial surface, an area corresponding to the marginal zone (MZ) in the wild type explants. The presence of an exclusion zone was also indicated in the orientation of mutant primary neurite and neuronal somata, which failed to adopt angles within ~20˚ of the radial line to the pial surface. Injection of recombinant Reelin to reeler, but not scrambler, mutant cortices fully rescued soma orientation, Golgi organization, and dendritic projection defects within four hrs. These findings indicate Reelin promotes directional dendritic growth into

  5. Crystallization and preliminary X-ray analysis of human S100A13

    International Nuclear Information System (INIS)

    Imai, Fabiana Lica; Nagata, Koji; Yonezawa, Naoto; Yu, Jinyan; Ito, Eriko; Kanai, Saeko; Tanokura, Masaru; Nakano, Minoru

    2006-01-01

    Human S100A13 protein was cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals obtained belonged to space group P2 1 2 1 2 1 and diffracted to a resolution of 1.8 Å. S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins and plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1α, two pro-angiogenic factors released by the endoplasmic reticulum/Golgi-independent non-classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.8 Å resolution and the space group was assigned as primitive orthorhombic P2 1 2 1 2 1

  6. Alpha-fetoprotein-L3 and Golgi protein 73 may serve as candidate biomarkers for diagnosing alpha-fetoprotein-negative hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Zhang ZG

    2015-12-01

    Full Text Available Zhiguo Zhang,1 Yanying Zhang,2 Yeying Wang,1 Lingling Xu,3 Wanju Xu3 1Department of Clinical Laboratory, Zhangqiu Maternity and Child Care Hospital, Zhangqiu, 2Department of Clinical Laboratory, Zaozhuang City Wangkai Infection Hospital, Zaozhuang, 3Department of Clinical Laboratory, Qianfoshan Hospital, Jinan, People’s Republic of China Abstract: Currently, there is no reliable biomarker for use in diagnosing alpha-fetoprotein (AFP-negative hepatocellular carcinoma (HCC. Such a biomarker would aid in making an early diagnosis of AFP-negative HCC, ensuring the timely initiation of treatment. This study examined AFP-L3 and Golgi protein 73 (GP73 as candidate biomarkers for AFP-negative HCC. The affinity adsorption method and enzyme-linked immunoassays were separately used to determine serum levels of AFP-L3 and GP73 in 50 patients with AFP-negative HCC, 30 non-HCC patients, and 50 healthy subjects. Fifty percent of patients with AFP-negative HCC tested positive for AFP-L3, while 3.33% of non-HCC patients and 2.00% of healthy subjects were AFP-L3 positive. Patients with AFP-negative HCC had significantly higher serum levels of AFP-L3 compared to non-HCC patients and healthy individuals; however, there was no significant difference in the AFP-L3 levels of non-HCC patients and healthy subjects. Sixty-six percent of patients with AFP-negative HCC tested positive for GP73, while 10% of non-HCC patients and 0% of healthy subjects were GP73-positive. Patients with AFP-negative HCC had significantly higher serum levels of GP73 compared to non-HCC patients and healthy subjects, but there was no significant difference between the GP73 levels of non-HCC patients and healthy individuals. Moreover, 20 patients with AFP-negative HCC were both AFP-L3- and GP73-positive, while no non-HCC patients or healthy subjects tested positive for both markers. Either AFP-L3 or GP73 may be used as a biomarker for diagnosing AFP-negative HCC, while their combined use

  7. The 33.1 kDa Excretory/secretory Protein Produced by Toxo-cara canis Larvae Serves as a Potential Common Biomarker for Serodiagnosis of Toxocariasis in Paratenic Animals and Human

    Directory of Open Access Journals (Sweden)

    Huu-Hung NGUYEN

    2017-02-01

    Full Text Available Background: Toxocariasis is a prevalent zoonosis disease caused by the closely related nematode species Toxocara canis and Toxocara cati which parasitise Canidae and Felidae respectively. In paratenic hosts, larvae of these worms cause multiple organ damage. However, how these paratenic hosts response to these worms and whether any common biomarker can be applied for diagnosis are still unclear.Methods: Excreted/secreted (E/S antigens were prepared by culture of T. canis larvae in vitro. Using a western blot (WB assay the humoral IgG responses, induced by Toxocara spp. larvae to the worm’s E/S antigens in different infected hosts including mice, rabbits and human, were examined.Results: In a mouse model of toxocariasis, intraperitoneal injection of T. canis larvae induces inflammatory leukocyte accumulation in the liver and the lungs but not in the brain, although a remarkable number of larvae were detected in this organ. Mice and rabbits responded differently to Toxocara spp. resulting in distinct heterogenous WB band patterns. Mice and rabbits both responded to a 33.1 kDa E/S constituent that turned out to be the most sensitive protein for serodiagnosis. Sera from human toxocariasis patients showed heterogenous WB band patterns similar to those observed in rabbits and all responded to the 33.1 kDa band. Conclusion: 33.1 kDa E/S protein can be considered as a critical common biomarker for toxocariasis immuno-diagnosis in both paratenic animals and human and its specificity requires further investigation.

  8. A single amino acid substitution within the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein renders simian-human immunodeficiency virus (SHIVKU-1bMC33) susceptible to rimantadine

    International Nuclear Information System (INIS)

    Hout, David R.; Gomez, Lisa M.; Pacyniak, Erik; Miller, Jean-Marie; Hill, M. Sarah; Stephens, Edward B.

    2006-01-01

    Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIV KU-1bMC33 in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIV M2 ) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M., Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIV KU-1bMC33 ) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIV VpuA19H replicated with similar kinetics as the parental SHIV KU-1bMC33 and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIV KU-1bMC33 . This SHIV VpuA19H virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIV M2 . Electron microscopic examination of SHIV VpuA19H -infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIV M2 -infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel with the alteration of one amino acid and provide

  9. A33 antibody-functionalized exosomes for targeted delivery of doxorubicin against colorectal Cancer.

    Science.gov (United States)

    Li, Yan; Gao, Yuan; Gong, Chunai; Wang, Zhuo; Xia, Qingming; Gu, Fenfen; Hu, Chuling; Zhang, Lijuan; Guo, Huiling; Gao, Shen

    2018-06-20

    Exosomes have emerged as a promising drug carrier with low immunogenicity, high biocompatibility and delivery efficiency. Here in, we isolated exosomes from A33-positive LIM1215 cells (A33-Exo) and loaded them with doxorubicin (Dox). Furthermore, we coated surface-carboxyl superparamagnetic iron oxide nanoparticles (US) with A33 antibodies (A33Ab-US), expecting that these A33 antibodies on the surface of the nanoparticles could bind to A33-positive exosomes and form a complex (A33Ab-US-Exo/Dox) to target A33-positive colon cancer cells. The results showed that A33Ab-US-Exo/Dox had good binding affinity and antiproliferative effect in LIM1215 cells, as shown by increased uptake of the complex. In vivo study showed that A33Ab-US-Exo/Dox had an excellent tumor targeting ability, and was able to inhibit tumor growth and prolong the survival of the mice with reduced cardiotoxicity. In summary, exosomes functionalized by targeting ligands through coating with high-density antibodies may prove to be a novel delivery system for targeted drugs against human cancers. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Papel del diacilglicerol en el tráfico de membranas en la zona entre el retículo endoplasmático y el complejo de Golgi, El

    OpenAIRE

    Fernández Ulibarri, Inés

    2008-01-01

    DE LA TESIS:El DAG es esencial para formar los intermediarios de transporte que se dirigen a la membrana plasmática. Sin embargo, no existen evidencias de su posible participación en las etapas tempranas de la vía secretora. Por tanto, nos centramos en averiguar la implicación del DAG en el transporte de proteínas en la zona del ER/Golgi. Para ello, utilizamos una variedad de fármacos conocidos que inhiben las enzimas responsables de la producción del DAG. Nuestros datos indican que el DAG es...

  11. El Papel del diacilglicerol en el tráfico de membranas en la zona entre el retículo endoplasmático y el complejo de Golgi

    OpenAIRE

    Fernández Ulibarri, Inés

    2008-01-01

    [spa] DE LA TESIS: El DAG es esencial para formar los intermediarios de transporte que se dirigen a la membrana plasmática. Sin embargo, no existen evidencias de su posible participación en las etapas tempranas de la vía secretora. Por tanto, nos centramos en averiguar la implicación del DAG en el transporte de proteínas en la zona del ER/Golgi. Para ello, utilizamos una variedad de fármacos conocidos que inhiben las enzimas responsables de la producción del DAG. Nuestros datos indican que el...

  12. Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33.

    Directory of Open Access Journals (Sweden)

    Jordy Saravia

    2015-10-01

    Full Text Available Respiratory syncytial virus (RSV is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction; whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma.

  13. 33 CFR 385.33 - Revisions to models and analytical tools.

    Science.gov (United States)

    2010-07-01

    ... on a case-by-case basis what documentation is appropriate for revisions to models and analytic tools... analytical tools. 385.33 Section 385.33 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF THE... Incorporating New Information Into the Plan § 385.33 Revisions to models and analytical tools. (a) In carrying...

  14. Crystallization and preliminary X-ray analysis of human S100A13

    Energy Technology Data Exchange (ETDEWEB)

    Imai, Fabiana Lica [Department of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Nagata, Koji [Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Yonezawa, Naoto [Department of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Yu, Jinyan; Ito, Eriko; Kanai, Saeko [Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Tanokura, Masaru [Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Nakano, Minoru, E-mail: mnakano@faculty.chiba-u.jp [Department of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan)

    2006-11-01

    Human S100A13 protein was cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals obtained belonged to space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to a resolution of 1.8 Å. S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins and plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1α, two pro-angiogenic factors released by the endoplasmic reticulum/Golgi-independent non-classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.8 Å resolution and the space group was assigned as primitive orthorhombic P2{sub 1}2{sub 1}2{sub 1}.

  15. Lumen Formation Is an Intrinsic Property of Isolated Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Kenichiro Taniguchi

    2015-12-01

    Full Text Available We demonstrate that dissociated human pluripotent stem cells (PSCs are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi. Molecularly, actin polymerization, regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA, promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs.

  16. 33 CFR 5.33 - Training, examination, and assignment.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Training, examination, and... GENERAL COAST GUARD AUXILIARY § 5.33 Training, examination, and assignment. The Commandant will prescribe the type of training, qualifications and examinations required before a member of the Auxiliary shall...

  17. TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER-Golgi intermediate compartments.

    Science.gov (United States)

    Hanna, Michael G; Block, Samuel; Frankel, E B; Hou, Feng; Johnson, Adam; Yuan, Lin; Knight, Gavin; Moresco, James J; Yates, John R; Ashton, Randolph; Schekman, Randy; Tong, Yufeng; Audhya, Anjon

    2017-09-12

    The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER-Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.

  18. SREBP-1c/MicroRNA 33b Genomic Loci Control Adipocyte Differentiation

    Science.gov (United States)

    Price, Nathan L.; Holtrup, Brandon; Kwei, Stephanie L.; Wabitsch, Martin; Rodeheffer, Matthew; Bianchini, Laurence; Suárez, Yajaira

    2016-01-01

    White adipose tissue (WAT) is essential for maintaining metabolic function, especially during obesity. The intronic microRNAs miR-33a and miR-33b, located within the genes encoding sterol regulatory element-binding protein 2 (SREBP-2) and SREBP-1, respectively, are transcribed in concert with their host genes and function alongside them to regulate cholesterol, fatty acid, and glucose metabolism. SREBP-1 is highly expressed in mature WAT and plays a critical role in promoting in vitro adipocyte differentiation. It is unknown whether miR-33b is induced during or involved in adipogenesis. This is in part due to loss of miR-33b in rodents, precluding in vivo assessment of the impact of miR-33b using standard mouse models. This work demonstrates that miR-33b is highly induced upon differentiation of human preadipocytes, along with SREBP-1. We further report that miR-33b is an important regulator of adipogenesis, as inhibition of miR-33b enhanced lipid droplet accumulation. Conversely, overexpression of miR-33b impaired preadipocyte proliferation and reduced lipid droplet formation and the induction of peroxisome proliferator-activated receptor γ (PPARγ) target genes during differentiation. These effects may be mediated by targeting of HMGA2, cyclin-dependent kinase 6 (CDK6), and other predicted miR-33b targets. Together, these findings demonstrate a novel role of miR-33b in the regulation of adipocyte differentiation, with important implications for the development of obesity and metabolic disease. PMID:26830228

  19. Expression and localization of GLUT1 and GLUT12 in prostate carcinoma.

    Science.gov (United States)

    Chandler, Jenalle D; Williams, Elizabeth D; Slavin, John L; Best, James D; Rogers, Suzanne

    2003-04-15

    Increased glucose consumption is a characteristic of malignant cells and in prostate carcinoma is associated with the proliferation of both androgen-dependent and independent cells. Transport of polar glucose across the nonpolar membrane relies on glucose transporter proteins, known as GLUTs. Increased expression of GLUT1 is a characteristic of many malignant cells. The authors characterized and cloned the cDNA for a novel glucose transporter, GLUT12, which was identified initially in malignant breast epithelial cells. To the authors' knowledge, there have been no reports on the expression of glucose transporters in the human prostate or human prostate carcinoma cells. The authors evaluated GLUT1 and GLUT12 expression in human prostate carcinoma cells. Reverse transcription-polymerase chain reaction was performed on total RNA extracted from cultured prostate carcinoma cells LNCaP, C4, C4-2, and C4-2B using primers to amplify GLUT1, GLUT12, or the housekeeping gene, 36B4. Total protein extracted from prostate carcinoma cell lines was assessed for GLUT12 protein by Western blot analysis. Cultured cell monolayers were incubated with antibodies to GLUT1 or GLUT12 and a peripheral Golgi protein, Golgi 58K, for detection by immunofluorescent confocal microscopy. Sections of benign prostatic hyperplasia and human prostate carcinoma were stained for immunohistochemical detection of GLUT1 and GLUT12. GLUT1 and GLUT12 mRNA and protein were detected in all cell lines evaluated. Immunofluorescence staining demonstrated both GLUT1 and GLUT12 on the plasma membrane and in the cytoplasm in all cultured prostate carcinoma cell lines, with GLUT1 but not GLUT12 appearing to colocalize with the Golgi. Immunohistochemical staining of benign prostatic hyperplasia indicated expression of GLUT1 but not GLUT12. Malignant tissue stained for GLUT12 but was negative for GLUT1. GLUT1 and GLUT12 are expressed in human prostate carcinoma cells. One possible rationale for the GLUT1 Golgi

  20. Lipopolysaccharide attenuates induction of pro-allergic cytokines, thymic stromal lymphopoietin and interleukin 33, in respiratory epithelial cells stimulated with polyI:C and human parechovirus

    Directory of Open Access Journals (Sweden)

    Tsang-Hsiung Lin

    2016-10-01

    Full Text Available Epidemiological studies based on the hygiene hypothesis declare that the level of childhood exposure to environmental microbial products is inversely related to the incidence of allergic diseases in later life. Multiple types of immune cell-mediated immune regulation networks support the hygiene hypothesis. Epithelial cells are the first line of response to microbial products in the environment and bridge the innate and adaptive immune systems; however, their role in the hygiene hypothesis is unknown. To demonstrate the hygiene hypothesis in airway epithelial cells, we examined the effect of lipopolysaccharide (LPS; Toll-like receptor 4 ligand on the expression of the proallergic cytokines thymic stromal lymphopoietin (TSLP and interleukin 33 (IL33 in H292 cells (pulmonary mucoepidermoid carcinoma cells. Stimulation with the TLR ligand polyI:C and human parechovirus type 1 (HPeV1 but not LPS induced TSLP and IL33 through interferon (IFN regulatory factor 3 (IRF3 and NFB activity, which was further validated by using inhibitors (dexamethasone and Bay 11-7082 and shRNA-mediated gene knockdown. Importantly, polyI:C and HPeV1-stimulated TSLP and IL33 induction was reduced by LPS treatment by attenuating TANK-binding kinase 1, IRF3 and NFB activation. Interestingly, the basal mRNA levels of TLR signaling proteins were downregulated with long-term LPS treatment of H292 cells, which suggests that such long-term exposure modulates the expression of innate immunity signaling molecules in airway epithelial cells to mitigate the allergic response. In contrast to the effects of LPS treatment, the alarmin HMBG1 (high mobility group protein B1 acts in synergy with polyI:C to promote TSLP and IL33 expression. Our data support part of the hygiene hypothesis in airway epithelia cells in vitro. In addition to therapeutic targeting of TSLP and IL33, local application of non-pathogenic LPS may be a rational strategy to prevent allergies.

  1. The truncated metabolite GLP-2 (3-33) interacts with the GLP-2 receptor as a partial agonist

    DEFF Research Database (Denmark)

    Thulesen, Jesper; Knudsen, Lotte Bjerre; Hartmann, Bolette

    2002-01-01

    . Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2...... with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth...

  2. Star Formation in M 33 (HerM33es)

    Science.gov (United States)

    Kramer, C.; Boquien, M.; Braine, J.; Buchbender, C.; Calzetti, D.; Gratier, P.; Mookerjea, B.; Relaño, M.; Verley, S.

    2011-11-01

    Within the key project "Herschel M 33 extended survey" (HerM33es), we are studying the physical and chemical processes driving star formation and galactic evolution in the nearby galaxy M 33, combining the study of local conditions affecting individual star formation with properties only becoming apparent on global scales. Here, we present recent results obtained by the HerM33es team. Combining Spitzer and Herschel data ranging from 3.6 μm to 500μm, along with H i, Hα, and GALEX UV data, we have studied the dust at high spatial resolutions of 150 pc, providing estimators of the total infrared (TIR) brightness and of the star formation rate. While the temperature of the warm dust at high brightness is driven by young massive stars, evolved stellar populations appear to drive the temperature of the cold dust. Plane-parallel models of photon dominated regions (PDRs) fail to reproduce fully the [C ii], [O i], and CO maps obtained in a first spectroscopic study of one 2' × 2' subregion of M 33, located on the inner, northern spiral arm and encompassing the H ii region BCLMP 302.

  3. A sensitive radioimmunoassay for measurements of 3,3'- diiodothyronine in unextracted serum

    International Nuclear Information System (INIS)

    Laurberg, P.

    1978-01-01

    A radioimmunoassay for measurements of 3,3'-diiodothyronine (3,3'-T2) in unextracted serum is described. Antisera to 3,3'-T2 were produced by immunization of rabbits with 3,3'-T2 coupled to human serum albumin. Scatchard plot analysis of the chosen antiserum gave an effective equilibrium constant of 1.1x 10 11 l/mol. The 3,3'-T2 binding capacity in undiluted serum was calculated to be 9.1 mmol/m. Monolabelled ( 125 I)3,3'T2 having a specific activity of 3800 mCi/mg was synthetized from 3-monoiodothyronine. The lowest concentration of standard inducing a consistent fall in per cent bound ( 125 I)3,3'-T2 was 6.0 pmol/l (0.24fmol/tube). Triiodothyronine (T3) cross-reacted significantly in the assay. At 50% inhibition of binding of ( 125 I)3,3'-T2, the cross-reaction was 0.29% (mol/mol), but at the low physiological concentrations of T3 it amounted to approximately 1%. T3 cross-reaction was corrected for in every assay. The interference of 3,3'-T2 binding proteins in serum was precluded by adding 8-anilino-1-napthalenesulphonic acid (ANS). Mean 3,3'-T2 concentrations were 38.9 pmol/l in fourteen normal young females and 48.9 pmol/l in fifteen normal young males. When 3,3'-T2 was measured(1) in extracts of serum, (2) in assays where T3 was added to standards in amounts equivalent to those present in the samples and T3 correction omitted, and (3) using a different 3,3'-T2 antibody, similar levels of 3,3'-T2 were found in normal serum. (Auth.)

  4. Different patterns of motor activity induce differential plastic changes in pyramidal neurons in the motor cortex of rats: A Golgi study.

    Science.gov (United States)

    Vázquez-Hernández, Nallely; González-Tapia, Diana C; Martínez-Torres, Nestor I; González-Tapia, David; González-Burgos, Ignacio

    2017-09-14

    Rehabilitation is a process which favors recovery after brain damage involving motor systems, and neural plasticity is the only real resource the brain has for inducing neurobiological events in order to bring about re-adaptation. Rats were placed on a treadmill and made to walk, in different groups, at different velocities and with varying degrees of inclination. Plastic changes in the spines of the apical and basal dendrites of fifth-layer pyramidal neurons in the motor cortices of the rats were detected after study with the Golgi method. Numbers of dendritic spines increased in the three experimental groups, and thin, mushroom, stubby, wide, and branched spines increased or decreased in proportion depending on the motor demands made of each group. Along with the numerical increase of spines, the present findings provide evidence that dendritic spines' geometrical plasticity is involved in the differential performance of motor activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. A human neurodevelopmental model for Williams syndrome.

    Science.gov (United States)

    Chailangkarn, Thanathom; Trujillo, Cleber A; Freitas, Beatriz C; Hrvoj-Mihic, Branka; Herai, Roberto H; Yu, Diana X; Brown, Timothy T; Marchetto, Maria C; Bardy, Cedric; McHenry, Lauren; Stefanacci, Lisa; Järvinen, Anna; Searcy, Yvonne M; DeWitt, Michelle; Wong, Wenny; Lai, Philip; Ard, M Colin; Hanson, Kari L; Romero, Sarah; Jacobs, Bob; Dale, Anders M; Dai, Li; Korenberg, Julie R; Gage, Fred H; Bellugi, Ursula; Halgren, Eric; Semendeferi, Katerina; Muotri, Alysson R

    2016-08-18

    Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes, with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations, leading to behavioural pathologies in humans, remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome, we narrowed this cellular phenotype to a single gene candidate, frizzled 9 (FZD9). At the neuronal stage, layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites, increased numbers of spines and synapses, aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain.

  6. Vps33b pathogenic mutations preferentially affect VIPAS39/SPE-39-positive endosomes.

    Science.gov (United States)

    Tornieri, Karine; Zlatic, Stephanie A; Mullin, Ariana P; Werner, Erica; Harrison, Robert; L'hernault, Steven W; Faundez, Victor

    2013-12-20

    Mutations in Vps33 isoforms cause pigment dilution in mice (Vps33a, buff) and Drosophila (car) and the neurogenic arthrogryposis, renal dysfunction and cholestasis syndrome in humans (ARC1, VPS33B). The later disease is also caused by mutations in VIPAS39, (Vps33b interacting protein, apical-basolateral polarity regulator, SPE-39 homolog; ARC2), a protein that interacts with the HOmotypic fusion and Protein Sorting (HOPS) complex, a tether necessary for endosome-lysosome traffic. These syndromes offer insight into fundamental endosome traffic processes unique to metazoans. However, the molecular and cellular mechanisms underlying these mutant phenotypes remain poorly understood. Here we investigate interactions of wild-type and disease-causing mutations in VIPAS39/SPE-39 and Vps33b by yeast two hybrid, immunoprecipitation and quantitative fluorescent microscopy. We find that although few mutations prevent interaction between VIPAS39/SPE-39 and Vps33b, some mutants fragment VIPAS39/SPE-39-positive endosomes, but all mutants alter the subcellular localization of Vps33b to VIPAS39/SPE-39-positive endosomes. Our data suggest that the ARC syndrome may result through impaired VIPAS39/SPE-39 and Vps33b-dependent endosomal maturation or fusion.

  7. GOLGA2/GM130, cis-Golgi matrix protein, is a novel target of anticancer gene therapy.

    Science.gov (United States)

    Chang, Seung-Hee; Hong, Seong-Ho; Jiang, Hu-Lin; Minai-Tehrani, Arash; Yu, Kyeong-Nam; Lee, Jae-Ho; Kim, Ji-Eun; Shin, Ji-Young; Kang, Bitna; Park, Sungjin; Han, Kiwon; Chae, Chanhee; Cho, Myung-Haing

    2012-11-01

    Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an important role in glycosylation and transport of protein in the secretory pathway. In this study, the effects of short hairpin RNA (shRNA) constructs targeting GOLGA2/GM130 (shGOLGA2) on autophagy and lung cancer growth were evaluated in vitro and in vivo. Downregulation of GOLGA2/GM130 led to induction of autophagy and inhibition of glycosylation in A549 cells and in the lungs of K-ras(LA1) mice. Furthermore, downregulation of GOLGA2/GM130 decreased angiogenesis and cancer cell invasion in vitro and suppressed tumorigenesis in lung cancer mice model. The tumor specificity of sequence targeting GOLGA2/GM130 was also demonstrated. Taken together, these results suggest that induction of autophagy by shGOLGA2 may induce cell death rather than cell survival. Therefore, downregulation of GOLGA2/GM130 may be a potential therapeutic option for lung cancer.

  8. The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

    Science.gov (United States)

    van Tiel, Claudia M; Westerman, Jan; Paasman, Marten A; Hoebens, Martha M; Wirtz, Karel W A; Snoek, Gerry T

    2002-06-21

    Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

  9. A golgi study of the optic tectum of the tegu lizard, Tupinambis nigropunctatus.

    Science.gov (United States)

    Butler, A B; Ebbesson, O E

    1975-06-01

    The dendritic patterns of cells in the optic tectum of the tegu lizard, Tupinambis nigropunctatus, were analyzed with the Ramon-Moliner modification of the Golgi-Cox technique. Cell types were compared with those described by other authors in the tectum of other reptiles; particular comparisons of our results were made with the description of cell types in the chameleon (Ramń, 1896), as the latter is the most complete analysis in the literature. The periventricular gray layers 3 and 5 consist primarily of two cell types--piriform or pyramidal shaped cells and horizontal cells. Cells in the medial portion of the tectum, in an area coextensive with the bilateral spinal projection zone, possess dendrites that extend across the midline. The latter cells have either fusiform or pyramidal shaped somas. The central white zone, layer 6, contains fibers, large fusiform or pyramidal shaped cells, fusiform cells, and small horizontal cells. The central gray zone, layer 7, is composed predominately of fusiform cells which have dendrites extending to the superficial optic layers, large polygonal cells, and horizontal cells. The superficial gray and white layers, layers 8-13, contain polygonal, fusiform, stellate, and horizontal elements. Layer 14 is composed solely of afferent optic tract fibers. Several differences in the occurrence and distribution of cell types between the tegu and the other reptiles studied are noted. Additionally, the laminar distribution of retinal, tectotectal, telencephalic, and spinal projections in the tegutectum can be related to the distribution of cell types, and those cells which may be postsynaptic to specific inputs can be identified. The highly differentiated laminar structure of the reptilian optic tectum, both in regard to cell type and to afferent and efferent connections, may serve as a model for studying some functional properties of lamination common to cortical structures.

  10. Clusterin in human gut-associated lymphoid tissue, tonsils, and adenoids: localization to M cells and follicular dendritic cells.

    Science.gov (United States)

    Verbrugghe, Phebe; Kujala, Pekka; Waelput, Wim; Peters, Peter J; Cuvelier, Claude A

    2008-03-01

    The follicle-associated epithelium (FAE) overlying the follicles of mucosa-associated lymphoid tissue is a key player in the initiation of mucosal immune responses. We recently reported strong clusterin expression in the FAE of murine Peyer's patches. In this study, we examined the expression of clusterin in the human gut-associated lymphoid tissue (GALT) and Waldeyer's ring. Immunohistochemistry for clusterin in human Peyer's patches, appendix and colon lymphoid follicles revealed expression in M cells and in follicular dendritic cells (FDCs). Using cryo-immunogold electron microscopy in Peyer's patches, we observed cytosolic immunoreactivity in M cells and labeling in the ER/Golgi biosynthetic pathway in FDCs. In palatine tonsils and adenoids, we demonstrated clusterin expression in germinal centers and in the lymphoepithelium in the crypts where M cells are localized. In conclusion, clusterin is expressed in M cells and follicular dendritic cells at inductive sites of human mucosa-associated lymphoid tissue suggesting a role for this protein in innate immune responses. Moreover, the use of clusterin as a human M cell marker could prove to be a valuable tool in future M cell research.

  11. Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Maaike Kockx

    Full Text Available Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs or directly target the GTPase domain (Dyngo or Dynole series, dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

  12. Electron microscopy of CO/sub 2/-laser-induced effects in human fibrocartilage

    Energy Technology Data Exchange (ETDEWEB)

    Whipple, T.L.; Marotta, J.J.; May, T.C.; Caspari, R.B.; Meyers, J.F.

    1987-01-01

    Previous reports of effects of CO/sub 2/ laser energy on human fibrocartilage suggest thermal injury extends to a depth of approximately 70 microns from the target surface with power settings of 35 W and exposure times of 0.5 seconds. The present study was undertaken to look for more subtle evidence of thermal alteration of human fibrocartilage treated with CO/sub 2/ laser irradiation. Fifteen human menisci were irradiated at power settings of 10, 20, and 30 W with exposure times of 0.1 and 0.5 seconds. The specimens were immediately fixed and sectioned for electron microscopic examination. Loss of a normal cross banding, and marginal clarity of individual collagen fibers were observed in the extracellular matrix and were observed at distances up to 300 microns from the exposed tissue surface. In addition, cellular changes at similar tissue depth consisted of cell membrane invaginations, clumping of nuclear chromatin, breakdown of endoplasmic reticulum architecture, and loss of mitochondria and Golgi complexes from the cytoplasm were observed. This study demonstrates deeper penetration of a radiation that was previously appreciated by light microscopy in irradiated human fibrocartilage, although the implications with respect to contraside viability and healing potential of the tissue in vivo is not known.

  13. Profiling of Human Molecular Pathways Affected by Retrotransposons at the Level of Regulation by Transcription Factor Proteins

    Science.gov (United States)

    Nikitin, Daniil; Penzar, Dmitry; Garazha, Andrew; Sorokin, Maxim; Tkachev, Victor; Borisov, Nicolas; Poltorak, Alexander; Prassolov, Vladimir; Buzdin, Anton A.

    2018-01-01

    Endogenous retroviruses and retrotransposons also termed retroelements (REs) are mobile genetic elements that were active until recently in human genome evolution. REs regulate gene expression by actively reshaping chromatin structure or by directly providing transcription factor binding sites (TFBSs). We aimed to identify molecular processes most deeply impacted by the REs in human cells at the level of TFBS regulation. By using ENCODE data, we identified ~2 million TFBS overlapping with putatively regulation-competent human REs located in 5-kb gene promoter neighborhood (~17% of all TFBS in promoter neighborhoods; ~9% of all RE-linked TFBS). Most of REs hosting TFBS were highly diverged repeats, and for the evolutionary young (0–8% diverged) elements we identified only ~7% of all RE-linked TFBS. The gene-specific distributions of RE-linked TFBS generally correlated with the distributions for all TFBS. However, several groups of molecular processes were highly enriched in the RE-linked TFBS regulation. They were strongly connected with the immunity and response to pathogens, with the negative regulation of gene transcription, ubiquitination, and protein degradation, extracellular matrix organization, regulation of STAT signaling, fatty acids metabolism, regulation of GTPase activity, protein targeting to Golgi, regulation of cell division and differentiation, development and functioning of perception organs and reproductive system. By contrast, the processes most weakly affected by the REs were linked with the conservative aspects of embryo development. We also identified differences in the regulation features by the younger and older fractions of the REs. The regulation by the older fraction of the REs was linked mainly with the immunity, cell adhesion, cAMP, IGF1R, Notch, Wnt, and integrin signaling, neuronal development, chondroitin sulfate and heparin metabolism, and endocytosis. The younger REs regulate other aspects of immunity, cell cycle progression and

  14. Profiling of Human Molecular Pathways Affected by Retrotransposons at the Level of Regulation by Transcription Factor Proteins

    Directory of Open Access Journals (Sweden)

    Daniil Nikitin

    2018-01-01

    Full Text Available Endogenous retroviruses and retrotransposons also termed retroelements (REs are mobile genetic elements that were active until recently in human genome evolution. REs regulate gene expression by actively reshaping chromatin structure or by directly providing transcription factor binding sites (TFBSs. We aimed to identify molecular processes most deeply impacted by the REs in human cells at the level of TFBS regulation. By using ENCODE data, we identified ~2 million TFBS overlapping with putatively regulation-competent human REs located in 5-kb gene promoter neighborhood (~17% of all TFBS in promoter neighborhoods; ~9% of all RE-linked TFBS. Most of REs hosting TFBS were highly diverged repeats, and for the evolutionary young (0–8% diverged elements we identified only ~7% of all RE-linked TFBS. The gene-specific distributions of RE-linked TFBS generally correlated with the distributions for all TFBS. However, several groups of molecular processes were highly enriched in the RE-linked TFBS regulation. They were strongly connected with the immunity and response to pathogens, with the negative regulation of gene transcription, ubiquitination, and protein degradation, extracellular matrix organization, regulation of STAT signaling, fatty acids metabolism, regulation of GTPase activity, protein targeting to Golgi, regulation of cell division and differentiation, development and functioning of perception organs and reproductive system. By contrast, the processes most weakly affected by the REs were linked with the conservative aspects of embryo development. We also identified differences in the regulation features by the younger and older fractions of the REs. The regulation by the older fraction of the REs was linked mainly with the immunity, cell adhesion, cAMP, IGF1R, Notch, Wnt, and integrin signaling, neuronal development, chondroitin sulfate and heparin metabolism, and endocytosis. The younger REs regulate other aspects of immunity, cell cycle

  15. Serum Golgi protein 73 is a prognostic rather than diagnostic marker in hepatocellular carcinoma.

    Science.gov (United States)

    Dong, Min; Chen, Zhan-Hong; Li, Xing; Li, Xiao-Yun; Wen, Jing-Yun; Lin, Qu; Ma, Xiao-Kun; Wei, Li; Chen, Jie; Ruan, Dan-Yun; Lin, Ze-Xiao; Wang, Tian-Tian; Wu, Dong-Hao; Wu, Xiang-Yuan

    2017-11-01

    Serum Golgi protein 73 (sGP73) is a candidate diagnostic biomarker for hepatocellular carcinoma (HCC). However, current evidence of its diagnostic value is conflicting, primarily due to the small sample sizes of previous studies, and its prognostic role in HCC also remains unclear. In the present study, sGP73 levels in 462 patients with HCC, 186 patients with liver cirrhosis, and 83 healthy controls were evaluated using ELISA, and it was identified that the median sGP73 levels were significantly higher in the HCC (18.7 ng/ml) and liver cirrhosis (18.5 ng/ml) patients than in the healthy controls (0 ng/ml; both P<0.001); however, the levels did not significantly differ between the HCC and liver cirrhosis groups (P=0.632). sGP73 had an inferior sensitivity and specificity for HCC diagnosis (27.79 and 77.96%, respectively) compared with α-fetoprotein (57.36 and 90.96%, respectively; P<0.001). In the HCC group, a high level of sGP73 was associated with aggressive clinicopathological features and independently predicted poor overall survival (OS) time (P<0.001). Additionally, in patients with resectable HCC, a high level of sGP73 was associated with significantly decreased disease-free survival (P<0.001) and OS (P=0.039) times compared with a low level of sGP73. This study demonstrated that sGP73 is unsuitable as a diagnostic marker for the early detection of HCC; however, it is an independent negative prognostic marker, providing a novel risk stratification factor and a potential therapeutic molecular target for HCC.

  16. Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain.

    Science.gov (United States)

    Pichery, Mélanie; Mirey, Emilie; Mercier, Pascale; Lefrancais, Emma; Dujardin, Arnaud; Ortega, Nathalie; Girard, Jean-Philippe

    2012-04-01

    IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33-LacZ gene trap reporter strain (Il-33(Gt/Gt)) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti-IL-33 Abs, using Il-33(Gt/Gt) (Il-33-deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33-LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues.

  17. Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking.

    Science.gov (United States)

    Budayeva, Hanna G; Cristea, Ileana M

    2016-10-01

    Human sirtuin 2 (SIRT2) is an NAD + -dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a

  18. Interleukin-33 (IL-33): A nuclear cytokine from the IL-1 family.

    Science.gov (United States)

    Cayrol, Corinne; Girard, Jean-Philippe

    2018-01-01

    Interleukin-33 (IL-33) is a tissue-derived nuclear cytokine from the IL-1 family abundantly expressed in endothelial cells, epithelial cells and fibroblast-like cells, both during homeostasis and inflammation. It functions as an alarm signal (alarmin) released upon cell injury or tissue damage to alert immune cells expressing the ST2 receptor (IL-1RL1). The major targets of IL-33 in vivo are tissue-resident immune cells such as mast cells, group 2 innate lymphoid cells (ILC2s) and regulatory T cells (Tregs). Other cellular targets include T helper 2 (Th2) cells, eosinophils, basophils, dendritic cells, Th1 cells, CD8 + T cells, NK cells, iNKT cells, B cells, neutrophils and macrophages. IL-33 is thus emerging as a crucial immune modulator with pleiotropic activities in type-2, type-1 and regulatory immune responses, and important roles in allergic, fibrotic, infectious, and chronic inflammatory diseases. The critical function of IL-33/ST2 signaling in allergic inflammation is illustrated by the fact that IL33 and IL1RL1 are among the most highly replicated susceptibility loci for asthma. In this review, we highlight 15 years of discoveries on IL-33 protein, including its molecular characteristics, nuclear localization, bioactive forms, cellular sources, mechanisms of release and regulation by proteases. Importantly, we emphasize data that have been validated using IL-33-deficient cells. © 2017 The Authors. Immunological Reviews Published by John Wiley & Sons Ltd.

  19. Syntenic homology of human unique DNA sequences within chromossome regions 5q31, 10q22, 13q32-33 and 19q13.1 in the great apes

    Directory of Open Access Journals (Sweden)

    Rhea U. Vallente-Samonte

    2000-09-01

    Full Text Available Homologies between chromosome banding patterns and DNA sequences in the great apes and humans suggest an apparent common origin for these two lineages. The availability of DNA probes for specific regions of human chromosomes (5q31, 10q22, 13q32-33 and 19q13.1 led us to cross-hybridize these to chimpanzee (Pan troglodytes, PTR, gorilla (Gorilla gorilla, GGO and orangutan (Pongo pygmaeus, PPY chromosomes in a search for equivalent regions in the great apes. Positive hybridization signals to the chromosome 5q31-specific DNA probe were observed at HSA 5q31, PTR 4q31, GGO 4q31 and PPY 4q31, while fluorescent signals using the chromosome 10q22-specific DNA probe were noted at HSA 10q22, PTR 8q22, GGO 8q22 and PPY 7q22. The chromosome arms showing hybridization signals to the Quint-EssentialTM 13-specific DNA probe were identified as HSA 13q32-33, PTR 14q32-33, GGO 14q32-33 and PPY 14q32-33, while those presenting hybridization signals to the chromosome 19q13.1-specific DNA probe were identified as HSA 19q13.1, PTR 20q13, GGO 20q13 and PPY 20q13. All four probes presumably hybridized to homologous chromosomal locations in the apes, which suggests a homology of certain unique DNA sequences among hominoid species.Homologias entre os padrões de bandamento de cromossomos e seqüências de DNA em grandes macacos e humanos sugerem uma aparente origem comum para estas duas linhagens. A disponibilidade de sondas de DNA para regiões específicas de cromossomos humanos (5q31, 10q22, 13q32-33 e 19q13.1 nos levou a realizar hibridação cruzada com cromossomos de chimpanzé (Pan troglodytes, PTR, gorila (Gorilla gorilla, GGO e orangotango (Pongo pygmaeus, PPY em um pesquisa de regiões equivalentes em grandes macacos. Sinais positivos de hibridação para a sonda de DNA específica para o cromossomo 5q31 foram observados em HSA 5q31, PTR 4q31, GGO 4q31 e PPY 4q31, enquanto que sinais fluorescentes usando a sonda de DNA específica para o cromossomo 10q22 foram

  20. 3,3′,4,4′,5-Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujise

    2004-01-01

    Full Text Available The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126, the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs, were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine (KB3-Phe61. In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In KB3-Phe61, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 μM PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in KB3-Phe61. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

  1. IL33 Is a Stomach Alarmin That Initiates a Skewed Th2 Response to Injury and InfectionSummary

    Directory of Open Access Journals (Sweden)

    Jon N. Buzzelli

    2015-03-01

    Full Text Available Background & Aims: Interleukin (IL33 is a recently described alarmin that is highly expressed in the gastric mucosa and potently activates Th2 immunity. It may play a pivotal role during Helicobacter pylori infection. Here, we delineate the role of IL33 in the normal gastric mucosa and in response to gastropathy. Methods: IL33 expression was evaluated in mice and human biopsy specimens infected with H pylori and in mice after dosing with aspirin. IL33 expression was localized in the gastric mucosa using immunofluorescence. Mice were given 1 or 7 daily doses of recombinant IL33 (1 μg/dose, and the stomach and the spleen responses were quantified morphologically, by flow cytometry and using quantitative reverse-transcription polymerase chain reaction and immunoblotting. Results: In mice, the IL33 protein was localized to the nucleus of a subpopulation of surface mucus cells, and co-localized with the surface mucus cell markers Ulex Europaeus 1 (UEA1, and Mucin 5AC (Muc5AC. A small proportion of IL33-positive epithelial cells also were Ki-67 positive. IL33 and its receptor Interleukin 1 receptor-like 1 (ST2 were increased 4-fold after acute (1-day H pylori infection, however, this increase was not apparent after 7 days and IL33 expression was reduced 2-fold after 2 months. Similarly, human biopsy specimens positive for H pylori had a reduced IL33 expression. Chronic IL33 treatment in mice caused systemic activation of innate lymphoid cell 2 and polarization of macrophages to the M2 phenotype. In the stomach, IL33-treated mice developed transmural inflammation and mucous metaplasia that was mediated by Th2/signal transducer and activator of transcription 3 signaling. Rag-1-/- mice, lacking mature lymphocytes, were protected from IL33-induced gastric pathology. Conclusions: IL33 is highly expressed in the gastric mucosa and promotes the activation of T helper 2–cytokine–expressing cells. The loss of IL33 expression after prolonged H

  2. 45 CFR 33.11 - Salary offset when the Department is the creditor agency but not the paying agency.

    Science.gov (United States)

    2010-10-01

    ... agency but not the paying agency. 33.11 Section 33.11 Public Welfare DEPARTMENT OF HEALTH AND HUMAN..., the date the Government's right to collect the debt first accrued, and that the Departmental... Retirement and Disability Fund, or other similar payments that may be due the employee from other Federal...

  3. Algorithm of Golgi protein 73 and liver stiffness accurately diagnoses significant fibrosis in chronic HBV infection.

    Science.gov (United States)

    Cao, Zhujun; Li, Ziqiang; Wang, Hui; Liu, Yuhan; Xu, Yumin; Mo, Ruidong; Ren, Peipei; Chen, Lichang; Lu, Jie; Li, Hong; Zhuang, Yan; Liu, Yunye; Wang, Xiaolin; Zhao, Gangde; Tang, Weiliang; Xiang, Xiaogang; Cai, Wei; Liu, Longgen; Bao, Shisan; Xie, Qing

    2017-11-01

    Serum Golgi protein 73 (GP73) is a potential biomarker for fibrosis assessment. We aimed to develop an algorithm based on GP73 and liver stiffness (LS) for further improvement of accuracy for significant fibrosis in patients with antiviral-naïve chronic hepatitis B virus (HBV) infection. Diagnostic accuracy evaluation of GP73 and development of GP73-LS algorithm was performed in training cohort (n = 267) with an independent cohort (n = 133) for validation. A stepwise increasing pattern of serum GP73 was observed across fibrosis stages in patients with antiviral-naïve chronic HBV infection. Serum GP73 significantly correlated (rho = 0.48, P 73, accuracy: 63.6%). Using GP73-LS algorithm, GP73 < 63 in agreement with LS < 8.5 provided accuracy of 81.7% to excluded significant fibrosis. GP73 ≥ 63 in agreement with LS ≥ 8.5 provided accuracy of 93.3% to confirm significant fibrosis. Almost 64% or 68% of patients in the training or validation cohort could be accurately classified. Serum GP73 is a robust biomarker for significant fibrosis diagnosis. GP73-LS algorithm provided better diagnostic accuracy than currently available approaches. More than 60% antiviral naïve CHB patients could use this algorithm without resorting to liver biopsy. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells

    Directory of Open Access Journals (Sweden)

    Andres Marilou A

    2007-09-01

    Full Text Available Abstract Background The cyclic nucleotide-gated ion channels (CNGCs maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots. Results AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50–150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents. Conclusion AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported

  5. Ultrastructure of Cajal-like interstitial cells in the human detrusor

    DEFF Research Database (Denmark)

    Rasmussen, Helle; Rumessen, Jüri J; Hansen, Alastair

    2009-01-01

    reticulum and Golgi apparatus were observed in the cell somata and cytoplasmic processes. Intermediate filaments were abundant but no thick filaments were found. ICC-L were interconnected by close appositions, gap junctions and peg-and-socket junctions (PSJ) but no specialised contacts to smooth muscle...

  6. Accumulation of histone variant H3.3 with age is associated with profound changes in the histone methylation landscape

    DEFF Research Database (Denmark)

    Tvardovskiy, Andrey; Schwämmle, Veit; Kempf, Stefan J

    2017-01-01

    a causal relationship between H3 variant replacement and age-dependent changes in H3 methylation. Furthermore, the H3.3 level is drastically reduced in human hepatocarcinoma cells as compared to nontumoral hepatocytes, suggesting the potential utility of the H3.3 relative abundance as a biomarker...

  7. Targeting a Novel Androgen Receptor-Repressed Pathway in Prostate Cancer Therapy

    Science.gov (United States)

    2017-09-01

    receptor; TNF, tumor necrosis factor; TGN, trans-Golgi network; EMT, epithelial-to-mesenchymal transition; MMP, matrix metalloprotease; HDAC, histone...such as bioactive peptides [31], lipids [32], growth factors [33], tumor necrosis factor (TNF) [34], chemo- kines, and many of which act through...of prostate cancer [91]. It has also been shown that vascular endothelial growth factors (VEGFs) promote cell proliferation and migration via PKD

  8. Interleukin-33 is expressed in the lesional epidermis in herpes virus infection but not in verruca vulgaris.

    Science.gov (United States)

    Jin, Meijuan; Komine, Mayumi; Tsuda, Hidetoshi; Oshio, Tomoyuki; Ohtsuki, Mamitaro

    2018-04-25

    Interleukin (IL)-33 is released on cell injury and activates the immune reaction. IL-33 is involved in antiviral reaction in herpes virus infection, but the source that secretes IL-33 has not been identified. We speculate that keratinocytes injured in herpes virus infection secrete IL-33. In order to detect IL-33 in the lesional epidermis of patients with herpes virus infection, we immunostained several cutaneous herpes virus infection samples with an anti-IL-33 antibody, and compared them with cutaneous human papilloma virus (HPV) infection samples. We observed strong nuclear and mild cytoplasmic staining in epidermal keratinocytes of the lesional skin samples with herpes simplex virus and varicella zoster virus infections. However, staining was not observed in the epidermis of verruca vulgaris (VV) samples. We assumed that the strong immune reaction to herpes virus infection may depend on strong IL-33 expression in the epidermis, while very weak immune reaction in samples from patients with VV may be due to low or no expression of IL-33 in the lesional epidermis. © 2018 Japanese Dermatological Association.

  9. Golga5 is dispensable for mouse embryonic development and postnatal survival.

    Science.gov (United States)

    McGee, Lynessa J; Jiang, Alex L; Lan, Yu

    2017-07-01

    Golgins are a family of coiled-coil proteins located at the cytoplasmic surface of the Golgi apparatus and have been implicated in maintaining Golgi structural integrity through acting as tethering factors for retrograde vesicle transport. Whereas knockdown of several individual golgins in cultured cells caused Golgi fragmentation and disruption of vesicle trafficking, analysis of mutant mouse models lacking individual golgins have discovered tissue-specific developmental functions. Recently, homozygous loss of function of GOLGA2, of which previous in vitro studies suggested an essential role in maintenance of Golgi structure and in mitosis, has been associated with a neuromuscular disorder in human patients, which highlights the need for understanding the developmental roles of the golgins in vivo. We report here generation of Golga5-deficient mice using CRISPR/Cas9-mediated genome editing. Although knockdown studies in cultured cells have implicated Golga5 in maintenance of Golgi organization, we show that Golga5 is not required for mouse embryonic development, postnatal survival, or fertility. Moreover, whereas Golga5 is structurally closely related to Golgb1, we show that inactivation of Golga5 does not enhance the severity of developmental defects in Golgb1-deficient mice. The Golga5-deficient mice enable further investigation of the roles and functional specificity of golgins in development and diseases. © 2017 Wiley Periodicals, Inc.

  10. Topographical distribution and morphology of NADPH-diaphorase-stained neurons in the human claustrum

    Science.gov (United States)

    Hinova-Palova, Dimka V.; Edelstein, Lawrence; Landzhov, Boycho; Minkov, Minko; Malinova, Lina; Hristov, Stanislav; Denaro, Frank J.; Alexandrov, Alexandar; Kiriakova, Teodora; Brainova, Ilina; Paloff, Adrian; Ovtscharoff, Wladimir

    2014-01-01

    We studied the topographical distribution and morphological characteristics of NADPH-diaphorase-positive neurons and fibers in the human claustrum. These neurons were seen to be heterogeneously distributed throughout the claustrum. Taking into account the size and shape of stained perikarya as well as dendritic and axonal characteristics, Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd)-positive neurons were categorized by diameter into three types: large, medium and small. Large neurons ranged from 25 to 35 μm in diameter and typically displayed elliptical or multipolar cell bodies. Medium neurons ranged from 20 to 25 μm in diameter and displayed multipolar, bipolar and irregular cell bodies. Small neurons ranged from 14 to 20 μm in diameter and most often displayed oval or elliptical cell bodies. Based on dendritic characteristics, these neurons were divided into spiny and aspiny subtypes. Our findings reveal two populations of NADPHd-positive neurons in the human claustrum—one comprised of large and medium cells consistent with a projection neuron phenotype, the other represented by small cells resembling the interneuron phenotype as defined by previous Golgi impregnation studies. PMID:24904317

  11. CERN: SC-33

    Energy Technology Data Exchange (ETDEWEB)

    Anon.

    1991-06-15

    On 22 April a forward-looking CERN also looked back for a day, when the 'SC-33' event reviewed the achievements of CERN's first machine, the 600 MeV SynchroCyclotron (SC), which closed down on 17 December after 33 years of valiant service.

  12. Development of an alpha-fetoprotein and Golgi protein 73 multiplex detection assay using xMAP technology.

    Science.gov (United States)

    Wu, Yun; Ma, Jie; Wang, Yipeng; Zhang, Yonghong; Hou, Yongjin; Zhang, Chunming; Sun, Huanqin; Sun, Jianping; Wang, Zikang; Li, Ning

    2018-04-06

    Development of a new method to simultaneously detect Alpha-fetoprotein (AFP) and Golgi protein 73 (GP73) from peripheral blood. Anti human AFP and GP73 monoclonal antibodies was used to develop a sandwich immunity reaction using xMAP technology for the simultaneous detection of plasma AFP and GP73. The assay evaluated the sensitivity, cross reactivity, range of detection, precision, recovery and linearity dilution effect. The assay utilized plasma samples and compared its performance with commercially available Enzyme Linked Immunosorbent Assay (ELISA) kits. The assay was successful in detecting AFP and GP73 simultaneously. Validation experiments demonstrated the limit of detection was AFP 0.006 μg/l and GP73 0.482 μg/l. The functional sensitivity was AFP 0.010 μg/l and GP73 0.640 μg/l. The range of detection was AFP 0.01-50 μg/l and GP73 0.64-100 μg/l. No cross reactivity was observed. The intra- and inter-assay variation for AFP was 0.19-3.46% and 3.1-5.8% and for GP73 was 1.5-3.2% and 1.1-7.6% respectively. The recovery for AFP was 96-106% and GP73 was 89-110%. 80 clinical plasma samples from healthy controls, and patients with liver cirrhosis and Hepatocellular Carcinoma (HCC) were evaluated. For healthy controls (n = 25), the AFP was 2.40 (1.55, 3.30) μg/l and GP73 was 42.60 (39.10, 57.40) μg/l. For patients with liver cirrhosis (n = 19), the AFP was 2.60 (1.70, 4.20) μg/l and GP73 was 136.10 (92.10, 261.70) μg/l, and for HCC patients (n = 36), the AFP was 13.65 (3.35, 158.88) μg/l and GP73 was 186.25 (96.73, 262.03) μg/l. The new assay demonstrated a good correlation with commercially available ELISA kits (correlation coefficients (r) were 0.997 (AFP, p < 0.001) and 0.959 (GP73, p < 0.001). The method demonstrated a sensitive, effective and accurate method for the simultaneous detection of AFP and GP73, and could be used clinically for routine detection and monitoring of patients with chronic hepatitis B

  13. Dendritic Kv3.3 potassium channels in cerebellar purkinje cells regulate generation and spatial dynamics of dendritic Ca2+ spikes.

    Science.gov (United States)

    Zagha, Edward; Manita, Satoshi; Ross, William N; Rudy, Bernardo

    2010-06-01

    Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

  14. Unusual armadillo fold in the human general vesicular transport factor p115.

    Directory of Open Access Journals (Sweden)

    Harald Striegl

    Full Text Available The golgin family gives identity and structure to the Golgi apparatus and is part of a complex protein network at the Golgi membrane. The golgin p115 is targeted by the GTPase Rab1a, contains a large globular head region and a long region of coiled-coil which forms an extended rod-like structure. p115 serves as vesicle tethering factor and plays an important role at different steps of vesicular transport. Here we present the 2.2 A-resolution X-ray structure of the globular head region of p115. The structure exhibits an armadillo fold that is decorated by elongated loops and carries a C-terminal non-canonical repeat. This terminal repeat folds into the armadillo superhelical groove and allows homodimeric association with important implications for p115 mediated multiple protein interactions and tethering.

  15. Partial functional complementation between human and mouse cytomegalovirus chemokine receptor homologues

    DEFF Research Database (Denmark)

    Farrell, Helen E; Abraham, Alexander M; Cardin, Rhonda D

    2011-01-01

    The human cytomegalovirus (CMV) proteins US28 and UL33 are homologous to chemokine receptors (CKRs). Knockout of the mouse CMV M33 protein (UL33 homologue) results in substantial attenuation of salivary gland infection/replication and reduced efficiency of reactivation from tissue explants. M33-m...

  16. Identification and characterization of an inner ear-expressed human melanoma inhibitory activity (MIA)-like gene (MIAL) with a frequent polymorphism that abolishes translation

    DEFF Research Database (Denmark)

    Rendtorff, Nanna Dahl; Frödin, M; Attié-Bitach, T

    2001-01-01

    hybridization or reverse transcription-polymerase chain reaction. The cDNA of the mouse homologue was also cloned and mapped about 80 cM from the top of mouse chromosome 2. In mouse, Mial was also expressed in the cochlea and the vestibule of the inner ear, as well as in brain, eye, limb, and ovary. Expression...... in mammalian cell cultures showed that MIAL is translated as an approximately 15-kDa polypeptide that is assembled into a covalently linked homodimer, modified by sulfation, and secreted from the cells via the Golgi apparatus. In the human MIAL gene, a frequent polymorphism was discovered in the translation...

  17. Predictive value of serum Golgi protein 73 for prominent hepatic necroinflammation in chronic HBV infection.

    Science.gov (United States)

    Xu, Zhengju; Shen, Jiankun; Pan, Xingnan; Wei, Meijuan; Liu, Liguan; Wei, Kaipeng; Liu, Lifei; Yang, Huanwen; Huang, Jinfa

    2018-06-01

    As a noninvasive marker, serum alanine aminotransferase (ALT) has limitations, because a large proportion of patients chronically infected with hepatitis B virus (HBV) suffer from severe hepatic necroinflammation, but have normal or mildly elevated ALT. In the present study, we aimed to investigate the potential value of serum Golgi protein 73 (GP73) in predicting significant hepatic necroinflamation among chronic HBV infected patients. A cohort of 497 chronic HBV infected patients was retrospectively recruited. Liver biopsy was performed in all patients and serum GP73 levels were measured by enzyme-linked immunosorbent assay. Serum GP73 increased in parallel with the increase in hepatic necroinflammatory activity grade (r = 0.682) and the stage of liver fibrosis (r = 0.539). The positive correlation of serum GP73 with the degree of hepatic necroinflammatory activity was statistically significant, while serum GP73 with the stage of liver fibrosis was weaker than that with hepatic necroinflammation. Furthermore, serum GP73 levels were significantly greater in patients with normal or mildly elevated ALT and significant hepatic necroinflammation (≥G2) than in patients with minimal to mild hepatic necroinflammation. The sensitivity and specificity of GP73 for the diagnosis of G2 hepatic necroinflammation was 42.35% and 95.0%, respectively, at a cut-off value of 88.38 ng/mL. When the cut-off value was set at 124.76 ng/mL, the sensitivity and specificity of GP73 for the diagnosis of G3 hepatic necroinflammation was 55.56% and 97.29%, respectively. These findings indicate that GP73 holds promise as an important candidate for diagnosing significant hepatic necroinflammation. © 2018 Wiley Periodicals, Inc.

  18. CERN: SC-33

    International Nuclear Information System (INIS)

    Anon.

    1991-01-01

    On 22 April a forward-looking CERN also looked back for a day, when the 'SC-33' event reviewed the achievements of CERN's first machine, the 600 MeV SynchroCyclotron (SC), which closed down on 17 December after 33 years of valiant service

  19. Nucleobindin co-localizes and associates with cyclooxygenase (COX-2 in human neutrophils.

    Directory of Open Access Journals (Sweden)

    Patrick Leclerc

    2008-05-01

    Full Text Available The inducible cyclooxygenase isoform (COX-2 is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc, a ubiquitous Ca(2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE(2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE(2 biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.

  20. Nuclear Hyperfine Structure in the Donor – Acceptor Complexes (CH3)3N-BF3 and (CH)33N-B(CH3)3

    Science.gov (United States)

    The donor-acceptor complexes (CH3)3N-BF3 and (CH3)3N-B(CH3)3 have been reinvestigated at high resolution by rotational spectroscopy in a supersonic jet. Nuclear hyperfine structure resulting from both nitrogen and boron has been resolved and quadrupole coupling constants have bee...

  1. Geoditin A Induces Oxidative Stress and Apoptosis on Human Colon HT29 Cells

    Directory of Open Access Journals (Sweden)

    Wing-Keung Liu

    2010-01-01

    Full Text Available Geoditin A, an isomalabaricane triterpene isolated from the marine sponge Geodia japonica, has been demonstrated to dissipate mitochondrial membrane potential, activate caspase 3, decrease cytoplasmic proliferating cell nuclear antigen (PCNA, and induce apoptosis of leukemia cells, but the underlying mechanism remains unclear [1]. In this study, we found fragmentation of Golgi structure, suppression of transferrin receptor expression, production of oxidants, and DNA fragmentation in human colon cancer HT29 cells after treatment with geoditin A for 24 h. This apoptosis was not abrogated by chelation of intracellular iron with salicylaldehyde isonicotinoyl hydrazone (SIH, but suppressed by N-acetylcysteine (NAC, a thiol antioxidant and GSH precursor, indicating that the cytotoxic effect of geoditin A is likely mediated by a NAC-inhibitable oxidative stress. Our results provide a better understanding of the apoptotic properties and chemotherapeutical potential of this marine triterpene.

  2. Downstream targets of WRKY33

    DEFF Research Database (Denmark)

    Petersen, Klaus; Fiil, Berthe Katrine; Mundy, John

    2008-01-01

    Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. In a recent publication we show that MPK4 and its substrate MKS1 interact with WRKY33 in vivo, and that WRKY33 is released from complexes with MPK4 upon infection....... Transcriptome analysis of a wrky33 loss-of-function mutant identified a subset of defense-related genes as putative targets of WRKY33. These genes include PAD3 and CYP71A13, which encode cytochrome P450 monoxygenases required for synthesis of the antimicrobial phytoalexin camalexin. Chromatin...... immunoprecipitation confirmed that WRKY33 bound the promoter of PAD3 when plants were inoculated with pathogens. Here we further discuss the involvement of two other targets of WRKY33, NUDT6 and ROF2 in defense responses against invading pathogens....

  3. Characteristics of a broad lytic spectrum endolysin from phage BtCS33 of Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Yuan Yihui

    2012-12-01

    Full Text Available Abstract Background Endolysins produced by bacteriophages lyse bacteria, and are thus considered a novel type of antimicrobial agent. Several endolysins from Bacillus phages or prophages have previously been characterized and used to target Bacillus strains that cause disease in animals and humans. B. thuringiensis phage BtCS33 is a Siphoviridae family phage and its genome has been sequenced and analyzed. In the BtCS33 genome, orf18 was found to encode an endolysin protein (PlyBt33. Results Bioinformatic analyses showed that endolysin PlyBt33 was composed of two functional domains, the N-terminal catalytic domain and the C-terminal cell wall binding domain. In this study, the entire endolysin PlyBt33, and both the N- and C-termini,were expressed in Escherichia coli and then purified. The lytic activities of PlyBt33 and its N-terminus were tested on bacteria. Both regions exhibited lytic activity, although PlyBt33 showed a higher lytic activity than the N-terminus. PlyBt33 exhibited activity against all Bacillus strains tested from five different species, but was not active against Gram-negative bacteria. Optimal conditions for PlyBt33 reactivity were pH 9.0 and 50°C. PlyBt33 showed high thermostability, with 40% of initial activity remaining following 1 h of treatment at 60°C. The C-terminus of PlyBt33 bound to B. thuringiensis strain HD-73 and Bacillus subtilis strain 168. This cell wall binding domain might be novel, as its amino acid sequence showed little similarity to previously reported endolysins. Conclusions PlyBt33 showed potential as a novel antimicrobial agent at a relatively high temperature and had a broad lytic spectrum within the Bacillus genus. The C-terminus of PlyBt33 might be a novel kind of cell wall binding domain.

  4. DAXX envelops a histone H3.3-H4 dimer for H3.3-specific recognition

    Energy Technology Data Exchange (ETDEWEB)

    Elsässer, Simon J; Huang, Hongda; Lewis, Peter W; Chin, Jason W; Allis, C David; Patel, Dinshaw J [MSKCC; (Rockefeller); (MRC)

    2013-01-24

    Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3–H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3–H4 dimer, with complex formation accompanied by structural transitions in the H3.3–H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly90 in H3.3 and Glu225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.

  5. STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability.

    Science.gov (United States)

    Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D; Silkworth, Whitney; Ross, Nathan T; Scherer, Christina A; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A; Stern, Andrew M; Schreiber, Stuart L; Golub, Todd R

    2012-02-21

    Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.

  6. Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

    International Nuclear Information System (INIS)

    Germann, U.A.; Willingham, M.C.; Pastan, I.; Gottesman, M.M.

    1990-01-01

    The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. The authors expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by [ 3 H]azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents compete with the [ 3 H]azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart

  7. 7 CFR 33.3 - Secretary.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Secretary. 33.3 Section 33.3 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.3 Secretary. Secretary means the...

  8. 7 CFR 2201.33 - Defaults.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Defaults. 2201.33 Section 2201.33 Agriculture Regulations of the Department of Agriculture (Continued) LOCAL TELEVISION LOAN GUARANTEE BOARD LOCAL TELEVISION LOAN GUARANTEE PROGRAM-PROGRAM REGULATIONS Loan Guarantees § 2201.33 Defaults. (a) In determining...

  9. 7 CFR 33.4 - Carrier.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Carrier. 33.4 Section 33.4 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.4 Carrier. Carrier means any common or...

  10. 7 CFR 33.5 - Apples.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Apples. 33.5 Section 33.5 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.5 Apples. Apples mean fresh whole apples...

  11. 7 CFR 33.2 - Person.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Person. 33.2 Section 33.2 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.2 Person. Person means an individual...

  12. Interferon γ-Induced Nuclear Interleukin-33 Potentiates the Release of Esophageal Epithelial Derived Cytokines.

    Directory of Open Access Journals (Sweden)

    Jing Shan

    Full Text Available Esophageal epithelial cells are an initiating cell type in esophageal inflammation, playing an essential role in the pathogenesis of gastroesophageal reflux disease (GERD. A new tissue-derived cytokine, interleukin-33 (IL-33, has been shown to be upregulated in esophageal epithelial cell nuclei in GERD, taking part in mucosal inflammation. Here, inflammatory cytokines secreted by esophageal epithelial cells, and their regulation by IL-33, were investigated.In an in vitro stratified squamous epithelial model, IL-33 expression was examined using quantitative RT-PCR, western blot, ELISA, and immunofluorescence. Epithelial cell secreted inflammatory cytokines were examined using multiplex flow immunoassay. IL-33 was knocked down with small interfering RNA (siRNA in normal human esophageal epithelial cells (HEECs. Pharmacological inhibitors and signal transducers and activators of transcription 1 (STAT1 siRNA were used to explore the signaling pathways.Interferon (IFNγ treatment upregulated nuclear IL-33 in HEECs. Furthermore, HEECs can produce various inflammatory cytokines, such as IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1, regulated on activation normal T-cell expressed and presumably secreted (RANTES, and granulocyte-macrophage colony-stimulating factor (GM-CSF in response to IFNγ. Nuclear, but not exogenous IL-33, amplified IFN induction of these cytokines. P38 mitogen-activated protein kinase (MAPK and janus protein tyrosine kinases (JAK/STAT1 were the common signaling pathways of IFNγ-mediated induction of IL-33 and other cytokines.Esophageal epithelial cells can actively participate in GERD pathogenesis through the production of various cytokines, and epithelial-derived IL-33 might play a central role in the production of these cytokines.

  13. 28 CFR 33.60 - General.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false General. 33.60 Section 33.60 Judicial Administration DEPARTMENT OF JUSTICE BUREAU OF JUSTICE ASSISTANCE GRANT PROGRAMS Criminal Justice Block Grants Submission and Review of Applications § 33.60 General. This subpart describes the process and criteria for...

  14. 28 CFR 552.33 - Media.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Media. 552.33 Section 552.33 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT CUSTODY Hostage Situation Management § 552.33 Media. The Warden shall assign staff to handle all news releases and news media inquiries...

  15. 22 CFR 218.33 - Mediation.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Mediation. 218.33 Section 218.33 Foreign... § 218.33 Mediation. (a) Referral of complaints for mediation. The agency will refer to the Federal Mediation and Conciliation Service all complaints that: (1) fall within the jurisdiction of these...

  16. 7 CFR 906.33 - Budget.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Budget. 906.33 Section 906.33 Agriculture Regulations... GRANDE VALLEY IN TEXAS Order Regulating Handling Expenses and Assessments § 906.33 Budget. At the... budget of income and expenditures necessary for the administration of this part. The committee shall...

  17. The truncated metabolite GLP-2 (3-33) interacts with the GLP-2 receptor as a partial agonist.

    Science.gov (United States)

    Thulesen, Jesper; Knudsen, Lotte Bjerre; Hartmann, Bolette; Hastrup, Sven; Kissow, Hannelouise; Jeppesen, Palle Bekker; Ørskov, Cathrine; Holst, Jens Juul; Poulsen, Steen Seier

    2002-01-15

    The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.

  18. Disturbances of ligand potency and enhanced degradation of the human glycine receptor at affected positions G160 and T162 originally identified in patients suffering from hyperekplexia

    Directory of Open Access Journals (Sweden)

    Sinem eAtak

    2015-12-01

    Full Text Available Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GlyR undergoes direct interaction with the incoming ligand via cation-π interactions. Recently we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GlyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GlyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER towards ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is conducted to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, T162 assigns primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof.

  19. 14 CFR 171.33 - Reports.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Reports. 171.33 Section 171.33 Aeronautics... FACILITIES NON-FEDERAL NAVIGATION FACILITIES Nondirectional Radio Beacon Facilities § 171.33 Reports. The owner of each facility to which this subpart applies shall make the following reports, at the times...

  20. 34 CFR 33.21 - Discovery.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Discovery. 33.21 Section 33.21 Education Office of the Secretary, Department of Education PROGRAM FRAUD CIVIL REMEDIES ACT § 33.21 Discovery. (a) The following types of discovery are authorized: (1) Requests for production of documents for inspection and copying...

  1. 45 CFR 602.33 - Supplies.

    Science.gov (United States)

    2010-10-01

    ... unused supplies exceeding $5,000 in total aggregate fair market value upon termination or completion of... 45 Public Welfare 3 2010-10-01 2010-10-01 false Supplies. 602.33 Section 602.33 Public Welfare....33 Supplies. (a) Title. Title to supplies acquired under a grant or subgrant will vest, upon...

  2. 14 CFR 1273.33 - Supplies.

    Science.gov (United States)

    2010-01-01

    ... residual inventory of unused supplies exceeding $5,000 in total aggregate fair market value upon... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Supplies. 1273.33 Section 1273.33..., Property, and Subawards § 1273.33 Supplies. (a) Title. Title to supplies acquired under a grant or subgrant...

  3. 7 CFR 3016.33 - Supplies.

    Science.gov (United States)

    2010-01-01

    ... aggregate fair market value upon termination or completion of the award, and if the supplies are not needed... 7 Agriculture 15 2010-01-01 2010-01-01 false Supplies. 3016.33 Section 3016.33 Agriculture... GOVERNMENTS Post-Award Requirements Changes, Property, and Subawards § 3016.33 Supplies. (a) Title. Title to...

  4. 28 CFR 33.51 - Audit.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Audit. 33.51 Section 33.51 Judicial... Additional Requirements § 33.51 Audit. Pursuant to Office of Management and Budget Circular A-128 “Audits of State and Local Governments,” all grantees and subgrantees must provide for an independent audit of...

  5. 40 CFR 31.33 - Supplies.

    Science.gov (United States)

    2010-07-01

    ...) Disposition. If there is a residual inventory of unused supplies exceeding $5,000 in total aggregate fair... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Supplies. 31.33 Section 31.33... Requirements Changes, Property, and Subawards § 31.33 Supplies. (a) Title. Title to supplies acquired under a...

  6. 13 CFR 143.33 - Supplies.

    Science.gov (United States)

    2010-01-01

    ... residual inventory of unused supplies exceeding $5,000 in total aggregate fair market value upon... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Supplies. 143.33 Section 143.33..., and Subawards § 143.33 Supplies. (a) Title. Title to supplies acquired under a grant or subgrant will...

  7. 29 CFR 1470.33 - Supplies.

    Science.gov (United States)

    2010-07-01

    .... If there is a residual inventory of unused supplies exceeding $5,000 in total aggregate fair market... 29 Labor 4 2010-07-01 2010-07-01 false Supplies. 1470.33 Section 1470.33 Labor Regulations... Changes, Property, and Subawards § 1470.33 Supplies. (a) Title. Title to supplies acquired under a grant...

  8. 45 CFR 92.33 - Supplies.

    Science.gov (United States)

    2010-10-01

    ... residual inventory of unused supplies exceeding $5,000 in total aggregate fair market value upon... 45 Public Welfare 1 2010-10-01 2010-10-01 false Supplies. 92.33 Section 92.33 Public Welfare..., Property, and Subawards § 92.33 Supplies. (a) Title. Title to supplies acquired under a grant or subgrant...

  9. 15 CFR 24.33 - Supplies.

    Science.gov (United States)

    2010-01-01

    ... residual inventory of unused supplies exceeding $5,000 in total aggregate fair market value upon... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Supplies. 24.33 Section 24.33 Commerce..., and Subawards § 24.33 Supplies. (a) Title. Title to supplies acquired under a grant or subgrant will...

  10. 44 CFR 13.33 - Supplies.

    Science.gov (United States)

    2010-10-01

    ... aggregate fair market value upon termination or completion of the award, and if the supplies are not needed... 44 Emergency Management and Assistance 1 2010-10-01 2010-10-01 false Supplies. 13.33 Section 13.33... GOVERNMENTS Post-Award Requirements Changes, Property, and Subawards § 13.33 Supplies. (a) Title. Title to...

  11. 7 CFR 3.3 - Definitions.

    Science.gov (United States)

    2010-01-01

    ... (Federal agencies and private sector entities) into their respective databases for the purpose of... 7 Agriculture 1 2010-01-01 2010-01-01 false Definitions. 3.3 Section 3.3 Agriculture Office of the Secretary of Agriculture DEBT MANAGEMENT General § 3.3 Definitions. For the purpose of this part, except as...

  12. 14 CFR 33.69 - Ignitions system.

    Science.gov (United States)

    2010-01-01

    ... STANDARDS: AIRCRAFT ENGINES Design and Construction; Turbine Aircraft Engines § 33.69 Ignitions system. Each..., except that only one igniter is required for fuel burning augmentation systems. [Amdt. 33-6, 39 FR 35466... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Ignitions system. 33.69 Section 33.69...

  13. α-Defensin HD5 Inhibits Human Papillomavirus 16 Infection via Capsid Stabilization and Redirection to the Lysosome

    Directory of Open Access Journals (Sweden)

    Mayim E. Wiens

    2017-01-01

    Full Text Available α-Defensins are an important class of abundant innate immune effectors that are potently antiviral against a number of nonenveloped viral pathogens; however, a common mechanism to explain their ability to block infection by these unrelated viruses is lacking. We previously found that human defensin 5 (HD5 blocks a critical host-mediated proteolytic processing step required for human papillomavirus (HPV infection. Here, we show that bypassing the requirement for this cleavage failed to abrogate HD5 inhibition. Instead, HD5 altered HPV trafficking in the cell. In the presence of an inhibitory concentration of HD5, HPV was internalized and reached the early endosome. The internalized capsid became permeable to antibodies and proteases; however, HD5 prevented dissociation of the viral capsid from the genome, reduced viral trafficking to the trans-Golgi network, redirected the incoming viral particle to the lysosome, and accelerated the degradation of internalized capsid proteins. This mechanism is equivalent to the mechanism by which HD5 inhibits human adenovirus. Thus, our data support capsid stabilization and redirection to the lysosome during infection as a general antiviral mechanism of α-defensins against nonenveloped viruses.

  14. 6 CFR 13.33 - Witnesses.

    Science.gov (United States)

    2010-01-01

    ... 6 Domestic Security 1 2010-01-01 2010-01-01 false Witnesses. 13.33 Section 13.33 Domestic Security DEPARTMENT OF HOMELAND SECURITY, OFFICE OF THE SECRETARY PROGRAM FRAUD CIVIL REMEDIES § 13.33 Witnesses. (a) Except as provided in paragraph (b) of this section, testimony at the hearing will be given orally by...

  15. 29 CFR 97.33 - Supplies.

    Science.gov (United States)

    2010-07-01

    ... aggregate fair market value upon termination or completion of the award, and if the supplies are not needed... 29 Labor 1 2010-07-01 2010-07-01 true Supplies. 97.33 Section 97.33 Labor Office of the Secretary... GOVERNMENTS Post-Award Requirements Changes, Property, and Subawards § 97.33 Supplies. (a) Title. Title to...

  16. PACS and SPIRE photometer maps of M33 : First results of the HERschel M33 Extended Survey (HERM33ES)

    NARCIS (Netherlands)

    Kramer, C.; Buchbender, C.; Xilouris, E. M.; Boquien, M.; Braine, J.; Calzetti, D.; Lord, S.; Mookerjea, B.; Quintana-Lacaci, G.; Relano, M.; Stacey, G.; Tabatabaei, F. S.; Verley, S.; Aalto, S.; Akras, S.; Albrecht, M.; Anderl, S.; Beck, R.; Bertoldi, F.; Combes, F.; Dumke, M.; Garcia-Burillo, S.; Gonzalez, M.; Gratier, P.; Guesten, R.; Henkel, C.; Israel, F. P.; Koribalski, B.; Lundgren, A.; Martin-Pintado, J.; Roellig, M.; Rosolowsky, E.; Schuster, K. F.; Sheth, K.; Sievers, A.; Stutzki, J.; Tilanus, R.P.J.; van der Tak, F.; Wiedner, M. C.; van der Werf, Paul P.

    Context. Within the framework of the HERM33ES key program, we are studying the star forming interstellar medium in the nearby, metal-poor spiral galaxy M33, exploiting the high resolution and sensitivity of Herschel. Aims. We use PACS and SPIRE maps at 100, 160, 250, 350, and 500 mu m wavelength, to

  17. 34 CFR 33.2 - Definitions.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Definitions. 33.2 Section 33.2 Education Office of the Secretary, Department of Education PROGRAM FRAUD CIVIL REMEDIES ACT § 33.2 Definitions. As used in this part..., record, or accounting or bookkeeping entry made: (a) With respect to a claim or to obtain the approval or...

  18. Manganese modified CdTe/CdS quantum dots as an immunoassay biosensor for the detection of Golgi protein-73.

    Science.gov (United States)

    Liu, Wei; Zhang, Aixia; Xu, Guanhong; Wei, Fangdi; Yang, Jing; Hu, Qin

    2016-01-05

    In this paper, a new fluorescence bioassay for Golgi protein-73 (GP73), a promising marker for monitoring liver tumor, was developed by using anti-GP73 antibody (GP73 Ab) capped quantum dots (QDs) coupled with protein A/G agarose beads in an attempt to improve the analysis time, cost and operation. First, carboxylic-functionalized Mn modified CdTe/CdS QDs were synthesized and covalently conjugated with GP73 Ab, then protein A/G agarose beads were specifically combined with the QDs-conjugated Ab to form the QDs-Ab-beads conjugate, which could capture and separate GP73 from the sample through simple centrifugation. It was found that the fluorescence intensity of the above QDs-Ab-beads biosensor could be specifically quenched by GP73 added. A simple, rapid and specific quantitative method for GP73 protein was proposed using the as-prepared QDs-Ab-beads as a biosensor. Under the optimized conditions, the calibration curve of the proposed assay showed good linearity with a correlation coefficient of 0.9935 in the concentration range of 20-150 ng/mL of GP73 protein. The limit of detection (defined as 3σ/K) was 10 ng/mL. The method built exhibited a great potential in the clinic test of GP73. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Subcellular localization of human neutral ceramidase expressed in HEK293 cells

    International Nuclear Information System (INIS)

    Hwang, Young-ha; Tani, Motohiro; Nakagawa, Tetsuto; Okino, Nozomu; Ito, Makoto

    2005-01-01

    We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence

  20. Comparative Analysis of Human and Canine Campylobacter upsaliensis Isolates by Amplified Fragment Length Polymorphism

    DEFF Research Database (Denmark)

    Damborg, Peter; Guardabassi, Luca; Pedersen, Karl

    2008-01-01

    Human (n = 33) and canine (n = 53) Campylobacter upsaliensis isolates from seven countries were genotyped by a new amplified fragment length polymorphism method. We observed 100% typeability and high overall diversity. The majority of human strains (23/33) clustered separately from canine strains...

  1. Mast cell chymase degrades the alarmins heat shock protein 70, biglycan, HMGB1, and interleukin-33 (IL-33) and limits danger-induced inflammation.

    Science.gov (United States)

    Roy, Ananya; Ganesh, Goutham; Sippola, Helena; Bolin, Sara; Sawesi, Osama; Dagälv, Anders; Schlenner, Susan M; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Kjellén, Lena; Hellman, Lars; Åbrink, Magnus

    2014-01-03

    During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(W(sash))-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation.

  2. Mast Cell Chymase Degrades the Alarmins Heat Shock Protein 70, Biglycan, HMGB1, and Interleukin-33 (IL-33) and Limits Danger-induced Inflammation*

    Science.gov (United States)

    Roy, Ananya; Ganesh, Goutham; Sippola, Helena; Bolin, Sara; Sawesi, Osama; Dagälv, Anders; Schlenner, Susan M.; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Kjellén, Lena; Hellman, Lars; Åbrink, Magnus

    2014-01-01

    During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(Wsash)-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation. PMID:24257755

  3. Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways.

    Science.gov (United States)

    Mallet, W G; Maxfield, F R

    1999-07-26

    Furin and TGN38 are menbrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N.,W.G. Mallet,T.T. Soe,T.E.McGraw, and F.R. Maxfield.1998.J. Cell Biol.142:923-936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independentpathways for endosomal transport of proteins to the TGN from the plasma membrane.

  4. Expression of μ, κ, and δ opioid receptor messenger RNA in the human CNS: a 33P in situ hybridization study

    International Nuclear Information System (INIS)

    Peckys, D.; Landwehrmeyer, G.B.

    1999-01-01

    The existence of at least three opioid receptor types, referred to as μ, κ, and δ, is well established. Complementary DNAs corresponding to the pharmacologically defined μ, κ, and δ opioid receptors have been isolated in various species including man. The expression patterns of opioid receptor transcripts in human brain has not been established with a cellular resolution, in part because of the low apparent abundance of opioid receptor messenger RNAs in human brain. To visualize opioid receptor messenger RNAs we developed a sensitive in situ hybridization histochemistry method using 33 P-labelled RNA probes. In the present study we report the regional and cellular expression of μ, κ, and δ opioid receptor messenger RNAs in selected areas of the human brain. Hybridization of the different opioid receptor probes resulted in distinct labelling patterns. For the μ and κ opioid receptor probes, the most intense regional signals were observed in striatum, thalamus, hypothalamus, cerebral cortex, cerebellum and certain brainstem areas as well as the spinal cord. The most intense signals for the δ opioid receptor probe were found in cerebral cortex. Expression of opioid receptor transcripts was restricted to subpopulations of neurons within most regions studied demonstrating differences in the cellular expression patterns of μ, κ, and δ opioid receptor messenger RNAs in numerous brain regions. The messenger RNA distribution patterns for each opioid receptor corresponded in general to the distribution of opioid receptor binding sites as visualized by receptor autoradiography. However, some mismatches, for instance between μ opioid receptor receptor binding and μ opioid receptor messenger RNA expression in the anterior striatum, were observed. A comparison of the distribution patterns of opioid receptor messenger RNAs in the human brain and that reported for the rat suggests a homologous expression pattern in many regions. However, in the human brain, κ

  5. A Tyrosine-Based Trafficking Motif of the Tegument Protein pUL71 Is Crucial for Human Cytomegalovirus Secondary Envelopment.

    Science.gov (United States)

    Dietz, Andrea N; Villinger, Clarissa; Becker, Stefan; Frick, Manfred; von Einem, Jens

    2018-01-01

    The human cytomegalovirus (HCMV) tegument protein pUL71 is required for efficient secondary envelopment and accumulates at the Golgi compartment-derived viral assembly complex (vAC) during infection. Analysis of various C-terminally truncated pUL71 proteins fused to enhanced green fluorescent protein (eGFP) identified amino acids 23 to 34 as important determinants for its Golgi complex localization. Sequence analysis and mutational verification revealed the presence of an N-terminal tyrosine-based trafficking motif (YXXΦ) in pUL71. This led us to hypothesize a requirement of the YXXΦ motif for the function of pUL71 in infection. Mutation of both the tyrosine residue and the entire YXXΦ motif resulted in an altered distribution of mutant pUL71 at the plasma membrane and in the cytoplasm during infection. Both YXXΦ mutant viruses exhibited similarly decreased focal growth and reduced virus yields in supernatants. Ultrastructurally, mutant-virus-infected cells exhibited impaired secondary envelopment manifested by accumulations of capsids undergoing an envelopment process. Additionally, clusters of capsid accumulations surrounding the vAC were observed, similar to the ultrastructural phenotype of a UL71-deficient mutant. The importance of endocytosis and thus the YXXΦ motif for targeting pUL71 to the Golgi complex was further demonstrated when clathrin-mediated endocytosis was inhibited either by coexpression of the C-terminal part of cellular AP180 (AP180-C) or by treatment with methyl-β-cyclodextrin. Both conditions resulted in a plasma membrane accumulation of pUL71. Altogether, these data reveal the presence of a functional N-terminal endocytosis motif that is an important determinant for intracellular localization of pUL71 and that is furthermore required for the function of pUL71 during secondary envelopment of HCMV capsids at the vAC. IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of birth defects among congenital virus infections and can

  6. Cellular interactions of a lipid-based nanocarrier model with human keratinocytes: Unravelling transport mechanisms.

    Science.gov (United States)

    Silva, Elisabete; Barreiros, Luísa; Segundo, Marcela A; Costa Lima, Sofia A; Reis, Salette

    2017-04-15

    Knowledge of delivery system transport through epidermal cell monolayer is vital to improve skin permeation and bioavailability. Recently, nanostructured lipid carriers (NLCs) have gained great attention for transdermal delivery due to their biocompatibility, high drug payload, occlusive properties and skin hydration effect. However, the nanocarriers transport related mechanisms in epidermal epithelial cells are not yet understood. In this research, the internalization and transport pathways of the NLCs across the epidermal epithelial cell monolayer (HaCaT cells) were investigated. The 250nm sized witepsol/miglyol NLCs, prepared by hot homogenization had reduced cytotoxicity and no effect on the integrity of cell membrane in human HaCaT keratinocytes. The internalization was time-, concentration- and energy-dependent, and the uptake of NLCs was a vesicle-mediated process by macropinocytosis and clathrin-mediated pathways. 3% of NLCs were found at the apical membrane side of the HaCaT monolayer through exocytosis mechanism. Additionally, the endoplasmic reticulum, Golgi apparatus and microtubules played crucial roles in the transport of NLCs out of HaCaT cells. NLCs were transported intact across the human keratinocytes monolayer, without disturbing the tight junction's structure. From the transcytosis data only approximately 12% of the internalized NLCs were passed from the apical to the basolateral side. The transcytosis of NLCs throughout the HaCaT cell monolayer towards the basolateral membrane side requires the involvement of the endoplasmic reticulum, Golgi apparatus and microtubules. Our findings may contribute to a systematic understanding of NLCs transport across epidermal epithelial cell monolayers and their optimization for clinical transdermal application. Transdermal drug delivery is a challenging and growing area of clinical application. Lipid nanoparticles such as nanostructured lipid carriers (NLCs) have gained wide interest for transdermal drug

  7. RLV/X-33 operations overview

    Science.gov (United States)

    Black, Stephen T.; Eshleman, Wally

    1997-01-01

    This paper describes the VentureStar™ SSTO RLV and X-33 operations concepts. Applications of advanced technologies, automated ground support systems, advanced aircraft and launch vehicle lessons learned have been integrated to develop a streamlined vehicle and mission processing concept necessary to meet the goals of a commercial SSTO RLV. These concepts will be validated by the X-33 flight test program where financial and technical risk mitigation are required. The X-33 flight test program totally demonstrates the vehicle performance, technology, and efficient ground operations at the lowest possible cost. The Skunk Work's test program approach and test site proximity to the production plant are keys. The X-33 integrated flight and ground test program incrementally expands the knowledge base of the overall system allowing minimum risk progression to the next flight test program milestone. Subsequent X-33 turnaround processing flows will be performed with an aircraft operations philosophy. The differences will be based on research and development, component reliability and flight test requirements.

  8. Targeting the IL-33/IL-13 Axis for Respiratory Viral Infections.

    Science.gov (United States)

    Donovan, Chantal; Bourke, Jane E; Vlahos, Ross

    2016-04-01

    Lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD), are highly prevalent worldwide. One of the major factors that limits the efficacy of current medication in these patients are viral infections, leading to exacerbations of symptoms and decreased quality of life. Current pharmacological strategies targeting virus-induced lung disease are problematic due to antiviral resistance and the requirement for strain-specific vaccination. Thus, new therapeutic strategies are urgently required. In this Opinion article, we provide state-of-the-art evidence from humans and preclinical animal models implicating the interleukin (IL)-33/IL-13 axis in virus-induced lung disease. Thus, targeting the IL-33/IL-13 axis may be a feasible way to overcome the limitations of current therapy used to treat virus-induced exacerbations of lung disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. 33 CFR 164.33 - Charts and publications.

    Science.gov (United States)

    2010-07-01

    ...) PORTS AND WATERWAYS SAFETY NAVIGATION SAFETY REGULATIONS § 164.33 Charts and publications. (a) Each vessel must have the following: (1) Marine charts of the area to be transited, published by the National... tables published by private entities using data provided by the National Ocean Service. (ii) Tidal...

  10. Effect of cobalt-60 gamma irradiation on viability of soybean (glycine max. l) seed

    International Nuclear Information System (INIS)

    Addai, Isaac Kwahene

    2001-12-01

    The rapid rate of deterioration and low viability of soybean seeds particularly in storage is a major constraint to its production in the subtropical and tropical areas. Various approaches have been made to solve this problem but they have centred mainly on the control of the environment. Experimental mutagenesis is one of the ways by which genetic variability could be created to serve as basis for selection. In this study, induced mutations were used to create genetic variation and mutants with improved storability selected to provide a more lasting solution to this problem. Seeds of three soybean varieties - Gmx 92-6-10, Gmx 92-5-4E and TGX 87D- 1303 were subjected to four (4) months ambient storage and three accelerated ageing tests. The germination percentages computed 7 days after planting showed that 20% ethanol solution better mimicked ambient storage than 20% methanol solution and the 75°C hot water. In the radiation dosage response studies, dry seeds containing 10% moisture from the three varieties were subjected to 0, 50, 100, 150, 200 and 250 Gy doses of 60Co gamma rays at Ghana Atomic Energy Commission, Kwabenya and sown. The 250 Gy dose reduced both germination percentage and plant height by about 50% relative to the control and was used as the dosage appropriate for induced mutations for the varieties. Five thousand M2 plants were harvested and screened individually for improved storability using 20% ethanol solution, storing threshed seeds at ambient condition and storing seeds in pods also at ambient conditions. The 20% ethanol solution was less effective in selecting putative in mutants compared to the two ambient storage screening methods, but could be used as an initial screening method. The variety Gmx 92-6-10 was generally considered to give the greatest response to irradiation because it produce the largest proportion of putative mutant improved storability in two out of the three screening methods used at the M2 generation. Since TGX 87D 1

  11. Planck Observations of M33

    Science.gov (United States)

    Tibbs, C. T.; Israel, F. P.; Laureijs, R. J.; Tauber, J. A.; Partridge, B.; Peel, M. W.; Fauvet, L.

    2018-03-01

    We have performed a comprehensive investigation of the global integrated flux density of M33 from radio to ultraviolet wavelengths, finding that the data between ˜100 GHz and 3 THz are accurately described by a single modified blackbody curve with a dust temperature of Tdust = 21.67 ± 0.30 K and an effective dust emissivity index of βeff = 1.35 ± 0.10, with no indication of an excess of emission at millimeter/sub-millimeter wavelengths. However, sub-dividing M33 into three radial annuli, we found that the global emission curve is highly degenerate with the constituent curves representing the sub-regions of M33. We also found gradients in Tdust and βeff across the disk of M33, with both quantities decreasing with increasing radius. Comparing the M33 dust emissivity with that of other Local Group members, we find that M33 resembles the Magellanic Clouds rather than the larger galaxies, i.e., the Milky Way and M31. In the Local Group sample, we find a clear correlation between global dust emissivity and metallicity, with dust emissivity increasing with metallicity. A major aspect of this analysis is the investigation into the impact of fluctuations in the Cosmic Microwave Background (CMB) on the integrated flux density spectrum of M33. We found that failing to account for these CMB fluctuations would result in a significant over-estimate of Tdust by ˜5 K and an under-estimate of βeff by ˜0.4.

  12. Acute ethanol exposure inhibits silencing of cerebellar Golgi cell firing induced by granule cell axon input

    Directory of Open Access Journals (Sweden)

    Paolo eBotta

    2014-02-01

    Full Text Available Golgi cells (GoCs are specialized interneurons that provide inhibitory input to granule cells in the cerebellar cortex. GoCs are pacemaker neurons that spontaneously fire action potentials, triggering spontaneous inhibitory postsynaptic currents in granule cells and also contributing to the generation tonic GABAA receptor-mediated currents in granule cells. In turn, granule cell axons provide feedback glutamatergic input to GoCs. It has been shown that high frequency stimulation of granule cell axons induces a transient pause in GoC firing in a type 2-metabotropic glutamate receptor (mGluR2-dependent manner. Here, we investigated the effect ethanol on the pause of GoC firing induced by high frequency stimulation of granule cell axons. GoC electrophysiological recordings were performed in parasagittal cerebellar vermis slices from postnatal day 23 to 26 rats. Loose-patch cell-attached recordings revealed that ethanol (40 mM reversibly decreases the pause duration. An antagonist of mGluR2 reduced the pause duration but did not affect the effect of ethanol. Whole-cell voltage-clamp recordings showed that currents evoked by an mGluR2 agonist were not significantly affected by ethanol. Perforated-patch experiments in which hyperpolarizing and depolarizing currents were injected into GoCs demonstrated that there is an inverse relationship between spontaneous firing and pause duration. Slight inhibition of the Na+/K+ pump mimicked the effect of ethanol on pause duration. In conclusion, ethanol reduces the granule cell axon-mediated feedback mechanism by reducing the input responsiveness of GoCs. This would result in a transient increase of GABAA receptor-mediated inhibition of granule cells, limiting information flow at the input stage of the cerebellar cortex.

  13. 14 CFR 33.21 - Engine cooling.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Engine cooling. 33.21 Section 33.21 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Design and Construction; General § 33.21 Engine cooling. Engine design and...

  14. 32 CFR 989.33 - Environmental justice.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false Environmental justice. 989.33 Section 989.33 National Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE ENVIRONMENTAL PROTECTION ENVIRONMENTAL IMPACT ANALYSIS PROCESS (EIAP) § 989.33 Environmental justice. During the preparation of...

  15. 14 CFR 33.47 - Detonation test.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Detonation test. 33.47 Section 33.47 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Block Tests; Reciprocating Aircraft Engines § 33.47 Detonation test. Each engine...

  16. Poly[3-(3, 4-dihydroxyphenyl) glyceric acid] from Comfrey exerts anti-cancer efficacy against human prostate cancer via targeting androgen receptor, cell cycle arrest and apoptosis.

    Science.gov (United States)

    Shrotriya, Sangeeta; Gagan, Deep; Ramasamy, Kumaraguruparan; Raina, Komal; Barbakadze, Vakhtang; Merlani, Maia; Gogilashvili, Lali; Amiranashvili, Lela; Mulkijanyan, Karen; Papadopoulos, Kyriakos; Agarwal, Chapla; Agarwal, Rajesh

    2012-08-01

    The major obstacles in human prostate cancer (PCA) treatment are the development of resistance to androgen ablation therapy leading to hormone-refractory state and the toxicity associated with chemotherapeutic drugs. Thus, the identification of additional non-toxic agents that are effective against both androgen-dependent and androgen-independent PCA is needed. In the present study, we investigated the efficacy of a novel phytochemical poly[3-(3, 4-dihydroxyphenyl)glyceric acid] (p-DGA) from Caucasian species of comfrey (Symphytum caucasicum) and its synthetic derivative syn-2, 3-dihydroxy-3-(3, 4-dihydroxyphenyl) propionic acid (m-DGA) against PCA LNCaP and 22Rv1 cells. We found that both p-DGA and m-DGA suppressed the growth and induced death in PCA cells, with comparatively lesser cytotoxicity towards non-neoplastic human prostate epithelial cells. Furthermore, we also found that both p-DGA and m-DGA caused G(1) arrest in PCA cells through modulating the expression of cell cycle regulators, especially an increase in CDKIs (p21 and p27). In addition, p-DGA and m-DGA induced apoptotic death by activating caspases, and also strongly decreased AR and PSA expression. Consistent with in vitro results, our in vivo study showed that p-DGA feeding strongly inhibited 22Rv1 tumors growth by 76% and 88% at 2.5 and 5mg/kg body weight doses, respectively, without any toxicity, together with a strong decrease in PSA level in plasma; and a decrease in PCNA, AR and PSA expression but increase in p21/p27 expression and apoptosis in tumor tissues from p-DGA-fed mice. Overall, present study identifies p-DGA as a potent agent against PCA without any toxicity, and supports its clinical application.

  17. The international Hip Outcome Tool-33 (iHOT-33): multicenter validation and translation to Spanish.

    Science.gov (United States)

    Ruiz-Ibán, Miguel Angel; Seijas, Roberto; Sallent, Andrea; Ares, Oscar; Marín-Peña, Oliver; Muriel, Alfonso; Cuéllar, Ricardo

    2015-05-20

    The international Hip Outcome Tool-33 (iHOT-33) is a 33-item self administered outcome measure based on a Visual Analogue Scale response format designed for young and active population with hip pathology. The aim of the present study is to translate and validate the iHOT-33 into Spanish. 97 patients undergoing hip arthroscopy were included in this prospective and multicenter study performed between January 2012 and May 2014. Crosscultural adaptation was used to translate iHOT-33 into Spanish. Patients completed the questionnaire before and after surgery. Feasibility, reliability, internal consistency, construct validity (correlation with Western Ontario and McMaster Universities Osteoarthritis Index), ceiling and floor effects and sensitivity to change were assessed for the present study. Mean age was 48 years old. Feasibility: 41.2 % patients had no blank questions, and 71.3 % of patients had fulfilled all but one or two questions. Reliability: ICC for the global questionnaire was 0.97, showing that the questionnaire is highly reproducible. Internal consistency: Cronbach's alpha was 0.98 for the global questionnaire. Construct validity: there was a high correlation with WOMAC (correlation coefficient >0.5). The Ceiling effect (taking into account the minimum detectable change) was 12.1 % and the floor effect was 21.6 %, for the global questionnaire. Large sensitivity to change was shown. the Spanish version of iHOT-33 has shown to be feasible, reliable and sensible to changes for patients undergoing hip arthroscopy. This validated translation of iHOT-33 allows for comparisons between studies involving either Spanish- or English-speaking patients. Prognostic study, Level I.

  18. 45 CFR 98.33 - Consumer education.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Consumer education. 98.33 Section 98.33 Public... Program Operations (Child Care Services)-Parental Rights and Responsibilities § 98.33 Consumer education... public consumer education information that will promote informed child care choices including, at a...

  19. 22 CFR 42.33 - Diversity immigrants.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Diversity immigrants. 42.33 Section 42.33 Foreign Relations DEPARTMENT OF STATE VISAS VISAS: DOCUMENTATION OF IMMIGRANTS UNDER THE IMMIGRATION AND NATIONALITY ACT, AS AMENDED Immigrants Subject to Numerical Limitations § 42.33 Diversity immigrants. (a...

  20. 14 CFR 33.51 - Operation test.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Operation test. 33.51 Section 33.51... STANDARDS: AIRCRAFT ENGINES Block Tests; Reciprocating Aircraft Engines § 33.51 Operation test. The operation test must include the testing found necessary by the Administrator to demonstrate backfire...

  1. 45 CFR 1170.33 - New construction.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false New construction. 1170.33 Section 1170.33 Public... ASSISTED PROGRAMS OR ACTIVITIES Accessibility § 1170.33 New construction. (a) Design, construction, and... Federal Accessibility Standards. (1) Effective as of January 18, 1991, design, construction, or alteration...

  2. 7 CFR 550.33 - Administrative supervision.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 6 2010-01-01 2010-01-01 false Administrative supervision. 550.33 Section 550.33 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL RESEARCH SERVICE, DEPARTMENT OF AGRICULTURE GENERAL ADMINISTRATIVE POLICY FOR NON-ASSISTANCE COOPERATIVE AGREEMENTS Management of Agreements Program Management § 550.33...

  3. 49 CFR 178.33-6 - Manufacture.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Manufacture. 178.33-6 Section 178.33-6 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Inside Containers, and Linings § 178.33-6 Manufacture. (a) By appliances and methods that will...

  4. The novel cytokine interleukin-33 activates acinar cell proinflammatory pathways and induces acute pancreatic inflammation in mice.

    Directory of Open Access Journals (Sweden)

    Duraisamy Kempuraj

    Full Text Available Acute pancreatitis is potentially fatal but treatment options are limited as disease pathogenesis is poorly understood. IL-33, a novel IL-1 cytokine family member, plays a role in various inflammatory conditions but its role in acute pancreatitis is not well understood. Specifically, whether pancreatic acinar cells produce IL-33 when stressed or respond to IL-33 stimulation, and whether IL-33 exacerbates acute pancreatic inflammation is unknown.In duct ligation-induced acute pancreatitis in mice and rats, we found that (a IL-33 concentration was increased in the pancreas; (b mast cells, which secrete and also respond to IL-33, showed degranulation in the pancreas and lung; (c plasma histamine and pancreatic substance P concentrations were increased; and (d pancreatic and pulmonary proinflammatory cytokine concentrations were increased. In isolated mouse pancreatic acinar cells, TNF-α stimulation increased IL-33 release while IL-33 stimulation increased proinflammatory cytokine release, both involving the ERK MAP kinase pathway; the flavonoid luteolin inhibited IL-33-stimulated IL-6 and CCL2/MCP-1 release. In mice without duct ligation, exogenous IL-33 administration induced pancreatic inflammation without mast cell degranulation or jejunal inflammation; pancreatic changes included multifocal edema and perivascular infiltration by neutrophils and some macrophages. ERK MAP kinase (but not p38 or JNK and NF-kB subunit p65 were activated in the pancreas of mice receiving exogenous IL-33, and acinar cells isolated from the pancreas of these mice showed increased spontaneous cytokine release (IL-6, CXCL2/MIP-2α. Also, IL-33 activated ERK in human pancreatic tissue.As exogenous IL-33 does not induce jejunal inflammation in the same mice in which it induces pancreatic inflammation, we have discovered a potential role for an IL-33/acinar cell axis in the recruitment of neutrophils and macrophages and the exacerbation of acute pancreatic inflammation

  5. Water quality criteria for colored smokes: Solvent Yellow 33, Final report. [Contains glossary

    Energy Technology Data Exchange (ETDEWEB)

    Davidson, K.A.; Hovatter, P.S.

    1987-11-01

    The available data on the environmental fate, aquatic toxicity, and mammalian toxicity of Solvent Yellow 33, a quinoline dye used in colored smoke grenades, were reviewed. The US Environmental Protection Agency (USEPA) guidelines were used in an attempt to generate water quality criteria for the protection of aquatic life and its use and of human health. 87 refs., 2 figs., 13 tabs.

  6. 10 CFR 95.33 - Security education.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Security education. 95.33 Section 95.33 Energy NUCLEAR... INFORMATION AND RESTRICTED DATA Physical Security § 95.33 Security education. All cleared employees must be... information. The facility may obtain defensive security, threat awareness, and other education and training...

  7. 10 CFR 75.33 - Accounting reports.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Accounting reports. 75.33 Section 75.33 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) SAFEGUARDS ON NUCLEAR MATERIAL-IMPLEMENTATION OF US/IAEA AGREEMENT Reports § 75.33 Accounting reports. (a)(1) The accounting reports for each IAEA material balance area must...

  8. 7 CFR 1779.33 - Interest rates.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 12 2010-01-01 2010-01-01 false Interest rates. 1779.33 Section 1779.33 Agriculture... (CONTINUED) WATER AND WASTE DISPOSAL PROGRAMS GUARANTEED LOANS § 1779.33 Interest rates. (a) General. Rates.... Interest rates will be those rates customarily charged borrowers in similar circumstances in the ordinary...

  9. 18 CFR 33.10 - Additional information.

    Science.gov (United States)

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Additional information. 33.10 Section 33.10 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION... § 33.10 Additional information. The Director of the Office of Energy Market Regulation, or his designee...

  10. 32 CFR 33.25 - Program income.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 1 2010-07-01 2010-07-01 false Program income. 33.25 Section 33.25 National... Post-Award Requirements Financial Administration § 33.25 Program income. (a) General. Grantees are encouraged to earn income to defray program costs. Program income includes income from fees for services...

  11. 14 CFR 33.89 - Operation test.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Operation test. 33.89 Section 33.89... STANDARDS: AIRCRAFT ENGINES Block Tests; Turbine Aircraft Engines § 33.89 Operation test. (a) The operation test must include testing found necessary by the Administrator to demonstrate— (1) Starting, idling...

  12. 14 CFR 415.33 - Safety organization.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Safety organization. 415.33 Section 415.33....33 Safety organization. (a) An applicant shall maintain a safety organization and document it by... communication, both within the applicant's organization and between the applicant and any federal launch range...

  13. 20 CFR 234.33 - Survivor annuities.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Survivor annuities. 234.33 Section 234.33 Employees' Benefits RAILROAD RETIREMENT BOARD REGULATIONS UNDER THE RAILROAD RETIREMENT ACT LUMP-SUM PAYMENTS Annuities Due but Unpaid at Death § 234.33 Survivor annuities. Any survivor annuity which is...

  14. Benzyl alcohol induces a reversible fragmentation of the Golgi apparatus and inhibits membrane trafficking between endosomes and the trans-Golgi network

    DEFF Research Database (Denmark)

    Simm, Roger; Kvalvaag, Audun Sverre; van Deurs, Bo

    2017-01-01

    Benzyl alcohol (BnOH) is widely used as a component of foods, cosmetics, household products and medical products. It is generally considered to be safe for human use, however, it has been connected to a number of adverse effects, including hypersensitivity reactions and neonatal deaths. Bn...

  15. 7 CFR 3575.33 - Interest rates.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Interest rates. 3575.33 Section 3575.33 Agriculture... GENERAL Community Programs Guaranteed Loans § 3575.33 Interest rates. (a) General. Rates will be negotiated between the lender and the borrower. They may be either fixed or variable rates. Interest rates...

  16. 14 CFR 33.62 - Stress analysis.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Stress analysis. 33.62 Section 33.62... STANDARDS: AIRCRAFT ENGINES Design and Construction; Turbine Aircraft Engines § 33.62 Stress analysis. A stress analysis must be performed on each turbine engine showing the design safety margin of each turbine...

  17. 14 CFR 33.83 - Vibration test.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Vibration test. 33.83 Section 33.83... STANDARDS: AIRCRAFT ENGINES Block Tests; Turbine Aircraft Engines § 33.83 Vibration test. (a) Each engine... experience, analysis, and component test and shall address, as a minimum, blades, vanes, rotor discs, spacers...

  18. 14 CFR 431.33 - Safety organization.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Safety organization. 431.33 Section 431.33... Launch and Reentry of a Reusable Launch Vehicle § 431.33 Safety organization. (a) An applicant shall maintain a safety organization and document it by identifying lines of communication and approval authority...

  19. 7 CFR 15b.33 - Housing.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Housing. 15b.33 Section 15b.33 Agriculture Office of... RECEIVING FEDERAL FINANCIAL ASSISTANCE Postsecondary Education § 15b.33 Housing. (a) Housing provided by the recipient. A recipient that provides housing to its nonhandicapped students shall provide comparable...

  20. 50 CFR 27.33 - Water skiing.

    Science.gov (United States)

    2010-10-01

    ... 50 Wildlife and Fisheries 6 2010-10-01 2010-10-01 false Water skiing. 27.33 Section 27.33 Wildlife... NATIONAL WILDLIFE REFUGE SYSTEM PROHIBITED ACTS Disturbing Violations: With Vehicles § 27.33 Water skiing. When water skiing is permitted upon national wildlife refuge waters, the public will be notified under...

  1. 32 CFR 33.41 - Financial reporting.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 1 2010-07-01 2010-07-01 false Financial reporting. 33.41 Section 33.41... GOVERNMENTS Post-Award Requirements Reports, Records, Retention, and Enforcement § 33.41 Financial reporting... forms as may from time to time be authorized by OMB, for: (i) Submitting financial reports to Federal...

  2. A Potent and Selective Quinoxalinone-Based STK33 Inhibitor Does Not Show Synthetic Lethality in KRAS-Dependent Cells

    Science.gov (United States)

    2012-01-01

    The KRAS oncogene is found in up to 30% of all human tumors. In 2009, RNAi experiments revealed that lowering mRNA levels of a transcript encoding the serine/threonine kinase STK33 was selectively toxic to KRAS-dependent cancer cell lines, suggesting that small-molecule inhibitors of STK33 might selectively target KRAS-dependent cancers. To test this hypothesis, we initiated a high-throughput screen using compounds in the Molecular Libraries Small Molecule Repository (MLSMR). Several hits were identified, and one of these, a quinoxalinone derivative, was optimized. Extensive SAR studies were performed and led to the chemical probe ML281 that showed low nanomolar inhibition of purified recombinant STK33 and a distinct selectivity profile as compared to other STK33 inhibitors that were reported in the course of these studies. Even at the highest concentration tested (10 μM), ML281 had no effect on the viability of KRAS-dependent cancer cells. These results are consistent with other recent reports using small-molecule STK33 inhibitors. Small molecules having different chemical structures and kinase-selectivity profiles are needed to fully understand the role of STK33 in KRAS-dependent cancers. In this regard, ML281 is a valuable addition to small-molecule probes of STK33. PMID:23256033

  3. 34 CFR 106.33 - Comparable facilities.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Comparable facilities. 106.33 Section 106.33 Education... Discrimination on the Basis of Sex in Education Programs or Activities Prohibited § 106.33 Comparable facilities. A recipient may provide separate toilet, locker room, and shower facilities on the basis of sex, but...

  4. 14 CFR 33.75 - Safety analysis.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Safety analysis. 33.75 Section 33.75... STANDARDS: AIRCRAFT ENGINES Design and Construction; Turbine Aircraft Engines § 33.75 Safety analysis. (a... consequences of all failures that can reasonably be expected to occur. This analysis will take into account, if...

  5. 31 CFR 3.3 - Legal review.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Legal review. 3.3 Section 3.3 Money and Finance: Treasury Office of the Secretary of the Treasury CLAIMS REGULATIONS AND INDEMNIFICATION OF DEPARTMENT OF TREASURY EMPLOYEES Claims Under the Federal Tort Claims Act § 3.3 Legal review. Any...

  6. 19 CFR 10.33 - Theatrical effects.

    Science.gov (United States)

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Theatrical effects. 10.33 Section 10.33 Customs... Under Bond § 10.33 Theatrical effects. For purposes of the entry of theatrical scenery, properties and... films. For provisions relating to the return without formal entry of theatrical effects taken from the...

  7. 14 CFR 33.43 - Vibration test.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Vibration test. 33.43 Section 33.43... STANDARDS: AIRCRAFT ENGINES Block Tests; Reciprocating Aircraft Engines § 33.43 Vibration test. (a) Each... configuration of the propeller type which is used for the endurance test, and using, for other engines, the same...

  8. 14 CFR 33.49 - Endurance test.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Endurance test. 33.49 Section 33.49... STANDARDS: AIRCRAFT ENGINES Block Tests; Reciprocating Aircraft Engines § 33.49 Endurance test. (a) General. Each engine must be subjected to an endurance test that includes a total of 150 hours of operation...

  9. 14 CFR 33.17 - Fire protection.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Fire protection. 33.17 Section 33.17... STANDARDS: AIRCRAFT ENGINES Design and Construction; General § 33.17 Fire protection. (a) The design and... protection unless damage by fire will not cause leakage or spillage of a hazardous quantity of flammable...

  10. 28 CFR 33.52 - Civil rights.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Civil rights. 33.52 Section 33.52... Block Grants Additional Requirements § 33.52 Civil rights. The Justice Assistance Act provides that “no... the provisions of title VI of the Civil Rights Act of 1964; section 504 of the Rehabilitation Act of...

  11. 10 CFR 820.33 - Default order.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Default order. 820.33 Section 820.33 Energy DEPARTMENT OF ENERGY PROCEDURAL RULES FOR DOE NUCLEAR ACTIVITIES Enforcement Process § 820.33 Default order. (a) Default. The Presiding Officer, upon motion by a party or the filing of a Notice of Intent to issue a...

  12. 25 CFR 226.33 - Line drilling.

    Science.gov (United States)

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Line drilling. 226.33 Section 226.33 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR ENERGY AND MINERALS LEASING OF OSAGE RESERVATION LANDS FOR OIL AND GAS MINING Requirements of Lessees § 226.33 Line drilling. Lessee shall not drill within 300 feet...

  13. 30 CFR 33.34 - Drilling test.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Drilling test. 33.34 Section 33.34 Mineral... MINING PRODUCTS DUST COLLECTORS FOR USE IN CONNECTION WITH ROCK DRILLING IN COAL MINES Test Requirements § 33.34 Drilling test. (a) A drilling test shall consist of drilling a set of 10 test holes, without...

  14. 14 CFR 33.87 - Endurance test.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Endurance test. 33.87 Section 33.87... STANDARDS: AIRCRAFT ENGINES Block Tests; Turbine Aircraft Engines § 33.87 Endurance test. (a) General. Each engine must be subjected to an endurance test that includes a total of at least 150 hours of operation...

  15. Pyramidal neurons in the septal and temporal CA1 field of the human and hedgehog tenrec hippocampus.

    Science.gov (United States)

    Liagkouras, Ioannis; Michaloudi, Helen; Batzios, Christos; Psaroulis, Dimitrios; Georgiadis, Marios; Künzle, Heinz; Papadopoulos, Georgios C

    2008-07-07

    The present study examines comparatively the cellular density of disector-counted/Nissl-stained CA1 pyramidal neurons and the morphometric characteristics (dendritic number/length, spine number/density and Sholl-counted dendritic branch points/20 microm) of the basal and apical dendritic systems of Golgi-impregnated CA1 neurons, in the septal and temporal hippocampus of the human and hedgehog tenrec brain. The obtained results indicate that in both hippocampal parts the cellular density of the CA1 pyramidal neurons is lower in human than in tenrec. However, while the human pyramidal cell density is higher in the septal hippocampal part than in the temporal one, in the tenrec the density of these cells is higher in the temporal part. The dendritic tree of the CA1 pyramidal cells, more developed in the septal than in temporal hippocampus in both species studied, is in general more complex in the human hippocampus. The basal and the apical dendritic systems exhibit species related morphometric differences, while dendrites of different orders exhibit differences in their number and length, and in their spine density. Finally, in both species, as well as hippocampal parts and dendritic systems, changes of dendritic morphometric features along ascending dendritic orders fluctuate in a similar way, as do the number of dendritic branch points in relation to the distance from the neuron soma.

  16. 30 CFR 33.31 - Test space.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Test space. 33.31 Section 33.31 Mineral... § 33.31 Test space. (a) Drilling tests shall be conducted in a test space formed by two curtains suspended across a mine opening in such a manner that the volume of the test space shall be approximately 2...

  17. The art of human anatomy: Renaissance to 21st century.

    Science.gov (United States)

    Van Hee, Robrecht; Wells, F C; Ballestriero, Roberta; Richardson, Ruth; Mazzarello, Paolo; Cani, Valentina; Catani, Marco

    2014-01-01

    This session examines the relationship between the art and science of anatomy from the time of Vesalius to the present with particular emphasis on the role of the medical artist and the changing nature of anatomical illustration over the last five centuries. Pivotal changes in the art of anatomy will be examined including the evolution of media and brain imaging from Golgi to Geschwind.

  18. Human calumenin localizes to the secretory pathway and is secreted to the medium

    DEFF Research Database (Denmark)

    Vorum, H; Hager, H; Christensen, Birgitte Mønster

    1999-01-01

    Calumenin belongs to a family of multiple EF-hand proteins that include reticulocalbin, ERC-55, and Cab45. Reticulocalbin and ERC-55 localize to the ER due to a C-terminal HDEL retrieval signal. Cab45 contains a HEEF C-terminal sequence and is localized to the Golgi apparatus. The murine homologue...

  19. IL-33 and IgE stimulate mast cell production of IL-2 and regulatory T cell expansion in allergic dermatitis.

    Science.gov (United States)

    Salamon, P; Shefler, I; Moshkovits, I; Munitz, A; Horwitz Klotzman, D; Mekori, Y A; Hershko, A Y

    2017-11-01

    We have previously shown that mast cells (MCs) suppress chronic allergic dermatitis in mice. The underlying mechanism involves MC-derived IL-2, which supports regulatory T cell (Treg) response at the site of inflammation. However, it is not clear what are the factors that drive MCs to produce IL-2. To understand the mechanisms that lead to IL-2 production from MCs in chronic allergic dermatitis. Isolated murine bone marrow-derived MCs (BMMCs) were incubated with various stimulators, and IL-2 production was assessed by RT-PCR and ELISA. The response of signalling pathways was evaluated by MAPK inhibitors and Western blot analysis. The effect of MC-IL-2 on Tregs was studied by incubation of splenic T cells with conditioned media obtained from activated BMMCs. Dermatitis was elicited by repeated exposures of mouse ears to oxazolone. MCs in mouse and human skin samples were evaluated by immunostaining. BMMCs released IL-2 in response to IL-33, and IL-2 production was further enhanced by concomitant FcεRI activation. The effect of IL-33 was mediated by activation of the MAPK family members. IL-2 in conditioned media from IL-33 and IgE-stimulated BMMCs led to considerable expansion of Tregs in vitro. IL-33 levels were elevated in oxazolone-challenged ears along with increased numbers of IL-2-expressing MCs. Human skin with chronic inflammation also contained IL-2-expressing MCs that colocalized with IL-33 staining in the dermis. IL-33, in collaboration with IgE, is critical for MC-IL-2 production in allergic skin disease, thus leading to Treg stimulation and suppression of allergic dermatitis. © 2017 John Wiley & Sons Ltd.

  20. 40 CFR 407.33 - [Reserved

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true [Reserved] 407.33 Section 407.33 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS CANNED AND PRESERVED FRUITS AND VEGETABLES PROCESSING POINT SOURCE CATEGORY Citrus Products Subcategory...

  1. A new member of the GM130 golgin subfamily is expressed in the optic lobe anlagen of the metamorphosing brain of Manduca sexta

    Directory of Open Access Journals (Sweden)

    Chiou-Miin Wang

    2003-12-01

    Full Text Available During metamorphosis of the insect brain, the optic lobe anlagen generate the proliferation centers for the visual cortices. We show here that, in the moth Manduca sexta, an 80 kDa Golgi complex protein (Ms-golgin80 is abundantly expressed in the cytoplasm of neuroblasts and ganglion mother cells in the optic lobe anlagen and proliferation centers. The predicted amino acid sequence for Ms-golgin80 is similar to that of several members of the GM130 subfamily of Golgi-associated proteins, including rat GM130 and human golgin-95. Homologs of Ms-golgin80 from Drosophila melanogaster, Caenorhabditis elegans, and Brugia malayi were identified through homology sequence search. Sequence similarities are present in three regions: the N-terminus, an internal domain of 89 amino acids, and another domain of 89 amino acids near the C-terminus. Structural similarities further suggest that these molecules play the same cellular role as GM130. GM130 is involved in the docking and fusion of coatomer (COP I coated vesicles to the Golgi membranes; it also regulates the fragmentation and subsequent reassembly of the Golgi complex during mitosis. Abundant expression of Ms-golgin80 in neuroblasts and ganglion mother cells and its reduced expression in the neuronal progeny of these cells suggest that this protein may be involved in the maintenance of the proliferative state.

  2. 48 CFR 33.201 - Definitions.

    Science.gov (United States)

    2010-10-01

    .... Alternative dispute resolution (ADR) means any type of procedure or combination of procedures voluntarily used... REQUIREMENTS PROTESTS, DISPUTES, AND APPEALS Disputes and Appeals 33.201 Definitions. As used in this subpart... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Definitions. 33.201...

  3. 32 CFR 33.32 - Equipment.

    Science.gov (United States)

    2010-07-01

    ... awarding agency. (d) Management requirements. Procedures for managing equipment (including replacement... return. (e) Disposition. When original or replacement equipment acquired under a grant or subgrant is no... 32 National Defense 1 2010-07-01 2010-07-01 false Equipment. 33.32 Section 33.32 National Defense...

  4. Expression of peanut agglutinin-binding mucin-type glycoprotein in human esophageal squamous cell carcinoma as a marker

    Directory of Open Access Journals (Sweden)

    Balakrishnan Ramathilakam

    2003-11-01

    Full Text Available Abstract Background The TF (Thomson – Friedenreich blood group antigen behaves as an onco-foetal carcinoma-associated antigen, showing increased expression in malignancies and its detection and quantification can be used in serologic diagnosis mainly in adenocarcinomas. This study was undertaken to analyze the sera and tissue level detectable mucin-type glycoprotein (TF-antigen by Peanut agglutinin (PNA and its diagnostic index in serum as well tissues of human esophageal squamous cell carcinoma as marker. Results We examined 100 patients for serological analysis by Enzyme Linked Lectin Assay (ELISA and demonstrated a sensitivity of 87.5%, specificity of 90% and a positive predictive value of 95%. The immuno-histochemical localization of TF antigen by Fluorescence Antigen Technique (FAT in 25 specimens of normal esophageal squamous epithelium specimens and 92 specimens with different grades of, allowed a quicker and more precise identification of its increased expression and this did not correlate with gender and tumor size. There was a positive correlation between membrane bound TF antigen expression with different histological progression, from well differentiated to poorly differentiated, determined by PNA binding. Specimens showed morphological changes and a pronounced increase in PNA binding in Golgi apparatus, secretory granules of the cytosol of well differentiated and an increased cell membrane labeling in moderately and poorly differentiated, when compared with ESCC and normal tissues. Conclusion The authors propose that the expression of TF-antigen in human may play an important role during tumorigenesis establishing it as a chemically well-defined carcinoma-associated antigen. Identification of the circulating TF-antigen as a reactive form and as a cryptic form in the healthy individuals, using PNA-ELLA and Immunohistochemical analysis of TF antigen by FAT is positively correlated with the different histological grades as a simple

  5. Depleted uranium determination at the Novi Sad low level facility

    International Nuclear Information System (INIS)

    Bikit, I.; Slivka, J.; Krmar, M.; Veskovic, M.; Conkic, Lj.; Varga, E.

    2002-01-01

    Natural uranium determination in environmental samples at the low-level gamma-spectroscopy laboratory of the Faculty of Science in Novi Sad has more than 20 years long tradition. When the issue of depleted uranium emerged the experimental advantages of the measuring equipment (GMX type of HPGe detector with enhanced efficiency below 100 keV, and iron low level shielding) where fully exploited. A detection technique selective for depleted uranium was developed. The details of this method together with the results for about 100 samples (soil, plants, water, food) are presented, and discussed. (author)

  6. A Recombinant Adenovirus Expressing Ovine Interferon Tau Prevents Influenza Virus-Induced Lethality in Mice.

    Science.gov (United States)

    Martín, V; Pascual, E; Avia, M; Rangel, G; de Molina, A; Alejo, A; Sevilla, N

    2016-01-06

    Ovine interferon tau (IFN-τ) is a unique type I interferon with low toxicity and a broad host range in vivo. We report the generation of a nonreplicative recombinant adenovirus expressing biologically active IFN-τ. Using the B6.A2G-Mx1 mouse model, we showed that single-dose intranasal administration of recombinant Ad5-IFN-τ can effectively prevent lethality and disease induced by highly virulent hv-PR8 influenza virus by activating the interferon response and preventing viral replication. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. N-Glycosylation of an IgG antibody secreted by Nicotiana tabacum BY-2 cells can be modulated through co-expression of human β-1,4-galactosyltransferase.

    Science.gov (United States)

    Navarre, Catherine; Smargiasso, Nicolas; Duvivier, Laurent; Nader, Joseph; Far, Johann; De Pauw, Edwin; Boutry, Marc

    2017-06-01

    Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.

  8. 22 CFR 192.33 - Dispute.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Dispute. 192.33 Section 192.33 Foreign Relations DEPARTMENT OF STATE HOSTAGE RELIEF VICTIMS OF TERRORISM COMPENSATION Medical Benefits for Captive... Director, Department of State, for a determination. If the person bringing the claim is not satisfied with...

  9. 46 CFR 67.33 - Individual.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Individual. 67.33 Section 67.33 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) DOCUMENTATION AND MEASUREMENT OF VESSELS DOCUMENTATION OF VESSELS...-born, naturalized, or a derivative citizen of the United States, or otherwise qualifies as a United...

  10. 17 CFR 33.7 - Disclosure.

    Science.gov (United States)

    2010-04-01

    ... DIFFICULT OR IMPOSSIBLE TO OFFSET AN EXISTING OPTION POSITION ON AN EXCHANGE. THE GRANTOR OF AN OPTION... 17 Commodity and Securities Exchanges 1 2010-04-01 2010-04-01 false Disclosure. 33.7 Section 33.7 Commodity and Securities Exchanges COMMODITY FUTURES TRADING COMMISSION REGULATION OF DOMESTIC EXCHANGE...

  11. Dasatinib-Induced Rhabdomyolysis in a 33-Year-Old Patient with Chronic Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Andrew Stevenson Joel Chandranesan

    2018-01-01

    Full Text Available Rhabdomyolysis is a life-threatening syndrome due to breakdown of the skeletal muscle. It can be caused by massive trauma and crush injuries or occur as a side effect of medications. Here, we describe a case of a 33-year-old male with human immunodeficiency virus (HIV and newly diagnosed chronic myeloid leukemia (CML with severe life-threatening rhabdomyolysis due to a rare offending agent.

  12. 49 CFR 37.33 - Airport transportation systems.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Airport transportation systems. 37.33 Section 37.33 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Applicability § 37.33 Airport transportation systems. (a) Transportation...

  13. Golgi Analysis of Neuron Morphology in the Presumptive Somatosensory Cortex and Visual Cortex of the Florida Manatee (Trichechus manatus latirostris).

    Science.gov (United States)

    Reyes, Laura D; Harland, Tessa; Reep, Roger L; Sherwood, Chet C; Jacobs, Bob

    2016-01-01

    The current study investigates neuron morphology in presumptive primary somatosensory (S1) and primary visual (V1) cortices of the Florida manatee (Trichechus manatus latirostris) as revealed by Golgi impregnation. Sirenians, including manatees, have an aquatic lifestyle, a large body size, and a relatively large lissencephalic brain. The present study examines neuron morphology in 3 cortical areas: in S1, dorsolateral cortex area 1 (DL1) and cluster cortex area 2 (CL2) and in V1, dorsolateral cortex area 4 (DL4). Neurons exhibited a variety of morphological types, with pyramidal neurons being the most common. The large variety of neuron types present in the manatee cortex was comparable to that seen in other eutherian mammals, except for rodents and primates, where pyramid-shaped neurons predominate. A comparison between pyramidal neurons in S1 and V1 indicated relatively greater dendritic branching in S1. Across all 3 areas, the dendritic arborization pattern of pyramidal neurons was also similar to that observed previously in the afrotherian rock hyrax, cetartiodactyls, opossums, and echidnas but did not resemble the widely bifurcated dendrites seen in the large-brained African elephant. Despite adaptations for an aquatic environment, manatees did not share specific neuron types such as tritufted and star-like neurons that have been found in cetaceans. Manatees exhibit an evolutionarily primitive pattern of cortical neuron morphology shared with most other mammals and do not appear to have neuronal specializations for an aquatic niche. © 2016 S. Karger AG, Basel.

  14. Identification of rice cornichon as a possible cargo receptor for the Golgi-localized sodium transporter OsHKT1;3

    Science.gov (United States)

    Rosas-Santiago, Paul; Lagunas-Gómez, Daniel; Barkla, Bronwyn J.; Vera-Estrella, Rosario; Lalonde, Sylvie; Jones, Alexander; Frommer, Wolf B.; Zimmermannova, Olga; Sychrová, Hana; Pantoja, Omar

    2015-01-01

    Membrane proteins are synthesized and folded in the endoplasmic reticulum (ER), and continue their path to their site of residence along the secretory pathway. The COPII system has been identified as a key player for selecting and directing the fate of membrane and secretory cargo proteins. Selection of cargo proteins within the COPII vesicles is achieved by cargo receptors. The cornichon cargo receptor belongs to a conserved protein family found in eukaryotes that has been demonstrated to participate in the selection of integral membrane proteins as cargo for their correct targeting. Here it is demonstrated at the cellular level that rice cornichon OsCNIH1 interacts with OsHKT1;3 and, in yeast cells, enables the expression of the sodium transporter to the Golgi apparatus. Physical and functional HKT–cornichon interactions are confirmed by the mating-based split ubiquitin system, bimolecular fluorescence complementation, and Xenopus oocyte and yeast expression systems. The interaction between the two proteins occurs in the ER of plant cells and their co-expression in oocytes leads to the sequestration of the transporter in the ER. In the yeast cornichon mutant erv14, OsHKT1;3 is mistargeted, preventing the toxic effects of sodium transport in the cell observed in wild-type cells or in the erv14 mutant that co-expressed OsHKT1;3 with either OsCNIH1 or Erv14p. Identification and characterization of rice cornichon as a possible cargo receptor opens up the opportunity to improve our knowledge on membrane protein targeting in plant cells. PMID:25750424

  15. 40 CFR 73.33 - Authorized account representative.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Authorized account representative. 73.33 Section 73.33 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) SULFUR DIOXIDE ALLOWANCE SYSTEM Allowance Tracking System § 73.33 Authorized account...

  16. Exploring M33 Through RR Lyrae Stars

    OpenAIRE

    Pritzl, Barton J.

    2013-01-01

    Recent surveys have detected RR Lyrae stars in M33, the Triangulum Galaxy. These variable stars are excellent tracers of ancient stellar populations. The RR Lyrae stars have been used to estimate metallicities at various locations within M33, as well as determining the distance to the galaxy. A summary of the M33 RR Lyrae stars is presented here as well as an analysis on what their properties imply for the unique M33 galaxy

  17. 36 CFR 1207.33 - Supplies.

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Supplies. 1207.33 Section 1207.33 Parks, Forests, and Public Property NATIONAL ARCHIVES AND RECORDS ADMINISTRATION GENERAL RULES... aggregate fair market value upon termination or completion of the award, and if the supplies are not needed...

  18. 22 CFR 143.33 - Mediation.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Mediation. 143.33 Section 143.33 Foreign... Mediation. (a) Referral of complaints for mediation. The agency will refer to the Federal Mediation and... participate in the mediation process to the extent necessary to reach an agreement or make an informed...

  19. 49 CFR 18.33 - Supplies.

    Science.gov (United States)

    2010-10-01

    ... supplies exceeding $5,000 in total aggregate fair market value upon termination or completion of the award... 49 Transportation 1 2010-10-01 2010-10-01 false Supplies. 18.33 Section 18.33 Transportation... Supplies. (a) Title. Title to supplies acquired under a grant or subgrant will vest, upon acquisition, in...

  20. Increased copper toxicity in Saccharomyces cerevisiae lacking VPS35, a component of the retromer and monogenic Parkinson disease gene in humans.

    Science.gov (United States)

    Sowada, Nadine; Stiller, Barbara; Kubisch, Christian

    2016-08-05

    The Saccharomyces cerevisiae gene VPS35 encodes a component of the retromer complex which is involved in vesicle transport from endosomes to the trans-Golgi network. Yeast and human VPS35 orthologs are highly conserved and mutations in human VPS35 cause an autosomal dominant form of late-onset Parkinson disease (PD). We now show that deletion of VPS35 in yeast (vps35Δ) leads to a dose-dependent growth defect towards copper. This increased sensitivity could be rescued by transformation with yeast wild-type VPS35 but not by the expression of a construct harboring the yeast equivalent (i.e. D686N) of the most commonly identified VPS35-associated PD mutation, p.D620N. In addition, we show that expression of one copy of α-synuclein, which is known to directly interact with copper, leads to a pronounced aggravation of copper toxicity in vps35Δ cells, thereby linking the regulation of copper homeostasis by Vps35p in yeast to one of the key molecules in PD pathophysiology. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Upregulation of Interleukin-33 in obstructive renal injury

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Wei-Yu, E-mail: wychen624@cgmh.org.tw [Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan (China); Chang, Ya-Jen [Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (China); Su, Chia-Hao [Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan (China); Tsai, Tzu-Hsien [Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan (China); Chen, Shang-Der [Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan (China); Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Kaohsiung, Taiwan (China); Hsing, Chung-Hsi [Department of Anesthesiology, Chi-Mei Medical Center, Tainan, Taiwan (China); Yang, Jenq-Lin, E-mail: jyang@adm.cgmh.org.tw [Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan (China)

    2016-05-13

    Interstitial fibrosis and loss of parenchymal tubular cells are the common outcomes of progressive renal diseases. Pro-inflammatory cytokines have been known contributing to the damage of tubular cells and fibrosis responses after renal injury. Interleukin (IL)-33 is a tissue-derived nucleus alarmin that drives inflammatory responses. The regulation and function of IL-33 in renal injury, however, is not well understood. To investigate the involvement of cytokines in the pathogenesis of renal injury and fibrosis, we performed the mouse renal injury model induced by unilateral urinary obstruction (UUO) and analyze the differentially upregulated genes between the obstructed and the contralateral unobstructed kidneys using RNA sequencing (RNAseq). Our RNAseq data identified IL33 and its receptor ST2 were upregulated in the UUO kidney. Quantitative analysis confirmed that transcripts of IL33 and ST2 were upregulated in the obstructed kidneys. Immunofluorescent staining revealed that IL-33 was upregulated in Vimentin- and alpha-SMA-positive interstitial cells. By using genetically knockout mice, deletion of IL33 reduced UUO-induced renal fibrosis. Moreover, in combination with BrdU labeling technique, we observed that the numbers of proliferating tubular epithelial cells were increased in the UUO kidneys from IL33-or ST2-deficient mice compared to wild type mice. Collectively, our study demonstrated the upregulation of IL-33/ST2 signaling in the obstructed kidney may promote tubular cell injury and interstitial fibrosis. IL-33 may serve as a biomarker to detect renal injury and that IL-33/ST2 signaling may represent a novel target for treating renal diseases. -- Highlights: •Interleukin (IL)-33 was upregulated in obstructed kidneys. •Interstitial myofibroblasts expressed IL-33 after UUO-induced renal injury. •Deficiency of IL33 reduced interstitial fibrosis and promoted tubular cell proliferation.

  2. 38 CFR 10.33 - Determination of dependency.

    Science.gov (United States)

    2010-07-01

    ... dependency. 10.33 Section 10.33 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUSTED COMPENSATION Adjusted Compensation; General § 10.33 Determination of dependency. A determination of the existence of the alleged dependency will be made upon consideration of all facts relating to...

  3. 45 CFR 1183.33 - Supplies.

    Science.gov (United States)

    2010-10-01

    ... supplies exceeding $5,000 in total aggregate fair market value upon termination or completion of the award... 45 Public Welfare 3 2010-10-01 2010-10-01 false Supplies. 1183.33 Section 1183.33 Public Welfare... Supplies. (a) Title. Title to supplies acquired under a grant or subgrant will vest, upon acquisition, in...

  4. 38 CFR 43.33 - Supplies.

    Science.gov (United States)

    2010-07-01

    ...) Disposition. If there is a residual inventory of unused supplies exceeding $5,000 in total aggregate fair... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Supplies. 43.33 Section... Requirements Changes, Property, and Subawards § 43.33 Supplies. (a) Title. Title to supplies acquired under a...

  5. 20 CFR 437.33 - Supplies.

    Science.gov (United States)

    2010-04-01

    ... supplies exceeding $5,000 in total aggregate fair market value upon termination or completion of the award... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Supplies. 437.33 Section 437.33 Employees... Supplies. (a) Title. Title to supplies acquired under a grant or subgrant will vest, upon acquisition, in...

  6. 22 CFR 135.33 - Supplies.

    Science.gov (United States)

    2010-04-01

    ... supplies exceeding $5,000 in total aggregate fair market value upon termination or completion of the award... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Supplies. 135.33 Section 135.33 Foreign... Supplies. (a) Title. Title to supplies acquired under a grant or subgrant will vest, upon acquisition, in...

  7. 28 CFR 66.33 - Supplies.

    Science.gov (United States)

    2010-07-01

    ... is a residual inventory of unused supplies exceeding $5,000 in total aggregate fair market value upon... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Supplies. 66.33 Section 66.33 Judicial... Supplies. (a) The Omnibus Crime Control and Safe Streets Act of 1968, as amended, Public Law 90-351...

  8. Disruption of the ECM33 Gene in Candida albicans Prevents Biofilm Formation, Engineered Human Oral Mucosa Tissue Damage and Gingival Cell Necrosis/Apoptosis

    Directory of Open Access Journals (Sweden)

    Mahmoud Rouabhia

    2012-01-01

    Full Text Available In this study we demonstrated that ΔCaecm33 double mutant showed reduced biofilm formation and causes less damage to gingival mucosa tissues. This was confirmed by the reduced level of necrotic cells and Bax/Bcl2 gene expression as apoptotic markers. In contrast, parental and Caecm33 mutant strains decreased basement membrane protein production (laminin 5 and type IV collagen. We thus propose that ECM33 gene/protein represents a novel target for the prevention and treatment of infections caused by Candida.

  9. New IPSAS 33-38

    OpenAIRE

    Christiaens, Johan; Vandendriessche, Freya

    2015-01-01

    The evolution of the development of the IPSAS has already been discussed a few times in TAA (2006 and 2010). This article analyzes and summarizes the recent IPSAS standards 33-38. Especially IPSAS 33 is an important standard as it discusses the first time adoption of IPSAS accrual basis. Furthermore, the new IPSAS 34-38 replace IPSAS 6, 7 and 8, and mainly deal with consolidation, investments and joint arrangements. The IPSAS are still in evolution, especially considering the potential develo...

  10. 7 CFR 1700.33 - Financial Services Staff.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 11 2010-01-01 2010-01-01 false Financial Services Staff. 1700.33 Section 1700.33... AGRICULTURE GENERAL INFORMATION Agency Organization and Functions § 1700.33 Financial Services Staff. The Financial Services Staff evaluates the financial condition of financially troubled borrowers in order to...

  11. 19 CFR 181.33 - Customs processing procedures.

    Science.gov (United States)

    2010-04-01

    ... 19 Customs Duties 2 2010-04-01 2010-04-01 false Customs processing procedures. 181.33 Section 181.33 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF....33 Customs processing procedures. (a) Status determination. After receipt of a post-importation claim...

  12. 21 CFR 1403.33 - Supplies.

    Science.gov (United States)

    2010-04-01

    ... supplies exceeding $5,000 in total aggregate fair market value upon termination or completion of the award... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Supplies. 1403.33 Section 1403.33 Food and Drugs... Supplies. (a) Title. Title to supplies acquired under a grant or subgrant will vest, upon acquisition, in...

  13. 34 CFR 80.33 - Supplies.

    Science.gov (United States)

    2010-07-01

    ... supplies exceeding $5,000 in total aggregate fair market value upon termination or completion of the award... 34 Education 1 2010-07-01 2010-07-01 false Supplies. 80.33 Section 80.33 Education Office of the... Supplies. (a) Title. Title to supplies acquired under a grant or subgrant will vest, upon acquisition, in...

  14. Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells.

    Science.gov (United States)

    Akita, Kae; Kobayashi, Megumi; Sato, Mayuko; Kutsuna, Natsumaro; Ueda, Takashi; Toyooka, Kiminori; Nagata, Noriko; Hasezawa, Seiichiro; Higaki, Takumi

    2017-01-01

    In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.

  15. 7 CFR 33.50 - Apples for processing.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Apples for processing. 33.50 Section 33.50 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Interpretive Rules § 33.50 Apples for processing. The...

  16. 29 CFR 34.33 - Methods of Administration.

    Science.gov (United States)

    2010-07-01

    ... OF THE JOB TRAINING PARTNERSHIP ACT OF 1982, AS AMENDED (JTPA) Governor's Responsibilities To Implement the Nondiscrimination and Equal Opportunity Requirements of JTPA § 34.33 Methods of Administration... 29 Labor 1 2010-07-01 2010-07-01 true Methods of Administration. 34.33 Section 34.33 Labor Office...

  17. 49 CFR 178.33a-6 - Manufacture.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Manufacture. 178.33a-6 Section 178.33a-6 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Inside Containers, and Linings § 178.33a-6 Manufacture. (a) By appliances and methods that...

  18. 49 CFR 178.33b-6 - Manufacture.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Manufacture. 178.33b-6 Section 178.33b-6 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Inside Containers, and Linings § 178.33b-6 Manufacture. (a) Each container must be...

  19. Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion

    NARCIS (Netherlands)

    Roelofsen, H; Wolters, H; Van Luyn, MJA; Miura, N; Kuipers, F; Vonk, RJ

    Background & Aims: Mutations in the ATP7B gene, encoding a copper-transporting P-type adenosine triphosphatase, lead to excessive hepatic copper accumulation because of impaired biliary copper excretion in Wilson's disease. In human liver, ATP7B is predominantly localized to the trans-Golgi network,

  20. 34 CFR 33.41 - Stay pending appeal.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Stay pending appeal. 33.41 Section 33.41 Education Office of the Secretary, Department of Education PROGRAM FRAUD CIVIL REMEDIES ACT § 33.41 Stay pending appeal. (a) An initial decision is stayed automatically pending disposition of a motion for...

  1. Autophagy as an ultrastructural marker of heavy metal toxicity in human cord blood hematopoietic stem cells

    International Nuclear Information System (INIS)

    Di Gioacchino, Mario; Petrarca, Claudia; Perrone, Angela; Farina, Massimo; Sabbioni, Enrico; Hartung, Thomas; Martino, Simone; Esposito, Diana L.; Lotti, Lavinia Vittoria; Mariani-Costantini, Renato

    2008-01-01

    Stem cells are a key target of environmental toxicants, but little is known about their toxicological responses. We aimed at developing an in-vitro model based on adult human stem cells to identify biomarkers of heavy metal exposure. To this end we investigated the responses of human CD34+ hematopoietic progenitor cells to hexavalent chromium (Cr[VI]) and cadmium (Cd). Parallel cultures of CD34+ cells isolated from umbilical cord blood were exposed for 48 h to 0.1 μM and 10 μM Cr(VI) or Cd. Cultures treated with 10 μM Cr(VI) or Cd showed marked cell loss. Ultrastructural analysis of surviving cells revealed prominent autophagosomes/autophagolysosomes, which is diagnostic of autophagy, associated with mitochondrial damage and replication, dilatation of the rough endoplasmic reticulum and Golgi complex, cytoplasmic lipid droplets and chromatin condensation. Treated cells did not show the morphologic hallmarks of apoptosis. Treatment with 0.1 μM Cr(VI) or Cd did not result in cell loss, but at the ultrastructural level cells showed dilated endoplasmic reticulum and evidence of mitochondrial damage. We conclude that autophagy is implicated in the response of human hematopoietic stem cells to toxic concentrations of Cr(VI) and Cd. Autophagy, which mediates cell survival and death under stress, deserves further evaluation to be established as biomarker of metal exposure

  2. Autophagy as an ultrastructural marker of heavy metal toxicity in human cord blood hematopoietic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Di Gioacchino, Mario [Aging Research Center, ' G. d' Annunzio' University Foundation, Via Colle dell' Ara, 66100 Chieti (Italy); Medicine and Science of Ageing University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti (Italy)], E-mail: m.digioacchino@unich.it; Petrarca, Claudia; Perrone, Angela [Aging Research Center, ' G. d' Annunzio' University Foundation, Via Colle dell' Ara, 66100 Chieti (Italy); Medicine and Science of Ageing University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti (Italy); Farina, Massimo; Sabbioni, Enrico; Hartung, Thomas [Oncology and Neurosciences University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti (Italy); Martino, Simone [Department of Experimental Medicine, University La Sapienza, Viale Regina Elena 324, 00161 Rome (Italy); Esposito, Diana L. [Aging Research Center, ' G. d' Annunzio' University Foundation, Via Colle dell' Ara, 66100 Chieti (Italy); Oncology and Neurosciences University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti (Italy); Lotti, Lavinia Vittoria [Department of Experimental Medicine, University La Sapienza, Viale Regina Elena 324, 00161 Rome (Italy); Mariani-Costantini, Renato [Aging Research Center, ' G. d' Annunzio' University Foundation, Via Colle dell' Ara, 66100 Chieti (Italy); Oncology and Neurosciences University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti (Italy)

    2008-03-15

    Stem cells are a key target of environmental toxicants, but little is known about their toxicological responses. We aimed at developing an in-vitro model based on adult human stem cells to identify biomarkers of heavy metal exposure. To this end we investigated the responses of human CD34+ hematopoietic progenitor cells to hexavalent chromium (Cr[VI]) and cadmium (Cd). Parallel cultures of CD34+ cells isolated from umbilical cord blood were exposed for 48 h to 0.1 {mu}M and 10 {mu}M Cr(VI) or Cd. Cultures treated with 10 {mu}M Cr(VI) or Cd showed marked cell loss. Ultrastructural analysis of surviving cells revealed prominent autophagosomes/autophagolysosomes, which is diagnostic of autophagy, associated with mitochondrial damage and replication, dilatation of the rough endoplasmic reticulum and Golgi complex, cytoplasmic lipid droplets and chromatin condensation. Treated cells did not show the morphologic hallmarks of apoptosis. Treatment with 0.1 {mu}M Cr(VI) or Cd did not result in cell loss, but at the ultrastructural level cells showed dilated endoplasmic reticulum and evidence of mitochondrial damage. We conclude that autophagy is implicated in the response of human hematopoietic stem cells to toxic concentrations of Cr(VI) and Cd. Autophagy, which mediates cell survival and death under stress, deserves further evaluation to be established as biomarker of metal exposure.

  3. 40 CFR 503.33 - Vector attraction reduction.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 29 2010-07-01 2010-07-01 false Vector attraction reduction. 503.33... STANDARDS FOR THE USE OR DISPOSAL OF SEWAGE SLUDGE Pathogens and Vector Attraction Reduction § 503.33 Vector attraction reduction. (a)(1) One of the vector attraction reduction requirements in § 503.33 (b)(1) through...

  4. 23 CFR 1.33 - Conflicts of interest.

    Science.gov (United States)

    2010-04-01

    ... 23 Highways 1 2010-04-01 2010-04-01 false Conflicts of interest. 1.33 Section 1.33 Highways... § 1.33 Conflicts of interest. No official or employee of a State or any other governmental... project shall have, directly or indirectly, any financial or other personal interest in any such contract...

  5. 9 CFR 113.33 - Mouse safety tests.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Mouse safety tests. 113.33 Section 113.33 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE... Procedures § 113.33 Mouse safety tests. One of the mouse safety tests provided in this section shall be...

  6. An integrative in silico approach to the structure of Omp33-36 in Acinetobacter baumannii.

    Science.gov (United States)

    Jahangiri, Abolfazl; Rasooli, Iraj; Owlia, Parviz; Fooladi, Abbas Ali Imani; Salimian, Jafar

    2018-02-01

    Omp33-36 in A. baumannii, a bacterium causing serious nosocomial infections, is a virulence factor associated with the pathogen metabolic fitness as well as its adherence and invasion to human epithelial cells. This protein is also involved in interaction of the bacteria with host cells by binding to fibronectin. Moreover, Omp33-36 renders cytotoxicity to A. baumannii in addition to inducing apoptosis and modulation of autophagy. In the present study, an integrated strategy is launched to pierce into the 3D structure of Omp33-36 protein. The signal peptide within the sequence was determined, then, topology as well as secondary and tertiary structures of the protein were predicted. The mature protein assigned as a 14-stranded barrel in which residues 1-19 is removed as signal peptide. The obtained 3D models were evaluated in terms of quality; and then, served as queries to find similar protein structures. The hits were analyzed regarding topology among which 14-stranded were considered. The most qualified model was refined and then its sequence aligned to its counterpart similar structure protein (CymA from Klebsiella oxytoca). The determined structure of Omp33-36 could justify its porin function and carbapenem-resistance associated with the loss of this protein. Copyright © 2018. Published by Elsevier Ltd.

  7. 25 CFR 700.33 - Act (The Act).

    Science.gov (United States)

    2010-04-01

    ... 25 Indians 2 2010-04-01 2010-04-01 false Act (The Act). 700.33 Section 700.33 Indians THE OFFICE OF NAVAJO AND HOPI INDIAN RELOCATION COMMISSION OPERATIONS AND RELOCATION PROCEDURES General Policies and Instructions Definitions § 700.33 Act (The Act). (a) The Act. The Act is Pub. L. 93-531, (88 Stat...

  8. Map-based cloning and characterization of Zea mays male sterility33 (ZmMs33) gene, encoding a glycerol-3-phosphate acyltransferase.

    Science.gov (United States)

    Xie, Ke; Wu, Suowei; Li, Ziwen; Zhou, Yan; Zhang, Danfeng; Dong, Zhenying; An, Xueli; Zhu, Taotao; Zhang, Simiao; Liu, Shuangshuang; Li, Jinping; Wan, Xiangyuan

    2018-06-01

    Map-based cloning of maize ms33 gene showed that ZmMs33 encodes a sn-2 glycerol-3-phosphate acyltransferase, the ortholog of rice OsGPAT3, and it is essential for male fertility in maize. Genetic male sterility has been widely studied for its biological significance and commercial value in hybrid seed production. Although many male-sterile mutants have been identified in maize (Zea mays L.), it is likely that most genes that cause male sterility are unknown. Here, we report a recessive genetic male-sterile mutant, male sterility33 (ms33), which displays small, pale yellow anthers, and complete male sterility. Using a map-based cloning approach, maize GRMZM2G070304 was identified as the ms33 gene (ZmMs33). ZmMs33 encodes a novel sn-2 glycerol-3-phosphate acyltransferase (GPAT) in maize. A functional complementation experiment showed that GRMZM2G070304 can rescue the male-sterile phenotype of the ms33-6029 mutant. GRMZM2G070304 was further confirmed to be the ms33 gene via targeted knockouts induced by the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system. ZmMs33 is preferentially expressed in the immature anther from the quartet to early-vacuolate microspore stages and in root tissues at the fifth leaf growth stage. Phylogenetic analysis indicated that ZmMs33 and OsGPAT3 are evolutionarily conserved for anther and pollen development in monocot species. This study reveals that the monocot-specific GPAT3 protein plays an important role in male fertility in maize, and ZmMs33 and mutants in this gene may have value in maize male-sterile line breeding and hybrid seed production.

  9. B. abortus RNA is the component involved in the down-modulation of MHC-I expression on human monocytes via TLR8 and the EGFR pathway

    Science.gov (United States)

    Milillo, M. Ayelén; Velásquez, Lis N.; Trotta, Aldana; Delpino, M. Victoria; Balboa, Luciana; Vermeulen, Mónica; Espindola, Sonia L.; Rodriguez-Rodrigues, Nahuel; Fernández, Gabriela C.; Oliveira, Sergio Costa; Giambartolomei, Guillermo H.

    2017-01-01

    Despite eliciting a potent CD8+ T cell response, Brucella abortus is able to persist and establish a chronic infection inside its host. We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR) pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP). Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and avoid the

  10. B. abortus RNA is the component involved in the down-modulation of MHC-I expression on human monocytes via TLR8 and the EGFR pathway.

    Directory of Open Access Journals (Sweden)

    M Ayelén Milillo

    2017-08-01

    Full Text Available Despite eliciting a potent CD8+ T cell response, Brucella abortus is able to persist and establish a chronic infection inside its host. We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP. Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and

  11. 32 CFR 33.44 - Termination for convenience.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 1 2010-07-01 2010-07-01 false Termination for convenience. 33.44 Section 33.44 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE DoD GRANT AND AGREEMENT... convenience. Except as provided in § 33.43 awards may be terminated in whole or in part only as follows: (a...

  12. Glycoprotein A33 deficiency: a new mouse model of impaired intestinal epithelial barrier function and inflammatory disease

    Directory of Open Access Journals (Sweden)

    Benjamin B. Williams

    2015-08-01

    Full Text Available The cells of the intestinal epithelium provide a selectively permeable barrier between the external environment and internal tissues. The integrity of this barrier is maintained by tight junctions, specialised cell-cell contacts that permit the absorption of water and nutrients while excluding microbes, toxins and dietary antigens. Impairment of intestinal barrier function contributes to multiple gastrointestinal disorders, including food hypersensitivity, inflammatory bowel disease (IBD and colitis-associated cancer (CAC. Glycoprotein A33 (GPA33 is an intestinal epithelium-specific cell surface marker and member of the CTX group of transmembrane proteins. Roles in cell-cell adhesion have been demonstrated for multiple CTX family members, suggesting a similar function for GPA33 within the gastrointestinal tract. To test a potential requirement for GPA33 in intestinal barrier function, we generated Gpa33−/− mice and subjected them to experimental regimens designed to produce food hypersensitivity, colitis and CAC. Gpa33−/− mice exhibited impaired intestinal barrier function. This was shown by elevated steady-state immunosurveillance in the colonic mucosa and leakiness to oral TRITC-labelled dextran after short-term exposure to dextran sodium sulphate (DSS to injure the intestinal epithelium. Gpa33−/− mice also exhibited rapid onset and reduced resolution of DSS-induced colitis, and a striking increase in the number of colitis-associated tumours produced by treatment with the colon-specific mutagen azoxymethane (AOM followed by two cycles of DSS. In contrast, Gpa33−/− mice treated with AOM alone showed no increase in sporadic tumour formation, indicating that their increased tumour susceptibility is dependent on inflammatory stimuli. Finally, Gpa33−/− mice displayed hypersensitivity to food allergens, a common co-morbidity in humans with IBD. We propose that Gpa33−/− mice provide a valuable model to study the mechanisms

  13. Candidate glutamatergic neurons in the visual system of Drosophila.

    Directory of Open Access Journals (Sweden)

    Shamprasad Varija Raghu

    Full Text Available The visual system of Drosophila contains approximately 60,000 neurons that are organized in parallel, retinotopically arranged columns. A large number of these neurons have been characterized in great anatomical detail. However, studies providing direct evidence for synaptic signaling and the neurotransmitter used by individual neurons are relatively sparse. Here we present a first layout of neurons in the Drosophila visual system that likely release glutamate as their major neurotransmitter. We identified 33 different types of neurons of the lamina, medulla, lobula and lobula plate. Based on the previous Golgi-staining analysis, the identified neurons are further classified into 16 major subgroups representing lamina monopolar (L, transmedullary (Tm, transmedullary Y (TmY, Y, medulla intrinsic (Mi, Mt, Pm, Dm, Mi Am, bushy T (T, translobula plate (Tlp, lobula intrinsic (Lcn, Lt, Li, lobula plate tangential (LPTCs and lobula plate intrinsic (LPi cell types. In addition, we found 11 cell types that were not described by the previous Golgi analysis. This classification of candidate glutamatergic neurons fosters the future neurogenetic dissection of information processing in circuits of the fly visual system.

  14. Gene : CBRC-CFAM-33-0017 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available inernema carpocapsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carp...CBRC-CFAM-33-0017 Novel 33 C UNKNOWN NU1M_ANOGA 0.003 25% ref|YP_026090.1| NADH dehydrogenase subunit 5 [Ste

  15. TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

    Science.gov (United States)

    Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M; Koenig, Sabine; Eimer, Stefan

    2012-01-01

    Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

  16. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling.

    Science.gov (United States)

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2015-01-15

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation. Copyright © 2015 by The American Association of Immunologists, Inc.

  17. The phase transition of the incommensurate phases β-Ln(PO3)3(Ln=Y,Tb…Yb), crystal structures of α-Ln(PO3)3(Ln=Y,Tb…Yb) and Sc(PO3)3

    Science.gov (United States)

    Höppe, Hennig A.

    2009-07-01

    The incommensurately modulated room-temperature phases β-Ln(PO3)3(Ln=Y,Tb…Yb) undergo a topotactic phase transition monitored by vibrational spectroscopy below 180 K leading to α-Ln(PO3)3(Ln=Y,Dy…Yb), above 200 K the incommensurate phases are reobtained. The low-temperature phases exhibit a new structure type (α-Dy(PO3)3, P21/c, Z=12,a=14.1422(6), b=20.0793(9),c=10.1018(4) A˚, β=127.532(3)∘). α-Tb(PO3)3 is isotypic with Gd(PO3)3(α-Tb(PO3)3, I2/a,Z=16,a=25.875(6), b=13.460(3), c=10.044(2) A˚, β=119.13(3)∘). The symmetry relations between the involved phases of the phase transition are discussed. The crystal structure of Sc(PO3)3 is isotypic with that of Lu(PO3)3 and C-type phosphates. The polyphosphates consist of infinite zig-zag chains of corner-sharing PO4 tetrahedra, the cations are coordinated sixfold in an almost octahedral arrangement. To confirm the quality of the determined crystal structures the deviation of the phosphate tetrahedra from ideal symmetry was determined and discussed.

  18. Human renin biosynthesis and secretion in normal and ischemic kidneys

    International Nuclear Information System (INIS)

    Pratt, R.E.; Carleton, J.E.; Richie, J.P.; Heusser, C.; Dzau, V.J.

    1987-01-01

    The pathway of renin biosynthesis and secretion in normal and ischemic human kidneys has been investigated by pulse-labeling experiments. The results indicate that in normal human kidney, preprorenin is rapidly processed to 47-kDa prorenin. Microradiosequencing showed that this molecule was generated by cleavage between Gly-23 and Leu-24, yielding a 43-amino acid proregion. Analysis of prorenin secreted by the kidney tissue yielded an identical sequence, indicating that prorenin is secreted without any further proteolysis. An examination of the kinetics of processing and secretion suggested that a majority of the newly synthesized prorenin is quickly secreted, while only a small fraction is processed intracellularly to the mature renin. The differences in secretion kinetics between prorenin and mature renin and the selective inhibition of prorenin secretion by monensin suggest that they are secreted independently via two pathways: a constitutive pathway probably from the Golgi or protogranules that rapidly release prorenin and a regulated pathway that secretes mature renin from the mature granules. A comparison of the kinetics of processing between normal and ischemic tissues suggests that renal ischemia leads to an overall increase in the rate of processing or prorenin to mature renin. In addition, prolonged biosynthetic labeling of renin in the ischemic kidney yielded two smaller molecular weight immunoreactive forms suggestive of renin fragments that may be degradative products. These fragments were not detected in normal kidney tissue labeled for similar lengths of time

  19. Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis.

    Science.gov (United States)

    DiGiuseppe, Stephen; Luszczek, Wioleta; Keiffer, Timothy R; Bienkowska-Haba, Malgorzata; Guion, Lucile G M; Sapp, Martin J

    2016-05-31

    During the entry process, the human papillomavirus (HPV) capsid is trafficked to the trans-Golgi network (TGN), whereupon it enters the nucleus during mitosis. We previously demonstrated that the minor capsid protein L2 assumes a transmembranous conformation in the TGN. Here we provide evidence that the incoming viral genome dissociates from the TGN and associates with microtubules after the onset of mitosis. Deposition onto mitotic chromosomes is L2-mediated. Using differential staining of an incoming viral genome by small molecular dyes in selectively permeabilized cells, nuclease protection, and flotation assays, we found that HPV resides in a membrane-bound vesicle until mitosis is completed and the nuclear envelope has reformed. As a result, expression of the incoming viral genome is delayed. Taken together, these data provide evidence that HPV has evolved a unique strategy for delivering the viral genome to the nucleus of dividing cells. Furthermore, it is unlikely that nuclear vesicles are unique to HPV, and thus we may have uncovered a hitherto unrecognized cellular pathway that may be of interest for future cell biological studies.

  20. Yeast Interacting Proteins Database: YHL002W, YNR006W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ycling of Golgi proteins and formation of lumenal membranes Rows with this bait as bait (1) Rows with this b...required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins destined...on, as well as for recycling of Golgi proteins and formation of lumenal membranes...ith Hse1p; required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated protei