WorldWideScience

Sample records for rapid golgi study

  1. Methods to study signaling at the Golgi apparatus.

    Science.gov (United States)

    Reitere, Veronika; Baschieri, Francesco; Millarte, Valentina; Farhan, Hesso

    2013-01-01

    Research on the secretory pathway in the past three decades accounts for our known knowledge about the composition and architecture of organelles and about the machinery that regulates membrane transport. An emerging topic in the past few years was the discovery that the secretory pathway is regulated by signaling, and in this regard, the Golgi apparatus received major attention. In the current chapter, we will highlight various techniques that are used by us and others to study signaling at the Golgi. We describe methods to study lipid and protein phosphorylation at the Golgi and various techniques for studying spatial activation of GTPases at this organelle. We also discuss how combining these techniques and improving their limitations is important for gaining a better understanding of how the Golgi intersects with various signal transduction pathways. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Study of GOLPH3: a potential stress-inducible protein from Golgi apparatus.

    Science.gov (United States)

    Li, Ting; You, Hong; Zhang, Jie; Mo, Xiaoye; He, Wenfang; Chen, Yang; Tang, Xiangqi; Jiang, Zheng; Tu, Ranran; Zeng, Liuwang; Lu, Wei; Hu, Zhiping

    2014-06-01

    Although the Golgi apparatus has been studied extensively for over 100 years, the complex structure-function relationships have yet to be elucidated. It is well known that the Golgi complex plays an important role in the transport, processing, sorting, and targeting of numerous proteins and lipids destined for secretion, plasma membrane, and lysosomes. Increasing evidence suggests that the Golgi apparatus is a sensor and common downstream effector of stress signals in cell death pathways. It undergoes disassembly and fragmentation in several neurological disorders. Recent studies indicate that Golgi phosphoprotein 3 (GOLPH3 also known as GPP34/GMx33/MIDAS), a peripheral membrane protein of trans-Golgi network, represents an exciting new class of oncoproteins involved in cell signal transduction and is potentially mobilized by stress. In this review, we focus on the importance of GOLPH3 in vesicular trafficking, Golgi architecture maintenance, receptor sorting, protein glycosylation, and further discuss its potential in signal sensing in stress response.

  3. 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

  4. The acoustic cortex in frontotemporal dementia: a Golgi and electron microscope study.

    Science.gov (United States)

    Baloyannis, Stavros J; Mauroudis, Ioannis; Manolides, Spyros L; Manolides, Leonidas S

    2011-04-01

    The neuronal loss and the alteration of the synapses in the acoustic cortex in frontotemporal dementia (FTD) may be related to the impairment of communication and symbolic sound perception, which is noticed in the majority of the cases. FTD is a heterogeneous neurodegenerative disorder, causing progressive decline of intellectual faculties, impairment of behavior and social performance, and impairment of speech eloquence, associated with various neurological manifestations based on a variable neuropathological background. We attempted to determine the morphological alterations of the dendrites and the dendritic spines in the acoustic cortex of 10 cases who fulfilled the diagnostic criteria for FTD. For the histological study we applied (a) routine neuropathological techniques and (b) rapid Golgi method. We proceeded to electron microscopy for the ultrastructural study of the synapses and the morphological and morphometric study of the organelles, the dendrites, and the dendritic spines. The morphological and morphometric analysis revealed substantial neuronal loss and synaptic alterations in the acoustic cortex in all the cases of FTD and particularly in Pick disease and in primary progressive aphasia. Mitochondria alterations and changes of the Golgi apparatus were seen mostly in Pick disease.

  5. The yeast Golgi apparatus.

    Science.gov (United States)

    Suda, Yasuyuki; Nakano, Akihiko

    2012-04-01

    The Golgi apparatus is an organelle that has been extensively studied in the model eukaryote, yeast. Its morphology varies among yeast species; the Golgi exists as a system of dispersed cisternae in the case of the budding yeast Saccharomyces cerevisiae, whereas the Golgi cisternae in Pichia pastoris and Schizosaccharomyces pombe are organized into stacks. In spite of the different organization, the mechanism of trafficking through the Golgi apparatus is believed to be similar, involving cisternal maturation, in which the resident Golgi proteins are transported backwards while secretory cargo proteins can stay in the cisternae. Questions remain regarding the organization of the yeast Golgi, the regulatory mechanisms that underlie cisternal maturation of the Golgi and transport machinery of cargo proteins through this organelle. Studies using different yeast species have provided hints to these mechanisms. © 2011 John Wiley & Sons A/S.

  6. The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid

    Science.gov (United States)

    2014-01-01

    Background Alzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid peptide (Aβ). Aβ is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate Aβ production, although the exact organelle in which Aβ is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating Aβ production, but these pathways are relatively under-studied. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing. Results We have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein (paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the Golgi to downstream compartments identified with compartment markers tagged with red fluorescent protein (mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because γ-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of fluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that APP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the highly selective γ-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is being cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal APP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3 (AP-3; a heterotetrameric protein complex required for trafficking many proteins to

  7. Golgi apparatus dis- and reorganizations studied with the aid of 2-deoxy-D-glucose and visualized by 3D-electron tomography.

    Science.gov (United States)

    Ranftler, Carmen; Meisslitzer-Ruppitsch, Claudia; Neumüller, Josef; Ellinger, Adolf; Pavelka, Margit

    2017-04-01

    We studied Golgi apparatus disorganizations and reorganizations in human HepG2 hepatoblastoma cells by using the nonmetabolizable glucose analogue 2-deoxy-D-glucose (2DG) and analyzing the changes in Golgi stack architectures by 3D-electron tomography. Golgi stacks remodel in response to 2DG-treatment and are replaced by tubulo-glomerular Golgi bodies, from which mini-Golgi stacks emerge again after removal of 2DG. The Golgi stack changes correlate with the measured ATP-values. Our findings indicate that the classic Golgi stack architecture is impeded, while cells are under the influence of 2DG at constantly low ATP-levels, but the Golgi apparatus is maintained in forms of the Golgi bodies and Golgi stacks can be rebuilt as soon as 2DG is removed. The 3D-electron microscopic results highlight connecting regions that interlink membrane compartments in all phases of Golgi stack reorganizations and show that the compact Golgi bodies mainly consist of continuous intertwined tubules. Connections and continuities point to possible new transport pathways that could substitute for other modes of traffic. The changing architectures visualized in this work reflect Golgi stack dynamics that may be essential for basic cell physiologic and pathologic processes and help to learn, how cells respond to conditions of stress.

  8. Synaptic alterations in the medial geniculate bodies and the inferior colliculi in Alzheimer's disease: a Golgi and electron microscope study.

    Science.gov (United States)

    Baloyannis, Stavros J; Mauroudis, Ioannis; Manolides, Spyros L; Manolides, Leonidas S

    2009-04-01

    The neuronal loss and the alteration of the synapses in the medial geniculate bodies and the inferior colliculi may be involved in the impairment of communication and symbolic sound perception, which is noticed even in the early stages of Alzheimer's disease. Alzheimer's disease (AD) is a neurodegenerative disorder, causing a progressive decline of intellectual faculties, gradual impairment of behavior and social performance, impairment of communication and speech eloquence, and various neurological manifestations. We attempted to figure out the synaptic alterations in the medial geniculate bodies and the inferior colliculi in 12 early cases of Alzheimer's disease, who fulfilled the clinical, and laboratory diagnostic criteria of Alzheimer's disease. For the histological study we applied routine neuropathological techniques as well as Bodian staining and rapid Golgi method. We proceeded to electron microscopy for the ultrastructural study of synapses and dendritic spines. The morphological and morphometric analysis revealed substantial neuronal loss and synaptic alterations in the medial geniculate bodies as well as in inferior colliculi. Dendritic spines of the polyhedral and elongated cells of the medial geniculate bodies were decreased in number. Mitochondrial alterations and fragmentation of Golgi apparatus were seen in 15% of the neurons of the medial geniculate bodies and in 5% of the neurons of the inferior colliculi. Senile plaques and neurofibrillary tangles were not seen in either the medial geniculate bodies or the inferior colliculi.

  9. Protein-protein interactions in the plant Golgi apparatus, studied with FRET acceptor photobleaching technique

    DEFF Research Database (Denmark)

    Poulsen, Christian Peter

    The focus of this Ph.D. study has primarily been to utilize and adapt the acceptor photobleaching technique for measuring of Förster resonance energy transfer (FRET) to tudy proteinprotein interactions (PPIs) among glycosyltranseferases (GTs) and nucleotide ugar transporters (NSTs) localized...... of the actinomyosin based movement of Golgi vesicles, and was proved to be superior to commonly used fixatives such as the cross-linking agent paraformaldehyde which causes quenching of the fluorophores. According to FRET analysis, the results showed association between two galactosyltransferases, AtGALT29A and At...

  10. The study of the Golgi apparatus in blood--basic science and clinical applications.

    Science.gov (United States)

    Zeng, Liuwang; Hu, Zhiping; Lu, Wei; Tang, Xiangqi; Zhang, Jie; Li, Ting; Yang, Binbin

    2010-01-01

    The Golgi apparatus (GA) is a cytoplasmic organelle that is of great interest to all scientists for its key role in the biosynthesis, transporting and sorting of both lipids and proteins located at the intersection of the secretory and endocytic pathways. Recently, more and more evidence shows that changes in the Golgi apparatus play an important role in the clinical progression and pathological development of many diseases. In this review, we will summarize the alteration of the Golgi apparatus in blood cells and anti-Golgi complex antibodies in blood serum under different conditions and further clarify the contribution of the Golgi apparatus dysfunction to the course of these diseases and its pathophysiological basis, which will significantly improve our understanding and impact our ability to develop more effective therapies for these diseases.

  11. Camillo Golgi and the discovery of the Golgi apparatus.

    Science.gov (United States)

    Dröscher, A

    1998-01-01

    Camillo Golgi (1843-1926) was born at Corteno, near Brescia, in northern Italy. After graduating in Medicine at the ancient University of Pavia, the former seat of great scientists and naturalists, Golgi continued a long-standing Italian tradition by studying the histology of the nervous system. While working as a modest physician at Abbiategrasso, a small town near Pavia, he developed a silver-osmium technique, the "reazione nera" (black reaction), for which he was awarded the Nobel Prize in 1906. In the late 1890's, 25 years after the publication of his black reaction and while Professor of General Pathology in Pavia, Golgi noticed a fine internal network in only partially silver-osmium-blackened Purkinje cells. Following confirmation by his assistant Emilio Veratti, Golgi published the discovery, called the "apparato reticolare interno", in the Bollettino della Società medico-chirurgica di Pavia in 1898, which is now considered the birthday of the "Golgi apparatus". The discovery of the Golgi apparatus can be added to the long list of accidental discoveries. The man after whom it is named was not a cytologist engaged in studying the inner structure of the cell, but a pathologist searching to prove a neuroanatomical theory.

  12. Protein-protein interactions in the plant Golgi apparatus, studied with FRET acceptor photobleaching technique

    DEFF Research Database (Denmark)

    Poulsen, Christian Peter

    to the plant Golgi apparatus and involved mainly in arabinogalactan protein (AGP) biosynthesis. Co-expression analysis identified 4 GTs and 4 NSTs possibly involved in AGP biosynthesis. As part of the method development, the cytoskeleton-acting agent Cytochalasin D was tested as an inhibitor...... of the actinomyosin based movement of Golgi vesicles, and was proved to be superior to commonly used fixatives such as the cross-linking agent paraformaldehyde which causes quenching of the fluorophores. According to FRET analysis, the results showed association between two galactosyltransferases, AtGALT29A and At...

  13. The neuronal structure of the substantia nigra in the guinea pig: Nissl and Golgi study.

    Science.gov (United States)

    Bogus-Nowakowska, K; Szteyn, S; Robak, A

    2000-01-01

    The studies were carried out on the mesencephalos of adult guinea pigs. The preparations were made by means of the Golgi technique, as well as the Nissl and Klüver-Barrera methods. Four types of neurons were distinguished in the substantia nigra (SN) of the guinea pig: 1. Bipolar neurons of two kinds: the neurons of the first kind have elongated, fusiform perikarya (25-40 microns), whereas the cells of the second kind have rounded and oval perikarya (15-22 microns). These neurons possess two dendritic trunks which arise from the opposite poles of the cell body and run for a relatively long distance. The bipolar neurons are the most numerous in the pars compacta of SN. 2. Triangular neurons with three primary dendrites arising conically from a perikaryon (20-35 microns). They are the most often observed type of neurons in the pars reticulata of SN. 3. Multipolar neurons with quadrangular or oval perikarya (22-35 microns) and 4-5 dendritic trunks which spread out in all directions. 4. Pear-shaped neurons (perikarya 15-25 microns), which have one or two primary dendritic trunks arising from one pole of the cell body. In all the types of neurons an axon originates either from the dendritic trunk or from the soma and is observed only in its initial segment.

  14. The neuronal structure of the medial geniculate body in the pig--Nissi and Golgi study.

    Science.gov (United States)

    Bogus-Nowakowska, Krystyna; Szteyn, Stanisław; Robak, Anna

    2002-01-01

    The studies were carried out on the brains of adult pigs. The preparations were made by means of the Golgi technique as well as the Nissl and Klüver-Barrera methods. Four types of neurons were described in the medial geniculate body (MGB) of the pig: 1. Multipolar neurons (perikarya 30-45 microm) with rounded, oval or quadrangular perikarya from which arise 4-7 dendritic trunks. The dendrites divide dichotomically twice, may send out collaterals and give off ramifications. The dendritic branches possess varicosities and knob-like spines. These neurons predominate in MGB. 2. Pear-shaped neurons (20-35 microm) with one or two dendritic trunks arising from one pole of the cell body. These dendrites have a tufted appearance. 3. Triangular neurons (30-45 microm) possess three thick dendrites which first bifurcate near the soma and then divide profusely into daughter branches. 4. Fusiform neurons (30-50 microm) have usually two dendritic trunks which arise from the opposite poles of the cell body and divide dichotomically twice. The fusiform neurons are the least numerous in MGB. Most MGB neurons have on the secondary tertiary dendrites and on their ramifications have delicate varicose or bead-like appendages and spine-like protrusions. In all types of neurons an axon arises either from the soma or from the initial portion of the dendritic trunk.

  15. How Do Rab Proteins Determine Golgi Structure?

    Science.gov (United States)

    Liu, Shijie; Storrie, Brian

    2015-01-01

    Rab proteins, small GTPases, are key regulators of mammalian Golgi apparatus organization. Based on the effect of Rab activation state, Rab proteins fall into two functional classes. In Class1, inactivation induces Golgi ribbon fragmentation and/or redistribution of Golgi enzymes to the ER, while overexpression of wild type or activation has little, if any, effect on Golgi ribbon organization. In Class 2, the reverse is true. We give emphasis to Rab6, the most abundant Golgi-associated Rab protein. Rab6 depletion in HeLa cells causes an increase in Golgi cisternal number, longer, more continuous cisternae, and a pronounced accumulation of vesicles; the effect of Rab6 on Golgi ribbon organization is probably through regulation of vesicle transport. In effector studies, motor proteins and their regulators are found to be key Rab6 effectors. A related Rab, Rab41, affects Golgi ribbon organization in a contrasting manner. The balance between minus- and plus-end directed motor recruitment may well be the major Rab-dependent factor in Golgi ribbon organization. PMID:25708460

  16. A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus.

    Science.gov (United States)

    Lund, Christian H; Bromley, Jennifer R; Stenbæk, Anne; Rasmussen, Randi E; Scheller, Henrik V; Sakuragi, Yumiko

    2015-01-01

    A growing body of evidence suggests that protein-protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. Our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  17. The Golgi apparatus: insights from filamentous fungi.

    Science.gov (United States)

    Pantazopoulou, Areti

    2016-01-01

    Cargo passage through the Golgi, albeit an undoubtedly essential cellular function, is a mechanistically unresolved and much debated process. Although the main molecular players are conserved, diversification of the Golgi among different eukaryotic lineages is providing us with tools to resolve standing controversies. During the past decade the Golgi apparatus of model filamentous fungi, mainly Aspergillus nidulans, has been intensively studied. Here an overview of the most important findings in the field is provided. Golgi architecture and dynamics, as well as the novel cell biology tools that were developed in filamentous fungi in these studies, are addressed. An emphasis is placed on the central role the Golgi has as a crossroads in the endocytic and secretory-traffic pathways in hyphae. Finally the major advances that the A. nidulans Golgi biology has yielded so far regarding our understanding of key Golgi regulators, such as the Rab GTPases RabC(Rab6) and RabE(Rab11), the oligomeric transport protein particle, TRAPPII, and the Golgi guanine nucleotide exchange factors of Arf1, GeaA(GBF1/Gea1) and HypB(BIG/Sec7), are highlighted. © 2016 by The Mycological Society of America.

  18. Synchronous intra-Golgi transport induces the release of Ca{sup 2+} from the Golgi apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Micaroni, Massimo, E-mail: m.micaroni@imb.uq.edu.au [Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Italy); Perinetti, Giuseppe; Di Giandomenico, Daniele [Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Italy); Bianchi, Katiuscia [Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, 44100 Ferrara (Italy); Spaar, Alexander [Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Italy); Mironov, Alexander A., E-mail: mironov@negrisud.it [Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Italy)

    2010-08-01

    The mechanisms of secretory transport through the Golgi apparatus remain an issue of debate. The precise functional importance of calcium ions (Ca{sup 2+}) for intra-Golgi transport has also been poorly studied. Here, using different approaches to measure free Ca{sup 2+} concentrations in the cell cytosol ([Ca{sup 2+}]{sub cyt}) and inside the lumen of the Golgi apparatus ([Ca{sup 2+}]{sub GA}), we have revealed transient increases in [Ca{sup 2+}]{sub cyt} during the late phase of intra-Golgi transport that are concomitant with a decline in the maximal [Ca{sup 2+}]{sub GA} restoration ability. Thus, this redistribution of Ca{sup 2+} from the Golgi apparatus into the cytosol during the movement of cargo through the Golgi apparatus appears to have a role in intra-Golgi transport, and mainly in the late Ca{sup 2+}-dependent phase of SNARE-regulated fusion of Golgi compartments.

  19. The basilar pontine gray in the adult monkey (Macaca mulatta): a Golgi study.

    Science.gov (United States)

    Copper, M H; Fox, C A

    1976-07-01

    In Golgi preparations of the adult monkey (Macaca mulatta) two types of neurons are distinguished in the pontine gray: (1) larger neurons which impregnate most frequently and (2) smaller neurons which impregnate rarely. The former are judged to be projection neurons with myelinated axons because only the initial segments of their axons impregnate, while the latter are judged to be intrinsic neurons since they appear to participate only in the local circuitry of the pontine gray. The projection neurons show a variety of sizes and shapes and are the equivalent of the large, medium and small neurons that Ramón y Cajal ('09) illustrated in the pons of the 5-day-old infant. Their cell bodies are rounded, polygonal, triangular, egg-, pear-, and spindle-shaped. Some have somatic spines. Usually four to seven dendrites issue from the cell body and as they branch they attentuate. The dendrites have knobby, nodular protuberances which give them a gnarled appearance. Also the dendrites have a few scattered spines. In most instances the dendrites have a wavy recurring pattern. Neurons pressed against the corticospinal, corticopontine fiber bundles frequently have elongated cell bodies and the dendrites sprouting from them form, tight, brush-like arrays. The intrinsic neurons have small ellipssoid or pear-shaped cell bodies and two or three long dendrites, which do not taper. In some impregnations short axons issuing from the cell body were found and in other impregnations several widely separated, shor axon-like processes were found on dendrites. A striking feature of the intrinsic neurons is the presence of stalked dendritic appendages bearing one or more bulbous bodies, 1 to 3 microns in diameter. The intrinsic neurons in no way resemble the hairy or mossy cells with short axis cylinders that Ramón y Cajal (09) described in the pons of the 5-day-old infant. The latter were not found in the present material.

  20. The types of neurons of the somatic oculomotor nucleus in the European bison. Nissl and Golgi studies.

    Science.gov (United States)

    Szteyn, S; Robak, A; Równiak, M

    1997-01-01

    The neuronal structure of the somatic oculomotor nucleus (SON) was studied on the basis of Nissl and Golgi preparations, obtained from mesencephalons of 4 European bisons. We distinguished four types of neurons in the investigated nucleus: 1. The large multipolar nerve cells with 5-8 thick dendritic trunks and a thin axon which emerges directly from the soma. These are the most numerous neurons in the SON. 2. The small multipolar neurons. These cells have 4-6 thick dendritic trunks. An axon arises mostly from initial segment of one of the dendrites. This type represents about 8% neurons of SON. 3. The triangular neurons. From perikaryon 3 thick dendritic trunks emerge. A thin axon arises directly from the cell body. These cells make about 10% neurons of SON. 4. The pear-shaped cells which have 1 or 2 dendritic trunks concentrate at one pole of the neurons. In the SON there are about 2% pear-shaped cells. Their features correspond to the features attributed by many authors to the interneurons.

  1. 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...... and characterize components of these complex organelles by mass spectrometry in the model plant Arabidopsis thaliana. Collectively, these studies have increased the number of Golgi and vesicular localized proteins identified by mass spectrometry to nearly 500 proteins. We have sought to provide a brief overview...

  2. Rab30 is required for the morphological integrity of the Golgi apparatus.

    Science.gov (United States)

    Kelly, Eoin E; Giordano, Francesca; Horgan, Conor P; Jollivet, Florence; Raposo, Graça; McCaffrey, Mary W

    2012-02-01

    Rab GTPases are key coordinators of eukaryotic intracellular membrane trafficking. In their active states, Rabs localise to the cytoplasmic face of intracellular compartments where they regulate membrane trafficking processes. Many Rabs have been extensively characterised whereas others, such as Rab30, have to date received relatively little attention. Here, we demonstrate that Rab30 is primarily associated with the secretory pathway, displaying predominant localisation to the Golgi apparatus. We find by time-lapse microscopy and fluorescence recovery after photobleaching studies that Rab30 is rapidly and continuously recruited to the Golgi. We also show that Rab30 function is required for the morphological integrity of the Golgi. Finally, we demonstrate that inactivation of Rab30 does not impair anterograde or retrograde transport through the Golgi. Taken together, these data illustrate that Rab30 primarily localises to the Golgi apparatus and is required for the structural integrity of this organelle. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

  3. Specific organization of Golgi apparatus in plant cells.

    Science.gov (United States)

    Vildanova, M S; Wang, W; Smirnova, E A

    2014-09-01

    Microtubules, actin filaments, and Golgi apparatus are connected both directly and indirectly, but it is manifested differently depending on the cell organization and specialization, and these connections are considered in many original studies and reviews. In this review we would like to discuss what underlies differences in the structural organization of the Golgi apparatus in animal and plant cells: specific features of the microtubule cytoskeleton organization, the use of different cytoskeleton components for Golgi apparatus movement and maintenance of its integrity, or specific features of synthetic and secretory processes. We suppose that a dispersed state of the Golgi apparatus in higher plant cells cannot be explained only by specific features of the microtubule system organization and by the absence of centrosome as an active center of their organization because the Golgi apparatus is organized similarly in the cells of other organisms that possess the centrosome and centrosomal microtubules. One of the key factors determining the Golgi apparatus state in plant cells is the functional uniformity or functional specialization of stacks. The functional specialization does not suggest the joining of the stacks to form a ribbon; therefore, the disperse state of the Golgi apparatus needs to be supported, but it also can exist "by default". We believe that the dispersed state of the Golgi apparatus in plants is supported, on one hand, by dynamic connections of the Golgi apparatus stacks with the actin filament system and, on the other hand, with the endoplasmic reticulum exit sites distributed throughout the endoplasmic reticulum.

  4. Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Paiement, J.; Jolicoeur, M.; Fazel, A.; Bergeron, J.J.

    1989-04-01

    We have studied the reconstitution of the Golgi apparatus in vivo using an heterologous membrane transplant system. Endogenous glycopeptides of rat hepatic Golgi fragments were radiolabeled in vitro with (3H)sialic acid using detergent-free conditions. The Golgi fragments consisting of dispersed vesicles and tubules with intraluminal lipoprotein-like particles were then microinjected into Xenopus oocytes and their fate studied by light (LM) and electron microscope (EM) radioautography. 3 h after microinjection, radiolabel was observed by LM radioautography over yolk platelet-free cytoplasmic regions near the injection site. EM radioautography revealed label over Golgi stacked saccules containing the hepatic marker of intraluminal lipoprotein-like particles. At 14 h after injection, LM radioautographs revealed label in the superficial cortex of the oocytes between the yolk platelets and at the oocyte surface. EM radioautography identified the labeled structures as the stacked saccules of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma, indicating that a proportion of microinjected material was transferred to the surface via the secretion pathway of the oocyte. The efficiency of transport was low, however, as biochemical studies failed to show extensive secretion of radiolabel into the extracellular medium by 14 h with approximately half the microinjected radiolabeled constituents degraded. Vinblastine (50 microM) administered to oocytes led to the formation of tubulin paracrystals. Although microinjected Golgi fragments were able to effect the formation of stacked saccules in vinblastine-treated oocytes, negligible transfer of heterologous material to the oocyte surface could be detected by radioautography.

  5. Golgi GRASPs: moonlighting membrane tethers

    Directory of Open Access Journals (Sweden)

    Jarvela T

    2012-05-01

    Full Text Available Timothy Jarvela, Adam D LinstedtDepartment of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USAAbstract: The identification of mammalian Golgi reassembly stacking proteins (GRASPs 15 years ago was followed by experiments implicating them in diverse functions, including two differing structural roles in Golgi biogenesis and at least two distinct roles in the secretion of proteins. GRASP55 and GRASP65 are localized to cis and medial/trans Golgi cisternae, respectively. They are both required for stacking of Golgi membranes in a Golgi reassembly assay. Depletion of either GRASP from cultured cells prevents the linking of Golgi membranes into their normal ribbon-like network. While GRASPs are not required for transport of secretory cargo per se, they are required for ER-to-Golgi transport of certain specific cargo, such as those containing a C-terminal valine motif. Surprisingly, GRASPs also promote secretion of cargo by the so-called unconventional secretory pathway, which bypasses the Golgi apparatus where the GRASPs reside. Furthermore, regulation of GRASP activity is now recognized for its connections to cell cycle control, development, and disease. Underlying these diverse activities is the structurally conserved N-terminal GRASP domain whose crystal structure was recently determined. It consists of a tandem array of atypical PSD95–DlgA–Zo–1 (PDZ domains, which are well-known protein–protein interaction motifs. The GRASP PDZ domains are used to localize the proteins to the Golgi as well as GRASP-mediated membrane tethering and cargo interactions. These activities are regulated, in part, by phosphorylation of the large unstructured C-terminal domain.Keywords: GRASP, review, membrane, tether, PDZ domain, secretory chaperone, unconventional secretion

  6. Discovery and rediscoveries of Golgi cells

    NARCIS (Netherlands)

    E. Galliano (Elisa); P. Mazzarello (Paolo); E. D'Angelo (Egidio)

    2010-01-01

    textabstractWhen Camillo Golgi invented the black reaction in 1873 and first described the fine anatomical structure of the nervous system, he described a 'big nerve cell' that later took his name, the Golgi cell of cerebellum ('Golgi'schen Zellen', Gustaf Retzius, 1892). The Golgi cell was then

  7. New components of the Golgi matrix

    Science.gov (United States)

    Xiang, Yi; Wang, Yanzhuang

    2012-01-01

    The eukaryotic Golgi apparatus is characterized by a stack of flattened cisternae that are surrounded by transport vesicles. The organization and function of the Golgi require Golgi matrix proteins, including GRASPs and golgins, which exist primarily as fiber-like bridges between Golgi cisternae or between cisternae and vesicles. In this review, we highlight recent findings on Golgi matrix proteins, including their roles in maintaining the Golgi structure, vesicle tethering, and novel, unexpected functions. These new discoveries further our understanding of the molecular mechanisms that maintain the structure and the function of the Golgi, as well as its relationship with other cellular organelles such as the centrosome. PMID:21494806

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

    Directory of Open Access Journals (Sweden)

    White Jamie

    2001-10-01

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

  9. Membrane Traffic Within the Golgi Apparatus

    OpenAIRE

    Glick, Benjamin S.; Nakano, Akihiko

    2009-01-01

    Newly synthesized secretory cargo molecules pass through the Golgi apparatus while resident Golgi proteins remain in the organelle. However, the pathways of membrane traffic within the Golgi are still uncertain. Most of the available data can be accommodated by the cisternal maturation model, which postulates that Golgi cisternae form de novo, carry the secretory cargoes forward, and ultimately disappear. The entry face of the Golgi receives material that has been exported from transitional E...

  10. Actin acting at the Golgi.

    Science.gov (United States)

    Egea, Gustavo; Serra-Peinado, Carla; Salcedo-Sicilia, Laia; Gutiérrez-Martínez, Enric

    2013-09-01

    The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in trafficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers.

  11. [Relationship between Golgi apparatus and cell migration direction in vivo and in vitro].

    Science.gov (United States)

    Liu, Bin; Liu, Zhi-feng; Ren, Bing-cheng; Chen, Cong; Ming, Hao-lang; Wang, Lei-lei; Zhao, Kai; Yang, Xue-jun

    2013-07-02

    To explore the relationship between Golgi apparatus and the direction of tumor cell migration in vivo and in vitro. Cell migration assays were conducted with rat C6 glioma cells, human U251 and SNB19 glioma cells respectively. Then immunofluorescence was used to detect the position of Golgi apparatus in migrating cells. The percentage of cells with Golgi apparatus facing towards wound edge was calculated. Cell pseudopodium was stained with TRITC-phalloidin and the relationship between Golgi apparatus and pseudopodium detected. Immunohistochemistry was used to reveal the Golgi apparatus in tumor tissue samples. And the percentage of cells with Golgi apparatus facing opposite to the necrotic zones was calculated. In cells located at wound edge, the Golgi apparatus was found facing towards the wound in the vast majority of cells (C6 83% ± 6%, U251 80% ± 7%, SNB19 82% ± 6%). In U251 and SNB19 cells, the golgi apparatus was located in the same direction with cellular pseudopodium. Immunohistochemical staining showed that in cells located around the necrotic zone, the Golgi apparatus faced opposite to the necrotic zones in most cells (rat tissue samples 80% ± 7%, human tissue samples 82% ± 6%). The Golgi apparatus is closely correlated with cell migration and it may be considered as a direction indicator of cell migration. And it provides an important index for the study of tumor cell invasion both in vivo and in vitro.

  12. Rab41 Is a Novel Regulator of Golgi Apparatus Organization That Is Needed for ER-To-Golgi Trafficking and Cell Growth

    Science.gov (United States)

    Liu, Shijie; Hunt, Lauren; Storrie, Brian

    2013-01-01

    Background The 60+ members of the mammalian Rab protein family group into subfamilies postulated to share common functionality. The Rab VI subfamily contains 5 Rab proteins, Rab6a/a’, Rab6b, Rab6c and Rab41. High-level knockdown of Rab6a/a’ has little effect on the tightly organized Golgi ribbon in HeLa cells as seen by fluorescence microscopy. In striking contrast, we found Rab41 was strongly required for normal Golgi ribbon organization. Methods/Results Treatment of HeLa cells with Rab41 siRNAs scattered the Golgi ribbon into clustered, punctate Golgi elements. Overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a similar Golgi phenotype. By electron microscopy, Rab41 depletion produced short, isolated Golgi stacks. Golgi-associated vesicles accumulated. At low expression levels, wild type and GTP-locked Rab41 showed little concentration in the Golgi region, but puncta were observed and most were in ruffled regions at the cell periphery. There was 25% co-localization of GTP-locked Rab41 with the ER marker, Sec61p. GDP-locked Rab41, as expected, displayed an entirely diffuse cytoplasmic distribution. Depletion of Rab41 or overexpression of GDP-locked Rab41 partially inhibited ER-to-Golgi transport of VSV-G protein. However, Rab41 knockdown had little, if any, effect on endosome-to-Golgi transport of SLTB. Additionally, after a 2-day delay, treatment with Rab41 siRNA inhibited cell growth, while overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a rapid, progressive cell loss. In double knockdown experiments with Rab6, the Golgi ribbon was fragmented, a result consistent with Rab41 and Rab6 acting in parallel. Conclusion We provide the first evidence for distinctive Rab41 effects on Golgi organization, ER-to-Golgi trafficking and cell growth. When combined with the evidence that Rab6a/a’ and Rab6b have diverse roles in Golgi function, while Rab6c regulates mitotic function, our data indicate

  13. Lipids of the Golgi membrane

    NARCIS (Netherlands)

    van Meer, G.

    1998-01-01

    The thin membrane of the endoplasmic reticulum matures into the thick plasma membrane in the Golgi apparatus. Along the way, the concentrations of cholesterol and sphingolipids increase. Here, Gerrit van Meer discusses how this phenomenon may reflect an intricate lipid-protein sorting machinery.

  14. Influenza infection modulates vesicular trafficking and induces Golgi complex disruption.

    Science.gov (United States)

    Yadav, Vibha; Panganiban, Antonito T; Honer Zu Bentrup, Kerstin; Voss, Thomas G

    2016-12-01

    Influenza A virus (IFV) replicates its genome in the nucleus of infected cells and uses the cellular protein transport system for genome trafficking from the nucleus to the plasma membrane. However, many details of the mechanism of this process, and its relationship to subsequent cytoplasmic virus trafficking, have not been elucidated. We examined the effect of nuclear transport inhibitors Leptomycin B (LB), 5,6 dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), the vesicular transport inhibitor Brefeldin A (BFA), the caspase inhibitor ZWEHD, and microtubule inhibitor Nocodazole (NOC) on virus replication and intracellular trafficking of viral nucleoprotein (NP) from the nucleus to the ER and Golgi. Also, we carried out complementary studies to determine the effect of IFV on intracellular membranes. Inhibition of the CRM1 and TAP-P15 nuclear transport pathways by DRB and LB blocked completely the export of virus. Inhibition of vesicular trafficking by BFA, NOC, and ZWEHD also affected influenza infection. Interestingly, IFV infection induced fragmentation of the Golgi complex resulting in diffuse distribution of large and small vesicles throughout the cytoplasm. Live-cell microscopy revealed expansion of Golgi localization signals indicating progressive dispersion of Golgi positive structures, resulting in the disassembly of the Golgi ribbon structure. Other vesicular components (Rab1b, ARF1 and GBF1) were also found to be required for IFV infection. Furthermore, the exact step at which IFV infection disrupts vesicle trafficking was identified as the ER-Golgi intermediate compartment. These findings suggest that IFV NP is trafficked from the nucleus via the CRM1 and TAP pathways. IFV modulates vesicular trafficking inducing disruption of the Golgi complex. These studies provide insight on the ways in which IFV affects intracellular trafficking of different host proteins and will facilitate identification of useful pharmaceutical targets to abrogate virus

  15. [From endoplasmic reticulum to Golgi apparatus: a secretory pathway controlled by signal molecules].

    Science.gov (United States)

    Wang, Jiasheng; Luo, Jianhong; Zhang, Xiaomin

    2013-07-01

    Protein transport from endoplasmic reticulum (ER) to Golgi apparatus has long been known to be a central process for protein quality control and sorting. Recent studies have revealed that a large number of signal molecules are involved in regulation of membrane trafficking through ER, ER-Golgi intermediate compartment and Golgi apparatus. These molecules can significantly change the transport rate of proteins by regulating vesicle budding and fusion. Protein transport from ER to Golgi apparatus is not only controlled by signal pathways triggered from outside the cell, it is also regulated by feedback signals from the transport pathway.

  16. Reduction in Golgi apparatus dimension in the absence of a residential protein, N-acetylglucosaminyltransferase V.

    Science.gov (United States)

    Dong, Zhizhong; Zuber, Christian; Pierce, Michael; Stanley, Pamela; Roth, Jürgen

    2014-02-01

    Various proteins are involved in the generation and maintenance of the membrane complex known as the Golgi apparatus. We have used mutant Chinese hamster ovary (CHO) cell lines Lec4 and Lec4A lacking N-acetylglucosaminyltransferase V (GlcNAcT-V, MGAT5) activity and protein in the Golgi apparatus to study the effects of the absence of a single glycosyltransferase on the Golgi apparatus dimension. Quantification of immunofluorescence in serial confocal sections for Golgi α-mannosidase II and electron microscopic morphometry revealed a reduction in Golgi volume density up to 49 % in CHO Lec4 and CHO Lec4A cells compared to parental CHO cells. This reduction in Golgi volume density could be reversed by stable transfection of Lec4 cells with a cDNA encoding Mgat5. Inhibition of the synthesis of β1,6-branched N-glycans by swainsonine had no effect on Golgi volume density. In addition, no effect on Golgi volume density was observed in CHO Lec1 cells that contain enzymatically active GlcNAcT-V, but cannot synthesize β1,6-branched glycans due to an inactive GlcNAcT-I in their Golgi apparatus. These results indicate that it may be the absence of the GlcNAcT-V protein that is the determining factor in reducing Golgi volume density. No dimensional differences existed in cross-sectioned cisternal stacks between Lec4 and control CHO cells, but significantly reduced Golgi stack hits were observed in cross-sectioned Lec4 cells. Therefore, the Golgi apparatus dimensional change in Lec4 and Lec4A cells may be due to a compaction of the organelle.

  17. Cell cycle regulation of Golgi membrane dynamics

    Science.gov (United States)

    Tang, Danming; Wang, Yanzhuang

    2013-01-01

    The Golgi apparatus is a membranous organelle in the cell that plays essential roles in protein and lipid trafficking, sorting, processing and modification. Its basic structure is a stack of closely aligned flattened cisternae. In mammalian cells, dozens of Golgi stacks are often laterally linked into a ribbon-like structure. Biogenesis of the Golgi during cell division occurs through a sophisticated disassembly and reassembly process that can be divided into three distinct but cooperative steps, including the deformation and reformation of the Golgi cisternae, stacks and ribbon. Here, we review our current understanding of the protein machineries that control these three steps in the cycle of mammalian cell division: GRASP65 and GRASP55 in Golgi stack and ribbon formation; ubiquitin and AAA ATPases in post-mitotic Golgi membrane fusion; and golgins and cytoskeleton in Golgi ribbon formation. PMID:23453991

  18. Three-dimensional shape of the Golgi apparatus in different cell types: serial section scanning electron microscopy of the osmium-impregnated Golgi apparatus.

    Science.gov (United States)

    Koga, Daisuke; Kusumi, Satoshi; Ushiki, Tatsuo

    2016-04-01

    Although many studies of the Golgi apparatus structure have been performed by light and electron microscopy, the full shape of the Golgi apparatus remained unclear due to the technical limitations of the previously applied microscopy techniques. In this study, we used serial section scanning electron microscopy (SEM) for the morphological study of the Golgi apparatus. This method is useful for three-dimensional (3D) reconstruction of cellular structures without requiring specialized instruments, unlike focused ion beam SEM (FIB-SEM) and serial block face SEM (SBF-SEM). Using the serial section SEM method developed by our laboratory, we investigate the 3D shape of the osmium-impregnated Golgi apparatus in rat epididymal cells, pancreatic acinar cells and gonadotropes. The combination of serial section SEM and a 3D reconstruction technique enabled us to elucidate the entire shape of the Golgi apparatus in these cells. The full shape of the Golgi apparatus in epididymal cells formed a basket-like structure with oval-shaped cisterns, while the Golgi apparatus in an acinar cell from the pancreas was composed of elongated ribbon-like structures that were connected to each other, making a coarse network. The overall image of the Golgi apparatus cisterns from a gonadotrope looked like a spherical cage. This study has clearly shown that entire 3D shape of the Golgi apparatus varies depending on the cell type and that the Golgi cisterns network appears as a single mass located in the large region of the cytoplasm. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Stacks off tracks: a role for the golgin AtCASP in plant endoplasmic reticulum-Golgi apparatus tethering.

    Science.gov (United States)

    Osterrieder, Anne; Sparkes, Imogen A; Botchway, Stan W; Ward, Andy; Ketelaar, Tijs; de Ruijter, Norbert; Hawes, Chris

    2017-06-15

    The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER) and synthesizes cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role for cis-Golgi plant golgins, which are long coiled-coil domain proteins anchored to Golgi membranes, in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC and suggests that this connection is most likely maintained by a golgin-mediated tethering complex. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  20. Golgi localized barley MTP8 proteins facilitate Mn transport

    DEFF Research Database (Denmark)

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

    2014-01-01

    8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi...

  1. Actin microfilaments are essential for the cytological positioning and morphology of the Golgi complex

    NARCIS (Netherlands)

    Valderrama, F; Babia, T; Ayala, [No Value; Kok, JW; Renau-Piqueras, J; Egea, G

    The organization and function of the Golgi complex was studied in normal rat kidney cells following disruption of the actin cytoskeleton induced by cytochalasin D. In cells treated with these reagents, the reticular and perinuclear Golgi morphology acquired a cluster shape restricted to the

  2. 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

  3. Aβ-induced Golgi fragmentation in Alzheimer’s disease enhances Aβ production

    Science.gov (United States)

    Joshi, Gunjan; Chi, Youjian; Huang, Zheping; Wang, Yanzhuang

    2014-01-01

    Golgi fragmentation occurs in neurons of patients with Alzheimer’s disease (AD), but the underlying molecular mechanism causing the defects and the subsequent effects on disease development remain unknown. In this study, we examined the Golgi structure in APPswe/PS1∆E9 transgenic mouse and tissue culture models. Our results show that accumulation of amyloid beta peptides (Aβ) leads to Golgi fragmentation. Further biochemistry and cell biology studies revealed that Golgi fragmentation in AD is caused by phosphorylation of Golgi structural proteins, such as GRASP65, which is induced by Aβ-triggered cyclin-dependent kinase-5 activation. Significantly, both inhibition of cyclin-dependent kinase-5 and expression of nonphosphorylatable GRASP65 mutants rescued the Golgi structure and reduced Aβ secretion by elevating α-cleavage of the amyloid precursor protein. Our study demonstrates a molecular mechanism for Golgi fragmentation and its effects on amyloid precursor protein trafficking and processing in AD, suggesting Golgi as a potential drug target for AD treatment. PMID:24639524

  4. Phospholipid synthesis participates in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex.

    Science.gov (United States)

    Sarri, Elisabet; Sicart, Adrià; Lázaro-Diéguez, Francisco; Egea, Gustavo

    2011-08-12

    The lipid metabolite diacylglycerol (DAG) is required for transport carrier biogenesis at the Golgi, although how cells regulate its levels is not well understood. Phospholipid synthesis involves highly regulated pathways that consume DAG and can contribute to its regulation. Here we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the post-Golgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking.

  5. Morphology of platelet Golgi apparatus and their significance after acute cerebral infarction.

    Science.gov (United States)

    Lu, Wei; Xu, Dong; Tu, Ranran; Hu, Zhiping

    2013-08-15

    Blood samples were harvested from the antecubital vein of 20 fasting patients with acute cerebral infarction at 1, 7 and 15 days after onset to prepare blood platelet suspension. Fasting antecubital vein blood was collected from an additional 20 normal adults as controls. Under transmission tron microscope, platelet Golgi tubules and vesicles became significantly thickened, enlarged, and irregular after acute cerebral infarction. Alpha granules in platelets significantly reduced in number, especially 1 day after cerebral infarction. Under immunoelectron microscopy, a few alpha granules aggregated around Golgi tubules and vesicles after infarction. These results suggested that platelet Golgi apparatus displayed significant morphological changes, which were possibly associated with enhanced synthetic and secretory functions of activated platelets after acute cerebral infarction. This study used Golgi apparatus blocking agent Brefeldin A to block Golgi apparatus in an aim to study the effects of Golgi apparatus on CD40L expression on the surface of activated platelets. Flow cytometry revealed that CD40L expression on activated platelet surfaces decreased significantly when Golgi apparatus was blocked, which indicated that Golgi apparatus participated in the synthesis and transport of CD40L to the platelet surface.

  6. GRASPs in Golgi Structure and Function

    Science.gov (United States)

    Zhang, Xiaoyan; Wang, Yanzhuang

    2016-01-01

    The Golgi apparatus is a central intracellular membrane organelle for trafficking and modification of proteins and lipids. Its basic structure is a stack of tightly aligned flat cisternae. In mammalian cells, dozens of stacks are concentrated in the pericentriolar region and laterally connected to form a ribbon. Despite extensive research in the last decades, how this unique structure is formed and why its formation is important for proper Golgi functioning remain largely unknown. The Golgi ReAssembly Stacking Proteins, GRASP65, and GRASP55, are so far the only proteins shown to function in Golgi stacking. They are peripheral membrane proteins on the cytoplasmic face of the Golgi cisternae that form trans-oligomers through their N-terminal GRASP domain, and thereby function as the “glue” to stick adjacent cisternae together into a stack and to link Golgi stacks into a ribbon. Depletion of GRASPs in cells disrupts the Golgi structure and results in accelerated protein trafficking and defective glycosylation. In this minireview we summarize our current knowledge on how GRASPs function in Golgi structure formation and discuss why Golgi structure formation is important for its function. PMID:26779480

  7. GRASPs in Golgi Structure and Function

    Directory of Open Access Journals (Sweden)

    Xiaoyan eZhang

    2016-01-01

    Full Text Available The Golgi apparatus is a central intracellular membrane organelle for trafficking and modification of proteins and lipids. Its basic structure is a stack of tightly aligned flat cisternae. In mammalian cells, dozens of stacks are concentrated in the pericentriolar region and laterally connected to form a ribbon. Despite extensive research in the last decades, how this unique structure is formed and why its formation is important for proper Golgi functioning remain largely unknown. The Golgi ReAssembly Stacking Proteins, GRASP65 and GRASP55, are so far the only proteins shown to function in Golgi stacking. They are peripheral membrane proteins on the cytoplasmic face of the Golgi cisternae that form trans-oligomers through their N-terminal GRASP domain, and thereby function as the glue to stick adjacent cisternae together into a stack and to link Golgi stacks into a ribbon. Depletion of GRASPs in cells disrupts the Golgi structure and results in accelerated protein trafficking and defective glycosylation. In this minireview we summarize our current knowledge on how GRASPs function in Golgi structure formation and discuss why Golgi structure formation is important for its function.

  8. Grab a Golgi: Laser trapping of golgi bodies reveals in vivo Interactions with the endoplasmic reticulum

    NARCIS (Netherlands)

    Sparkes, I.A.; Ketelaar, T.; Ruijter, de N.C.A.; Hawes, C.

    2009-01-01

    In many vacuolate plant cells individual Golgi bodies appear to be attached to tubules of the pleiomorphic cortical endoplasmic reticulum (ER) network. Such observations culminated in the controversial mobile secretory unit hypothesis to explain transport of cargo from the ER to Golgi via Golgi

  9. 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.

  10. Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells.

    Science.gov (United States)

    Taverna, Elena; Mora-Bermúdez, Felipe; Strzyz, Paulina J; Florio, Marta; Icha, Jaroslav; Haffner, Christiane; Norden, Caren; Wilsch-Bräuninger, Michaela; Huttner, Wieland B

    2016-02-16

    Apical radial glia (aRG), the stem cells in developing neocortex, are unique bipolar epithelial cells, extending an apical process to the ventricle and a basal process to the basal lamina. Here, we report novel features of the Golgi apparatus, a central organelle for cell polarity, in mouse aRGs. The Golgi was confined to the apical process but not associated with apical centrosome(s). In contrast, in aRG-derived, delaminating basal progenitors that lose apical polarity, the Golgi became pericentrosomal. The aRG Golgi underwent evolutionarily conserved, accordion-like compression and extension concomitant with cell cycle-dependent nuclear migration. Importantly, in line with endoplasmic reticulum but not Golgi being present in the aRG basal process, its plasma membrane contained glycans lacking Golgi processing, consistent with direct ER-to-cell surface membrane traffic. Our study reveals hitherto unknown complexity of neural stem cell polarity, differential Golgi contribution to their specific architecture, and fundamental Golgi re-organization upon cell fate change.

  11. Gβ1γ2 Activates Phospholipase A2-Dependent Golgi Membrane Tubule Formation

    Directory of Open Access Journals (Sweden)

    William J Brown

    2014-02-01

    Full Text Available Heterotrimeric G proteins transduce the ligand binding of transmembrane G protein coupled receptors into a variety of intracellular signaling pathways. Recently, heterotrimeric Gβγ subunit signaling at the Golgi complex has been shown to regulate the formation of vesicular transport carriers that deliver cargo from the Golgi to the plasma membrane. In addition to vesicles, membrane tubules have also been shown to mediate export from the Golgi complex, which requires the activity of cytoplasmic phospholipase A2 (PLA2 enzyme activity. Through the use of an in vitro reconstitution assay with isolated Golgi complexes, we provide evidence that Gβ1γ2 signaling also stimulates Golgi membrane tubule formation. In addition, we show that an inhibitor of Gβγ activation of PLA2 enzymes inhibits in vitro Golgi membrane tubule formation. Additionally, purified Gβγ protein stimulates membrane tubules in the presence of low (sub-threshold cytosol concentrations. Importantly, this Gβγ stimulation of Golgi membrane tubule formation was inhibited by treatment with the PLA2 antagonist ONO-RS-082. These studies indicate that Gβ1γ2 signaling activates PLA2 enzymes required for Golgi membrane tubule formation, thus establishing a new layer of regulation for this process.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kaneko, Yayoi [Department of Molecular Cell Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511 (Japan); Tamura, Kaori [Department of Molecular Cell Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Totsukawa, Go [Department of Molecular Cell Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511 (Japan); Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp [Department of Molecular Cell Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan)

    2010-11-05

    Research highlights: {yields} p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis. {yields} Phosphorylated p37 does not bind to Golgi membranes. {yields} 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.

  13. Rapid Capability Fielding Toolbox Study

    Science.gov (United States)

    2010-03-01

    skipped  or  attenuated, depending on the specific effort) are as follows:   The  process  is  initiated  with  the  identification  and  careful ...O.L., “Modeling Methods and Conceptual Design Principles for Reconfigurable Systems”,  Journal  of Mechanical Design, 139, 101102, October 2008.  15...Motorola  Dennis Roberson is Vice Provost and Executive Director of the Institute of Business and  Interprofessional   Studies, as well as Acting

  14. 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…

  15. [From cellular biology to molecular biology: Golgi apparatus from the discovery to nowadays].

    Science.gov (United States)

    Falchetti, Mario; Lupi, Ramona; Ottini, Laura

    2007-01-01

    On April the 9th 1898 Golgi presented the discovery of the Apparato Reticolare Interno or internal reticular apparatus to the Società Medico-Chirurgica in Pavia. The internal reticular apparatus was described as "a fine and elegant network within the cell body" of Purkinje cells. The discovery of this new intracellular structure can be considered a byproduct of Golgi studies devoted to the analysis of the nervous system histology. Golgi and his co-workers detected the internal reticular apparatus in many cell types and described the organelle pleiomorphism due to specific physiological or pathological conditions. However, the real existence of the apparatus was questioned until the organelle was finally identified by electron microscopy in 1954. At this point Golgi apparatus became an actual intracellular structure without any clear function. The involvement in cell secretion processes was verified by using biochemical and molecular investigations from the 1960s. Nowadays, Golgi apparatus is clearly known to be involved in different cell functions as growth, homeostasis and division. The correct execution of these functions lies on the ability to maintain an equilibrated balance between the proteins therein resident. Recently, Golgi apparatus has been involved also in human pathology as mutations in proteins localized in the organelle are linked to some hereditary disorders like the Lowe syndrome. Golgi apparatus has been debated since its discovery. From the Golgi milestones discussed here it is evident that controversies that have arisen were often resolved by information resulting from the application of new technical developments. Indeed the compound dynamic structure and the relevance in cell physiology and in human pathology render Golgi apparatus an open object for future studies. Overall, the history of the Golgi apparatus represents an excellent model not only to follow the transition of the study approaches from cellular biology to molecular cell biology

  16. Remodeling of the Golgi structure by ERK signaling

    OpenAIRE

    Wei, Jen-Hsuan; Seemann, Joachim

    2009-01-01

    Emerging evidence suggests that the Golgi functions as a regulatory node for various signaling cascades. Modules of the MAPK pathway are targeted to the Golgi upon stimulation of cells with mitogens. The target for activated ERK on the Golgi membranes is GRASP65, a peripheral membrane protein required for Golgi cisternal stacking. Phosphorylation of GRASP65 at Serine 277 results in a loss of its oligomerization and causes unstacking of Golgi cisternae. This reorganization of the Golgi structu...

  17. Types of neurons of the claustrum in the rabbit--Nissl, Klüver-Barrera and Golgi studies.

    Science.gov (United States)

    Wasilewska, B; Najdzion, J

    2001-01-01

    The studies were carried out on the claustrum of 8 adult rabbits. Four types of neurons were distinguished: 1. Multipolar neurons, which have dendritic trunks either with conus (multipolar polygonal perikarya) or without conus (multipolar rounded perikarya). Both subdivisions of the multipolar neurons have 3-6 dendritic trunks. Only some branches of these trunks have spines. An axon emerges mainly from the cell body, rarely from the initial part of the dendritic trunk. 2. Bipolar neurons with fusiform or rounded perikarya; they have two dendrites covered with spines. An axon originates directly from the cell body or from one of the dendritic trunks. 3. Triangular neurons, which have three dendritic branches with spines. An axon emerges directly from the soma, often near the primary dendritic trunk. 4. Pear-shaped neurons with one or two dendritic trunks arise from one pole of the cell body and with an axon that originates from the opposite side of the perikaryon. The dendrites are covered with spines.

  18. The endoplasmic reticulum-resident chaperone heat shock protein 47 protects the Golgi apparatus from the effects of O-glycosylation inhibition.

    Science.gov (United States)

    Miyata, Shingo; Mizuno, Tatsunori; Koyama, Yoshihisa; Katayama, Taiichi; Tohyama, Masaya

    2013-01-01

    The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of O-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of O-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of O-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria.

  19. Early Golgi Abnormalities and Neurodegeneration upon Loss of Presynaptic Proteins Munc18-1, Syntaxin-1, or SNAP-25.

    Science.gov (United States)

    Santos, Tatiana C; Wierda, Keimpe; Broeke, Jurjen H; Toonen, Ruud F; Verhage, Matthijs

    2017-04-26

    The loss of presynaptic proteins Munc18-1, syntaxin-1, or SNAP-25 is known to produce cell death, but the underlying features have not been compared experimentally. Here, we investigated these features in cultured mouse CNS and DRG neurons. Side-by-side comparisons confirmed massive cell death, before synaptogenesis, within 1-4 DIV upon loss of t-SNAREs (syntaxin-1, SNAP-25) or Munc18-1, but not v-SNAREs (synaptobrevins/VAMP1/2/3 using tetanus neurotoxin (TeNT), also in TI-VAMP/VAMP7 knock-out (KO) neurons). A condensed cis- Golgi was the first abnormality observed upon Munc18-1 or SNAP-25 loss within 3 DIV. This phenotype was distinct from the Golgi fragmentation observed in apoptosis. Cell death was too rapid after syntaxin-1 loss to study Golgi abnormalities. Syntaxin-1 and Munc18-1 depend on each other for normal cellular levels. We observed that endogenous syntaxin-1 accumulates at the Golgi of Munc18-1 KO neurons. However, expression of a non-neuronal Munc18 isoform that does not bind syntaxin-1, Munc18-3, in Munc18-1 KO neurons prevented cell death and restored normal cis- Golgi morphology, but not synaptic transmission or syntaxin-1 targeting. Finally, we observed that DRG neurons are the only Munc18-1 KO neurons that do not degenerate in vivo or in vitro In these neurons, cis- Golgi abnormalities were less severe, with no changes in Golgi shape. Together, these data demonstrate that cell death upon Munc18-1, syntaxin-1, or SNAP-25 loss occurs via a degenerative pathway unrelated to the known synapse function of these proteins and involving early cis- Golgi abnormalities, distinct from apoptosis. SIGNIFICANCE STATEMENT This study provides new insights in a neurodegeneration pathway triggered by the absence of specific proteins involved in synaptic transmission (syntaxin-1, Munc18-1, SNAP-25), whereas other proteins involved in the same molecular process (synaptobrevins, Munc13-1/2) do not cause degeneration. Massive cell death occurs in cultured neurons

  20. Phospholipase A2 Antagonists Inhibit Nocodazole-induced Golgi Ministack Formation: Evidence of an ER Intermediate and Constitutive Cycling

    OpenAIRE

    Drecktrah, Daniel; Brown, William J.

    1999-01-01

    Evidence has been presented both for and against obligate retrograde movement of resident Golgi proteins through the endoplasmic reticulum (ER) during nocodazole-induced Golgi ministack formation. Here, we studied the nocodazole-induced formation of ministacks using phospholipase A2 (PLA2) antagonists, which have been shown previously to inhibit brefeldin A–stimulated Golgi-to-ER retrograde transport. Examination of clone 9 rat hepatocytes by immunofluorescence and immunoelectron microscopy r...

  1. YIPF1, YIPF2, and YIPF6 are medial-/trans-Golgi and trans-Golgi network-localized Yip domain family proteins, which play a role in the Golgi reassembly and glycan synthesis.

    Science.gov (United States)

    Soonthornsit, Jeerawat; Sakai, Noriko; Sasaki, Yurika; Watanabe, Ryota; Osako, Shiho; Nakamura, Nobuhiro

    2017-04-15

    In this study, we attempted to explore the function of three uncharacterized mammalian homologs of yeast Yip domain family proteins-YIPF6, a homolog of Yip1p, and YIPF1 and YIPF2, which are homologs of Yif1p. Immunofluorescence staining revealed that YIPF1, YIPF2, and YIPF6 mainly localize in the medial-/trans-Golgi and also partially in the trans-Golgi network (TGN). On treatment with brefeldin A (BFA), the homologs co-migrated partly with medial-/trans-Golgi markers and also with a TGN marker in earlier time point, but finally redistributed within cytoplasmic punctate structures that were distinct from medial-/trans-Golgi and the TGN markers. YIPF6 formed a stable complex separately with YIPF1 and YIPF2, and knockdown of YIPF6 reduced YIPF1 and YIPF2 levels. These results suggest that YIPF6 forms complexes with YIPF1 and YIPF2 for their stable expression and localization within the Golgi apparatus. Knockdown experiments showed that YIPF1 and YIPF2, by contrast, are not necessary for the expression and localization of YIPF6. The structure of the Golgi apparatus and its disassembly after BFA treatment were not significantly affected by the knockdown of YIPF1, YIPF2, or YIPF6. However, reassembly of the Golgi apparatus after the removal of BFA was markedly delayed by the knockdown of YIPF1 and YIPF2, but not by that of YIPF6. These results strongly suggest that free YIPF6 after disassociating with YIPF1 and YIPF2 interferes with the reassembly of the Golgi apparatus. Knockdown of YIPF1 and YIPF2, but not that of YIPF6, also reduced intracellular glycans in HT-29 cells. Thus, we confirmed that YIPF1, YIPF2, and YIPF6 play a significant role in supporting normal glycan synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Ca(2+) signalling in the Golgi apparatus.

    Science.gov (United States)

    Pizzo, Paola; Lissandron, Valentina; Capitanio, Paola; Pozzan, Tullio

    2011-08-01

    The Golgi apparatus plays a central role in lipid and protein post-translational modification and sorting. Morphologically the organelle is heterogeneous and it is possible to distinguish stacks of flat cysternae (cis- and medial Golgi), tubular-reticular networks and vesicles (trans-Golgi). These morphological differences parallel a distinct functionality with a selective distribution and complementary roles of the enzymes found in the different compartments. The Golgi apparatus has been also shown to be involved in Ca(2+) signalling: it is indeed endowed with Ca(2+) pumps, Ca(2+) release channels and Ca(2+) binding proteins and is thought to participate in determining the spatio-temporal complexity of the Ca(2+) signal within the cell, though this role is still poorly understood. Recently, it has been demonstrated that the organelle is heterogeneous in terms of Ca(2+) handling and selective reduction of Ca(2+) concentration, both in vitro and in a genetic human disease, within one of its sub-compartment results in alterations of protein trafficking within the secretory pathway and of the entire Golgi morphology. In this paper we review the available information on the Ca(2+) toolkit within the Golgi, its heterogeneous distribution in the organelle sub-compartments and discuss the implications of these characteristics for the physiopathology of the Golgi apparatus. 2011 Elsevier Ltd. All rights reserved.

  3. Golgi localized barley MTP8 proteins facilitate Mn transport.

    Directory of Open Access Journals (Sweden)

    Pai Pedas

    Full Text Available 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-bound proteins belonging to the cation diffusion facilitator (CDF family in the cereal species barley (Hordeum vulgare. Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP are localized to Golgi. When heterologously expressed 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 decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species.

  4. 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.

  5. Breaking the COPI monopoly on Golgi recycling.

    Science.gov (United States)

    Storrie, B; Pepperkok, R; Nilsson, T

    2000-09-01

    The unexpected discovery of a transport pathway from the Golgi to the endoplasmic reticulum (ER) independent of COPI coat proteins sheds light on how Golgi resident enzymes and protein toxins gain access to the ER from as far as the trans Golgi network. This new pathway provides an explanation for how membrane is recycled to allow for an apparent concentration of anterograde cargo at distinct stages of the secretory pathway. As signal-mediated COPI-dependent recycling also involves the concentration of resident proteins into retrograde COPI vesicles, the main bulk of lipids must be recycled, possibly through a COPI-independent pathway.

  6. GRASP65 controls the cis Golgi integrity in vivo

    NARCIS (Netherlands)

    Veenendaal, Tineke; Jarvela, Tim; Grieve, Adam G; van Es, Johan H; Linstedt, Adam D; Rabouille, Catherine

    2014-01-01

    GRASP65 and GRASP55 are peripheral Golgi proteins localized to cis and medial/trans cisternae, respectively. They are implicated in diverse aspects of protein transport and structure related to the Golgi complex, including the stacking of the Golgi stack and/or the linking of mammalian Golgi stacks

  7. A Unique Ball-Shaped Golgi Apparatus in the Rat Pituitary Gonadotrope

    Science.gov (United States)

    Sakai, Yuko; Koga, Daisuke; Bochimoto, Hiroki; Hira, Yoshiki; Hosaka, Masahiro; Ushiki, Tatsuo

    2012-01-01

    In polarized exocrine cells, the Golgi apparatus is cup-shaped and its convex and concave surfaces are designated as cis and trans faces, functionally confronting the rough endoplasmic reticulum and the cell surface, respectively. To clarify the morphological characteristics of the Golgi apparatus in non-polarized endocrine cells, the investigators immunocytochemically examined its precise architecture in pituitary gonadotropes, especially in relation to the arrangement of the intracellular microtubule network. The Golgi apparatus in the gonadotropes was not cup-shaped but ball-shaped or spherical, and its outer and inner surfaces were the cis and trans faces, respectively. Centrioles were situated at the center of the Golgi apparatus, from which radiating microtubules isotropically extended to the cell periphery through the gaps in the spherical wall of the Golgi stack. The shape of the Golgi apparatus and the arrangement of microtubules demonstrated in the present study could explain the microtubule-dependent movements of tubulovesicular carriers and granules within the gonadotropes. Furthermore, the spherical shape of the Golgi apparatus possibly reflects the highly symmetrical arrangement of microtubule arrays, as well as the poor polarity in the cell surface of pituitary gonadotropes. PMID:22562559

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

    Science.gov (United States)

    Xu, Haijiao; Su, Weiheng; Cai, Mingjun; Jiang, Junguang; Zeng, Xianlu; Wang, Hongda

    2013-01-01

    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.

  9. 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.

  10. 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.

  11. How Golgi glycosylation meets and needs trafficking: the case of the COG complex.

    Science.gov (United States)

    Reynders, Ellen; Foulquier, François; Annaert, Wim; Matthijs, Gert

    2011-07-01

    Protein glycosylation is one of the major biosynthetic functions occurring in the endoplasmic reticulum and Golgi compartments. It requires an amazing number of enzymes, chaperones, lectins and transporters whose actions delicately secure the fidelity of glycan structures. Over the past 30 years, glycobiologists hammered that glycan structures are not mere decorative elements but serve crucial cellular functions. This becomes dramatically illustrated by a group of mostly severe, inherited human disorders named congenital disorders of glycosylation (CDG). To date, many types of CDG have been defined genetically and most of the time the defects impair the biosynthesis, transfer and remodeling of N-glycans. Recently, the identification of the several types of CDG caused by deficiencies in the conserved oligomeric Golgi (COG) complex, a complex involved in vesicular Golgi trafficking, expanded the field of CDG but also brought novel insights in glycosylation. The molecular mechanisms underlying the complex pathway of N-glycosylation in the Golgi are far from understood. The availability of COG-deficient CDG patients and patients' cells offered a new way to study how COG, and its different subunits, could influence the Golgi N-glycosylation machinery and localization. This review summarizes the recent findings on the implication of COG in Golgi glycosylation. It highlights the need for a dynamic, finely tuned balance between anterograde and retrograde trafficking for the correct localization of Golgi enzymes to assure the stepwise maturation of N-glycan chains.

  12. 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.

  13. 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 H2O2-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 H2O2 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. H2O2 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 H2O2 treatment. The expression of GRASP65 showed no significant changes at different time points; the phosphorylated GRASP65 level was significantly increased after H2O2 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 H2O2 treatment, while its role in H2O2-induced Golgi morphological changes may be complex.

  14. Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors.

    Science.gov (United States)

    Obata, Y; Horikawa, K; Takahashi, T; Akieda, Y; Tsujimoto, M; Fletcher, J A; Esumi, H; Nishida, T; Abe, R

    2017-06-29

    Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek-Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)'s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.

  15. Golgi localisation of GMAP210 requires two distinct cis-membrane binding mechanisms

    Directory of Open Access Journals (Sweden)

    Goud Bruno

    2009-08-01

    Full Text Available Abstract Background The Golgi apparatus in mammals appears as a ribbon made up of interconnected stacks of flattened cisternae that is positioned close to the centrosome in a microtubule-dependent manner. How this organisation is achieved and retained is not well understood. GMAP210 is a long coiled-coil cis-Golgi associated protein that plays a role in maintaining Golgi ribbon integrity and position and contributes to the formation of the primary cilium. An amphipathic alpha-helix able to bind liposomes in vitro has been recently identified at the first 38 amino acids of the protein (amphipathic lipid-packing sensor motif, and an ARF1-binding domain (Grip-related Arf-binding domain was found at the C-terminus. To which type of membranes these two GMAP210 regions bind in vivo and how this contributes to GMAP210 localisation and function remains to be investigated. Results By using truncated as well as chimeric mutants and videomicroscopy we found that both the N-terminus and the C-terminus of GMAP210 are targeted to the cis-Golgi in vivo. The ALPS motif was identified as the N-terminal binding motif and appeared concentrated in the periphery of Golgi elements and between Golgi stacks. On the contrary, the C-terminal domain appeared uniformly distributed in the cis-cisternae of the Golgi apparatus. Strikingly, the two ends of the protein also behave differently in response to the drug Brefeldin A. The N-terminal domain redistributed to the endoplasmic reticulum (ER exit sites, as does the full-length protein, whereas the C-terminal domain rapidly dissociated from the Golgi apparatus to the cytosol. Mutants comprising the full-length protein but lacking one of the terminal motifs also associated with the cis-Golgi with distribution patterns similar to those of the corresponding terminal end whereas a mutant consisting in fused N- and C-terminal ends exhibits identical localisation as the endogenous protein. Conclusion We conclude that the Golgi

  16. Oligomerization of a trans Golgi/trans Golgi network retained protein occurs in the Golgi complex and may be part of its retention

    NARCIS (Netherlands)

    Horzinek, M.C.; Locker, J.K.; Opstelten, D.J.; Ericsson, M.; Rottier, P.J.M.

    1995-01-01

    The mouse hepatitis virus M protein is a triple spanning membrane glycoprotein that, when expressed independently, localizes to trans-Golgi as well as to the trans-Golgi network (TGN). Passage of this protein from the endoplasmic reticulum through the intermediate compartment to the late Golgi and

  17. High-content analysis of Rab protein function at the ER-Golgi interface

    Science.gov (United States)

    Galea, George; Simpson, Jeremy C

    2015-01-01

    ABSTRACT The Rab family of small GTPases play fundamental roles in the regulation of trafficking pathways between intracellular membranes in eukaryotic cells. In this short commentary we highlight a recent high-content screening study that investigates the roles of Rab proteins in retrograde trafficking from the Golgi complex to the endoplasmic reticulum, and we discuss how the findings of this work and other literature might influence our thoughts on how the architecture of the Golgi complex is regulated. PMID:26693811

  18. 2-Deoxy-D-glucose treatment changes the Golgi apparatus architecture without blocking synthesis of complex lipids.

    Science.gov (United States)

    Ranftler, Carmen; Meisslitzer-Ruppitsch, Claudia; Stangl, Herbert; Röhrl, Clemens; Fruhwürth, Stefanie; Neumüller, Josef; Pavelka, Margit; Ellinger, Adolf

    2015-04-01

    The classic Golgi apparatus organization, an arrangement of highly ordered cisternal stacks with tubular-vesicular membrane specializations on both sides, is the functional image of a continuous flow of contents and membranes with input, metabolization, and output in a dynamic steady state. In response to treatment with 2-deoxy-D-glucose (2-DG), which lowers the cellular ATP level by about 70% within minutes, this organization is rapidly replaced by tubular-glomerular membrane convolutes described as Golgi networks and bodies. 2-DG is a non-metabolizable glucose analogue and competitive inhibitor of glycolysis, which has become attractive in the context of therapeutic approaches for several kinds of tumors specifically targeting glycolysis in cancer. With the question of whether the functions of the Golgi apparatus in lipid synthesis would be influenced by the 2-DG-induced Golgi apparatus reorganization, we focused on lipid metabolism within the Golgi bodies. For this, we applied a fluorophore-labeled short-chain ceramide (BODIPY-Cer) in various combinations with 2-DG treatment to HepG2 cell cultures and followed uptake, enrichment and metabolization to higher ordered lipids. The cellular ATP status in each experiment was controlled with a bioluminescence assay, and the response of the Golgi apparatus was tracked by immunostaining of the trans-Golgi network protein TGN46. For electron microscopy, the fluorescent BODIPY-Cer signals were converted into electron-dense precipitates by photooxidation of diaminobenzidine (DAB); DAB precipitates labeled trans-Golgi areas in control cultures but also compartments at the periphery of the Golgi bodies formed in response to 2-DG treatment, thus indicating that concentration of ceramide takes place in spite of the Golgi apparatus reorganization. Lipid analyses by thin-layer chromatography (TLC) performed in parallel showed that BODIPY-Cer is not only concentrated in compartments of the 2-DG-induced Golgi bodies but is partly

  19. Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery

    Science.gov (United States)

    Pokrovskaya, Irina D; Willett, Rose; Smith, Richard D; Morelle, Willy; Kudlyk, Tetyana; Lupashin, Vladimir V

    2011-01-01

    Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery. PMID:21421995

  20. Golgi phosphoprotein 2 in physiology and in diseases

    Directory of Open Access Journals (Sweden)

    Kim Ha-Jeong

    2012-09-01

    Full Text Available Abstract Golgi phosphoprotein 2 (GOLPH2, also termed GP73 and GOLM1 is a type II transmembrane protein residing in the cis and medial-Golgi cisternae. GOLPH2 is predominantly expressed in the epithelial cells of many human tissues. Under poorly defined circumstances, GOLPH2 can be cleaved and released to the extracellular space. Despite of its relatively “young age” since the first description in 2000, the physiological and pathological roles of GOLPH2 have been the subject that has attracted considerable amount of attention in recent years. Here, we review the history of GOLPH2’s discovery and the multitude of studies by many groups around the world aimed at understanding its molecular, cellular, physiological, and pathogenic activities in various settings.

  1. The centrosome-Golgi apparatus nexus.

    Science.gov (United States)

    Rios, Rosa M

    2014-09-05

    A shared feature among all microtubule (MT)-dependent processes is the requirement for MTs to be organized in arrays of defined geometry. At a fundamental level, this is achieved by precisely controlling the timing and localization of the nucleation events that give rise to new MTs. To this end, MT nucleation is restricted to specific subcellular sites called MT-organizing centres. The primary MT-organizing centre in proliferating animal cells is the centrosome. However, the discovery of MT nucleation capacity of the Golgi apparatus (GA) has substantially changed our understanding of MT network organization in interphase cells. Interestingly, MT nucleation at the Golgi apparently relies on multiprotein complexes, similar to those present at the centrosome, that assemble at the cis-face of the organelle. In this process, AKAP450 plays a central role, acting as a scaffold to recruit other centrosomal proteins important for MT generation. MT arrays derived from either the centrosome or the GA differ in their geometry, probably reflecting their different, yet complementary, functions. Here, I review our current understanding of the molecular mechanisms involved in MT nucleation at the GA and how Golgi- and centrosome-based MT arrays work in concert to ensure the formation of a pericentrosomal polarized continuous Golgi ribbon structure, a critical feature for cell polarity in mammalian cells. In addition, I comment on the important role of the Golgi-nucleated MTs in organizing specialized MT arrays that serve specific functions in terminally differentiated cells.

  2. Alpha- and gamma-motoneurons in the adult human spinal cord and somatic cranial nerve nuclei: the significance of dendroachitectonics studied by the Golgi method.

    Science.gov (United States)

    Abdel-Maguid, T E; Bowsher, D

    1979-07-15

    A modified Golgi method (Vaisamruat and Hess, '53) was found to give satisfactory impregnation of cell bodies and dendrites, but not of dendritic spines and axons, in adult human material fixed by immersion in formalin. Examination of the motor columns in the spinal cord intumescences and of the third and twelfth cranial nerve nuclei revealed four neuron types, based on dendritic field size and dendritic branching pattern. Two of these were recognized as alpha-motoneurons; one of them was seen only in the medial motor column of the spinal ventral horn, while the other was observed in the cranial motor nuclei as well as the spinal lateral motor column. Differences in somadendritic dimensions in this neuron type were thought to reflect motor unit size, and thus terminal axon field dimensions. Of the two types of gamma-motoneurons recognized in the spinal cord and oculomotor nucleus, one was a miniature version of the commoner type of alpha-motoneuron. On this basis, it is proposed that it may give rise to fusimotor axons with plate endings. The second type of gamma-motoneuron does not resemble any of the other motoneuron types, and its axons may therefore be thought to terminate in trail endings.

  3. Dynamic changes of the Golgi apparatus during bovine in vitro oocyte maturation.

    Science.gov (United States)

    Racedo, S E; Rawe, V Y; Niemann, H

    2012-04-01

    For successful fertilization by the male gamete, oocyte cytoplasmic organelles such as the Golgi apparatus have to undergo specific changes: the entire process is known as cytoplasmic maturation. The goal of this study was to unravel the dynamics of the Golgi apparatus in bovine oocytes at critical stages of in vitro maturation, i.e. germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI) and metaphase II, and to investigate the role of various molecules critically involved therein. The cytoplasmic distribution of proteins was assessed by immunocytochemistry and laser confocal microscopy. We applied specific inhibitors, including nocodazole to unravel the functional role of the microtubular elements; sodium orthovanadate, which primarily inhibits cytoplasmic dynein ATPase activity; monastrol which inhibits the kinesin EG5; and roscovitine to inhibit the kinase cyclin-dependent kinase 2A (CDC2A). Prior to GVBD, the Golgi apparatus was translocated from the centre of the cytoplasm to the cortical area in the periphery, where it underwent fragmentation. A second translocation was observed between GVBD and MI stages, when the Golgi apparatus was moved from the cortex to the centre of the cytoplasm. Incubation with the specific inhibitors revealed that microtubules played an active role in the final localization at GVBD, while CDC2A was essential for Golgi fragmentation at GVBD stage. This partitioning was a precondition for the second movement. In conclusion, for the first time we show basic mechanisms critically involved in the regulation of the dynamic changes of Golgi apparatus during meiosis of the bovine oocyte.

  4. Evidence for a Golgi-to-endosome protein sorting pathway in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Priscilla Krai

    Full Text Available During the asexual intraerythrocytic stage, the malaria parasite Plasmodium falciparum must traffic newly-synthesized proteins to a broad array of destinations within and beyond the parasite's plasma membrane. In this study, we have localized two well-conserved protein components of eukaryotic endosomes, the retromer complex and the small GTPase Rab7, to define a previously-undescribed endosomal compartment in P. falciparum. Retromer and Rab7 co-localized to a small number of punctate structures within parasites. These structures, which we refer to as endosomes, lie in close proximity to the Golgi apparatus and, like the Golgi apparatus, are inherited by daughter merozoites. However, the endosome is clearly distinct from the Golgi apparatus as neither retromer nor Rab7 redistributed to the endoplasmic reticulum upon brefeldin A treatment. Nascent rhoptries (specialized secretory organelles required for invasion developed adjacent to endosomes, an observation that suggests a role for the endosome in rhoptry biogenesis. A P. falciparum homolog of the sortilin family of protein sorting receptors (PfSortilin was localized to the Golgi apparatus. Together, these results elaborate a putative Golgi-to-endosome protein sorting pathway in asexual blood stage parasites and suggest that one role of retromer is to mediate the retrograde transport of PfSortilin from the endosome to 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. Formation and maintenance of the Golgi apparatus in plant cells.

    Science.gov (United States)

    Ito, Yoko; Uemura, Tomohiro; Nakano, Akihiko

    2014-01-01

    The Golgi apparatus plays essential roles in intracellular trafficking, protein and lipid modification, and polysaccharide synthesis in eukaryotic cells. It is well known for its unique stacked structure, which is conserved among most eukaryotes. However, the mechanisms of biogenesis and maintenance of the structure, which are deeply related to ER-Golgi and intra-Golgi transport systems, have long been mysterious. Now having extremely powerful microscopic technologies developed for live-cell imaging, the plant Golgi apparatus provides an ideal system to resolve the question. The plant Golgi apparatus has unique features that are not conserved in other kingdoms, which will also give new insights into the Golgi functions in plant life. In this review, we will summarize the features of the plant Golgi apparatus and transport mechanisms around it, with a focus on recent advances in Golgi biogenesis by live imaging of plants cells. © 2014 Elsevier Inc. All rights reserved.

  7. Golgi cell activity during eyeblink conditioning in decerebrate ferrets.

    Science.gov (United States)

    Rasmussen, A; Zucca, R; Jirenhed, D-A; Johansson, F; Ortenblad, C; Svensson, P; Hesslow, G

    2014-02-01

    Golgi cells have a central position in the cerebellar cortical network and are indirectly connected to Purkinje cells, which are important for the acquisition of learned responses in classical conditioning. In order to clarify the role of Golgi cells in classical conditioning, we made extracellular Golgi cell recordings during different stages of conditioning, using four different conditional stimuli. Our results show that forelimb and superior colliculus stimulation, but not mossy fiber stimulation, evokes a short latency increase in Golgi cell firing. These results suggest that Golgi cells are involved in modulating input to the cerebellar cortex. There were however no differences in Golgi cell activity between naïve and trained animals, which suggests that Golgi cells are not intimately involved in the plastic changes that occur during classical conditioning. The absence of long latency effects of the conditional stimulus also questions whether Golgi cells contribute to the generation of a temporal code in the granule cells.

  8. ER trapping reveals Golgi enzymes continually revisit the ER through a recycling pathway that controls Golgi organization

    Science.gov (United States)

    Sengupta, Prabuddha; Satpute-Krishnan, Prasanna; Seo, Arnold Y.; Burnette, Dylan T.; Patterson, George H.; Lippincott-Schwartz, Jennifer

    2015-01-01

    Whether Golgi enzymes remain localized within the Golgi or constitutively cycle through the endoplasmic reticulum (ER) is unclear, yet is important for understanding Golgi dependence on the ER. Here, we demonstrate that the previously reported inefficient ER trapping of Golgi enzymes in a rapamycin-based assay results from an artifact involving an endogenous ER-localized 13-kD FK506 binding protein (FKBP13) competing with the FKBP12-tagged Golgi enzyme for binding to an FKBP-rapamycin binding domain (FRB)-tagged ER trap. When we express an FKBP12-tagged ER trap and FRB-tagged Golgi enzymes, conditions precluding such competition, the Golgi enzymes completely redistribute to the ER upon rapamycin treatment. A photoactivatable FRB-Golgi enzyme, highlighted only in the Golgi, likewise redistributes to the ER. These data establish Golgi enzymes constitutively cycle through the ER. Using our trapping scheme, we identify roles of rab6a and calcium-independent phospholipase A2 (iPLA2) in Golgi enzyme recycling, and show that retrograde transport of Golgi membrane underlies Golgi dispersal during microtubule depolymerization and mitosis. PMID:26598700

  9. Rab6a/a' are important Golgi regulators of pro-inflammatory TNF secretion in macrophages.

    Science.gov (United States)

    Micaroni, Massimo; Stanley, Amanda C; Khromykh, Tatiana; Venturato, Juliana; Wong, Colin X F; Lim, Jet P; Marsh, Brad J; Storrie, Brian; Gleeson, Paul A; Stow, Jennifer L

    2013-01-01

    Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6-GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages.

  10. Human rhinovirus 16 causes Golgi apparatus fragmentation without blocking protein secretion.

    Science.gov (United States)

    Mousnier, Aurelie; Swieboda, Dawid; Pinto, Anaïs; Guedán, Anabel; Rogers, Andrew V; Walton, Ross; Johnston, Sebastian L; Solari, Roberto

    2014-10-01

    The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. Importance: The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be

  11. Molecular Pathway of Microtubule Organization at the Golgi Apparatus

    NARCIS (Netherlands)

    Wu, Jingchao; de Heus, Cecilia; Liu, Qingyang|info:eu-repo/dai/nl/375265147; Bouchet, Benjamin P|info:eu-repo/dai/nl/371636019; Noordstra, Ivar; Jiang, Kai|info:eu-repo/dai/nl/374338094; Hua, Shasha|info:eu-repo/dai/nl/377295698; Martin, Maud; Yang, Chao; Grigoriev, Ilya; Katrukha, Eugene A; Altelaar, A F Maarten|info:eu-repo/dai/nl/304833517; Hoogenraad, Casper C|info:eu-repo/dai/nl/227263502; Qi, Robert Z; Klumperman, Judith; Akhmanova, Anna|info:eu-repo/dai/nl/156410591

    2016-01-01

    The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and

  12. 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

  13. Golgi maturation visualized in living yeast.

    Science.gov (United States)

    Losev, Eugene; Reinke, Catherine A; Jellen, Jennifer; Strongin, Daniel E; Bevis, Brooke J; Glick, Benjamin S

    2006-06-22

    The Golgi apparatus is composed of biochemically distinct early (cis, medial) and late (trans, TGN) cisternae. There is debate about the nature of these cisternae. The stable compartments model predicts that each cisterna is a long-lived structure that retains a characteristic set of Golgi-resident proteins. In this view, secretory cargo proteins are transported by vesicles from one cisterna to the next. The cisternal maturation model predicts that each cisterna is a transient structure that matures from early to late by acquiring and then losing specific Golgi-resident proteins. In this view, secretory cargo proteins traverse the Golgi by remaining within the maturing cisternae. Various observations have been interpreted as supporting one or the other mechanism. Here we provide a direct test of the two models using three-dimensional time-lapse fluorescence microscopy of the yeast Saccharomyces cerevisiae. This approach reveals that individual cisternae mature, and do so at a consistent rate. In parallel, we used pulse-chase analysis to measure the transport of two secretory cargo proteins. The rate of cisternal maturation matches the rate of protein transport through the secretory pathway, suggesting that cisternal maturation can account for the kinetics of secretory traffic.

  14. The Compartmental Organization of the Golgi Apparatus.

    Science.gov (United States)

    Rothman, James E.

    1985-01-01

    Relations between structure and function of the Golgi apparatus are emerging from recent laboratory work on this cellular organelle which modifies proteins, sorts them, and packages them for delivery. The structure's three specialized compartments are explained through discussions of the glycosylation pathway, density-gradient experiments,…

  15. A polygalacturonase localized in the Golgi apparatus in Pisum sativum.

    Science.gov (United States)

    Ohashi, Takao; Jinno, Jun; Inoue, Yoshiyuki; Ito, Shoko; Fujiyama, Kazuhito; Ishimizu, Takeshi

    2017-09-01

    Pectin is a plant cell wall constituent that is mainly composed of polygalacturonic acid (PGA), a linear α1,4-d-galacturonic acid (GalUA) backbone. Polygalacturonase (PG) hydrolyzes the α1,4-linkages in PGA. Nearly all plant PGs identified thus far are secreted as soluble proteins. Here we describe the microsomal PG activity in pea (Pisum sativum) epicotyls and present biochemical evidence that it was localized to the Golgi apparatus, where pectins are biosynthesized. The microsomal PG was purified, and it was enzymatically characterized. The purified enzyme showed maximum activity towards pyridylaminated oligogalacturonic acids with six degrees of polymerization (PA-GalUA6), with a Km value of 11 μM for PA-GalUA6. The substrate preference of the enzyme was complementary to that of PGA synthase. The main PG activity in microsomes was detected in the Golgi fraction by sucrose density gradient ultracentrifugation. The activity of the microsomal PG was lower in rapidly growing epicotyls, in contrast to the high expression of PGA synthase. The role of this PG in the regulation of pectin biosynthesis or plant growth is discussed. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  16. How the 1906 Nobel Prize in Physiology or Medicine was shared between Golgi and Cajal.

    Science.gov (United States)

    Grant, Gunnar

    2007-10-01

    In 1906 the Nobel Prize in Physiology or Medicine was shared between Camillo Golgi and Ramón y Cajal in recognition of their work on the structure of the nervous system. Golgi's most impressive contribution was his method, described in 1873. This was applied in studies of the cerebellum, the olfactory bulb, hippocampus and the spinal cord. These studies together with his earlier work were included in his Opera Omnia, published in 1903. His method was highly praised by Cajal. His adherence to the reticular theory was opposed by Cajal, however, who had spelled out the neuron theory already in the late 1800s. Cajal's extraordinary contributions to the structure of the nervous system, based largely on the Golgi method and Ehrlich's methylene blue stain, were published in his Textura del Sistema Nerviosa de Hombre y de los Vertebrados, three volumes published from 1897 to 1904. Documents from the Nobel Archives reveal that Kölliker, Retzius and Fürst were the ones who proposed Golgi and Cajal for a shared prize. Golgi was nominated by Hertwig, as well. Cajal was proposed by Ziehen and Holmgren, and also by Retzius, as an alternative to a shared prize. Holmgren, who was commissioned to write the report to the Nobel Committee, found Cajal far superior to Golgi. Sundberg, asked for another evaluation, was more positive to Golgi's contributions than Holmgren. Gadelius supported Holmgren's views. The final vote gave a majority for a shared prize. The prize ceremony and the lectures were described in detail in Cajal's autobiography.

  17. PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus.

    Science.gov (United States)

    Jin, Ting; Ding, Qiurong; Huang, Heng; Xu, Daqian; Jiang, Yuhui; Zhou, Ben; Li, Zhenghu; Jiang, Xiaomeng; He, Jing; Liu, Weizhong; Zhang, Yixuan; Pan, Yi; Wang, Zhenzhen; Thomas, Walter G; Chen, Yan

    2012-04-01

    Ras plays a pivotal role in many cellular activities, and its subcellular compartmentalization provides spatial and temporal selectivity. Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family. PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus. Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner. Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas, NRas and KRas4A, but not KRas4B. PAQR10 and PAQR11 can also interact with HRas, NRas and KRas4A, but not KRas4B. The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state. Consistently, knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus. Intriguingly, PAQR10 and PAQR11 are able to interact with RasGRP1, a guanine nucleotide exchange protein of Ras, and increase Golgi localization of RasGRP1. The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11. The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1. Furthermore, differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11. Collectively, this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11.

  18. Molecular Pathway of Microtubule Organization at the Golgi Apparatus.

    Science.gov (United States)

    Wu, Jingchao; de Heus, Cecilia; Liu, Qingyang; Bouchet, Benjamin P; Noordstra, Ivar; Jiang, Kai; Hua, Shasha; Martin, Maud; Yang, Chao; Grigoriev, Ilya; Katrukha, Eugene A; Altelaar, A F Maarten; Hoogenraad, Casper C; Qi, Robert Z; Klumperman, Judith; Akhmanova, Anna

    2016-10-10

    The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myomegalin. CLASPs stabilize CAMSAP2-decorated microtubules but are not required for their Golgi tethering. AKAP450 is also essential for Golgi microtubule nucleation, and myomegalin and CDK5RAP2 but not CAMSAP2 contribute to this function. In the absence of centrosomes, AKAP450- and CAMSAP2-dependent pathways of microtubule minus-end organization become dominant, and the presence of at least one of them is needed to maintain microtubule density. Strikingly, a compact Golgi can be assembled in the absence of both centrosomal and Golgi microtubules. However, CAMSAP2- and AKAP450-dependent Golgi microtubules facilitate Golgi reorientation and cell invasion in a 3D matrix. We propose that Golgi-anchored microtubules are important for polarized cell movement but not for coalescence of Golgi membranes. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Inter-Golgi transport mediated by COPI-containing vesicles carrying small cargoes

    Science.gov (United States)

    Pellett, Patrina A; Dietrich, Felix; Bewersdorf, Jörg; Rothman, James E; Lavieu, Grégory

    2013-01-01

    A core prediction of the vesicular transport model is that COPI vesicles are responsible for trafficking anterograde cargoes forward. In this study, we test this prediction by examining the properties and requirements of inter-Golgi transport within fused cells, which requires mobile carriers in order for exchange of constituents to occur. We report that both small soluble and membrane-bound secretory cargo and exogenous Golgi resident glycosyl-transferases are exchanged between separated Golgi. Large soluble aggregates, which traverse individual stacks, do not transfer between Golgi, implying that small cargoes (which can fit in a typical transport vesicle) are transported by a different mechanism. Super-resolution microscopy reveals that the carriers of both anterograde and retrograde cargoes are the size of COPI vesicles, contain coatomer, and functionally require ARF1 and coatomer for transport. The data suggest that COPI vesicles traffic both small secretory cargo and steady-state Golgi resident enzymes among stacked cisternae that are stationary. DOI: http://dx.doi.org/10.7554/eLife.01296.001 PMID:24137546

  20. The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate

    Directory of Open Access Journals (Sweden)

    C Soldani

    2009-06-01

    Full Text Available The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vescicle traffic (i.e., microtubules might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells.

  1. Golgi disruption and early embryonic lethality in mice lacking USO1.

    Directory of Open Access Journals (Sweden)

    Susie Kim

    Full Text Available Golgins are a family of long rod-like proteins characterized by the presence of central coiled-coil domains. Members of the golgin family have important roles in membrane trafficking, where they function as tethering factors that capture transport vesicles and facilitate membrane fusion. Golgin family members also have essential roles in maintaining the organization of the Golgi apparatus. Knockdown of individual golgins in cultured cells resulted in the disruption of the Golgi structure and the dispersal of Golgi marker proteins throughout the cytoplasm. However, these cellular phenotypes have not always been recapitulated in vivo. For example, embryonic development proceeds much further than expected and Golgi disruption was observed in only a subset of cell types in mice lacking the ubiquitously expressed golgin GMAP-210. Cell-type specific functional compensation among golgins may explain the absence of global cell lethality when a ubiquitously expressed golgin is missing. In this study we show that functional compensation does not occur for the golgin USO1. Mice lacking this ubiquitously expressed protein exhibit disruption of Golgi structure and early embryonic lethality, indicating that USO1 is indispensable for early embryonic development.

  2. 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.

  3. C11ORF24 is a novel type I membrane protein that cycles between the Golgi apparatus and the plasma membrane in Rab6-positive vesicles.

    Science.gov (United States)

    Fraisier, Vincent; Kasri, Amal; Miserey-Lenkei, Stéphanie; Sibarita, Jean-Baptiste; Nair, Deepak; Mayeux, Adeline; Bardin, Sabine; Toyoda, Yusuke; Poser, Ina; Poznyakovskiy, Andrei; Goud, Bruno; Hyman, Anthony A; Dimitrov, Ariane

    2013-01-01

    The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.

  4. A non-enzymatic function of Golgi glycosyltransferases: mediation of Golgi fragmentation by interaction with non-muscle myosin IIA.

    Science.gov (United States)

    Petrosyan, Armen; Cheng, Pi-Wan

    2013-06-01

    The Golgi apparatus undergoes morphological changes under stress or malignant transformation, but the precise mechanisms are not known. We recently showed that non-muscle myosin IIA (NMIIA) binds to the cytoplasmic tail of Core 2 N-acetylglucosaminyltransferase mucus-type (C2GnT-M) and transports it to the endoplasmic reticulum for recycling. Here, we report that Golgi fragmentation induced by brefeldin A (BFA) or coatomer protein (β-COP) knockdown (KD) in Panc1-bC2GnT-M (c-Myc) cells is accompanied by the increased association of NMIIA with C2GnT-M and its degradation by proteasomes. Golgi fragmentation is prevented by inhibition or KD of NMIIA. Using multiple approaches, we have shown that the speed of BFA-induced Golgi fragmentation is positively correlated with the levels of this enzyme in the Golgi. The observation is reproduced in LNCaP cells which express high levels of two endogenous glycosyltransferases--C2GnT-L and β-galactoside α2,3 sialyltransferase 1. NMIIA is found to form complexes with these two enzymes but not Golgi matrix proteins. The KD of both enzymes or the prevention of Golgi glycosyltransferases from exiting endoplasmic reticulum reduced Golgi-associated NMIIA and decreased the BFA-induced fragmentation. Interestingly, the fragmented Golgi detected in colon cancer HT-29 cells can be restored to a compact morphology after inhibition or KD of NMIIA. The Golgi disorganization induced by the microtubule or actin destructive agent is NMIIA-independent and does not affect the levels of glycosyltransferases. We conclude that NMIIA interacts with Golgi residential but not matrix proteins, and this interaction is responsible for Golgi fragmentation induced by β-COP KD or BFA treatment. This is a novel non-enzymatic function of Golgi glycosyltransferases.

  5. A non-enzymatic function of Golgi glycosyltransferases: Mediation of Golgi fragmentation by interaction with non-muscle myosin IIA

    Science.gov (United States)

    Petrosyan, Armen; Cheng, Pi-Wan

    2013-01-01

    The Golgi apparatus undergoes morphological changes under stress or malignant transformation, but the precise mechanisms are not known. We recently showed that non-muscle myosin IIA (NMIIA) binds to the cytoplasmic tail of Core 2 N-acetylglucosaminyltransferase mucus-type (C2GnT-M) and transports it to the endoplasmic reticulum for recycling. Here, we report that Golgi fragmentation induced by brefeldin A (BFA) or coatomer protein (β-COP) knockdown (KD) in Panc1-bC2GnT-M (c-Myc) cells is accompanied by the increased association of NMIIA with C2GnT-M and its degradation by proteasomes. Golgi fragmentation is prevented by inhibition or KD of NMIIA. Using multiple approaches, we have shown that the speed of BFA-induced Golgi fragmentation is positively correlated with the levels of this enzyme in the Golgi. The observation is reproduced in LNCaP cells which express high levels of two endogenous glycosyltransferases—C2GnT-L and β-galactoside α2,3 sialyltransferase 1. NMIIA is found to form complexes with these two enzymes but not Golgi matrix proteins. The KD of both enzymes or the prevention of Golgi glycosyltransferases from exiting endoplasmic reticulum reduced Golgi-associated NMIIA and decreased the BFA-induced fragmentation. Interestingly, the fragmented Golgi detected in colon cancer HT-29 cells can be restored to a compact morphology after inhibition or KD of NMIIA. The Golgi disorganization induced by the microtubule or actin destructive agent is NMIIA-independent and does not affect the levels of glycosyltransferases. We conclude that NMIIA interacts with Golgi residential but not matrix proteins, and this interaction is responsible for Golgi fragmentation induced by β-COP KD or BFA treatment. This is a novel non-enzymatic function of Golgi glycosyltransferases. PMID:23396488

  6. Recognition and tethering of transport vesicles at the Golgi apparatus.

    Science.gov (United States)

    Witkos, Tomasz M; Lowe, Martin

    2017-08-01

    The Golgi apparatus occupies a central position within the secretory pathway where it is a hub for vesicle trafficking. Distinct classes of transport vesicles traffic diverse cargoes into and out of this organelle, as well as between the different Golgi subcompartments. A key feature of Golgi trafficking is the specific recognition of transport vesicles at the different regions of the Golgi apparatus, required for the correct cargo delivery. Specificity is ensured by coiled-coil golgins and multi-subunit tethering complexes (MTCs), which act together to capture vesicles and promote their subsequent fusion with the Golgi membrane. In this review we discuss our current understanding of how golgins and MTCs function together to mediate the specific recognition of vesicles at the Golgi apparatus. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. 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.

  8. The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells.

    Science.gov (United States)

    Cruz-García, David; Díaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R; Gracia-Navarro, Francisco; Castaño, Justo P; Montero-Hadjadje, Maité; Tonon, Marie-Christine; Vaudry, Hubert; Anouar, Youssef; Vázquez-Martínez, Rafael; Malagón, María M

    2012-04-15

    Golgi-associated long coiled-coil proteins, often referred to as golgins, are involved in the maintenance of the structural organization of the Golgi apparatus and the regulation of membrane traffic events occurring in this organelle. Little information is available on the contribution of golgins to Golgi function in cells specialized in secretion such as endocrine cells or neurons. In the present study, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, NECC1 (neuroendocrine long coiled-coil protein 1). The present study shows that NECC1 is a peripheral membrane protein displaying high stability to detergent extraction, which distributes across the Golgi apparatus in neuroendocrine cells. In addition, NECC1 partially localizes to post-Golgi carriers containing secretory cargo in PC12 cells. Overexpression of NECC1 resulted in the formation of juxtanuclear aggregates together with a slight fragmentation of the Golgi and a decrease in K+-stimulated hormone release. In contrast, NECC1 silencing did not alter Golgi architecture, but enhanced K+-stimulated hormone secretion in PC12 cells. In all, the results of the present study identify NECC1 as a novel component of the Golgi matrix and support a role for this protein as a negative modulator of the regulated trafficking of secretory cargo in neuroendocrine cells.

  9. Growth of the Mammalian Golgi Apparatus during Interphase.

    Science.gov (United States)

    Sin, Alex T-W; Harrison, Rene E

    2016-09-15

    During the cell cycle, genetic materials and organelles are duplicated to ensure that there is sufficient cellular content for daughter cells. While Golgi growth in interphase has been observed in lower eukaryotes, the elaborate ribbon structure of the mammalian Golgi apparatus has made it challenging to monitor. Here we demonstrate the growth of the mammalian Golgi apparatus in its protein content and volume during interphase. Through ultrastructural analyses, physical growth of the Golgi apparatus was revealed to occur by cisternal elongation of the individual Golgi stacks. By examining the timing and regulation of Golgi growth, we established that Golgi growth starts after passage through the cell growth checkpoint at late G1 phase and continues in a manner highly correlated with cell size growth. Finally, by identifying S6 kinase 1 as a major player in Golgi growth, we revealed the coordination between cell size and Golgi growth via activation of the protein synthesis machinery in early interphase. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. Putative glycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics.

    Science.gov (United States)

    Nikolovski, Nino; Rubtsov, Denis; Segura, Marcelo P; Miles, Godfrey P; Stevens, Tim J; Dunkley, Tom P J; Munro, Sean; Lilley, Kathryn S; Dupree, Paul

    2012-10-01

    The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains of the localized proteins showed that the single-span transmembrane domains have unique properties in each organelle. Many of the novel Golgi-localized proteins belong to uncharacterized protein families. Structure-based homology analysis identified 12 putative Golgi glycosyltransferase (GT) families that have no functionally characterized members and, therefore, are not yet assigned to a Carbohydrate-Active Enzymes database GT family. The substantial numbers of these putative GTs lead us to estimate that the true number of plant Golgi GTs might be one-third above those currently annotated. Other newly identified proteins are likely to be involved in the transport and interconversion of nucleotide sugar substrates as well as polysaccharide and protein modification.

  11. Subcortical auditory structures in the Mongolian gerbil: I. Golgi architecture.

    Science.gov (United States)

    Mylius, Judith; Brosch, Michael; Scheich, Henning; Budinger, Eike

    2013-04-15

    By means of the Golgi-Cox and Nissl methods we investigated the cyto- and fiberarchitecture as well as the morphology of neurons in the subcortical auditory structures of the Mongolian gerbil (Meriones unguiculatus), a frequently used animal model in auditory neuroscience. We describe the divisions and subdivisions of the auditory thalamus including the medial geniculate body, suprageniculate nucleus, and reticular thalamic nucleus, as well as of the inferior colliculi, nuclei of the lateral lemniscus, superior olivary complex, and cochlear nuclear complex. In this study, we 1) confirm previous results about the organization of the gerbil's subcortical auditory pathway using other anatomical staining methods (e.g., Budinger et al. [2000] Eur J Neurosci 12:2452-2474); 2) add substantially to the knowledge about the laminar and cellular organization of the gerbil's subcortical auditory structures, in particular about the orientation of their fibrodendritic laminae and about the morphology of their most distinctive neuron types; and 3) demonstrate that the cellular organization of these structures, as seen by the Golgi technique, corresponds generally to that of other mammalian species, in particular to that of rodents. Copyright © 2012 Wiley Periodicals, Inc.

  12. Golgi apparatus: finally mechanics comes to play in the secretory pathway.

    Science.gov (United States)

    Egea, Gustavo; Serra-Peinado, Carla

    2014-08-18

    New findings report a mechanical role for actin in Golgi organization and vesicular trafficking. An elegant study uses optical tweezers and live-cell imaging to demonstrate the effects of a mechanical constraint on the dynamics of secretory membrane trafficking, combining physical experimental approaches with in cellulo studies of endomembranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Implications of the Golgi apparatus in prostate cancer.

    Science.gov (United States)

    Migita, Toshiro; Inoue, Satoshi

    2012-11-01

    The classical view of the Golgi apparatus is of a small membranous organelle involved in protein transport and secretion. Recent descriptions of the molecular network connecting the Golgi to other organelles demonstrate the essential roles of the Golgi in cellular activities as a stress sensor, apoptosis trigger, lipid/protein modifier, mitotic checkpoint, and a mediator of malignant transformation. Thus, the Golgi function should have a fundamental impact on cancer cell survival. Prostate cancer is initially responsive to androgenic hormones; however, it almost invariably progresses to a castration-refractory or hormone-insensitive state. Nevertheless, androgen signaling remains active at this stage and is important as a therapeutic target. Certain Golgi-associated molecules have recently been demonstrated to be regulated by androgen action, and the Golgi is emerging as a new therapeutic target in prostate cancer. The key Golgi-associated molecules essential for prostate cancer development and the potential therapeutic options targeting the Golgi apparatus are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Golgi Localization of Glycosyltransferases Requires a Vps74p Oligomer

    Energy Technology Data Exchange (ETDEWEB)

    Schmitz, Karl R.; Liu, Jingxuan; Li, Shiqing; Setty, Thanuja Gangi; Wood, Christopher S.; Burd, Christopher G.; Ferguson, Kathryn M. (UPENN-MED)

    2010-02-19

    The mechanism of glycosyltransferase localization to the Golgi apparatus is a long-standing question in secretory cell biology. All Golgi glycosyltransferases are type II membrane proteins with small cytosolic domains that contribute to Golgi localization. To date, no protein has been identified that recognizes the cytosolic domains of Golgi enzymes and contributes to their localization. Here, we report that yeast Vps74p directly binds to the cytosolic domains of cis and medial Golgi mannosyltransferases and that loss of this interaction correlates with loss of Golgi localization of these enzymes. We have solved the X-ray crystal structure of Vps74p and find that it forms a tetramer, which we also observe in solution. Deletion of a critical structural motif disrupts tetramer formation and results in loss of Vps74p localization and function. Vps74p is highly homologous to the human GMx33 Golgi matrix proteins, suggesting a conserved function for these proteins in the Golgi enzyme localization machinery.

  15. Golgi Fragmentation in ALS Motor Neurons. New Mechanisms Targeting Microtubules, Tethers, and Transport Vesicles

    NARCIS (Netherlands)

    Haase, Georg; Rabouille, Catherine

    2015-01-01

    Pathological alterations of the Golgi apparatus, such as its fragmentation represent an early pre-clinical feature of many neurodegenerative diseases and have been widely studied in the motor neuron disease amyotrophic lateral sclerosis (ALS). Yet, the underlying molecular mechanisms have remained

  16. Staining of dead neurons by the Golgi method in autopsy material.

    Science.gov (United States)

    Baloyannis, Stavros J

    2015-01-01

    Golgi silver impregnation techniques remain ideal methods for the visualization of the neurons as a whole in formalin fixed brains and paraffin sections, enabling to obtain insight into the morphological and morphometric characters of the dendritic arbor, and the estimation of the morphology of the spines and the spinal density, since they delineate the profile of nerve cells with unique clarity and precision. In addition, the Golgi technique enables the study of the topographic relationships between neurons and neuronal circuits in normal conditions, and the following of the spatiotemporal morphological alterations occurring during degenerative processes. The Golgi technique has undergone many modifications in order to be enhanced and to obtain the optimal and maximal visualization of neurons and neuronal processes, the minimal precipitations, the abbreviation of the time required for the procedure, enabling the accurate study and description of specific structures of the brain. In the visualization of the sequential stages of the neuronal degeneration and death, the Golgi method plays a prominent role in the visualization of degenerating axons and dendrites, synaptic “boutons,” and axonal terminals and organelles of the cell body. In addition, new versions of the techniques increases the capacity of precise observation of the neurofibrillary degeneration, the proliferation of astrocytes, the activation of the microglia, and the morphology of capillaries in autopsy material of debilitating diseases of the central nervous system.

  17. Rapid sulfur capture studies at high temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Richards, G.A.; Lawson, W.F.; Maloney, D.J.; Shaw, D.W.

    1990-12-01

    Determine conditions that would reproduce optimum sulfur capture ( super-equilibrium'') behavior. No attempt was made to extract kinetic data for calcination or sulfur capture, as might be done in a comprehensive study of sorbent behavior. While some interesting anomalies are present in the calcination data and in the limited surface area data, no attempt was made to pursue those issues. Since little sulfur capture was observed at operating conditions where super-equilibrium'' might be expected to occur, tests were stopped when the wide range of parameters that were studied failed to produce significant sulfur capture via the super-equilibrium mechanism. Considerable space in this report is devoted to a description of the experiment, including details of the GTRC construction. This description is included because we have received requests for a detailed description of the GTRC itself, as well as the pressurized dry powder feed system. In addition, many questions about accurately sampling the sulfur species from a high-temperature, high-pressure reactor were raised during the course of this investigation. A full account of the development of the gas and particulate sampling train in thus provided. 8 refs., 17 figs., 2 tabs.

  18. Golgi sorting regulates organization and activity of GPI-proteins at apical membranes

    Science.gov (United States)

    Tivodar, Simona; Formiggini, Fabio; Ossato, Giulia; Gratton, Enrico; Tramier, Marc; Coppey-Moisan, Maïté; Zurzolo, Chiara

    2014-01-01

    Here, we combined classical biochemistry with novel biophysical approaches to study with high spatial and temporal resolution the organization of GPI-anchored proteins (GPI-APs) at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, following sorting in the Golgi, each GPI-AP reaches the apical surface in homo-clusters. Golgi-derived homo-clusters are required for their subsequent plasma membrane organization into cholesterol-dependent hetero-clusters. By contrast, in non-polarized MDCK cells GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form hetero-clusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, different from fibroblasts, in polarized epithelial cells a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and the function of GPI-APs at the apical surface. PMID:24681536

  19. 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

  20. A KDEL Retrieval System for ER-Golgi Transport of Japanese Encephalitis Viral Particles.

    Science.gov (United States)

    Wang, Robert Y L; Wu, Yu-Jen; Chen, Han-Shan; Chen, Chih-Jung

    2016-02-05

    Evidence has emerged that RNA viruses utilize the host secretory pathway for processing and trafficking mature viral particles and for exiting the infected cells. Upon completing the complex assembly process, the viral particles take advantage of the cellular secretory trafficking machinery for their intracellular trafficking toward the Golgi organelle and budding or export of virions. In this study, we showed that Japanese encephalitis virus (JEV)-induced extracellular GRP78 contains no KDEL motif using an anti-KDEL-specific antibody. Overexpression of the KDEL-truncated GRP78 in the GPR78 knocked down cells significantly reduced JEV infectivity, suggesting that the KDEL motif is required for GRP78 function in the release of JE viral particles. In addition, we demonstrated the KDELR protein, an ER-Golgi retrieval system component, is associated with viral envelope proteins and is engaged in the subcellular localization of viral particles in Golgi. More importantly, accumulation of intracellular virions was observed in the KDELR knocked down cells, indicating that the KDELR protein mediated the intracellular trafficking of JE viral particles. Altogether, we demonstrated that intracellular trafficking of JE assembled viral particles was mediated by the host ER-Golgi retrieval system prior to exit by the secretory pathway.

  1. The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis.

    Science.gov (United States)

    Kato, Shin; Chen, Jingsi; Cornog, Katherine H; Zhang, Huili; Roberts, Jesse D

    2015-06-01

    cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction. Copyright © 2015 the American Physiological Society.

  2. Proteoglycan synthesis and Golgi organization in polarized epithelial cells.

    Science.gov (United States)

    Dick, Gunnar; Akslen-Hoel, Linn K; Grøndahl, Frøy; Kjos, Ingrid; Prydz, Kristian

    2012-12-01

    A large number of complex glycosylation mechanisms take place in the Golgi apparatus. In epithelial cells, glycosylated protein molecules are transported to both the apical and the basolateral surface domains. Although the prevailing view is that the Golgi apparatus provides the same lumenal environment for glycosylation of apical and basolateral cargo proteins, there are indications that proteoglycans destined for the two opposite epithelial surfaces are exposed to different conditions in transit through the Golgi apparatus. We will here review data relating proteoglycan and glycoprotein synthesis to characteristics of the apical and basolateral secretory pathways in epithelial cells.

  3. A unique ball-shaped Golgi apparatus in the rat pituitary gonadotrope: its functional implications in relation to the arrangement of the microtubule network.

    Science.gov (United States)

    Watanabe, Tsuyoshi; Sakai, Yuko; Koga, Daisuke; Bochimoto, Hiroki; Hira, Yoshiki; Hosaka, Masahiro; Ushiki, Tatsuo

    2012-08-01

    In polarized exocrine cells, the Golgi apparatus is cup-shaped and its convex and concave surfaces are designated as cis and trans faces, functionally confronting the rough endoplasmic reticulum and the cell surface, respectively. To clarify the morphological characteristics of the Golgi apparatus in non-polarized endocrine cells, the investigators immunocytochemically examined its precise architecture in pituitary gonadotropes, especially in relation to the arrangement of the intracellular microtubule network. The Golgi apparatus in the gonadotropes was not cup-shaped but ball-shaped or spherical, and its outer and inner surfaces were the cis and trans faces, respectively. Centrioles were situated at the center of the Golgi apparatus, from which radiating microtubules isotropically extended to the cell periphery through the gaps in the spherical wall of the Golgi stack. The shape of the Golgi apparatus and the arrangement of microtubules demonstrated in the present study could explain the microtubule-dependent movements of tubulovesicular carriers and granules within the gonadotropes. Furthermore, the spherical shape of the Golgi apparatus possibly reflects the highly symmetrical arrangement of microtubule arrays, as well as the poor polarity in the cell surface of pituitary gonadotropes.

  4. Reconstitution of the targeting of Rab6A to the Golgi apparatus in semi-intact HeLa cells: A role of BICD2 in stabilizing Rab6A on Golgi membranes and a concerted role of Rab6A/BICD2 interactions in Golgi-to-ER retrograde transport.

    Science.gov (United States)

    Matsuto, Mariko; Kano, Fumi; Murata, Masayuki

    2015-10-01

    Rab is a small GTP-binding protein family that regulates various pathways of vesicular transport. Although more than 60 Rab proteins are targeted to specific organelles in mammalian cells, the mechanisms underlying the specificity of Rab proteins for the respective organelles remain unknown. In this study, we reconstituted the Golgi targeting of Rab6A in streptolysin O (SLO)-permeabilized HeLa cells in a cytosol-dependent manner and investigated the biochemical requirements of targeting. Golgi-targeting assays identified Bicaudal-D (BICD)2, which is reportedly involved in the dynein-mediated transport of mRNAs during oogenesis and embryogenesis in Drosophila, as a cytosolic factor for the Golgi targeting of Rab6A in SLO-permeabilized HeLa cells. Subsequent immunofluorescence analyses indicated decreased amounts of the GTP-bound active form of Rab6 in BICD2-knockdown cells. In addition, fluorescence recovery after photobleaching (FRAP) analyses revealed that overexpression of the C-terminal region of BICD2 decreased the exchange rate of GFP-Rab6A between the Golgi membrane and the cytosol. Collectively, these results indicated that BICD2 facilitates the binding of Rab6A to the Golgi by stabilizing its GTP-bound form. Moreover, several analyses of vesicular transport demonstrated that Rab6A and BICD2 play crucial roles in Golgi tubule fusion with the endoplasmic reticulum (ER) in brefeldin A (BFA)-treated cells, indicating that BICD2 is involved in coat protein I (COPI)-independent Golgi-to-ER retrograde vesicular transport. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. A review of rapid solidification studies of intermetallic compounds

    Science.gov (United States)

    Koch, C. C.

    1985-01-01

    A review of rapid solidification studies of high-temperature ordered intermetallic compounds is presented. Emphasis is on the nickel - and iron- aluminides which are of potential interest as structural materials. The nickel-base aluminides which have been rapidly solidified exhibit changes in grain size, compositional segregation, and degree of long range order (as reflected in APB size and distribution) which markedly affect mechanical properties. Some experiments indicate the formation of a metastable L1(2) phase in rapidly solidified Fe-(Ni,Mn)-Al-C alloys, while other work observes only a metastable fcc phase in the same composition range. The metastable phases and/or microstructures in both nickel and iron aluminides are destroyed by annealing at temperatures above 750 K, with subsequent degradation of mechanical properties. Rapid solidification studies of several other intermetallic compounds are briefly noted.

  6. Atlastin GTPases are required for Golgi apparatus and ER morphogenesis

    National Research Council Canada - National Science Library

    Rismanchi, Neggy; Soderblom, Cynthia; Stadler, Julia; Zhu, Peng-Peng; Blackstone, Craig

    2008-01-01

    .... Interestingly, while atlastin-1 is predominantly localized to vesicular tubular complexes and cis-Golgi cisternae, mostly in brain, atlastin-2 and -3 are localized to the endoplasmic reticulum (ER...

  7. Rapid HIV-1 testing during labor: a multicenter study.

    Science.gov (United States)

    Bulterys, Marc; Jamieson, Denise J; O'Sullivan, Mary Jo; Cohen, Mardge H; Maupin, Robert; Nesheim, Steven; Webber, Mayris P; Van Dyke, Russell; Wiener, Jeffrey; Branson, Bernard M

    2004-07-14

    Timely testing of women in labor with undocumented human immunodeficiency virus (HIV) status could enable immediate provision of antiretroviral prophylaxis. To determine the feasibility and acceptance of rapid HIV testing among women in labor and to assess rapid HIV assay performance. The Mother-Infant Rapid Intervention At Delivery (MIRIAD) study implemented 24-hour counseling and voluntary rapid HIV testing for women in labor at 16 US hospitals from November 16, 2001, through November 15, 2003. A rapid HIV-1 antibody test for whole blood was used. Acceptance of HIV testing; sensitivity, specificity, and predictive value of the rapid test; time from blood collection to patient notification of results. There were 91,707 visits to the labor and delivery units in the study, 7381 of which were by eligible women without documentation of HIV testing. Of these, 5744 (78%) women were approached for rapid HIV testing and 4849 (84%) consented. HIV-1 test results were positive for 34 women (prevalence = 7/1000). Sensitivity and specificity of the rapid test were 100% and 99.9%, respectively; positive predictive value was 90% compared with 76% for enzyme immunoassay (EIA). Factors independently associated with higher test acceptance included younger age, being black or Hispanic, gestational age less than 32 weeks, and having had no prenatal care. Lower acceptance was associated with being admitted between 4 pm and midnight, particularly on Friday nights, but this may be explained in part by fewer available personnel. Median time from blood collection to patient notification of result was 66 minutes (interquartile range, 45-120 minutes), compared with 28 hours for EIA (PHIV testing is feasible and delivers accurate and timely test results for women in labor. It provides HIV-positive women prompt access to intrapartum and neonatal antiretroviral prophylaxis, proven to reduce perinatal HIV transmission, and may be particularly applicable to higher-risk populations.

  8. Repositioning of Somatic Golgi Apparatus Is Essential for the Dendritic Establishment of Adult-Born Hippocampal Neurons.

    Science.gov (United States)

    Rao, Sneha; Kirschen, Gregory W; Szczurkowska, Joanna; Di Antonio, Adrian; Wang, Jia; Ge, Shaoyu; Shelly, Maya

    2018-01-17

    functional contribution of these newborn neurons to the existing hippocampal circuit and associated behaviors, while the molecular mechanisms controlling their early morphological integration are less well understood. Dentate granule cells (DGCs) have a single, complex, apical dendrite. The events leading adult-born DGCs' to transition from simple spindle-like morphology to mature dendrite morphology are largely unknown. We studied establishment of newborn DGCs dendritic pattern and found it was mediated by a signaling pathway regulating precise localization of the Golgi apparatus. Furthermore, this Golgi-associated mechanism for dendrite establishment might be impaired in a human genetic epilepsy syndrome, polyhydramnios, megalencephaly, and symptomatic epilepsy. Copyright © 2018 the authors 0270-6474/18/380632-17$15.00/0.

  9. GPHR-dependent functions of the Golgi apparatus are essential for the formation of lamellar granules and the skin barrier.

    Science.gov (United States)

    Tarutani, Masahito; Nakajima, Kimiko; Uchida, Yoshikazu; Takaishi, Mikiro; Goto-Inoue, Naoko; Ikawa, Masahito; Setou, Mitsutoshi; Kinoshita, Taroh; Elias, Peter M; Sano, Shigetoshi; Maeda, Yusuke

    2012-08-01

    The lumen of the Golgi apparatus is regulated to be weakly acidic, which is critical for its functions. The Golgi pH regulator (GPHR) is an anion channel essential for normal acidification of the Golgi apparatus, and is therefore required for its functions. The Golgi apparatus has been thought to be the origin of lamellar granules in the skin. To study the functional role(s) of GPHR in the skin, we established keratinocyte-specific GPHR-knockout mice using the Cre-loxP system. These mutant mice exhibited hypopigmented skin, hair loss, and scaliness. Histological examination of GPHR-knockout mice showed ballooning of the basal cells and follicular dysplasia. In addition, inflammatory cells were seen in the dermis. The expression of trans-Golgi network 46, a marker for lamellar bodies, and kallikrein 7, a protein within lamellar bodies, is diminished in GPHR-knockout mouse skin. Examination by electron microscopy revealed that keratinocytes produced aberrant lamellar bodies. The transepidermal water loss of these knockout mice was increased compared with wild-type mice. Moreover, expression of cathelicidin-related antimicrobial peptide (CRAMP) in the skin was diminished. These results suggest that GPHR is essential for the homeostasis of the epidermis including the formation of lamellar bodies and for the barrier function.

  10. Besnoitia besnoiti and Toxoplasma gondii: two apicomplexan strategies to manipulate the host cell centrosome and Golgi apparatus.

    Science.gov (United States)

    Cardoso, Rita; Nolasco, Sofia; Gonçalves, João; Cortes, Helder C; Leitão, Alexandre; Soares, Helena

    2014-09-01

    Besnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.

  11. Phosphatidylinositol 4-phosphate in the Golgi apparatus regulates cell-cell adhesion and invasive cell migration in human breast cancer.

    Science.gov (United States)

    Tokuda, Emi; Itoh, Toshiki; Hasegawa, Junya; Ijuin, Takeshi; Takeuchi, Yukiko; Irino, Yasuhiro; Fukumoto, Miki; Takenawa, Tadaomi

    2014-06-01

    Downregulation of cell-cell adhesion and upregulation of cell migration play critical roles in the conversion of benign tumors to aggressive invasive cancers. In this study, we show that changes in cell-cell adhesion and cancer cell migration/invasion capacity depend on the level of phosphatidylinositol 4-phosphate [PI(4)P] in the Golgi apparatus in breast cancer cells. Attenuating SAC1, a PI(4)P phosphatase localized in the Golgi apparatus, resulted in decreased cell-cell adhesion and increased cell migration in weakly invasive cells. In contrast, silencing phosphatidylinositol 4-kinase IIIβ, which generates PI(4)P in the Golgi apparatus, increased cell-cell adhesion and decreased invasion in highly invasive cells. Furthermore, a PI(4)P effector, Golgi phosphoprotein 3, was found to be involved in the generation of these phenotypes in a manner that depends on its PI(4)P-binding ability. Our results provide a new model for breast cancer cell progression in which progression is controlled by PI(4)P levels in the Golgi apparatus. ©2014 American Association for Cancer Research.

  12. Unconjugated secondary bile acids activate the unfolded protein response and induce golgi fragmentation via a src-kinase-dependant mechanism

    Science.gov (United States)

    Sharma, Ruchika; Quilty, Francis; Gilmer, John F.; Long, Aideen; Byrne, Anne-Marie

    2017-01-01

    Bile acids are components of gastro-duodenal refluxate and regarded as causative agents in oesophageal disease but the precise mechanisms are unknown. Here we demonstrate that a specific subset of physiological bile acids affect the protein secretory pathway by inducing ER stress, activating the Unfolded Protein Response (UPR) and causing disassembly of the Golgi apparatus in oesophageal cells. Deoxycholic acid (DCA), Chemodeoxycholic acid (CDCA) and Lithocholic acid (LCA) activated the PERK arm of the UPR, via phosphorylation of eIF2α and up-regulation of ATF3, CHOP and BiP/GRP78. UPR activation by these bile acids is mechanistically linked with Golgi fragmentation, as modulating the UPR using a PERK inhibitor (GSK2606414) or salubrinal attenuated bile acid-induced effects on Golgi structure. Furthermore we demonstrate that DCA, CDCA and LA activate Src kinase and that inhibition of this kinase attenuated both bile acid-induced BiP/GRP78 expression and Golgi fragmentation. This study highlights a novel mechanism whereby environmental factors (bile acids) impact important cellular processes regulating cell homeostasis, including the UPR and Golgi structure, which may contribute to cancer progression in the oesophagus. PMID:27888615

  13. 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.

  14. Crn7 interacts with AP-1 and is required for the maintenance of Golgi morphology and protein export from the Golgi

    NARCIS (Netherlands)

    Rybakin, Vasily; Gounko, Natalia V.; Spaete, Kira; Hoening, Stefan; Majoul, Irina V.; Duden, Rainer; Noegel, Angelika A.

    2006-01-01

    Crn7 is a novel cytosolic mammalian WD-repeat protein of unknown function that associates with Golgi membranes. Here, we demonstrate that Crn7 knockdown by small interfering-RNA results in dramatic changes in the Golgi morphology and function. First, the Golgi ribbon is disorganized in Crn7 KD

  15. 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...

  16. Sequential Depletion and Acquisition of Proteins during Golgi Stack Disassembly and Reformation

    Science.gov (United States)

    Schoberer, Jennifer; Runions, John; Steinkellner, Herta; Strasser, Richard; Hawes, Chris; Osterrieder, Anne

    2010-01-01

    Herein, we report the stepwise transport of multiple plant Golgi membrane markers during disassembly of the Golgi apparatus in tobacco leaf epidermal cells in response to the induced expression of the GTP-locked Sar1p or Brefeldin A (BFA), and reassembly on BFA washout. The distribution of fluorescent Golgi-resident N-glycan processing enzymes and matrix proteins (golgins) with specific cis–trans-Golgi sub-locations was followed by confocal microscopy during disassembly and reassembly. The first event during Golgi disassembly was the loss of trans-Golgi enzymes and golgins from Golgi membranes, followed by a sequential redistribution of medial and cis-Golgi enzymes into the endoplasmic reticulum (ER), whilst golgins were relocated to the ER or cytoplasm. This event was confirmed by fractionation and immuno-blotting. The sequential redistribution of Golgi components in a trans–cis sequence may highlight a novel retrograde trafficking pathway between the trans-Golgi and the ER in plants. Release of Golgi markers from the ER upon BFA washout occurred in the opposite sequence, with cis-matrix proteins labelling Golgi-like structures before cis/medial enzymes. Trans-enzyme location was preceded by trans-matrix proteins being recruited back to Golgi membranes. Our results show that Golgi disassembly and reassembly occur in a highly ordered fashion in plants. PMID:20716110

  17. Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M

    1985-01-01

    the Mg2+-precipitated fraction were equally well protected from proteolytic cleavage (in the absence of Triton X-100). This indicates that the basolateral plasma membrane is unlikely to be involved in the post-Golgi transport of newly synthesized aminopeptidase N and suggests instead a direct delivery...... that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex....

  18. Comparative study of blood smears microscopy and rapid test strips ...

    African Journals Online (AJOL)

    To evaluate two of the currently available assay methods, specimen from 200 patients admitted on provisional diagnosis of malaria were screened in this study and compared with the smear microscopy method. Our results showed a statistical significant difference (p< 0.05) between the two rapid strip methods of ACON and ...

  19. Experimental Study of Cloud Formation from Rapidly Opened Containers

    Energy Technology Data Exchange (ETDEWEB)

    Wooldridge, C.E. [Stanford Research Institute; Amaro, A.J. [Stanford Research Institute; Kier, R.J. [Stanford Research Institute

    1969-08-01

    This report presents the results of an experimental study of droplet size emanating from a rapidly-opened container of volatile liquid, of the internal dynamics of the cavitation process inside such a container, & of the evaporation time of propane drops.

  20. [Centennial of the nobel prize for Golgi and Cajal--founding of modern neuroscience and irony of discovery].

    Science.gov (United States)

    Chu, Nai-Shin

    2006-09-01

    In 1906, Golgi and Ramón y Cajal shared the Nobel Prize in Physiology or Medicine "in recognition of their work on the structure of the nervous system". However, it was an unusual occasion in the history of Nobel Prize award because their views on the structure of the nervous system were not only different but even opposite, creating the "storm center of histological controversy". Furthermore, the new staining method Cajal had employed to study the nervous system was developed by Golgi, creating an irony of discovery. In 1873, Golgi revolutionized the histological study of the nervous system by developing a new staining method, "la reazione nera" or black reaction, which allowed good visualization of axons, dendrites and glia. But because his stain was so selective, staining only about 3 percent of neurons, he was unable to see clearly how the neuronal processes ended as they approached other neurons. Consequently, he embraced the popular belief that neuronal processes physically fuse with each other--the "reticular theory". On the other hand, Cajal was incidentally introduced to the Golgi stain 14 years after its discovery and immediately realized its beauty. He found that better results could be produced by staining more intensely and cutting thicker sections. He further observed that the Golgi stain worked best on non-myelinated axons. The search for brains containing non-myelinated axons led him to study birds and very young mammals, including embryos. Cajal obtained fascinating results by modifying the Golgi stain and by studying avian and young mammalian brains. From those studies, Cajal was able to infer that axons and dendrites ended freely and did not physically anastomose. Therefore, he strongly advocated the "neuron theory". Golgi seemed to be too headstrong and too conservative to relinquish his belief that neurons constitute a network which reacts as a whole. On the other hand, Cajal's hard work using the Golgi stain led to new understanding on the

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

    Science.gov (United States)

    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 cytokinesis. The precise location of Golgi membranes and resident proteins during mitosis remains unclear, partly due to limitations of molecular markers and the resolution of light microscopy. We generated a fusion consisting of the first 117 residues of α-mannosidase II tagged with a fluorescent protein and a tetracysteine motif. The mannosidase component guarantees docking into the Golgi membrane, with the tags exposed in the lumen. The fluorescent protein is optically visible without further treatment, whereas the tetracysteine tag can be reduced acutely with a membrane-permeant phosphine, labeled with ReAsH, monitored in the light microscope, and used to trigger the photoconversion of diaminobenzidine, allowing 4D optical recording on live cells and correlated ultrastructural analysis by electron microscopy. These methods reveal that Golgi reassembly is preceded by the formation of four colinear clusters at telophase, two per daughter cell. Within each daughter, the smaller cluster near the midbody gradually migrates to rejoin the major cluster on the far side of the nucleus and asymmetrically reconstitutes a single Golgi apparatus, first in one daughter cell and then in the other. Our studies provide previously undescribed insights into Golgi disassociation and reassembly during mitosis and offer a powerful approach to follow recombinant protein distribution in 4D imaging and correlated high-resolution analysis. PMID:17101980

  2. 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

  3. 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

    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...

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

    Science.gov (United States)

    Jensen, Camilla Stampe; Misonou, Hiroaki

    2015-01-01

    The subcellular localization of neuronal membrane signaling molecules such as receptors and ion channels depends on intracellular trafficking mechanisms. Essentially, vesicular trafficking mechanisms ensure that a large number of membrane proteins are correctly targeted to different subcellular 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 characteristics of different types of transport vesicles. We demonstrate the application of our protocol to investigate whether ion channels are sorted into distinct vesicular populations at the Golgi apparatus. We also demonstrate how these techniques are suitable for pharmacological dissection of the transport 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 neurons. We believe that the procedures described here are useful for researchers who are interested in studying molecular mechanisms of protein trafficking in neurons.

  5. Discrete and continuous models of protein sorting in the Golgi

    Science.gov (United States)

    Gong, Haijun; Schwartz, Russell

    2009-03-01

    The Golgi apparatus plays an important role in processing and sorting proteins and lipids. Golgi compartments constantly exchange material with each other and with other cellular components, allowing them to maintain and reform distinct identities despite dramatic changes in structure and size during cell division, development and osmotic stress. We have developed two minimal models of membrane and protein exchange in the Golgi --- a discrete, stochastic model [1] and a continuous ordinary differential equation (ODE) model --- both based on two fundamental mechanisms: vesicle-coat-mediated selective concentration of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins during vesicle formation and SNARE-mediated selective fusion of vesicles. Both show similar ability to establish and maintain distinct identities over broad parameter ranges, but they diverge in extreme conditions where Golgi collapse and reassembly may be observed. By exploring where the models differ, we hope to better identify those features essential to minimal models of various Golgi behaviors. [1] H. Gong, D. Sengupta, A. D. Linstedt, R. Schwartz. Biophys J. 95: 1674-1688, 2008.

  6. 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.

  7. Okadaic acid disrupts Golgi structure and impairs enzyme synthesis and secretion in the rat pancreas.

    Science.gov (United States)

    Waschulewski, I H; Kruse, M L; Agricola, B; Kern, H F; Schmidt, W E

    1996-06-01

    Okadaic acid, a serine/threonine phosphatase inhibitor, has been shown to inhibit rat pancreatic enzyme secretion by interference with late processes in stimulus-secretion coupling. To further characterize its action, we studied the effect of okadaic acid on secretion of newly synthesized proteins, protein synthesis, and cellular ultrastructure in pancreatic lobules derived from rats stimulated in vivo by feeding the synthetic proteinase inhibitor FOY-305. Okadaic acid completely blocked protein secretion at concentrations that inhibit the Ca2+/calmodulin-dependent protein phosphatase 2b, calcineurin. Protein synthesis was abolished at 10(-6) mol/l and reduced by 60% at 5 x 10(-7) mol/l okadaic acid. Pancreatic lobules exposed to 5 x 10(-7) mol/l okadaic acid for 20 min fully restored their secretory capacity on removal of the drug; whereas, after a preincubation with okadaic acid for > 40 min, protein secretion remained impaired during the recovery period. Electron microscopic examination of pancreatic acinar cells treated with 5 x 10(-7) mol/l okadaic acid revealed a dilated Golgi complex after 15 and 30 min and a subsequent fragmentation of Golgi cisternae into clouds of small uniform vesicles after 60 min. Reassembly of Golgi stacks occurred after a 60-min recovery without okadaic acid. These data indicate that serine/threonine phosphatases play an important role not only in the regulation of pancreatic enzyme synthesis and exocytosis but also are crucial for the maintenance of normal Golgi architecture and function in the exocrine rat pancreas. These effects are probably not exclusively mediated via type 2b calcineurin-like protein phosphatases.

  8. Rice Stripe Tenuivirus NSvc2 Glycoproteins Targeted to the Golgi Body by the N-Terminal Transmembrane Domain and Adjacent Cytosolic 24 Amino Acids via the COP I- and COP II-Dependent Secretion Pathway

    Science.gov (United States)

    Yao, Min; Liu, Xiaofan; Li, Shuo; Xu, Yi; Zhou, Yijun

    2014-01-01

    ABSTRACT The NSvc2 glycoproteins encoded by Rice stripe tenuivirus (RSV) share many characteristics common to the glycoproteins found among Bunyaviridae. Within this viral family, glycoproteins targeting to the Golgi apparatus play a pivotal role in the maturation of the enveloped spherical particles. RSV particles, however, adopt a long filamentous morphology. Recently, RSV NSvc2 glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. Here, we demonstrate that the amino-terminal NSvc2 (NSvc2-N) targets to the Golgi apparatus in Nicotiana benthamiana cells, whereas the carboxyl-terminal NSvc2 (NSvc2-C) accumulates in the endoplasmic reticulum (ER). Upon coexpression, NSvc2-N redirects NSvc2-C from the ER to the Golgi bodies. The NSvc2 glycoproteins move together with the Golgi stacks along the ER/actin network. The targeting of the NSvc2 glycoproteins to the Golgi bodies was strictly dependent on functional anterograde traffic out of the ER to the Golgi bodies or on a retrograde transport route from the Golgi apparatus. The analysis of truncated and chimeric NSvc2 proteins demonstrates that the Golgi targeting signal comprises amino acids 269 to 315 of NSvc2-N, encompassing the transmembrane domain and 24 adjacent amino acids in the cytosolic tail. Our findings demonstrate for the first time that the glycoproteins from an unenveloped Tenuivirus could target Golgi bodies in plant cells. IMPORTANCE NSvc2 glycoprotein encoded by unenveloped Rice stripe tenuivirus (RSV) share many characteristics in common with glycoprotein found among Bunyaviridae in which all members have membrane-enveloped sphere particle. Recently, RSV NSvc2 glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. In this study, we demonstrated that the RSV glycoproteins could target Golgi bodies in plant cells. The targeting of NSvc2 glycoproteins to the Golgi bodies was dependent on active COP II or COP I. The Golgi targeting signal was mapped to the

  9. Transmembrane domain quality control systems operate at the endoplasmic reticulum and Golgi apparatus.

    Science.gov (United States)

    Briant, Kit; Johnson, Nicholas; Swanton, Eileithyia

    2017-01-01

    Multiple protein quality control systems operate to ensure that misfolded proteins are efficiently cleared from the cell. While quality control systems that assess the folding status of soluble domains have been extensively studied, transmembrane domain (TMD) quality control mechanisms are poorly understood. Here, we have used chimeras based on the type I plasma membrane protein CD8 in which the endogenous TMD was substituted with transmembrane sequences derived from different polytopic membrane proteins as a mode to investigate the quality control of unassembled TMDs along the secretory pathway. We find that the three TMDs examined prevent trafficking of CD8 to the cell surface via potentially distinct mechanisms. CD8 containing two distinct non-native transmembrane sequences escape the ER and are subsequently retrieved from the Golgi, possibly via Rer1, leading to ER localisation at steady state. A third chimera, containing an altered transmembrane domain, was predominantly localised to the Golgi at steady state, indicating the existence of an additional quality control checkpoint that identifies non-native transmembrane domains that have escaped ER retention and retrieval. Preliminary experiments indicate that protein retained by quality control mechanisms at the Golgi are targeted to lysosomes for degradation.

  10. RNAi screening reveals a large signaling network controlling the Golgi apparatus in human cells.

    Science.gov (United States)

    Chia, Joanne; Goh, Germaine; Racine, Victor; Ng, Susanne; Kumar, Pankaj; Bard, Frederic

    2012-01-01

    The Golgi apparatus has many important physiological functions, including sorting of secretory cargo and biosynthesis of complex glycans. These functions depend on the intricate and compartmentalized organization of the Golgi apparatus. To investigate the mechanisms that regulate Golgi architecture, we developed a quantitative morphological assay using three different Golgi compartment markers and quantitative image analysis, and performed a kinome- and phosphatome-wide RNAi screen in HeLa cells. Depletion of 159 signaling genes, nearly 20% of genes assayed, induced strong and varied perturbations in Golgi morphology. Using bioinformatics data, a large regulatory network could be constructed. Specific subnetworks are involved in phosphoinositides regulation, acto-myosin dynamics and mitogen activated protein kinase signaling. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades can control glycosylation directly at the Golgi level. Our results provide a genetic overview of the signaling pathways that control the Golgi apparatus in human cells.

  11. 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

  12. A serial section Golgi analysis of the primate claustrum.

    Science.gov (United States)

    Brand, S

    1981-01-01

    The cellular composition of the primate claustrum was analyzed using serially sectioned Golgi impregnated neurons. The tissue used in this study was embedded in a soft resin mixture and cut with 25 mm long glass knives. The resin embedding allowed the sections to be cut serially at a thickness of only 3 micrometers. A camera lucida was employed for drawing the cellular processes from selected impregnated neurons; these drawings were later incorporated into a single composite picture of the neuron. Three types of neurons were observed in the primate claustrum. The largest of these neurons (Type 1) had a cell body and spine-laden dendritic arborization that varied in size and shape according to the neuron's position in the claustrum. The axons of Type I neurons were successfully impregnated in 25-day-old animals and were found to form collaterals within the claustrum. The collaterals from the axons of these cells appeared to leave the claustrum through both the external and extreme capsules. A second neuron found in the claustrum (Type II) had a round cell body with smooth beaded dendrites which radiated in all directions. The axon of the Type II neuron appeared to give off numerous collaterals that were not observed to leave the claustrum. A third type of neuron (Type III) had a small pear shaped cell body and a sparse dendritic tree. The axon and its collaterals appeared to remain within the dendritic circumference of the Type III neuron.

  13. Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis

    Science.gov (United States)

    Au, Catherine E.; Hermo, Louis; Byrne, Elliot; Smirle, Jeffrey; Fazel, Ali; Simon, Paul H. G.; Kearney, Robert E.; Cameron, Pamela H.; Smith, Charles E.; Vali, Hojatollah; Fernandez-Rodriguez, Julia; Ma, Kewei; Nilsson, Tommy; Bergeron, John J. M.

    2015-01-01

    The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell–specific Golgi-localized type II integral membrane glycoprotein. TM9SF3, also of unknown function, was revealed to be a universal Golgi marker for both somatic and germ cells. During acrosome formation, several Golgi proteins (GBF1, GPP34, GRASP55) localize to both the acrosome and Golgi, while GL54D, TM9SF3, and the Golgi trafficking protein TMED7/p27 are segregated from the acrosome. After acrosome formation, GL54D, TM9SF3, TMED4/p25, and TMED7/p27 continue to mark Golgi identity as it migrates away from the acrosome, while the others (GBF1, GPP34, GRASP55) remain in the acrosome and are progressively lost in later steps of differentiation. Cytoplasmic HSP70.2 and the endoplasmic reticulum luminal protein-folding enzyme PDILT are also Golgi recruited but only during acrosome formation. This resource identifies abundant Golgi proteins that are expressed differentially during mitosis, meiosis, and postacrosome Golgi migration, including the last step of differentiation. PMID:25808494

  14. 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

    with Cdc42. Moreover, Golgi association of FMNL2 or -3 induced a phalloidin-detectable actin meshwork around the Golgi. Importantly, functional interference with FMNL2/3 formins by RNAi or CRISPR/Cas9-mediated gene deletion invariably induced Golgi fragmentation in different cell lines. Furthermore...

  15. 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

  16. Golgi apparatus analyzed by cryo-electron microscopy.

    Science.gov (United States)

    Han, Hong-Mei; Bouchet-Marquis, Cedric; Huebinger, Jan; Grabenbauer, Markus

    2013-10-01

    In 1898, the Golgi apparatus was discovered by light microscopy, and since the 1950s, the ultrastructure composition is known by electron microscopic investigation. The complex three-dimensional morphology fascinated researchers and was sometimes even the driving force to develop novel visualization techniques. However, the highly dynamic membrane systems of Golgi apparatus are delicate and prone to fixation artifacts. Therefore, the understanding of Golgi morphology and its function has been improved significantly with the development of better preparation methods. Nowadays, cryo-fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in amorphous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hydrated state by cryo-electron microscopy and tomography. Even though the overall morphology of vitrified Golgi stacks is comparable to well-prepared and resin-embedded samples, previously unknown structural details can be observed solely based on their native density. At this point, any further improvement of sample preparation would gain novel insights, perhaps not in terms of general morphology, but on fine structural details of this dynamic organelle.

  17. 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.

  18. Nitric oxide scavenging causes remodeling of the endoplasmic reticulum, Golgi apparatus and mitochondria in pulmonary arterial endothelial cells.

    Science.gov (United States)

    Lee, Jason E; Yuan, Huijuan; Liang, Feng-Xia; Sehgal, Pravin B

    2013-09-01

    The dependence of the structure and function of cytoplasmic organelles in endothelial cells on constitutively produced intracellular nitric oxide (NO) remains largely unexplored. We previously reported fragmentation of the Golgi apparatus in cells exposed to NO scavengers or after siRNA-mediated knockdown of eNOS. Others have reported increased mitochondrial fission in response to an NO donor. Functionally, we previously reported that bovine pulmonary arterial endothelial cells (PAECs) exposed to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) developed a prosecretory phenotype characterized by prolonged secretion of soluble proteins. In the present study, we investigated whether NO scavenging led to remodeling of the endoplasmic reticulum (ER). Live-cell DAF-2DA imaging confirmed the presence of intracellular NO in association with the BODIPY C5-ceramide-labeled Golgi apparatus. Untreated human PAECs displayed a pattern of peripheral tubulo-reticular ER with a juxtanuclear accumulation of ER sheets. Cells exposed to c-PTIO showed a dramatic increase in ER sheets as assayed using immunofluorescence for the ER structural protein reticulon-4b/Nogo-B and the ER-resident GTPase atlastin-3, live-cell fluorescence assays using RTN4-GFP and KDEL-mCherry, and electron microscopy methods. These ER changes were inhibited by the NO donor diethylamine NONOate, and also produced by L-NAME, but not D-NAME or 8-br-cGMP. This ER remodeling was accompanied by Golgi fragmentation and increased fibrillarity and function of mitochondria (uptake of tetramethyl-rhodamine, TMRE). Despite Golgi fragmentation the functional ER/Golgi trafficking unit was preserved as seen by the accumulation of Sec31A ER exit sites adjacent to the dispersed Golgi elements and a 1.8-fold increase in secretion of soluble cargo. Western blotting and immunopanning data showed that RTN4b was increasingly ubiquitinated following c-PTIO exposure, especially in the

  19. Golgi apparatus and protein trafficking in Alzheimer's disease.

    Science.gov (United States)

    Baloyannis, Stavros J

    2014-01-01

    Alzheimer's disease (AD) is a progressive degeneration of the brain, inducing memory decline, inability in learning, and behavioral alterations, resulting progressively in a marked deterioration of all mental activities and eventually a vegetative state. The main causative factor, however, is still unclear. The implication of amyloid-β, AβPP, tau protein, the selective loss of neurons, the alteration of the synapses, the cytoskeletal changes, and the morphological alterations of the brain capillaries contribute substantially to the pathogenetic profile of the disease, without sufficiently enlightening the initial steps of the pathological procedures. The ultrastructure of the neuronal organelles as well as histochemical studies revealed substantial alterations, primarily concerning mitochondria. In this study, the morphological and morphometric alterations of the Golgi apparatus (GA) are described in the Purkinje cells of the cerebellum in twenty AD brains, studied with electron microscopy. As it is well established, GA has a very important role to play in many procedures such as glycosylation, sulfation, and proteolysis of protein systems, which are synthesized in the endoplasmic reticulum of nerve cells and glia. GA may also play a crucial role in protein trafficking and in misfolding of protein aggregates. In addition, the hyperphosphorylation of tau protein is closely related with the pathology of GA. In AD cases, described in this study, an obvious fragmentation of the cisternae of GA was observed in the Purkinje cells of the vermis and the cerebellar hemispheres. This alteration of GA may be associated with alterations of microtubules, impaired protein trafficking, and dendritic, spinal, and synaptic pathology, since protein trafficking plays an essential role in the three dimensional organization of the dendritic arbor and in the integrity of the synaptic components.

  20. Phospholipase D Is Involved in the Formation of Golgi Associated Clathrin Coated Vesicles in Human Parotid Duct Cells

    Science.gov (United States)

    Brito de Souza, Lorena; Pinto da Silva, Luis Lamberti; Jamur, Maria Célia; Oliver, Constance

    2014-01-01

    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. PMID:24618697

  1. Vesicular trafficking of incoming human papillomavirus 16 to the Golgi apparatus and endoplasmic reticulum requires γ-secretase activity.

    Science.gov (United States)

    Zhang, Wei; Kazakov, Teymur; Popa, Andreea; DiMaio, Daniel

    2014-09-16

    The route taken by papillomaviruses from the cell surface to the nucleus during infection is incompletely understood. Here, we developed a novel human papillomavirus 16 (HPV16) pseudovirus in which the carboxy terminus of the minor capsid protein L2 is exposed on the exterior of the intact capsid prior to cell binding. With this pseudovirus, we used the proximity ligation assay immune detection technique to demonstrate that during entry HPV16 L2 traffics into and out of the early endosome prior to Golgi localization, and we demonstrated that L2 enters the endoplasmic reticulum during entry. The cellular membrane-associated protease, γ-secretase, is required for infection by HPV16 pseudovirus and authentic HPV16. We also showed that inhibition of γ-secretase does not interfere substantively with virus internalization, initiation of capsid disassembly, entry into the early endosome, or exit from this compartment, but γ-secretase is required for localization of L2 and viral DNA to the Golgi apparatus and the endoplasmic reticulum. These results show that incoming HPV16 traffics sequentially from the cell surface to the endosome and then to the Golgi apparatus and the endoplasmic reticulum prior to nuclear entry. The human papillomaviruses are small nonenveloped DNA viruses responsible for approximately 5% of all human cancer deaths, but little is known about the process by which these viruses transit from the cell surface to the nucleus. Here we show that incoming HPV16, the most common high-risk HPV, traffics though a series of vesicular compartments during infectious entry, including the endosome, Golgi apparatus, and endoplasmic reticulum. Furthermore, we show that γ-secretase, a cellular membrane-associated protease, is required for entry of the L2 minor capsid protein and viral DNA into the Golgi apparatus and endoplasmic reticulum. These studies reveal a new pathway of cell entry by DNA viruses and suggest that components of this pathway are candidate

  2. 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.

  3. [The isolation and assessment of Golgi apparatus from gastric cancer cells SGC7901].

    Science.gov (United States)

    He, Tingting; Yi, Yongfen; Li, Yanqing; Xiao, Zhong

    2010-10-01

    The Golgi complex is the central organelle of the secretory pathway and has many complicate functions. The endeavours to isolate and purify the Golgi apparatus from cultured cells will benefit further investigation of Golgi. A large number of gastric cancer cells SGC7901 were cultivated in vitro, then Golgi apparatus were isolated from the cells by differential centrifugation combined with sucrose density gradient ultra-centrifugation. Its purity was characterized biochemically by enzymatic assays, morphologically by electron microscopy (EM) and neutral red supravital staining. Finally the Golgi complex was successfully fractionated from gastric cancer cells SGC7901. The first successful isolation of Golgi apparatus from gastric cancer cells SGC7901 by using ultra-centrifugation will lead to research into the function of Golgi apparatus.

  4. Starvation-Dependent Regulation of Golgi Quality Control Links the TOR Signaling and Vacuolar Protein Sorting Pathways

    Directory of Open Access Journals (Sweden)

    Niv Dobzinski

    2015-09-01

    Full Text Available Upon amino acid (AA starvation and TOR inactivation, plasma-membrane-localized permeases rapidly undergo ubiquitination and internalization via the vacuolar protein sorting/multivesicular body (VPS-MVB pathway and are degraded in the yeast vacuole. We now show that specific Golgi proteins are also directed to the vacuole under these conditions as part of a Golgi quality-control (GQC process. The degradation of GQC substrates is dependent upon ubiquitination by the defective-for-SREBP-cleavage (DSC complex, which was identified via genetic screening and includes the Tul1 E3 ligase. Using a model GQC substrate, GFP-tagged Yif1, we show that vacuolar targeting necessitates upregulation of the VPS pathway via proteasome-mediated degradation of the initial endosomal sorting complex required for transport, ESCRT-0, but not downstream ESCRT components. Thus, early cellular responses to starvation include the targeting of specific Golgi proteins for degradation, a phenomenon reminiscent of the inactivation of BTN1, the yeast Batten disease gene ortholog.

  5. α-Synuclein Delays Endoplasmic Reticulum (ER)-to-Golgi Transport in Mammalian Cells by Antagonizing ER/Golgi SNAREs

    Science.gov (United States)

    Thayanidhi, Nandhakumar; Helm, Jared R.; Nycz, Deborah C.; Bentley, Marvin; Liang, Yingjian

    2010-01-01

    Toxicity of human α-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human α-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant α-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble α-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble α-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that α-synuclein antagonizes SNARE function. Ykt6 reversed α-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified α-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble α-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway. PMID:20392839

  6. Alpha-synuclein delays endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells by antagonizing ER/Golgi SNAREs.

    Science.gov (United States)

    Thayanidhi, Nandhakumar; Helm, Jared R; Nycz, Deborah C; Bentley, Marvin; Liang, Yingjian; Hay, Jesse C

    2010-06-01

    Toxicity of human alpha-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human alpha-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant alpha-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble alpha-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble alpha-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that alpha-synuclein antagonizes SNARE function. Ykt6 reversed alpha-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified alpha-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble alpha-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.

  7. Benzyl alcohol induces a reversible fragmentation of the Golgi apparatus and inhibits membrane trafficking between endosomes and the trans-Golgi network.

    Science.gov (United States)

    Simm, Roger; Kvalvaag, Audun Sverre; van Deurs, Bo; Lindbäck, Toril; Sandvig, Kirsten

    2017-08-01

    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. BnOH is a membrane fluidizing agent that can affect membrane protein activity and cellular processes such as ligand binding to cell surface receptors, endocytosis and degradation of lysosomal cargo. In this study, we examined the effects of BnOH on intracellular transport using Shiga toxin (Stx), diphtheria toxin (DT) and ricin. BnOH caused reduced toxicity of all three toxins at BnOH concentrations that cause membrane fluidization. The reduced toxicity of Stx and ricin was mainly due to inhibition of retrograde transport between endosomes and the trans-Golgi network as BnOH had small effects on cell association and endocytosis of ricin and Stx. Strikingly, BnOH also induced a reversible fragmentation of the Golgi apparatus. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. 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.

  9. Conserved Molecular Mechanisms Underlying Homeostasis of the Golgi Complex

    Directory of Open Access Journals (Sweden)

    Cathal Wilson

    2010-01-01

    Full Text Available The Golgi complex performs a central function in the secretory pathway in the sorting and sequential processing of a large number of proteins destined for other endomembrane organelles, the plasma membrane, or secretion from the cell, in addition to lipid metabolism and signaling. The Golgi apparatus can be regarded as a self-organizing system that maintains a relatively stable morphofunctional organization in the face of an enormous flux of lipids and proteins. A large number of the molecular players that operate in these processes have been identified, their functions and interactions defined, but there is still debate about many aspects that regulate protein trafficking and, in particular, the maintenance of these highly dynamic structures and processes. Here, we consider how an evolutionarily conserved underlying mechanism based on retrograde trafficking that uses lipids, COPI, SNAREs, and tethers could maintain such a homeodynamic system.

  10. Retrograde transport of protein toxins through the Golgi apparatus

    DEFF Research Database (Denmark)

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

    2013-01-01

    A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins...... 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......, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through...

  11. Molecular determinants of the N-terminal acetyltransferase Naa60 anchoring to the Golgi membrane.

    Science.gov (United States)

    Aksnes, Henriette; Goris, Marianne; Strømland, Øyvind; Drazic, Adrian; Waheed, Qaiser; Reuter, Nathalie; Arnesen, Thomas

    2017-04-21

    Nα-Acetyltransferase 60 (Naa60 or NatF) was recently identified as an unconventional N-terminal acetyltransferase (NAT) because it localizes to organelles, in particular the Golgi apparatus, and has a preference for acetylating N termini of the transmembrane proteins. This knowledge challenged the prevailing view of N-terminal acetylation as a co-translational ribosome-associated process and suggested a new mechanistic functioning for the enzymes responsible for this increasingly recognized protein modification. Crystallography studies on Naa60 were unable to resolve the C-terminal tail of Naa60, which is responsible for the organellar localization. Here, we combined modeling, in vitro assays, and cellular localization studies to investigate the secondary structure and membrane interacting capacity of Naa60. The results show that Naa60 is a peripheral membrane protein. Two amphipathic helices within the Naa60 C terminus bind the membrane directly in a parallel position relative to the lipid bilayer via hydrophobic and electrostatic interactions. A peptide corresponding to the C terminus was unstructured in solution and only folded into an α-helical conformation in the presence of liposomes. Computational modeling and cellular mutational analysis revealed the hydrophobic face of two α-helices to be critical for membranous localization. Furthermore, we found a strong and specific binding preference of Naa60 toward membranes containing the phosphatidylinositol PI(4)P, thus possibly explaining the primary residency of Naa60 at the PI(4)P-rich Golgi. In conclusion, we have defined the mode of cytosolic Naa60 anchoring to the Golgi apparatus, most likely occurring post-translationally and specifically facilitating post-translational N-terminal acetylation of many transmembrane proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Detection of rapid-eye movements in sleep studies.

    Science.gov (United States)

    Agarwal, Rajeev; Takeuchi, Tomoka; Laroche, Suzie; Gotman, Jean

    2005-08-01

    One of the key features of rapid-eye movement (REM) sleep is the presence of bursts of REMs. Sleep studies routinely use REMs to classify sleep stages. Moreover, REM count or density has been used in studies involving learning and various psychiatric disorders. Most of these studies have been based on the visual identification of REMs, which is generally a very time-consuming task. This and the varying definitions of REMs across scorers have warranted the development of automatic REM detection methodologies. In this paper, we present a new detection scheme that combines many of the intrinsic properties of REMs and requires minimal parameter adjustments. In the proposed method, a single parameter can be used to control the REM detection sensitivity and specificity tradeoff. Manually scored training data are used to develop the method. We assess the performance of the method against manual scoring of individual REM events and present validation results using a separate data set. The ability of the method to discriminate fast horizontal ocular movement in REM sleep from other types of events is highlighted. A key advantage of the presented method is the minimal a priori information requirement. The results of training data (recordings from five subjects) show an overall sensitivity of 78.8% and specificity of 81.6%. The performance on the testing data (recording from five subjects different from the training data) showed overall sensitivity of 67.2% and specificity of 77.5%.

  13. Analysis of site-specific N-glycan remodeling in the endoplasmic reticulum and the Golgi

    Science.gov (United States)

    Hang, Ivan; Lin, Chia-wei; Grant, Oliver C; Fleurkens, Susanna; Villiger, Thomas K; Soos, Miroslav; Morbidelli, Massimo; Woods, Robert J; Gauss, Robert; Aebi, Markus

    2015-01-01

    The hallmark of N-linked protein glycosylation is the generation of diverse glycan structures in the secretory pathway. Dynamic, non-template-driven processes of N-glycan remodeling in the endoplasmic reticulum and the Golgi provide the cellular setting for structural diversity. We applied newly developed mass spectrometry-based analytics to quantify site-specific N-glycan remodeling of the model protein Pdi1p expressed in insect cells. Molecular dynamics simulation, mutational analysis, kinetic studies of in vitro processing events and glycan flux analysis supported the defining role of the protein in N-glycan processing. PMID:26240167

  14. Connecting the cytoskeleton to the endoplasmic reticulum and Golgi.

    Science.gov (United States)

    Gurel, Pinar S; Hatch, Anna L; Higgs, Henry N

    2014-07-21

    A tendency in cell biology is to divide and conquer. For example, decades of painstaking work have led to an understanding of endoplasmic reticulum (ER) and Golgi structure, dynamics, and transport. In parallel, cytoskeletal researchers have revealed a fantastic diversity of structure and cellular function in both actin and microtubules. Increasingly, these areas overlap, necessitating an understanding of both organelle and cytoskeletal biology. This review addresses connections between the actin/microtubule cytoskeletons and organelles in animal cells, focusing on three key areas: ER structure and function; ER-to-Golgi transport; and Golgi structure and function. Making these connections has been challenging for several reasons: the small sizes and dynamic characteristics of some components; the fact that organelle-specific cytoskeletal elements can easily be obscured by more abundant cytoskeletal structures; and the difficulties in imaging membranes and cytoskeleton simultaneously, especially at the ultrastructural level. One major concept is that the cytoskeleton is frequently used to generate force for membrane movement, with two potential consequences: translocation of the organelle, or deformation of the organelle membrane. While initially discussing issues common to metazoan cells in general, we subsequently highlight specific features of neurons, since these highly polarized cells present unique challenges for organellar distribution and dynamics. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. A new insight into the three-dimensional architecture of the Golgi complex: Characterization of unusual structures in epididymal principal cells.

    Science.gov (United States)

    Martínez-Martínez, Narcisa; Martínez-Alonso, Emma; Tomás, Mónica; Neumüller, Josef; Pavelka, Margit; Martínez-Menárguez, José A

    2017-01-01

    Principal epididymal cells have one of the largest and more developed Golgi complex of mammalian cells. In the present study, we have used this cell as model for the study of the three-dimensional architecture of the Golgi complex of highly secretory and endocytic cells. Electron tomography demonstrated the presence in this cell type of some unknown or very unusual Golgi structures such as branched cisternae, pocket-like cisternal invaginations or tubular connections. In addition, we have used this methodology and immunoelectron microscopy to analyze the close relationship between this organelle and both the endoplasmic reticulum and microtubules, and to describe in detail how these elements interact with compact and non-compact regions of the ribbon.

  16. Rapidly improving stroke symptoms: a pilot, prospective study.

    Science.gov (United States)

    Balucani, Clotilde; Bianchi, Riccardo; Ramkishun, Charles; Weedon, Jeremy; Law, Susan; Szarek, Michael; Rojas-Soto, Diana; Tariq, Sara; Levine, Steven R

    2015-06-01

    Rapidly improving stroke symptoms (RISSs) are a controversial exclusion for intravenous recombinant tissue plasminogen activator (rt-PA) for acute ischemic stroke (AIS). We estimated the frequency of 4 prespecified RISS definitions and explored their relationship to clinical outcome. Pilot, prospective study of AIS patients admitted within 4.5 hours of symptom onset. Serial assessments using National Institute of Health Stroke Scale (NIHSS) were performed every 20 ± 5 minutes until a rt-PA treatment decision was made, independent of the study. Improvement was calculated as the difference between baseline NIHSS and treatment decision NIHSS. RISS was defined as a 4-point or greater improvement, 25% or greater, 50% or greater, and according to the previously reported TREAT (The Re-examining Acute Eligibility for Thrombolysis) criteria. Unfavorable outcome was defined as modified Rankin Scale score more than 1 at 90 days after stroke. Logistic regression determined if RISS definition(s) related to the outcome. Fifty patients with AIS were enrolled: mean age 65 years; median baseline NIHSS score 5 (interquartile range, 2-11). RISS frequencies were 10%-22% based on definition. Median treatment decision NIHSS score is 5 (interquartile range, 2-9). Twenty-three (46%) patients received rt-PA. None of the 3 non-TREAT RISS definitions was independently associated with the outcome. Five of fifty (10%) were RISS according to the TREAT criteria, all 5 had good outcome without rt-PA. A Serial NIHSS assessment before treatment decision is feasible and may help determine the frequency and magnitude of RISS. This is the first prospective estimate of RISS frequency and outcome according to various prespecified definitions. The TREAT RISS frequency as a more restrictive definition may better predict good outcome of RISS in future, larger studies. Copyright © 2015 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  17. 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.

  18. Persistent and acute chlamydial infections induce different structural changes in the Golgi apparatus.

    Science.gov (United States)

    Zhu, Huiling; Li, Hongmei; Wang, Pu; Chen, Mukai; Huang, Zengwei; Li, Kunpeng; Li, Yinyin; He, Jian; Han, Jiande; Zhang, Qinfen

    2014-07-01

    Chlamydia trachomatis causes a wide range of diseases that have a significant impact on public health. Acute chlamydial infections can cause fragmentation of the Golgi compartment ensuring the lipid transportation from the host cell. However, the changes that occur in the host cell Golgi apparatus after persistent infections are unclear. Here, we examined Golgi-associated gene (golga5) transcription and expression along with the structure of the Golgi apparatus in cells persistently infected with Chlamydia trachomatis. The results showed that persistent infections caused little fragmentation of the Golgi. The results also revealed that Golgi fragmentation might be associated with the suppression of transcription of the gene golga5. Copyright © 2014 Elsevier GmbH. All rights reserved.

  19. Optineurin associates with the podocyte Golgi complex to maintain its structure.

    Science.gov (United States)

    Sippl, Christiane; Zeilbeck, Ludwig F; Fuchshofer, Rudolf; Tamm, Ernst R

    2014-11-01

    Optineurin, a cytosolic protein associated with the actin cytoskeleton, microtubules, and the Golgi complex, appears to have an important function in neurons, as mutations in its gene are causative for neurodegenerative diseases such as primary open-angle glaucoma and amyotrophic lateral sclerosis. Here, we report that optineurin is localized in podocytes of the kidney and induced upon injury following treatment with puromycin aminonucleoside. In cultured human podocytes, optineurin localizes to the Golgi complex. Optineurin depletion by RNA interference causes Golgi fragmentation. Moreover, if the Golgi complex is fragmented following microtubule destabilization induced by nocodazole treatment, optineurin dissociates from Golgi vesicles. Furthermore, optineurin colocalizes with vinculin-labeled focal contacts of cultured podocytes and with lysosome-like structures. Optineurin is essential for the survival of cultured podocytes, as optineurin depletion causes cell death. Thus, optineurin appears to play an important role in the maintenance of the podocyte Golgi complex and in the trafficking of vesicles to focal contacts and lysosomes.

  20. TMF/ARA160 Governs the Dynamic Spatial Orientation of the Golgi Apparatus during Sperm Development.

    Science.gov (United States)

    Elkis, Yoav; Bel, Shai; Rahimi, Roni; Lerer-Goldstein, Tali; Levin-Zaidman, Smadar; Babushkin, Tatiana; Shpungin, Sally; Nir, Uri

    2015-01-01

    TMF/ARA160 is known to be a TATA element Modulatory Factor (TMF). It was initially identified as a DNA-binding factor and a coactivator of the Androgen receptor. It was also characterized as a Golgi-associated protein, which is essential for acrosome formation during functional sperm development. However, the molecular roles of TMF in this intricate process have not been revealed. Here, we show that during spermiogenesis, TMF undergoes a dynamic change of localization throughout the Golgi apparatus. Specifically, TMF translocates from the cis-Golgi to the trans-Golgi network and to the emerging vesicles surface, as the round spermatids develop. Notably, lack of TMF led to an abnormal spatial orientation of the Golgi and to the deviation of the trans-Golgi surface away from the nucleus of the developing round spermatids. Concomitantly, pro-acrosomal vesicles derived from the TMF-/- Golgi lacked targeting properties and did not tether to the spermatid nuclear membrane thereby failing to form the acrosome anchoring scaffold, the acroplaxome, around the cell-nucleus. Absence of TMF also perturbed the positioning of microtubules, which normally lie in proximity to the Golgi and are important for maintaining Golgi spatial orientation and dynamics and for chromatoid body formation, which is impaired in TMF-/- spermatids. In-silico evaluation combined with molecular and electron microscopic analyses revealed the presence of a microtubule interacting domain (MIT) in TMF, and confirmed the association of TMF with microtubules in spermatogenic cells. Furthermore, the MIT domain in TMF, along with microtubules integrity, are required for stable association of TMF with the Golgi apparatus. Collectively, we show here for the first time that a Golgi and microtubules associated protein is crucial for maintaining proper Golgi orientation during a cell developmental process.

  1. Loss of the golgin GM130 causes Golgi disruption, Purkinje neuron loss, and ataxia in mice

    OpenAIRE

    Liu, Chunyi; Mei, Mei; Li, Qiuling; Roboti, Peristera; Pang, Qianqian; Ying, Zhengzhou; Gao, Fei; Lowe, Martin; Bao, Shilai

    2017-01-01

    The Golgi apparatus lies at the heart of the secretory pathway where it is required for secretory trafficking and cargo modification. Disruption of Golgi architecture and function has been widely observed in neurodegenerative disease, but whether Golgi dysfunction is causal with regard to the neurodegenerative process, or is simply a manifestation of neuronal death, remains unclear. Here we report that targeted loss of the golgin GM130 leads to a profound neurological phenotype in mice. Globa...

  2. The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus.

    Science.gov (United States)

    Lee, Wan Yin; Goh, Germaine; Chia, Joanne; Boey, Adrian; Gunko, Natalia V; Bard, Frederic

    2015-01-01

    The Golgi apparatus plays a pivotal role in the sorting and post-translational modifications of secreted and membrane proteins. In mammalian cells, the Golgi is organized in stacks of cisternae linked together to form a network with a ribbon shape. Regulation of Golgi ribbon formation is poorly understood. Here we find in an image-based RNAi screen that depletion of the ubiquitin-ligase CBLC induces Golgi fragmentation. Depletions of the close homologues CBL and CBLB do not induce any visible defects. In CBLC-depleted cells, Golgi stacks appear relatively unperturbed at both the light and electron microscopy levels, suggesting that CBLC controls mostly network organization. CBLC partially localizes on Golgi membranes and this localization is enhanced after activation of the SRC kinase. Inhibition of SRC reverts CBLC depletion effects, suggesting interplay between the two. CBLC's regulation of Golgi network requires its ubiquitin ligase activity. However, SRC levels are not significantly affected by CBLC, and CBLC knockdown does not phenocopy SRC activation, suggesting that CBLC's action at the Golgi is not direct downregulation of SRC. Altogether, our results demonstrate a role of CBLC in regulating Golgi ribbon by antagonizing the SRC tyrosine kinase.

  3. VAMP4 is required to maintain the ribbon structure of the Golgi apparatus.

    Science.gov (United States)

    Shitara, Akiko; Shibui, Toru; Okayama, Miki; Arakawa, Toshiya; Mizoguchi, Itaru; Sakakura, Yasunori; Shakakura, Yasunori; Takuma, Taishin

    2013-08-01

    The Golgi apparatus forms a twisted ribbon-like network in the juxtanuclear region of vertebrate cells. Vesicle-associated membrane protein 4 (VAMP4), a v-SNARE protein expressed exclusively in the vertebrate trans-Golgi network (TGN), plays a role in retrograde trafficking from the early endosome to the TGN, although its precise function within the Golgi apparatus remains unclear. To determine whether VAMP4 plays a functional role in maintaining the structure of the Golgi apparatus, we depleted VAMP4 gene expression using RNA interference technology. Depletion of VAMP4 from HeLa cells led to fragmentation of the Golgi ribbon. These fragments were not uniformly distributed throughout the cytoplasm, but remained in the juxtanuclear area. Electron microscopy and immunohistochemistry showed that in the absence of VAMP4, the length of the Golgi stack was shortened, but Golgi stacking was normal. Anterograde trafficking was not impaired in VAMP4-depleted cells, which contained intact microtubule arrays. Depletion of the cognate SNARE partners of VAMP4, syntaxin 6, syntaxin 16, and Vti1a also disrupted the Golgi ribbon structure. Our findings suggested that the maintenance of Golgi ribbon structure requires normal retrograde trafficking from the early endosome to the TGN, which is likely to be mediated by the formation of VAMP4-containing SNARE complexes.

  4. Actin- and microtubule-dependent regulation of Golgi morphology by FHDC1

    Science.gov (United States)

    Copeland, Sarah J.; Thurston, Susan F.; Copeland, John W.

    2016-01-01

    The Golgi apparatus is the central hub of intracellular trafficking and consists of tethered stacks of cis, medial, and trans cisternae. In mammalian cells, these cisternae are stitched together as a perinuclear Golgi ribbon, which is required for the establishment of cell polarity and normal subcellular organization. We previously identified FHDC1 (also known as INF1) as a unique microtubule-binding member of the formin family of cytoskeletal-remodeling proteins. We show here that endogenous FHDC1 regulates Golgi ribbon formation and has an apparent preferential association with the Golgi-derived microtubule network. Knockdown of FHDC1 expression results in defective Golgi assembly and suggests a role for FHDC1 in maintenance of the Golgi-derived microtubule network. Similarly, overexpression of FHDC1 induces dispersion of the Golgi ribbon into functional ministacks. This effect is independent of centrosome-derived microtubules and instead likely requires the interaction between the FHDC1 microtubule-binding domain and the Golgi-derived microtubule network. These effects also depend on the interaction between the FHDC1 FH2 domain and the actin cytoskeleton. Thus our results suggest that the coordination of actin and microtubule dynamics by FHDC1 is required for normal Golgi ribbon formation. PMID:26564798

  5. The golgin tether giantin regulates the secretory pathway by controlling stack organization within Golgi apparatus.

    Science.gov (United States)

    Koreishi, Mayuko; Gniadek, Thomas J; Yu, Sidney; Masuda, Junko; Honjo, Yasuko; Satoh, Ayano

    2013-01-01

    Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications.

  6. The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus.

    Directory of Open Access Journals (Sweden)

    Wan Yin Lee

    Full Text Available The Golgi apparatus plays a pivotal role in the sorting and post-translational modifications of secreted and membrane proteins. In mammalian cells, the Golgi is organized in stacks of cisternae linked together to form a network with a ribbon shape. Regulation of Golgi ribbon formation is poorly understood. Here we find in an image-based RNAi screen that depletion of the ubiquitin-ligase CBLC induces Golgi fragmentation. Depletions of the close homologues CBL and CBLB do not induce any visible defects. In CBLC-depleted cells, Golgi stacks appear relatively unperturbed at both the light and electron microscopy levels, suggesting that CBLC controls mostly network organization. CBLC partially localizes on Golgi membranes and this localization is enhanced after activation of the SRC kinase. Inhibition of SRC reverts CBLC depletion effects, suggesting interplay between the two. CBLC's regulation of Golgi network requires its ubiquitin ligase activity. However, SRC levels are not significantly affected by CBLC, and CBLC knockdown does not phenocopy SRC activation, suggesting that CBLC's action at the Golgi is not direct downregulation of SRC. Altogether, our results demonstrate a role of CBLC in regulating Golgi ribbon by antagonizing the SRC tyrosine kinase.

  7. The first transmembrane domain of lipid phosphatase SAC1 promotes Golgi localization.

    Directory of Open Access Journals (Sweden)

    Jinzhi Wang

    Full Text Available The lipid phosphatase Sac1 cycles between endoplasmic reticulum and cisternal Golgi compartments. In proliferating mammalian cells, a canonical dilysine motif at the C-terminus of Sac1 is required for coatomer complex-I (COP-I-binding and continuous retrieval to the ER. Starvation triggers accumulation of Sac1 at the Golgi. The mechanism responsible for Golgi retention of Sac1 is unknown. Here we show that the first of the two transmembrane regions in human SAC1 (TM1 functions in Golgi localization. A minimal construct containing only TM1 and the adjacent flanking sequences is concentrated at the Golgi. Transplanting TM1 into transferrin receptor 2 (TfR2 induces Golgi accumulation of this normally plasma membrane and endosomal protein, indicating that TM1 is sufficient for Golgi localization. In addition, we determined that the N-terminal cytoplasmic domain of SAC1 also promotes Golgi localization, even when TM1 is mutated or absent. We conclude that the distribution of SAC1 within the Golgi is controlled via both passive membrane thickness-dependent partitioning of TM1 and a retention mechanism that requires the N-terminal cytoplasmic region.

  8. Changes in the Golgi apparatus of neocortical and hippocampal neurons in the hibernating hamster

    Directory of Open Access Journals (Sweden)

    Alejandro eAntón

    2015-12-01

    Full Text Available Hibernating animals have been used as models to study several aspects of the plastic changes that occur in the metabolism and physiology of neurons. These models are also of interest in the study of Alzheimer’s disease because the microtubule-associated protein tau is hyperphosphorylated during the hibernation state known as torpor, similar to the pretangle stage of Alzheimer’s disease. Hibernating animals undergo torpor periods with drops in body temperature and metabolic rate, and a virtual cessation of neural activity. These processes are accompanied by morphological and neurochemical changes in neurons, which reverse a few hours after coming out of the torpor state. Since tau has been implicated in the structural regulation of the neuronal Golgi apparatus (GA we have used Western Blot and immunocytochemistry to analyze whether the GA is modified in cortical neurons of the Syrian hamster at different hibernation stages. The results show that, during the hibernation cycle, the GA undergo important structural changes along with differential modifications in expression levels and distribution patterns of Golgi structural proteins. These changes were accompanied by significant transitory reductions in the volume and surface area of the GA elements during torpor and arousal stages as compared with euthermic animals

  9. Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Poulsen, S S

    1983-01-01

    The effect of monensin and colchicine on the biogenesis of aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) was studied in organ-cultured pig small-intestina......The effect of monensin and colchicine on the biogenesis of aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) was studied in organ-cultured pig small...... destination. These findings suggest the involvement of the Golgi complex in the post-translational processing and transport of microvillar enzymes. The presence in the growth medium of colchicine (50 micrograms/ml) caused a significant inhibition of the appearance of newly synthesized enzymes...... in the microvillar membrane during a 3 h labelling period. Since synthesis and post-translational modification of the microvillar enzymes were largely unaffected by colchicine, the results obtained suggest that microtubules play a role in the final transport of the enzymes from the Golgi complex to the microvillar...

  10. Protein Kinase D and Gβγ Subunits Mediate Agonist-evoked Translocation of Protease-activated Receptor-2 from the Golgi Apparatus to the Plasma Membrane.

    Science.gov (United States)

    Jensen, Dane D; Zhao, Peishen; Jimenez-Vargas, Nestor N; Lieu, TinaMarie; Gerges, Marina; Yeatman, Holly R; Canals, Meritxell; Vanner, Stephen J; Poole, Daniel P; Bunnett, Nigel W

    2016-05-20

    Agonist-evoked endocytosis of G protein-coupled receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled receptors from intracellular stores are unexplored. Protease-activated receptor-2 (PAR2) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2-stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca(2+) signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Backscattered electron image of osmium-impregnated/macerated tissues as a novel technique for identifying the cis-face of the Golgi apparatus by high-resolution scanning electron microscopy.

    Science.gov (United States)

    Koga, D; Bochimoto, H; Watanabe, T; Ushiki, T

    2016-07-01

    The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three-dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis-trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis- or trans-faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis-Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis-elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron-mode SEM. Prolonged osmication by osmium impregnation (2% OsO4 solution at 40°C for 40 h) and osmium maceration (0.1% OsO4 solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  12. Distribution and morphological changes of the Golgi apparatus during Drosophila spermatogenesis.

    Science.gov (United States)

    Yasuno, Yusaku; Kawano, Jun-ichi; Inoue, Yoshihiro H; Yamamoto, Masa-Toshi

    2013-08-01

    In spermatogenesis, the Golgi apparatus is important for the formation of the acrosome, which is a sperm-specific organelle essential for fertilization. Comprehensive examinations of the spatiotemporal distribution and morphological characterizations of the Golgi in various cells during spermatogenesis are necessary for functional analyses and mutant screenings in the model eukaryote Drosophila. Here, we examined the distribution and morphology of the Golgi during Drosophila spermatogenesis with immunofluorescence and electron microscopy. In pre-meiotic germ cells, the Golgi apparatuses were distributed evenly in the cytoplasm. In contrast, they were located exclusively in two regions near the poles during the meiotic metaphase, where they were segregated prior to the chromosomes. In cells in anaphase to telophase, the Golgi were predominantly left behind in the equatorial region between the separating daughter nuclei. After completion of meiosis, the dispersed Golgi were assembled at the apical side of the spermatid nucleus to form the acrosome. Further investigation of the Golgi distribution in β2-tubulin mutants showed aberrant and uneven distributions of the Golgi among sister cells in the meiotic spermatocytes and in the post-meiotic spermatids. At the ultrastructural level, the Golgi apparatus in pre-meiotic spermatocytes comprised a pair of stacks. The two stacks were situated adjacent to each other, as if they had duplicated before entering into meiotic division. These results highlight the dynamic nature of the Golgi during spermatogenesis and provide a framework for analyzing the correlations between the dynamics of the Golgi and its function in sperm development. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  13. Feasibility study of rapid opioid rotation and titration.

    Science.gov (United States)

    Korkmazsky, Marina; Ghandehari, Javid; Sanchez, Angela; Lin, Hung-Mo; Lin, Huong-Mo; Pappagallo, Marco

    2011-01-01

    Opioid guidelines recommend opioid rotation and switching for patients who do not achieve adequate pain relief or who experience intolerable adverse events (AEs) with their current opioid. However, specific recommendations and protocols for opioid rotation are lacking, making the practice time consuming and difficult for primary care physicians to accomplish independently or coordinate with a pain specialist. To assess the safety and feasibility of using 24-hour intravenous patient-controlled analgesia (IV-PCA) to achieve rapid opioid rotation and titration (RORT). Open-label pilot study. Hospital research center. At admission, patients (aged ≥ 18 years) with treatment-refractory chronic pain who were taking morphine or oxycodone for ≥ 3 months and had pain scores ≥ 4 on a 10-point scale, underwent opioid rotation to oral oxymorphone extended release (ER). They also received IV-PCA oxymorphone for 24 hours as needed. At discharge, the participants were taking oral oxymorphone ER with oxymorphone immediate release (IR) as needed based on their total 24-hour oral plus IV-PCA oxymorphone use. During a 2-week follow-up, their oxymorphone usage was titrated as needed. Main outcome measures were AEs, Patient Global Impression of Change (PGIC), Brief Pain Inventory (0 = no pain/interference, 10 = worst pain/complete interference), treatment satisfaction, and change in oxymorphone dose. Twelve patients enrolled and completed the 24-hour IV-PCA; 10 completed the 2-week follow-up post-24-hour IV-PCA. PGIC status improved by 12 hours (odds ratio [OR], 0.19, 95% CI, 0.08 - 0.44; P < 0.001), and both PGIC status and activity scores improved by 24 hours (OR, 0.23, 95% CI, 0.09 - 0.55; P = 0.001; OR, 0.49, 95% CI, 0.25 - 0.96; P = 0.04, respectively) and 2 weeks (OR, 0.14, 95% CI, 0.04 - 0.46; P = 0.001; OR, 0.21, 95% CI, 0.06 - 0.72; P = 0.01) versus 6 hours. During the 24-hour IV-PCA time period, 6 of 10 patients accomplished ≥ 50% of their overall dose titration. At 2

  14. Ethanol-induced disruption of Golgi apparatus morphology, primary neurite number and cellular orientation in developing cortical neurons.

    Science.gov (United States)

    Powrozek, Teresa A; Olson, Eric C

    2012-11-01

    Prenatal ethanol exposure disrupts cortical neurite initiation and outgrowth, but prior studies have reported both ethanol-dependent growth promotion and inhibition. To resolve this ambiguity and better approximate in vivo conditions, we quantitatively analyzed neuronal morphology using a new, whole hemisphere explant model. In this model, Layer 6 (L6) cortical neurons migrate, laminate and extend neurites in an organotypic fashion. To selectively label L6 neurons, we performed ex utero electroporation of a GFP expression construct at embryonic day 13 and allowed the explants to develop for 2 days in vitro. Explants were exposed to (400 mg/dL) ethanol for either 4 or 24 h prior to fixation. Complete 3-D reconstructions were made of >80 GFP-positive neurons in each experimental condition. Acute responses to ethanol exposure included compaction of the Golgi apparatus accompanied by elaboration of supernumerary primary apical neurites, as well as a modest (∼15%) increase in higher order apical neurite length. With longer exposure time, ethanol exposure leads to a consistent, significant disorientation of the cell (cell body, primary apical neurite, and Golgi) with respect to the pial surface. The effects on cellular orientation were accompanied by decreased expression of cytoskeletal elements, microtubule-associated protein 2 and F-actin. These findings indicate that upon exposure to ethanol, developing L6 neurons manifest disruptions in Golgi apparatus and cytoskeletal elements which may in turn trigger selective and significant perturbations to primary neurite formation and neuronal polarity. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Golgi fragmentation precedes neuromuscular denervation and is associated with endosome abnormalities in SOD1-ALS mouse motor neurons

    NARCIS (Netherlands)

    van Dis, Vera; Kuijpers, Marijn; Haasdijk, Elize D; Teuling, Eva; Oakes, Scott A; Hoogenraad, Casper C; Jaarsma, Dick

    2014-01-01

    BACKGROUND: Fragmentation of stacked cisterns of the Golgi apparatus into dispersed smaller elements is a feature associated with degeneration of neurons in amyotrophic lateral sclerosis (ALS) and some other neurodegenerative disorders. However, the role of Golgi fragmentation in motor neuron

  16. Aquaporin-3 and aquaporin-4 are sorted differently and separately in the trans-Golgi network

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Sundbye, Sabrina Maria Gade; Nelson, W. James

    2013-01-01

    observed mostly in separate post-Golgi carriers, and spinning disk microscopy showed that most of AQP3 and AQP4 were delivered to the plasma membrane in separate vesicles. In contrast, VSV-G and LDL-R, two well-charcterized basolateral proteins, co-localized to a high degree in the same post-Golgi carriers...

  17. N-Ras induces alterations in Golgi complex architecture and in constitutive protein transport

    NARCIS (Netherlands)

    Babia, T; Ayala, [No Value; Valderrama, F; Mato, E; Bosch, M; Santaren, JF; Renau-Piqueras, J; Kok, JW; Thomsen, TM; Egea, G

    Aberrant glycosylation of proteins and lipids is a common feature of many tumor cell types, and is often accompanied by alterations in membrane traffic and an anomalous localization of Golgi-resident proteins and glycans. These observations suggest that the Golgi complex is a key organelle for at

  18. In vivo analysis of the calcium signature in the plant Golgi apparatus reveals unique dynamics.

    Science.gov (United States)

    Ordenes, Viviana R; Moreno, Ignacio; Maturana, Daniel; Norambuena, Lorena; Trewavas, Anthony J; Orellana, Ariel

    2012-11-01

    The Golgi apparatus is thought to play a role in calcium homeostasis in plant cells. However, the calcium dynamics in this organelle is unknown in plants. To monitor the [Ca2+]Golgiin vivo, we obtained and analyzed Arabidopsis thaliana plants that express aequorin in the Golgi. Our results show that free [Ca2+] levels in the Golgi are higher than in the cytosol (0.70 μM vs. 0.05 μM, respectively). Stimuli such as cold shock, mechanical stimulation and hyperosmotic stress, led to a transient increase in cytosolic calcium; however, no instant change in the [Ca2+]Golgi concentration was detected. Nevertheless, a delayed increase in the [Ca2+]Golgi up to 2-3 μM was observed. Cyclopiazonic acid and thapsigargin inhibited the stimuli-induced [Ca2+]Golgi increase, suggesting that [Ca2+]Golgi levels are dependent upon the activity of Ca2+-ATPases. Treatment of these plants with the synthetic auxin analog, 2,4-dichlorophenoxy acetic acid (2,4-D), produced a slow decrease of free calcium in the organelle. Our results indicate that the plant Golgi apparatus is not involved in the generation of cytosolic calcium transients and exhibits its own dynamics modulated in part by the activity of Ca2+ pumps and hormones. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    NARCIS (Netherlands)

    Jansen, J.C.; Cirak, S.; Scherpenzeel, M. van; Timal, S.; Reunert, J.; Rust, S.; Perez, B.; Vicogne, D.; Krawitz, P.; Wada, Y.; Ashikov, A.M.; Perez-Cerda, C.; Medrano, C.; Arnoldy, A.; Hoischen, A.; Huijben, K.; Steenbergen, G.; Quelhas, D.; Diogo, L.; Rymen, D.; Jaeken, J.; Guffon, N.; Cheillan, D.; Heuvel, B. van den; Maeda, Y.; Kaiser, O.; Schara, U.; Gerner, P.; Boogert, M.A. van den; Holleboom, A.G.; Nassogne, M.C.; Sokal, E.; Salomon, J.; Bogaart, G. van den; Drenth, J.P.; Huynen, M.A.; Veltman, J.A.; Wevers, R.A.; Morava, E.; Matthijs, G.; Foulquier, F.; Marquardt, T.; Lefeber, D.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. Live cell assays to identify regulators of ER to Golgi trafficking

    Science.gov (United States)

    Lisauskas, Tautvydas; Matula, Petr; Claas, Christoph; Reusing, Susanne; Wiemann, Stefan; Erfle, Holger; Lehmann, Lars; Fischer, Peter; Eils, Roland; Rohr, Karl; Storrie, Brian; Starkuviene, Vytaute

    2013-01-01

    We applied fluorescence microscopy based quantitative assays to living cells to identify regulators of ER to Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors, which influence Golgi to ER re-localization of GalT-CFP after brefeldin A (BFA) addition and/or wash-out. We then tested 14 proteins that localize to the ER and/or Golgi complex when over-expressed for a role in ER to Golgi trafficking. Nine of them interfered with the rate of BFA induced redistribution of GalT-CFP from the Golgi complex to the ER, 6 of them interfered with GalT-CFP redistribution from the ER to a juxtanuclear region (i.e., Golgi complex) after BFA wash-out, and 6 of them were positive effectors in both assays. Notably, our live cell approach captures regulator function in ER to Golgi trafficking, that were missed in previous fixed cell assays; as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens. PMID:22132776

  1. Golgi apparatus self-organizes into the characteristic shape via postmitotic reassembly dynamics.

    Science.gov (United States)

    Tachikawa, Masashi; Mochizuki, Atsushi

    2017-05-16

    The Golgi apparatus is a membrane-bounded organelle with the characteristic shape of a series of stacked flat cisternae. During mitosis in mammalian cells, the Golgi apparatus is once fragmented into small vesicles and then reassembled to form the characteristic shape again in each daughter cell. The mechanism and details of the reassembly process remain elusive. Here, by the physical simulation of a coarse-grained membrane model, we reconstructed the three-dimensional morphological dynamics of the Golgi reassembly process. Considering the stability of the interphase Golgi shape, we introduce two hypothetical mechanisms-the Golgi rim stabilizer protein and curvature-dependent restriction on membrane fusion-into the general biomembrane model. We show that the characteristic Golgi shape is spontaneously organized from the assembly of vesicles by proper tuning of the two additional mechanisms, i.e., the Golgi reassembly process is modeled as self-organization. We also demonstrate that the fine Golgi shape forms via a balance of three reaction speeds: vesicle aggregation, membrane fusion, and shape relaxation. Moreover, the membrane fusion activity decreases thickness and the number of stacked cisternae of the emerging shapes.

  2. Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders

    NARCIS (Netherlands)

    D. Jaarsma (Dick); C.C. Hoogenraad (Casper)

    2015-01-01

    textabstractThe Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly complex polarized morphology. The motor protein complex

  3. Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders

    NARCIS (Netherlands)

    Jaarsma, Dick; Hoogenraad, Casper C

    2015-01-01

    The Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly complex polarized morphology. The motor protein complex cytoplasmic dynein has

  4. Membrane flow in plants: Fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and Golgi apparatus with (/sup 3/H)leucine

    Energy Technology Data Exchange (ETDEWEB)

    Kappler, R.; Kristen, U.; Morre, D.J.

    1986-01-01

    Tobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture,was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of (/sup 3/H)leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth.

  5. Increased metabolic activity in nucleus basalis of Meynert neurons in elderly individuals with mild cognitive impairment as indicated by the size of the Golgi apparatus.

    NARCIS (Netherlands)

    Dubelaar, E.J.G.; Mufson, E.J.; Meulen, W.G. ter; Heerikhuize, J.J. van; Verwer, R.W.H.; Swaab, D.F.

    2006-01-01

    In this study, we examined the metabolic activity of nucleus basalis of Meynert (NBM) neurons in individuals clinically diagnosed with no cognitive impairment (NCI, n = 8), mild cognitive impairment (MCI, n = 9), and subjects with moderate Alzheimer disease (AD, n = 7). We used Golgi apparatus (GA)

  6. PDMP blocks brefeldin A-induced retrograde membrane transport from Golgi to ER : Evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism

    NARCIS (Netherlands)

    Kok, JW; Babia, T; Filipeanu, CM; Nelemans, A; Egea, G; Hoekstra, D

    1998-01-01

    In this study, we show that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP

  7. 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.

  8. Golgi proteins in circulating human platelets are distributed across non-stacked, scattered structures.

    Science.gov (United States)

    Yadav, Shilpi; Williamson, Jonathan K; Aronova, Maria A; Prince, Andrew A; Pokrovskaya, Irina D; Leapman, Richard D; Storrie, Brian

    2017-06-01

    Platelets are small, anucleate cell fragments that are central to hemostasis, thrombosis, and inflammation. They are derived from megakaryocytes from which they inherit their organelles. As platelets can synthesize proteins and contain many of the enzymes of the secretory pathway, one might expect all mature human platelets to contain a stacked Golgi apparatus, the central organelle of the secretory pathway. By thin section electron microscopy, stacked membranes resembling the stacked Golgi compartment in megakaryocytes and other nucleated cells can be detected in both proplatelets and platelets. However, the incidence of such structures is low and whether each and every platelet contains such a structure remains an open question. By single-label, immunofluorescence staining, Golgi glycosyltransferases are found within each platelet and map to scattered structures. Whether these structures are positive for marker proteins from multiple Golgi subcompartments remains unknown. Here, we have applied state-of-the-art techniques to probe the organization state of the Golgi apparatus in resting human platelets. By the whole cell volume technique of serial-block-face scanning electron microscopy (SBF-SEM), we failed to observe stacked, Golgi-like structures in any of the 65 platelets scored. When antibodies directed against Golgi proteins were tested against HeLa cells, labeling was restricted to an elongated juxtanuclear ribbon characteristic of a stacked Golgi apparatus. By multi-label immunofluorescence microscopy, we found that each and every resting human platelet was positive for cis, trans, and trans Golgi network (TGN) proteins. However, in each case, the proteins were found in small puncta scattered about the platelet. At the resolution of deconvolved, widefield fluorescence microscopy, these proteins had limited tendency to map adjacent to one another. When the results of 3D structured illumination microscopy (3D SIM), a super resolution technique, were scored

  9. Transport vesicle tethering at the trans Golgi network: coiled coil proteins in action

    Directory of Open Access Journals (Sweden)

    Pak-yan Patricia Cheung

    2016-03-01

    Full Text Available The Golgi complex is decorated with so-called Golgin proteins that share a common feature: a large proportion of their amino acid sequences are predicted to form coiled-coil structures. The possible presence of extensive coiled coils implies that these proteins are highly elongated molecules that can extend a significant distance from the Golgi surface. This property would help them to capture or trap inbound transport vesicles and to tether Golgi mini-stacks together. This review will summarize our current understanding of coiled coil tethers that are needed for the receipt of transport vesicles at the trans Golgi network. How do long tethering proteins actually catch vesicles? Golgi-associated, coiled coil tethers contain numerous binding sites for small GTPases, SNARE proteins, and vesicle coat proteins. How are these interactions coordinated and are any or all of them important for the tethering process? Progress towards understanding these questions and remaining, unresolved mysteries will be discussed.

  10. Golgi Fragmentation in Amyotrophic Lateral Sclerosis, an Overview of Possible Triggers and Consequences

    Directory of Open Access Journals (Sweden)

    Vinod eSundaramoorthy

    2015-10-01

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is an invariably fatal neurodegenerative disorder, which specifically targets motor neurons in the brain, brain stem and spinal cord. Whilst the etiology of ALS remains unknown, fragmentation of the Golgi apparatus is detected in ALS patient motor neurons and in animal/cellular disease models. The Golgi is a highly dynamic organelle that acts as a dispatching station for the vesicular transport of secretory/transmembrane proteins. It also mediates autophagy and maintains endoplasmic reticulum (ER and axonal homeostasis. Both the trigger for Golgi fragmentation and the functional consequences of a fragmented Golgi apparatus in ALS remain unclear. However recent evidence has highlighted defects in vesicular trafficking as a pathogenic mechanism in ALS. This review summarises the evidence describing Golgi fragmentation in ALS, with possible links to other disease processes including cellular trafficking, ER stress, defective autophagy and axonal degeneration.

  11. Recruitment of Arf1-GDP to Golgi by Glo3p-Type ArfGAPs Is Crucial for Golgi Maintenance and Plant Growth1[W][OA

    Science.gov (United States)

    Min, Myung Ki; Jang, Mihue; Lee, Myounghui; Lee, Junho; Song, Kyungyoung; Lee, Yongjik; Choi, Kwan Yong; Robinson, David G.; Hwang, Inhwan

    2013-01-01

    ADP-ribosylation factor1 (Arf1), a member of the small GTP-binding proteins, plays a pivotal role in protein trafficking to multiple organelles. In its GDP-bound form, Arf1 is recruited from the cytosol to organelle membranes, where it functions in vesicle-mediated protein trafficking. However, the mechanism of Arf1-GDP recruitment remains unknown. Here, we provide evidence that two Glo3p-type Arf GTPase-activating proteins (ArfGAPs), ArfGAP domain8 (AGD8) and AGD9, are involved in the recruitment of Arf1-GDP to the Golgi apparatus in Arabidopsis (Arabidopsis thaliana). RNA interference plants expressing low levels of AGD8 and AGD9 exhibited abnormal Golgi morphology, inhibition of protein trafficking, and arrest of plant growth and development. In RNA interference plants, Arf1 was poorly recruited to the Golgi apparatus. Conversely, high levels of AGD8 and AGD9 induced Arf1 accumulation at the Golgi and suppressed Golgi disruption and inhibition of vacuolar trafficking that was caused by overexpression of AGD7. Based on these results, we propose that the Glo3p-type ArfGAPs AGD8 and AGD9 recruit Arf1-GDP from the cytosol to the Golgi for Arf1-mediated protein trafficking, which is essential for plant development and growth. PMID:23266962

  12. Retrograde Transport from Early Endosomes to the trans-Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions.

    Science.gov (United States)

    Sivan, Gilad; Weisberg, Andrea S; Americo, Jeffrey L; Moss, Bernard

    2016-10-01

    The anterograde pathway, from the endoplasmic reticulum through the trans-Golgi network to the cell surface, is utilized by trans-membrane and secretory proteins. The retrograde pathway, which directs traffic in the opposite direction, is used following endocytosis of exogenous molecules and recycling of membrane proteins. Microbes exploit both routes: viruses typically use the anterograde pathway for envelope formation prior to exiting the cell, whereas ricin and Shiga-like toxins and some nonenveloped viruses use the retrograde pathway for cell entry. Mining a human genome-wide RNA interference (RNAi) screen revealed a need for multiple retrograde pathway components for cell-to-cell spread of vaccinia virus. We confirmed and extended these results while discovering that retrograde trafficking was required for virus egress rather than entry. Retro-2, a specific retrograde trafficking inhibitor of protein toxins, potently prevented spread of vaccinia virus as well as monkeypox virus, a human pathogen. Electron and confocal microscopy studies revealed that Retro-2 prevented wrapping of virions with an additional double-membrane envelope that enables microtubular transport, exocytosis, and actin polymerization. The viral B5 and F13 protein components of this membrane, which are required for wrapping, normally colocalize in the trans-Golgi network. However, only B5 traffics through the secretory pathway, suggesting that F13 uses another route to the trans-Golgi network. The retrograde route was demonstrated by finding that F13 was largely confined to early endosomes and failed to colocalize with B5 in the presence of Retro-2. Thus, vaccinia virus makes novel use of the retrograde transport system for formation of the viral wrapping membrane. Efficient cell-to-cell spread of vaccinia virus and other orthopoxviruses depends on the wrapping of infectious particles with a double membrane that enables microtubular transport, exocytosis, and actin polymerization

  13. Insights into the Localization and Function of the Membrane Trafficking Regulator GNOM ARF-GEF at the Golgi Apparatus in Arabidopsis[W

    Science.gov (United States)

    Naramoto, Satoshi; Otegui, Marisa S.; Kutsuna, Natsumaro; de Rycke, Riet; Dainobu, Tomoko; Karampelias, Michael; Fujimoto, Masaru; Feraru, Elena; Miki, Daisuke; Fukuda, Hiroo; Nakano, Akihiko; Friml, Jiří

    2014-01-01

    GNOM is one of the most characterized membrane trafficking regulators in plants, with crucial roles in development. GNOM encodes an ARF-guanine nucleotide exchange factor (ARF-GEF) that activates small GTPases of the ARF (ADP ribosylation factor) class to mediate vesicle budding at endomembranes. The crucial role of GNOM in recycling of PIN auxin transporters and other proteins to the plasma membrane was identified in studies using the ARF-GEF inhibitor brefeldin A (BFA). GNOM, the most prominent regulator of recycling in plants, has been proposed to act and localize at so far elusive recycling endosomes. Here, we report the GNOM localization in context of its cellular function in Arabidopsis thaliana. State-of-the-art imaging, pharmacological interference, and ultrastructure analysis show that GNOM predominantly localizes to Golgi apparatus. Super-resolution confocal live imaging microscopy identified GNOM and its closest homolog GNOM-like 1 at distinct subdomains on Golgi cisternae. Short-term BFA treatment stabilizes GNOM at the Golgi apparatus, whereas prolonged exposures results in GNOM translocation to trans-Golgi network (TGN)/early endosomes (EEs). Malformed TGN/EE in gnom mutants suggests a role for GNOM in maintaining TGN/EE function. Our results redefine the subcellular action of GNOM and reevaluate the identity and function of recycling endosomes in plants. PMID:25012191

  14. Insights into the localization and function of the membrane trafficking regulator GNOM ARF-GEF at the Golgi apparatus in Arabidopsis.

    Science.gov (United States)

    Naramoto, Satoshi; Otegui, Marisa S; Kutsuna, Natsumaro; de Rycke, Riet; Dainobu, Tomoko; Karampelias, Michael; Fujimoto, Masaru; Feraru, Elena; Miki, Daisuke; Fukuda, Hiroo; Nakano, Akihiko; Friml, Jiří

    2014-07-01

    GNOM is one of the most characterized membrane trafficking regulators in plants, with crucial roles in development. GNOM encodes an ARF-guanine nucleotide exchange factor (ARF-GEF) that activates small GTPases of the ARF (ADP ribosylation factor) class to mediate vesicle budding at endomembranes. The crucial role of GNOM in recycling of PIN auxin transporters and other proteins to the plasma membrane was identified in studies using the ARF-GEF inhibitor brefeldin A (BFA). GNOM, the most prominent regulator of recycling in plants, has been proposed to act and localize at so far elusive recycling endosomes. Here, we report the GNOM localization in context of its cellular function in Arabidopsis thaliana. State-of-the-art imaging, pharmacological interference, and ultrastructure analysis show that GNOM predominantly localizes to Golgi apparatus. Super-resolution confocal live imaging microscopy identified GNOM and its closest homolog GNOM-like 1 at distinct subdomains on Golgi cisternae. Short-term BFA treatment stabilizes GNOM at the Golgi apparatus, whereas prolonged exposures results in GNOM translocation to trans-Golgi network (TGN)/early endosomes (EEs). Malformed TGN/EE in gnom mutants suggests a role for GNOM in maintaining TGN/EE function. Our results redefine the subcellular action of GNOM and reevaluate the identity and function of recycling endosomes in plants. © 2014 American Society of Plant Biologists. All rights reserved.

  15. βIII spectrin regulates the structural integrity and the secretory protein transport of the Golgi complex.

    Science.gov (United States)

    Salcedo-Sicilia, Laia; Granell, Susana; Jovic, Marko; Sicart, Adrià; Mato, Eugenia; Johannes, Ludger; Balla, Tamas; Egea, Gustavo

    2013-01-25

    A spectrin-based cytoskeleton is associated with endomembranes, including the Golgi complex and cytoplasmic vesicles, but its role remains poorly understood. Using new generated antibodies to specific peptide sequences of the human βIII spectrin, we here show its distribution in the Golgi complex, where it is enriched in the trans-Golgi and trans-Golgi network. The use of a drug-inducible enzymatic assay that depletes the Golgi-associated pool of PI4P as well as the expression of PH domains of Golgi proteins that specifically recognize this phosphoinositide both displaced βIII spectrin from the Golgi. However, the interference with actin dynamics using actin toxins did not affect the localization of βIII spectrin to Golgi membranes. Depletion of βIII spectrin using siRNA technology and the microinjection of anti-βIII spectrin antibodies into the cytoplasm lead to the fragmentation of the Golgi. At ultrastructural level, Golgi fragments showed swollen distal Golgi cisternae and vesicular structures. Using a variety of protein transport assays, we show that the endoplasmic reticulum-to-Golgi and post-Golgi protein transports were impaired in βIII spectrin-depleted cells. However, the internalization of the Shiga toxin subunit B to the endoplasmic reticulum was unaffected. We state that βIII spectrin constitutes a major skeletal component of distal Golgi compartments, where it is necessary to maintain its structural integrity and secretory activity, and unlike actin, PI4P appears to be highly relevant for the association of βIII spectrin the Golgi complex.

  16. TMEM165 deficiencies in Congenital Disorders of Glycosylation type II (CDG-II): Clues and evidences for roles of the protein in Golgi functions and ion homeostasis.

    Science.gov (United States)

    Dulary, Eudoxie; Potelle, Sven; Legrand, Dominique; Foulquier, François

    2017-04-01

    Congenital Disorders of Glycosylation (CDG) are rare inherited diseases causing glycosylation defects responsible for severe growth and psychomotor retardations in patients. Whereas most genetic defects affect enzymes directly involved in the glycosylation process, like glycosyltransferases or sugar transporters, recent findings revealed the impact of gene mutations on proteins implicated in both Golgi vesicular trafficking and ion homeostasis. TMEM165 is one of these deficient Golgi proteins found in CDG patients whose function in the secretory pathway has been deduced from several recent studies using TMEM165 deficient mammalian cells or yeast cells deficient in Gtd1p, the yeast TMEM165 ortholog. These studies actually confirm previous observations based on both sequence and predicted topology of this transmembrane protein and the phenotypes of human and yeast cells, namely that TMEM165 is very probably a transporter involved in ion homeostasis. Whereas the exact function of TMEM165 remains to be fully characterized, several studies hypothesize that TMEM165 could be a Golgi localized Ca2+/H+ antiporter. However, recent data also support the role of TMEM165 in Golgi Mn2+ homeostasis then arguing for a putative role of Mn2+ transporter for TMEM165 essential to achieve the correct N-glycosylation process of proteins in the secretory pathway. This manuscript is a review of the current state of knowledge on TMEM165 deficiencies in Congenital Disorders of Glycosylation as well as new data on function of TMEM165 and some speculative models on TMEM165/Golgi functions are discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Discrete, continuous, and stochastic models of protein sorting in the Golgi apparatus

    Science.gov (United States)

    Gong, Haijun; Guo, Yusong; Linstedt, Adam; Schwartz, Russell

    2010-01-01

    The Golgi apparatus plays a central role in processing and sorting proteins and lipids in eukaryotic cells. Golgi compartments constantly exchange material with each other and with other cellular components, allowing them to maintain and reform distinct identities despite dramatic changes in structure and size during cell division, development, and osmotic stress. We have developed three minimal models of membrane and protein exchange in the Golgi—a discrete, stochastic model, a continuous ordinary differential equation model, and a continuous stochastic differential equation model—each based on two fundamental mechanisms: vesicle-coat-mediated selective concentration of cargoes and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins during vesicle formation and SNARE-mediated selective fusion of vesicles. By exploring where the models differ, we hope to discover whether the discrete, stochastic nature of vesicle-mediated transport is likely to have appreciable functional consequences for the Golgi. All three models show similar ability to restore and maintain distinct identities over broad parameter ranges. They diverge, however, in conditions corresponding to collapse and reassembly of the Golgi. The results suggest that a continuum model provides a good description of Golgi maintenance but that considering the discrete nature of vesicle-based traffic is important to understanding assembly and disassembly of the Golgi. Experimental analysis validates a prediction of the models that altering guanine nucleotide exchange factor expression levels will modulate Golgi size.

  18. Novel scanning electron microscopy methods for analyzing the 3D structure of the Golgi apparatus.

    Science.gov (United States)

    Koga, Daisuke; Ushiki, Tatsuo; Watanabe, Tsuyoshi

    2017-01-01

    The structure of the Golgi apparatus has been extensively examined by light and electron microscopy, but details of its three-dimensional (3D) structure have remained unclear because of the technical limitations of conventional microscopy techniques. To overcome this problem, we have developed several novel scanning electron microscopy (SEM) methods for observing the 3D structure of subcellular organelles including the Golgi apparatus: (1) an osmium maceration method that facilitates SEM observation of membranous organelles, including the Golgi apparatus, by selectively removing soluble cytoplasmic proteins, (2) an osmium impregnation/maceration method that combines an osmium impregnation method with the osmium maceration method to determine the polarity of the Golgi apparatus by SEM, (3) a correlative light and SEM method that combines a cryosectioning technique with the osmium maceration method to enable correlation of the immunocytochemical distribution of molecules with the 3D ultrastructure of the Golgi apparatus, and (4) array tomography based on the systematic collection and integration of SEM images of serial ultrathin sections on glass slides for revealing the 3D ultrastructure of the entire Golgi apparatus. Together, the novel SEM techniques listed above can reveal the complete 3D structure of the Golgi apparatus in different cell types.

  19. Mutant SOD1 inhibits ER-Golgi transport in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Atkin, Julie D; Farg, Manal A; Soo, Kai Ying; Walker, Adam K; Halloran, Mark; Turner, Bradley J; Nagley, Phillip; Horne, Malcolm K

    2014-04-01

    Cu/Zn-superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER-Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER-Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER-Golgi transport by over-expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER-Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells. © 2013 International Society for Neurochemistry.

  20. ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER

    Science.gov (United States)

    Cheetham, R. D.; Morré, D. James; Pannek, Carol; Friend, Daniel S.

    1971-01-01

    The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue. PMID:5092211

  1. 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.

  2. Decoupling polarization of the Golgi apparatus and GM1 in the plasma membrane.

    Science.gov (United States)

    Bisel, Blaine; Calamai, Martino; Vanzi, Francesco; Pavone, Francesco Saverio

    2013-01-01

    Cell polarization is a process of coordinated cellular rearrangements that prepare the cell for migration. GM1 is synthesized in the Golgi apparatus and localized in membrane microdomains that appear at the leading edge of polarized cells, but the mechanism by which GM1 accumulates asymmetrically is unknown. The Golgi apparatus itself becomes oriented toward the leading edge during cell polarization, which is thought to contribute to plasma membrane asymmetry. Using quantitative image analysis techniques, we measure the extent of polarization of the Golgi apparatus and GM1 in the plasma membrane simultaneously in individual cells subject to a wound assay. We find that GM1 polarization starts just 10 min after stimulation with growth factors, while Golgi apparatus polarization takes 30 min. Drugs that block Golgi polarization or function have no effect on GM1 polarization, and, conversely, inhibiting GM1 polarization does not affect Golgi apparatus polarization. Evaluation of Golgi apparatus and GM1 polarization in single cells reveals no correlation between the two events. Our results indicate that Golgi apparatus and GM1 polarization are controlled by distinct intracellular cascades involving the Ras/Raf/MEK/ERK and the PI3K/Akt/mTOR pathways, respectively. Analysis of cell migration and invasion suggest that MEK/ERK activation is crucial for two dimensional migration, while PI3K activation drives three dimensional invasion, and no cumulative effect is observed from blocking both simultaneously. The independent biochemical control of GM1 polarity by PI3K and Golgi apparatus polarity by MEK/ERK may act synergistically to regulate and reinforce directional selection in cell migration.

  3. Transport of the GlcNAc-1-phosphotransferase α/β-subunit precursor protein to the Golgi apparatus requires a combinatorial sorting motif.

    Science.gov (United States)

    Franke, Mine; Braulke, Thomas; Storch, Stephan

    2013-01-11

    The Golgi-resident N-acetylglucosamine-1-phosphotransferase (PT) complex is composed of two α-, β-, and γ-subunits and represents the key enzyme for the biosynthesis of mannose 6-phosphate recognition marker on soluble lysosomal proteins. Mutations in the PT complex cause the lysosomal storage diseases mucolipidosis II and III. A prerequisite for the enzymatic activity is the site-1 protease-mediated cleavage of the PT α/β-subunit precursor protein in the Golgi apparatus. Here, we have investigated structural requirements of the PT α/β-subunit precursor protein for its efficient export from the endoplasmic reticulum (ER). Both wild-type and a cleavage-resistant type III membrane PT α/β-subunit precursor protein are exported whereas coexpressed separate α- and β-subunits failed to reach the cis-Golgi compartment. Mutational analyses revealed combinatorial, non-exchangeable dileucine and dibasic motifs located in a defined sequence context in the cytosolic N- and C-terminal domains that are required for efficient ER exit and subsequent proteolytic activation of the α/β-subunit precursor protein in the Golgi. In the presence of a dominant negative Sar1 mutant the ER exit of the PT α/β-subunit precursor protein is inhibited indicating its transport in coat protein complex II-coated vesicles. Expression studies of missense mutations identified in mucolipidosis III patients that alter amino acids in the N- and C-terminal domains demonstrated that the substitution of a lysine residue in close proximity to the dileucine sorting motif impaired ER-Golgi transport and subsequent activation of the PT α/β-subunit precursor protein. The data suggest that the oligomeric type III membrane protein PT complex requires a combinatorial sorting motif that forms a tertiary epitope to be recognized by distinct sites within the coat protein complex II machinery.

  4. Role of the Golgi Apparatus in the Blood-Brain Barrier: Golgi Protection May Be a Targeted Therapy for Neurological Diseases.

    Science.gov (United States)

    Deng, Shuwen; Liu, Hui; Qiu, Ke; You, Hong; Lei, Qiang; Lu, Wei

    2017-07-20

    The blood-brain barrier (BBB) protects the brain from toxic material in the blood, provides nutrients for brain tissues, and screens harmful substances from the brain. The specific brain microvascular endothelial cells (BMVECs), tight junction between endothelial cells, and astrocytes ensure proper function of the central nervous system (CNS). Pathological factors disrupt the integrity of the BBB by destroying the normal function of endothelial cells and decreasing the production of tight junction proteins or the expression of proteins specifically localized on astrocytes. Interestingly, fragmentation of the Golgi apparatus is observed in neurological diseases and is involved in the destruction of the BBB function. The Golgi acts as a processing center in which proteins are transported after being processed in the endoplasmic reticulum. Besides reprocessing, classifying, and packaging proteins, the Golgi apparatus (GA) also acts as a signaling platform and calcium pool. In this review, we summarized the current literature on the potential relationship between the Golgi and endothelial cells, tight junction, and astrocytes. The normal function of the BBB is maintained as long as the normal function and morphology of the GA are not disturbed. Furthermore, we speculate that protecting the Golgi may be a novel therapeutic approach to protect the BBB and treat neurological diseases due to BBB dysfunction.

  5. Functional analysis of putative phosphoenolpyruvate transporters localized to the Golgi apparatus in Schizosaccharomyces pombe.

    Science.gov (United States)

    Yoritsune, Ken-ichi; Higuchi, Yujiro; Matsuzawa, Tomohiko; Takegawa, Kaoru

    2014-11-01

    The cell surface of Schizosaccharomyces pombe is negatively charged due to the presence of pyruvylated oligosaccharides, which is important for cell-cell recognition. However, the mechanism of pyruvate supply to oligosaccharides is not clearly understood. Here, we analyzed three putative phosphoenolpyruvate (PEP) transporter genes (pet1(+) , pet2(+) , and pet3(+) ) in S. pombe, identified by sequence homology search against the Arabidopsis thaliana PEP transporter AtPPT1. Schizosaccharomyces pombe strain carrying a disruption in pet1(+) (pet1Δ) or in pet2(+) (pet2Δ), but not the strain carrying a disruption in pet3(+) (pet3Δ), showed reduced pyruvate level on the cell surface. This reduction in pyruvate level was restored to the control level by expressing green fluorescent protein (GFP)-tagged Pet1p and Pet2p in respective disruptants. Fluorescence microscope studies revealed that GFP-tagged Pet1p and Pet2p were localized to the Golgi apparatus. Although expression of neither AtPPT1 nor AtPPT2 suppressed the pet1Δ phenotype, that of chimeric constructs, where the N-terminal regions of AtPPT1 and AtPPT2 were replaced by the N-terminal region of Pet1p, partially suppressed the pet1Δ phenotype. Furthermore, the reduction in cell surface negative charge in pet1Δ cells was restored by incubating these cells with recombinant Pvg1p and PEP. Thus, Pet1p and Pet2p are likely involved in transporting PEP from the cytoplasm into the Golgi. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  6. Differential calcium handling by the cis and trans regions of the Golgi apparatus.

    Science.gov (United States)

    Aulestia, Francisco J; Alonso, María Teresa; García-Sancho, Javier

    2015-03-15

    High Ca2+ content in the Golgi apparatus (Go) is essential for protein processing and sorting. In addition, the Go can shape the cytosolic Ca2+ signals by releasing or sequestering Ca2+. We generated two new aequorin-based Ca2+ probes to specifically measure Ca2+ in the cis/cis-to-medial-Go (cGo) or the trans-Go (tGo). Ca2+ homoeostasis in these compartments and in the endoplasmic reticulum (ER) has been studied and compared. Moreover, the relative size of each subcompartment was estimated from aequorin consumption. We found that the cGo accumulates Ca2+ to high concentrations (150-300 μM) through the sarco plasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). The tGo, in turn, is divided into two subcompartments: tGo1 and tGo2. The subcompartment tGo1 contains 20% of the aequorin and has a high internal [Ca2+]; Ca2+ is accumulated in this subcompartment via the secretory pathway Ca2+-ATPase 1 (SPCA-1) at a very high affinity (K50=30 nM). The subcompartment tGo2 contains 80% of aequorin, has a lower [Ca2+] and no SPCA-1 activity; Ca2+ uptake happens through SERCA and is slower than in tGo1. The two tGo subcompartments, tGo1 and tGo2, are diffusionally isolated. Inositol trisphosphate mobilizes Ca2+ from the cGo and tGo2, but not from tGo1, whereas caffeine releases Ca2+ from all the Golgi regions, and nicotinic acid dinucleotide phosphate and cADP ribose from none.

  7. 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

  8. Cell biology of the endoplasmic reticulum and the Golgi apparatus through proteomics.

    Science.gov (United States)

    Smirle, Jeffrey; Au, Catherine E; Jain, Michael; Dejgaard, Kurt; Nilsson, Tommy; Bergeron, John

    2013-01-01

    Enriched endoplasmic reticulum (ER) and Golgi membranes subjected to mass spectrometry have uncovered over a thousand different proteins assigned to the ER and Golgi apparatus of rat liver. This, in turn, led to the uncovering of several hundred proteins of poorly understood function and, through hierarchical clustering, showed that proteins distributed in patterns suggestive of microdomains in cognate organelles. This has led to new insights with respect to their intracellular localization and function. Another outcome has been the critical testing of the cisternal maturation hypothesis showing overwhelming support for a predominant role of COPI vesicles in the transport of resident proteins of the ER and Golgi apparatus (as opposed to biosynthetic cargo). Here we will discuss new insights gained and also highlight new avenues undertaken to further explore the cell biology of the ER and the Golgi apparatus through tandem mass spectrometry.

  9. Sec16 determines the size and functioning of the Golgi in the protist parasite, Trypanosoma brucei.

    Science.gov (United States)

    Sealey-Cardona, Marco; Schmidt, Katy; Demmel, Lars; Hirschmugl, Tatjana; Gesell, Tanja; Dong, Gang; Warren, Graham

    2014-06-01

    The Sec16 homologue in Trypanosoma brucei has been identified and characterized. TbSec16 colocalizes with COPII components at the single endoplasmic reticulum exit site (ERES), which is next to the single Golgi stack in the insect (procyclic) form of this organism. Depletion of TbSec16 reduces the size of the ERES and the Golgi, and slows growth and transport of a secretory marker to the cell surface; conversely, overexpression of TbSec16 increases the size of the ERES and Golgi but has no effect on growth or secretion. Together these data suggest that TbSec16 regulates the size of the ERES and Golgi and this size is set for optimal growth of the organism. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. 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...

  11. 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

  12. Wolbachia bacteria reside in host Golgi-related vesicles whose position is regulated by polarity proteins.

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Cho

    Full Text Available Wolbachia pipientis are intracellular symbiotic bacteria extremely common in various organisms including Drosophila melanogaster, and are known for their ability to induce changes in host reproduction. These bacteria are present in astral microtubule-associated vesicular structures in host cytoplasm, but little is known about the identity of these vesicles. We report here that Wolbachia are restricted only to a group of Golgi-related vesicles concentrated near the site of membrane biogenesis and minus-ends of microtubules. The Wolbachia vesicles were significantly mislocalized in mutant embryos defective in cell/planar polarity genes suggesting that cell/tissue polarity genes are required for apical localization of these Golgi-related vesicles. Furthermore, two of the polarity proteins, Van Gogh/Strabismus and Scribble, appeared to be present in these Golgi-related vesicles. Thus, establishment of polarity may be closely linked to the precise insertion of Golgi vesicles into the new membrane addition site.

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

    Directory of Open Access Journals (Sweden)

    Maria Rödiger

    2018-02-01

    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.

  14. Study on rapid evacuation in high-rise buildings

    Directory of Open Access Journals (Sweden)

    Xin Zhang

    2017-06-01

    Full Text Available More and more high rising buildings emerged in modern cities, but emergency evacuation of tall buildings has been a worldwide difficult problem. In this paper, a new evacuation device for high rising buildings in fire accident was proposed and studied. This device mainly consisted of special spiral slideway and shunt valve. People in this device could fast slide down to the first floor under gravity without any electric power and physical strength, which is suitable for various emergency evacuation including mobility-impaired persons. The plane simulation test has shown that human being in alternative clockwise and counterclockwise movement will not become dizzy. The evacuated people should wear protection pad, which can prevent slider from being injured by surface friction with the slide, and eliminate the friction coefficient difference caused by different clothes and slide surface. The calculation results show that the evacuation speed of the new device is much faster than traditional staircases. Moreover, such new evacuation device can also be used as a means of vertical transportation in high-rise buildings partly. People can take it from any floor to ground floor directly, which not only save time for waiting for the lifts but also save the power. The new evacuation system is of simple structure, easy to use, and suitable for evacuation and partly used as vertical downwards traffic, which shows light on solving world-wide difficulties on fast evacuation in high-rise buildings.

  15. A cyclooxygenase-2-dependent prostaglandin E2 biosynthetic system in the Golgi apparatus.

    Science.gov (United States)

    Yuan, Chong; Smith, William L

    2015-02-27

    Cyclooxygenases (COXs) catalyze the committed step in prostaglandin (PG) biosynthesis. COX-1 is constitutively expressed and stable, whereas COX-2 is inducible and short lived. COX-2 is degraded via endoplasmic reticulum (ER)-associated degradation (ERAD) following post-translational glycosylation of Asn-594. COX-1 and COX-2 are found in abundance on the luminal surfaces of the ER and inner membrane of the nuclear envelope. Using confocal immunocytofluorescence, we detected both COX-2 and microsomal PGE synthase-1 (mPGES-1) but not COX-1 in the Golgi apparatus. Inhibition of trafficking between the ER and Golgi retarded COX-2 ERAD. COX-2 has a C-terminal STEL sequence, which is an inefficient ER retention signal. Substituting this sequence with KDEL, a robust ER retention signal, concentrated COX-2 in the ER where it was stable and slowly glycosylated on Asn-594. Native COX-2 and a recombinant COX-2 having a Golgi targeting signal but not native COX-1 exhibited efficient catalytic coupling to mPGES-1. We conclude that N-glycosylation of Asn-594 of COX-2 occurs in the ER, leading to anterograde movement of COX-2 to the Golgi where the Asn-594-linked glycan is trimmed prior to retrograde COX-2 transport to the ER for ERAD. Having an inefficient ER retention signal leads to sluggish Golgi to ER transit of COX-2. This permits significant Golgi residence time during which COX-2 can function catalytically. Cytosolic phospholipase A2α, which mobilizes arachidonic acid for PG synthesis, preferentially translocates to the Golgi in response to physiologic Ca(2+) mobilization. We propose that cytosolic phospholipase A2α, COX-2, and mPGES-1 in the Golgi comprise a dedicated system for COX-2-dependent PGE2 biosynthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ying-Nai; Wang, Hongmei; Yamaguchi, Hirohito [Department of Molecular and Cellular Oncology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030 (United States); Lee, Hong-Jen; Lee, Heng-Huan [Department of Molecular and Cellular Oncology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030 (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030 (United States); Hung, Mien-Chie, E-mail: mhung@mdanderson.org [Department of Molecular and Cellular Oncology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030 (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030 (United States); Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University and Hospital, Taichung 404, Taiwan (China); Asia University, Taichung 413, Taiwan (China)

    2010-09-03

    Research highlights: {yields} ARF1 activation is involved in the EGFR transport to the ER and the nucleus. {yields} Assembly of {gamma}-COP coatomer mediates EGFR transport to the ER and the nucleus. {yields} 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{sub 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 {gamma}-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.

  17. An OBSL1-Cul7Fbxw8 Ubiquitin Ligase Signaling Mechanism Regulates Golgi Morphology and Dendrite Patterning

    Science.gov (United States)

    Litterman, Nadia; Ikeuchi, Yoshiho; Gallardo, Gilbert; O'Connell, Brenda C.; Sowa, Mathew E.; Gygi, Steven P.; Harper, J. Wade; Bonni, Azad

    2011-01-01

    The elaboration of dendrites in neurons requires secretory trafficking through the Golgi apparatus, but the mechanisms that govern Golgi function in neuronal morphogenesis in the brain have remained largely unexplored. Here, we report that the E3 ubiquitin ligase Cul7Fbxw8 localizes to the Golgi complex in mammalian brain neurons. Inhibition of Cul7Fbxw8 by independent approaches including Fbxw8 knockdown reveals that Cul7Fbxw8 is selectively required for the growth and elaboration of dendrites but not axons in primary neurons and in the developing rat cerebellum in vivo. Inhibition of Cul7Fbxw8 also dramatically impairs the morphology of the Golgi complex, leading to deficient secretory trafficking in neurons. Using an immunoprecipitation/mass spectrometry screening approach, we also uncover the cytoskeletal adaptor protein OBSL1 as a critical regulator of Cul7Fbxw8 in Golgi morphogenesis and dendrite elaboration. OBSL1 forms a physical complex with the scaffold protein Cul7 and thereby localizes Cul7 at the Golgi apparatus. Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites. Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7Fbxw8 in the control of Golgi and dendrite morphogenesis in neurons. Collectively, these findings define a novel OBSL1-regulated Cul7Fbxw8 ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development. PMID:21572988

  18. Loss of the golgin GM130 causes Golgi disruption, Purkinje neuron loss, and ataxia in mice.

    Science.gov (United States)

    Liu, Chunyi; Mei, Mei; Li, Qiuling; Roboti, Peristera; Pang, Qianqian; Ying, Zhengzhou; Gao, Fei; Lowe, Martin; Bao, Shilai

    2017-01-10

    The Golgi apparatus lies at the heart of the secretory pathway where it is required for secretory trafficking and cargo modification. Disruption of Golgi architecture and function has been widely observed in neurodegenerative disease, but whether Golgi dysfunction is causal with regard to the neurodegenerative process, or is simply a manifestation of neuronal death, remains unclear. Here we report that targeted loss of the golgin GM130 leads to a profound neurological phenotype in mice. Global KO of mouse GM130 results in developmental delay, severe ataxia, and postnatal death. We further show that selective deletion of GM130 in neurons causes fragmentation and defective positioning of the Golgi apparatus, impaired secretory trafficking, and dendritic atrophy in Purkinje cells. These cellular defects manifest as reduced cerebellar size and Purkinje cell number, leading to ataxia. Purkinje cell loss and ataxia first appear during postnatal development but progressively worsen with age. Our data therefore indicate that targeted disruption of the mammalian Golgi apparatus and secretory traffic results in neuronal degeneration in vivo, supporting the view that Golgi dysfunction can play a causative role in neurodegeneration.

  19. PKCδ and ε regulate the morphological integrity of the ER-Golgi intermediate compartment (ERGIC) but not the anterograde and retrograde transports via the Golgi apparatus.

    Science.gov (United States)

    Sugawara, Taichi; Nakatsu, Daiki; Kii, Hiroaki; Maiya, Nobuhiko; Adachi, Atsuhiro; Yamamoto, Akitsugu; Kano, Fumi; Murata, Masayuki

    2012-04-01

    The ER-Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Golgi fragmentation precedes neuromuscular denervation and is associated with endosome abnormalities in SOD1-ALS mouse motor neurons

    Science.gov (United States)

    2014-01-01

    Background Fragmentation of stacked cisterns of the Golgi apparatus into dispersed smaller elements is a feature associated with degeneration of neurons in amyotrophic lateral sclerosis (ALS) and some other neurodegenerative disorders. However, the role of Golgi fragmentation in motor neuron degeneration is not well understood. Results Here we use a SOD1-ALS mouse model (low-copy Gurney G93A-SOD1 mouse) to show that motor neurons with Golgi fragmentation are retrogradely labeled by intramuscularly injected CTB (beta subunit of cholera toxin), indicating that Golgi fragmentation precedes neuromuscular denervation and axon retraction. We further show that Golgi fragmentation may occur in the absence of and precede two other pathological markers, i.e. somatodendritic SOD1 inclusions, and the induction of ATF3 expression. In addition, we show that Golgi fragmentation is associated with an altered dendritic organization of the Golgi apparatus, does not depend on intact apoptotic machinery, and is facilitated in transgenic mice with impaired retrograde dynein-dependent transport (BICD2-N mice). A connection to altered dynein-dependent transport also is suggested by reduced expression of endosomal markers in neurons with Golgi fragmentation, which also occurs in neurons with impaired dynein function. Conclusions Together the data indicate that Golgi fragmentation is a very early event in the pathological cascade in ALS that is associated with altered organization of intracellular trafficking. PMID:24708899

  1. 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.

  2. 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...... have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19...

  3. Total dural irradiation: RapidArc versus static-field IMRT: A case study

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, Paul J., E-mail: paulj.kelly@hse.ie [Department of Radiation Oncology, Dana Farber/Brigham and Women' s Cancer Center, Harvard Medical School, Boston, MA (United States); Mannarino, Edward; Lewis, John Henry; Baldini, Elizabeth H.; Hacker, Fred L. [Department of Radiation Oncology, Dana Farber/Brigham and Women' s Cancer Center, Harvard Medical School, Boston, MA (United States)

    2012-07-01

    The purpose of this study was to compare conventional fixed-gantry angle intensity-modulated radiation therapy (IMRT) with RapidArc for total dural irradiation. We also hypothesize that target volume-individualized collimator angles may produce substantial normal tissue sparing when planning with RapidArc. Five-, 7-, and 9-field fixed-gantry angle sliding-window IMRT plans were generated for comparison with RapidArc plans. Optimization and normal tissue constraints were constant for all plans. All plans were normalized so that 95% of the planning target volume (PTV) received at least 100% of the dose. RapidArc was delivered using 350 Degree-Sign clockwise and counterclockwise arcs. Conventional collimator angles of 45 Degree-Sign and 315 Degree-Sign were compared with 90 Degree-Sign on both arcs. Dose prescription was 59.4 Gy in 33 fractions. PTV metrics used for comparison were coverage, V{sub 107}%, D1%, conformality index (CI{sub 95}%), and heterogeneity index (D{sub 5}%-D{sub 95}%). Brain dose, the main challenge of this case, was compared using D{sub 1}%, Dmean, and V{sub 5} Gy. Dose to optic chiasm, optic nerves, globes, and lenses was also compared. The use of unconventional collimator angles (90 Degree-Sign on both arcs) substantially reduced dose to normal brain. All plans achieved acceptable target coverage. Homogeneity was similar for RapidArc and 9-field IMRT plans. However, heterogeneity increased with decreasing number of IMRT fields, resulting in unacceptable hotspots within the brain. Conformality was marginally better with RapidArc relative to IMRT. Low dose to brain, as indicated by V5Gy, was comparable in all plans. Doses to organs at risk (OARs) showed no clinically meaningful differences. The number of monitor units was lower and delivery time was reduced with RapidArc. The case-individualized RapidArc plan compared favorably with the 9-field conventional IMRT plan. In view of lower monitor unit requirements and shorter delivery time, Rapid

  4. 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

  5. Aquaporin-3 and aquaporin-4 are sorted differently and separately in the trans-Golgi network.

    Directory of Open Access Journals (Sweden)

    Eva C Arnspang

    Full Text Available Aquaporin-3 (AQP3 and aquaporin-4 (AQP4 are homologous proteins expressed in the basolateral plasma membrane of kidney collecting duct principal cells, where they mediate the exit pathway for apically reabsorbed water. Although both proteins are localized to the same plasma membrane domain, it is unknown if they are sorted together in the Golgi, or arrive in the same or different vesicles at the plasma membrane. We addressed these questions using high resolution deconvolution imaging, spinning disk and laser scanning confocal microscopy of cells expressing AQP3 and AQP4. AQP3 and AQP4 were observed mostly in separate post-Golgi carriers, and spinning disk microscopy showed that most of AQP3 and AQP4 were delivered to the plasma membrane in separate vesicles. In contrast, VSV-G and LDL-R, two well-characterized basolateral proteins, co-localized to a high degree in the same post-Golgi carriers, indicating that the differential sorting of AQP3 and AQP4 is specific and regulated. Significantly, a chimeric AQP3 containing the AQP4 cytoplasmic tails co-localized with AQP4 in post-Golgi vesicles. These results indicate that AQP3 and AQP4 are separated into different post-Golgi carriers based on different cytoplasmic domain sorting signals, and are then delivered separately to the plasma membrane.

  6. The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing.

    Science.gov (United States)

    Pourcelot, Marie; Zemirli, Naima; Silva Da Costa, Leandro; Loyant, Roxane; Garcin, Dominique; Vitour, Damien; Munitic, Ivana; Vazquez, Aimé; Arnoult, Damien

    2016-08-18

    After viral infection and the stimulation of some pattern-recognition receptors, TANK-binding kinase I (TBK1) is activated by K63-linked polyubiquitination followed by trans-autophosphorylation. While the activated TBK1 induces type I interferon production by phosphorylating the transcription factor IRF3, the precise molecular mechanisms underlying TBK1 activation remain unclear. We report here the localization of the ubiquitinated and phosphorylated active form of TBK1 to the Golgi apparatus after the stimulation of RIG-I-like receptors (RLRs) or Toll-like receptor-3 (TLR3), due to TBK1 K63-linked ubiquitination on lysine residues 30 and 401. The ubiquitin-binding protein optineurin (OPTN) recruits ubiquitinated TBK1 to the Golgi apparatus, leading to the formation of complexes in which TBK1 is activated by trans-autophosphorylation. Indeed, OPTN deficiency in various cell lines and primary cells impairs TBK1 targeting to the Golgi apparatus and its activation following RLR or TLR3 stimulation. Interestingly, the Bluetongue virus NS3 protein binds OPTN at the Golgi apparatus, neutralizing its activity and thereby decreasing TBK1 activation and downstream signaling. Our results highlight an unexpected role of the Golgi apparatus in innate immunity as a key subcellular gateway for TBK1 activation after RNA virus infection.

  7. PAQR3 modulates cholesterol homeostasis by anchoring Scap/SREBP complex to the Golgi apparatus.

    Science.gov (United States)

    Xu, Daqian; Wang, Zheng; Zhang, Yuxue; Jiang, Wei; Pan, Yi; Song, Bao-Liang; Chen, Yan

    2015-08-27

    Cholesterol biosynthesis is regulated by transcription factors SREBPs and their escort protein Scap. On sterol depletion, Scap/SREBP complex is transported from endoplasmic reticulum (ER) to the Golgi apparatus where SREBP is activated. Under cholesterol sufficient condition, Insigs act as anchor proteins to retain Scap/SREBP in the ER. However, the anchor protein of Scap/SREBP in the Golgi is unknown. Here we report that a Golgi-localized membrane protein progestin and adipoQ receptors 3 (PAQR3) interacts with Scap and SREBP and tethers them to the Golgi. PAQR3 promotes Scap/SREBP complex formation, potentiates SREBP processing and enhances lipid synthesis. The mutually exclusive interaction between Scap and PAQR3 or Insig-1 is regulated by cholesterol level. PAQR3 knockdown in liver blunts SREBP pathway and decreases hepatic cholesterol content. Disrupting the interaction of PAQR3 with Scap/SREBP by a synthetic peptide inhibits SREBP processing and activation. Thus, PAQR3 regulates cholesterol homeostasis by anchoring Scap/SREBP to the Golgi and disruption of such function reduces cholesterol biosynthesis.

  8. The role of GRASPs in morphological alterations of Golgi apparatus: mechanisms and effects.

    Science.gov (United States)

    Ji, Guang; Ji, Hui; Mo, Xiaoye; Li, Ting; Yu, Yaduo; Hu, Zhiping

    2013-01-01

    The Golgi apparatus (GA) is a pivotal organelle in cell metabolism, functioning not only in the processing and transportation of cargoes but also in ion homeostasis, cell apoptosis, and stress sensing. We are interested in the intricate role of GA and the recently present novel concept of 'GA stress'. GA shows various morphological alterations in many neurodegenerative diseases and cell apoptosis induced by biochemical reagents, mechanisms in which oxidative stress is strongly involved. In turn, the structural changes and morphological alterations of the GA could also transduce stress signals. Therefore, besides the biochemical changes, more attention should be paid to the morphological alterations of the GA itself during pathological processes and diseases. The Golgi reassembly and stacking proteins (GRASPs) have been identified as important components acting in the transformation of Golgi structure, and they may thus affect the Golgi functions and cell behavior. In this review, we will discuss the intricate role of the GRASPs in remodeling the GA morphology and focus on their mechanisms and effects in the processes of Golgi stacking, mitosis, cell apoptosis, and cargo secretion. We would also like to provide a further prospective of their potential biological values in neurodegenerative diseases.

  9. Atypical protein kinase C regulates primary dendrite specification of cerebellar Purkinje cells by localizing Golgi apparatus.

    Science.gov (United States)

    Tanabe, Koji; Kani, Shuichi; Shimizu, Takashi; Bae, Young-Ki; Abe, Takaya; Hibi, Masahiko

    2010-12-15

    Neurons have highly polarized structures that determine what parts of the soma elaborate the axon and dendrites. However, little is known about the mechanisms that establish neuronal polarity in vivo. Cerebellar Purkinje cells extend a single primary dendrite from the soma that ramifies into a highly branched dendritic arbor. We used the zebrafish cerebellum to investigate the mechanisms by which Purkinje cells acquire these characteristics. To examine dendritic morphogenesis in individual Purkinje cells, we marked the cell membrane using a Purkinje cell-specific promoter to drive membrane-targeted fluorescent proteins. We found that zebrafish Purkinje cells initially extend multiple neurites from the soma and subsequently retract all but one, which becomes the primary dendrite. In addition, the Golgi apparatus specifically locates to the root of the primary dendrite, and its localization is already established in immature Purkinje cells that have multiple neurites. Inhibiting secretory trafficking through the Golgi apparatus reduces dendritic growth, suggesting that the Golgi apparatus is involved in the dendritic morphogenesis. We also demonstrated that in a mutant of an atypical protein kinase C (aPKC), Prkci, Purkinje cells retain multiple primary dendrites and show disrupted localization of the Golgi apparatus. Furthermore, a mosaic inhibition of Prkci in Purkinje cells recapitulates the aPKC mutant phenotype. These results suggest that the aPKC cell autonomously controls the Golgi localization and thereby regulates the specification of the primary dendrite of Purkinje cells.

  10. 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.

  11. Myomegalin is necessary for the formation of centrosomal and Golgi-derived microtubules

    Directory of Open Access Journals (Sweden)

    Régine Roubin

    2012-12-01

    The generation of cellular microtubules is initiated at specific sites such as the centrosome and the Golgi apparatus that contain nucleation complexes rich in γ-tubulin. The microtubule growing plus-ends are stabilized by plus-end tracking proteins (+TIPs, mainly EB1 and associated proteins. Myomegalin was identified as a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterase. We show here that Myomegalin exists as several isoforms. We characterize two of them. One isoform, CM-MMG, harbors a conserved domain (CM1, recently described as a nucleation activator, and is related to a family of γ-tubulin binding proteins, which includes Drosophila centrosomin. It localizes at the centrosome and at the cis-Golgi in an AKAP450-dependent manner. It recruits γ-tubulin nucleating complexes and promotes microtubule nucleation. The second isoform, EB-MMG, is devoid of CM1 domain and has a unique N-terminus with potential EB1-binding sites. It localizes at the cis-Golgi and can localize to microtubule plus-ends. EB-MMG binds EB1 and affects its loading on microtubules and microtubule growth. Depletion of Myomegalin by small interfering RNA delays microtubule growth from the centrosome and Golgi apparatus, and decreases directional migration of RPE1 cells. In conclusion, the Myomegalin gene encodes different isoforms that regulate microtubules. At least two of these have different roles, demonstrating a previously unknown mechanism to control microtubules in vertebrate cells.

  12. Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models.

    Science.gov (United States)

    Višnjar, Tanja; Chesi, Giancarlo; Iacobacci, Simona; Polishchuk, Elena; Resnik, Nataša; Robenek, Horst; Kreft, Marko; Romih, Rok; Polishchuk, Roman; Kreft, Mateja Erdani

    2017-10-09

    Uroplakins (UPs) play an essential role in maintaining an effective urothelial permeability barrier at the level of superficial urothelial cell (UC) layer. Although the organization of UPs in the apical plasma membrane (PM) of UCs is well known, their transport in UCs is only partially understood. Here, we dissected trafficking of UPs and its differentiation-dependent impact on Golgi apparatus (GA) architecture. We demonstrated that individual subunits UPIb and UPIIIa are capable of trafficking from the endoplasmic reticulum to the GA in UCs. Moreover, UPIb, UPIIIa or UPIb/UPIIIa expressing UCs revealed fragmentation and peripheral redistribution of Golgi-units. Notably, expression of UPIb or UPIb/UPIIIa triggered similar GA fragmentation in MDCK and HeLa cells that do not express UPs endogenously. The colocalization analysis of UPIb/UPIIIa-EGFP and COPI, COPII or clathrin suggested that UPs follow constitutively the post-Golgi route to the apical PM. Depolymerisation of microtubules leads to complete blockade of the UPIb/UPIIIa-EGFP post-Golgi transport, while disassembly of actin filaments shows significantly reduced delivery of UPIb/UPIIIa-EGFP to the PM. Our findings show the significant effect of the UPs expression on the GA fragmentation, which enables secretory Golgi-outpost to be distributed as close as possible to the sites of cargo delivery at the PM.

  13. 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.

  14. 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.

  15. 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

  16. A Rapid Compression Expansion Machine (RCEM) for studying chemical kinetics: Experimental principle and first applications

    CERN Document Server

    Werler, Marc; Maas, Ulrich

    2016-01-01

    A novel extension of a rapid compression machine (RCM), namely a Rapid Compression Expansion Machine (RCEM), is described and its use for studying chemical kinetics is demonstrated. Like conventional RCMs, the RCEM quickly compresses a fuel/air mixture by pushing a piston into a cylinder; the resulting high temperatures and pressures initiate chemical reactions. In addition, the machine can rapidly expand the compressed gas in a controlled way by pulling the piston outwards again. This freezes chemical activity after a pre-defined reaction duration, and therefore allows a convenient probe sampling and ex-situ gas analysis of stable species. The RCEM therefore is a promising instrument for studying chemical kinetics, including also partially reacted fuel/air mixtures. The setup of the RCEM, its experimental characteristics and its use for studying chemical reactions are outlined in detail. To allow comparisons of RCEM results with predictions of chemical reaction mechanisms, a simple numerical model of the RCE...

  17. The first images of nerve cells: Golgi on the olfactory bulb 1875.

    Science.gov (United States)

    Shepherd, Gordon M; Greer, Charles A; Mazzarello, Paolo; Sassoè-Pognetto, Marco

    2011-01-07

    The third paper by Camillo Golgi on his new method was on the olfactory bulb. This paper has never been translated into English, but is of special interest both for its pioneering description of olfactory bulb cells and for containing the first illustration by Golgi of cells stained with his new method. A translation into English is provided in this paper, together with commentaries on the significant points in his descriptions. These results are placed in the perspective of Cajal's subsequent first publication on the olfactory bulb and brief mention of the work of other early histologists. This perspective allows one to see more clearly Golgi's fundamental contributions to the olfactory bulb in particular and to the description of the neuronal architecture of the brain in general. Copyright © 2010. Published by Elsevier B.V.

  18. Diacylglycerol is required for the formation of COPI vesicles in the Golgi-to-ER transport pathway.

    Science.gov (United States)

    Fernández-Ulibarri, Inés; Vilella, Montserrat; Lázaro-Diéguez, Francisco; Sarri, Elisabet; Martínez, Susana E; Jiménez, Nuria; Claro, Enrique; Mérida, Isabel; Burger, Koert N J; Egea, Gustavo

    2007-09-01

    Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation.

  19. GO-PROMTO illuminates protein membrane topologies of glycan biosynthetic enzymes in the Golgi apparatus of living tissues.

    Science.gov (United States)

    Søgaard, Casper; Stenbæk, Anne; Bernard, Sophie; Hadi, Masood; Driouich, Azeddine; Scheller, Henrik Vibe; Sakuragi, Yumiko

    2012-01-01

    The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes.

  20. PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity

    Science.gov (United States)

    Matsui, Toshinori; Watanabe, Takashi; Matsuzawa, Kenji; Kakeno, Mai; Okumura, Nobumasa; Sugiyama, Ikuko; Itoh, Norimichi; Kaibuchi, Kozo

    2015-01-01

    The organization of the Golgi apparatus is essential for cell polarization and its maintenance. The polarity regulator PAR complex (PAR3, PAR6, and aPKC) plays critical roles in several processes of cell polarization. However, how the PAR complex participates in regulating the organization of the Golgi remains largely unknown. Here we demonstrate the functional cross-talk of the PAR complex with CLASP2, which is a microtubule plus-end–tracking protein and is involved in organizing the Golgi ribbon. CLASP2 directly interacted with PAR3 and was phosphorylated by aPKC. In epithelial cells, knockdown of either PAR3 or aPKC induced the aberrant accumulation of CLASP2 at the trans-Golgi network (TGN) concomitantly with disruption of the Golgi ribbon organization. The expression of a CLASP2 mutant that inhibited the PAR3-CLASP2 interaction disrupted the organization of the Golgi ribbon. CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185. This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2. Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization. On the basis of these observations, we propose that PAR3 and aPKC control the organization of the Golgi through CLASP2 phosphorylation. PMID:25518939

  1. 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.

  2. Topology of sphingolipid galactosyltransferases in ER and Golgi: Transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis

    NARCIS (Netherlands)

    Burger, K.N.J.; Bijl, P.; van Meer, G.

    1996-01-01

    Glucosylceramide (GlcCer) is synthesized at the cytosolic surface of the Golgi complex while enzymes acting in late steps of glycosphingolipid biosynthesis have their active centers in the Golgi lumen. However, the topology of the 'early' galactose-transferring enzymes is largely unknown. We used

  3. Golgi fragmentation precedes neuromuscular denervation and is associated with endosome abnormalities in SOD1-ALS mouse motor neurons

    NARCIS (Netherlands)

    V. van Dis (Vera); M. Kuijpers (Marijn); E.D. Haasdijk (Elize); E. Teuling (Eva); S.A. Oakes (Scott A.); C.C. Hoogenraad (Casper); D. Jaarsma (Dick)

    2014-01-01

    textabstractBackground: Fragmentation of stacked cisterns of the Golgi apparatus into dispersed smaller elements is a feature associated with degeneration of neurons in amyotrophic lateral sclerosis (ALS) and some other neurodegenerative disorders. However, the role of Golgi fragmentation in motor

  4. Traffic jams in fish bones: ER-to-Golgi protein transport during zebrafish development.

    Science.gov (United States)

    Melville, David B; Knapik, Ela W

    2011-01-01

    Extracellular matrix (ECM) proteins, cell adhesion molecules, cytokines, morphogens and membrane receptors are synthesized in the ER and transported through the Golgi complex to the cell surface and the extracellular space. The first leg in this journey from the ER to Golgi is facilitated by the Coat Protein II (COPII) vesicular carriers. Genetic defects in genes encoding various COPII components cause a broad spectrum of human diseases, from anemia to skeletal deformities. Here, we summarize our findings in zebrafish and discuss how mutations in COPII elements may cause specific cellular and developmental defects.

  5. Dependence of Golgi apparatus integrity on nitric oxide in vascular cells: implications in pulmonary arterial hypertension

    Science.gov (United States)

    Lee, Jason E.; Patel, Kirit; Almodóvar, Sharilyn; Tuder, Rubin M.; Flores, Sonia C.

    2011-01-01

    Although reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), its consequences on organellar structure and function within vascular cells is largely unexplored. We investigated the effect of reduced NO on the structure of the Golgi apparatus as assayed by giantin or GM130 immunofluorescence in human pulmonary arterial endothelial (HPAECs) and smooth muscle (HPASMCs) cells, bovine PAECs, and human EA.hy926 endothelial cells. Golgi structure was also investigated in cells in tissue sections of pulmonary vascular lesions in idiopathic PAH (IPAH) and in macaques infected with a chimeric simian immunodeficiency virus containing the human immunodeficiency virus (HIV)-nef gene (SHIV-nef) with subcellular three-dimensional (3D) immunoimaging. Compounds with NO scavenging activity including 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), methylene blue, N-acetylcysteine, and hemoglobin markedly fragmented the Golgi in all cell types evaluated as did monocrotaline pyrrole, while LY-83583, sildenafil, fasudil, Y-27632, Tiron, Tempol, or H2O2 did not. Golgi fragmentation by NO scavengers was inhibited by diethylamine NONOate, was evident in HPAECs after selective knockdown of endothelial nitric oxide synthase using small interfering RNA (siRNA), was independent of microtubule organization, required the GTPase dynamin 2, and was accompanied by depletion of α-soluble N-ethylmaleimide-sensitive factor (NSF) acceptor protein (α-SNAP) from Golgi membranes and codispersal of the SNAP receptor (SNARE) Vti1a with giantin. Golgi fragmentation was confirmed in endothelial and smooth muscle cells in pulmonary arterial lesions in IPAH and the SHIV-nef-infected macaque with subcellular 3D immunoimaging. In SHIV-nef-infected macaques Golgi fragmentation was observed in cells containing HIV-nef-bearing endosomes. The observed Golgi fragmentation suggests that NO plays a significant role in

  6. 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......-of-function and overexpression lines for two of these transporters identified biochemical alterations supporting their roles in the biosynthesis of Rha- and Gal-containing polysaccharides. Thus, cell wall polysaccharide biosynthesis in the Golgi apparatus of plants is likely also regulated by substrate transport mechanisms....

  7. Localization and function of cytosolic phospholipase A2α at the Golgi

    OpenAIRE

    Leslie, Christina C.; Gangelhoff, Todd A.; Gelb, Michael H.

    2010-01-01

    Cytosolic phospholipase A2α (cPLA2α, Group IVA phospholipase A2) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA2α translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme’s calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA2α translocation to the Golgi. However, other features of cPLA2α regulation are incompletely un...

  8. 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...... of the hindlimb muscles, GC elements as well as microtubules converge toward a common pattern, that of the slow-twitch fibers, in all fibers. Our data suggest that innervation regulates the distribution of microtubules, which in turn organize the Golgi complex according to muscle fiber type....

  9. A rapid assay for on-site monitoring of infliximab trough levels: a feasibility study

    NARCIS (Netherlands)

    Corstjens, Paul L. A. M.; Fidder, Herma H.; Wiesmeijer, Karien C.; de Dood, Claudia J.; Rispens, Theo; Wolbink, Gert-Jan; Hommes, Daniel W.; Tanke, Hans J.

    2013-01-01

    Monitoring levels of biologicals against tumor necrosis factor (TNF) has been suggested to improve therapeutic outcomes in inflammatory bowel diseases (IBDs). This pilot study describes a rapid lateral flow (LF)-based assay for on-site monitoring of serum trough levels of humanized monoclonal

  10. Glucose regulates clathrin adaptors at the trans-Golgi network and endosomes

    Science.gov (United States)

    Aoh, Quyen L.; Graves, Lee M.; Duncan, Mara C.

    2011-01-01

    Glucose is a rich source of energy and the raw material for biomass increase. Many eukaryotic cells remodel their physiology in the presence and absence of glucose. The yeast Saccharomyces cerevisiae undergoes changes in transcription, translation, metabolism, and cell polarity in response to glucose availability. Upon glucose starvation, translation initiation and cell polarity are immediately inhibited, and then gradually recover. In this paper, we provide evidence that, as in cell polarity and translation, traffic at the trans-Golgi network (TGN) and endosomes is regulated by glucose via an unknown mechanism that depends on protein kinase A (PKA). Upon glucose withdrawal, clathrin adaptors exhibit a biphasic change in localization: they initially delocalize from the membrane within minutes and later partially recover onto membranes. Additionally, the removal of glucose induces changes in posttranslational modifications of adaptors. Ras and Gpr1 signaling pathways, which converge on PKA, are required for changes in adaptor localization and changes in posttranslational modifications. Acute inhibition of PKA demonstrates that inhibition of PKA prior to glucose withdrawal prevents several adaptor responses to starvation. This study demonstrates that PKA activity prior to glucose starvation primes membrane traffic at the TGN and endosomes in response to glucose starvation. PMID:21832155

  11. Gravitropism and lateral root emergence are dependent on the trans-Golgi network protein TNO1

    Directory of Open Access Journals (Sweden)

    Rahul eRoy

    2015-11-01

    Full Text Available The trans-Golgi network (TGN is a dynamic organelle that functions as a relay station for receiving endocytosed cargo, directing secretory cargo, and trafficking to the vacuole. TGN-LOCALIZED SYP41-INTERACTING PROTEIN (TNO1 is a large, TGN-localized, coiled-coil protein that associates with the membrane fusion protein SYP41, a t-SNARE, and is required for efficient protein trafficking to the vacuole. Here, we show that a tno1 mutant has auxin transport-related defects. Mutant roots have delayed lateral root emergence, decreased gravitropic bending of plant organs and increased sensitivity to the auxin analog 2,4-Dichlorophenoxyacetic acid. Auxin asymmetry at the tips of elongating stage II lateral roots was reduced in the tno1 mutant, suggesting a role for TNO1 in cellular auxin transport during lateral root emergence. During gravistimulation, tno1 roots exhibited delayed auxin transport from the columella to the basal epidermal cells. Endocytosis to the TGN was unaffected in the mutant, indicating that bulk endocytic defects are not responsible for the observed phenotypes. Together these studies demonstrate a role for TNO1 in mediating auxin responses during root development and gravistimulation, potentially through trafficking of auxin transport proteins.

  12. STEM Tomography Imaging of Hypertrophied Golgi Stacks in Mucilage-Secreting Cells.

    Science.gov (United States)

    Kang, Byung-Ho

    2016-01-01

    Because of the weak penetrating power of electrons, the signal-to-noise ratio of a transmission electron micrograph (TEM) worsens as section thickness increases. This problem is alleviated by the use of the scanning transmission electron microscopy (STEM). Tomography analyses using STEM of thick sections from yeast and mammalian cells are of higher quality than are bright-field (BF) images. In this study, we compared regular BF tomograms and STEM tomograms from 500-nm thick sections from hypertrophied Golgi stacks of alfalfa root cap cells. Due to their thickness and intense heavy metal staining, BF tomograms of the thick sections suffer from poor contrast and high noise levels. We were able to mitigate these drawbacks by using STEM tomography. When we performed STEM tomography of densely stained chloroplasts of Arabidopsis cotyledon, we observed similar improvements relative to BF tomograms. A longer time is required to collect a STEM tilt series than similar BF TEM images, and dynamic autofocusing required for STEM imaging often fails at high tilt angles. Despite these limitations, STEM tomography is a powerful method for analyzing structures of large or dense organelles of plant cells.

  13. Poliovirus infection and expression of the poliovirus protein 2B provoke the disassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179.

    Science.gov (United States)

    Sandoval, I V; Carrasco, L

    1997-06-01

    Infection of Vero cells with poliovirus results in complete disassembly of the Golgi complex. Milestones of the process of disassembly are the release to the cytosol of the beta-COP bound to Golgi membranes, the disruption of the cis-Golgi network into fragments scattered throughout the cytoplasm, and the disassembly of the stacked cisternae by a process mediated by long tubular structures. Transient expression of the viral protein 2B in COS-7 cells also causes the disassembly of the Golgi complex by a process preceded by the accumulation of the protein in the Golgi area. Vero cells infected for 3 h show no recognizable Golgi complexes at the ultrastructural level and display an enormously swollen endoplasmic reticulum (ER) with extensive areas of its surface heavily coated. Ro-090179 (Ro), a flavonoid isolated from the herb Agastache rugosa, provokes the specific swelling and disruption of the Golgi complex and strongly inhibits poliovirus infection. Ro provokes the swelling and the disruption of the stacked cisternae and trans-Golgi elements without affecting the cis-most Golgi cisternae much. Moreover, Ro inhibits the fusion of the Golgi complex with the ER in cells treated with brefeldin A and provokes the accumulation of the intermediate compartment membrane protein p58 into ERD2-positive Golgi elements but has no effect on the anterograde transport involved in protein secretion. Our results indicate that the secretory pathway and specifically the Golgi complex are preferential targets of poliovirus.

  14. 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.

  15. 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

  16. Specific Sorting and Post-Golgi trafficking of Dendritic Potassium Channels in Living Neurons

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Watanabe, Shoji; Rasmussen, Hanne Borger

    2014-01-01

    localization in distinct dendritic sub-compartments are largely unknown. Here, we developed a quantitative live-cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels which exhibit distinct localizations; Kv2...

  17. PDMP blocks the BFA-induced ADP-ribosylation of BARS-50 in isolated Golgi membranes

    NARCIS (Netherlands)

    De Matteis, MA; Luna, A; Di Tullio, G; Corda, D; Kok, JW; Luini, A; Egea, G

    1999-01-01

    We reported that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2-decanoylamino-3-morpholinol-1-propanol (PDMP), blocks brefeldin A (BFA)-induced retrograde membrane transport from the Golgi complex to the endoplasmic reticulum (ER) (Kok et al,, 1998, J. Cell Biol. 142, 25-38), We now

  18. 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

  19. Cytoskeleton and Golgi-apparatus interactions: a two-way road of function and structure

    Directory of Open Access Journals (Sweden)

    Egea G

    2015-01-01

    Full Text Available Gustavo Egea,1 Carla Serra-Peinado,1 María P Gavilan,2 Rosa M Rios21Departament de Biologia Cel·lular, Immulogia i Neurociències, Facultat de Medicina and Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS, Universitat de Barcelona, Barcelona, Spain; 2Departamento de Señalización Celular, CSIC-Centro Andaluz de Biomedicina y Medicina Regenerativa (CABIMER, Seville, SpainAbstract: The Golgi apparatus is the result of a complex and dynamic interaction between a large variety of molecules that determine its architecture, protein and lipid transports, and those that integrate signals from outside and inside the cell. The cytoskeleton facilitates the functional integration of all these processes. Association and coordination between microtubules and actin filaments, as well as their respective binding and regulatory proteins, are clearly necessary for Golgi structure and function. Protein sorting, membrane fission and fusion, and the motion of Golgi-derived transport carriers are all affected by both cytoskeleton elements.Keywords: cytoskeleton, Golgi apparatus, membrane trafficking, secretory pathway, actin, microtubules

  20. Aquaporin-3 and aquaporin-4 are sorted differently and separately in the trans-Golgi network

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Sundbye, S.; Nelson, W. J.

    2013-01-01

    observed mostly in separate post-Golgi carriers, and spinning disk microscopy showed that most of AQP3 and AQP4 were delivered to the plasma membrane in separate vesicles. In contrast, VSV-G and LDL-R, two well-characterized basolateral proteins, co-localized to a high degree in the same post...

  1. Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis

    NARCIS (Netherlands)

    Halter, D.; Neumann, S.|info:eu-repo/dai/nl/304834513; van Dijk, S.M.; Wolthoorn, J.; de Maziere, A.M.G.L.; Vieira, O.V.; Mattjus, P.; Klumperman, J.; van Meer, G.; Sprong, H.|info:eu-repo/dai/nl/222364815

    2007-01-01

    Glycosphingolipids are controlled by the spatial organization of their metabolism and by trans port specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized

  2. 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.

  3. TRANSPORTE DE UDP-GALACTOSA EN EL APARATO DE GOLGI DE CELULAS VEGETALES.

    OpenAIRE

    NORAMBUENA MORALES, LORENA

    2004-01-01

    En vegetales, el aparato de Golgi existe en un gran número de organelos por célula, distribuyéndose homogéneamente por todo el citoplasma. La abundancia de este organelo está en directa relación con la necesidad de depósito de componentes de pared celular 106p.

  4. Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex.

    Science.gov (United States)

    Sprong, H; Degroote, S; Claessens, T; van Drunen, J; Oorschot, V; Westerink, B H; Hirabayashi, Y; Klumperman, J; van der Sluijs, P; van Meer, G

    2001-10-29

    Although glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.

  5. 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 ...

  6. The use of rapid review methods in health technology assessments: 3 case studies.

    Science.gov (United States)

    Kaltenthaler, Eva; Cooper, Katy; Pandor, Abdullah; Martyn-St James, Marrissa; Chatters, Robin; Wong, Ruth

    2016-08-26

    Rapid reviews are of increasing importance within health technology assessment due to time and resource constraints. There are many rapid review methods available although there is little guidance as to the most suitable methods. We present three case studies employing differing methods to suit the evidence base for each review and outline some issues to consider when selecting an appropriate method. Three recently completed systematic review short reports produced for the UK National Institute for Health Research were examined. Different approaches to rapid review methods were used in the three reports which were undertaken to inform the commissioning of services within the NHS and to inform future trial design. We describe the methods used, the reasoning behind the choice of methods and explore the strengths and weaknesses of each method. Rapid review methods were chosen to meet the needs of the review and each review had distinctly different challenges such as heterogeneity in terms of populations, interventions, comparators and outcome measures (PICO) and/or large numbers of relevant trials. All reviews included at least 10 randomised controlled trials (RCTs), each with numerous included outcomes. For the first case study (sexual health interventions), very diverse studies in terms of PICO were included. P-values and summary information only were presented due to substantial heterogeneity between studies and outcomes measured. For the second case study (premature ejaculation treatments), there were over 100 RCTs but also several existing systematic reviews. Data for meta-analyses were extracted directly from existing systematic reviews with new RCT data added where available. For the final case study (cannabis cessation therapies), studies included a wide range of interventions and considerable variation in study populations and outcomes. A brief summary of the key findings for each study was presented and narrative synthesis used to summarise results for each

  7. Reticular theory versus neuron theory in the work of Camillo Golgi.

    Science.gov (United States)

    Cimino, G

    1999-01-01

    In 1873 Golgi invented a revolutionary method for microscopic research of the nervous system, based on a particular technique for staining nerve cells, which came to be known as "black reaction". Thanks to this method, he was able to provide a thorough and precise description of nerve cells in various regions of the cerebro-spinal axis, clearly distinguishing the axon from the dendrites. He drew up a new classification of cells on the basis of the structure of their nervous prolongation, and he criticized Gerlach's theory of the "protoplasmic network". Golgi claimed to observe in the gray matter an extremely dense and intricate network, composed of a web of intertwined branches of axons coming from different cell layers ("diffuse nervous network"). This structure, which emerges from the axons and is therefore essentially different from that hypothesized by Gerlach, appeared in his view to be the main organ of the nervous system, the organ that connected different cerebral areas both anatomically and functionally by means of the transmission of an electric nervous impulse. Golgi's reticular theory, along with the other reticular theories of the nervous system prevalent at the end of the nineteenth century, had in a certain sense overturned the 'atomistic-reductionist' principle that lay behind the cell theory. These theories were in fact based on a holistic model, according to which the cerebro-spinal axis was considered to be a continuous structure, and its functions the result of a collective action. At the end of the 1880's, Ramon y Cajal began to elaborate the neuron theory, using Golgi's microscopic technique. Golgi, however, did not accept this theory, and a controversy arose between the two scientists that was not put to rest even after the rivals were both awarded the Nobel Prize in 1906. If we look at the reasons for which Golgi opposed the neuron theory, we can see that they derived not so much from disagreement over the actual data observed, as from a

  8. Rapid elemental analysis and provenance study of Blumea balsamifera DC using laser-induced breakdown spectroscopy.

    Science.gov (United States)

    Liu, Xiaona; Zhang, Qiao; Wu, Zhisheng; Shi, Xinyuan; Zhao, Na; Qiao, Yanjiang

    2014-12-31

    Laser-induced breakdown spectroscopy (LIBS) was applied to perform a rapid elemental analysis and provenance study of Blumea balsamifera DC. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were implemented to exploit the multivariate nature of the LIBS data. Scores and loadings of computed principal components visually illustrated the differing spectral data. The PLS-DA algorithm showed good classification performance. The PLS-DA model using complete spectra as input variables had similar discrimination performance to using selected spectral lines as input variables. The down-selection of spectral lines was specifically focused on the major elements of B. balsamifera samples. Results indicated that LIBS could be used to rapidly analyze elements and to perform provenance study of B. balsamifera.

  9. Effect of a rapid maxillary expansion on snoring and sleep in children: a pilot study.

    Science.gov (United States)

    Giannasi, Lilian Chrystiane; Santos, Israel Reis; Alfaya, Thays Almeida; Bussadori, Sandra Kalil; Leitão-Filho, Fernando Studart; de Oliveira, Luis Vicente Franco

    2015-07-01

    The aim of this study was to assess the efficacy of the McNamara rapid palatal expansion device for the treatment of sleep disorders in children. The sample enrolled 12 children aged 4-11 years. Children with snoring and bruxism whose parents did not agree to tonsil surgery were included in the study. During the initial evaluation, a questionnaire addressing sleep was administered, and plaster models were made for the construction of the McNamara rapid maxillary expansion device. The expansion period was 7-15 days, and the McNamara device was removed after 6-8 months. The same questionnaire was administered again after 30 days of use of the orthopedic appliance. The data were analyzed using the McNemar test, with the level of significance set to 5% (Pmaxillary expansion, can be an effective treatment for snoring and other undesirable sleep behaviors in children.

  10. Rapid Elemental Analysis and Provenance Study of Blumea balsamifera DC Using Laser-Induced Breakdown Spectroscopy

    Directory of Open Access Journals (Sweden)

    Xiaona Liu

    2014-12-01

    Full Text Available Laser-induced breakdown spectroscopy (LIBS was applied to perform a rapid elemental analysis and provenance study of Blumea balsamifera DC. Principal component analysis (PCA and partial least squares discriminant analysis (PLS-DA were implemented to exploit the multivariate nature of the LIBS data. Scores and loadings of computed principal components visually illustrated the differing spectral data. The PLS-DA algorithm showed good classification performance. The PLS-DA model using complete spectra as input variables had similar discrimination performance to using selected spectral lines as input variables. The down-selection of spectral lines was specifically focused on the major elements of B. balsamifera samples. Results indicated that LIBS could be used to rapidly analyze elements and to perform provenance study of B. balsamifera.

  11. Minus end-directed kinesin-14 KIFC1 regulates the positioning and architecture of the Golgi apparatus.

    Science.gov (United States)

    She, Zhen-Yu; Pan, Meng-Ying; Tan, Fu-Qing; Yang, Wan-Xi

    2017-05-30

    The Golgi apparatus is the central organelle along the eukaryotic secretory and endocytic pathway. In non-polarized mammalian cells, the Golgi complex is usually located proximal to the nucleus at the cell center and is closely associated with the microtubule organizing center. Microtubule networks are essential in the organization and central localization of the Golgi apparatus, but the molecular basis underlying these processes are poorly understood. Here we reveal that minus end-directed kinesin-14 KIFC1 proteins are required for the structural integrity and positioning of the Golgi complex in non-polarized mammalian cells. Remarkably, we found that the motor domain of kinesin-14 KIFC1 regulates the recognition and binding of the Golgi and KIFC1 also statically binds to the microtubules via its tail domain. These findings reveal a new stationary binding model that kinesin-14 KIFC1 proteins function as crosslinkers between the Golgi apparatus and the microtubules and contribute to the central positioning and structural maintenance of the Golgi apparatus.

  12. Colorimetric deoxyribonucleic acid hybridization assay for rapid screening of Salmonella in foods: collaborative study.

    Science.gov (United States)

    Curiale, M S; Klatt, M J; Mozola, M A

    1990-01-01

    A collaborative study was performed in 11 laboratories to validate a colorimetric DNA hybridization (DNAH) method for rapid detection of Salmonella in foods. The method was compared to the standard culture method for detection of Salmonella in nonfat dry milk, milk chocolate, soy isolate, dried whole egg, ground black pepper, and raw ground turkey. Samples inoculated with high (0.4-2 cells/g) and low (0.04-0.2 cells/g) levels of Salmonella and uninoculated control samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by DNAH and culture procedure for any of the 6 foods. The colorimetric DNA hybridization assay screening method has been adopted official first action as a rapid screening method for detection of Salmonella in all foods.

  13. Fluorescent enzyme immunoassay for rapid screening of Salmonella in foods: collaborative study.

    Science.gov (United States)

    Flowers, R S; Klatt, M J; Keelan, S L; Swaminathan, B; Gehle, W D; Chandonnet, H E

    1989-01-01

    A collaborative study was performed in 13 laboratories to validate an enzyme immunoassay (EIA) procedure for rapid detection of Salmonella in foods. The EIA was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been adopted official first action as a rapid screening method for detection of Salmonella.

  14. Relationship of red splenic arteriolar hyaline with rapid death: a clinicopathological study of 82 autopsy cases.

    Science.gov (United States)

    Kotani, Hirokazu; Miyao, Masashi; Manabe, Sho; Ishida, Tokiko; Kawai, Chihiro; Abiru, Hitoshi; Tamaki, Keiji

    2012-12-31

    Little is known about the relationship between splenic arteriolar hyaline and cause of death. The purpose of this retrospective study was to evaluate the clinicopathological significance of splenic arteriolar hyaline in autopsy cases and estimate the applicability of hyaline for diagnosing the cause and rapidity of death. Archival data and histological slides from 82 cases were reviewed retrospectively. One section of each spleen was evaluated microscopically. The tinctorial pattern of splenic arteriolar hyaline was examined with Heidenhain's Azan trichrome stain, and the relationships between this pattern and age, cause of death, and rapidity of death were investigated. Fifty-four cases demonstrated hyaline change, with 3 different tinctorial patterns: red, blue, and a combination of red and blue. The 3 patterns coexisted in various proportions in each tissue section. Frequency of the blue pattern increased with age (P virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1132441651796836.

  15. Frightening music triggers rapid changes in brain monoamine receptors: a pilot PET study.

    Science.gov (United States)

    Zhang, Ying; Chen, Qiaozhen; Du, Fenglei; Hu, Yanni; Chao, Fangfang; Tian, Mei; Zhang, Hong

    2012-10-01

    Frightening music can rapidly arouse emotions in listeners that mimic those from actual life-threatening experiences. However, studies of the underlying mechanism for perceiving danger created by music are limited. We investigated monoamine receptor changes induced by frightening music using (11)C-N-methyl-spiperone ((11)C-NMSP) PET. Ten healthy male volunteers were included, and their psychophysiologic changes were evaluated. Compared with the baseline condition, listening to frightening music caused a significant decrease in (11)C-NMSP in the right and left caudate nuclei, right limbic region, and right paralimbic region; a particularly significant decrease in the right anterior cingulate cortex; but an increase in the right frontal occipital and left temporal lobes of the cerebral cortex. Transient fright triggers rapid changes in monoamine receptors, which decrease in the limbic and paralimbic regions but increase in the cerebral cortex.

  16. The N-terminus of Vps74p is essential for the retention of glycosyltransferases in the Golgi but not for the modulation of apical polarized growth in Saccharomyces cerevisiae.

    Science.gov (United States)

    Hsu, Jia-Wei; Chang, Lin-Chun; Jang, Li-Ting; Huang, Chun-Fang; Lee, Fang-Jen S

    2013-01-01

    Vps74p is a member of the PtdIns(4)P-binding protein family. Vps74p interacts with Golgi-resident glycosyltransferases and the coat protein COPI complex to modulate Golgi retention of glycosyltransferases and with the PtdIns(4)P phosphatase Sac1p to modulate PtdIns(4)P homeostasis at the Golgi. Genetic analysis has shown that Vps74p is required for the formation of abnormal elongated buds in cdc34-2 cells. The C-terminal region of Vps74p is required for Vps74p multimerization, Golgi localization, and glycosyltransferase interactions; however, the functional significance of the N-terminal region and three putative phosphorylation sites of Vps74p have not been well characterized. In this study, we demonstrate that Vps74p executes multiple cellular functions using different domains. We found that the N-terminal 66 amino acids of Vps74p are dispensable for its Golgi localization and modulation of cell wall integrity but are required for glycosyltransferase retention and glycoprotein processing. Deletion of the N-terminal 90 amino acids, but not the 66 amino acids, of Vps74p impaired its ability to restore the elongated bud phenotype in cdc34-2/vps74Δ cells. Deletion of Sac1p and Arf1p also specifically reduced the abnormal elongated bud phenotype in cdc34-2 cells. Furthermore, we found that three N-terminal phosphorylation sites contribute to rapamycin hypersensitivity, although these phosphorylation residues are not involved in Vps74p localization, ability to modulate glycosyltransferase retention, or elongated bud formation in cdc34-2 cells. Thus, we propose that Vps74p may use different domains to interact with specific effectors thereby differentially modulating a variety of cellular functions.

  17. The Golgi apparatus is a functionally distinct Ca2+ store regulated by PKA and Epac branches of the β1-adrenergic signaling pathway

    Science.gov (United States)

    Yang, Zhaokang.; Kirton, Hannah M.; MacDougall, David A.; Boyle, John P.; Deuchars, James; Frater, Brenda; Ponnambalam, Sreenivasan; Hardy, Matthew E.; White, Edward; Calaghan, Sarah C.; Peers, Chris; Steele, Derek S.

    2016-01-01

    Ca2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. However, the signaling pathways that control this form of Ca2+ release are poorly understood and evidence of discrete Golgi Ca2+ release events is lacking. Here, we identified the Golgi apparatus as the source of prolonged Ca2+ release events that originate from the nuclear ‘poles’ of primary cardiac cells. Once initiated, Golgi Ca2+ release was unaffected by global depletion of sarcoplasmic reticulum Ca2+, and disruption of the Golgi apparatus abolished Golgi Ca2+ release without affecting sarcoplasmic reticulum function, suggesting functional and anatomical independence of Golgi and sarcoplasmic reticulum Ca2+ stores. Maximal activation of β1-adrenoceptors had only a small stimulating effect on Golgi Ca2+ release. However, inhibition of phosphodiesterase (PDE) 3 or 4, or downregulation of PDE 3 and 4 in heart failure markedly potentiated β1-adrenergic stimulation of Golgi Ca2+ release, consistent with compartmentalization of cAMP signaling within the Golgi apparatus microenvironment. β1-adrenergic stimulation of Golgi Ca2+ release involved activation of both Epac and PKA signaling pathways and CaMKII. Interventions that stimulated Golgi Ca2+ release induced trafficking of vascular growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane. These data establish the Golgi apparatus as a juxtanuclear focal point for Ca2+ and β1-adrenergic signaling, which functions independently from the sarcoplasmic reticulum and the global Ca2+ transients that underlie the primary contractile function of the cell. PMID:26462734

  18. The Golgi apparatus is a functionally distinct Ca2+ store regulated by the PKA and Epac branches of the β1-adrenergic signaling pathway.

    Science.gov (United States)

    Yang, Zhaokang; Kirton, Hannah M; MacDougall, David A; Boyle, John P; Deuchars, James; Frater, Brenda; Ponnambalam, Sreenivasan; Hardy, Matthew E; White, Edward; Calaghan, Sarah C; Peers, Chris; Steele, Derek S

    2015-10-13

    Ca(2+) release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. We found that the Golgi apparatus was the source of prolonged Ca(2+) release events that originated near the nuclei of primary cardiomyocytes. Golgi Ca(2+) release was unaffected by depletion of sarcoplasmic reticulum Ca(2+), and disruption of the Golgi apparatus abolished Golgi Ca(2+) release without affecting sarcoplasmic reticulum function, suggesting functional and spatial independence of Golgi and sarcoplasmic reticulum Ca(2+) stores. β1-Adrenoceptor stimulation triggers the production of the second messenger cAMP, which activates the Epac family of Rap guanine nucleotide exchange factors and the kinase PKA (protein kinase A). Phosphodiesterases (PDEs), including those in the PDE3 and PDE4 families, degrade cAMP. Activation of β1-adrenoceptors stimulated Golgi Ca(2+) release, an effect that required activation of Epac, PKA, and the kinase CaMKII. Inhibition of PDE3s or PDE4s potentiated β1-adrenergic-induced Golgi Ca(2+) release, which is consistent with compartmentalization of cAMP signaling near the Golgi apparatus. Interventions that stimulated Golgi Ca(2+) release appeared to increase the trafficking of vascular endothelial growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane of cardiomyocytes. In cardiomyocytes from rats with heart failure, decreases in the abundance of PDE3s and PDE4s were associated with increased Golgi Ca(2+) release events. These data suggest that the Golgi apparatus is a focal point for β1-adrenergic-stimulated Ca(2+) signaling and that the Golgi Ca(2+) store functions independently from the sarcoplasmic reticulum and the global Ca(2+) transients that trigger contraction in cardiomyocytes. Copyright © 2015, American Association for the Advancement of Science.

  19. Relationship of red splenic arteriolar hyaline with rapid death: a clinicopathological study of 82 autopsy cases

    Directory of Open Access Journals (Sweden)

    Kotani Hirokazu

    2012-12-01

    Full Text Available Abstract Background Little is known about the relationship between splenic arteriolar hyaline and cause of death. The purpose of this retrospective study was to evaluate the clinicopathological significance of splenic arteriolar hyaline in autopsy cases and estimate the applicability of hyaline for diagnosing the cause and rapidity of death. Methods Archival data and histological slides from 82 cases were reviewed retrospectively. One section of each spleen was evaluated microscopically. The tinctorial pattern of splenic arteriolar hyaline was examined with Heidenhain’s Azan trichrome stain, and the relationships between this pattern and age, cause of death, and rapidity of death were investigated. Results Fifty-four cases demonstrated hyaline change, with 3 different tinctorial patterns: red, blue, and a combination of red and blue. The 3 patterns coexisted in various proportions in each tissue section. Frequency of the blue pattern increased with age (P P  Conclusions Estimation of splenic arteriolar hyaline with Heidenhain’s Azan trichrome stain is useful for assessment of the cause and rapidity of death. Virtual slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1132441651796836

  20. Rapid extraction of lexical tone phonology in Chinese characters: a visual mismatch negativity study.

    Directory of Open Access Journals (Sweden)

    Xiao-Dong Wang

    Full Text Available BACKGROUND: In alphabetic languages, emerging evidence from behavioral and neuroimaging studies shows the rapid and automatic activation of phonological information in visual word recognition. In the mapping from orthography to phonology, unlike most alphabetic languages in which there is a natural correspondence between the visual and phonological forms, in logographic Chinese, the mapping between visual and phonological forms is rather arbitrary and depends on learning and experience. The issue of whether the phonological information is rapidly and automatically extracted in Chinese characters by the brain has not yet been thoroughly addressed. METHODOLOGY/PRINCIPAL FINDINGS: We continuously presented Chinese characters differing in orthography and meaning to adult native Mandarin Chinese speakers to construct a constant varying visual stream. In the stream, most stimuli were homophones of Chinese characters: The phonological features embedded in these visual characters were the same, including consonants, vowels and the lexical tone. Occasionally, the rule of phonology was randomly violated by characters whose phonological features differed in the lexical tone. CONCLUSIONS/SIGNIFICANCE: We showed that the violation of the lexical tone phonology evoked an early, robust visual response, as revealed by whole-head electrical recordings of the visual mismatch negativity (vMMN, indicating the rapid extraction of phonological information embedded in Chinese characters. Source analysis revealed that the vMMN was involved in neural activations of the visual cortex, suggesting that the visual sensory memory is sensitive to phonological information embedded in visual words at an early processing stage.

  1. Study on rapid bio-drying technology of cow dung with CaO2

    Science.gov (United States)

    Chen, Xiaotian; Qu, Guangfei; Liu, Shugen; Xie, Ruosong; He, Yanhua

    2017-05-01

    Effect of CaO2 on cow dung rapid bio-drying technology was researched. A static aerobic composting system was applied to this experiment which combining natural ventilation with Turing in the process of composting. The physical characteristics of cow dung was observed and the compost temperature, moisture content, organic matter, total nitrogen, total phosphorus, potassium content was determined which in order to study the effect of CaO2 on rapid drying of cattle in the compost. In the initial stage of compost, adding CaO2 groups compared with the control group, the temperature rise faster, 4-6 days in advance to the thermophilic phase; at the end of composting, the CaO2 composition and moisture content decreased significantly to below 30%. The addition of CaO2 in fertilizer was shorten the composting time, extend the thermophilic phase, to provide sufficient oxygen meeting the growth needs of aerobic microorganisms. It convinced that the rapid bio-drying of dairy manure has a good effect and provided a new idea for the effective treatment of cow dung.

  2. Rapid extraction of lexical tone phonology in Chinese characters: a visual mismatch negativity study.

    Science.gov (United States)

    Wang, Xiao-Dong; Liu, A-Ping; Wu, Yin-Yuan; Wang, Peng

    2013-01-01

    In alphabetic languages, emerging evidence from behavioral and neuroimaging studies shows the rapid and automatic activation of phonological information in visual word recognition. In the mapping from orthography to phonology, unlike most alphabetic languages in which there is a natural correspondence between the visual and phonological forms, in logographic Chinese, the mapping between visual and phonological forms is rather arbitrary and depends on learning and experience. The issue of whether the phonological information is rapidly and automatically extracted in Chinese characters by the brain has not yet been thoroughly addressed. We continuously presented Chinese characters differing in orthography and meaning to adult native Mandarin Chinese speakers to construct a constant varying visual stream. In the stream, most stimuli were homophones of Chinese characters: The phonological features embedded in these visual characters were the same, including consonants, vowels and the lexical tone. Occasionally, the rule of phonology was randomly violated by characters whose phonological features differed in the lexical tone. We showed that the violation of the lexical tone phonology evoked an early, robust visual response, as revealed by whole-head electrical recordings of the visual mismatch negativity (vMMN), indicating the rapid extraction of phonological information embedded in Chinese characters. Source analysis revealed that the vMMN was involved in neural activations of the visual cortex, suggesting that the visual sensory memory is sensitive to phonological information embedded in visual words at an early processing stage.

  3. Study of a Rapid Cycling Synchrotron to replace the CERN PS Booster

    CERN Document Server

    Hanke, K; Angoletta, M E; Balhan, B; Bartmann, W; Benedikt, M; Borburgh, J; Bozzini, D; Carli, C; Dahlen, P; Dobers, T; Fitterer, M; Garoby, R; Gilardoni, S; Goddard, B; Hansen, J; Hermanns, T; Lopez-Hernandez, L A; Hourican, M; Jensen, S; Kosmicki, A; Meddahi, M; Mikulec, B; Newborough, A; Nonis, M; Olek, S; Paoluzzi, M; Pittet, S; Puccio, B; Raginel, V; Ruehl, I; Schönauer, H; Sermeus, L; Steerenberg, R; Tan, J; Tückmantel, J; Vretenar, M; Widorski, M

    2011-01-01

    CERN’s proton injector chain is undergoing a massive consolidation and upgrade program in order to deliver beams meeting the needs of the LHC Luminosity Upgrade. As an alternative to the upgrade of the existing Proton Synchrotron Booster (PSB), the construction of a Rapid Cycling Synchrotron (RCS) has been studied. This machine would replace the PSB and deliver beams to the LHC as well as to CERN’s rich fixed-target physics program. This paper summarizes the outcome of the feasibility study along with a tentative RCS design.

  4. Identification of a novel glycan processing enzyme with exo-acting β-allosidase activity in the Golgi apparatus using a new platform for the synthesis of fluorescent substrates.

    Science.gov (United States)

    Hakamata, Wataru; Miura, Kazuki; Hirano, Takako; Nishio, Toshiyuki

    2015-01-01

    The majority of eukaryotic proteins undergo post-translational modifications (PTMs) involving the attachment of complex glycans, predominantly through N-glycosylation and O-glycosylation. PTMs play important roles in virtually all cellular processes, and aberrant regulation of protein glycosylation and glycan processing has been implicated in various diseases. However, glycan processing on proteins in various cellular contexts has not been visualized. We had previously developed a quinone methide cleavage (QMC) platform for enhanced substrate design. This platform was applied here to screen for novel glycan-processing enzymes. We designed and synthesized fluorescent substrates with β-allopyranoside residues using the QMC platform. When applied in cell-based assays, the fluorescent substrates allowed rapid and clear visualization of β-allosidase activity in the Golgi apparatus of human cultured cells. The QMC platform will likely find broad applications in visualizing the activities of glycan processing enzymes in living cells and in studying PTMs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. 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)P2 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 Vo 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).

  6. Performance of an oral fluid rapid HIV-1/2 test: experience from four CDC studies.

    Science.gov (United States)

    Delaney, Kevin P; Branson, Bernard M; Uniyal, Apurva; Kerndt, Peter R; Keenan, Patrick A; Jafa, Krishna; Gardner, Ann D; Jamieson, Denise J; Bulterys, Marc

    2006-08-01

    To evaluate the performance of a rapid HIV antibody test used with whole blood and oral fluid in settings where the test is likely to be used. In four separate studies, we compared the accuracy of the rapid test performed on whole blood and oral fluid specimens with the results of conventional HIV tests. Oral fluid and whole blood from persons of unknown HIV status recruited from clinics, labor and delivery units, and outreach venues were tested with the OraQuick Advance rapid HIV-1/2 antibody test. Sensitivity and specificity were compared with results of the enzyme immunoassay (EIA) and Western blot algorithm used by the study sites. OraQuick sensitivity was 99.7% with whole blood and 99.1% with oral fluid from 327 persons who were HIV antibody positive by the conventional algorithm. OraQuick specificity was 99.9% with whole blood and 99.6% with oral fluid from 12 010 HIV-negative persons; EIA specificity was 99.7%. A cluster of 16 false-positive oral fluid tests occurred in one study, in which specificity was lower (99.0%) than in the other three studies (99.6-99.8%). In diverse settings in four studies, the OraQuick test showed high sensitivity and specificity for HIV antibody in whole blood and oral fluid specimens. Slightly more false-positive and false-negative results occurred with oral fluid than with whole blood, but performance with both specimen types was similar to, or better than, that of conventional EIAs.

  7. Experimental Study of Disruption of Columnar Grains During Rapid Solidification in Additive Manufacturing

    Science.gov (United States)

    Manogharan, Guha; Yelamanchi, Bharat; Aman, Ronald; Mahbooba, Zaynab

    2016-03-01

    Over the years, many studies have been conducted to study and analyze the grain structures of metal alloys during additive manufacturing to improve mechanical properties. In particular, columnar grains are observed predominantly during rapid solidification of molten metal. This leads to lower mechanical properties and requires expensive secondary heat-treatment processes. This study is aimed at disrupting the formation of columnar grain growth during rapid solidification using ultrasonic vibration and analyzes the effects on grain structure and mechanical properties. A gas-metal arc welder mounted on a Rep-Rap-based low-cost metal 3 Dimension printer was used to deposit ER70S-6 mild steel layers on a plate. A contact-type ultrasonic transducer with a control system to vary the frequency and power of the vibration was used. The effects of ultrasonic vibration were determined from the statistical analysis of microstructure and micro-indentation techniques on the deposited layer and heat-affected zone. It was found that both frequency and interaction between frequency and power had significant impact on the refinement of average grain size up to 10.64% and increased the number of grains by approximately 41.78%. Analysis of micro-indentation tests showed that there was an increase of approximately 14.30% in micro-hardness due to the applied frequency during rapid solidification. A pole diagram shows that application of vibration causes randomization of grain orientation. Along with the results from this study, further efforts in modeling and experimentation of multi-directional vibrations would lead to a better understanding of disrupting columnar grains in applications that use mechanical vibrations, such as welding, directed energy deposition, brazing, etc.

  8. Enabling rapid behavioral ecotoxicity studies using an integrated lab-on-a-chip systems

    Science.gov (United States)

    Huang, Yushi; Nugegoda, Dayanthi; Wlodkowic, Donald

    2015-12-01

    Behavioral ecotoxicity tests are gaining an increasing recognition in environmental toxicology. Behavior of sensitive bioindicator species can change rapidly in response to an acute exposure to contaminants and thus has a much higher sensitivity as compared to conventional LC50 mortality tests. Furthermore, behavioral endpoints seems to be very good candidates to develop early-warning biomonitoring systems needed for rapid chemical risk assessment. Behavioral tests are non-invasive, fast, do not harm indicator organisms (behavioural changes are very rapid) and are thus fully compatible with 3R (Replacement - Reduction - Refinement) principle encouraging alternatives to conventional animal testing. These characteristics are essential when designing improved ecotoxicity tests for chemical risk assessment. In this work, we present a pilot development of miniaturized Lab-on-a-Chip (LOC) devices for studying toxin avoidance behaviors of small aquatic crustaceans. As an investigative tool, LOCs represent a new direction that may miniaturize and revolutionize behavioral ecotoxicology. Specifically our innovative microfluidic prototype: (i) enables convening "caging" of specimens for real-time videomicroscopy; (ii) eliminates the evaporative water loss thus providing an opportunity for long-term behavioral studies; (iii) exploits laminar fluid flow under low Reynolds numbers to generate discrete domains and gradients enabling for the first time toxin avoidance studies on small aquatic crustaceans; (iv) integrates off-the-chip mechatronic interfaces and video analysis algorithms for single animal movement analysis. We provide evidence that by merging innovative bioelectronic and biomicrofluidic technologies we can deploy inexpensive and reliable systems for culture, electronic tracking and complex computational analysis of behavior of bioindicator organisms.

  9. The DCR protein TTC3 affects differentiation and Golgi compactness in neurons through specific actin-regulating pathways.

    Directory of Open Access Journals (Sweden)

    Gaia Elena Berto

    Full Text Available In neuronal cells, actin remodeling plays a well known role in neurite extension but is also deeply involved in the organization of intracellular structures, such as the Golgi apparatus. However, it is still not very clear which mechanisms may regulate actin dynamics at the different sites. In this report we show that high levels of the TTC3 protein, encoded by one of the genes of the Down Syndrome Critical Region (DCR, prevent neurite extension and disrupt Golgi compactness in differentiating primary neurons. These effects largely depend on the capability of TTC3 to promote actin polymerization through signaling pathways involving RhoA, ROCK, CIT-N and PIIa. However, the functional relationships between these molecules differ significantly if considering the TTC3 activity on neurite extension or on Golgi organization. Finally, our results reveal an unexpected stage-dependent requirement for F-actin in Golgi organization at different stages of neuronal differentiation.

  10. 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

    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...... deltaepsilon complex was allowed to exit the ER and was also predominantly located in the Golgi apparatus. However, neither hexameric TCR complexes nor TCRzeta chains were efficiently expressed at the cell surface without the other. The observations that TCRzeta and hexameric TCR complexes are transported from...... 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....

  11. Rapid Canine Retraction with Dentoalveolar Distraction Osteogenesis: An in vivo Study

    Directory of Open Access Journals (Sweden)

    Allwin Benjamin Raj

    2013-01-01

    Full Text Available The aim of this clinical study is to assess the effectiveness of a new technique of rapid canine retraction through distraction osteogenesis. The effects of dentoalveolar distraction on the dentofacial structures, the dental changes that has been produced by dentoalveolar distraction and the vitality of the distracted canine immediately after distraction and 3 months postdistraction using pulp vitality test were also evaluated. Custom made canine distractors were used for distraction. Pre and postdistraction lateral cephalogram, OPG, Model analysis and electrical pulp vitality testing is carried out and results were evaluated.

  12. Rapid Detection of Land Cover Changes Using Crowdsourced Geographic Information: A Case Study of Beijing, China

    Directory of Open Access Journals (Sweden)

    Yuan Meng

    2017-08-01

    Full Text Available Land cover change (LCC detection is a significant component of sustainability research including ecological economics and climate change. Due to the rapid variability of natural environment, effective LCC detection is required to capture sufficient change-related information. Although such information has been available through remotely sensed images, the complicated image processing and classification make it time consuming and labour intensive. In contrast, the freely available crowdsourced geographic information (CGI contains easily interpreted textual information, and thus has the potential to be applied for capturing effective change-related information. Therefore, this paper presents and evaluates a method using CGI for rapid LCC detection. As a case study, Beijing is chosen as the study area, and CGI is applied to monitor LCC information. As one kind of CGI which is generated from commercial Internet maps, points of interest (POIs with detailed textual information are utilised to detect land cover in 2016. Those POIs are first classified into land cover nomenclature based on their textual information. Then, a kernel density approach is proposed to effectively generate land cover regions in 2016. Finally, with GlobeLand30 in 2010 as baseline map, LCC is detected using the post-classification method in the period of 2010–2016 in Beijing. The result shows that an accuracy of 89.20% is achieved with land cover regions generated by POIs, indicating that POIs are reliable for rapid LCC detection. Additionally, an LCC detection comparison is proposed between remotely sensed images and CGI, revealing the advantages of POIs in terms of LCC efficiency. However, due to the uneven distribution, remotely sensed images are still required in areas with few POIs.

  13. Variable actin dynamics requirement for the exit of different cargo from the trans-Golgi network.

    Science.gov (United States)

    Lázaro-Diéguez, Francisco; Colonna, Cecilia; Cortegano, Miguel; Calvo, María; Martínez, Susana E; Egea, Gustavo

    2007-08-07

    Efficient post-Golgi trafficking depends on microtubules, but actin filaments and actin-associated proteins are also postulated. Here we examined, by inverse fluorescence recovery after photobleaching, the role of actin dynamics in the exit from the TGN of fluorescent-tagged apical or basolateral and raft or non-raft-associated cargoes. Either the actin-stabilizing jasplakinolide or the actin-depolymerising latrunculin B variably but significantly inhibited post-Golgi traffic of non-raft associated apical p75NTR and basolateral VSV-G cargoes. The TGN-exit of the apical-destined VSV-G mutant was impaired only by latrunculin B. Strikingly, the raft-associated GPI-anchor protein was not affected by either actin toxin. Results indicate that actin dynamics participates in the TGN egress of both apical- and basolateral-targeted proteins but is not needed for apical raft-associated cargo.

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

    Science.gov (United States)

    Nie, Hongyun; Liu, Yueyue; Yin, Xiaolei; Cao, Huiren; Wang, Yanfei; Xiong, Wei; Lin, Yushuang; Xu, Zhigang

    2016-01-01

    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.

  15. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex.

    Science.gov (United States)

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo

    2016-04-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The Golgi apparatus in the endomembrane-rich gastric parietal cells exist as functional stable mini-stacks dispersed throughout the cytoplasm.

    Science.gov (United States)

    Gunn, Priscilla A; Gliddon, Briony L; Londrigan, Sarah L; Lew, Andrew M; van Driel, Ian R; Gleeson, Paul A

    2011-12-01

    Acid-secreting gastric parietal cells are polarized epithelial cells that harbour highly abundant and specialized, H+,K+ ATPase-containing, tubulovesicular membranes in the apical cytoplasm. The Golgi apparatus has been implicated in the biogenesis of the tubulovesicular membranes; however, an unanswered question is how a typical Golgi organization could regulate normal membrane transport within the membrane-dense cytoplasm of parietal cells. Here, we demonstrate that the Golgi apparatus of parietal cells is not the typical juxta-nuclear ribbon of stacks, but rather individual Golgi units are scattered throughout the cytoplasm. The Golgi membrane structures labelled with markers of both cis- and trans-Golgi membrane, indicating the presence of intact Golgi stacks. The parietal cell Golgi stacks were closely aligned with the microtubule network and were shown to participate in both anterograde and retrograde transport pathways. Dispersed Golgi stacks were also observed in parietal cells from H+,K+ ATPase-deficient mice that lack tubulovesicular membranes. These results indicate that the unusual organization of individual Golgi stacks dispersed throughout the cytoplasm of these terminally differentiated cells is likely to be a developmentally regulated event.

  17. Golgi Apparatus-Localized Synaptotagmin 2 Is Required for Unconventional Secretion in Arabidopsis

    Science.gov (United States)

    Gao, Bin; Fan, Hai; Jin, Jingbo; Botella, Miguel A.; Jiang, Liwen; Lin, Jinxing

    2011-01-01

    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 (HYGR) 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 HYGR 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 HYGR caused hypersensitivity of the transgenic Arabidopsis plants to hygromycin B. HYGR, which lacks a signal sequence, was present in the cytoplasm as well as in the extracellular space in HYGR-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 HYGR-GFP was truncated at carboxyl terminus of HYGR shortly after its synthesis, and the cells deficient SYT2 failed to efficiently truncate HYGR-GFP,resulting in HYGR-GFP accumulated in prevacuoles/vacuoles, indicating that SYT2 was involved in HYGR-GFP trafficking and secretion. Conclusion/Significance These findings reveal for the first time that SYT2 is localized on the Golgi apparatus and regulates HYGR-GFP secretion via the unconventional protein transport from the cytosol to the extracelluar matrix in plant cells. PMID:22140429

  18. Content delivery to newly forming Weibel-Palade bodies is facilitated by multiple connections with the Golgi apparatus.

    Science.gov (United States)

    Mourik, Marjon J; Faas, Frank G A; Zimmermann, Hans; Voorberg, Jan; Koster, Abraham J; Eikenboom, Jeroen

    2015-05-28

    Weibel-Palade bodies (WPBs) comprise an on-demand storage organelle within vascular endothelial cells. It's major component, the hemostatic protein von Willebrand factor (VWF), is known to assemble into long helical tubules and is hypothesized to drive WPB biogenesis. However, electron micrographs of WPBs at the Golgi apparatus show that these forming WPBs contain very little tubular VWF compared with mature peripheral WPBs, which raises questions on the mechanisms that increase the VWF content and facilitate vesicle growth. Using correlative light and electron microscopy and electron tomography, we investigated WPB biogenesis in time. We reveal that forming WPBs maintain multiple connections to the Golgi apparatus throughout their biogenesis. Also by volume scanning electron microscopy, we confirmed the presence of these connections linking WPBs and the Golgi apparatus. From electron tomograms, we provided evidence that nontubular VWF is added to WPBs, which suggested that tubule formation occurs in the WPB lumen. During this process, the Golgi membrane and clathrin seem to provide a scaffold to align forming VWF tubules. Overall, our data show that multiple connections with the Golgi facilitate content delivery and indicate that the Golgi appears to provide a framework to determine the overall size and dimensions of newly forming WPBs. © 2015 by The American Society of Hematology.

  19. A Model for the Self-Organization of Vesicular Flux and Protein Distributions in the Golgi Apparatus

    Science.gov (United States)

    Ispolatov, Iaroslav; Müsch, Anne

    2013-01-01

    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 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. PMID:23874173

  20. Lessons Learned from Applying Design Thinking in a NASA Rapid Design Study in Aeronautics

    Science.gov (United States)

    McGowan, Anna-Maria; Bakula, Casey; Castner, Raymond

    2017-01-01

    In late 2015, NASA's Aeronautics Research Mission Directorate (ARMD) funded an experiment in rapid design and rapid teaming to explore new approaches to solving challenging design problems in aeronautics in an effort to cultivate and foster innovation. This report summarizes several lessons learned from the rapid design portion of the study. This effort entailed learning and applying design thinking, a human-centered design approach, to complete the conceptual design for an open-ended design challenge within six months. The design challenge focused on creating a capability to advance experimental testing of autonomous aeronautics systems, an area of great interest to NASA, the US government as a whole, and an entire ecosystem of users and developers around the globe. A team of nine civil servant researchers from three of NASA's aeronautics field centers with backgrounds in several disciplines was assembled and rapidly trained in design thinking under the guidance of the innovation and design firm IDEO. The design thinking process, while used extensively outside the aerospace industry, is less common and even counter to many practices within the aerospace industry. In this report, several contrasts between common aerospace research and development practices and design thinking are discussed, drawing upon the lessons learned from the NASA rapid design study. The lessons discussed included working towards a design solution without a set of detailed design requirements, which may not be practical or even feasible for management to ascertain for complex, challenging problems. This approach allowed for the possibility of redesigning the original problem statement to better meet the needs of the users. Another lesson learned was to approach problems holistically from the perspective of the needs of individuals that may be affected by advances in topic area instead of purely from a technological feasibility viewpoint. The interdisciplinary nature of the design team also

  1. Exploring interprofessional practices in rapid response systems: a case study protocol.

    Science.gov (United States)

    Allen, Emily; Jackson, Debra; Elliott, Doug

    2015-01-01

    To describe the development of a proposed case study protocol investigating interprofessional relationships in a rapid response system (RRS) in a socioculturally complex clinical environment. Suboptimal care of deteriorating ward patients remains a concern for many acute healthcare organisations. Despite the advent of RRSs, emergency response teams are not always used to their full potential. How and why interprofessional relationships influence practices associated with the care and management of ward patients at risk of clinical deterioration requires investigation. Theoretical and empirical literature describing case study research and RRSs. Review methods An integrative review approach of the literature, focusing on key terms relating to 'case study research' and 'rapid response system', provided context and informed development of the study protocol. A single-site mixed-method instrumental case study protocol was developed using methodological triangulation and a multi-level model to examine interprofessional relationships between a broad range of stakeholders. Concurrent data collection and analysis will occur using document review of clinical scenarios, non-participant observations and semi-structured interviews. Case study research is an effective method for investigating socioculturally complex clinical environments. A strength of this approach is the flexibility in the choice of methods, which allows the researcher to build the design most suitable for the subjects or phenomena being investigated. Although this flexibility may be considered a potential weakness, rigour can be achieved by application of the strategies described. Findings from this research will provide rich descriptive insights into RRS relationships and healthcare professional practices during day-to-day management of acute ward patients at risk of or experiencing clinical deterioration. Description of this structured case study research approach will also inform other researchers.

  2. DNA methylation profile associated with rapid decline in kidney function: findings from the CRIC Study

    Science.gov (United States)

    Wing, Maria R.; Devaney, Joseph M.; Joffe, Marshall M.; Xie, Dawei; Feldman, Harold I.; Dominic, Elizabeth A.; Guzman, Nicolas J.; Ramezani, Ali; Susztak, Katalin; Herman, James G.; Cope, Leslie; Harmon, Brennan; Kwabi-Addo, Bernard; Gordish-Dressman, Heather; Go, Alan S.; He, Jiang; Lash, James P.; Kusek, John W.; Raj, Dominic S.

    2014-01-01

    Background Epigenetic mechanisms may be important in the progression of chronic kidney disease (CKD). Methods We studied the genome-wide DNA methylation pattern associated with rapid loss of kidney function using the Infinium HumanMethylation 450 K BeadChip in 40 Chronic Renal Insufficiency (CRIC) study participants (n = 3939) with the highest and lowest rates of decline in estimated glomerular filtration rate. Results The mean eGFR slope was 2.2 (1.4) and −5.1 (1.2) mL/min/1.73 m2 in the stable kidney function group and the rapid progression group, respectively. CpG islands in NPHP4, IQSEC1 and TCF3 were hypermethylated to a larger extent in subjects with stable kidney function (P-values of 7.8E−05 to 9.5E−05). These genes are involved in pathways known to promote the epithelial to mesenchymal transition and renal fibrosis. Other CKD-related genes that were differentially methylated are NOS3, NFKBIL2, CLU, NFKBIB, TGFB3 and TGFBI, which are involved in oxidative stress and inflammatory pathways (P-values of 4.5E−03 to 0.046). Pathway analysis using Ingenuity Pathway Analysis showed that gene networks related to cell signaling, carbohydrate metabolism and human behavior are epigenetically regulated in CKD. Conclusions Epigenetic modifications may be important in determining the rate of loss of kidney function in patients with established CKD. PMID:24516231

  3. 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

  4. Palmitoylation of stathmin family proteins domain A controls Golgi versus mitochondrial subcellular targeting.

    Science.gov (United States)

    Chauvin, Stéphanie; Poulain, Fabienne E; Ozon, Sylvie; Sobel, André

    2008-10-01

    Precise localization of proteins to specialized subcellular domains is fundamental for proper neuronal development and function. The neural microtubule-regulatory phosphoproteins of the stathmin family are such proteins whose specific functions are controlled by subcellular localization. Whereas stathmin is cytosolic, SCG10, SCLIP and RB3/RB3'/RB3'' are localized to the Golgi and vesicle-like structures along neurites and at growth cones. We examined the molecular determinants involved in the regulation of this specific subcellular localization in hippocampal neurons in culture. We show that their conserved N-terminal domain A carrying two palmitoylation sites is dominant over the others for Golgi and vesicle-like localization. Using palmitoylation-deficient GFP (green fluorescent protein) fusion mutants, we demonstrate that domains A of stathmin proteins have the particular ability to control protein targeting to either Golgi or mitochondria, depending on their palmitoylation. This regulation involves the co-operation of two subdomains within domain A, and seems also to be under the control of its SLD (stathmin-like domain) extension. Our results unravel that, in specific biological conditions, palmitoylation of stathmin proteins might be able to control their targeting to express their functional activities at appropriate subcellular sites. They, more generally, open new perspectives regarding the role of palmitoylation as a signalling mechanism orienting proteins to their functional subcellular compartments.

  5. Curvature-driven lateral segregation of membrane constituents in Golgi cisternae

    Science.gov (United States)

    Derganc, Jure

    2007-12-01

    Lateral segregation of mobile membrane constituents (e.g. lipids, proteins or membrane domains) into the regions of their preferred curvature relaxes stresses in the membrane. The equilibrium distribution of the constituents in the membrane is thus a balance between the gains in the membrane elastic energy and the segregation-induced loss of entropy. The membrane in the Golgi cisternae is particularly susceptible to the curvature-driven segregation because it possesses two very different curvatures—the highly curved membrane in the cisternal rims and the flat membrane in the cisternal sides. In this work, we calculate the extent of lateral segregation in the Golgi cisternae in the case where the segregation is driven by the Helfrich bending energy. It is assumed that the membrane bending constant and spontaneous curvature depend on the local membrane composition. A simple analytical expression for the extent of the lateral segregation is derived. The results show that the segregation depends on the ratio between the bending constant and the thermal energy, the difference of the preferred curvatures of the constituents and the sizes of the constituents. Applying the model to a typical Golgi cisterna, it was found that entropy can effectively limit the extent of the curvature-driven lateral segregation.

  6. 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.

  7. Sac1--Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Yiying; Deng, Yongqiang; Horenkamp, Florian; Reinisch, Karin M.; Burd, Christopher G. [Yale-MED

    2014-08-25

    Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.

  8. Optical Coherence Tomography in the UK Biobank Study - Rapid Automated Analysis of Retinal Thickness for Large Population-Based Studies.

    Directory of Open Access Journals (Sweden)

    Pearse A Keane

    Full Text Available To describe an approach to the use of optical coherence tomography (OCT imaging in large, population-based studies, including methods for OCT image acquisition, storage, and the remote, rapid, automated analysis of retinal thickness.In UK Biobank, OCT images were acquired between 2009 and 2010 using a commercially available "spectral domain" OCT device (3D OCT-1000, Topcon. Images were obtained using a raster scan protocol, 6 mm x 6 mm in area, and consisting of 128 B-scans. OCT image sets were stored on UK Biobank servers in a central repository, adjacent to high performance computers. Rapid, automated analysis of retinal thickness was performed using custom image segmentation software developed by the Topcon Advanced Biomedical Imaging Laboratory (TABIL. This software employs dual-scale gradient information to allow for automated segmentation of nine intraretinal boundaries in a rapid fashion.67,321 participants (134,642 eyes in UK Biobank underwent OCT imaging of both eyes as part of the ocular module. 134,611 images were successfully processed with 31 images failing segmentation analysis due to corrupted OCT files or withdrawal of subject consent for UKBB study participation. Average time taken to call up an image from the database and complete segmentation analysis was approximately 120 seconds per data set per login, and analysis of the entire dataset was completed in approximately 28 days.We report an approach to the rapid, automated measurement of retinal thickness from nearly 140,000 OCT image sets from the UK Biobank. In the near future, these measurements will be publically available for utilization by researchers around the world, and thus for correlation with the wealth of other data collected in UK Biobank. The automated analysis approaches we describe may be of utility for future large population-based epidemiological studies, clinical trials, and screening programs that employ OCT imaging.

  9. Emotional noun processing: an ERP study with rapid serial visual presentation.

    Directory of Open Access Journals (Sweden)

    Shengnan Yi

    Full Text Available Reading is an important part of our daily life, and rapid responses to emotional words have received a great deal of research interest. Our study employed rapid serial visual presentation to detect the time course of emotional noun processing using event-related potentials. We performed a dual-task experiment, where subjects were required to judge whether a given number was odd or even, and the category into which each emotional noun fit. In terms of P1, we found that there was no negativity bias for emotional nouns. However, emotional nouns elicited larger amplitudes in the N170 component in the left hemisphere than did neutral nouns. This finding indicated that in later processing stages, emotional words can be discriminated from neutral words. Furthermore, positive, negative, and neutral words were different from each other in the late positive complex, indicating that in the third stage, even different emotions can be discerned. Thus, our results indicate that in a three-stage model the latter two stages are more stable and universal.

  10. Palliative care making a difference in rural Uganda, Kenya and Malawi: three rapid evaluation field studies.

    Science.gov (United States)

    Grant, Liz; Brown, Judith; Leng, Mhoira; Bettega, Nadia; Murray, Scott A

    2011-05-12

    Many people live and die in pain in Africa. We set out to describe patient, family and local community perspectives on the impact of three community based palliative care interventions in sub-Saharan Africa. Three palliative care programmes in Uganda, Kenya and Malawi were studied using rapid evaluation field techniques in each country, triangulating data from three sources: interviews with key informants, observations of clinical encounters and the local health and social care context, and routine data from local reports and statistics. We interviewed 33 patients with advanced illness, 27 family carers, 36 staff, 25 volunteers, and 29 community leaders and observed clinical care of 12 patients. In each site, oral morphine was being used effectively. Patients valued being treated with dignity and respect. Being supported at home reduced physical, emotional and financial burden of travel to, and care at health facilities. Practical support and instruction in feeding and bathing patients facilitated good deaths at home.In each country mobile phones enabled rapid access to clinical and social support networks. Staff and volunteers generally reported that caring for the dying in the face of poverty was stressful, but also rewarding, with resilience fostered by having effective analgesia, and community support networks. Programmes were reported to be successful because they integrated symptom control with practical and emotional care, education, and spiritual care. Holistic palliative care can be delivered effectively in the face of poverty, but a public health approach is needed to ensure equitable provision.

  11. Microfluidic device for rapid solution exchange to study kinetics of cell physiology

    Science.gov (United States)

    Hu, Howard; Honnatti, Meghana; Gillis, Kevin

    2006-11-01

    Exchanging the extracellular solution of the cell rapidly (less than 10ms) is an important requirement in study the kinetics of cell physiology. A microfluidic device is developed to exchange the solution around the cells as they flow through a junction at the intersection of two microfluidic channels. The solution exchange time is measured experimentally by fluorescently labeling the cell surface membranes with a styryl dye, FM1-43 or FM 2-10, and then observing the time course of cell fluorescence decay following the rapid drop in the extracellular concentration of the FM dye that occurs as the cell flows past the fluidic junction. A numerical model is developed to guide the experimental design of microfluidic device. In the model, the motion of a single cell through a fluid junction is simulated and the mixing process of the solutions is solved. The model also includes the kinetics of departitioning of FM dyes from the cell membrane. The departitioning time constants for the FM dyes are determined from fitting the measured data of the cell fluorescence decay. This departitioning kinetics is important as FM dyes are commonly used to label cell membranes for the purpose of measuring the release of neurotransmitter from synaptic vesicles via exocytosis and the subsequent reuptake of vesicular membrane by endocytosis.

  12. Three stages of emotional word processing: an ERP study with rapid serial visual presentation.

    Science.gov (United States)

    Zhang, Dandan; He, Weiqi; Wang, Ting; Luo, Wenbo; Zhu, Xiangru; Gu, Ruolei; Li, Hong; Luo, Yue-Jia

    2014-12-01

    Rapid responses to emotional words play a crucial role in social communication. This study employed event-related potentials to examine the time course of neural dynamics involved in emotional word processing. Participants performed a dual-target task in which positive, negative and neutral adjectives were rapidly presented. The early occipital P1 was found larger when elicited by negative words, indicating that the first stage of emotional word processing mainly differentiates between non-threatening and potentially threatening information. The N170 and the early posterior negativity were larger for positive and negative words, reflecting the emotional/non-emotional discrimination stage of word processing. The late positive component not only distinguished emotional words from neutral words, but also differentiated between positive and negative words. This represents the third stage of emotional word processing, the emotion separation. Present results indicated that, similar with the three-stage model of facial expression processing; the neural processing of emotional words can also be divided into three stages. These findings prompt us to believe that the nature of emotion can be analyzed by the brain independent of stimulus type, and that the three-stage scheme may be a common model for emotional information processing in the context of limited attentional resources. © The Author (2014). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  13. Palliative care making a difference in rural Uganda, Kenya and Malawi: three rapid evaluation field studies

    Directory of Open Access Journals (Sweden)

    Bettega Nadia

    2011-05-01

    Full Text Available Abstract Background Many people live and die in pain in Africa. We set out to describe patient, family and local community perspectives on the impact of three community based palliative care interventions in sub-Saharan Africa. Methods Three palliative care programmes in Uganda, Kenya and Malawi were studied using rapid evaluation field techniques in each country, triangulating data from three sources: interviews with key informants, observations of clinical encounters and the local health and social care context, and routine data from local reports and statistics. Results We interviewed 33 patients with advanced illness, 27 family carers, 36 staff, 25 volunteers, and 29 community leaders and observed clinical care of 12 patients. In each site, oral morphine was being used effectively. Patients valued being treated with dignity and respect. Being supported at home reduced physical, emotional and financial burden of travel to, and care at health facilities. Practical support and instruction in feeding and bathing patients facilitated good deaths at home. In each country mobile phones enabled rapid access to clinical and social support networks. Staff and volunteers generally reported that caring for the dying in the face of poverty was stressful, but also rewarding, with resilience fostered by having effective analgesia, and community support networks. Conclusions Programmes were reported to be successful because they integrated symptom control with practical and emotional care, education, and spiritual care. Holistic palliative care can be delivered effectively in the face of poverty, but a public health approach is needed to ensure equitable provision.

  14. End mill tools integration in CNC machining for rapid manufacturing processes: simulation studies

    Directory of Open Access Journals (Sweden)

    Muhammed Nafis Osman Zahid

    2015-01-01

    Full Text Available Computer numerical controlled (CNC machining has been recognized as a manufacturing process that is capable of producing metal parts with high precision and reliable quality, whereas many additive manufacturing methods are less capable in these respects. The introduction of a new layer-removal methodology that utilizes an indexing device to clamp the workpiece can be used to extend CNC applications into the realm of rapid manufacturing (CNC-RM processes. This study aims to improve the implementation of CNC machining for RM by formulating a distinct approach to integrate end mill tools during finishing processes. A main objective is to enhance process efficiency by minimizing the staircasing effect of layer removal so as to improve the quality of machined parts. In order to achieve this, different types of end mill tools are introduced to cater for specific part surfaces during finishing operations. Virtual machining simulations are executed to verify the method and the implications. The findings indicate the advantages of the approach in terms of cutting time and excess volume left on the parts. It is shown that using different tools for finishing operations will improve the capabilities of CNC machining for rapid manufacturing applications.

  15. TECRA Unique test for rapid detection of Salmonella in food: collaborative study.

    Science.gov (United States)

    Hughes, D; Dailianis, A E; Hill, L; McIntyre, D A; Anderson, A

    2001-01-01

    The TECRA Unique Salmonella test uses the principle of immunoenrichment to allow rapid detection of Salmonellae in food. A collaborative study was conducted to compare the TECRA Salmonella Unique test with the reference culture method given in the U.S. Food and Drug Administration's Bacteriological Analytical Manual. Three food types (milk powder, pepper, and soy flour) were analyzed in Australia and 2 food types (milk chocolate and dried egg) were analyzed in the United States. Forty-one collaborators participated in the study. For each of the 5 foods at each of the 3 levels, a comparison showed no significant differences (p > or = 0.05) in the proportion of positive test samples for Unique and that for the reference method using the Chi-square test for independence with continuity correction.

  16. Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

    Directory of Open Access Journals (Sweden)

    Supaporn Kradtap Hartwell

    2012-01-01

    Full Text Available Enzyme kinetics studies normally focus on the initial rate of enzymatic reaction. However, the manual operation of steps of the conventional enzyme kinetics method has some drawbacks. Errors can result from the imprecise time control and time necessary for manual changing the reaction cuvettes into and out of the detector. By using the automatic flow-based analytical systems, enzyme kinetics studies can be carried out at real-time initial rate avoiding the potential errors inherent in manual operation. Flow-based systems have been developed to provide rapid, low-volume, and high-precision analyses that effectively replace the many tedious and high volume requirements of conventional wet chemistry analyses. This article presents various arrangements of flow-based techniques and their potential use in future enzyme kinetics applications.

  17. LA SINTASSI DELLE LETTERE DI CAMILLO GOLGI: TRA GRAMMATICA EPISTOLARE, LINGUA SCIENTIFICA E LINGUAGGIO BUROCRATICO

    Directory of Open Access Journals (Sweden)

    Marta Damato

    2016-02-01

    Full Text Available L’articolo si propone di mettere in luce alcuni aspetti sintattici della lingua usata da Camillo Golgi, medico e ricercatore, nonché Premio Nobel 1906 per la Medicina o la Fisiologia, per come si offrono nella sua produzione epistolare ufficiale e privata.In prima istanza verrà fornita una duplice contestualizzazione dell’analisi linguistica da condurre: si descriverà la scrittura epistolare come genere peculiare, sia per la sua particolare collocazione al crocevia della diamesia (tra scritto e parlato, sia in relazione alla cosiddetta grammatica epistolare; in seguito, si presenterà la figura purtroppo ancora poco nota di Camillo Golgi, con tutti i suoi meriti scientifici e con l’auspicio di una sua riscoperta. Poi si giungerà alla vera e propria analisi linguistica, che verterà, come si è scritto, sulla sintassi delle lettere golgiane: l’analisi farà emergere la parziale adesione dello scrivente al canone dell’epistolografia, mettendo anche in luce alcuni scarti rispetto a questo, scarti collegati a precise scelte del Golgi che si allontanano dal parlato tipicamente riprodotto nella comunicazione epistolare, in direzione di un modello scritto formale e controllato, rispondente, in particolare, alle modalità espressive tipiche della prosa scientifica e della lingua della burocrazia. The syntax of Camillo Golgi’s letters: epistolary grammar, the language of science and bureaucracy The article intends to illustrate some prominent syntactical aspects of the language used by Camillo Golgi, researcher and famous doctor, winner of the Nobel Prize for Medicine and Physiology in 1906, as these aspects appear in his official and private correspondence. First the context of the linguistic analysis will be explained. Epistolary writing is a particular genre, halfway between written and spoken language, featuring a special grammar. Then the little-known figure of Camillo Golgi, with his many scientific merits, will be presented and

  18. 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......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...

  19. Physician counseling of young adults with rapid weight gain: a retrospective cohort study

    Directory of Open Access Journals (Sweden)

    Thompson Jason

    2010-04-01

    Full Text Available Abstract Background The incidence of weight gain is highest during young adulthood. Our study aims to describe weight gain patterns among young adults and to evaluate physician recognition of and counseling for rapid weight gain. Methods This retrospective cohort study included patients ages 18-35 at an academic internal medicine clinic between 2004-2008. We conducted chart reviews to determine weight change over time, whether weight gain greater than 3 lbs/year was documented, whether counseling was provided, and whether patients became overweight. We categorized weight gain documentation by location on the problem list, encounter diagnosis, or note text. We categorized counseling as weight-specific or general diet and exercise counseling. We used Chi-square tests to evaluate the relationship between weight change over time and the following variables: gender, diagnosis of weight gain, and counseling for weight gain. Fisher's Exact test was used to test for an association between diagnosis and counseling category. Results The study included 365 patients. Weight gain was greater than 3 lbs/year for 24% (90/365 of patients, of whom 56 (15% gained 3-5.9 lbs/year, and 34 (9% gained more than 6 lbs/year. Among patients gaining more than 3 lbs/year, physicians documented weight gain as a problem in only 10% (9/90. Of the 9 patients for whom weight gain was documented, physicians provided weight-specific counseling in three, and general diet and exercise counseling in four. Of the 81 individuals with no documented diagnosis of weight gain, 63% had no documented counseling, but 34% received general diet and exercise counseling. Among patients with over 180 days of follow-up, 8% (10/126 became overweight. Conclusions Physicians infrequently recognize or counsel for weight gain among young adult patients. Improving identification of patients with rapid weight gain can provide an opportunity for tailored weight-related counseling.

  20. γ-COPI mediates the retention of kAE1 G701D protein in Golgi apparatus - a mechanistic explanation of distal renal tubular acidosis associated with the G701D mutation.

    Science.gov (United States)

    Duangtum, Natapol; Junking, Mutita; Phadngam, Suratchanee; Sawasdee, Nunghathai; Castiglioni, Andrea; Charngkaew, Komgrid; Limjindaporn, Thawornchai; Isidoro, Ciro; Yenchitsomanus, Pa-Thai

    2017-07-17

    Mutations of the solute carrier family 4 member 1 (SLC4A1) gene encoding kidney anion (chloride/bicarbonate ion) exchanger 1 (kAE1) can cause genetic distal renal tubular acidosis (dRTA). Different SLC4A1 mutations give rise to mutant kAE1 proteins with distinct defects in protein trafficking. The mutant kAE1 protein may be retained in endoplasmic reticulum (ER) or Golgi apparatus, or mis-targeted to the apical membrane, failing to display its function at the baso-lateral membrane. The ER-retained mutant kAE1 interacts with calnexin chaperone protein; disruption of this interaction permits the mutant kAE1 to reach the cell surface and display anion exchange activity. However, the mechanism of Golgi retention of mutant kAE1 G701D protein, which is otherwise functional, is still unclear. In the present study, we show that Golgi retention of kAE1 G701D is due to a stable interaction with the Golgi-resident protein, coat protein complex I (COPI), that plays a role in retrograde vesicular trafficking and Golgi-based quality control. The interaction and co-localization of kAE1 G701D with the γ-COPI subunit were demonstrated in human embryonic kidney (HEK-293T) cells by co-immunoprecipitation and immunofluorescence staining. Small interference RNA (siRNA) silencing of COPI expression in the transfected HEK-293T cells increased the cell surface expression of transgenic kAE1 G701D, as shown by immunofluorescence staining. Our data unveil the molecular mechanism of Golgi retention of kAE1 G701D and suggest that disruption of the COPI-kAE1 G701D interaction could be a therapeutic strategy to treat dRTA caused by this mutant. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  1. Molecular dynamics simulation studies of structural and dynamical properties of rapidly quenched Al

    Energy Technology Data Exchange (ETDEWEB)

    Shen, B.; Liu, C. Y.; Jia, Y.; Yue, G. Q.; Ke, F. S.; Zhao, H. B.; Chen, L. Y.; Wang, S. Y.; Wang, C. Z.; Ho, K. M.

    2014-01-01

    The structural and dynamical properties of rapidly quenched Al are studied by molecular dynamics simulations. The pair-correlation function of high temperature liquid Al agrees well with the experimental results. Different cooling rates are applied with high cooling rates leading to glass formation, while low cooling rates leading to crystallization. The local structures are characterized by Honeycutt–Andersen indices and Voronoi tessellation analysis. The results show that for high cooling rates, the local structures of the liquid and glassy Al are predominated by icosahedral clusters, together with considerable amount of face-centered cubic and hexagonal close packed short-range orders. These short-range order results are further confirmed using the recently developed atomic cluster alignment method. Moreover, the atomic cluster alignment clearly shows the crystal nucleation process in supercooled liquid of Al. Finally, the mean square displacement for the liquid is also analyzed, and the corresponding diffusion coefficient as a function of temperature is calculated.

  2. Experimental study on fragmental behavior of coals and biomasses during rapid pyrolysis.

    Science.gov (United States)

    Cui, Tongmin; Xu, Jianliang; Fan, Wenke; Chang, Qinghua; Yu, Guangsuo; Wang, Fuchen

    2016-12-01

    In order to study the primary fragmentation behavior of coals and biomasses, experiments of rapid pyrolysis were carried out. This work focused on the devolatilization and fragmentation characteristics including the solid/gas yield, particle density/morphology, particle size and fragmental probability (S f ). The effects of temperature, time and solid property were investigated. The viscous flow model was employed to characterize the pressure difference (ΔP), which was considered as the driving force of diffusion and fragmentation. The Ohm principle was used to establish the linear relation of devolatilization rate and fragmentation rate. The result showed that temperature and time have positive contribution to the fragmentation. The occurrence of fragmentation was observed more apparently with the decreasing of the ash content in the biomass. The pressure difference has a positive correlation with the fragmental rate, which shows the validity of application Ohm principle in the prediction of fragmenting process. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Heavy flavor studies at forward and backward rapidities in Cu+Au collisions with PHENIX detector

    Science.gov (United States)

    da Silva, Cesar; Phenix Collaboration

    2016-09-01

    Asymmetric Cu+Au collisions at √{sNN}=200 GeV performed at RHIC in 2012 open an opportunity to study particle yields in the presence of different mixtures of initial and final state nuclear effects by using probes measured from negative to positive rapidity regions. Heavy flavor yields may be affected by initial state effects on gluons and energy loss in the final state hot medium. The 2012 run was the first where the Forward Vertex Detector (FVTX) was operating. This detector allows the identification of D and B mesons from displaced vertex measurements. Results on B-meson nuclear modification through its J/ ψ decay channel will be presented along with the status of the analysis of semi-leptonic decays of charm and bottom yields.

  4. Activity of Specific Lipid-regulated ADP Ribosylation Factor-GTPase–activating Proteins Is Required for Sec14p-dependent Golgi Secretory Function in Yeast

    OpenAIRE

    Yanagisawa, Lora L.; Marchena, Jennifer; Xie, Zhigang; Li, Xinmin; Poon, Pak P.; Singer, Richard A.; Johnston, Gerald C.; Randazzo, Paul A.; Bankaitis, Vytas A.

    2002-01-01

    Yeast phosphatidylinositol transfer protein (Sec14p) coordinates lipid metabolism with protein-trafficking events. This essential Sec14p requirement for Golgi function is bypassed by mutations in any one of seven genes that control phosphatidylcholine or phosphoinositide metabolism. In addition to these “bypass Sec14p” mutations, Sec14p-independent Golgi function requires phospholipase D activity. The identities of lipids that mediate Sec14p-dependent Golgi function, and the identity of the p...

  5. New insights from coral growth band studies in an era of rapid environmental change

    Science.gov (United States)

    Lough, Janice M.; Cooper, Timothy F.

    2011-10-01

    The rapid formation of calcium carbonate coral skeletons (calcification) fuelled by the coral-algal symbiosis is the backbone of tropical coral reef ecosystems. However, the efficacy of calcification is measurably influenced by the sea's physico-chemical environment, which is changing rapidly. Warming oceans have already led to increased frequency and severity of coral bleaching, and ocean acidification has a demonstrable potential to cause reduced rates of calcification. There is now general agreement that ocean warming and acidification are attributable to human activities increasing greenhouse gas concentrations in the atmosphere, and the large part of the extra carbon dioxide (the main greenhouse gas) that is absorbed by oceans. Certain massive corals provide historical perspectives on calcification through the presence of dateable annual density banding patterns. Each band is a page in an environmental archive that reveals past responses of growth (linear extension, skeletal density and calcification rate) and provides a basis for prediction of future of coral growth. A second major line of research focuses on the measurement of various geochemical tracers incorporated into the growth bands, allowing the reconstruction of past marine climate conditions (i.e. palaeoclimatology). Here, we focus on the structural properties of the annual density bands themselves (viz. density; linear extension), exploring their utility in providing both perspectives on the past and pointers to the future of calcification on coral reefs. We conclude that these types of coral growth records, though relatively neglected in recent years compared to the geochemical studies, remain immensely valuable aids to unravelling the consequences of anthropogenic climate change on coral reefs. Moreover, an understanding of coral growth processes is an essential pre-requisite for proper interpretation of studies of geochemical tracers in corals.

  6. Rapid spread of complex change: a case study in inpatient palliative care

    Directory of Open Access Journals (Sweden)

    Filipski Marta I

    2009-12-01

    Full Text Available Abstract Background Based on positive findings from a randomized controlled trial, Kaiser Permanente's national executive leadership group set an expectation that all Kaiser Permanente and partner hospitals would implement a consultative model of interdisciplinary, inpatient-based palliative care (IPC. Within one year, the number of IPC consultations program-wide increased almost tenfold from baseline, and the number of teams nearly doubled. We report here results from a qualitative evaluation of the IPC initiative after a year of implementation; our purpose was to understand factors supporting or impeding the rapid and consistent spread of a complex program. Methods Quality improvement study using a case study design and qualitative analysis of in-depth semi-structured interviews with 36 national, regional, and local leaders. Results Compelling evidence of impacts on patient satisfaction and quality of care generated 'pull' among adopters, expressed as a remarkably high degree of conviction about the value of the model. Broad leadership agreement gave rise to sponsorship and support that permeated the organization. A robust social network promoted knowledge exchange and built on an existing network with a strong interest in palliative care. Resource constraints, pre-existing programs of a different model, and ambiguous accountability for implementation impeded spread. Conclusions A complex, hospital-based, interdisciplinary intervention in a large health care organization spread rapidly due to a synergy between organizational 'push' strategies and grassroots-level pull. The combination of push and pull may be especially important when the organizational context or the practice to be spread is complex.

  7. Studi Bahaya Erosi Tanah Dengan Metode Pemetaan Cepat (Rapid Mapping di Sub DAS Cimanuk Hulu

    Directory of Open Access Journals (Sweden)

    Rahmat Razali

    2016-11-01

    Full Text Available ABSTRAK Erosi tanah merupakan salah satu isu ekosistem lingkungan yang banyak menjadi perhatian, dimana manusia berperan merubah lingkungan yang ada di pegunungan seperti kondisi saat ini Sub DAS Cimanuk Hulu cukup memprihatinkan apabila tidak dilakukan penanganan dan pengendalian pemanfaatan ruangnya. Kaitannya dalam memperoleh informasi pendugaan erosi, metode kualitatif dimana dalam analisisanya dibantu menggunakan Sistem Informasi Geografi (SIG lebih fleksibel dibandingkan model erosi lainnya dan penerapannya dapat disesuaikan dengan karakteristik daerah kajian dan ketersediaan data. Penelitian ini menggunakan Metode Pemetaan Cepat (Rapid Mapping untuk mengidentifikasi persebaran bahaya erosi berdasarkan faktor-faktor yang mempengaruhi erosi seperti penutupan lahan, prosentase tanah terbuka dan kemiringan lereng diturunkan dari analisis Citra dan DEM. Hasil menunjukkan bahwa dengan memanfaatkan analisis Citra dan DEM cukup efektif untuk mengetahui persebaran bahaya erosi dan dapat digunakan untuk menentukan arahan teknik konservasi tanah.   ABSTRACT Soil erosion is one of the issues that many ecosystems of concern, where people act to change the environment in the mountains as the current conditions sub watershed Cimanuk Hulu quite alarming if not the handling and control of the use of space. Relation to erosion prediction information, qualitative methods which aided in analyze using Geographic Information System (GIS is more flexible than other erosion models and their application can be tailored to the characteristics of the study area and data availability. This study used the Fast Mapping (Rapid Mapping to identify the distribution of erosion based on factors that affect erosion as land cover, percentage of bareland and slope derived from the analysis of the image and DEM.Results show that by using image analysis and DEM effectively enough to know of erosion hazard and the distribution can be used to determine the soil conservation

  8. Varicella-zoster virus ORF7 interacts with ORF53 and plays a role in its trans-Golgi network localization.

    Science.gov (United States)

    Wang, Wei; Fu, Wenkun; Pan, Dequan; Cai, Linli; Ye, Jianghui; Liu, Jian; Liu, Che; Que, Yuqiong; Xia, Ningshao; Zhu, Hua; Cheng, Tong

    2017-10-01

    Varicella-zoster virus (VZV) is a neurotropic alphaherpesvirus that causes chickenpox and shingles. ORF7 is an important virulence determinant of VZV in both human skin and nerve tissues, however, its specific function and involved molecular mechanism in VZV pathogenesis remain largely elusive. Previous yeast two-hybrid studies on intraviral protein-protein interaction network in herpesviruses have revealed that VZV ORF7 may interact with ORF53, which is a virtually unstudied but essential viral protein. The aim of this study is to identify and characterize VZV ORF53, and to investigate its relationship with ORF7. For this purpose, we prepared monoclonal antibodies against ORF53 and, for the first time, characterized it as a ~40 kDa viral protein predominantly localizing to the trans-Golgi network of the infected host cell. Next, we further confirmed the interaction between ORF7 and ORF53 by co-immunoprecipitation and co-localization studies in both plasmid-transfected and VZV-infected cells. Moreover, interestingly, we found that ORF53 lost its trans-Golgi network localization and became dispersed in the cytoplasm of host cells infected with an ORF7-deleted recombinant VZV, and thus ORF7 seems to play a role in normal subcellular localization of ORF53. Collectively, these results suggested that ORF7 and ORF53 may function as a complex during infection, which may be implicated in VZV pathogenesis.

  9. Implicit affectivity and rapid processing of affective body language: An fMRI study.

    Science.gov (United States)

    Suslow, Thomas; Ihme, Klas; Quirin, Markus; Lichev, Vladimir; Rosenberg, Nicole; Bauer, Jochen; Bomberg, Luise; Kersting, Anette; Hoffmann, Karl-Titus; Lobsien, Donald

    2015-10-01

    Previous research has revealed affect-congruity effects for the recognition of affects from faces. Little is known about the impact of affect on the perception of body language. The aim of the present study was to investigate the relationship of implicit (versus explicit) affectivity with the recognition of briefly presented affective body expressions. Implicit affectivity, which can be measured using indirect assessment methods, has been found to be more predictive of spontaneous physiological reactions than explicit (self-reported) affect. Thirty-four healthy women had to label the expression of body postures (angry, fearful, happy, or neutral) presented for 66 ms and masked by a neutral body posture in a forced-choice format while undergoing functional magnetic resonance imaging (fMRI). Participants' implicit affectivity was assessed using the Implicit Positive and Negative Affect Test. Measures of explicit state and trait affectivity were also administered. Analysis of the fMRI data was focused on a subcortical network involved in the rapid perception of affective body expressions. Only implicit negative affect (but not explicit affect) was correlated with correct labeling performance for angry body posture. As expected, implicit negative affect was positively associated with activation of the subcortical network in response to fearful and angry expression (compared to neutral expression). Responses of the caudate nucleus to affective body expression were especially associated with its recognition. It appears that processes of rapid recognition of affects from body postures could be facilitated by an individual's implicit negative affect. © 2015 Scandinavian Psychological Associations and John Wiley & Sons Ltd.

  10. Studies on the Process Parameters of Rapid Prototyping Technique (Stereolithography for the Betterment of Part Quality

    Directory of Open Access Journals (Sweden)

    Raju Bangalore Singe Gowda

    2014-01-01

    Full Text Available Rapid prototyping (RP has evolved as frontier technology in the recent times, which allows direct transformation of CAD files into functional prototypes where it tremendously reduces the lead time to produce physical prototypes necessary for design verification, fit, and functional analysis by generating the prototypes directly from the CAD data. Part quality in the rapid prototyping process is a function of build parameters such as hatch cure depth, layer thickness, orientation, and hatch spacing. Thus an attempt was made to identify, study, and optimize the process parameters governing the system which are related to part characteristics using Taguchi experimental design techniques quality. The part characteristics can be divided into physical part and mechanical part characteristics. The physical characteristics are surface finish, dimensional accuracy, distortion, layer thickness, hatch cure, and hatch file, whereas mechanical characteristics are flexural strength, ultimate tensile strength, and impact strength. Thus, this paper proposes to characterize the influence of the physical build parameters over the part quality. An L9 orthogonal array was designed with the minimum number of experimental runs with desired parameter settings and also by analysis tools such as ANOVA (analysis of variance. Establishment of experimentally verified correlations between the physical part characteristics and mechanical part characteristics to obtain an optimal process parameter level for betterment of part quality is obtained. The process model obtained by the empirical relation can be used to determine the strength of the prototype for the given set of parameters that shows the dependency of strength, which are essential for designers and RP machine users.

  11. Decreased levels of cell-division cycle 42 (Cdc42) protein in peripheral lymphocytes from ischaemic stroke patients are associated with Golgi apparatus function.

    Science.gov (United States)

    Mo, Xiao-Ye; Li, Ting; Hu, Zhi-Ping

    2013-06-01

    To investigate levels of cell-division cycle 42 (Cdc42) protein, and their relationship with Golgi apparatus function in peripheral lymphocytes, in patients following ischaemic stroke. Patients with acute cerebral ischaemic stroke (within 24-72 h of the onset of focal neurological symptoms) and healthy control subjects were enrolled in this prospective case-control study. The cellular location of Cdc42 in peripheral lymphocytes was demonstrated using immunofluorescence. Protein levels of Cdc42 and trans-golgi network protein 2 (TGN46) in peripheral lymphocytes were determined by immunocytochemical staining and Western blotting. A total of 38 patients with stroke and 38 control subjects were studied. The mean ± SD percentage of Cdc42-positive lymphocytes from patients with stroke was significantly lower than that in control subjects (39.53 ± 13.55% versus 66.61 ± 23.30%, respectively). Similar findings were demonstrated for TGN46. Cdc42 levels were positively correlated with TGN46 levels (r = 0.92). Acute ischaemic stroke was associated with reduced levels of Cdc42 protein. These findings might lead to the development of drugs that could have therapeutic benefits in patients with acute ischaemic stroke.

  12. Rapid, easy, and cheap randomization: prospective evaluation in a study cohort

    Directory of Open Access Journals (Sweden)

    Parker Melissa J

    2012-06-01

    Full Text Available Abstract Background When planning a randomized controlled trial (RCT, investigators must select randomization and allocation procedures based upon a variety of factors. While third party randomization is cited as being among the most desirable randomization processes, many third party randomization procedures are neither feasible nor cost-effective for small RCTs, including pilot RCTs. In this study we present our experience with a third party randomization and allocation procedure that utilizes current technology to achieve randomization in a rapid, reliable, and cost-effective manner. Methods This method was developed by the investigators for use in a small 48-participant parallel group RCT with four study arms. As a nested study, the reliability of this randomization procedure was prospectively evaluated in this cohort. The primary outcome of this nested study was the proportion of subjects for whom allocation information was obtained by the Research Assistant within 15 min of the initial participant randomization request. A secondary outcome was the average time for communicating participant group assignment back to the Research Assistant. Descriptive information regarding any failed attempts at participant randomization as well as costs attributable to use of this method were also recorded. Statistical analyses included the calculation of simple proportions and descriptive statistics. Results Forty-eight participants were successfully randomized and group allocation instruction was received for 46 (96% within 15 min of the Research Assistant placing the initial randomization request. Time elapsed in minutes until receipt of participant allocation instruction was Mean (SD 3.1 +/− 3.6; Median (IQR 2 (2,3; Range (1–20 for the entire cohort of 48. For the two participants for whom group allocation information was not received by the Research Assistant within the 15-min pass threshold, this information was obtained following a second

  13. Comparison of bipyridyl, maltol and kojic acid action as organic vanadium ligands on activity of galactosyltransferase (EC 2.4.1.38, some physiological parameters and ultrastructure of Golgi complexes in rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Wojciech Dabros

    2007-10-01

    Full Text Available The biochemical activity and morphology of control and streptozotocin-diabetic rat liver Golgi complexes were previously investigated by us under influence of some vanadium [V(IV] compounds. The effectiveness of these derivatives depends on the kind of complexing ligands. This paper presents the investigation of the effect of bipyridyl, the ligand of a new vanadium compound, tested by us with maltol and kojic acid (two ligands studied by the present and other authors. The three ligands alone action was tested under the same experimental conditions as in the case of whole compounds with vanadium and applied to liver Golgi complexes of control rats. A preliminary study for maltol and kojic acid had been previously carried out by us parallel with tests of whole vanadium complexes, but valuable differences in biological action found in our condition of experiments suggested the extension of studies to include the two above-mentioned ligands and to compare the effects of the three investigated ligands. The supplementary part of the experiment focused mainly on the ultrastructure of Golgi complexes in hepatocytes. Four groups of animals were used: C - control rats, C + M (maltol, C + (ka2 (kojic acid and C + (bpy2 (bipyridyl. The control rats received 0.09M NaCl as drinking liquid; all the other animals were given 3.6 mmol/L of appropriate ligand solution in 0.09M NaCl during 7 days. All the animals survived the experiments. Only in group C + (bpy2 did the authors observe statistically significant differences as compared with the controls (group C. The differences were detected in physiological studies and manifested as body weight decreased by approximately 20% during the experiment, lower liquid (p<0.001 and food (p<0.01 intake and increase of free blood sugar level (p<0.01. The yield of Golgi membrane isolation decreased in this group (p<0.01. The main investigated biochemical parameter, i.e. the activity of liver Golgi marker enzyme

  14. A New Approach of Short Wave Protection against Middle Cerebral Artery Occlusion/Reperfusion Injury via Attenuation of Golgi Apparatus Stress by Inhibition of Downregulation of Secretory Pathway Ca(2+)-ATPase Isoform 1 in Rats.

    Science.gov (United States)

    Fan, Yongmei; Zhang, Changjie; Li, Ting; Peng, Wenna; Yin, Jing; Li, Xiaofao; Kong, Ying; Lan, Chunna; Wang, Rumi; Hu, Zhiping

    2016-07-01

    Short wave (SW), a pattern of electromagnetic therapy, achieves an oscillating electromagnetic field. It has been reported that it may have a potential effect on cerebral injury. The present study was designed to investigate the potential role and possible mechanism of SW in focal cerebral ischemia/reperfusion (I/R) injury in rats. Secretory pathway Ca(2+)/Mn(2+) ATPase isoform 1 is a major component of Golgi apparatus stress. It has been reported as representative of Golgi apparatus stress. Up to 120 minutes of middle cerebral artery occlusion (MCAO) and reperfusion injury was induced in male Sprague-Dawley rats. Different sessions of SW daily were administered over head after reperfusion from day 1 to day 7. Functional recovery scores, survival rates, infarct volume analysis, electron microscope test, and western blotting studies were used to analyze the therapy. SW protected against neuronal death and apoptosis in cornu ammon 1 region of hippocampus by reducing neuronal deficit, infarct volume, and ultrastructure. SW partly inhibited upregulation of caspase3. In addition, the expression of secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) was upregulated by SW. Our data indicate that SW can be protected against focal cerebral I/R injury, and the influence on Golgi apparatus stress might provide us a new perspective in further study. To the authors' knowledge, this is the first report using SW to increase expression of SPCA1 indicating modulate Golgi apparatus stress in MCAO and reperfusion model. Copyright © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  15. Study of Jet Transverse Momentum and Jet Rapidity Dependence on Dijet Azimuthal Decorrelations

    Energy Technology Data Exchange (ETDEWEB)

    Chakravarthula, Kiran [Louisiana Tech Univ., Ruston, LA (United States)

    2012-01-01

    In a collision experiment involving highly energetic particles such as hadrons, processes at high momentum transfers can provide information useful for many studies involving Quantum Chromodynamics (QCD). One way of analyzing these interactions is through angular distributions. In hadron-hadron collisions, the angular distribution between the two leading jets with the largest transverse momentum (pT ) is affected by the production of additional jets. While soft radiation causes small differences in the azimuthal angular distribution of the two leading jets produced in a collision event, additional hard jets produced in the event have more pronounced influence on the distribution of the two leading jets produced in the collision. Thus, the dijet azimuthal angular distribution can serve as a variable that can be used to study the transition from soft to hard QCD processes in a collision event. This dissertation presents a triple-differential study involving the azimuthal angular distribution and the jet transverse momenta, and jet rapidities of the first two leading jets. The data used for this research are obtained from proton-antiproton (p$\\bar{p}$) collisions occurring at a center of mass energy of 1.96TeV, using the DØ detector in Run II of the Tevatron Collider at the Fermi National Accelerator Laboratory (FNAL) in Illinois, USA. Comparisons are made to perturbative QCD (pQCD) predictions at next-to-leading order (NLO).

  16. A simple and rapid approach for screening of SARS-coronavirus genotypes: an evaluation study

    Directory of Open Access Journals (Sweden)

    Jin Yongjie

    2005-10-01

    Full Text Available Abstract Background The Severe Acute Respiratory Syndrome (SARS was a newly emerged infectious disease which caused a global epidemic in 2002–2003. Sequence analysis of SARS-coronavirus isolates revealed that specific genotypes predominated at different periods of the epidemic. This information can be used as a footprint for tracing the epidemiology of infections and monitor viral evolution. However, direct sequencing analysis of a large number of clinical samples is cumbersome and time consuming. We present here a simple and rapid assay for the screening of SARS-coronavirus genotypes based on the use of fluorogenic oligonucleotide probes for allelic discrimination. Methods Thirty SARS patients were recruited. Allelic discrimination assays were developed based on the use of fluorogenic oligonucleotide probes (TaqMan. Genotyping of the SARS-coronavirus isolates obtained from these patients were carried out by the allelic discrimination assays and confirmed by direct sequencing. Results Genotyping based on the allelic discrimination assays were fully concordant with direct sequencing. All of the 30 SARS-coronavirus genotypes studied were characteristic of genotypes previously documented to be associated with the latter part of the epidemic. Seven of the isolates contained a previously reported major deletion but in patients not epidemiologically related to the previously studied cohort. Conclusion We have developed a simple and accurate method for the characterization and screening of SARS-coronavirus genotypes. It is a promising tool for the study of epidemiological relationships between documented cases during an outbreak.

  17. Intelligent computational model for classification of sub-Golgi protein using oversampling and fisher feature selection methods.

    Science.gov (United States)

    Ahmad, Jamal; Javed, Faisal; Hayat, Maqsood

    2017-05-01

    Golgi is one of the core proteins of a cell, constitutes in both plants and animals, which is involved in protein synthesis. Golgi is responsible for receiving and processing the macromolecules and trafficking of newly processed protein to its intended destination. Dysfunction in Golgi protein is expected to cause many neurodegenerative and inherited diseases that may be cured well if they are detected effectively and timely. Golgi protein is categorized into two parts cis-Golgi and trans-Golgi. The identification of Golgi protein via direct method is very hard due to limited available recognized structures. Therefore, the researchers divert their attention toward the sequences from structures. However, owing to technological advancement, exploration of huge amount of sequences was reported in the databases. So recognition of large amount of unprocessed data using conventional methods is very difficult. Therefore, the concept of intelligence was incorporated with computational model. Intelligence based computational model obtained reasonable results, but the gap of improvement is still under consideration. In this regard, an intelligent automatic recognition model is developed in order to enhance the true classification rate of sub-Golgi proteins. In this approach, discrete and evolutionary feature extraction methods are applied on the benchmark Golgi protein datasets to excerpt salient, propound and variant numerical descriptors. After that, an oversampling technique Syntactic Minority over Sampling Technique is employed to balance the data. Hybrid spaces are also generated with combination of these feature spaces. Further, Fisher feature selection method is utilized to reduce the extra noisy and redundant features from feature vector. Finally, k-nearest neighbor algorithm is used as learning hypothesis. Three distinct cross validation tests are used to examine the stability and efficiency of the proposed model. The predicted outcomes of proposed model are better

  18. Weight restoration therapy rapidly reverses cortical thinning in anorexia nervosa: A longitudinal study.

    Science.gov (United States)

    Bernardoni, Fabio; King, Joseph A; Geisler, Daniel; Stein, Elisa; Jaite, Charlotte; Nätsch, Dagmar; Tam, Friederike I; Boehm, Ilka; Seidel, Maria; Roessner, Veit; Ehrlich, Stefan

    2016-04-15

    Structural magnetic resonance imaging studies have documented reduced gray matter in acutely ill patients with anorexia nervosa to be at least partially reversible following weight restoration. However, few longitudinal studies exist and the underlying mechanisms of these structural changes are elusive. In particular, the relative speed and completeness of brain structure normalization during realimentation remain unknown. Here we report from a structural neuroimaging study including a sample of adolescent/young adult female patients with acute anorexia nervosa (n=47), long-term recovered patients (n=34), and healthy controls (n=75). The majority of acutely ill patients were scanned longitudinally (n=35): at the beginning of standardized weight restoration therapy and again after partial weight normalization (>10% body mass index increase). High-resolution structural images were processed and analyzed with the longitudinal stream of FreeSurfer software to test for changes in cortical thickness and volumes of select subcortical regions of interest. We found globally reduced cortical thickness in acutely ill patients to increase rapidly (0.06 mm/month) during brief weight restoration therapy (≈3 months). This significant increase was predicted by weight restoration alone and could not be ascribed to potentially mediating factors such as duration of illness, hydration status, or symptom improvements. By comparing cortical thickness in partially weight-restored patients with that measured in healthy controls, we confirmed that cortical thickness had normalized already at follow-up. This pattern of thinning in illness and rapid normalization during weight rehabilitation was largely mirrored in subcortical volumes. Together, our findings indicate that structural brain insults inflicted by starvation in anorexia nervosa may be reversed at a rate much faster than previously thought if interventions are successful before the disorder becomes chronic. This provides

  19. Cross-sectional study of possible association between rapid eating and high body fat rates among female Japanese college students.

    Science.gov (United States)

    Yaguchi-Tanaka, Yuri; Kawagoshi, Yumiko; Sasaki, Satoshi; Fukao, Akira

    2013-01-01

    The incidence of excessive body fat among young Japanese females with a normal BMI, which is referred to as normal weight obesity (NWO), has recently increased. Some studies have associated eating rates with BMI. However, an association between body fat rate and dietary habits has not been proven. We compared differences in dietary habits between 72 female Japanese junior college students with normal (Eating rapidly was significantly associated with body fat ratios. Our findings suggest that eating rapidly increases body fat ratios.

  20. Cdc1p is an endoplasmic reticulum-localized putative lipid phosphatase that affects Golgi inheritance and actin polarization by activating Ca2+ signaling.

    Science.gov (United States)

    Losev, Eugene; Papanikou, Effrosyni; Rossanese, Olivia W; Glick, Benjamin S

    2008-05-01

    In the budding yeast Saccharomyces cerevisiae, mutations in the essential gene CDC1 cause defects in Golgi inheritance and actin polarization. However, the biochemical function of Cdc1p is unknown. Previous work showed that cdc1 mutants accumulate intracellular Ca(2+) and display enhanced sensitivity to the extracellular Mn(2+) concentration, suggesting that Cdc1p might regulate divalent cation homeostasis. By contrast, our data indicate that Cdc1p is a Mn(2+)-dependent protein that can affect Ca(2+) levels. We identified a cdc1 allele that activates Ca(2+) signaling but does not show enhanced sensitivity to the Mn(2+) concentration. Furthermore, our studies show that Cdc1p is an endoplasmic reticulum-localized transmembrane protein with a putative phosphoesterase domain facing the lumen. cdc1 mutant cells accumulate an unidentified phospholipid, suggesting that Cdc1p may be a lipid phosphatase. Previous work showed that deletion of the plasma membrane Ca(2+) channel Cch1p partially suppressed the cdc1 growth phenotype, and we find that deletion of Cch1p also suppresses the Golgi inheritance and actin polarization phenotypes. The combined data fit a model in which the cdc1 mutant phenotypes result from accumulation of a phosphorylated lipid that activates Ca(2+) signaling.

  1. Cdc1p Is an Endoplasmic Reticulum-Localized Putative Lipid Phosphatase That Affects Golgi Inheritance and Actin Polarization by Activating Ca2+ Signaling ▿ †

    Science.gov (United States)

    Losev, Eugene; Papanikou, Effrosyni; Rossanese, Olivia W.; Glick, Benjamin S.

    2008-01-01

    In the budding yeast Saccharomyces cerevisiae, mutations in the essential gene CDC1 cause defects in Golgi inheritance and actin polarization. However, the biochemical function of Cdc1p is unknown. Previous work showed that cdc1 mutants accumulate intracellular Ca2+ and display enhanced sensitivity to the extracellular Mn2+ concentration, suggesting that Cdc1p might regulate divalent cation homeostasis. By contrast, our data indicate that Cdc1p is a Mn2+-dependent protein that can affect Ca2+ levels. We identified a cdc1 allele that activates Ca2+ signaling but does not show enhanced sensitivity to the Mn2+ concentration. Furthermore, our studies show that Cdc1p is an endoplasmic reticulum-localized transmembrane protein with a putative phosphoesterase domain facing the lumen. cdc1 mutant cells accumulate an unidentified phospholipid, suggesting that Cdc1p may be a lipid phosphatase. Previous work showed that deletion of the plasma membrane Ca2+ channel Cch1p partially suppressed the cdc1 growth phenotype, and we find that deletion of Cch1p also suppresses the Golgi inheritance and actin polarization phenotypes. The combined data fit a model in which the cdc1 mutant phenotypes result from accumulation of a phosphorylated lipid that activates Ca2+ signaling. PMID:18332110

  2. A role of Rab29 in the integrity of the trans-Golgi network and retrograde trafficking of mannose-6-phosphate receptor.

    Directory of Open Access Journals (Sweden)

    Shicong Wang

    Full Text Available Rab29 (also referred as Rab7L1 is a novel Rab protein, and is recently demonstrated to regulate phagocytosis and traffic from the Golgi to the lysosome. However, its roles in membrane trafficking have not been investigated extensively. Our results in this study revealed that Rab29 is associated with the trans-Golgi network (TGN, and is essential for maintaining the integrity of the TGN, because inhibition of the activity of Rab29 or depletion of Rab29 resulted in fragmentation of the TGN marked by TGN46. Expression of the dominant negative form Rab29T21N or shRNA-Rab29 also altered the distribution of mannose-6-phosphate receptor (M6PR, and interrupted the retrograde trafficking of M6PR through monitoring the endocytosis of CD8-tagged calcium dependent M6PR (cdM6PR or calcium independent M6PR (ciM6PR, but without significant effects on the anterograde trafficking of vesicular stomatitis virus G protein (VSV-G. Our results suggest that Rab29 is essential for the integrity of the TGN and participates in the retrograde trafficking of M6PRs.

  3. The microtubule cytoskeleton and pollen tube Golgi vesicle system are required for in vitro S-RNase internalization and gametic self-incompatibility in apple.

    Science.gov (United States)

    Meng, Dong; Gu, Zhaoyu; Yuan, Hui; Wang, Aide; Li, Wei; Yang, Qing; Zhu, Yuandi; Li, Tianzhong

    2014-05-01

    S-RNase is the female determinant of gametophytic self-incompatibility in apple and is usually considered to be the reason for rejection of pollen. In this study, we investigated the role of microtubules (MTs) in internalization of S-RNases by pollen tubes cultured in vitro. The results showed that S-RNase was imported into the pollen tube where it inhibits pollen tube growth, and that S-RNase is co-localized with the Golgi vesicle during the internalization process. Moreover, MT depolymerization is observed following accumulation of S-RNases in the pollen cytosol. On the other hand, S-RNase was prevented from entering the pollen tube when the pollen was treated with the actin filament (AF) inhibitor latrunculin A (LatA), the MT inhibitor oryzalin, or the MT stabilizer taxol at subtoxic concentrations. These hindered the construction of the MT, with pollen tubes capable of growth under these conditions. Pollen tubes showed improved growth in self-pollinated styles that were pre-treated with taxol. This suggests that cytoskeleton antagonists can prevent S-RNase-mediated inhibition of pollen tubes in vivo by blocking S-RNase internalization. These results suggest that an intact and dynamic cytoskeleton is required for the in vitro internalization of S-RNase, as shown by the effects of various cytoskeleton inhibitors. S-RNase internalization takes place via a membrane/cytoskeleton-based Golgi vesicle system, which can also affect self-incompatibility in apple.

  4. 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.

  5. Manganese accumulates within golgi apparatus in dopaminergic cells as revealed by synchrotron X-ray fluorescence nanoimaging.

    Science.gov (United States)

    Carmona, Asunción; Devès, Guillaume; Roudeau, Stéphane; Cloetens, Peter; Bohic, Sylvain; Ortega, Richard

    2010-03-17

    Chronic exposure to manganese results in neurological symptoms referred to as manganism and is identified as a risk factor for Parkinson's disease. In vitro, manganese induces cell death in the dopaminergic cells, but the mechanisms of manganese cytotoxicity are still unexplained. In particular, the subcellular distribution of manganese and its interaction with other trace elements needed to be assessed. Applying synchrotron X-ray fluorescence nanoimaging, we found that manganese was located within the Golgi apparatus of PC12 dopaminergic cells at physiologic concentrations. At increasing concentrations, manganese accumulates within the Golgi apparatus until cytotoxic concentrations are reached resulting in a higher cytoplasmic content probably after the Golgi apparatus storage capacity is exceeded. Cell exposure to manganese and brefeldin A, a molecule known to specifically cause the collapse of the Golgi apparatus, results in the striking intracellular redistribution of manganese, which accumulates in the cytoplasm and the nucleus. These results indicate that the Golgi apparatus plays an important role in the cellular detoxification of manganese. In addition manganese exposure induces a decrease in total iron content, which could contribute to the overall neurotoxicity.

  6. Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-gamma-inducible cofactor.

    Science.gov (United States)

    Modiano, Nir; Lu, Yanping E; Cresswell, Peter

    2005-06-14

    Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras/Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-gamma-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-gamma. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result.

  7. 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

    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.

  8. Combined momentum collimation studies in a high-intensity rapid cycling proton synchrotron

    Directory of Open Access Journals (Sweden)

    Jing-Yu Tang

    2011-05-01

    Full Text Available Momentum collimation in a high-intensity rapid cycling synchrotron (RCS is a very important issue. Based on the two-stage collimation principle, a combined momentum collimation method is proposed and studied in detail here. The method makes use of the combination of secondary collimators in both the longitudinal and transverse planes. The primary collimator is placed at a high-dispersion location of an arc, and the longitudinal and transverse secondary collimators are in the same arc and in the adjacent downstream dispersion-free long straight section, respectively. The particles with positive momentum deviations will be scattered and degraded by a carbon scraper and then collected mainly by the transverse collimators, whereas the particles with negative momentum deviations will be scattered by a tantalum scraper and mainly collected by the longitudinal secondary collimators. This is to benefit from the different effects of protons passing through a high atomic number material and a low atomic number material, as the former produces relatively more scattering than the latter for the same energy loss. The studies also reveal that momentum collimation is strongly dependent on the transverse beam correlation that comes from the injection painting. The relevant requirements on the lattice design are also discussed, especially for compact rings. The multiparticle simulations using both TURTLE and ORBIT codes are presented to show the physical images of the collimation method, which was carried out with the input of the RCS of China Spallation Neutron Source.

  9. A STUDY ON THE INTRODUCTION OF BUS RAPID TRANSIT SYSTEM IN ASIAN DEVELOPING CITIES

    Directory of Open Access Journals (Sweden)

    Thaned SATIENNAM

    2006-01-01

    Full Text Available Bus Rapid Transit (BRT has increasingly become an attractive urban transit alternative in many Asian developing cities due to its cost-effective and flexible implementation. However, it still seems to be difficult to introduce BRT to these cities because almost all of their city structures have been developed under solely a road transport development city plan and weakness of land use control gives rise to many problems, such as urban sprawl, traffic congestion, and air pollution. The purpose of this study was to introduce several strategies to support BRT implementation in Asian developing cities, such as a strategy to appropriately integrate the paratransit system into BRT system as being a feeder along a BRT corridor to supply demand. These proposed strategies were evaluated by applying demand forecasting and emission models to the BRT project plan of Bangkok Metropolitan Administration (BMA in Thailand. It was demonstrated that the proposed strategies could effectively improve the BRT ridership, traffic conditions, and air pollution emission of the entire system in Bangkok. This study could be further extended to include strategy recommendation if a BRT system were to be introduced to other Asian developing cities.

  10. Combustion Chemistry of Ethanol: Ignition and Speciation Studies in a Rapid Compression Facility.

    Science.gov (United States)

    Barraza-Botet, Cesar L; Wagnon, Scott W; Wooldridge, Margaret S

    2016-09-29

    Ethanol remains the most important alternative fuel for the transportation sector. This work presents new experimental data on ethanol ignition, including stable species measurements, obtained with the University of Michigan rapid compression facility. Ignition delay times were determined from pressure histories of ignition experiments with stoichiometric ethanol-air mixtures at pressures of ∼3-10 atm. Temperatures (880-1150 K) were controlled by varying buffer gas composition (Ar, N2, CO2). High-speed imaging was used to record chemiluminescence during the experiments, which showed homogeneous ignition events. The results for ignition delay time agreed well with trends on the basis of previous experimental measurements. Speciation experiments were performed using fast gas sampling and gas chromatography to identify and quantify ethanol and 11 stable intermediate species formed during the ignition delay period. Simulations were carried out using a chemical kinetic mechanism available in the literature, and the agreement with the experimental results for ignition delay time and the intermediate species measured was excellent for the majority of the conditions studied. From the simulation results, ethanol + HO2 was identified as an important reaction at the experimental conditions for both the ignition delay time and intermediate species measurements. Further studies to improve the accuracy of the rate coefficient for ethanol + HO2 would improve the predictive understanding of intermediate and low-temperature ethanol combustion.

  11. Rapid L2 Word Learning through High Constraint Sentence Context: An Event-Related Potential Study

    Directory of Open Access Journals (Sweden)

    Baoguo Chen

    2017-12-01

    Full Text Available Previous studies have found quantity of exposure, i.e., frequency of exposure (Horst et al., 1998; Webb, 2008; Pellicer-Sánchez and Schmitt, 2010, is important for second language (L2 contextual word learning. Besides this factor, context constraint and L2 proficiency level have also been found to affect contextual word learning (Pulido, 2003; Tekmen and Daloglu, 2006; Elgort et al., 2015; Ma et al., 2015. In the present study, we adopted the event-related potential (ERP technique and chose high constraint sentences as reading materials to further explore the effects of quantity of exposure and proficiency on L2 contextual word learning. Participants were Chinese learners of English with different English proficiency levels. For each novel word, there were four high constraint sentences with the critical word at the end of the sentence. Learners read sentences and made semantic relatedness judgment afterwards, with ERPs recorded. Results showed that in the high constraint condition where each pseudoword was embedded in four sentences with consistent meaning, N400 amplitude upon this pseudoword decreased significantly as learners read the first two sentences. High proficiency learners responded faster in the semantic relatedness judgment task. These results suggest that in high quality sentence contexts, L2 learners could rapidly acquire word meaning without multiple exposures, and L2 proficiency facilitated this learning process.

  12. Penta-EF-Hand Protein Peflin Is a Negative Regulator of ER-To-Golgi Transport.

    Directory of Open Access Journals (Sweden)

    Mariah Rayl

    Full Text Available Luminal calcium regulates vesicle transport early in the secretory pathway. In ER-to-Golgi transport, depletion of luminal calcium leads to significantly reduced transport and a buildup of budding and newly budded COPII vesicles and vesicle proteins. Effects of luminal calcium on transport may be mediated by cytoplasmic calcium sensors near ER exits sites (ERES. The penta-EF-hand (PEF protein apoptosis-linked gene 2 (ALG-2 stabilizes sec31A at ER exit sites (ERES and promotes the assembly of inner and outer shell COPII components. However, in vitro and intact cell approaches have not determined whether ALG-2 is a negative or positive regulator, or a regulator at all, under basal physiological conditions. ALG-2 interacts with another PEF protein, peflin, to form cytosolic heterodimers that dissociate in response to calcium. However, a biological function for peflin has not been demonstrated and whether peflin and the ALG-2/peflin interaction modulates transport has not been investigated. Using an intact, single cell, morphological assay for ER-to-Golgi transport in normal rat kidney (NRK cells, we found that depletion of peflin using siRNA resulted in significantly faster transport of the membrane cargo VSV-G. Double depletion of peflin and ALG-2 blocked the increased transport resulting from peflin depletion, demonstrating a role for ALG-2 in the increased transport. Furthermore, peflin depletion caused increased targeting of ALG-2 to ERES and increased ALG-2/sec31A interactions, suggesting that peflin may normally inhibit transport by preventing ALG-2/sec31A interactions. This work identifies for the first time a clear steady state role for a PEF protein in ER-to-Golgi transport-peflin is a negative regulator of transport.

  13. Fluorescence imaging of dendritic spines of Golgi-Cox-stained neurons using brightening background

    Science.gov (United States)

    Ai, Min; Xiong, Hanqing; Yang, Tao; Shang, Zhenhua; Chen, Muqing; Liu, Xiuli; Zeng, Shaoqun

    2015-01-01

    We report a novel fluorescence imaging approach to imaging nonfluorescence-labeled biological tissue samples. The method was demonstrated by imaging neurons in Golgi-Cox-stained and epoxy-resin-embedded samples through the excitation of the background fluorescence of the specimens. The dark neurons stood out clearly against background fluorescence in the images, enabling the tracing of a single dendritic spine using both confocal and wide-field fluorescence microscopy. The results suggest that the reported fluorescence imaging method would provide an effective alternative solution to image nonfluorescence-labeled samples, and it allows tracing the dendritic spine structure of neurons.

  14. Detecting the golgi protein 73 of liver cancer with micro cantilever

    Science.gov (United States)

    Thanh Tuyen Le, Thi; Pham, Van Tho; Nhat Khoa Phan, Thanh; Binh Pham, Van; Thao Le, Van; Hien Tong, Duy

    2014-12-01

    Golgi protein 73 (GP73) is a potential serum biomarker used in diagnosing human hepatocellular carcinoma (HCC). Compared to alpha-fetoprotein, detection of GP73 is expected to give better sensitivity and specificity and thus offers a better method for diagnosis of HCC at an early stage. In this paper, silicon nitride microcantilever was used to detect GP73. The cantilever was modified through many steps to contain antibody of GP73. The result shows that the cantilever can be used as a label-free sensor to detect this kind of biomarker.

  15. Rapid genetic diversification within dog breeds as evidenced by a case study on Schnauzers.

    Science.gov (United States)

    Streitberger, K; Schweizer, M; Kropatsch, R; Dekomien, G; Distl, O; Fischer, M S; Epplen, J T; Hertwig, S T

    2012-10-01

    As a result of strong artificial selection, the domesticated dog has arguably become one of the most morphologically diverse vertebrate species, which is mirrored in the classification of around 400 different breeds. To test the influence of breeding history on the genetic structure and variability of today's dog breeds, we investigated 12 dog breeds using a set of 19 microsatellite markers from a total of 597 individuals with about 50 individuals analysed per breed. High genetic diversity was noted over all breeds, with the ancient Asian breeds (Akita, Chow Chow, Shar Pei) exhibiting the highest variability, as was indicated chiefly by an extraordinarily high number of rare and private alleles. Using a Bayesian clustering method, we detected significant genetic stratification within the closely related Schnauzer breeds. The individuals of these three recently differentiated breeds (Miniature, Standard and Giant Schnauzer) could not be assigned to a single cluster each. This hidden genetic structure was probably caused by assortative mating owing to breeders' preferences regarding coat colour types and the underlying practice of breeding in separate lineages. Such processes of strong artificial disruptive selection for different morphological traits in isolated and relatively small lineages can result in the rapid creation of new dog types and potentially new breeds and represent a unique opportunity to study the evolution of genetic and morphological differences in recently diverged populations. © 2011 The Authors, Animal Genetics © 2011 Stichting International Foundation for Animal Genetics.

  16. Defining impact of a rapid response team: qualitative study with nurses, physicians and hospital administrators.

    Science.gov (United States)

    Benin, Andrea L; Borgstrom, Christopher P; Jenq, Grace Y; Roumanis, Sarah A; Horwitz, Leora I

    2012-05-01

    The objective of this study was to qualitatively describe the impact of a Rapid Response Team (RRT) at a 944-bed, university-affiliated hospital. We analysed 49 open-ended interviews with administrators, primary team attending physicians, trainees, RRT attending hospitalists, staff nurses, nurses and respiratory technicians. Themes elicited were categorised into the domains of (1) morale and teamwork, (2) education, (3) workload, (4) patient care, and (5) hospital administration. Positive implications beyond improved care for acutely ill patients were: increased morale and empowerment among nurses, real-time redistribution of workload for nurses (reducing neglect of non-acutely ill patients during emergencies), and immediate access to expert help. Negative implications were: increased tensions between nurses and physician teams, a burden on hospitalist RRT members, and reduced autonomy for trainees. The RRT provides advantages that extend well beyond a reduction in rates of transfers to intensive care units or codes but are balanced by certain disadvantages. The potential impact from these multiple sources should be evaluated to understand the utility of any RRT programme.

  17. Republished: Defining impact of a rapid response team: qualitative study with nurses, physicians and hospital administrators.

    Science.gov (United States)

    Benin, Andrea L; Borgstrom, Christopher P; Jenq, Grace Y; Roumanis, Sarah A; Horwitz, Leora I

    2012-10-01

    The objective of this study was to qualitatively describe the impact of a Rapid Response Team (RRT) at a 944-bed, university-affiliated hospital. We analysed 49 open-ended interviews with administrators, primary team attending physicians, trainees, RRT attending hospitalists, staff nurses, nurses and respiratory technicians. Themes elicited were categorised into the domains of (1) morale and teamwork, (2) education, (3) workload, (4) patient care, and (5) hospital administration. Positive implications beyond improved care for acutely ill patients were: increased morale and empowerment among nurses, real-time redistribution of workload for nurses (reducing neglect of non-acutely ill patients during emergencies), and immediate access to expert help. Negative implications were: increased tensions between nurses and physician teams, a burden on hospitalist RRT members, and reduced autonomy for trainees. The RRT provides advantages that extend well beyond a reduction in rates of transfers to intensive care units or codes but are balanced by certain disadvantages. The potential impact from these multiple sources should be evaluated to understand the utility of any RRT programme.

  18. Periodontal and dental effects of surgically assisted rapid maxillary expansion, assessed by using digital study models

    Directory of Open Access Journals (Sweden)

    Danilo Furquim Siqueira

    2015-06-01

    Full Text Available OBJECTIVE: The present study assessed the maxillary dental arch changes produced by surgically assisted rapid maxillary expansion (SARME. METHODS: Dental casts from 18 patients (mean age of 23.3 years were obtained at treatment onset (T1, three months after SARME (T2 and 6 months after expansion (T3. The casts were scanned in a 3D scanner (D-250, 3Shape, Copenhagen, Denmark. Maxillary dental arch width, dental crown tipping and height were measured and assessed by ANOVA and Tukey's test. RESULTS: Increased transversal widths from T1 and T2 and the maintenance of these values from T2 and T3 were observed. Buccal teeth tipping also showed statistically significant differences, with an increase in all teeth from T1 to T2 and a decrease from T2 to T3. No statistically significant difference was found for dental crown height, except for left first and second molars, although clinically irrelevant. CONCLUSION: SARME proved to be an effective and stable procedure, with minimum periodontal hazards.

  19. Rapid Elimination of Blood Alcohol Using Erythrocytes: Mathematical Modeling and In Vitro Study

    Directory of Open Access Journals (Sweden)

    Yuliya G. Alexandrovich

    2017-01-01

    Full Text Available Erythrocytes (RBCs loaded with alcohol dehydrogenase (ADH and aldehyde dehydrogenase (ALD can metabolize plasma ethanol and acetaldehyde but with low efficiency. We investigated the rate-limiting factors in ethanol oxidation by these enzymes loaded into RBCs. Mathematical modeling and in vitro experiments on human RBCs loaded simultaneously with ADH and ALD (by hypoosmotic dialysis were performed. The simulation showed that the rate of nicotinamide-adenine dinucleotide (NAD+ generation in RBC glycolysis, but not the activities of the loaded enzymes, is the rate-limiting step in external ethanol oxidation. The rate of oxidation could be increased if RBCs are supplemented by NAD+ and pyruvate. Our experimental data verified this theoretical conclusion. RBCs loaded with the complete system of ADH, ALD, NAD+, and pyruvate metabolized ethanol 20–40 times faster than reported in previous studies. The one-step procedure of hypoosmotic dialysis is the optimal method to encapsulate ADH and ALD in RBCs after cell recovery, encapsulation yield, osmotic resistance, and RBC-indexes. Consequently, transfusion of the RBCs loaded with the complete metabolic system, including ADH, ALD, pyruvate, and NAD+ in the patients with alcohol intoxication, may be a promising method for rapid detoxification of blood alcohol based on metabolism.

  20. Melanin binding study of clinical drugs with cassette dosing and rapid equilibrium dialysis inserts.

    Science.gov (United States)

    Pelkonen, Laura; Tengvall-Unadike, Unni; Ruponen, Marika; Kidron, Heidi; Del Amo, Eva M; Reinisalo, Mika; Urtti, Arto

    2017-11-15

    Melanin pigment is a negatively charged polymer found in pigmented human tissues. In the eye, iris, ciliary body, choroid and retinal pigment epithelium (RPE) are heavily pigmented. Several drug molecules are known to bind to melanin, but larger sets of drugs have not been compared often in similar test conditions. In this study, we introduce a powerful tool for screening of melanin binding. The binding of a set of 34 compounds to isolated porcine RPE melanin was determined by cassette (n-in-one) dosing in rapid equilibrium dialysis inserts and the binding was quantitated with LC-MS/MS analytics. The compounds represented large variety in melanin binding (from 8.6%, ganciclovir) to over 95% bound (ampicillin and ciprofloxacin). The data provides information on melanin binding of small molecular weight compounds that are used for ocular (e.g. brinzolamide, ganciclovir) and systemic (e.g. tizanidine, indomethacin) therapy. Interestingly, competition among compounds was seen for melanin binding and the binding did not show any correlation with plasma protein binding. These results increase the understanding of melanin binding of ocular drugs and can be further exploited to predict pharmacokinetics in the eye. Pigment binding provides an interesting option for improved drug distribution to retina and choroid that are difficult target tissues in drug delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Studying the Technology of Creating Cortical Electrode Instruments using the Rapid Prototyping Technology

    Directory of Open Access Journals (Sweden)

    Ablyaz T. R.

    2017-06-01

    Full Text Available This paper shows the results of studying the technology of manufacturing cortical electrode-instruments (EI with the use of indirect methods of the Rapid Prototyping technology. Functional EI prototypes were made by layered synthesis of the photopolymer material with the use of the stereolithography technology (SLA - Stereo Lithography Apparatus. The article is focused on two methods of indirect EI manufacturing. One of the EI prototypes was used for making a molded wax model for hot investment casting, followed by applying copper coating. The second prototype was used for applying copper plating to a prepared current-conductive layer. As a result of EDMing a steel workpiece, both EIs reached the desired depth, which is 1 mm. The copper plating applied to the EI preserves its integrity. Through the use of the casting technology, there is a possibility to cut the economic costs by 35%. Using a prototype with preliminarily applied conductive coating makes it possible to make geometrically-complex EIs.

  2. Rapid assessment procedures in environmental sanitation research: a case study from the northern border of Mexico.

    Science.gov (United States)

    Cifuentes, Enrique; Alamo, Urinda; Kendall, Tamil; Brunkard, Joan; Scrimshaw, Susan

    2006-01-01

    There is a need to enhance the quality and sustainability of environmental health programs in Mexico. What socio-cultural factors influenced the adoption or rejection of Clean Water in Homes programs in this population? We applied rapid appraisal procedures (RAP) to evaluate these community-based programs. Qualitative study conducted in communities along Mexico's northern border. We conducted informal dialogues, semi-structured interviews, field notes and observations. Home visits used a checklist to observe: sources of water, handwashing, as well as human waste and garbage disposal patterns. Data analysis was conducted using ATLAS.ti, which facilitated comparison and illustration of discrepancies, the elaboration of emerging issues and relationships between them. Community members perceived that the Clean Water program was a top-down intervention. Water is perceived as a political issue and a matter of corruption. Inequity also limits solidarity activities involved in environmental sanitation. Migration to the United States of America (US) contributes to community fragmentation, which in turn dilutes communal efforts to improve water and sanitation infrastructure. While targeting women as program "recipients", the Clean Water program did not take gendered spheres of decision-making into account. Community members and authorities discussed the main results in "assemblies", particularly addressing the needs of excluded groups. The oversight of not exploring community members' needs and priorities prior to program implementation resulted in interventions that did not address the structural (economic, infrastructure) and socio-cultural barriers faced by community members to undertake the health-promoting behaviour change, and provoked resentment.

  3. Biosynthesis of intestinal microvillar proteins. The intracellular transport of aminopeptidase N and sucrase-isomaltase occurs at different rates pre-Golgi but at the same rate post-Golgi

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M

    1985-01-01

    in the microvillar fraction at a slower rate than aminopeptidase N. The relative pool sizes of mature and transient forms of both enzymes in intracellular membranes (Mg2+-precipitated fraction) were determined to obtain information on the relative time, spent pre- and post-Golgi, respectively, prior to microvillar...... expression. This ratio was 0.24 +/- 0.06 (mean +/- SD) for sucrase-isomaltase as compared to 0.40 +/- 0.04 (mean +/- SD) for aminopeptidase N. Considering the slower rate of pre-Golgi transport for sucrase-isomaltase, this indicates that the two microvillar enzymes have rather similar if not identical rates...

  4. 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.

  5. 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

    OpenAIRE

    Orlando Torres-Fernández

    2006-01-01

    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...

  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

    2016-03-09

    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.

  7. 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....

  8. A Study of Postural Loading in Malaysian Mining Industry using Rapid Entire Body Assessment

    Directory of Open Access Journals (Sweden)

    Norhidayah M.S.

    2016-01-01

    Full Text Available The ergonomics and environment factors have been the core issue for the mining industry for many years, and its profiles are rising. To ensure an ergonomics work environment, it is possible to require specific attention especially in this industries sector. It is becoming increasingly difficult to ignore the essential issue in Malaysia due to lack of ergonomics knowledge and low awareness among the engineers in the mining sector. The focus of this study is to evaluate and validate the physical risk factor associated with work-related musculoskeletal disorder (WMSDs by using Rapid Entire Body Assessment (REBA among mining industry workers. All the physical risk factors involved the main body regions such as upper arm, lower arm, wrist, trunk, neck and leg that has been identified associated with WMSDs. There were 18 subjects were selected to involve in this study. Those subjects were chosen according to their job task. To increase the reliability of the result, each subject was evaluated thrice in the trials. From the analysis, the average of final score of the REBA is 8.24 indicates high risk and calls for engineering/or work method changes to reduce or eliminate muscular disorder risk. The results of the analysis were used to improve the process of work, design of workstation and also improving the work posture to enhance the comfort level of operators. This study is crucial among the mining industry that is a lack of the information and research about the ergonomics issues in the industry. The overall finding indicated that the whole process of selected work task will contribute to musculoskeletal disorder either for a short or long time exposure.

  9. A validation study of reconstructed rapid prototyping models produced by two technologies.

    Science.gov (United States)

    Dietrich, Christian Andreas; Ender, Andreas; Baumgartner, Stefan; Mehl, Albert

    2017-09-01

    To determine the accuracy (trueness and precision) of two different rapid prototyping (RP) techniques for the physical reproduction of three-dimensional (3D) digital orthodontic study casts, a comparative assessment using two 3D STL files of two different maxillary dentitions (two cases) as a reference was accomplished. Five RP replicas per case were fabricated using both stereolithography (SLA) and the PolyJet system. The 20 reproduced casts were digitized with a highly accurate reference scanner, and surface superimpositions were performed. Precision was measured by superimposing the digitized replicas within each case with themselves. Superimposing the digitized replicas with the corresponding STL reference files assessed trueness. Statistical significance between the two tested RP procedures was evaluated with independent-sample t-tests (P < .05). The SLA and PolyJet replicas showed statistically significant differences for trueness and precision. The precision of both tested RP systems was high, with mean deviations in stereolithographic models of 23 (±6) μm and in PolyJet replicas of 46 (±13) μm. The mean deviation for trueness in stereolithographic replicas was 109 (±4) μm, while in PolyJet replicas, it was 66 (±14) μm. Comparing the STL reference files, the PolyJet replicas showed higher trueness than the SLA models. But the precision measurements favored the SLA technique. The dimensional errors observed in this study were a maximum of 127 μm. In the present study, both types of reproduced digital orthodontic models are suitable for diagnostics and treatment planning.

  10. The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity

    Science.gov (United States)

    Hwang, Gi-Wook; Murai, Yasutaka; Takahashi, Tsutomu; Naganuma, Akira

    2014-07-01

    Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

  11. Dendritic and spinal pathology in the acoustic cortex in Alzheimer's disease: morphological estimation in Golgi technique and electron microscopy.

    Science.gov (United States)

    Baloyannis, Stavros J; Manolides, Spyros L; Manolides, Leonidas S

    2011-06-01

    The morphological and morphometric estimation of the dendrites and the dendritic spines in the acoustic cortex in Alzheimer's disease revealed substantial alterations of the dendritic arborization and marked loss of the dendritic spines, which may be related to communication impairment even in early cases of Alzheimer's disease. Alzheimer's disease is characterized by progressive loss of memory, impairment of judgment, and decline in communication and speech eloquence. In the present study we attempted to describe the morphological and morphometric alterations of the dendrites and the dendritic spines in the acoustic cortex in early cases of Alzheimer's disease, in order to approach the communication impairment of patients suffering from Alzheimer's disease from a neuropathological point of view. We studied the acoustic cortex in 22 cases of Alzheimer's disease by Golgi technique and electron microscopy. The morphological and morphometric estimation of the acoustic cortex revealed loss of Cajal-Retzius cells in layer I, as well as an impressive abbreviation of the dendritic fields associated with loss of dendritic spines in all the layers of the cortex. Numerous distorted, dystrophic, and degenerated dendritic spines were also seen, which were intermixed with a considerable number of giant spines. The dendritic and spinal alterations were closely associated with mitochondrial alterations.

  12. The Qb-SNARE Memb11 interacts specifically with Arf1 in the Golgi apparatus of Arabidopsis thaliana.

    Science.gov (United States)

    Marais, Claireline; Wattelet-Boyer, Valérie; Bouyssou, Guillaume; Hocquellet, Agnès; Dupuy, Jean-William; Batailler, Brigitte; Brocard, Lysiane; Boutté, Yohann; Maneta-Peyret, Lilly; Moreau, Patrick

    2015-11-01

    The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are critical for the function of the secretory pathway. The SNARE Memb11 is involved in membrane trafficking at the ER-Golgi interface. The aim of the work was to decipher molecular mechanisms acting in Memb11-mediated ER-Golgi traffic. In mammalian cells, the orthologue of Memb11 (membrin) is potentially involved in the recruitment of the GTPase Arf1 at the Golgi membrane. However molecular mechanisms associated to Memb11 remain unknown in plants. Memb11 was detected mainly at the cis-Golgi and co-immunoprecipitated with Arf1, suggesting that Arf1 may interact with Memb11. This interaction of Memb11 with Arf1 at the Golgi was confirmed by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was found to be specific to Memb11 as compared to either Memb12 or Sec22. Using a structural bioinformatic approach, several sequences in the N-ter part of Memb11 were hypothesized to be critical for this interaction and were tested by BiFC on corresponding mutants. Finally, by using both in vitro and in vivo approaches, we determined that only the GDP-bound form of Arf1 interacts with Memb11. Together, our results indicate that Memb11 interacts with the GDP-bound form of Arf1 in the Golgi apparatus. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  13. Assessment of the diagnostic value of a urinary adipsin rapid strip test for pre-eclampsia: A prospective multicenter study.

    Science.gov (United States)

    Peng, Bing; Zhang, Li; Yan, Jianying; Qi, Hongbo; Zhang, Weiyuan; Fan, Ling; Hu, Yayi; Lin, Li; Li, Xiaotian; Hu, Rong; Xie, Lan; Zhang, Jianping; Wu, Yanqiao; Li, Li; Zhou, Rong

    2017-01-01

    The purpose of the present study was to evaluate the clinical value of the rapid strip test of urinary adipsin for the quick diagnosis of pre-eclampsia. In a multicenter diagnostic test study, we studied the diagnostic accuracy of the rapid strip test of urinary adipsin in women presenting with pre-eclampsia. A total of 204 pre-eclampsia patients and 254 healthy pregnant women were recruited for this study, respectively. The rapid strip test of urinary adipsin was used to detect the adipsin in the urine of each patient. The diagnostic value of the rapid strip test of urinary adipsin for pre-eclampsia was demonstrated by its high sensitivity and specificity (95.10% and 97.64%, respectively). The diagnostic accuracy was 96.51%. The consistency analysis showed that the kappa value was 0.93 compared with the gold standard diagnosis of pre-eclampsia. The rapid strip test of urinary adipsin is a non-invasive test for the diagnosis of pre-eclampsia with high sensitivity and specificity. It could help the quick diagnosis of pre-eclampsia in clinical practice greatly. © 2016 Japan Society of Obstetrics and Gynecology.

  14. Rapid Syphilis Tests as Catalysts for Health Systems Strengthening: A Case Study from Peru.

    Science.gov (United States)

    García, Patricia J; Cárcamo, César P; Chiappe, Marina; Valderrama, Maria; La Rosa, Sayda; Holmes, King K; Mabey, David C W; Peeling, Rosanna W

    2013-01-01

    Untreated maternal syphilis leads to adverse pregnancy outcomes. The use of point of care tests (POCT) offers an opportunity to improve screening coverage for syphilis and other aspects of health systems. Our objective is to present the experience of the introduction of POCT for syphilis in Peru and describe how new technology can catalyze health system strengthening. The study was implemented from September 2009-November 2010 to assess the feasibility of the use of a POCT for syphilis for screening pregnant women in Lima, Peru. Outcomes measured included access to syphilis screening, treatment coverage, partner treatment, effect on patient flow and service efficiency, acceptability among providers and patients, and sustainability. Before the introduction of POCT, a pregnant woman needed 6 visits to the health center in 27 days before she received her syphilis result. We trained 604 health providers and implemented the POCT for syphilis as the "two for one strategy", offering with one finger stick both syphilis and HIV testing. Implementation of the POCT resulted in testing and treatment on the first visit. Screening and treatment coverages for syphilis improved significantly compared with the previous year. Implementation of POCT has been scaled up nationally since the study ended, and coverages for screening, treatment and partner treatment have remained over 92%. Implementation of POCT for syphilis proved feasible and acceptable, and led to improvement in several aspects of health services. For the process to be effective we highlight the importance of: (1) engaging the authorities; (2) dissipating tensions between providers and identifying champions; (3) training according to the needs; (4) providing monitoring, supervision, support and recognition; (5) sharing results and discussing actions together; (6) consulting and obtaining feedback from users; and (7) integrating with other services such as with rapid HIV testing.

  15. Golgi fragmentation in pmn mice is due to a defective ARF1/TBCE cross-talk that coordinates COPI vesicle formation and tubulin polymerization

    NARCIS (Netherlands)

    Bellouze, Sarah; Schäfer, Michael K; Buttigieg, Dorothée; Baillat, Gilbert; Rabouille, Catherine; Haase, Georg

    2014-01-01

    Golgi fragmentation is an early hallmark of many neurodegenerative diseases but its pathophysiological relevance and molecular mechanisms are unclear. We here demonstrate severe and progressive Golgi fragmentation in motor neurons of progressive motor neuronopathy (pmn) mice due to loss of the

  16. 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.

  17. El citoesqueleto de espectrina y el complejo de Golgi. Implicaciones en su arquitectura y funcionalidad en el transporte secretor

    OpenAIRE

    Salcedo Sicilia, Laia

    2012-01-01

    [spa] El complejo de Golgi (Golgi) es un orgánulo dinámico que modifica proteínas y lípidos sintetizados en el retículo endoplasmático (RE) y los clasifica para enviarlos a su destino final. Está formado por un conjunto de cisternas aplanadas y apiladas (stack), con una región central plana y otra lateral dilatada. Cada stack está polarizado, con una cara cis, que es donde se recibe la carga sintetizada, y una cara trans, que es donde se le da salida. Adyacente a esta zona hay la red trans-Go...

  18. Phosphatidylinositol 4-kinase III-beta is required for Golgi maintenance and cytokinesis in Trypanosoma brucei.

    Science.gov (United States)

    Rodgers, Melissa J; Albanesi, Joseph P; Phillips, Margaret A

    2007-07-01

    The parasitic protozoan Trypanosoma brucei contains two type III phosphatidylinositol 4-kinases (alpha and beta). We have cloned the gene encoding the T. brucei type III phosphatidylinositol 4-kinase beta (TbPI4KIII-beta), expressed the protein in COS-7 cells, and confirmed that the protein catalyzes the phosphorylation of phosphatidylinositol. Depletion of TbPI4KIII-beta in procyclic T. brucei by RNA interference (RNAi) resulted in inhibition of cell growth and a distorted cellular morphology. RNAi cells had a distorted Golgi apparatus, and lysosomal and flagellar pocket proteins were mislocalized. Ultrastructural analysis revealed the internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles, and a loss of cell polarity. Scanning electron microcopy revealed a twisted phenotype, and dividing cells often exhibited a detached daughter flagellum and lacked a cleavage furrow. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-beta have a postmitotic cytokinesis block that occurs after a single round of mitosis, suggestive of a specific cell cycle block. In summary, TbPI4KIII-beta is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis.

  19. CK2 phosphorylates Sec31 and regulates ER-To-Golgi trafficking.

    Directory of Open Access Journals (Sweden)

    Mayuko Koreishi

    Full Text Available Protein export from the endoplasmic reticulum (ER is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking.

  20. A functional splice variant of the human Golgi CMP-sialic acid transporter.

    Science.gov (United States)

    Salinas-Marín, Roberta; Mollicone, Rosella; Martínez-Duncker, Iván

    2016-12-01

    The human Golgi Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Sia) transporter SLC35A1, a member of the nucleotide sugar transporter family, translocates CMP-Sia from the cytosol into the Golgi lumen where sialyltransferases use it as donor substrate for the synthesis of sialoglycoconjugates. In 2005, we reported a novel Congenital Disorder of Glycosylation (CDG) termed CDG-IIf or SLC35A1-CDG, characterized by macrothrombocytopenia, neutropenia and complete lack of the sialyl-Lex antigen (NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc-R) on polymorphonuclear cells. This disease was caused by the presence of inactive SLC35A1 alleles. It was also found that the SLC35A1 generates additional isoforms through alternative splicing. In this work, we demonstrate that one of the reported isoforms, the del177 with exon 6 skipping, is able to maintain sialylation in HepG2 cells submitted to wt knockdown and restore sialylation to normal levels in the Chinese Hamester Ovary (CHO) cell line Lec2 mutant deficient in CMP-Sia transport. The characteristics of the alternatively spliced protein are discussed as well as therapeutic implications of this finding in CDGs caused by mutations in nucleotide sugar transporters (NSTs).

  1. Crystallographic analysis of murine p24γ2 Golgi dynamics domain.

    Science.gov (United States)

    Nagae, Masamichi; Liebschner, Dorothee; Yamada, Yusuke; Morita-Matsumoto, Kana; Matsugaki, Naohiro; Senda, Toshiya; Fujita, Morihisa; Kinoshita, Taroh; Yamaguchi, Yoshiki

    2017-04-01

    The p24 family proteins form homo- and hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. It consists of four subfamilies (p24α, p24β, p24γ, and p24δ). p24γ2 plays crucial roles in the selective transport of glycosylphosphatidylinositol-anchored proteins. Here, we determined the crystal structure of mouse p24γ2 Golgi dynamics (GOLD) domain at 2.8 Å resolution by the single anomalous diffraction method using intrinsic sulfur atoms. In spite of low sequence identity among p24 family proteins, p24γ2 GOLD domain assumes a β-sandwich fold, similar to that of p24β1 or p24δ1. An additional short α-helix is observed at the C-terminus of the p24γ2 GOLD domain. Intriguingly, p24γ2 GOLD domains crystallize as dimers, and dimer formation seems assisted by the short α-helix. Dimerization modes of GOLD domains are compared among p24 family proteins. Proteins 2017; 85:764-770. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Golgi-like staining of visual cortex cells obtained by extracellular biocytin application in vitro.

    Science.gov (United States)

    Kenan-Vaknin, G; Katz, H; Malach, R

    1992-02-07

    We report here the application of biocytin (a biotin-lysine complex) as an extracellular tracer in vitro. Biocytin was applied extracellularly, revealing Golgi-like staining of cells in the adult in vitro rat visual cortex. Micropipettes were filled with a solution of 2.3-2.6% biocytin dissolved in 0.05 M Tris buffer, pH 7.4. Biocytin was applied by one of 3 methods: diffusion, pressure injection or drop application. Cell bodies and dendrites around the application site and their efferent axonal processes were stained; dendritic spines were often visible. The injection sites varied in size from a single cell to a diameter of 400 microns. When applied in layer I-III, few filled cells were also seen in layers IV and V, outside the application site. The drop application (5-10 microliters) of biocytin resulted in filling of cells throughout the cortex. The combination of biocytin and the slice preparation was found to be very useful in revealing cell morphology and tracing interlaminar connections in the visual cortex. The advantages of this technique are its ease of application, the precise and restricted injection sites, and Golgi-like morphological detail.

  3. Association between microtubules and Golgi vesicles isolated from rat parotid glands.

    Science.gov (United States)

    Coffe, G; Raymond, M N

    1990-01-01

    We report an isolation procedure of trans-Golgi vesicles (GVs) from rat parotid glands. Various organelle markers were used, particularly galactosyl transferase as a trans-Golgi marker, to test the purity of the GV fraction. A quantitative in vitro binding assay between microtubules and GVs is described. The vesicles were incubated with taxol-induced microtubules, layered between 50% and 43% sucrose cushions and subjected to centrifugation. Unlike free microtubules which were sedimented, the GV-bound microtubules co-migrated upward with GVs. Quantification of these bound microtubules was carried out by densitometric scanning of Coomassie blue-stained gels. The association between microtubules and GVs followed a saturation curve, with a plateau value of 20 micrograms of microtubule protein bound to 500 micrograms of GV fraction. The half-saturation of the GV sites was obtained with a microtubule concentration of 20 micrograms/ml. Electron microscopy of negatively stained re-floated material showed numerous microtubule-vesicle complexes. Coating of microtubules with an excess of brain microtubule-associated proteins (MAPs) abolished binding. In the absence of exogenous microtubules, we showed that the GV fraction was already interacting with a class of endogenous rat parotid microtubules. This class of colcemid and cold-stable microtubules represents 10-20% of the total tubulin content of the parotid cell.

  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. Rapid eye movement sleep behavior disorder in Parkinson's disease: magnetic resonance imaging study.

    Science.gov (United States)

    Ford, Andrew H; Duncan, Gordon W; Firbank, Michael J; Yarnall, Alison J; Khoo, Tien K; Burn, David J; O'Brien, John T

    2013-06-01

    Rapid eye movement sleep behavior disorder has poor prognostic implications for Parkinson's disease. The authors recruited 124 patients with early Parkinson's disease to compare clinical and neuroimaging findings based on the presence of this sleep disorder. The presence of rapid eye movement sleep behavior disorder was assessed with the Mayo Sleep Questionnaire. Magnetic resonance imaging sequences were obtained for voxel-based morphometry and diffusion tensor imaging. Patients with sleep disorder had more advanced disease, but groups had similar clinical characteristics and cognitive performance. Those with sleep disorder had areas of reduced cortical grey matter volume and white matter changes compared with those who did not have sleep disorder. However, differences were slight and were not significant when the analyses were adjusted for multiple comparisons. Rapid eye movement sleep behavior disorder was associated with subtle changes in white matter integrity and grey matter volume in patients with early Parkinson's disease. Copyright © 2013 Movement Disorder Society.

  6. A Pilot Study of Rapid Hepatitis C Testing in Probation and Parole Populations in Rhode Island.

    Science.gov (United States)

    Zaller, Nickolas D; Patry, Emily J; Bazerman, Lauri B; Noska, Amanda; Kuo, Irene; Kurth, Ann; Beckwith, Curt G

    2016-01-01

    Hepatitis C virus (HCV) affects between five and seven million individuals in the United States and chronic infection can lead to liver disease, cirrhosis, and hepatocellular carcinoma. Probation/parole offices are a novel setting for rapid HCV testing, providing outreach to populations at increased risk for HCV infection and/or transmitting HCV to others. While some correctional facilities offer HCV testing, many individuals who present to probation/parole offices are never or briefly incarcerated and may not access medical services. We conducted a rapid HCV testing pilot at probation/parole offices in Rhode Island. Overall, 130 people accepted rapid HCV testing, of whom 12 had reactive tests. Only four of these individuals presented to a community-based clinic for confirmatory testing, despite being offered a monetary incentive. Identifying and addressing barriers to HCV confirmatory testing and follow-up care is critical to increasing the uptake of HCV care and treatment in this vulnerable population.

  7. A sensitive three monoclonal antibodies based automatic latex particle-enhanced turbidimetric immunoassay for Golgi protein 73 detection

    Science.gov (United States)

    Xia, Yanyan; Shen, Han; Zhu, Yefei; Xu, Hongpan; Li, Zhiyang; Si, Jin

    2017-01-01

    Golgi protein 73 (GP73) is a novel and potential marker for diagnosing hepatocellular carcinoma (HCC) that has been found to be abnormally elevated in liver disease. A latex particle-enhanced turbidimetric immunoassay (LTIA) was recently introduced and licensed for application in a variety of automated clinical chemistry analyzers. However, no studies have reported sufficient data on analytical performance of this method when using 3 monoclonal antibodies for GP73 measurement. The experimental conditions were firstly optimized and range of linearity, diagnostic potential, clinical relevance were compared with the LTIA based on polyclonal antibodies and ELISA. Dilution tests for the LTIA using 3 monoclonal antibodies produced a calibration curve from 10 to 350 ng/mL while the polyclonal antibodies produced the curve from 20 to 320 ng/mL. The detection limit was achieved at 1.82 ng/mL concentration. Within-run CV was obtained in the range of 1.5–2.9% and ROC curves indicated sensitivity and specificity of the LTIA based on 3 monoclonal antibodies were 96.7% and 93.3%, respectively, higher than for the polyclonal antibodies (94.6% and 72.4%) and ELISA (70.0% and 83.3%). Therefore, the LTIA assay based on 3 monoclonal antibodies is thus applicable in quantification of GP73 concentration in automated biochemistry analyzers. PMID:28054632

  8. A rapid assay for on-site monitoring of infliximab trough levels: a feasibility study.

    Science.gov (United States)

    Corstjens, Paul L A M; Fidder, Herma H; Wiesmeijer, Karien C; de Dood, Claudia J; Rispens, Theo; Wolbink, Gert-Jan; Hommes, Daniel W; Tanke, Hans J

    2013-09-01

    Monitoring levels of biologicals against tumor necrosis factor (TNF) has been suggested to improve therapeutic outcomes in inflammatory bowel diseases (IBDs). This pilot study describes a rapid lateral flow (LF)-based assay for on-site monitoring of serum trough levels of humanized monoclonal antibody infliximab (IFX). The applied chromatographic method utilizes sequential flows of diluted serum, wash buffer, and an immunoglobulin generic label on LF strips with a Test line comprised of TNF-α. The successive flows permitted enrichment of IFX at the Test line before the label was applied. The label, luminescent upconverting phosphor (UCP) particles coated with protein-A, emits a 550-nm visible light upon excitation with 980-nm infrared light. IFX concentrations were determined through measurement of UCP fluorescence at the Test line. The assay was optimized to detect IFX levels as low as 0.17 μg/mL in serum. For patients with IBD, this limit is appropriate to detect levels associated with loss of response (0.5 μg IFX/mL). The assay was evaluated with clinical samples from patients with Crohn's disease and correlated well within the physiologically relevant range from 0.17 to 10 μg/mL with an IFX-specific ELISA. Performance of the assay was further successfully validated with samples from blood donors, IFX negative IBD patients, and rheumatoid arthritis patients that had developed anti-IFX antibodies. Because of its generic nature, the assay is suited for detecting most therapeutic anti-TNF-α monoclonal antibodies.

  9. Rapid eye movement-sleep is reduced in patients with acute uncomplicated diverticulitis—an observational study

    DEFF Research Database (Denmark)

    Huang, Chenxi; Alamili, Mahdi; Nielsen, Claus Henrik

    2015-01-01

    responses are believed to cause postoperative sleep disturbances, as inflammatory responses can alter sleep architecture through cytokine-brain interactions. Our aim was to investigate alteration of sleep architecture during acute infection and its relationships to inflammation and clinical symptoms....... Materials & Methods. In this observational study, we included patients with acute uncomplicated diverticulitis as a model to investigate the isolated effects of inflammatory responses on sleep. Eleven patients completed the study. Patients were admitted and treated with antibiotics for two nights, during...... was reduced 4% and 7% the first (p = 0.006) and second (p = 0.014) nights of diverticulitis, compared to baseline, respectively. The rapid eye movement sleep was reduced 33% the first night (p = 0.016), compared to baseline. Moreover, plasma IL-6 levels were correlated to non-rapid eye movement sleep, rapid...

  10. Dental arch changes associated with rapid maxillary expansion: A retrospective model analysis study

    Directory of Open Access Journals (Sweden)

    Ivor M D′Souza

    2015-01-01

    Full Text Available Introduction: Transverse deficiency of the maxilla is a common clinical problem in orthodontics and dentofacial orthopedics. Transverse maxillary deficiency, isolated or associated with other dentofacial deformities, results in esthetic and functional impairment giving rise to several clinical manifestations such as asymmetrical facial growth, positional and functional mandibular deviations, altered dentofacial esthetics, adverse periodontal responses, unstable dental tipping, and other functional problems. Orthopedic maxillary expansion is the preferred treatment approach to increase the maxillary transverse dimension in young patients by splitting of the mid palatal suture. This orthopedic procedure has lately been subject of renewed interest in orthodontic treatment mechanics because of its potential for increasing arch perimeter to alleviate crowding in the maxillary arch without adversely affecting facial profile. Hence, the present investigation was conducted to establish a correlation between transverse expansion and changes in the arch perimeter, arch width and arch length. Methods: For this purpose, 10 subjects (five males, five females were selected who had been treated by rapid maxillary expansion (RME using hyrax rapid palatal expander followed by fixed mechanotherapy (PEA. Pretreatment (T1, postexpansion (T2, and posttreatment (T3 dental models were compared for dental changes brought about by RME treatment and its stability at the end of fixed mechanotherapy. After model measurements were made, the changes between T1-T2, T2-T3 and T1-T3 were determined for each patient. The mean difference between T1-T2, T2-T3 and T1-T3 were compared to assess the effects of RME on dental arch measurements. Results are expressed as mean ± standard deviation and are compared by repeated measures analysis of variance followed by a post-hoc test. Arch perimeter changes are correlated with changes in arch widths at the canine, premolar and molar

  11. The rapid FEV(1) decline in chronic obstructive pulmonary disease is associated with predominant emphysema: a longitudinal study.

    Science.gov (United States)

    Cerveri, Isa; Corsico, Angelo G; Grosso, Amelia; Albicini, Federica; Ronzoni, Vanessa; Tripon, Bianca; Imberti, Federica; Galasso, Thomas; Klersy, Catherine; Luisetti, Maurizio; Pistolesi, Massimo

    2013-02-01

    Early identification of patients with COPD and prone to more rapid decline in lung function is of particular interest from both a prognostic and therapeutic point of view. The aim of this study was to identify the clinical, functional and imaging characteristics associated with the rapid FEV(1) decline in COPD. Between 2001 and 2005, 131 outpatients with moderate COPD in stable condition under maximum inhaled therapy underwent clinical interview, pulmonary function tests and HRCT imaging of the chest and were followed for at least 3 years. Twenty-six percent of patients had emphysema detected visually using HRCT. The FEV(1) decline was 42 ± 66 mL/y in the total sample, 88 ± 76 mL/y among rapid decliners and 6 ± 54 mL/y among the other patients. In the univariable analysis, the decline of FEV(1) was positively associated with pack-years (p emphysema at HRCT (p emphysema proved to be an independent prognostic factor of rapid decline (p = 0.001). When emphysema was replaced by RV, the model still remained significant. The rapid decline in lung function may be identified by the presence of emphysema at HRCT or increased RV in patients with a long smoking history.

  12. Long-Term Follow-up Investigation of Isolated Rapid Eye Movement Sleep Without Atonia Without Rapid Eye Movement Sleep Behavior Disorder: A Pilot Study.

    Science.gov (United States)

    Stefani, Ambra; Gabelia, David; Högl, Birgit; Mitterling, Thomas; Mahlknecht, Philipp; Stockner, Heike; Poewe, Werner; Frauscher, Birgit

    2015-11-15

    Idiopathic rapid eye movement (REM) sleep behavior disorder (RBD) is a harbinger of synuclein-mediated neurodegenerative diseases. It is unknown if this also applies to isolated REM sleep without atonia (RWA). We performed a long-term follow-up investigation of subjects with isolated RWA. Participants were recruited from 50 subjects with isolated RWA who were identified at the sleep laboratory of the Department of Neurology at the Medical University of Innsbruck between 2003 and 2005. Eligible subjects underwent follow-up clinical examination, polysomnography, and assessment of neurodegenerative biomarkers (cognitive impairment, finger speed deficit, impaired color vision, olfactory dysfunction, orthostatic hypotension, and substantia nigra hyperechogenicity). After a mean of 8.6 ± 0.9 y, 1 of 14 participating subjects (7.3%) progressed to RBD. Ten of 14 RWA subjects (71.4%) were positive for at least one neurodegenerative biomarker. Substantia nigra hyperechogenicity and presence of mild cognitive impairment were both present in 4 of 14 subjects with isolated RWA. Electromyographic activity measures increased significantly from baseline to follow-up polysomnography ("any" mentalis and both anterior tibialis muscles: 32.5 ± 9.4 versus 52.2 ± 16.6%; p = 0.004). This study provides first evidence that isolated RWA is an early biomarker of synuclein-mediated neurodegeneration. These results will have to be replicated in larger studies with longer observational periods. If confirmed, these disease findings have implications for defining at-risk cohorts for Parkinson disease. © 2015 American Academy of Sleep Medicine.

  13. Validation Study of Rapid Assays of Bioburden, Endotoxins and Other Contamination.

    Science.gov (United States)

    Shintani, Hideharu

    2016-01-01

    Microbial testing performed in support of pharmaceutical and biopharmaceutical production falls into three main categories: detection (qualitative), enumeration (quantitative), and characterization/identification. Traditional microbiological methods are listed in the compendia and discussed by using the conventional growth-based techniques, which are labor intensive and time consuming. In general, such tests require several days of incubation for microbial contamination (bioburden) to be detected, and therefore management seldom is able to take proactive corrective measures. In addition, microbial growth is limited by the growth medium used and incubation conditions, thus impacting testing sensitivity, accuracy, and reproducibility.  For more than 20 years various technology platforms for rapid microbiological methods (RMM) have been developed, and many have been readily adopted by the food industry and clinical microbiology laboratories. Their use would certainly offer drug companies faster test turnaround times to accommodate the aggressive deadlines for manufacturing processes and product release. Some rapid methods also offer the possibility for real-time microbial analyses, enabling management to respond to microbial contamination events in a more timely fashion, and can provide cost savings and higher efficiencies in quality control testing laboratories. Despite the many proven business and quality benefits and the fact that the FDA's initiative to promote the use of process analytical technology (PAT) includes rapid microbial methods, pharmaceutical and biopharmaceutical industries have been somewhat slow to embrace alternative microbial methodologies for several reasons. The major reason is that the bioburden counts detected by the incubation method and rapid assay are greatly divergent.  The use of rapid methods is a dynamic field in applied microbiology and one that has gained increased attention nationally and internationally over time. This topic

  14. A Rapid Deployment Seismological network (RaDeSeis) for real time aftershock studies

    Science.gov (United States)

    Hloupis, G.; Vallianatos, F.; Makris, J. P.

    2009-04-01

    The understanding of earthquake faulting process is one of the main factors that contribute to earthquake damage. One of the most valuable and essential tools for the understanding of faulting process in the analysis of aftershocks. The critical point for successful aftershock studies is the mobile seismological network that will deployed in order to provide the required data. The main problem that arise for these networks is how fast the recorded data are available to data centres in order to estimate the focal mechanisms, the source parameters estimation as well as to examine microseismic activity. The ideal situation is to have these data available in real time but this is limited by the different telemetry requirements for every individual installation. Based on the experience gained from several installations in Hellenic Seismological Network of Crete (HSNC) we propose a mobile network scheme (called RaDeSeis) capable of installed in a limited amount of time and provide real time seismological data. RaDeSeis is an hybrid network based on VSAT and WiFi communication links between seismological stations and data centre. The network is deployed in star topology where the central station is the communication hub at the same time. Dedicated point-to-point links between central station and border station established using WiFi links. Communication between central station and data centre is established by VSAT. With appropriate routing on central station the data centre is collecting, control and monitor all the stations from the area of interest in real time. In order to decrease the time needed for each installation a specific software (RaLiEs - Rapid Link Establishment) is originated for the quicker link establishment between border stations and central station (with an average distance of 40km LOS) as well as to data centre. By using this software each telecommunication installation needs less than half an hour to complete the necessary link adjustments

  15. Rapid maxillary expansion screws on the test bench--a pilot study.

    Science.gov (United States)

    Muchitsch, Alfred Peter; Wendl, B; Winsauer, H; Pichelmayer, M; Payer, M

    2011-06-01

    In order to apply high, short-term forces during rapid maxillary expansion (RME) to the sutures of the maxilla with minimum loss of force and without causing unwanted side-effects (dentoalveolar tipping, etc.), the appliance should be as rigid as possible. The retention arms of the RME screws, representing a particularly vulnerable and stressed weak point of RME appliances, were the focus of this laboratory technical study. Retention arms of 16 types of RME screws comprising four arms and one with eight arms were examined using a three-point bending test. According to their ability to absorb the applied bending loads, the screws were classified in product groups from 1 (highest) to 6 (lowest). Fifteen of the tested retention arms (stainless steel), despite having the same diameter (1.48-1.49 mm), differed up to 69.81 per cent between the highest (288.0 N) and lowest (169.6 N) maximum force parameters and up to 66.40 per cent between the highest (3325.9 N/mm(2)) and lowest (1998.7 N/mm(2)) maximum bending stress parameters. Due to optimum formability, though reduced rigidity, a titanium screw for nickel-sensitive patients (group 6) displayed the lowest force and bending tension values. The stainless steel double arms of the eight-arm screw device welded on both ends displayed the highest force data. The mean ductilities of the groups with the most and least rigid single steel arms differed by 22.77 per cent. Statistical analysis using the Pearson correlation coefficient revealed a significant indirect correlation between ductility and both maximum force (r = -0.780, P < 0.001) and maximum bending stress (r = -0.778, P < 0.001). The SUPERscrews, the Tiger Dental four-arm screw (group 1), and the eight-arm screw displayed the highest capacity to absorb an applied bending load. The screws in groups 3-6 appear acceptable for RME during the pre-pubertal period, whereas in the pubertal and post-pubertal period, groups 1 and 2 are sufficient. In early adulthood only the

  16. Potential of short-column liquid chromatographia with tandem mass spectrometric detection for the rapid study of pesticide degradation

    NARCIS (Netherlands)

    Hogenboom, A.C.; Steen, R.J.C.A.; Niessen, W.M.A.; Brinkman, U.A.T.

    1998-01-01

    The applicability of solid-phase extraction-LC using two short columns (SPE-LC) and/or single-short-column liquid chromatography (SSC) combined on- line with tandem mass spectrometry (MS) was demonstrated for the rapid study of pesticide degradation. A fast analytical procedure was developed to

  17. EXCIMER-LASER ABLATION OF SOFT-TISSUE - A STUDY OF THE CONTENT OF RAPIDLY EXPANDING AND COLLAPSING BUBBLES

    NARCIS (Netherlands)

    van Leeuwen, T. G.; Jansen, E. D.; Motamedi, M.; Welch, A. J.; Borst, C.

    1994-01-01

    Both holmium (lambda = 2.09 mum) and excimer (lambda = 308 nm) lasers are used for ablation of tissue. In a previous study, excimer laser ablation of aorta produced rapidly expanding and collapsing vapor bubbles. To investigate whether the excimer-induced bubble is caused by vaporization of (tissue)

  18. Pilot plant study of alternative filter media for rapid gravity filtration.

    Science.gov (United States)

    Davies, P D; Wheatley, A D

    2012-01-01

    Sand has been the main filter media used in rapid gravity filtration since its introduction. The dominance of sand has been due to its low cost and availability. Extensive experience has led to sand filters with a dependable and predictable performance. Sand remains the preferred filter medium but usually with a larger sized anthracite capping to reduce the onset of head loss. Other approved filter media are now commercially available and this paper compares sand with recycled glass, Filtralite(®) and slate at pilot scale. The results have reaffirmed the basic importance of particle size on head loss and turbidity performance rather than surface activity or specific surface area. The results did suggest, however, that particle shape and packing exerted a stronger influence on performance than previously acknowledged. These could be used to improve the design and the contribution to sustainability made by rapid gravity filters.

  19. Toxoplasma gondii Syntaxin 6 Is Required for Vesicular Transport Between Endosomal-Like Compartments and the Golgi Complex

    Science.gov (United States)

    Jackson, Allison J; Clucas, Caroline; Mamczur, Nicola J; Ferguson, David J; Meissner, Markus

    2013-01-01

    Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post-Golgi and it has been speculated that they pass through endosomal-like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well-established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant-like secretory pathway. PMID:23962112

  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. Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells.

    Science.gov (United States)

    Lajevardipour, Alireza; Chon, James W M; Chattopadhyay, Amitabha; Clayton, Andrew H A

    2016-11-22

    Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

  2. Saccharomyces cerevisiae depend on vesicular traffic between Golgi and vacuole when Inositolphosphorylceramide synthase Aur1 is inactivated

    DEFF Research Database (Denmark)

    Voynova, Natalia S; Roubaty, Carole; Vazquez, Hector M

    2015-01-01

    that vesicle mediated transport between Golgi, endosomes and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and Quinacrine uptake into vacuoles shows that Ab...

  3. Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

    Science.gov (United States)

    Lajevardipour, Alireza; Chon, James W. M.; Chattopadhyay, Amitabha; Clayton, Andrew H. A.

    2016-11-01

    Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

  4. 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.

  5. Organelle-cytoskeleton relationships in fibroblasts: mitochondria, Golgi apparatus, and endoplasmic reticulum in phases of movement and growth

    DEFF Research Database (Denmark)

    Couchman, J R; Rees, D A

    1982-01-01

    by the actions of both colchicine and dihydrocytochalasin B showing that orientation and translocation depend on a co-ordinate interaction of microtubules and microfilamentous meshwork around the centrioles as origin. The Golgi apparatus and endoplasmic reticulum do not rearrange dramatically during...

  6. Feasibility Study of Carbon Nanotube Microneedles for Rapid Transdermal Drug Delivery

    OpenAIRE

    Lyon, Bradley J.; Aria, Adrianus I.; Gharib, Morteza

    2013-01-01

    We introduce a new approach for fabricating hollow microneedles using vertically-aligned carbon nanotubes (VA-CNTs) for rapid transdermal drug delivery. Here, we discuss the fabrication of the microneedles emphasizing the overall simplicity and flexibility of the method to allow for potential industrial application. By capitalizing on the nanoporosity of the CNT bundles, uncured polymer can be wicked into the needles ultimately creating a high strength composite of aligned nanotubes and polym...

  7. Rapid assessment of visual impairment (RAVI in marine fishing communities in South India - study protocol and main findings

    Directory of Open Access Journals (Sweden)

    Madala Sreenivas R

    2011-09-01

    Full Text Available Abstract Background Reliable data are a pre-requisite for planning eye care services. Though conventional cross sectional studies provide reliable information, they are resource intensive. A novel rapid assessment method was used to investigate the prevalence and causes of visual impairment and presbyopia in subjects aged 40 years and older. This paper describes the detailed methodology and study procedures of Rapid Assessment of Visual Impairment (RAVI project. Methods A population-based cross-sectional study was conducted using cluster random sampling in the coastal region of Prakasam district of Andhra Pradesh in India, predominantly inhabited by fishing communities. Unaided, aided and pinhole visual acuity (VA was assessed using a Snellen chart at a distance of 6 meters. The VA was re-assessed using a pinhole, if VA was Results The data collection was completed in Conclusion There is a high prevalence of visual impairment in marine fishing communities in Prakasam district in India. The data from this rapid assessment survey can now be used as a baseline to start eye care services in this region. The rapid assessment methodology (RAVI reported in this paper is robust, quick and has the potential to be replicated in other areas.

  8. Rapid eye movement-sleep is reduced in patients with acute uncomplicated diverticulitis—an observational study

    Directory of Open Access Journals (Sweden)

    Chenxi Huang

    2015-08-01

    Full Text Available Introduction. Sleep disturbances are commonly found in patients in the postoperative period. Sleep disturbances may give rise to several complications including cardiopulmonary instability, transient cognitive dysfunction and prolonged convalescence. Many factors including host inflammatory responses are believed to cause postoperative sleep disturbances, as inflammatory responses can alter sleep architecture through cytokine-brain interactions. Our aim was to investigate alteration of sleep architecture during acute infection and its relationships to inflammation and clinical symptoms.Materials & Methods. In this observational study, we included patients with acute uncomplicated diverticulitis as a model to investigate the isolated effects of inflammatory responses on sleep. Eleven patients completed the study. Patients were admitted and treated with antibiotics for two nights, during which study endpoints were measured by polysomnography recordings, self-reported discomfort scores and blood samples of cytokines. One month later, the patients, who now were in complete remission, were readmitted and the endpoints were re-measured (the baseline values.Results. Total sleep time was reduced 4% and 7% the first (p = 0.006 and second (p = 0.014 nights of diverticulitis, compared to baseline, respectively. The rapid eye movement sleep was reduced 33% the first night (p = 0.016, compared to baseline. Moreover, plasma IL-6 levels were correlated to non-rapid eye movement sleep, rapid eye movement sleep and fatigue.Conclusion. Total sleep time and rapid eye movement sleep were reduced during nights with active diverticulitis and correlated with markers of inflammation.

  9. Development of an OP9 derived cell line as a robust model to rapidly study adipocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Jacqueline M Lane

    Full Text Available One hallmark of obesity is adipocyte hypertrophy and hyperplasia. To gain novel insights into adipose biology and therapeutics, there is a pressing need for a robust, rapid, and informative cell model of adipocyte differentiation for potential RNAi and drug screens. Current models are prohibitive for drug and RNAi screens due to a slow differentiation time course and resistance to transfection. We asked if we could create a rapid, robust model of adipogenesis to potentially enable rapid functional and obesity therapeutic screens. We generated the clonal population OP9-K, which differentiates rapidly and reproducibly, and displays classic adipocyte morphology: rounded cell shape, lipid accumulation, and coalescence of lipids into a large droplet. We further validate the OP9-K cells as an adipocyte model system by microarray analysis of the differentiating transcriptome. OP9-K differentiates via known adipogenic pathways, involving the transcriptional activation and repression of common adipose markers Plin1, Gata2, C/Ebpα and C/Ebpβ and biological pathways, such as lipid metabolism, PPARγ signaling, and osteogenesis. We implemented a method to quantify lipid accumulation using automated microscopy and tested the ability of our model to detect alterations in lipid accumulation by reducing levels of the known master adipogenic regulator Pparγ. We further utilized our model to query the effects of a novel obesity therapeutic target, the transcription factor SPI1. We determine that reduction in levels of Spi1 leads to an increase in lipid accumulation. We demonstrate rapid, robust differentiation and efficient transfectability of the OP9-K cell model of adipogenesis. Together with our microscopy based lipid accumulation assay, adipogenesis assays can be achieved in just four days' time. The results of this study can contribute to the development of rapid screens with the potential to deepen our understanding of adipose biology and efficiently

  10. Yeast and Mammals Utilize Similar Cytosolic Components to Drive Protein Transport through the Golgi Complex

    Science.gov (United States)

    Dunphy, William G.; Pfeffer, Suzanne R.; Clary, Douglas O.; Wattenberg, Binks W.; Glick, Benjamin S.; Rothman, James E.

    1986-03-01

    Vesicular transport between successive compartments of the mammalian Golgi apparatus has recently been reconstituted in a cell-free system. In addition to ATP, transport requires both membrane-bound and cytosolic proteins. Here we report that the cytosol fraction from yeast will efficiently substitute for mammalian cytosol. Mammalian cytosol contains several distinct transport factors, which we have distinguished on the basis of gel filtration and ion-exchange chromatography. Yeast cytosol appears to contain the same collection of transport factors. Resolved cytosol factors from yeast and mammals complement each other in a synergistic manner. These findings suggest that the molecular mechanisms of intracellular protein transport have been conserved throughout evolution. Moreover, this hybrid cell-free system will enable the application of yeast genetics to the identification and isolation of cytosolic proteins that sustain intracellular protein transport.

  11. Nonequilibrium description of de novo biogenesis and transport through Golgi-like cisternae

    Science.gov (United States)

    Sachdeva, Himani; Barma, Mustansir; Rao, Madan

    2016-12-01

    A central issue in cell biology is the physico-chemical basis of organelle biogenesis in intracellular trafficking pathways, its most impressive manifestation being the biogenesis of Golgi cisternae. At a basic level, such morphologically and chemically distinct compartments should arise from an interplay between the molecular transport and chemical maturation. Here, we formulate analytically tractable, minimalist models, that incorporate this interplay between transport and chemical progression in physical space, and explore the conditions for de novo biogenesis of distinct cisternae. We propose new quantitative measures that can discriminate between the various models of transport in a qualitative manner-this includes measures of the dynamics in steady state and the dynamical response to perturbations of the kind amenable to live-cell imaging.

  12. 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.

  13. PtdIns4P recognition by Vps74/GOLPH3 links PtdIns 4-kinase signaling to retrograde Golgi trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Wood, Christopher S.; Schmitz, Karl R.; Bessman, Nicholas J.; Setty, Thanuja Gangi; Ferguson, Kathryn M.; Burd, Christopher G.; (UPENN-MED)

    2010-02-11

    Targeting and retention of resident integral membrane proteins of the Golgi apparatus underly the function of the Golgi in glycoprotein and glycolipid processing and sorting. In yeast, steady-state Golgi localization of multiple mannosyltransferases requires recognition of their cytosolic domains by the peripheral Golgi membrane protein Vps74, an orthologue of human GOLPH3/GPP34/GMx33/MIDAS (mitochondrial DNA absence sensitive factor). We show that targeting of Vps74 and GOLPH3 to the Golgi apparatus requires ongoing synthesis of phosphatidylinositol (PtdIns) 4-phosphate (PtdIns4P) by the Pik1 PtdIns 4-kinase and that modulation of the levels and cellular location of PtdIns4P leads to mislocalization of these proteins. Vps74 and GOLPH3 bind specifically to PtdIns4P, and a sulfate ion in a crystal structure of GOLPH3 indicates a possible phosphoinositide-binding site that is conserved in Vps74. Alterations in this site abolish phosphoinositide binding in vitro and Vps74 function in vivo. These results implicate Pik1 signaling in retention of Golgi-resident proteins via Vps74 and show that GOLPH3 family proteins are effectors of Golgi PtdIns 4-kinases.

  14. Localization and trafficking of an isoform of the AtPRA1 family to the Golgi apparatus depend on both N- and C-terminal sequence motifs.

    Science.gov (United States)

    Jung, Chan Jin; Lee, Myoung Hui; Min, Myung Ki; Hwang, Inhwan

    2011-02-01

    Prenylated Rab acceptors (PRAs) bind to prenylated Rab proteins and possibly aid in targeting Rabs to their respective compartments. In Arabidopsis, 19 isoforms of PRA1 have been identified and, depending upon the isoforms, they localize to the endoplasmic reticulum (ER), Golgi apparatus and endosomes. Here, we investigated the localization and trafficking of AtPRA1.B6, an isoform of the Arabidopsis PRA1 family. In colocalization experiments with various organellar markers, AtPRA1.B6 tagged with hemagglutinin (HA) at the N-terminus localized to the Golgi apparatus in protoplasts and transgenic plants. The valine residue at the C-terminal end and an EEE motif in the C-terminal cytoplasmic domain were critical for anterograde trafficking from the ER to the Golgi apparatus. The N-terminal region contained a sequence motif for retention of AtPRA1.B6 at the Golgi apparatus. In addition, anterograde trafficking of AtPRA1.B6 from the ER to the Golgi apparatus was highly sensitive to the HA:AtPRA1.B6 level. The region that contains the sequence motif for Golgi retention also conferred the abundance-dependent trafficking inhibition. On the basis of these results, we propose that AtPRA1.B6 localizes to the Golgi apparatus and its ER-to-Golgi trafficking and localization to the Golgi apparatus are regulated by multiple sequence motifs in both the C- and N-terminal cytoplasmic domains. © 2010 John Wiley & Sons A/S.

  15. Papel de la actividad de la proteína quinasa A(PKA) en el mantenimiento estructural y biogénesis del complejo de Golgi

    OpenAIRE

    Bejarano Fernández, Eloy

    2007-01-01

    El complejo de Golgi es un lugar de concentración preferente de PKA en células mamíferos y su actividad modula diferentes pasos del tráfico intracelular. Dado que la arquitectura del orgánulo depende del balance entre transporte molecular anterógrado y retrógrado, PKA podría estar involucrada en la organización estructural y funcional del complejo de Golgi. Además, las evidencias disponibles sugieren que podría existir un ciclo de asociación/disociación de PKA a las membranas de Golgi. Por ot...

  16. Valoración funcional de la actividad de la proteína quinasa A (PKA) adscrita al complejo de Golgi

    OpenAIRE

    Mavillard Saborido, Fabiola

    2010-01-01

    La finalidad fundamental del presente estudio ha sido determinar el significado funcional de la presencia de PKA en el complejo de Golgi. Concretamente, hemos pretendido:1. Evaluar las condiciones en las que se produce la activación del complejo enzimático PKA asociado al Golgi2. Determinar el destino subcelular de las subunidades Cα activadas.3. Establecer las consecuencias de tal activación, ya sea para la fisiología de la célula, o bien para la dinámica morfofuncional del Golgi.

  17. Rapid prototyping compliant arterial phantoms for in-vitro studies and device testing

    Directory of Open Access Journals (Sweden)

    Biglino Giovanni

    2013-01-01

    Full Text Available Abstract Background Compliant vascular phantoms are desirable for in-vitro patient-specific experiments and device testing. TangoPlus FullCure 930® is a commercially available rubber-like material that can be used for PolyJet rapid prototyping. This work aims to gather preliminary data on the distensibility of this material, in order to assess the feasibility of its use in the context of experimental cardiovascular modelling. Methods The descending aorta anatomy of a volunteer was modelled in 3D from cardiovascular magnetic resonance (CMR images and rapid prototyped using TangoPlus. The model was printed with a range of increasing wall thicknesses (0.6, 0.7, 0.8, 1.0 and 1.5 mm, keeping the lumen of the vessel constant. Models were also printed in both vertical and horizontal orientations, thus resulting in a total of ten specimens. Compliance tests were performed by monitoring pressure variations while gradually increasing and decreasing internal volume. Knowledge of distensibility was thus derived and then implemented with CMR data to test two applications. Firstly, a patient-specific compliant model of hypoplastic aorta suitable for connection in a mock circulatory loop for in-vitro tests was manufactured. Secondly, the right ventricular outflow tract (RVOT of a patient necessitating pulmonary valve replacement was printed in order to physically test device insertion and assess patient’s suitability for percutaneous pulmonary valve intervention. Results The distensibility of the material was identified in a range from 6.5 × 10-3 mmHg-1 for the 0.6 mm case, to 3.0 × 10-3 mmHg-1 for the 1.5 mm case. The models printed in the vertical orientation were always more compliant than their horizontal counterpart. Rapid prototyping of a compliant hypoplastic aorta and of a RVOT anatomical model were both feasible. Device insertion in the RVOT model was successful. Conclusion Values of distensibility, compared with literature data, show that Tango

  18. Rapid forest carbon assessments of oceanic islands: a case study of the Hawaiian archipelago

    Directory of Open Access Journals (Sweden)

    Gregory P. Asner

    2016-01-01

    Full Text Available Abstract Background Spatially explicit forest carbon (C monitoring aids conservation and climate change mitigation efforts, yet few approaches have been developed specifically for the highly heterogeneous landscapes of oceanic island chains that continue to undergo rapid and extensive forest C change. We developed an approach for rapid mapping of aboveground C density (ACD; units = Mg or metric tons C ha−1 on islands at a spatial resolution of 30 m (0.09 ha using a combination of cost-effective airborne LiDAR data and full-coverage satellite data. We used the approach to map forest ACD across the main Hawaiian Islands, comparing C stocks within and among islands, in protected and unprotected areas, and among forests dominated by native and invasive species. Results Total forest aboveground C stock of the Hawaiian Islands was 36 Tg, and ACD distributions were extremely heterogeneous both within and across islands. Remotely sensed ACD was validated against U.S. Forest Service FIA plot inventory data (R2 = 0.67; RMSE = 30.4 Mg C ha−1. Geospatial analyses indicated the critical importance of forest type and canopy cover as predictors of mapped ACD patterns. Protection status was a strong determinant of forest C stock and density, but we found complex environmentally mediated responses of forest ACD to alien plant invasion. Conclusions A combination of one-time airborne LiDAR data acquisition and satellite monitoring provides effective forest C mapping in the highly heterogeneous landscapes of the Hawaiian Islands. Our statistical approach yielded key insights into the drivers of ACD variation, and also makes possible future assessments of C storage change, derived on a repeat basis from free satellite data, without the need for additional LiDAR data. Changes in C stocks and densities of oceanic islands can thus be continually assessed in the face of rapid environmental changes such as biological invasions, drought, fire and land use

  19. Rapid forest carbon assessments of oceanic islands: a case study of the Hawaiian archipelago.

    Science.gov (United States)

    Asner, Gregory P; Sousan, Sinan; Knapp, David E; Selmants, Paul C; Martin, Roberta E; Hughes, R Flint; Giardina, Christian P

    2016-12-01

    Spatially explicit forest carbon (C) monitoring aids conservation and climate change mitigation efforts, yet few approaches have been developed specifically for the highly heterogeneous landscapes of oceanic island chains that continue to undergo rapid and extensive forest C change. We developed an approach for rapid mapping of aboveground C density (ACD; units = Mg or metric tons C ha-1) on islands at a spatial resolution of 30 m (0.09 ha) using a combination of cost-effective airborne LiDAR data and full-coverage satellite data. We used the approach to map forest ACD across the main Hawaiian Islands, comparing C stocks within and among islands, in protected and unprotected areas, and among forests dominated by native and invasive species. Total forest aboveground C stock of the Hawaiian Islands was 36 Tg, and ACD distributions were extremely heterogeneous both within and across islands. Remotely sensed ACD was validated against U.S. Forest Service FIA plot inventory data (R2 = 0.67; RMSE = 30.4 Mg C ha-1). Geospatial analyses indicated the critical importance of forest type and canopy cover as predictors of mapped ACD patterns. Protection status was a strong determinant of forest C stock and density, but we found complex environmentally mediated responses of forest ACD to alien plant invasion. A combination of one-time airborne LiDAR data acquisition and satellite monitoring provides effective forest C mapping in the highly heterogeneous landscapes of the Hawaiian Islands. Our statistical approach yielded key insights into the drivers of ACD variation, and also makes possible future assessments of C storage change, derived on a repeat basis from free satellite data, without the need for additional LiDAR data. Changes in C stocks and densities of oceanic islands can thus be continually assessed in the face of rapid environmental changes such as biological invasions, drought, fire and land use. Such forest monitoring information can be used to promote

  20. Cystamine-mediated inhibition of protein disulfide isomerase triggers aggregation of misfolded orexin-A in the Golgi apparatus and prevents extracellular secretion of orexin-A.

    Science.gov (United States)

    Fujita, Issei; Nobunaga, Mizuki; Seki, Takahiro; Kurauchi, Yuki; Hisatsune, Akinori; Katsuki, Hiroshi

    2017-07-22

    Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy. Copyright © 2017. Published by Elsevier Inc.

  1. A participatory action research pilot study of urban health disparities using rapid assessment response and evaluation.

    Science.gov (United States)

    Brown, David Richard; Hernández, Agueda; Saint-Jean, Gilbert; Evans, Siân; Tafari, Ida; Brewster, Luther G; Celestin, Michel J; Gómez-Estefan, Carlos; Regalado, Fernando; Akal, Siri; Nierenberg, Barry; Kauschinger, Elaine D; Schwartz, Robert; Page, J Bryan

    2008-01-01

    Healthy People 2010 made it a priority to eliminate health disparities. We used a rapid assessment response and evaluation (RARE) to launch a program of participatory action research focused on health disparities in an urban, disadvantaged Black community serviced by a major south Florida health center. We formed partnerships with community members, identified local health disparities, and guided interventions targeting health disparities. We describe the RARE structure used to triangulate data sources and guide intervention plans as well as findings and conclusions drawn from scientific literature and epidemiological, historic, planning, clinical, and ethnographic data. Disenfranchisement and socioeconomic deprivation emerged as the principal determinants of local health disparities and the most appropriate targets for intervention.

  2. [Study on rapid determination and analysis of rocket kerosene by near infrared spectrum and chemometrics].

    Science.gov (United States)

    Xia, Ben-Li; Cong, Ji-Xin; Li, Xia; Wang, Xuan-Jun

    2011-06-01

    The rocket kerosene quality properties such as density, distillation range, viscosity and iodine value were successfully measured based on their near-infrared spectrum (NIRS) and chemometrics. In the present paper, more than 70 rocket kerosene samples were determined by near infrared spectrum, the models were built using the partial least squares method within the appropriate wavelength range. The correlation coefficients (R2) of every rocket kerosene's quality properties ranged from 0.862 to 0.999. Ten unknown samples were determined with the model, and the result showed that the prediction accuracy of near infrared spectrum method accords with standard analysis requirements. The new method is well suitable for replacing the traditional standard method to rapidly determine the properties of the rocket kerosene.

  3. Rapid thermal annealing and crystallization mechanisms study of silicon nanocrystal in silicon carbide matrix

    Directory of Open Access Journals (Sweden)

    Wan Zhenyu

    2011-01-01

    Full Text Available Abstract In this paper, a positive effect of rapid thermal annealing (RTA technique has been researched and compared with conventional furnace annealing for Si nanocrystalline in silicon carbide (SiC matrix system. Amorphous Si-rich SiC layer has been deposited by co-sputtering in different Si concentrations (50 to approximately 80 v%. Si nanocrystals (Si-NC containing different grain sizes have been fabricated within the SiC matrix under two different annealing conditions: furnace annealing and RTA both at 1,100°C. HRTEM image clearly reveals both Si and SiC-NC formed in the films. Much better "degree of crystallization" of Si-NC can be achieved in RTA than furnace annealing from the research of GIXRD and Raman analysis, especially in high-Si-concentration situation. Differences from the two annealing procedures and the crystallization mechanism have been discussed based on the experimental results.

  4. Non-inflammatory cerebral amyloid angiopathy as a cause of rapidly progressive dementia: A case study

    Directory of Open Access Journals (Sweden)

    Leonel Tadao Takada

    Full Text Available Abstract A 77 year-old men developed a subacute-onset, rapidly progressive cognitive decline. After 6 months of evolution, he scored 6 on the Mini-Mental State Examination and had left hemiparesis and hemineglect. The patient died 11 months after the onset of cognitive symptoms. Brain MRI showed microhemorrhages on gradient-echo sequence and confluent areas of white matter hyperintensities on T2-weighted images. Brain biopsy revealed amyloid-b peptide deposition in vessel walls, some of them surrounded by micro-bleeds. In this case report, we discuss the role of cerebral amyloid angiopathy (CAA in cognitive decline, due to structural lesions associated with hemorrhages and infarcts, white matter lesions and co-morbidity of Alzheimer's disease, as well as the most recently described amyloid angiopathy-related inflammation.

  5. Electronic vending machines for dispensing rapid HIV self-testing kits: a case study.

    Science.gov (United States)

    Young, Sean D; Klausner, Jeffrey; Fynn, Risa; Bolan, Robert

    2014-02-01

    This short report evaluates the feasibility of using electronic vending machines for dispensing oral, fluid, rapid HIV self-testing kits in Los Angeles County. Feasibility criteria that needed to be addressed were defined as: (1) ability to find a manufacturer who would allow dispensing of HIV testing kits and could fit them to the dimensions of a vending machine, (2) ability to identify and address potential initial obstacles, trade-offs in choosing a machine location, and (3) ability to gain community approval for implementing this approach in a community setting. To address these issues, we contracted a vending machine company who could supply a customized, Internet-enabled machine that could dispense HIV kits and partnered with a local health center available to host the machine onsite and provide counseling to participants, if needed. Vending machines appear to be feasible technologies that can be used to distribute HIV testing kits.

  6. Rapid analysis of vessel elements (RAVE): a tool for studying physiologic, pathologic and tumor angiogenesis.

    Science.gov (United States)

    Seaman, Marc E; Peirce, Shayn M; Kelly, Kimberly

    2011-01-01

    Quantification of microvascular network structure is important in a myriad of emerging research fields including microvessel remodeling in response to ischemia and drug therapy, tumor angiogenesis, and retinopathy. To mitigate analyst-specific variation in measurements and to ensure that measurements represent actual changes in vessel network structure and morphology, a reliable and automatic tool for quantifying microvascular network architecture is needed. Moreover, an analysis tool capable of acquiring and processing large data sets will facilitate advanced computational analysis and simulation of microvascular growth and remodeling processes and enable more high throughput discovery. To this end, we have produced an automatic and rapid vessel detection and quantification system using a MATLAB graphical user interface (GUI) that vastly reduces time spent on analysis and greatly increases repeatability. Analysis yields numerical measures of vessel volume fraction, vessel length density, fractal dimension (a measure of tortuosity), and radii of murine vascular networks. Because our GUI is open sourced to all, it can be easily modified to measure parameters such as percent coverage of non-endothelial cells, number of loops in a vascular bed, amount of perfusion and two-dimensional branch angle. Importantly, the GUI is compatible with standard fluorescent staining and imaging protocols, but also has utility analyzing brightfield vascular images, obtained, for example, in dorsal skinfold chambers. A manually measured image can be typically completed in 20 minutes to 1 hour. In stark comparison, using our GUI, image analysis time is reduced to around 1 minute. This drastic reduction in analysis time coupled with increased repeatability makes this tool valuable for all vessel research especially those requiring rapid and reproducible results, such as anti-angiogenic drug screening.

  7. Rapid analysis of vessel elements (RAVE: a tool for studying physiologic, pathologic and tumor angiogenesis.

    Directory of Open Access Journals (Sweden)

    Marc E Seaman

    Full Text Available Quantification of microvascular network structure is important in a myriad of emerging research fields including microvessel remodeling in response to ischemia and drug therapy, tumor angiogenesis, and retinopathy. To mitigate analyst-specific variation in measurements and to ensure that measurements represent actual changes in vessel network structure and morphology, a reliable and automatic tool for quantifying microvascular network architecture is needed. Moreover, an analysis tool capable of acquiring and processing large data sets will facilitate advanced computational analysis and simulation of microvascular growth and remodeling processes and enable more high throughput discovery. To this end, we have produced an automatic and rapid vessel detection and quantification system using a MATLAB graphical user interface (GUI that vastly reduces time spent on analysis and greatly increases repeatability. Analysis yields numerical measures of vessel volume fraction, vessel length density, fractal dimension (a measure of tortuosity, and radii of murine vascular networks. Because our GUI is open sourced to all, it can be easily modified to measure parameters such as percent coverage of non-endothelial cells, number of loops in a vascular bed, amount of perfusion and two-dimensional branch angle. Importantly, the GUI is compatible with standard fluorescent staining and imaging protocols, but also has utility analyzing brightfield vascular images, obtained, for example, in dorsal skinfold chambers. A manually measured image can be typically completed in 20 minutes to 1 hour. In stark comparison, using our GUI, image analysis time is reduced to around 1 minute. This drastic reduction in analysis time coupled with increased repeatability makes this tool valuable for all vessel research especially those requiring rapid and reproducible results, such as anti-angiogenic drug screening.

  8. Rapid Offline-Online Post-Disaster Landslide Mapping Tool: A case study from Nepal

    Science.gov (United States)

    Olyazadeh, Roya; Jaboyedoff, Michel; Sudmeier-Rieux, Karen; Derron, Marc-Henri; Devkota, Sanjaya

    2016-04-01

    One of the crucial components of post disaster management is the efficient mapping of impacted areas. Here we present a tool designed to map landslides and affected objects after the earthquakes of 2015 in Nepal as well as for intense rainfall impact. Because internet is not available in many rural areas of Nepal, we developed an offline-online prototype based on Open-Source WebGIS technologies to make data on hazard impacts, including damaged infrastructure, landslides or flooding events available to authorities and the general public. This mobile application was designed as a low-cost, rapid and participatory method for recording impacts from hazard events. It is possible to record such events offline and upload them through a server, where internet connection is available. This application allows user authentication, image capturing, and information collation such as geolocation, event description, interactive mapping and finally storing all the data in the server for further analysis and visualisation. This application can be accessed by a mobile phone (Android) or a tablet as a hybrid version for both offline and online versions. The offline version has an interactive-offline map function which allows users to upload satellites image in order to improve ground truthing interpretation. After geolocation, the user can start mapping and then save recorded data into Geojson-TXT files that can be easily uploaded to the server whenever internet is available. This prototype was tested specifically for a rapid assessment of landslides and relevant land use characteristics such as roads, forest area, rivers in the Phewa Lake watershed near Pokhara, Nepal where a large number landslides were activated or reactivated after the 2015 monsoon season. More than 60 landslides were recorded during two days of field trip. Besides, it is possible to use this application for any other kind of hazard event like flood, avalanche, etc. Keywords: Offline, Online, Open source, Web

  9. Palliative care making a difference in rural Uganda, Kenya and Malawi:three rapid evaluation field studies

    OpenAIRE

    Bettega Nadia; Leng Mhoira; Brown Judith; Grant Liz; Murray Scott A

    2011-01-01

    Abstract Background Many people live and die in pain in Africa. We set out to describe patient, family and local community perspectives on the impact of three community based palliative care interventions in sub-Saharan Africa. Methods Three palliative care programmes in Uganda, Kenya and Malawi were studied using rapid evaluation field techniques in each country, triangulating data from three sources: interviews with key informants, observations of clinical encounters and the local health an...

  10. Bedside Availability of Prepared Oxytocin and Rapid Administration After Delivery to Prevent Postpartum Hemorrhage: An Observational Study in Karnataka, India

    OpenAIRE

    Moucheraud, Corrina; Gass, Jonathon; Lipsitz, Stuart; Spector, Jonathan; Agrawal, Priya; Hirschhorn, Lisa R; Gawande, Atul; Kodkany, Bhala

    2015-01-01

    Postpartum hemorrhage is a leading cause of maternal death worldwide. Rapid provision of uterotonics after childbirth is recommended to reduce the incidence and severity of postpartum hemorrhage. Data obtained through direct observation of childbirth practices, collected in a study of the World Health Organization?s Safe Childbirth Checklist in Karnataka, India, were used to measure if oxytocin prepared for administration and available at the bedside before birth was associated with decreased...

  11. Pompe Disease Results in a Golgi-based Glycosylation Deficit in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes*

    Science.gov (United States)

    Raval, Kunil K.; Tao, Ran; White, Brent E.; De Lange, Willem J.; Koonce, Chad H.; Yu, Junying; Kishnani, Priya S.; Thomson, James A.; Mosher, Deane F.; Ralphe, John C.; Kamp, Timothy J.

    2015-01-01

    Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation. PMID:25488666

  12. Pompe disease results in a Golgi-based glycosylation deficit in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Raval, Kunil K; Tao, Ran; White, Brent E; De Lange, Willem J; Koonce, Chad H; Yu, Junying; Kishnani, Priya S; Thomson, James A; Mosher, Deane F; Ralphe, John C; Kamp, Timothy J

    2015-01-30

    Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Performance Assessment of GPS-Sensed Precipitable Water Vapor using IGS Ultra-Rapid Orbits: A Preliminary Study in Thailand

    Directory of Open Access Journals (Sweden)

    Yoon-Soo Choi

    2011-01-01

    Full Text Available Precipitable Water Vapor (PWV is a significant variable used for climate change studies. Currently PWV can be derived from the Global Positioning System (GPS observation in addition to the specific instruments such as Radiosondes (RS, Microwave Radiometers (MWR and Meteorological Satellites. To accurately derive PWV from GPS data, long periods of observation time in conjunction with final orbit data have to be applied in the data processing steps. This final orbit data can be acquired from the International GNSS Service (IGS with 13 days latency, which is not practical in climate change studies or meteorological forecasting. Alternatively, real-time ultra-rapid orbits are more suitable for this application but with lower orbit accuracy. It is therefore interesting to evaluate the impact of using different orbits in the estimation of PWV. In this study, data from permanent GPS base stations in Thailand were processed using Bernese 5.0 software to derive near real-time PWV values. Ultra-rapid orbit data have been introduced in the data processing step with different time windows and compared to that using final orbit data with the 24-hr time window. The results have shown that 1.0 mm and 2.9 mm biases can be achieved using 24-hr and 12-hr time windows, respectively. These results therefore address the potential use of ultra-rapid orbits for a near real-time estimation of PWV.

  14. Study on rapid eye movement sleep behavior disorder in patients with Parkinson's disease at early stage

    Directory of Open Access Journals (Sweden)

    Hui-li ZOU

    2017-10-01

    Full Text Available Objective To investigate the incidence, occurrence time and electrophysiological characteristics of rapid eye movement behavior disorder (RBD in patients with early Parkinson's disease (PD, and the characteristics of motor symptoms and non . motor symtoms (NMS. Methods Sixty PD patients were divided into PD + RBD group (N = 42 and control group (N = 18 according to whether they were complicated with RBD or not. Unified Parkinson's Disease Rating Scale (UPDRSⅡ andⅢ, Hoehn-Yahr Stage, Montreal Cognitive Assessment (MoCA, Hamilton Anxiety Rating Scale (HAMA, Hamilton Depression Rating Scale (HAMD, RBD Screening Questionnaire (RBDSQ, Epworth Sleepiness Scale (ESS and polysomnography (PSG were used in 60 patients. Results Among 60 patients, 42 (70% were accompanied by RBD. PSG showed that PD + RBD patients mainly presented upper limb stretching and gripping, body twitching, laughing, shouting, cursing and other non.violent actions, except 2 cases presented violent actions, such as hitting, kicking, etc. In PD + RBD group, the age was older (P = 0.024, duration was longer (P = 0.000, and UPDRSⅡ (P = 0.005,UPDRSⅢ(P = 0.001, the scale values of Hoehn-Yahr Sotage 2 (P = 0.007, anxiety (P = 0.044 and depression (P = 0.001 ratio were all higher than control group. There were significant differences in density of mandible myoelectric activity (P = 0.000 and ratio of rapid eye movement (REM without atonia (P = 0.000 between 2 groups. In PD + RBD group, 16 patients (38.10% had symptoms of RBD, earlier than PD occurred 5.20 (3.91, 6.51 years. Conclusions PD patients with older age, longer duration, more severe motor symptoms and non?motor symptoms were more likely to be accompanied by RBD. The severity of RBD in PD patients accompanied with RBD is higher than that in PD without RBD. RBD may be the early manifestation of PD. PSG has important value in the diagnosis of PD with RBD. DOI: 10.3969/j.issn.1672-6731.2017.10.006

  15. Immediate facial rehabilitation in cancer patients using CAD-CAM and rapid prototyping technology: a pilot study.

    Science.gov (United States)

    Ciocca, Leonardo; Fantini, Massimiliano; Marchetti, Claudio; Scotti, Roberto; Monaco, Carlo

    2010-06-01

    This study describes the workflow in a procedure to create a provisional facial prosthesis for cancer patients using digital and rapid prototyping technologies without the need for supporting craniofacial implants. An integrated workflow procedure aimed at the construction of provisional silicone prosthesis was used to rehabilitate a facial disfigurement in a patient who had undergone ablative surgery of the midface. A laser scan of the defect was obtained, and a digital model of the patient's face was constructed using virtual mirroring of the healthy side and referencing the "Nose Digital Library." The missing volume of the face was reconstructed, and a rapid-prototyped mold was devised to process the silicone prosthesis. A provisional eyeglasses-supported prosthesis designed with a CAD/CAM-projected titanium substructure was connected using the micro-components of implant prosthetic devices. The workflow described herein offers a viable procedure for quickly restoring facial defects by means of provisional prosthetic rehabilitation.

  16. Rapidly solidified Ag-Cu eutectics: A comparative study using drop-tube and melt fluxing techniques

    Science.gov (United States)

    Yu, Y.; Mullis, A. M.; Cochrane, R. F.

    2016-03-01

    A comparative study of rapid solidification of Ag-Cu eutectic alloy processed via melt fluxing and drop-tube techniques is presented. A computational model is used to estimate the cooling rate and undercooling of the free fall droplets as this cannot be determined directly. SEM micrographs show that both materials consist of lamellar and anomalous eutectic structures. However, below the critical undercooling the morphologies of each are different in respect of the distribution and volume of anomalous eutectic. The anomalous eutectic in flux- undercooled samples preferentially forms at cell boundaries around the lamellar eutectic in the cell body. In drop-tube processed samples it tends to distribute randomly inside the droplets and at much smaller volume fractions. That the formation of the anomalous eutectic can, at least in part, be suppressed in the drop-tube is strongly suggestive that the formation of anomalous eutectic occurs via remelting process, which is suppressed by rapid cooling during solidification.

  17. Fat Content Modulates Rapid Detection of Food: A Visual Search Study Using Fast Food and Japanese Diet

    Directory of Open Access Journals (Sweden)

    Reiko Sawada

    2017-06-01

    Full Text Available Rapid detection of food is crucial for the survival of organisms. However, previous visual search studies have reported discrepant results regarding the detection speeds for food vs. non-food items; some experiments showed faster detection of food than non-food, whereas others reported null findings concerning any speed advantage for the detection of food vs. non-food. Moreover, although some previous studies showed that fat content can affect visual attention for food, the effect of fat content on the detection of food remains unclear. To investigate these issues, we measured reaction times (RTs during a visual search task in which participants with normal weight detected high-fat food (i.e., fast food, low-fat food (i.e., Japanese diet, and non-food (i.e., kitchen utensils targets within crowds of non-food distractors (i.e., cars. Results showed that RTs for food targets were shorter than those for non-food targets. Moreover, the RTs for high-fat food were shorter than those for low-fat food. These results suggest that food is more rapidly detected than non-food within the environment and that a higher fat content in food facilitates rapid detection.

  18. Fat Content Modulates Rapid Detection of Food: A Visual Search Study Using Fast Food and Japanese Diet

    Science.gov (United States)

    Sawada, Reiko; Sato, Wataru; Toichi, Motomi; Fushiki, Tohru

    2017-01-01

    Rapid detection of food is crucial for the survival of organisms. However, previous visual search studies have reported discrepant results regarding the detection speeds for food vs. non-food items; some experiments showed faster detection of food than non-food, whereas others reported null findings concerning any speed advantage for the detection of food vs. non-food. Moreover, although some previous studies showed that fat content can affect visual attention for food, the effect of fat content on the detection of food remains unclear. To investigate these issues, we measured reaction times (RTs) during a visual search task in which participants with normal weight detected high-fat food (i.e., fast food), low-fat food (i.e., Japanese diet), and non-food (i.e., kitchen utensils) targets within crowds of non-food distractors (i.e., cars). Results showed that RTs for food targets were shorter than those for non-food targets. Moreover, the RTs for high-fat food were shorter than those for low-fat food. These results suggest that food is more rapidly detected than non-food within the environment and that a higher fat content in food facilitates rapid detection. PMID:28690568

  19. Fat Content Modulates Rapid Detection of Food: A Visual Search Study Using Fast Food and Japanese Diet.

    Science.gov (United States)

    Sawada, Reiko; Sato, Wataru; Toichi, Motomi; Fushiki, Tohru

    2017-01-01

    Rapid detection of food is crucial for the survival of organisms. However, previous visual search studies have reported discrepant results regarding the detection speeds for food vs. non-food items; some experiments showed faster detection of food than non-food, whereas others reported null findings concerning any speed advantage for the detection of food vs. non-food. Moreover, although some previous studies showed that fat content can affect visual attention for food, the effect of fat content on the detection of food remains unclear. To investigate these issues, we measured reaction times (RTs) during a visual search task in which participants with normal weight detected high-fat food (i.e., fast food), low-fat food (i.e., Japanese diet), and non-food (i.e., kitchen utensils) targets within crowds of non-food distractors (i.e., cars). Results showed that RTs for food targets were shorter than those for non-food targets. Moreover, the RTs for high-fat food were shorter than those for low-fat food. These results suggest that food is more rapidly detected than non-food within the environment and that a higher fat content in food facilitates rapid detection.

  20. Furin and proprotein convertase 7 (PC7)/lymphoma PC endogenously expressed in rat liver can be resolved into distinct post-Golgi compartments.

    Science.gov (United States)

    Wouters, S; Leruth, M; Decroly, E; Vandenbranden, M; Creemers, J W; van de Loo, J W; Ruysschaert, J M; Courtoy, P J

    1998-01-01

    The intracellular compartmentalization in rat liver of the membrane-associated convertases furin and proprotein convertase 7 (PC7)/lymphoma PC (LPC) was investigated by analytical subcellular fractionation. In control animals, both enzymes were found to localize in fractions depleted of endoplasmic reticulum, cis-Golgi and lysosomal markers, but to co-distribute with the Golgi marker galactosyltransferase and the trans-Golgi network (TGN) marker TGN38. After overloading Golgi-derived vesicles with very-low-density lipoproteins (VLDL) by feeding rats with ethanol, the distribution of PC7/LPC was shifted markedly towards lower densities, in contrast with those of furin and the TGN marker. This provides support for the TGN localization of endogenously expressed furin and indicates that, at steady state, a considerable proportion of PC7/LPC may be associated with vesicles derived from the TGN. PMID:9820806

  1. Suppression of interictal spikes during phasic rapid eye movement sleep: a quantitative stereo-electroencephalography study.

    Science.gov (United States)

    Campana, C; Zubler, F; Gibbs, S; de Carli, F; Proserpio, P; Rubino, A; Cossu, M; Tassi, L; Schindler, K; Nobili, L

    2017-10-01

    Tonic and phasic rapid eye movement (REM) sleep seem to represent two different brain states exerting different effects on epileptic activity. In particular, interictal spikes are suppressed strongly during phasic REM sleep. The reason for this effect is not understood completely. A different level of synchronization in phasic and tonic REM sleep has been postulated, yet never measured directly. Here we assessed the interictal spike rate across non-REM (NREM) sleep, phasic and tonic REM sleep in nine patients affected by drug resistant focal epilepsy: five with type II focal cortical dysplasia and four with hippocampal sclerosis. Moreover, we applied different quantitative measures to evaluate the level of synchronization at the local and global scale during phasic and tonic REM sleep. We found a lower spike rate in phasic REM sleep, both within and outside the seizure onset zone. This effect seems to be independent from the histopathological substrate and from the brain region, where epileptic activity is produced (temporal versus extra-temporal). A higher level of synchronization was observed during tonic REM sleep both on a large (global) and small (local) spatial scale. Phasic REM sleep appears to be an interesting model for understanding the mechanisms of suppression of epileptic activity. © 2017 European Sleep Research Society.

  2. Developing a Natural Gas-Powered Bus Rapid Transit Service: A Case Study

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, G.

    2015-11-03

    The Roaring Fork Transit Authority (RFTA) and its VelociRFTA Bus Rapid Transit (BRT) program are unique in many ways. For example, VelociRFTA was the first rural BRT system in the United States and the operational environment of the VelociRFTA BRT is one of the most severe in the country, with extreme winter temperatures and altitudes close to 8,000 feet. RFTA viewed high altitude operation as the most challenging characteristic when it began considering the use of natural gas. RFTA is the second-largest public transit system in Colorado behind Denver's Regional Transportation District (RTD), and it is one of the largest rural public transit systems in the country. In 2013, RFTA accepted delivery of 22 new compressed natural gas (CNG) buses that went into service after completion of maintenance and refueling facilities earlier that year. This paper examines the lessons learned from RFTA's experience of investigating--and ultimately choosing--CNG for their new BRT program and focuses on the unique environment of RFTA's BRT application; the decision process to include CNG fueling in the project; unforeseen difficulties encountered in the operation of CNG buses; public perception; cost comparison to competing fuels; and considerations for indoor fueling facilities and project funding.

  3. Developing a Natural Gas-Powered Bus Rapid Transit Service. A Case Study

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, George [National Renewable Energy Lab. (NREL), Golden, CO (United States)

    2015-11-01

    The Roaring Fork Transit Authority (RFTA) and its VelociRFTA Bus Rapid Transit (BRT) program are unique in many ways. For example, VelociRFTA was the first rural BRT system in the United States and the operational environment of the VelociRFTA BRT is one of the most severe in the country, with extreme winter temperatures and altitudes close to 8,000 feet. RFTA viewed high altitude operation as the most challenging characteristic when it began considering the use of natural gas. RFTA is the second-largest public transit system in Colorado behind Denver's Regional Transportation District (RTD), and it is one of the largest rural public transit systems in the country. In 2013, RFTA accepted delivery of 22 new compressed natural gas (CNG) buses that went into service after completion of maintenance and refueling facilities earlier that year. This paper examines the lessons learned from RFTA's experience of investigating--and ultimately choosing--CNG for their new BRT program and focuses on the unique environment of RFTA's BRT application; the decision process to include CNG fueling in the project; unforeseen difficulties encountered in the operation of CNG buses; public perception; cost comparison to competing fuels; and considerations for indoor fueling facilities and project funding.

  4. Organizational Perspectives on Rapid Response Team Structure, Function, and Cost: A Qualitative Study.

    Science.gov (United States)

    Smith, Patricia L; McSweeney, Jean

    Understanding how an organization determines structure and function of a rapid response team (RRT), as well as cost evaluation and implications, can provide foundational knowledge to guide decisions about RRTs. The objectives were to (1) identify influencing factors in organizational development of RRT structure and function and (2) describe evaluation of RRT costs. Using a qualitative, ethnographic design, nurse executives and experts in 15 moderate-size hospitals were interviewed to explore their decision-making processes in determining RRT structure and function. Face-to-face interviews were audio recorded and transcribed verbatim and verified for accurateness. Using content analysis and constant comparison, interview data were analyzed. Demographic data were analyzed using descriptive statistics. The sample included 27 participants from 15 hospitals in 5 south-central states. They described a variety of RRT responders and functions, with the majority of hospitals having a critical care charge nurse attending all RRT calls for assistance. Others described a designated RRT nurse with primary RRT duties as responder to all RRT calls. Themes of RRT development from the data included influencers, decision processes, and thoughts about cost. It is important to understand how hospitals determine optimal structure and function to enhance support of quality nursing care. Determining the impact of an RRT on costs and benefits is vital in balancing patient safety and limited resources. Future research should focus on clarifying differences between team structure and function in outcomes as well as the most effective means to estimate costs and benefits.

  5. Direct loading of polymer matrices in plastic microchips for rapid DNA analysis: a comparative study.

    Science.gov (United States)

    Hurth, Cedric; Gu, Jian; Aboud, Maurice; Estes, Matthew D; Nordquist, Alan R; McCord, Bruce; Zenhausern, Frederic

    2012-08-01

    We report the design and performance validation of microfluidic separation technologies for human identification using a disposable plastic device suitable for integration into an automated rapid DNA analysis system. A fabrication process for a 15-cm long hot-embossed plastic microfluidic devices with a smooth semielliptical cross section out of cyclic olefin copolymer is presented. We propose a mixed polymer solution of 95% w/v hydroxyethylcellulose and 5% w/v polyvinylpyrrolidone for a final polymer concentration of 2.5 or 3.0% to be used as coating and sieving matrix for DNA separation. This formulation allows preparing the microchip without pretreatment in a single-loading step and provides high-resolution separation (≈1.2 bp for fragments <200 bp), which is superior to existing commercial matrices under the same conditions. The hot-embossed device performance is characterized and compared to injection-molded devices made out of cyclic olefin copolymer based on their respective injector geometry, channel shape, and surface charges. Each device design is assessed by fluorescence videomicroscopy to evaluate the formation of injection plugs, then by comparing electropherograms for the separation of a DNA size standard relevant to human identification. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Accuracy Assessment of Using Rapid Prototyping Drill Templates for Atlantoaxial Screw Placement: A Cadaver Study

    Directory of Open Access Journals (Sweden)

    Shuai Guo

    2016-01-01

    Full Text Available Purpose. To preliminarily evaluate the feasibility and accuracy of using rapid prototyping drill templates (RPDTs for C1 lateral mass screw (C1-LMS and C2 pedicle screw (C2-PS placement. Methods. 23 formalin-fixed craniocervical cadaver specimens were randomly divided into two groups. In the conventional method group, intraoperative fluoroscopy was used to assist the screw placement. In the RPDT navigation group, specific RPDTs were constructed for each specimen and were used intraoperatively for screw placement navigation. The screw position, the operating time, and the fluoroscopy time for each screw placement were compared between the 2 groups. Results. Compared with the conventional method, the RPDT technique significantly increased the placement accuracy of the C2-PS (p0.05. Moreover, the RPDT technique significantly decreased the operating and fluoroscopy times. Conclusion. Using RPDTs significantly increases the accuracy of C1-LMS and C2-PS placement while decreasing the screw placement time and the radiation exposure. Due to these advantages, this approach is worth promoting for use in the Harms technique.

  7. Structure of the Membrane-tethering GRASP Domain Reveals a Unique PDZ Ligand Interaction That Mediates Golgi Biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Truschel, S.T.; Heroux, A.; Sengupta, D.; Foote, A.; Macbeth, M. R.; Linstedt, A. D.

    2011-06-10

    Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.

  8. Structure of the Membrane-tethering GRASP Domain Reveals a Unique PDZ Ligand Interaction That Mediates Golgi Biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    S Truschel; D Sengupta; A Foote; A Heroux; M Macbeth; A Linstedt

    2011-12-31

    Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.

  9. Study on transcranial sonography in patients with idiopathic rapid eye movement sleep behavior disorder

    Directory of Open Access Journals (Sweden)

    Xu-dong LI

    2016-04-01

    Full Text Available Objective To investigate the changes of transcranial sonography (TCS in patients with rapid eye movement sleep behavior disorder (RBD.  Methods Fifteen patients fulfilled the diagnostic criteria of RBD according to International Classification of Sleep Disorders (ICSD, 2nd edition. Under the monitor of polysomnography (PSG, the sleep architectures of all RBD cases were evaluated by Polysmith software and visual analysis. Furthermore, all RBD patients and 15 normal controls underwent TCS. Cases with substantia nigra echo intensity over Ⅲ grade and substantia nigra area over 0.20 cm2 were supposed to be hyperechogenicity. Additionally, the width of the third ventricle was measured and whether there was hyperechogenicity in basal ganglia was evaluated. The cognitive functions were evaluated by Mini-Mental State Examination (MMSE and Montreal Cognitive Assessment (MoCA.  Results RBD patients presented typical clinical manifestations and electrophysiologic changes. No significant difference (P = 0.080, 0.109 was found in the comparison of hyperechogenicity rate on substantia nigra (6/15 and basal ganglia (7/15 in RBD patients and normal controls (1/15, 2/15. No significant difference in the comparison of MoCA was found in RBD patients with or without substantia nigra hyperechogenicity (P = 0.075. The RBD patients with hyperechogenicity on basal ganglia had higher MMSE scores than those without hyperechogenicity on basal ganglia, and the difference was significant (P = 0.021.  Conclusions RBD which is suggested as the prodromal period of synucleinopathy may present hyperechogenicity in substantia nigra and basal ganglia on TCS. TCS could detect subclinical changes of brain and evaluate the risk of synucleinopathy. DOI: 10.3969/j.issn.1672-6731.2016.04.010

  10. Asymmetric rapid maxillary expansion in true unilateral crossbite malocclusion: a prospective controlled clinical study.

    Science.gov (United States)

    Ileri, Zehra; Basciftci, Faruk Ayhan

    2015-03-01

    To investigate the short-term effects of the asymmetric rapid maxillary (ARME) appliance on the vertical, sagittal, and transverse planes in patients with true unilateral posterior crossbite. Subjects were divided into two groups. The treatment group was comprised of 21 patients with unilateral posterior crossbite (mean age  =  13.3 ± 2.1 years). Members of this group were treated with the ARME appliance. The control group was comprised of 17 patients with Angle Class I who were kept under observation (mean age  =  12.3 ± 0.8 years). Lateral and frontal cephalograms were taken before the expansion (T1), immediately after expansion (T2), and at postexpansion retention (T3) in the treatment group and at preobservation (T1) and postobservation (T2) in the control group. A total of 34 measurements were assessed on cephalograms. For statistical analysis, the Wilcoxon test and analysis of covariance were used. The ARME appliance produced significant increases in nasal, maxillary base, upper arch, and lower arch dimensions (P ARME appliance created asymmetric increments in the transversal dimensions of the nose, maxilla, and upper arch in the short term. Asymmetric expansion therapy for subjects with unilateral maxillary deficiency may provide satisfactory outcomes in adolescents, with the exception of mandibular arch expansion. The triangular pattern of expansion caused clockwise rotation of the mandible and the occlusal plane and produced significant alterations in the vertical facial dimensions, whereas it created no displacement in maxilla in the sagittal plane.

  11. Object-based change detection in rapid urbanization regions with remotely sensed observations: a case study of Shenzhen, China

    Science.gov (United States)

    He, Lihuang; Dong, Guihua; Wang, Wei-Min; Yang, Lijun; Liang, Hong

    2013-10-01

    China, the most populous country on Earth, has experienced rapid urbanization which is one of the main causes of many environmental and ecological problems. Therefore, the monitoring of rapid urbanization regions and the environment is of critical importance for their sustainable development. In this study, the object-based classification is employed to detect the change of land cover in Shenzhen, which is located in South China and has been urbanized rapidly in recent three decades. First, four Landsat TM images, which were acquired on 1990, 2000 and 2010, respectively, are selected from the image database. Atmospheric corrections are conducted on these images with improved dark-object subtraction technique and surface meteorological observations. Geometric correction is processed with ground control points derived from topographic maps. Second, a region growing multi-resolution segmentation and a soft nearest neighbour classifier are used to finish object-based classification. After analyzing the fraction of difference classes over time series, we conclude that the comparison of derived land cover classes with socio-economic statistics demonstrates the strong positive correlation between built-up classes and urban population as well as gross GDP and GDPs in second and tertiary industries. Two different mechanisms of urbanization, namely new land development and redevelopment, are revealed. Consequently, we found that, the districts of Shenzhen were urbanized through different mechanisms.

  12. A Comparative Study with RapidMiner and WEKA Tools over some Classification Techniques for SMS Spam

    Science.gov (United States)

    Foozy, Cik Feresa Mohd; Ahmad, Rabiah; Faizal Abdollah, M. A.; Chai Wen, Chuah

    2017-08-01

    SMS Spamming is a serious attack that can manipulate the use of the SMS by spreading the advertisement in bulk. By sending the unwanted SMS that contain advertisement can make the users feeling disturb and this against the privacy of the mobile users. To overcome these issues, many studies have proposed to detect SMS Spam by using data mining tools. This paper will do a comparative study using five machine learning techniques such as Naïve Bayes, K-NN (K-Nearest Neighbour Algorithm), Decision Tree, Random Forest and Decision Stumps to observe the accuracy result between RapidMiner and WEKA for dataset SMS Spam UCI Machine Learning repository.

  13. Rapidly resorbable vs. non-resorbable suture for experimental colonic anastomoses in rats--a randomized experimental study

    DEFF Research Database (Denmark)

    Klein, Mads; Pommergaard, Hans-Christian; Gögenur, Ismail

    2011-01-01

    Anastomotic dehiscence remains an important challenge for colorectal surgeons worldwide. Extensive research focused on performing a safe anastomosis is conducted with rats being the most used model when examining colorectal anastomoses. In daily clinical practice resorbable sutures are used when...... hand-sewn anastomoses are performed. However, in the experimental studies examining colorectal anastomoses, non-resorbable sutures have predominantly been used. The aim of this study was to compare a rapidly resorbable suture with a non-resorbable suture in experimental colorectal anastomoses....

  14. Development of a Rapid Microbore Metabolic Profiling Ultraperformance Liquid Chromatography-Mass Spectrometry Approach for High-Throughput Phenotyping Studies.

    Science.gov (United States)

    Gray, Nicola; Adesina-Georgiadis, Kyrillos; Chekmeneva, Elena; Plumb, Robert S; Wilson, Ian D; Nicholson, Jeremy K

    2016-06-07

    A rapid gradient microbore ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method has been developed to provide a high-throughput analytical platform for the metabolic phenotyping of urine from large sample cohorts. The rapid microbore metabolic profiling (RAMMP) approach was based on scaling a conventional reversed-phase UPLC-MS method for urinary profiling from 2.1 mm × 100 mm columns to 1 mm × 50 mm columns, increasing the linear velocity of the solvent, and decreasing the gradient time to provide an analysis time of 2.5 min/sample. Comparison showed that conventional UPLC-MS and rapid gradient approaches provided peak capacities of 150 and 50, respectively, with the conventional method detecting approximately 19 000 features compared to the ∼6 000 found using the rapid gradient method. Similar levels of repeatability were seen for both methods. Despite the reduced peak capacity and the reduction in ions detected, the RAMMP method was able to achieve similar levels of group discrimination as conventional UPLC-MS when applied to rat urine samples obtained from investigative studies on the effects of acute 2-bromophenol and chronic acetaminophen administration. When compared to a direct infusion MS method of similar analysis time the RAMMP method provided superior selectivity. The RAMMP approach provides a robust and sensitive method that is well suited to high-throughput metabonomic analysis of complex mixtures such as urine combined with a 5-fold reduction in analysis time compared with the conventional UPLC-MS method.

  15. Three-dimensional assessment of buccal alveolar bone after rapid and slow maxillary expansion: a clinical trial study.

    Science.gov (United States)

    Brunetto, Mauricio; Andriani, Juliana da Silva Pereira; Ribeiro, Gerson Luiz Ulema; Locks, Arno; Correa, Marcio; Correa, Letícia Ruhland

    2013-05-01

    The purposes of this study were to analyze and compare the immediate effects of rapid and slow maxillary expansion protocols, accomplished by Haas-type palatal expanders activated in different frequencies of activation on the positioning of the maxillary first permanent molars and on the buccal alveolar bones of these teeth with cone-beam computerized tomography. The sample consisted of 33 children (18 girls, 15 boys; mean age, 9 years) randomly distributed into 2 groups: rapid maxillary expansion (n = 17) and slow maxillary expansion (n = 16). Patients in the rapid maxillary expansion group received 2 turns of activation (0.4 mm) per day, and those in the slow maxillary expansion group received 2 turns of activation (0.4 mm) per week until 8 mm of expansion was achieved in both groups. Cone-beam computerized tomography images were taken before treatment and after stabilization of the jackscrews. Data were gathered through a standardized analysis of cone-beam computerized tomography images. Intragroup statistical analysis was accomplished with the Wilcoxon matched-pairs test, and intergroup statistical analysis was accomplished with analysis of variance. Linear relationships, among all variables, were determined by Spearman correlation. Both protocols caused buccal displacement of the maxillary first permanent molars, which had more bodily displacement in the slow maxillary expansion group, whereas more inclination was observed in the rapid maxillary expansion group. Vertical and horizontal bone losses were found in both groups; however, the slow maxillary expansion group had major bone loss. Periodontal modifications in both groups should be carefully considered because of the reduction of spatial resolution in the cone-beam computerized tomography examinations after stabilization of the jackscrews. Modifications in the frequency of activation of the palatal expander might influence the dental and periodontal effects of palatal expansion. Copyright © 2013

  16. 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.

  17. Cell type-specific post-Golgi apparatus localization of a "resident" endoplasmic reticulum glycoprotein, glucosidase II

    OpenAIRE

    1990-01-01

    Glucosidase II, an asparagine-linked oligosaccharide processing enzyme, is a resident glycoprotein of the endoplasmic reticulum. In kidney tubular cells, in contrast to previous findings on hepatocytes, we found by light and electron microscopy immunoreactivity for glucosidase II predominantly in post-Golgi apparatus structures. The majority of immunolabel was in endocytotic structures beneath the plasma membrane. Immunoprecipitation confirmed presence of the glucosidase II subunit in purifie...

  18. Binding of AP-1 Golgi adaptors to membranes requires phosphorylated cytoplasmic domains of the mannose 6-phosphate/insulin-like growth factor II receptor.

    Science.gov (United States)

    Le Borgne, R; Schmidt, A; Mauxion, F; Griffiths, G; Hoflack, B

    1993-10-25

    In mammalian cells, clathrin-coated vesicles mediate transport of the lysosomal enzyme receptors from the trans-Golgi network to the endocytic pathway. A critical step of this process is the recruitment of Golgi-specific adaptors onto Golgi membranes for efficient clathrin polymerization. An in vitro assay was used here to quantitate this event in streptolysin-O-permeabilized NRK cells. At 37 degrees C, these interactions are cytosol- and energy-dependent, sensitive to GTP gamma S (guanosine 5'-O-(thiotriphosphate)) and brefeldin A. We report that Golgi-specific adaptor binding is enhanced in mannose 6-phosphate/insulin-like growth factor II (IGF II) receptor-overexpressing cells and reduced in mannose 6-phosphate receptor-deficient cells. Furthermore, adaptor binding is partially inhibited after addition of soluble cytoplasmic domains of the mannose 6-phosphate/IGF II receptor. Almost complete inhibition is only observed when this domain is phosphorylated on serines 2421 and 2492, a major modification acquired during exit of the receptor from the Golgi. These results show that the mannose 6-phosphate/IGF II receptor is part of the components that recruit the Golgi-specific adaptors and that its phosphorylation is an important feature for high affinity interactions with sorting components.

  19. Actin remodeling by ADF/cofilin is required for cargo sorting at the trans-Golgi network.

    Science.gov (United States)

    von Blume, Julia; Duran, Juan M; Forlanelli, Elena; Alleaume, Anne-Marie; Egorov, Mikhail; Polishchuk, Roman; Molina, Henrik; Malhotra, Vivek

    2009-12-28

    Knockdown of the actin-severing protein actin-depolymerizing factor (ADF)/cofilin inhibited export of an exogenously expressed soluble secretory protein from Golgi membranes in Drosophila melanogaster and mammalian tissue culture cells. A stable isotope labeling by amino acids in cell culture mass spectrometry-based protein profiling revealed that a large number of endogenous secretory proteins in mammalian cells were not secreted upon ADF/cofilin knockdown. Although many secretory proteins were retained, a Golgi-resident protein and a lysosomal hydrolase were aberrantly secreted upon ADF/cofilin knockdown. Overall, our findings indicate that inactivation of ADF/cofilin perturbed the sorting of a subset of both soluble and integral membrane proteins at the trans-Golgi network (TGN). We suggest that ADF/cofilin-dependent actin trimming generates a sorting domain at the TGN, which filters secretory cargo for export, and that uncontrolled growth of this domain causes missorting of proteins. This type of actin-dependent compartmentalization and filtering of secretory cargo at the TGN by ADF/cofilin could explain sorting of proteins that are destined to the cell surface.

  20. Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle.

    Science.gov (United States)

    Hallée, Stéphanie; Richard, Dave

    2015-01-01

    Invasion of a red blood cell by Plasmodium falciparum merozoites is an essential step in the malaria lifecycle. Several of the proteins involved in this process are stored in the apical complex of the merozoite, a structure containing secretory organelles that are released at specific times during invasion. The molecular players involved in erythrocyte invasion thus represent potential key targets for both therapeutic and vaccine-based strategies to block parasite development. In our quest to identify and characterize new effectors of invasion, we investigated the P. falciparum homologue of a P. berghei protein putatively localiz