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Sample records for activity-dependent bulk endocytosis

  1. The Molecular Physiology of Activity-Dependent Bulk Endocytosis of Synaptic Vesicles

    Clayton, Emma L.; Cousin, Michael A.

    2010-01-01

    Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Since maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions single SV retrieval modes such as clathrin-mediated endocytosis (CME) predominate. However during increased neuronal activity additional SV retrieval capacity is required, which is provided by activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mechanism during elevated neuronal activity. It is a high capacity SV retrieval mode that is immediately triggered during such stimulation conditions. This review will summarise the current knowledge regarding the molecular mechanism of ADBE, including molecules required for its triggering and subsequent steps, including SV budding from bulk endosomes. The molecular relationship between ADBE and the SV reserve pool will also be discussed. It is becoming clear that an understanding of the molecular physiology of ADBE will be of critical importance in attempts to modulate both normal and abnormal synaptic function during intense neuronal activity. PMID:19765184

  2. The molecular physiology of activity-dependent bulk endocytosis of synaptic vesicles.

    Clayton, E. L.; Cousin, M. A.

    2009-01-01

    Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Because maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions, single SV retrieval modes such as clathrin-mediated endocytosis predominate. However, during increased neuronal activity, additional SV retrieval capacit...

  3. The Molecular Physiology of Activity-Dependent Bulk Endocytosis of Synaptic Vesicles

    Clayton, Emma L.; Cousin, Michael A

    2009-01-01

    Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Since maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions single SV retrieval modes such as clathrin-mediated endocytosis (CME) predominate. However during increased neuronal activity additional SV retrieval capaci...

  4. Activity-dependent acceleration of endocytosis at a central synapse.

    Wu, Wei; Xu, Jianhua; Wu, Xin-Sheng; Wu, Ling-Gang

    2005-12-14

    Accumulated evidence indicates the existence of rapid and slow endocytosis at many synapses. It has been proposed that rapid endocytosis is activated by intense stimulation when vesicle recycling needs to be speeded up to supply vesicles at hippocampal synapses. However, the evidence, as obtained with imaging techniques, which are somewhat indirect in indicating rapid endocytosis, is controversial. Furthermore, a slower time course of endocytosis is often found after more intense nerve activity, casting doubt on the role of rapid endocytosis at synapses. Here, we addressed this issue at a mammalian central synapse, the calyx of Held, using a capacitance measurement technique that provides a higher time resolution than imaging techniques. We found that rapid endocytosis with a time constant of approximately 1-2 s was activated during intense nerve activity. Reducing the presynaptic calcium current or buffering the intracellular calcium with EGTA significantly inhibited rapid endocytosis, suggesting that calcium triggers rapid endocytosis. During intense stimulation, rapid endocytosis retrieved up to approximately eight vesicles per second per active zone, approximately eightfold larger than reported in the hippocampus, and thus played a dominant role during and within 3 s after intense stimulation. Slow endocytosis became dominant 3 s after intense stimulation likely because of the fall of the intracellular calcium level that deactivated rapid endocytosis. These results underscore the importance of calcium-triggered rapid endocytosis, which offers the nerve terminal the plasticity to speed up vesicle cycling during intense nerve activity. PMID:16354926

  5. CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors.

    Loebrich, Sven; Benoit, Marc Robert; Konopka, Jaclyn Aleksandra; Cottrell, Jeffrey Richard; Gibson, Joanne; Nedivi, Elly

    2016-02-01

    Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors. PMID:26776730

  6. Endocytosis in filamentous fungi

    Kalkman, Edward R I C

    2007-01-01

    Endocytosis is little understood in filamentous fungi. For some time it has been controversial as to whether endocytosis occurs in filamentous fungi. A comparative genomics analysis between Saccharomyces cerevisiae and 10 genomes of filamentous fungal species showed that filamentous fungi possess complex endocytic machineries. The use of the endocytic marker dye FM4-64, and various vesicle trafficking inhibitors revealed many similarities between endocytosis in the filamentous ...

  7. Exocytosis and Endocytosis: Modes, Functions, and Coupling Mechanisms*

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

    2016-01-01

    Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis. PMID:24274740

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

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

    2014-01-01

    Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis. PMID:24274740

  9. Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

    Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

    2016-07-01

    At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as

  10. A neurotoxic phospholipase A2 impairs yeast amphiphysin activity and reduces endocytosis.

    Mojca Mattiazzi

    Full Text Available BACKGROUND: Presynaptically neurotoxic phospholipases A(2 inhibit synaptic vesicle recycling through endocytosis. PRINCIPAL FINDINGS: Here we provide insight into the action of a presynaptically neurotoxic phospholipase A(2 ammodytoxin A (AtxA on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin. CONCLUSIONS: We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein-protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A(2 work can open new ways to regulate endocytosis.

  11. Endocytosis of influenza viruses

    Lakadamyali, Melike; Rust, Michael J.; Zhuang, Xiaowei

    2004-01-01

    Receptor-mediated endocytosis is known to play an important role in the entry of many viruses into host cells. However, the exact internalization mechanism has, until recently, remained poorly understood for many medically important viruses, including influenza. Developments in real-time imaging of single viruses as well as the use of dominant negative mutants to selectively block specific endocytic pathways, have improved our understanding of the influenza infection process.

  12. Endosome-mediated endocytic mechanism replenishes the majority of synaptic vesicles at mature CNS synapses in an activity-dependent manner.

    Park, Joohyun; Cho, Oh Yeon; Kim, Jung Ah; Chang, Sunghoe

    2016-01-01

    Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity. PMID:27534442

  13. Endosomes Derived from Clathrin-Independent Endocytosis Serve as Precursors for Endothelial Lumen Formation

    Porat-Shliom, Natalie; Weigert, Roberto; Donaldson, Julie G.

    2013-01-01

    Clathrin-independent endocytosis (CIE) is a form of bulk plasma membrane (PM) endocytosis that allows cells to sample and evaluate PM composition. Once in endosomes, the internalized proteins and lipids can be recycled back to the PM or delivered to lysosomes for degradation. Endosomes arising from CIE contain lipid and signaling molecules suggesting that they might be involved in important biological processes. During vasculogenesis, new blood vessels are formed from precursor cells in a pro...

  14. Ricin transport into cells: studies of endocytosis and intercellular transport

    Sandvig, Kirsten; Grimmer, S.; Iversen, T.G.; Rodal, S.K.; Torgersen, Maria Lyngaas; Nicoziani, Paolo; van Deurs, Bo

    Cell Biology, ricin, endocytosis, Golgi apparatus, cholesterol, clathrin, toxin, Rab, endoplasmic reticulum......Cell Biology, ricin, endocytosis, Golgi apparatus, cholesterol, clathrin, toxin, Rab, endoplasmic reticulum...

  15. Multiple Functions of Sterols in Yeast Endocytosis

    Heese-Peck, Antje; Pichler, Harald; Zanolari, Bettina; Watanabe, Reika; Daum, Günther; Riezman, Howard

    2002-01-01

    Sterols are essential factors for endocytosis in animals and yeast. To investigate the sterol structural requirements for yeast endocytosis, we created a variety of ergΔ mutants, each accumulating a distinct set of sterols different from ergosterol. Mutant erg2Δerg6Δ and erg3Δerg6Δ cells exhibit a strong internalization defect of the α-factor receptor (Ste2p). Specific sterol structures are necessary for pheromone-dependent receptor hyperphosphorylation, a prerequisite for internalization. Th...

  16. Activity Dependent Regulation of Inhibitory Circuitry

    Sharma, Nikhil

    2015-01-01

    Inhibition controls information flow through a neural circuit by modulating synaptic integration, restricting action potentials, and coordinating the activity of ensembles of neurons. These functions are mediated by a diverse array of inhibitory neuron subtypes that synapse on defined domains of a postsynaptic neuron. Activity-dependent transcription controls inhibitory synapse number and function, but how this transcription program affects the inhibitory inputs that form on di...

  17. Nanomechanics of magnetically driven cellular endocytosis

    Zablotskyy, Vitaliy A.; Lunov, O.; Dejneka, Alexandr; Jastrabík, Lubomír; Polyakova, T.; Syrovets, T.; Simmet, T.

    2011-01-01

    Roč. 99, č. 18 (2011), 183701/1-183701/3. ISSN 0003-6951 R&D Projects: GA AV ČR KAN301370701; GA MŠk(CZ) 1M06002 Institutional research plan: CEZ:AV0Z10100522 Keywords : magnetically controled endocytosis * cell membranes * iron oxide nanoparticles Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 3.844, year: 2011

  18. Endosomes derived from clathrin-independent endocytosis serve as precursors for endothelial lumen formation.

    Natalie Porat-Shliom

    Full Text Available Clathrin-independent endocytosis (CIE is a form of bulk plasma membrane (PM endocytosis that allows cells to sample and evaluate PM composition. Once in endosomes, the internalized proteins and lipids can be recycled back to the PM or delivered to lysosomes for degradation. Endosomes arising from CIE contain lipid and signaling molecules suggesting that they might be involved in important biological processes. During vasculogenesis, new blood vessels are formed from precursor cells in a process involving internalization and accumulation of endocytic vesicles. Here, we found that CIE has a role in endothelial lumen formation. Specifically, we found that human vascular endothelial cells (HUVECs utilize CIE for internalization of distinct cargo molecules and that in three-dimensional cultures CIE membranes are delivered to the newly formed lumen.

  19. Interaction among Saccharomyces cerevisiae pheromone receptors during endocytosis

    Chien-I Chang

    2014-03-01

    Full Text Available This study investigates endocytosis of Saccharomyces cerevisiae α-factor receptor and the role that receptor oligomerization plays in this process. α-factor receptor contains signal sequences in the cytoplasmic C-terminal domain that are essential for ligand-mediated endocytosis. In an endocytosis complementation assay, we found that oligomeric complexes of the receptor undergo ligand-mediated endocytosis when the α-factor binding site and the endocytosis signal sequences are located in different receptors. Both in vitro and in vivo assays suggested that ligand-induced conformational changes in one Ste2 subunit do not affect neighboring subunits. Therefore, recognition of the endocytosis signal sequence and recognition of the ligand-induced conformational change are likely to be two independent events.

  20. An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.

    Soheil Aghamohammadzadeh

    Full Text Available Clathrin-mediated endocytosis (CME is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

  1. An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.

    Aghamohammadzadeh, Soheil; Smaczynska-de Rooij, Iwona I; Ayscough, Kathryn R

    2014-01-01

    Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions. PMID:25072293

  2. Cell mobility after endocytosis of carbon nanotubes

    Pirbhai, Massooma; Flores, Thomas; Jedlicka, Sabrina; Rotkin, Slava

    2013-03-01

    Directed cell movement plays a crucial role in cellular behaviors such as neuronal cell division, cell migration, and cell differentiation. There is evidence in preclinical in vivo studies that small fields have successfully been used to enhance regrowth of damages spinal cord axons but with a small success rate. Fortunately, the evolution of functional biomaterials and nanotechnology may provide promising solutions for enhancing the application of electric fields in guiding neuron migration and neurogenesis within the central nervous system. In this work, we studied how endocytosis and subsequent retention of carbon nanotubes affects the mobility of cells under the influence of an electric field, including the directed cell movement.

  3. Nanomechanics of magnetically driven cellular endocytosis

    Zablotskii, V.; Lunov, O.; Dejneka, A.; Jastrabík, L.; Polyakova, T.; Syrovets, T.; Simmet, Th.

    2011-10-01

    Being essential for many pharmacodynamic and pharmacokinetic processes and playing a crucial role in regulating substrate detachment that enables cellular locomotion, endocytotic mechanisms in many aspects still remain a mystery and therefore can hardly be controlled. Here, we report on experimental and modeling studies of the magnetically assisted endocytosis of functionalized superparamagnetic iron oxide nanoparticles by prostate cancer cells (PC-3) and characterize the time and force scales of the cellular uptake machinery. The results indicate how the cellular uptake rate could be controlled by applied magnetic field, membrane elasticity, and nanoparticle magnetic moment.

  4. Yeast Exocytic v-SNAREs Confer Endocytosis

    Gurunathan, Sangiliyandi; Chapman-Shimshoni, Daphne; Trajkovic, Selena; Gerst, Jeffrey E.

    2000-01-01

    In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1 (SNC1ala43), are deficient in the endocytic uptake of components from the cell surface. We found that both SNC and temperature-shifted SNC1ala43 yeast are d...

  5. Exocytosis and endocytosis in juxtaglomerular cells.

    Friis, U G; Jensen, B L; Hansen, P B; Andreasen, D; Skøtt, O

    2000-01-01

    The cellular events related to secretion of renin are not well understood. Here we review some of the evidence that has led to the current understanding of renin secretion as a process that involves exocytosis as the predominant mode of secretion. This is based on the observation of occasional fusion events between secretory granules and cell membrane and measurement of intermittent secretion of renin from single afferent arterioles, with a renin content of each secretion episode that corresponds to the renin content of one secretory granule. More recently it has been demonstrated that the afferent arterioles lose a large number of renin granules after acute stimulation without changing the average granular volume. Current electrophysiological techniques have now permitted direct measurements of cell membrane capacitance in juxtaglomerular (JG) cells as a measure of net addition (exocytosis) or removal (endocytosis) of membrane material. With this technique we have shown that cAMP, which is a vasodilator and stimulates renin secretion, enhances net exocytosis at low concentrations, while at higher concentrations membrane retrieval processes are also stimulated. We suggest that both exocytosis and endocytosis are regulated processes in the JG-cells and both may be important for the long-term control of renin secretion at the single cell level. PMID:10691785

  6. Endocytosis of Integrin-Binding Human Picornaviruses

    Pirjo Merilahti

    2012-01-01

    Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  7. Endocytosis and Membrane Turnover in the Germ Tube of Uromyces fabae

    Hoffmann, Jochen; Mendgen, Kurt

    1998-01-01

    We have used the fluorescent dye FM4-64 as a tracer to demonstrate bulk membrane internalization (endocytosis) and redistribution of the dye within the cytoplasm of the germ tube of the rust fungus Uromyces fabae. Staining of the hyphal membrane was detected 4 s after application of FM4-64 and reached a maximum after 1 min. The highest fluorescence intensity occurred in the apex. Subsequently, staining of the plasma membrane decreased and a subapical region of the fungal protoplast (5 20 µm f...

  8. Differential requirements for actin during yeast and mammalian endocytosis.

    Aghamohammadzadeh, Soheil; Ayscough, Kathryn R

    2009-08-01

    Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination. PMID:19597484

  9. Clathrin-independent endocytosis: mechanisms and function

    Sandvig, Kirsten; Pust, Sascha; Skotland, Tore;

    2011-01-01

    It is now about 20 years since we first wrote reviews about clathrin-independent endocytosis. The challenge at the time was to convince the reader about its existence. Then the suggestion came up that caveolae might be responsible for the uptake. However, clearly this could not be the case since a...... large fraction of the clathrin-independent uptake is dynamin-independent. Today, two decades later, the field has developed considerably. New techniques have enabled a detailed analysis of several clathrin-independent endocytic mechanisms, and caveolae have been found to be mostly stable structures...... having several functions of their own. This article aims at providing a brief update on the importance of clathrin-independent endocytic mechanisms, how the processes are regulated differentially, for instance on the poles of polarized cells, and the challenges in studying them....

  10. Exocytosis and endocytosis in juxtaglomerular cells

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

    2000-01-01

    that the afferent arterioles lose a large number of renin granules after acute stimulation without changing the average granular volume. Current electrophysiological techniques have now permitted direct measurements of cell membrane capacitance in juxtaglomerular (JG) cells as a measure of net addition (exocytosis...... fusion events between secretory granules and cell membrane and measurement of intermittent secretion of renin from single afferent arterioles, with a renin content of each secretion episode that corresponds to the renin content of one secretory granule. More recently it has been demonstrated...... and endocytosis are regulated processes in the JG-cells and both may be important for the long-term control of renin secretion at the single cell level....

  11. Imaging the Dynamics of Endocytosis in Live Mammalian Tissues

    Weigert, Roberto

    2014-01-01

    In mammalian cells, endocytosis plays a pivotal role in regulating several basic cellular functions. Up to now, the dynamics and the organization of the endocytic pathways have been primarily investigated in reductionist model systems such as cell and organ cultures. Although these experimental models have been fully successful in unraveling the endocytic machinery at a molecular level, our understanding of the regulation and the role of endocytosis in vivo has been limited. Recently, advance...

  12. Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds

    Pagnussat, Luciana; Burbach, Christian; Baluška, František; de la Canal, Laura

    2012-01-01

    During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4–64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunoloc...

  13. Ankyrin-G Inhibits Endocytosis of Cadherin Dimers.

    Cadwell, Chantel M; Jenkins, Paul M; Bennett, Vann; Kowalczyk, Andrew P

    2016-01-01

    Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions. PMID:26574545

  14. Clathrin-mediated endocytosis: the gateway into plant cells.

    Chen, Xu; Irani, Niloufer G; Friml, Jiří

    2011-12-01

    Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, hormonal signaling and communication with the environment including nutrient delivery, toxin avoidance, and pathogen defense. The major endocytic mechanism in plants depends on the coat protein clathrin. It starts by clathrin-coated vesicle formation at the plasma membrane, where specific cargoes are recognized and packaged for internalization. Recently, genetic, biochemical and advanced microscopy studies provided initial insights into mechanisms and roles of clathrin-mediated endocytosis in plants. Here we summarize the present state of knowledge and compare mechanisms of clathrin-mediated endocytosis in plants with animal and yeast paradigms as well as review plant-specific regulations and roles of this process. PMID:21945181

  15. Bulk undercooling

    Kattamis, T. Z.

    1984-01-01

    Bulk undercooling methods and procedures will first be reviewed. Measurement of various parameters which are necessary to understand the solidification mechanism during and after recalescence will be discussed. During recalescence of levitated, glass-encased large droplets (5 to 8 mm diam) high speed temperature sensing devices coupled with a rapid response oscilloscope are now being used at MIT to measure local thermal behavior in hypoeutectic and eutectic binary Ni-Sn alloys. Dendrite tip velocities were measured by various investigators using thermal sensors or high speed cinematography. The confirmation of the validity of solidification models of bulk-undercooled melts is made difficult by the fineness of the final microstructure, the ultra-rapid evolution of the solidifying system which makes measurements very awkward, and the continuous modification of the microstructure which formed during recalescence because of precipitation, remelting and rapid coarsening.

  16. A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15

    1996-01-01

    A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and ...

  17. Cdk5 is essential for synaptic vesicle endocytosis

    Tan, Timothy C; Valova, Valentina A; Malladi, Chandra S;

    2003-01-01

    Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynami...

  18. OUABAIN AND INSULIN INDUCE SODIUM PUMP ENDOCYTOSIS IN RENAL EPITHELIUM

    Gupta, Shalini; Yan, Yanling; Malhotra, Deepak; Liu, Jiang; Xie, Zijian; Najjar, Sonia M.; Shapiro, Joseph I

    2012-01-01

    Cardiotonic steroids signaling through the basolateral sodium pump (Na/KATPase) have been shown to alter renal salt handling in intact animals. As the relationship between renal salt handling and blood pressure is a key determinant of hypertension, and patients with insulin resistance are frequently hypertensive, we chose to examine whether there might be competition for resources necessary for receptor mediated endocytosis.

  19. Bladder uptake of liposomes after intravesical administration occurs by endocytosis.

    Bharathi Raja Rajaganapathy

    Full Text Available Liposomes have been used therapeutically and as a local drug delivery system in the bladder. However, the exact mechanism for the uptake of liposomes by bladder cells is unclear. In the present study, we investigated the role of endocytosis in the uptake of liposomes by cultured human UROtsa cells of urothelium and rat bladder. UROtsa cells were incubated in serum-free media with liposomes containing colloidal gold particles for 2 h either at 37°C or at 4°C. Transmission Electron Microscopy (TEM images of cells incubated at 37°C found endocytic vesicles containing gold inside the cells. In contrast, only extracellular binding was noticed in cells incubated with liposomes at 4°C. Absence of liposome internalization at 4°C indicates the need of energy dependent endocytosis as the primary mechanism of entry of liposomes into the urothelium. Flow cytometry analysis revealed that the uptake of liposomes at 37°C occurs via clathrin mediated endocytosis. Based on these observations, we propose that clathrin mediated endocytosis is the main route of entry for liposomes into the urothelial layer of the bladder and the findings here support the usefulness of liposomes in intravesical drug delivery.

  20. Constitutive Endocytosis of VEGFR2 Protects the Receptor against Shedding.

    Basagiannis, Dimitris; Christoforidis, Savvas

    2016-08-01

    VEGFR2 plays a fundamental role in blood vessel formation and in life threatening diseases, such as cancer angiogenesis and cardiovascular disorders. Although inactive growth factor receptors are mainly localized at the plasma membrane, VEGFR2 undergoes constitutive endocytosis (in the absence of ligand) and recycling. Intriguingly, the significance of these futile transport cycles of VEGFR2 remains unclear. Here we found that, unexpectedly, the function of constitutive endocytosis of VEGFR2 is to protect the receptor against plasma membrane cleavage (shedding), thereby preserving the functional state of the receptor until the time of activation by VEGF. Inhibition of constitutive endocytosis of VEGFR2, by interference with the function of clathrin, dynamin, or Rab5, increases dramatically the cleavage/shedding of VEGFR2. Shedding of VEGFR2 produces an N-terminal soluble fragment (100 kDa, s100), which is released in the extracellular space, and a residual C-terminal part (130 kDa, p130) that remains integrated at the plasma membrane. The released soluble fragment (s100) co-immunoprecipitates with VEGF, in line with the topology of the VEGF-binding domain at the N terminus of VEGFR2. Increased shedding of VEGFR2 (via inhibition of constitutive endocytosis) results in reduced response to VEGF, consistently with the loss of the VEGF-binding domain from the membrane remnant of VEGFR2. These data suggest that constitutive internalization of VEGFR2 protects the receptor against shedding and provides evidence for an unprecedented mechanism via which endocytosis can regulate the fate and activity of growth factor receptors. PMID:27298320

  1. Activity-dependent plasticity of hippocampal place maps.

    Schoenenberger, Philipp; O'Neill, Joseph; Csicsvari, Jozsef

    2016-01-01

    Hippocampal neurons encode a cognitive map of space. These maps are thought to be updated during learning and in response to changes in the environment through activity-dependent synaptic plasticity. Here we examine how changes in activity influence spatial coding in rats using halorhodopsin-mediated, spatially selective optogenetic silencing. Halorhoposin stimulation leads to light-induced suppression in many place cells and interneurons; some place cells increase their firing through disinhibition, whereas some show no effect. We find that place fields of the unaffected subpopulation remain stable. On the other hand, place fields of suppressed place cells were unstable, showing remapping across sessions before and after optogenetic inhibition. Disinhibited place cells had stable maps but sustained an elevated firing rate. These findings suggest that place representation in the hippocampus is constantly governed by activity-dependent processes, and that disinhibition may provide a mechanism for rate remapping. PMID:27282121

  2. Activity-dependent modulation of neural circuit synaptic connectivity

    Tessier, Charles R.; Kendal Broadie

    2009-01-01

    In many nervous systems, the establishment of neural circuits is known to proceed via a two-stage process; 1) early, activity-independent wiring to produce a rough map characterized by excessive synaptic connections, and 2) subsequent, use-dependent pruning to eliminate inappropriate connections and reinforce maintained synapses. In invertebrates, however, evidence of the activity-dependent phase of synaptic refinement has been elusive, and the dogma has long been that invertebrate circ...

  3. Activity-dependent modulation of neural circuit synaptic connectivity

    Charles R Tessier

    2009-07-01

    Full Text Available In many nervous systems, the establishment of neural circuits is known to proceed via a two-stage process; 1 early, activity-independent wiring to produce a rough map characterized by excessive synaptic connections, and 2 subsequent, use-dependent pruning to eliminate inappropriate connections and reinforce maintained synapses. In invertebrates, however, evidence of the activity-dependent phase of synaptic refinement has been elusive, and the dogma has long been that invertebrate circuits are “hard-wired” in a purely activity-independent manner. This conclusion has been challenged recently through the use of new transgenic tools employed in the powerful Drosophila system, which have allowed unprecedented temporal control and single neuron imaging resolution. These recent studies reveal that activity-dependent mechanisms are indeed required to refine circuit maps in Drosophila during precise, restricted windows of late-phase development. Such mechanisms of circuit refinement may be key to understanding a number of human neurological diseases, including developmental disorders such as Fragile X syndrome (FXS and autism, which are hypothesized to result from defects in synaptic connectivity and activity-dependent circuit function. This review focuses on our current understanding of activity-dependent synaptic connectivity in Drosophila, primarily through analyzing the role of the fragile X mental retardation protein (FMRP in the Drosophila FXS disease model. The particular emphasis of this review is on the expanding array of new genetically-encoded tools that are allowing cellular events and molecular players to be dissected with ever greater precision and detail.

  4. Activity-dependent neuronal signalling and autism spectrum disorder

    Ebert, Daniel H.; Greenberg, Michael E.

    2013-01-01

    Neuronal activity induces the post-translational modification of synaptic molecules, promotes localized protein synthesis within dendrites and activates gene transcription, thereby regulating synaptic function and allowing neuronal circuits to respond dynamically to experience. Evidence indicates that many of the genes that are mutated in autism spectrum disorders are crucial components of the activity-dependent signalling networks that regulate synapse development and plasticity. Dysregulati...

  5. Membrane Mechanics of Endocytosis in Cells with Turgor

    Dmitrieff, Serge

    2015-01-01

    Endocytosis is an essential process by which cells internalize a piece of plasma membrane and material from the outside. In cells with turgor, pressure opposes membrane defor- mations, and increases the amount of force that has to be generated by the endocytic machinery. To determine this force, and calculate the shape of the membrane, we used physical theory to model an elastic surface under pressure. Accurate fits of experimental profiles are obtained assuming that the coated membrane is highly rigid and preferentially curved at the endocytic site. The forces required from the actin machinery peaks at the onset of deformation, indicating that once invagination has been initiated, endocytosis is unlikely to stall before completion. Coat proteins do not lower the initiation force but may affect the process by the curvature they induce. In the presence of isotropic curvature inducers, pulling the tip of the invagination can trigger the formation of a neck at the base of the invagination. Hence direct neck cons...

  6. Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

    Singh, Bijay; Maharjan, Sushila; Kim, You-Kyoung; Jiang, Tai; Islam, Mohammad Ariful; Kang, Sang-Kee; Cho, Myung-Haing; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-01

    Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational

  7. Signaling induced by hop/STI-1 depends on endocytosis

    The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrPC), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrPC and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter's activity. The data indicate that signaling induced by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrPC by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases

  8. A memristor SPICE model accounting for synaptic activity dependence.

    Qingjiang Li

    Full Text Available In this work, we propose a new memristor SPICE model that accounts for the typical synaptic characteristics that have been previously demonstrated with practical memristive devices. We show that this model could account for both volatile and non-volatile memristance changes under distinct stimuli. We then demonstrate that our model is capable of supporting typical STDP with simple non-overlapping digital pulse pairs. Finally, we investigate the capability of our model to simulate the activity dependence dynamics of synaptic modification and present simulated results that are in excellent agreement with biological results.

  9. A role for the dynamin-like protein Vps1 during endocytosis in yeast

    Rooij, Iwona I. Smaczynska-de; Allwood, Ellen G.; Aghamohammadzadeh, Soheil; Hettema, Ewald H.; Goldberg, Martin W.; Ayscough, Kathryn R.

    2010-01-01

    Dynamins are a conserved family of proteins involved in membrane fusion and fission. Although mammalian dynamins are known to be involved in several membrane-trafficking events, the role of dynamin-1 in endocytosis is the best-characterised role of this protein family. Despite many similarities between endocytosis in yeast and mammalian cells, a comparable role for dynamins in yeast has not previously been demonstrated. The reported lack of involvement of dynamins in yeast endocytosis has rai...

  10. Endocytosis Is Crucial for Cell Polarity and Apical Membrane Recycling in the Filamentous Fungus Aspergillus oryzae▿

    Higuchi, Yujiro; Shoji, Jun-ya; Arioka, Manabu; Kitamoto, Katsuhiko

    2008-01-01

    Establishing the occurrence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis. Recently, however, it was shown that the fluorescent dye N-(3-triethylammoniumpropyl)-4-(p-diethyl-aminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) and the plasma membrane protein AoUapC (Aspergillus oryzae UapC) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis. Although the occurr...

  11. Activity-dependent neuronal model on complex networks.

    de Arcangelis, Lucilla; Herrmann, Hans J

    2012-01-01

    Neuronal avalanches are a novel mode of activity in neuronal networks, experimentally found in vitro and in vivo, and exhibit a robust critical behavior: these avalanches are characterized by a power law distribution for the size and duration, features found in other problems in the context of the physics of complex systems. We present a recent model inspired in self-organized criticality, which consists of an electrical network with threshold firing, refractory period, and activity-dependent synaptic plasticity. The model reproduces the critical behavior of the distribution of avalanche sizes and durations measured experimentally. Moreover, the power spectra of the electrical signal reproduce very robustly the power law behavior found in human electroencephalogram (EEG) spectra. We implement this model on a variety of complex networks, i.e., regular, small-world, and scale-free and verify the robustness of the critical behavior. PMID:22470347

  12. Activity-dependent neural plasticity from bench to bedside.

    Ganguly, Karunesh; Poo, Mu-Ming

    2013-10-30

    Much progress has been made in understanding how behavioral experience and neural activity can modify the structure and function of neural circuits during development and in the adult brain. Studies of physiological and molecular mechanisms underlying activity-dependent plasticity in animal models have suggested potential therapeutic approaches for a wide range of brain disorders in humans. Physiological and electrical stimulations as well as plasticity-modifying molecular agents may facilitate functional recovery by selectively enhancing existing neural circuits or promoting the formation of new functional circuits. Here, we review the advances in basic studies of neural plasticity mechanisms in developing and adult nervous systems and current clinical treatments that harness neural plasticity, and we offer perspectives on future development of plasticity-based therapy. PMID:24183023

  13. Human SCARB2-mediated entry and endocytosis of EV71.

    Yi-Wen Lin

    Full Text Available Enterovirus (EV 71 infection is known to cause hand-foot-and-mouth disease (HFMD and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2 has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2 to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus.

  14. Clathrin-mediated endocytosis in budding yeast at a glance.

    Lu, Rebecca; Drubin, David G; Sun, Yidi

    2016-04-15

    Clathrin-mediated endocytosis is an essential cellular process that involves the concerted assembly and disassembly of many different proteins at the plasma membrane. In yeast, live-cell imaging has shown that the spatiotemporal dynamics of these proteins is highly stereotypical. Recent work has focused on determining how the timing and functions of endocytic proteins are regulated. In this Cell Science at a Glance article and accompanying poster, we review our current knowledge of the timeline of endocytic site maturation and discuss recent works focusing on how phosphorylation, ubiquitylation and lipids regulate various aspects of the process. PMID:27084361

  15. Clostridial Glucosylating Toxins Enter Cells via Clathrin-Mediated Endocytosis

    Papatheodorou, Panagiotis; Zamboglou, Constantinos; Genisyuerek, Selda; Guttenberg, Gregor; Aktories, Klaus

    2010-01-01

    Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi α-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and α-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an ...

  16. The use of FM dyes to analyze plant endocytosis

    Malínská, Kateřina; Jelínková, Adriana; Petrášek, Jan

    Vol. 1209. Totowa: Humana Press, 2014 - (Otegui, M.), s. 1-11. (Methods in Molecular Biology). ISBN 978-1-4939-1419-7 R&D Projects: GA ČR(CZ) GAP305/11/2476; GA ČR GPP305/11/P797 Institutional support: RVO:61389030 Keywords : Endocytosis * Endomembranes * FM 1-43 Subject RIV: EB - Genetics ; Molecular Biology http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Alerting&SrcApp=Alerting&DestApp=MEDLINE&DestLinkType=FullRecord&UT=25117271

  17. Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds.

    Pagnussat, Luciana; Burbach, Christian; Baluška, František; de la Canal, Laura

    2012-03-01

    During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4-64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination. PMID:22476454

  18. Ricin A chain reaches the endoplasmic reticulum after endocytosis

    Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purified by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum

  19. Bile acids reduce endocytosis of high-density lipoprotein (HDL in HepG2 cells.

    Clemens Röhrl

    Full Text Available High-density lipoprotein (HDL transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence of high concentrations of taurocholate, a natural non-cell-permeable bile acid, in human hepatic HepG2 and HuH7 cells. In contrast, selective cholesteryl-ester (CE uptake was increased. Taurocholate exerted these effects extracellularly and independently of HDL modification, cell membrane perturbation or blocking of endocytic trafficking. Instead, this reduction of endocytosis and increase in selective uptake was dependent on SR-BI. In addition, cell-permeable bile acids reduced HDL endocytosis by farnesoid X receptor (FXR activation: chenodeoxycholate and the non-steroidal FXR agonist GW4064 reduced HDL endocytosis, whereas selective CE uptake was unaltered. Reduced HDL endocytosis by FXR activation was independent of SR-BI and was likely mediated by impaired expression of the scavenger receptor cluster of differentiation 36 (CD36. Taken together we have shown that bile acids reduce HDL endocytosis by transcriptional and non-transcriptional mechanisms. Further, we suggest that HDL endocytosis and selective lipid uptake are not necessarily tightly linked to each other.

  20. Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

    Wüstner, Daniel; Faergeman, Nils J

    2008-01-01

    regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin...

  1. The TPLATE Adaptor Complex Drives Clathrin-Mediated Endocytosis in Plants

    Gadeyne, A.; Sanchez-Rodriguez, C.; Rubbo, Di S.; Ketelaar, T.

    2014-01-01

    Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure.

  2. Solar activity dependence of nightside aurora in winter conditions

    Zhou, Su; Luan, Xiaoli; Dou, Xiankang

    2016-02-01

    The dependence of the nightside (21:00-03:00 MLT; magnetic local time) auroral energy flux on solar activity was quantitatively studied for winter/dark and geomagnetically quiet conditions. Using data combined from Thermosphere, Ionosphere, Mesosphere Energetics and Dynamics/Global Ultraviolet Imager and Defense Meteorological Satellite Program/Special Sensor Ultraviolet Spectrographic Imager observations, we separated the effects of geomagnetic activity from those of solar flux on the nightside auroral precipitation. The results showed that the nightside auroral power was reduced by ~42% in solar maximum (F10.7 = 200 sfu; solar flux unit 1 sfu = 10-22 W m-2 Hz-1) with respect to that under solar minimum (F10.7 = 70 sfu) for the Kp = 1 condition, and this change rate became less (~21%) for the Kp = 3 condition. In addition, the solar cycle dependence of nightside auroral power was similar with that from both the premidnight (21:00-23:00 MLT) and postmidnight (01:00-03:00 MLT) sectors. These results indicated that as the ionospheric ionization increases with the enhanced auroral and geomagnetic activities, the solar activity dependences of nightside auroral power become weaker, at least under geomagnetically quiet conditions.

  3. Shaping inhibition: activity dependent structural plasticity of GABAergic synapses

    Pablo Mendez

    2014-10-01

    Full Text Available Inhibitory transmission through the neurotransmitter Ɣ-aminobutyric acid (GABA shapes network activity in the mammalian cerebral cortex by filtering synaptic incoming information and dictating the activity of principal cells. The incredibly diverse population of cortical neurons that use GABA as neurotransmitter shows an equally diverse range of mechanisms that regulate changes in the strength of GABAergic synaptic transmission and allow them to dynamically follow and command the activity of neuronal ensembles. Similarly to glutamatergic synaptic transmission, activity-dependent functional changes in inhibitory neurotransmission are accompanied by alterations in GABAergic synapse structure that range from morphological reorganization of postsynaptic density to de novo formation and elimination of inhibitory contacts. Here we review several aspects of structural plasticity of inhibitory synapses, including its induction by different forms of neuronal activity, behavioral and sensory experience and the molecular mechanisms and signaling pathways involved. We discuss the functional consequences of GABAergic synapse structural plasticity for information processing and memory formation in view of the heterogenous nature of the structural plasticity phenomena affecting inhibitory synapses impinging on somatic and dendritic compartments of cortical and hippocampal neurons.

  4. Endocytosis of simian virus 40 into the endoplasmic reticulum

    The endocytosis of SV-40 into CV-1 cells we studied using biochemical and ultrastructural techniques. The half-time of binding of [35S]methionine-radiolabeled SV-40 to CV-1 cells was 25 min. Most of the incoming virus particles remained undegraded for several hours. Electron microscopy showed that some virus entered the endosomal/lysosomal pathway via coated vesicles, while the majority were endocytosed via small uncoated vesicles. After infection at high multiplicity, one third of total cell-associated virus was observed to enter the ER, starting 1-2 h after virus application. The viruses were present in large, tubular, smooth membrane networks generated as extentions of the ER. The results describe a novel and unique membrane transport pathway that allows endocytosed viral particles to be targeted from the plasma membrane to the ER

  5. Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis.

    Papatheodorou, Panagiotis; Zamboglou, Constantinos; Genisyuerek, Selda; Guttenberg, Gregor; Aktories, Klaus

    2010-01-01

    Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi alpha-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and alpha-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin. PMID:20498856

  6. Temperature effect on endocytosis and exocytosis by rabbit alveolar macrophages

    Endocytosis of 125I-mannose-bovine serum albumin (BSA) and exocytosis of 125I-mannose-poly-D-lysine by rabbit alveolar macrophages were examined as a function of temperature. A plot for total ligand uptake (cell-associated ligand plus degraded ligand) versus time shows a single inflection point at 20 degrees C. Ligand degradation does not occur below 20 degrees C. Internalization of surface-bound 125I-mannose-BSA is negligible below 10 degrees C. The rate constant for internalization increases dramatically above 20 degrees C: 0.02 min-1 at 20 degrees C, 0.05 min-1 at 25 degrees C, 0.13 min-1 at 30 degrees C, and 0.29 min-1 at 35 degrees C. 125I-Mannose-N-acetyl-poly-D-lysine preloaded in lysosomes is exocytosed in a temperature and time-dependent fashion. Even at lower temperatures (2-10 degrees C), secretion of 125I-mannose-N-acetyl-poly-D-lysine was detected, indicating that movement of lysosomal content to plasma membrane and beyond cannot be suppressed at these temperatures. Thus, the temperature dependence of exocytosis of an 125I-labeled ligand is quite different from that of endocytosis, suggesting that the two processes are controlled by different mechanisms. Stimulation of secretion of preloaded 125I-mannose-N-acetyl-poly-D-lysine by mannose-BSA was more pronounced at lower temperatures with a sharp inflection point at 10 degrees C. These findings suggest that endosomes containing newly internalized mannose-BSA interact with the exocytosis pathway and enhance secretion of 125I-mannose-N-acetyl-poly-D-lysine from lysosomes

  7. A role for the dynamin-like protein Vps1 during endocytosis in yeast.

    Smaczynska-de Rooij, Iwona I; Allwood, Ellen G; Aghamohammadzadeh, Soheil; Hettema, Ewald H; Goldberg, Martin W; Ayscough, Kathryn R

    2010-10-15

    Dynamins are a conserved family of proteins involved in membrane fusion and fission. Although mammalian dynamins are known to be involved in several membrane-trafficking events, the role of dynamin-1 in endocytosis is the best-characterised role of this protein family. Despite many similarities between endocytosis in yeast and mammalian cells, a comparable role for dynamins in yeast has not previously been demonstrated. The reported lack of involvement of dynamins in yeast endocytosis has raised questions over the general applicability of the current yeast model of endocytosis, and has also precluded studies using well-developed methods in yeast, to further our understanding of the mechanism of dynamin function during endocytosis. Here, we investigate the yeast dynamin-like protein Vps1 and demonstrate a transient burst of localisation to sites of endocytosis. Using live-cell imaging of endocytic reporters in strains lacking vps1, and also electron microscopy and biochemical approaches, we demonstrate a role for Vps1 in facilitating endocytic invagination. Vps1 mutants were generated, and analysis in several assays reveals a role for the C-terminal self-assembly domain in endocytosis but not in other membrane fission events with which Vps1 has previously been associated. PMID:20841380

  8. Clathrin- and dynamin-independent endocytosis of FGFR3--implications for signalling.

    Ellen Margrethe Haugsten

    Full Text Available Endocytosis of tyrosine kinase receptors can influence both the duration and the specificity of the signal emitted. We have investigated the mechanisms of internalization of fibroblast growth factor receptor 3 (FGFR3 and compared it to that of FGFR1 which is internalized predominantly through clathrin-mediated endocytosis. Interestingly, we observed that FGFR3 was internalized at a slower rate than FGFR1 indicating that it may use a different endocytic mechanism than FGFR1. Indeed, after depletion of cells for clathrin, internalization of FGFR3 was only partly inhibited while endocytosis of FGFR1 was almost completely abolished. Similarly, expression of dominant negative mutants of dynamin resulted in partial inhibition of the endocytosis of FGFR3 whereas internalization of FGFR1 was blocked. Interfering with proposed regulators of clathrin-independent endocytosis such as Arf6, flotillin 1 and 2 and Cdc42 did not affect the endocytosis of FGFR1 or FGFR3. Furthermore, depletion of clathrin decreased the degradation of FGFR1 resulting in sustained signalling. In the case of FGFR3, both the degradation and the signalling were only slightly affected by clathrin depletion. The data indicate that clathrin-mediated endocytosis is required for efficient internalization and downregulation of FGFR1 while FGFR3, however, is internalized by both clathrin-dependent and clathrin-independent mechanisms.

  9. Endocytosis of adiponectin receptor 1 through a clathrin-and Rab5-dependent pathway

    Qiurong Ding; Zhenzhen Wang; Yan Chen

    2009-01-01

    In eukaryotic cells, receptor endocytosis is a key event regulating signaling transduction. Adiponectin receptors belong to a new receptor family that is distinct from G-protein-coupled receptors and has critical roles in the pathogen-esis of diabetes and metabolic syndrome. Here, we analyzed the endocytosis of adiponectin and adiponectin receptor 1 (AdipoR1) and found that they are both internalized into transferrin-positive compartments that follow similar traffic routes. Blocking clathrin-mediated endocytosis by expressing Epsl5 mutants or depleting K+ trapped AdipoRl at the plasma membrane, and K+ depletion abolished adiponectin internalization, indicating that the endocytosis of AdipoRl and adiponectin is clathrin-dependent. Depletion of K+ and overexpression of Eps15 mutants enhance adiponectin-stimulated AMP-activated protein kinase phosphorylation, suggesting that the endocytosis of AdipoR1 might down-regulate adiponectin signaling. In addition, AdipoR1 colocalizes with the small GTPase Rab5, and a dominant negative Rab5 abrogates AdipoR1 endocytosis. These data indicate that AdipoRl is internalized through a clathrin- and Rab5-dependent pathway and that endocytosis may play a role in the regulation of adiponectin signaling.

  10. Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

    Fekri, Farnaz; Delos Santos, Ralph Christian; Karshafian, Raffi

    2016-01-01

    Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted

  11. Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms.

    Fekri, Farnaz; Delos Santos, Ralph Christian; Karshafian, Raffi; Antonescu, Costin N

    2016-01-01

    Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted

  12. [Molecular physiology of receptor mediated endocytosis and its role in overcoming multidrug resistance].

    Severin, E S; Posypanova, G A

    2011-06-01

    Receptor-mediated endocytosis plays important role in the selective uptake of proteins at the plasma membrane of eukaryotic cells. Endocytosis regulates many processes of cell signalling by controlling the number of functional receptors on the cell surface. The article reviews the mechanism of clathrin-dependent endocytosis and the possibility of using this phenomenon for the targeted delivery of drugs. Use of certain proteins as targeting component of drug delivery systems can significantly improve the selectivity of this drug, as well as to overcome the multidrug resistance of cells resulting from the activity of the ABC-transporters. PMID:21874867

  13. A luminescent assay for real-time measurements of receptor endocytosis in living cells.

    Robers, Matthew B; Binkowski, Brock F; Cong, Mei; Zimprich, Chad; Corona, Cesear; McDougall, Mark; Otto, George; Eggers, Christopher T; Hartnett, Jim; Machleidt, Thomas; Fan, Frank; Wood, Keith V

    2015-11-15

    Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs. PMID:26278171

  14. Sensing the delivery and endocytosis of nanoparticles using magneto-photo-acoustic imaging

    M. Qu

    2015-09-01

    Full Text Available Many biomedical applications necessitate a targeted intracellular delivery of the nanomaterial to specific cells. Therefore, a non-invasive and reliable imaging tool is required to detect both the delivery and cellular endocytosis of the nanoparticles. Herein, we demonstrate that magneto-photo-acoustic (MPA imaging can be used to monitor the delivery and to identify endocytosis of magnetic and optically absorbing nanoparticles. The relationship between photoacoustic (PA and magneto-motive ultrasound (MMUS signals from the in vitro samples were analyzed to identify the delivery and endocytosis of nanoparticles. The results indicated that during the delivery of nanoparticles to the vicinity of the cells, both PA and MMUS signals are almost linearly proportional. However, accumulation of nanoparticles within the cells leads to nonlinear MMUS-PA relationship, due to non-linear MMUS signal amplification. Therefore, through longitudinal MPA imaging, it is possible to monitor the delivery of nanoparticles and identify the endocytosis of the nanoparticles by living cells.

  15. Bradykinin release avoids high molecular weight kininogen endocytosis.

    Igor Z Damasceno

    Full Text Available Human H-kininogen (120 kDa plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type and CHO-745 (mutant deficient in proteoglycans biosynthesis cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular

  16. Bile Acids Reduce Endocytosis of High-Density Lipoprotein (HDL) in HepG2 Cells

    Clemens Röhrl; Karin Eigner; Stefanie Fruhwürth; Herbert Stangl

    2014-01-01

    High-density lipoprotein (HDL) transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence...

  17. Clathrin-mediated endocytosis at the synaptic terminal: bridging the gap between physiology and molecules

    Royle, Stephen J; Lagnado, Leon

    2010-01-01

    It has long been known that the maintenance of fast communication between neurons requires that presynaptic terminals recycle the small vesicles from which neurotransmitter is released. But the mechanisms that retrieve vesicles from the cell surface are still not understood. Although we have a wealth of information about the molecular details of endocytosis in non-neuronal cells, it is clear that endocytosis at the synapse is faster and regulated in distinct ways. A satisfying understanding o...

  18. Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors

    1992-01-01

    Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-...

  19. Discovery of New Cargo Proteins that enter Cells through Clathrin-Independent Endocytosis

    Eyster, Craig A.; Higginson, Jason D.; Huebner, Robert; Porat-Shliom, Natalie; Weigert, Roberto; Wu, Wells W.; Shen, Rong-Fong; Donaldson, Julie G.

    2009-01-01

    Clathrin-independent endocytosis (CIE) allows internalization of plasma membrane proteins lacking clathrin-targeting sequences, such as the major histocompatibility complex Class I protein (MHCI), into cells. After internalization, vesicles containing MHCI fuse with transferrin-containing endosomes generated from clathrin-dependent endocytosis. In HeLa cells, MHCI is subsequently routed to late endosomes or recycled back out to the plasma membrane (PM) in distinctive tubular carriers. Arf6 is...

  20. Mechanistic analysis of massive endocytosis in relation to functionally defined surface membrane domains

    Hilgemann, Donald W.; Fine, Michael

    2011-01-01

    A large fraction of endocytosis in eukaryotic cells occurs without adaptors or dynamins. Here, we present evidence for the involvement of lipid domains in massive endocytosis (MEND) activated by both large Ca transients and amphipathic compounds in baby hamster kidney and HEK293 cells. First, we demonstrate functional coupling of the two MEND types. Ca transients can strongly facilitate detergent-activated MEND. Conversely, an amphipath with dual alkyl chains, ditridecylphthalate, is without ...

  1. Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform.

    Sandoval, Sergio; Mendez, Natalie; Alfaro, Jesus G; Yang, Jian; Aschemeyer, Sharraya; Liberman, Alex; Trogler, William C; Kummel, Andrew C

    2015-08-01

    A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N -hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg , respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization. PMID:26315280

  2. A yeast t-SNARE involved in endocytosis.

    Séron, K; Tieaho, V; Prescianotto-Baschong, C; Aust, T; Blondel, M O; Guillaud, P; Devilliers, G; Rossanese, O W; Glick, B S; Riezman, H; Keränen, S; Haguenauer-Tsapis, R

    1998-10-01

    The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease. In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis. PMID:9763449

  3. Ouabain uptake by endocytosis in isolated guinea pig atria

    Mammalian cells specifically internalize some molecular species through receptor-mediated endocytosis (RME). The authors have used four different experimental protocols to investigate whether ouabain enters cardiac cells of guinea pig atrium through this pathway. First, by electron microscope morphometry the authors found that ouabain increased endocytic vesicles in atrial cells. Second, by scintillation counting they found that [3H]ouabain uptake by the tissue is decreased by three treatments that decrease RME, i.e., NH4Cl, trifluoperazine, and 16 mM [K+]0. Third, by radioautography at the electron microscope level, they checked that in preceding experiments [3H]ouabain was washed out of plasma membrane after 60-min rinse and interiorized into the cardiac cells. Fourth, isometric tension recordings showed that the positive inotropic effect of ouabain was diminished in the presence of inhibitors, whereas that of a hydrophobic analogue, ouabagenin, was not affected. These results suggest that ouabain enters cardiac cells through RME and also that an intracellular site may, at least in part, be responsible for its inotropic effect

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

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

    2016-02-01

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

  5. Calcyon is Necessary for Activity Dependent AMPA Receptor Internalization and LTD in CA1 Neurons of Hippocampus

    Davidson, Heather Trantham; Xiao, Jiping; Dai, Rujuan; Bergson, Clare

    2009-01-01

    Calcyon is a single transmembrane endocytic protein that regulates clathrin assembly and clathrin mediated endocytosis in brain. Ultrastructural studies indicate that calcyon localizes to spines, but whether it regulates glutamate neurotransmission is not known. Here, we show that deletion of the calcyon gene in mice inhibits agonist stimulated endocytosis of AMPA receptors, without altering basal surface levels of the GluR1 or GluR2 subunits. Whole cell patch clamp studies of hippocampal neu...

  6. Endocytosis and intracellular protein degradation in cystic fibrosis fibroblasts

    Normal rates of pinocytosis of [3H]sucrose were measured in cystic fibrosis fibroblasts, and were not affected by the addition of cystic fibrosis serum. Bulk protein degradation (a significant proportion of which occurs intralysosomally following autophagy) and its regulation by growth state were apparently identical in normal and cystic fibrosis cultures. (Auth.)

  7. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

    Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M

    2016-01-01

    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment. PMID:26459807

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

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

    2010-09-03

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

  9. Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy.

    Víctor M Campa

    Full Text Available The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution.

  10. On the ultrastructure of Trichomonas vaginalis: cytoskeleton, endocytosis and hydrogenosomes

    SIXTO RAUL COSTAMAGNA

    2001-07-01

    Full Text Available This paper is focused on the study of the ultrastructure of Trichomonas vaginalis in liquid cultures. Its cytoskeleton, the morphology of its hydrogenosomes, and endocytosis phenomena have been observed by scanning electron microscopy (SEM and transmission electron microscopy (TEM. For the present study, the traditional techniques for SEM and TEM have been slightly modified for the processing of this flagellate. Following our experiments, it can be concluded that: 1. The modified techniques are adequate for the ultrastructural study of this protozoon. 2. There are no mitochondria in T. vaginalis. 3. T. vaginalis might use micropinocytosis processes related to coated vesicles as a habitual endo- and exocytosis mechanism, while phagocytosis is observed for major vesicles. 4. As to the cytoskeleton, microtubules are numerous and display different structures, which are analyzed in this paper. 5. Many hydrogenosomes are found in the cytoplasm of T. vaginalis underneath the undulating membrane and along the axostyle, each with electron-dense deposits in the manner of «operculums.»El presente estudio está referido a la ultraestructura de Trichomonas vaginalis, cultivadas en medio líquido. Su citoesqueleto, fenómenos de endocitosis y la morfología de sus hidrogenosomas fueron observados por microscopía electrónica de barrido y de transmisión. Las técnicas clásicas para el procesamiento al SEM y TEM de este flagelado, fueron discreta y sutilmente modificadas por nosotros. Como resultado de nuestras experiencias se concluye que: 1. Las técnicas modificadas empleadas son adecuadas para el estudio morfológico y ultraestructural de este Protozoo. 2. Al TEM su citoplasma no muestra mitocondrias. 3. Utiliza los fenómenos de micropinocitosis asociados con vesículas con cubierta como mecanismo habitual de endo y exocitosis selectiva, mientras que para partículas mayores la fagocitosis es frecuentemente vista. 4. Con referencia al citoesqueleto

  11. Caveolae-mediated endocytosis of biocompatible gold nanoparticles in living Hela cells

    Efficient intracellular delivery of gold nanoparticles (AuNPs) and unraveling the mechanism underlying the intracellular delivery are essential for advancing the applications of AuNPs toward in vivo imaging and therapeutic interventions. We employed fluorescence microscopy to investigate the internalization mechanism of small-size AuNPs by living Hela cells. Herein, we found that the caveolae-mediated endocytosis was the dominant pathway for the intracellular delivery of small-size AuNPs. The intracellular delivery was suppressed when we depleted the cholesterol with methyl-β-cyclodextrin (MβCD); in contrast, the sucrose that disrupts the formation of clathrin-mediated endocytosis did not block the endocytosis of AuNPs. Meanwhile, we examined the intracellular localization of AuNPs in endocytic vesicles by fluorescent colocalization. This work would provide a potential technique to study the intracellular delivery of small-size nanoparticles for biomedical applications. (paper)

  12. Impaired activity-dependent neural circuit assembly and refinement in autism spectrum disorder genetic models

    Caleb Andrew Doll; Kendal eBroadie

    2014-01-01

    Early-use activity during circuit-specific critical periods refines brain circuitry by the coupled processes of eliminating inappropriate synapses and strengthening maintained synapses. We theorize these activity-dependent developmental processes are specifically impaired in autism spectrum disorders (ASDs). ASD genetic models in both mouse and Drosophila have pioneered our insights into normal activity-dependent neural circuit assembly and consolidation, and how these developmental mechanism...

  13. Drosophila king tubby (ktub mediates light-induced rhodopsin endocytosis and retinal degeneration

    Chen Shu-Fen

    2012-12-01

    Full Text Available Background The tubby (tub and tubby-like protein (tulp genes encode a small family of proteins found in many organisms. Previous studies have shown that TUB and TULP genes in mammalian involve in obesity, neural development, and retinal degeneration. The purpose of this study was to investigate the role of Drosophila king tubby (ktub in rhodopsin 1 (Rh1 endocytosis and retinal degeneration upon light stimulation. Results Drosophila ktub mutants were generated using imprecise excision. Wild type and mutant flies were raised in dark or constant light conditions. After a period of light stimulation, retinas were dissected, fixed and stained with anti-Rh1 antibody to reveal Rh1 endocytosis. Confocal and transmission electron microscope were used to examine the retinal degeneration. Immunocytochemical analysis shows that Ktub is expressed in the rhabdomere domain under dark conditions. When flies receive light stimulation, the Ktub translocates from the rhabdomere to the cytoplasm and the nucleus of the photoreceptor cells. Wild type photoreceptors form Rh1-immunopositive large vesicles (RLVs shortly after light stimulation. In light-induced ktub mutants, the majority of Rh1 remains at the rhabdomere, and only a few RLVs appear in the cytoplasm of photoreceptor cells. Mutation of norpA allele causes massive Rh1 endocytosis in light stimulation. In ktub and norpA double mutants, however, Rh1 endocytosis is blocked under light stimulation. This study also shows that ktub and norpA double mutants rescue the light-induced norpA retinal degeneration. Deletion constructs further demonstrate that the Tubby domain of the Ktub protein participates in an important role in Rh1 endocytosis. Conclusions The results in this study delimit the novel function of Ktub in Rh1 endocytosis and retinal degeneration.

  14. Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis

    Highlights: •Prominin-2 expression induced protrusions that co-localized with lipid raft markers. •Prominin-2 expression decreased caveolae, caveolar endocytosis and increased pCav1. •Prominin-2 expression inhibited fluid phase endocytosis by inactivation of Cdc42. •These endocytic effects can be reversed by adding exogenous cholesterol. •Caveolin1 knockdown restored fluid phase endocytosis in Prominin2 expressing cells. -- Abstract: Background: Membrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins that bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. Aim: To characterize the effects of Prom-2 expression on PM microdomain organization. Methods: Prom2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. Results: Prom2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. Conclusions: Prom2 protrusions primarily

  15. Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis

    Singh, Raman Deep, E-mail: Takhter.Ramandeep@mayo.edu; Schroeder, Andreas S.; Scheffer, Luana; Holicky, Eileen L.; Wheatley, Christine L.; Marks, David L., E-mail: Marks.david@mayo.edu; Pagano, Richard E.

    2013-05-10

    Highlights: •Prominin-2 expression induced protrusions that co-localized with lipid raft markers. •Prominin-2 expression decreased caveolae, caveolar endocytosis and increased pCav1. •Prominin-2 expression inhibited fluid phase endocytosis by inactivation of Cdc42. •These endocytic effects can be reversed by adding exogenous cholesterol. •Caveolin1 knockdown restored fluid phase endocytosis in Prominin2 expressing cells. -- Abstract: Background: Membrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins that bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. Aim: To characterize the effects of Prom-2 expression on PM microdomain organization. Methods: Prom2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. Results: Prom2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. Conclusions: Prom2 protrusions primarily

  16. Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae

    Fischer-Parton, S; Parton, R M; Hickey, P C; Dijksterhuis, J; Atkinson, H A; Read, N D

    2000-01-01

    Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were

  17. Trypanosoma cruzi: antigen-receptor mediated endocytosis of antibody

    Judith Abelha

    1981-06-01

    Full Text Available Trypanomastigote forms of Trypanosoma cruzi were derived from tissue culture and incubated with immune and non-immune human sera. All immune sera showed high titers of specific humoral antibodies of the IgM or the IgG type. Agglutination and swelling of parasites were observed after incubation at 37ºC, but many trypomastigotes remained free-swimming in the sera for two to three days. The quantitiy of immune serum capable of lysing a maximum of 10 x 10 [raised to the power of 6] sensitized red cells was not capable of lysing 4 x 10 [raised to the power of 3] tripomastigotes. Typically, the parasites underwent cyclical changes with the formation of clumps of amastigotes and the appearance of epimastigote forms. Multiplication of the parasites was observed in immune sera. Further, the infectivity of the parasites to susceptible mice was not lost. All sera used produced similar general effects on the growth of the parasite. The antibody bound to T. cruzi appeard to enter cells by antigen-receptor mediated endocytosis. The ferritin-conjugated antibody was internalized and delivered to phagolysosomes where they might be completely degraded to amino-acids. This seemed to be a coupled process by which the immunoglobulin is first bound to specific parasite surface receptor and then rapidly endocytosed by the cell.Formas tripomastigotas de Trypanosoma cruzi derivadas de cultura de tecido foram encubadas com soros humanos imunes e não-imunes.Todos os soros humanos usados tinham títulos elevados de anticorpos das classes IgM ou IgG. Aglutinação e entumescimento dos parasitos eram observados apos encubação a 37ºC mas muitos tripomastigotas permaneceram circulando livremente nos soros por dois a três dias. A quantidade de soro imune capaz de lisar um máximo de 10 x 10 [elevado a 6] hemácias sensibilizadas não foi capaz de lisar 4 x 10 [elevado a 3] tripomastigotas. Tipicamente, os parasitos apresentavam alterações cíclicas com formação de

  18. Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release

    Verstreken, Patrik; Kjaerulff, Ole; Lloyd, Thomas E;

    2002-01-01

    We have identified mutations in Drosophila endophilin to study its function in vivo. Endophilin is required presynaptically at the neuromuscular junction, and absence of Endophilin dramatically impairs endocytosis in vivo. Mutant larvae that lack Endophilin fail to take up FM1-43 dye in synaptic ...

  19. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    George Kourouniotis

    2016-07-01

    Full Text Available The binding of epidermal growth factor (EGF to EGF receptor (EGFR stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ and tagged a green fluorescent protein (GFP at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc, extracellular signal-regulated kinase (ERK and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

  20. Silence of synaptotagmin I in INS-1 cells inhibits fast exocytosis and fast endocytosis

    Synaptotagmin I (Syt I) is a Ca2+ sensor for triggering fast synchronized release of neurotransmitters. However, controversy remains whether Syt I is also obligatory for the exocytosis and endocytosis of larger dense core vesicles (LDCVs) in endocrine cells. In this study, we used a short hairpin RNA (shRNA) to silence the expression of Syt I and investigated the roles of Syt I on exocytosis and endocytosis in INS-1 cells. Our results demonstrated that expression of Syt I is remarkably reduced by the Syt I gene targeting shRNA. Using high-time resolution capacitance measurement, we found that the silence of Syt I decreased the calcium sensitivity of fusion of insulin granules and therefore reduced the exocytotic burst triggered by step-like [Ca2+] i elevation. In addition, the occurrence frequency and amplitude of fast endocytosis were remarkably reduced in the silenced cells. We conclude that Syt I not only participates in the Ca2+-sensing of LDCV fusion with plasmalemma, but also plays a crucial role in fast endocytosis in INS-1 cells

  1. NtGNL1a ARF-GEF acts in endocytosis in tobacco cells

    Jelínková, Adriana; Müller, Karel; Pařezová, Markéta; Petrášek, Jan

    2015-01-01

    Roč. 15, NOV 5 (2015), s. 272. ISSN 1471-2229 R&D Projects: GA ČR GPP305/11/P797 Institutional support: RVO:61389030 Keywords : Endocytosis * PIN1 protein trafficking * Inhibitors of endomembrane trafficking Subject RIV: EA - Cell Biology Impact factor: 3.813, year: 2014

  2. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

    2016-01-01

    The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis. PMID:27463710

  3. Activity-dependent brain-derived neurotrophic factor expression regulates cortistatin-interneurons and sleep behavior

    Martinowich Keri

    2011-03-01

    Full Text Available Abstract Background Sleep homeostasis is characterized by a positive correlation between sleep length and intensity with the duration of the prior waking period. A causal role for brain-derived neurotrophic factor (BDNF in sleep homeostasis has been suggested, but the underlying mechanisms remain unclear. Cortistatin, a neuropeptide expressed primarily in a subset of cortical GABAergic interneurons, is another molecule implicated in sleep homeostasis. Results We confirmed that sleep deprivation leads to an increase in cortical cortistatin mRNA expression. Disruption of activity-dependent BDNF expression in a genetically modified mouse line impairs both baseline levels of cortistatin mRNA as well as its levels following sleep deprivation. Disruption of activity-dependent BDNF also leads to a decrease in sleep time during the active (dark phase. Conclusion Our studies suggest that regulation of cortistatin-expressing interneurons by activity-dependent BDNF expression may contribute to regulation of sleep behavior.

  4. Definition of a Bidirectional Activity-Dependent Pathway Involving BDNF and Narp

    Abigail Mariga

    2015-12-01

    Full Text Available One of the cardinal features of neural development and adult plasticity is the contribution of activity-dependent signaling pathways. However, the interrelationships between different activity-dependent genes are not well understood. The immediate early gene neuronal-activity-regulated pentraxin (NPTX2 or Narp encodes a protein that has been associated with excitatory synaptogenesis, AMPA receptor aggregation, and the onset of critical periods. Here, we show that Narp is a direct transcriptional target of brain-derived neurotrophic factor (BDNF, another highly regulated activity-dependent gene involved in synaptic plasticity. Unexpectedly, Narp is bidirectionally regulated by BDNF. Acute BDNF withdrawal results in downregulation of Narp, whereas transcription of Narp is greatly enhanced by BDNF. Furthermore, our results show that BDNF directly regulates Narp to mediate glutamatergic transmission and mossy fiber plasticity. Hence, Narp serves as a significant epistatic target of BDNF to regulate synaptic plasticity during periods of dynamic activity.

  5. Effect of sulfur dioxide on pulmonary macrophage endocytosis at rest and during exercise

    Inhaled SO2 may cause damage by injuring upper airways. To what extent can SO2 also alter pulmonary macrophage function in the parenchyma and what is the impact of exercise? We studied the effect of SO2 on pulmonary macrophage endocytosis in resting and in exercising animals by measuring the rates of macrophage endocytosis in situ for 1 h of a test particle of insoluble radioactive colloidal gold (198Au), 1, 24, or 48 h after inhalation exposure to SO2. Resting hamsters exposed for 4 h to 50 ppm SO2 had no significant reduction in macrophage endocytosis compared with air-breathing control hamsters. However, if hamsters were exposed to the same concentration of SO2 while continuously running (40 min at 0.9 km/h), macrophage endocytosis was significantly reduced 1 h after exposure even though the exposure time was only one-sixth as long. Twenty-four hours later, the percentage of gold ingested by pulmonary macrophages remained significantly depressed. By 48 h, the rate had returned to control values. Exercise alone did not affect endocytosis. Hamsters exposed to 50 ppm SO2, with or without exercise, also showed significant reductions in the number of lavaged macrophages. This decrease was greatest and most persistent in the SO2 plus exercise group. These data indicate that even when animals are exposed to water-soluble gases, which are normally removed by the upper airways, exercise can potentiate damage to more peripheral components of the pulmonary defense system such as the macrophage

  6. Phosphorylation decreases ubiquitylation of the thiazide-sensitive cotransporter NCC and subsequent clathrin-mediated endocytosis.

    Rosenbaek, Lena L; Kortenoeven, Marleen L A; Aroankins, Takwa S; Fenton, Robert A

    2014-05-01

    The thiazide-sensitive sodium chloride cotransporter, NCC, is the major NaCl transport protein in the distal convoluted tubule (DCT). The transport activity of NCC can be regulated by phosphorylation, but knowledge of modulation of NCC trafficking by phosphorylation is limited. In this study, we generated novel tetracycline-inducible Madin-Darby canine kidney type I (MDCKI) cell lines expressing NCC to examine the role of NCC phosphorylation and ubiquitylation on NCC endocytosis. In MDCKI-NCC cells, NCC was highly glycosylated at molecular weights consistent with NCC monomers and dimers. NCC constitutively cycles to the apical plasma membrane of MDCKI-NCC cells, with 20-30% of the membrane pool of NCC internalized within 30 min. The use of dynasore, PitStop2, methyl-β-cyclodextrin, nystatin, and filipin (specific inhibitors of either clathrin-dependent or -independent endocytosis) demonstrated that NCC is internalized via a clathrin-mediated pathway. Reduction of endocytosis resulted in greater levels of NCC in the plasma membrane. Immunogold electron microscopy confirmed the association of NCC with the clathrin-mediated internalization pathway in rat DCT cells. Compared with controls, inducing phosphorylation of NCC via low chloride treatment or mimicking phosphorylation by replacing Thr-53, Thr-58, and Ser-71 residues with Asp resulted in increased membrane abundance and reduced rates of NCC internalization. NCC ubiquitylation was lowest in the conditions with greatest NCC phosphorylation, thus providing a mechanism for the reduced endocytosis. In conclusion, our data support a model where NCC is constitutively cycled to the plasma membrane, and upon stimulation, it can be phosphorylated to both increase NCC activity and decrease NCC endocytosis, together increasing NaCl transport in the DCT. PMID:24668812

  7. Listeria monocytogenes internalin B activates junctional endocytosis to accelerate intestinal invasion.

    Mickey Pentecost

    2010-05-01

    Full Text Available Listeria monocytogenes (Lm uses InlA to invade the tips of the intestinal villi, a location at which cell extrusion generates a transient defect in epithelial polarity that exposes the receptor for InlA, E-cadherin, on the cell surface. As the dying cell is removed from the epithelium, the surrounding cells reorganize to form a multicellular junction (MCJ that Lm exploits to find its basolateral receptor and invade. By examining individual infected villi using 3D-confocal imaging, we uncovered a novel role for the second major invasin, InlB, during invasion of the intestine. We infected mice intragastrically with isogenic strains of Lm that express or lack InlB and that have a modified InlA capable of binding murine E-cadherin and found that Lm lacking InlB invade the same number of villi but have decreased numbers of bacteria within each infected villus tip. We studied the mechanism of InlB action at the MCJs of polarized MDCK monolayers and find that InlB does not act as an adhesin, but instead accelerates bacterial internalization after attachment. InlB locally activates its receptor, c-Met, and increases endocytosis of junctional components, including E-cadherin. We show that MCJs are naturally more endocytic than other sites of the apical membrane, that endocytosis and Lm invasion of MCJs depends on functional dynamin, and that c-Met activation by soluble InlB or hepatocyte growth factor (HGF increases MCJ endocytosis. Also, in vivo, InlB applied through the intestinal lumen increases endocytosis at the villus tips. Our findings demonstrate a two-step mechanism of synergy between Lm's invasins: InlA provides the specificity of Lm adhesion to MCJs at the villus tips and InlB locally activates c-Met to accelerate junctional endocytosis and bacterial invasion of the intestine.

  8. Impaired activity-dependent neural circuit assembly and refinement in autism spectrum disorder genetic models

    Caleb Andrew Doll

    2014-02-01

    Full Text Available Early-use activity during circuit-specific critical periods refines brain circuitry by the coupled processes of eliminating inappropriate synapses and strengthening maintained synapses. We theorize these activity-dependent developmental processes are specifically impaired in autism spectrum disorders (ASDs. ASD genetic models in both mouse and Drosophila have pioneered our insights into normal activity-dependent neural circuit assembly and consolidation, and how these developmental mechanisms go awry in specific genetic conditions. The monogenic Fragile X syndrome (FXS, a common cause of heritable ASD and intellectual disability, has been particularly well linked to defects in activity-dependent critical period processes. The Fragile X Mental Retardation Protein (FMRP is positively activity-regulated in expression and function, in turn regulates excitability and activity in a negative feedback loop, and appears to be required for the activity-dependent remodeling of synaptic connectivity during early-use critical periods. The Drosophila FXS model has been shown to functionally conserve the roles of human FMRP in synaptogenesis, and has been centrally important in generating our current mechanistic understanding of the FXS disease state. Recent advances in Drosophila optogenetics, transgenic calcium reporters, highly-targeted transgenic drivers for individually-identified neurons, and a vastly improved connectome of the brain are now being combined to provide unparalleled opportunities to both manipulate and monitor activity-dependent processes during critical period brain development in defined neural circuits. The field is now poised to exploit this new Drosophila transgenic toolbox for the systematic dissection of activity-dependent mechanisms in normal versus ASD brain development, particularly utilizing the well-established Drosophila FXS disease model.

  9. Bulk chemicals from biomass

    Haveren, van J.; Scott, E.L.; Sanders, J.P.M.

    2008-01-01

    Given the current robust forces driving sustainable production, and available biomass conversion technologies, biomass-based routes are expected to make a significant impact on the production of bulk chemicals within 10 years, and a huge impact within 20-30 years. In the Port of Rotterdam there is a

  10. Ferromagnetic bulk glassy alloys

    This paper deals with the review on the formation, thermal stability and magnetic properties of the Fe-based bulk glassy alloys in as-cast bulk and melt-spun ribbon forms. A large supercooled liquid region over 50 K before crystallization was obtained in Fe-(Al, Ga)-(P, C, B, Si), Fe-(Cr, Mo, Nb)-(Al, Ga)-(P, C, B) and (Fe, Co, Ni)-Zr-M-B (M=Ti, Hf, V, Nb, Ta, Cr, Mo and W) systems and bulk glassy alloys were produced in a thickness range below 2 mm for the Fe-(Al, Ga)-(P, C, B, Si) system and 6 mm for the Fe-Co-(Zr, Nb, Ta)-(Mo, W)-B system by copper-mold casting. The ring-shaped glassy Fe-(Al, Ga)-(P, C, B, Si) alloys exhibit much better soft magnetic properties as compared with the ring-shaped alloy made from the melt-spun ribbon because of the formation of the unique domain structure. The good combination of high glass-forming ability and good soft magnetic properties indicates the possibility of future development as a new bulk glassy magnetic material

  11. Heat transport in bulk/nanoporous/bulk silicon devices

    Criado-Sancho, M. [Departamento de Ciencias y Técnicas Físicoquimicas, Facultad de Ciencias, UNED, Senda del Rey 9, 20040 Madrid (Spain); Jou, D., E-mail: David.Jou@uab.cat [Departament de Física, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia (Spain); Institut d' Estudis Catalans, Carme 47, 08001 Barcelona, Catalonia (Spain)

    2013-02-04

    We study heat transport in bulk/nanoporous/bulk silicon devices; we show that, despite bulk/nanoporous devices may act as thermal rectifiers, the non-linear aspects of their joint thermal conductance are not strong enough to lead to a negative differential thermal resistance, necessary to allow bulk/nanoporous/bulk Si devices to act as thermal transistors. Furthermore, we explicitly study the effective thermal conductivity of the mentioned devices for several temperatures, geometries, porosities, and pore size.

  12. Activity-dependent branching ratios in stocks, solar x-ray flux, and the Bak-Tang-Wiesenfeld sandpile model

    Martin, Elliot; Shreim, Amer; Paczuski, Maya

    2010-01-01

    We define an activity-dependent branching ratio that allows comparison of different time series Xt . The branching ratio bx is defined as bx=E[ξx/x] . The random variable ξx is the value of the next signal given that the previous one is equal to x , so ξx={Xt+1∣Xt=x} . If bx>1 , the process is on average supercritical when the signal is equal to x , while if bxefficient market hypothesis.” For stock volumes, solar x-ray flux intensities, and the Bak-Tang-Wiesenfeld (BTW) sandpile model, bx is supercritical for small values of activity and subcritical for the largest ones, indicating a tendency to return to a typical value. For stock volumes this tendency has an approximate power-law behavior. For solar x-ray flux and the BTW model, there is a broad regime of activity where bx≃1 , which we interpret as an indicator of critical behavior. This is true despite different underlying probability distributions for Xt and for ξx . For the BTW model the distribution of ξx is Gaussian, for x sufficiently larger than 1, and its variance grows linearly with x . Hence, the activity in the BTW model obeys a central limit theorem when sampling over past histories. The broad region of activity where bx is close to one disappears once bulk dissipation is introduced in the BTW model—supporting our hypothesis that it is an indicator of criticality.

  13. DREAM controls the on/off switch of specific activity-dependent transcription pathways.

    Mellström, Britt; Sahún, Ignasi; Ruiz-Nuño, Ana; Murtra, Patricia; Gomez-Villafuertes, Rosa; Savignac, Magali; Oliveros, Juan C; Gonzalez, Paz; Kastanauskaite, Asta; Knafo, Shira; Zhuo, Min; Higuera-Matas, Alejandro; Errington, Michael L; Maldonado, Rafael; DeFelipe, Javier; Jefferys, John G R; Bliss, Tim V P; Dierssen, Mara; Naranjo, Jose R

    2014-03-01

    Changes in nuclear Ca(2+) homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K(+) channel interacting protein 3), is a Ca(2+)-binding protein that binds DNA and represses transcription in a Ca(2+)-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca(2+)-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory. PMID:24366545

  14. Bulk materials handling review

    NONE

    2007-02-15

    The paper provides details of some of the most important coal handling projects and technologies worldwide. It describes development by Aubema Crushing Technology GmbH, Bedeschi, Cimbria Moduflex, DBT, Dynamic Air Conveying Systems, E & F Services, InBulk Technologies, Nord-Sen Metal Industries Ltd., Pebco Inc, Primasonics International Ltd., R.J.S. Silo Clean (International) Ltd., Takraf GmbH, and The ACT Group. 17 photos.

  15. pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

    Shen, Yi; Rosendale, Morgane; Robert E Campbell; Perrais, David

    2014-01-01

    Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultan...

  16. Binding of heparin and of the small proteoglycan decorin to the same endocytosis receptor proteins leads to different metabolic consequences

    1991-01-01

    Decorin, a small interstitial dermatan sulfate proteoglycan, is turned over in cultured cells of mesenchymal origin by receptor-mediated endocytosis followed by intralysosomal degradation. Two endosomal proteins of 51 and 26 kD have been implicated in the endocytotic process because of their interaction with decorin core protein. However, heparin and protein-free dermatan sulfate were able to inhibit endocytosis of decorin in a concentration-dependent manner. After Western blotting of endosom...

  17. Clathrin is Important for Normal Actin Dynamics and Progression of Sla2p-Containing Patches During Endocytosis in Yeast

    Newpher, Thomas M.; Lemmon, Sandra K.

    2006-01-01

    Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization. Here, we investigate the dynamics of clathrin relative to the early endocytic patch protein Sla2p. We find that clathrin is recruited to ...

  18. Capacitance measurements. An analysis of the phase detector technique used to study exocytosis and endocytosis.

    Joshi, C.; Fernandez, J M

    1988-01-01

    We have studied the admittance of patch-clamped mast cells during exocytosis and found that they are adequately described by a four parameter equivalent circuit. On the basis of these measurements, we show that, contrary to current belief, when using a phase sensitive detector, small capacitance changes due to exocytosis or endocytosis should be studied by measuring current 90 degrees out of phase, relative to the component that corresponds to changes in series resistance. We have extended th...

  19. Cyclin dependent kinase 5 regulates endocytosis in nerve terminals via dynamin I phosphorylation

    Full text: Synaptic vesicle endocytosis (SVE) in nerve terminals is essential for normal synaptic transmission and for memory retrieval. Dynamin I is a 96kDa nerve terminal phosphoprotein necessary for synaptic vesicle endocytosis in the nerve terminal. Dynamin I is dephosphorylated and rephosphorylated in a cyclical fashion with nerve terminal depolarisation and repolarisation. A number of kinases phosphorylate dynamin I in vitro including PKC, MAP kinase and cdc2. PKC phosphorylates dynamin in the proline rich domain on Ser 795 and is also thought to be the in vivo kinase for dynamin I. Another candidate is the neuron specific kinase cdk5, crucial for CNS development. The aim of this study is to identify the kinase which phosphorylates dynamin I in intact nerve terminals. Here we show that cyclin-dependent kinase 5 (cdk5) phosphorylates dynamin I in the proline-rich tail on Ser-774 or Ser-778. The phosphorylation of these sites but not Ser-795 also occurred in intact nerve terminals suggesting that cdk5 is the physiologically relevant enzyme for dynamin I. Synaptosomes prepared from rat brains (after cervical dislocations) and labelled with 32 Pi, were incubated with 100 M roscovitine (a selective inhibitor of cdks), 10 M Ro 31-8220 (a selective PKC inhibitor) and 100 M PD 98059 (a MEK kinase inhibitor). Dynamin rephosphorylation during repolarisation was reduced in synaptosomes treated with roscovitine and Ro 38-8220 but not in synaptosomes treated with PD 98059. Fluorimetric experiments on intact synaptosomes utilising FM-210 (a fluorescent dye) indicate that endocytosis was reduced in synaptosomes treated with 100 M roscovitine. Our results suggest that dynamin phosphorylation in intact nerve terminals may not be regulated by PKC or MAP kinase and that dynamin phosphorylation by cdk5 may regulate endocytosis. Copyright (2002) Australian Neuroscience Society

  20. Human vascular endothelial cells transport foreign exosomes from cow's milk by endocytosis.

    Kusuma, Rio Jati; Manca, Sonia; Friemel, Taylor; Sukreet, Sonal; Nguyen, Christopher; Zempleni, Janos

    2016-05-15

    Encapsulation of microRNAs in exosomes confers protection against degradation and a vehicle for shuttling of microRNAs between cells and tissues, and cellular uptake by endocytosis. Exosomes can be found in foods including milk. Humans absorb cow's milk exosomes and deliver the microRNA cargo to peripheral tissues, consistent with gene regulation by dietary nucleic acids across species boundaries. Here, we tested the hypothesis that human vascular endothelial cells transport milk exosomes by endocytosis, constituting a step crucial for the delivery of dietary exosomes and their cargo to peripheral tissues. We tested this hypothesis by using human umbilical vein endothelial cells and fluorophore-labeled exosomes isolated from cow's milk. Exosome uptake followed Michaelis-Menten kinetics (Vmax = 0.057 ± 0.004 ng exosome protein × 40,000 cells/h; Km = 17.97 ± 3.84 μg exosomal protein/200 μl media) and decreased by 80% when the incubation temperature was lowered from 37°C to 4°C. When exosome surface proteins were removed by treatment with proteinase K, or transport was measured in the presence of the carbohydrate competitor d-galactose or measured in the presence of excess unlabeled exosomes, transport rates decreased by 45% to 80% compared with controls. Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50% decrease in transport. When fluorophore-labeled exosomes were administered retro-orbitally, exosomes accumulated in liver, spleen, and lungs in mice. We conclude that human vascular endothelial cells transport bovine exosomes by endocytosis and propose that this is an important step in the delivery of dietary exosomes and their cargo to peripheral tissues. PMID:26984735

  1. Contributions of herpes simplex virus type 1 envelope proteins to entry by endocytosis

    Herpes simplex virus (HSV) proteins specifically required for endocytic entry but not direct penetration have not been identified. HSVs deleted of gE, gG, gI, gJ, gM, UL45, or Us9 entered cells via either pH-dependent or pH-independent endocytosis and were inactivated by mildly acidic pH. Thus, the ...

  2. Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

    Ghaddar, Kassem; Merhi, Ahmad; Saliba, Elie; Krammer, Eva-Maria; Prévost, Martine; André, Bruno

    2014-01-01

    Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport ...

  3. Enhanced endocytosis of nano-curcumin in nasopharyngeal cancer cells: An atomic force microscopy study

    Prasanth, R.; Nair, Greshma; Girish, C. M.

    2011-10-01

    Recent studies in drug development have shown that curcumin can be a good competent due to its improved anticancer, antioxidant, anti-proliferative, and anti-inflammatory activities. A detailed real time characterization of drug (curcumin)-cell interaction is carried out in human nasopharyngeal cancer cells using atomic force microscopy. Nanocurcumin shows an enhanced uptake over micron sized drugs attributed to the receptor mediated route. Cell membrane stiffness plays a critical role in the drug endocytosis in nasopharyngeal cancer cells.

  4. Human immunodeficiency virus infection of T cells and monocytes proceeds via receptor-mediated endocytosis

    1988-01-01

    The rates of internalization and uncoating of 32P-labelled human immunodeficiency virus (HIV) in the human T lymphoid cell line CEM are consonant with a receptor-mediated endocytosis mechanism of entry. This interpretation was affirmed by electron microscopic observation of virions within endosomes. Virus binding and infectivity were inhibited to the same extent by pretreatment with OKT4A antibody, therefore, the CD4 receptor-dependent pathway of internalization appears to be the infectious r...

  5. Parameter estimation with bio-inspired meta-heuristic optimization : modeling the dynamics of endocytosis.

    Tashkova Katerina; Korošec Peter; Šilc Jurij; Todorovski Ljupčo; Džeroski Sašo

    2011-01-01

    Abstract Background We address the task of parameter estimation in models of the dynamics of biological systems based on ordinary differential equations (ODEs) from measured data, where the models are typically non-linear and have many parameters, the measurements are imperfect due to noise, and the studied system can often be only partially observed. A representative task is to estimate the parameters in a model of the dynamics of endocytosis, i.e., endosome maturation, reflected in a cut-ou...

  6. Systemic blockade of the Hyaluronan Receptor for Endocytosis (HARE) prevents lymph node metastasis of prostate cancer

    Simpson, Melanie A.; Weigel, Janet A.; Weigel, Paul H.

    2012-01-01

    Tumor progression and metastasis are promoted by the remodeling of organized tissue architecture and engagement of molecular interactions that support tumor cell passage through endothelial barriers. Prostate tumor cells that secrete and turn over excessive quantities of pericellular hyaluronan (HA) exhibit accelerated growth kinetics and spontaneous lymph node metastasis in mice. The HA Receptor for Endocytosis (HARE) is an endocytic clearance receptor for HA in the liver that is also highly...

  7. Rosiglitazone Balances Insulin-Induced Exo- And Endocytosis In Single 3t3-L1 Adipocytes

    Velebit, Jelena; Chowdhury, Helena H.; Kreft, Marko; Zorec, Robert

    2011-01-01

    Abstract Rosiglitazone (Rosi) improves insulin sensitivity and increases the translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This involves the fusion of membrane-bound compartments with the plasma membrane, thus increasing the plasma membrane area. However, recent work has shown that in Rosi-pretreated 3T3-L1 adipocytes membrane area did not increase following insulin application, suggesting that the rates of exo- and endocytosis are balanced. Here we ex...

  8. Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination

    Qiao, Bo; Sugianto, Priscilla; Fung, Eileen; del-Castillo-Rueda, Alejandro; Moran-Jimenez, Maria-Josefa; Ganz, Tomas; Nemeth, Elizabeta

    2012-01-01

    Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing fer...

  9. Identification and Selected Reaction Monitoring (SRM) Quantification of Endocytosis Factors Associated with Numb*

    Krieger, Jonathan R; Taylor, Paul; Gajadhar, Aaron S.; Guha, Abhijit; Moran, Michael F.; McGlade, C. Jane

    2012-01-01

    Numb is an endocytic adaptor protein that regulates the endocytosis and trafficking of transmembrane receptors including Notch, E-cadherin, and integrins. Vertebrate Numb is alternatively spliced at exons 3 and 9 to give rise to four protein isoforms. Expression of these isoforms varies at different developmental stages, and although the function of Numb isoforms containing exon 3 has been studied, the role of exon 9 inclusion has not been shown. Here we use affinity purification and tandem m...

  10. Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.

    Sarah J Fletcher

    Full Text Available Tight junctions (TJs link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes. By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.

  11. Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis

    Kastner, Christian; Pohl, Marcus; Sendeski, Mauricio;

    2009-01-01

    Human glomerulonephritis (GN) is characterized by sustained proteinuria, sodium retention, hypertension, and edema formation. Increasing quantities of filtered protein enter the renal tubule, where they may alter epithelial transport functions. Exaggerated endocytosis and consequent protein...... overload may affect proximal tubules, but intrinsic malfunction of distal epithelia has also been reported. A straightforward assignment to a particular tubule segment causing salt retention in GN is still controversial. We hypothesized that 1) trafficking and surface expression of major transporters and...

  12. Endocytosis of activated receptors and clathrin-coated pit formation: deciphering the chicken or egg relationship

    1996-01-01

    The fundamental mechanisms by which receptors once targeted for endocytosis are found in coated pits is an important yet unresolved question. Specifically, are activated receptors simply trapped on encountering preexisting coated pits, subsequently being rapidly internalized? Or do the receptors themselves, by active recruitment, gather soluble coat and cytosolic components and initiate the rapid assembly of new coated pits that then mediate their internalization? To explore this question, we...

  13. Multiple GTP-binding proteins participate in clathrin-coated vesicle- mediated endocytosis

    1993-01-01

    We have examined the effects of various agonists and antagonists of GTP- binding proteins on receptor-mediated endocytosis in vitro. Stage- specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to ...

  14. HIV-1 Vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane.

    2006-05-01

    Full Text Available The human immunodeficiency virus (HIV type-1 viral protein U (Vpu protein enhances the release of diverse retroviruses from human, but not monkey, cells and is thought to do so by ablating a dominant restriction to particle release. Here, we determined how Vpu expression affects the subcellular distribution of HIV-1 and murine leukemia virus (MLV Gag proteins in human cells where Vpu is, or is not, required for efficient particle release. In HeLa cells, where Vpu enhances HIV-1 and MLV release approximately 10-fold, concentrations of HIV-1 Gag and MLV Gag fused to cyan fluorescent protein (CFP were initially detected at the plasma membrane, but then accumulated over time in early and late endosomes. Endosomal accumulation of Gag-CFP was prevented by Vpu expression and, importantly, inhibition of plasma membrane to early endosome transport by dominant negative mutants of Rab5a, dynamin, and EPS-15. Additionally, accumulation of both HIV and MLV Gag in endosomes required a functional late-budding domain. In human HOS cells, where HIV-1 and MLV release was efficient even in the absence of Vpu, Gag proteins were localized predominantly at the plasma membrane, irrespective of Vpu expression or manipulation of endocytic transport. While these data indicated that Vpu inhibits nascent virion endocytosis, Vpu did not affect transferrin endocytosis. Moreover, inhibition of endocytosis did not restore Vpu-defective HIV-1 release in HeLa cells, but instead resulted in accumulation of mature virions that could be released from the cell surface by protease treatment. Thus, these findings suggest that a specific activity that is present in HeLa cells, but not in HOS cells, and is counteracted by Vpu, traps assembled retrovirus particles at the cell surface. This entrapment leads to subsequent endocytosis by a Rab5a- and clathrin-dependent mechanism and intracellular sequestration of virions in endosomes.

  15. Hyaluronic acid binding, endocytosis and degradation by sinusoidal liver endothelial cells

    The binding, endocytosis, and degradation of 125I-hyaluronic acid (125I-HA) by liver endothelial cells (LEC) was studied under several conditions. The dissociation of receptor-bound 125I-HA was rapid, with a half time of ∼31 min and a Koff of 6.3 x 10-4/sec. A large reversible increase in 125I-HA binding to LEC at pH 5.0 was due to an increase in the observed affinity of the binding interaction. Pronase digestion suggested the protein nature of the receptor and the intracellular location of the digitonin exposed binding activity. Binding and endocytosis occur in the presence of 10 mM EGTA indicating that divalent cations are not required for receptor function. To study the degradation of 125I-HA by LEC, a cetylpyridinium chloride (CPC) precipitation assay was characterized. The minimum HA length required for precipitation was elucidated. The fate of the LEC HA receptor after endocytosis was examined

  16. Hyaluronic acid binding, endocytosis and degradation by sinusoidal liver endothelial cells

    McGary, C.T.

    1988-01-01

    The binding, endocytosis, and degradation of {sup 125}I-hyaluronic acid ({sup 125}I-HA) by liver endothelial cells (LEC) was studied under several conditions. The dissociation of receptor-bound {sup 125}I-HA was rapid, with a half time of {approx}31 min and a K{sub off} of 6.3 {times} 10{sup {minus}4}/sec. A large reversible increase in {sup 125}I-HA binding to LEC at pH 5.0 was due to an increase in the observed affinity of the binding interaction. Pronase digestion suggested the protein nature of the receptor and the intracellular location of the digitonin exposed binding activity. Binding and endocytosis occur in the presence of 10 mM EGTA indicating that divalent cations are not required for receptor function. To study the degradation of {sup 125}I-HA by LEC, a cetylpyridinium chloride (CPC) precipitation assay was characterized. The minimum HA length required for precipitation was elucidated. The fate of the LEC HA receptor after endocytosis was examined.

  17. Responses of plant calmodulin to endocytosis induced by rare earth elements.

    Wang, Lihong; Cheng, Mengzhu; Chu, Yunxia; Li, Xiaodong; Chen, David D Y; Huang, Xiaohua; Zhou, Qing

    2016-07-01

    The wide application of rare earth elements (REEs) have led to their diffusion and accumulation in the environment. The activation of endocytosis is the primary response of plant cells to REEs. Calmodulin (CaM), as an important substance in calcium (Ca) signaling systems, regulating almost all of the physiological activities in plants, such as cellular metabolism, cell growth and division. However, the response of CaM to endocytosis activated by REEs remains unknown. By using immunofluorescence labeling and a confocal laser scanning microscope, we found that trivalent lanthanum [La(III)], an REE ion, affected the expression of CaM in endocytosis. Using circular dichroism, X-ray photoelectron spectroscopy and computer simulations, we demonstrated that a low concentration of La(III) could interact with extracellular CaM by electrostatic attraction and was then bound to two Ca-binding sites of CaM, making the molecular structure more compact and orderly, whereas a high concentration of La(III) could be coordinated with cytoplasmic CaM or bound to other Ca-binding sites, making the molecular structure more loose and disorderly. Our results provide a reference for revealing the action mechanisms of REEs in plant cells. PMID:27081794

  18. The translocation of fullerenic nanoparticles into lysosome via the pathway of clathrin-mediated endocytosis

    Li Wei; Chen Chunying; Ye Chang; Zhao Yuliang; Chen Zhen; Meng Huan; Gao Yuxi; Yuan Hui; Xing Genmei; Zhao Feng; Chai Zhifang [Laboratory for Bio-Environmental Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Nanotechnology of China and Institute of High Energy Physics, Chinese Academy of Science, Yuquan Road 19B, Beijing 100049 (China); Wei Taotao; Zhang Xujia; Yang Fuyu [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China); Lao Fang; Han Dong [National Center for Nanoscience and Technology of China, No 2, Ist North Street Zhongguancun, Beijing 100080 (China); Tang Xianhua; Zhang Yingge [Chinese Academy of Military Medical Sciences, Beijing 100039 (China)], E-mail: chenchy@nanoctr.cn, E-mail: weitt@moon.ibp.ac.cn, E-mail: zhaoyuliang@ihep.ac.cn

    2008-04-09

    Manufactured fullerene nanoparticles easily enter into cells and hence have been rapidly developed for biomedical uses. However, it is generally unknown which route the nanoparticles undergo when crossing cell membranes and where they localize to the intracellular compartments. Herein we have used both microscopic imaging and biological techniques to explore the processes of [C{sub 60}(C(COOH){sub 2}){sub 2}]{sub n} nanoparticles across cellular membranes and their intracellular translocation in 3T3 L1 and RH-35 living cells. The fullerene nanoparticles are quickly internalized by the cells and then routed to the cytoplasm with punctate localization. Upon entering the cell, they are synchronized to lysosome-like vesicles. The [C{sub 60}(C(COOH){sub 2}){sub 2}]{sub n} nanoparticles entering cells are mainly via endocytosis with time-, temperature- and energy-dependent manners. The cellular uptake of [C{sub 60}(C(COOH){sub 2}){sub 2}]{sub n} nanoparticles was found to be clathrin-mediated but not caveolae-mediated endocytosis. The endocytosis mechanism and the subcellular target location provide key information for the better understanding and predicting of the biomedical function of fullerene nanoparticles inside cells.

  19. The translocation of fullerenic nanoparticles into lysosome via the pathway of clathrin-mediated endocytosis

    Li, Wei; Chen, Chunying; Ye, Chang; Wei, Taotao; Zhao, Yuliang; Lao, Fang; Chen, Zhen; Meng, Huan; Gao, Yuxi; Yuan, Hui; Xing, Genmei; Zhao, Feng; Chai, Zhifang; Zhang, Xujia; Yang, Fuyu; Han, Dong; Tang, Xianhua; Zhang, Yingge

    2008-04-01

    Manufactured fullerene nanoparticles easily enter into cells and hence have been rapidly developed for biomedical uses. However, it is generally unknown which route the nanoparticles undergo when crossing cell membranes and where they localize to the intracellular compartments. Herein we have used both microscopic imaging and biological techniques to explore the processes of [C60(C(COOH)2)2]n nanoparticles across cellular membranes and their intracellular translocation in 3T3 L1 and RH-35 living cells. The fullerene nanoparticles are quickly internalized by the cells and then routed to the cytoplasm with punctate localization. Upon entering the cell, they are synchronized to lysosome-like vesicles. The [C60(C(COOH)2)2]n nanoparticles entering cells are mainly via endocytosis with time-, temperature- and energy-dependent manners. The cellular uptake of [C60(C(COOH)2)2]n nanoparticles was found to be clathrin-mediated but not caveolae-mediated endocytosis. The endocytosis mechanism and the subcellular target location provide key information for the better understanding and predicting of the biomedical function of fullerene nanoparticles inside cells.

  20. Activity-dependent targeting of TRPV1 with a pore-permeating capsaicin analog

    Hui LI; Wang, Shu; Chuang, Alexander Y.; Cohen, Bruce E.; Chuang, Huai-hu

    2011-01-01

    The capsaicin receptor TRPV1 is the principal transduction channel for nociception. Excessive TRPV1 activation causes pathological pain. Ideal pain mangement requires selective inhibition of hyperactive pain-sensing neurons, but sparing normal nociception. We sought to determine whether it is possible to use activity-dependent TRPV1 agonists to identify nerves with excessive TRPV1 activity, as well as exploit the TRPV1 pore to deliver charged anesthetics for neuronal silencing. We synthesized...

  1. The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition

    Bloodgood, Brenda L.; Sharma, Nikhil; Browne, Heidi Adlman; Trepman, Alissa Z.; Greenberg, Michael E.

    2013-01-01

    A heterogeneous population of inhibitory neurons controls the flow of information through a neural circuit1–3. Inhibitory synapses that form on pyramidal neuron dendrites modulate the summation of excitatory synaptic potentials4–6 and prevent the generation of dendritic calcium spikes7,8. Precisely timed somatic inhibition limits both the number of action potentials and the time window during which firing can occur8,9. The activity-dependent transcription factor NPAS4 regulates inhibitory syn...

  2. Robustness and Enhancement of Neural Synchronization by Activity-Dependent Coupling

    Zhigulin, Valentin P.; Mikhail I. Rabinovich; Huerta, Ramon; Abarbanel, Henry D. I.

    2002-01-01

    We study the synchronization of two model neurons coupled through a synapse having an activity-dependent strength. Our synapse follows the rules of Spike-Timing Dependent Plasticity (STDP). We show that this plasticity of the coupling between neurons produces enlarged frequency locking zones and results in synchronization that is more rapid and much more robust against noise than classical synchronization arising from connections with constant strength. We also present a simple discrete map m...

  3. The Development and Activity-Dependent Expression of Aggrecan in the Cat Visual Cortex

    Kind, P. C.; Sengpiel, F.; Beaver, C. J.; Crocker-Buque, A.; Kelly, G. M.; R. T. Matthews; Mitchell, D E

    2012-01-01

    The Cat-301 monoclonal antibody identifies aggrecan, a chondroitin sulfate proteoglycan in the cat visual cortex and dorsal lateral geniculate nucleus (dLGN). During development, aggrecan expression increases in the dLGN with a time course that matches the decline in plasticity. Moreover, examination of tissue from selectively visually deprived cats shows that expression is activity dependent, suggesting a role for aggrecan in the termination of the sensitive period. Here, we demonstrate for ...

  4. Robustness and Enhancement of Neural Synchronization by Activity-Dependent Coupling

    Zhigulin, V P; Huerta, R; Abarbanel, Henry D I; Zhigulin, Valentin P.; Rabinovich, Mikhail I.; Huerta, Ramon; Abarbanel, Henry D I

    2003-01-01

    We study the synchronization of two model neurons coupled through a synapse having an activity-dependent strength. Our synapse follows the rules of Spike-Timing Dependent Plasticity (STDP). We show that this plasticity of the coupling between neurons produces enlarged frequency locking zones and results in synchronization that is more rapid and much more robust against noise than classical synchronization arising from connections with constant strength. We also present a simple discrete map model that demonstrates the generality of the phenomenon.

  5. Expression of activity-dependent neuroprotective protein in the brain of adult rats

    Gennet, N.; Herden, C.; Bubb, V J; Quinn, J P; Kipar, A.

    2008-01-01

    Activity-dependent neuroprotective protein (ADNP) is a VIP-regulated gene, which is essential for brain development. A synthetic peptide (NAP) derived from the ADNP sequence is highly neuroprotective, therefore it has been hypothesised that ADNP has a similar role. ADNP contains classical transcription factor motifs and nuclear localisation domains, but it has also been reported to be secreted and to co-localise with microtubules, indicating that ADNP may have multiple...

  6. Wormholes in Bulk Viscous Cosmology

    Jamil, Mubasher

    2008-01-01

    We investigate the effects of the accretion of phantom energy with non-zero bulk viscosity onto a Morris-Thorne wormhole. We have found that if the bulk viscosity is large then the mass of wormhole increases rapidly as compared to small or zero bulk viscosity.

  7. Creating bulk nanocrystalline metal.

    Fredenburg, D. Anthony (Georgia Institute of Technology, Atlanta, GA); Saldana, Christopher J. (Purdue University, West Lafayette, IN); Gill, David D.; Hall, Aaron Christopher; Roemer, Timothy John (Ktech Corporation, Albuquerque, NM); Vogler, Tracy John; Yang, Pin

    2008-10-01

    Nanocrystalline and nanostructured materials offer unique microstructure-dependent properties that are superior to coarse-grained materials. These materials have been shown to have very high hardness, strength, and wear resistance. However, most current methods of producing nanostructured materials in weapons-relevant materials create powdered metal that must be consolidated into bulk form to be useful. Conventional consolidation methods are not appropriate due to the need to maintain the nanocrystalline structure. This research investigated new ways of creating nanocrystalline material, new methods of consolidating nanocrystalline material, and an analysis of these different methods of creation and consolidation to evaluate their applicability to mesoscale weapons applications where part features are often under 100 {micro}m wide and the material's microstructure must be very small to give homogeneous properties across the feature.

  8. Explosive bulk charge

    Miller, Jacob Lee

    2015-04-21

    An explosive bulk charge, including: a first contact surface configured to be selectively disposed substantially adjacent to a structure or material; a second end surface configured to selectively receive a detonator; and a curvilinear side surface joining the first contact surface and the second end surface. The first contact surface, the second end surface, and the curvilinear side surface form a bi-truncated hemispherical structure. The first contact surface, the second end surface, and the curvilinear side surface are formed from an explosive material. Optionally, the first contact surface and the second end surface each have a substantially circular shape. Optionally, the first contact surface and the second end surface consist of planar structures that are aligned substantially parallel or slightly tilted with respect to one another. The curvilinear side surface has one of a smooth curved geometry, an elliptical geometry, and a parabolic geometry.

  9. The Incredible Bulk

    Fukushima, Keita; Kumar, Jason; Sandick, Pearl; Yamamoto, Takahiro

    2014-01-01

    Recent experimental results from the LHC have placed strong constraints on the masses of colored superpartners. The MSSM parameter space is also constrained by the measurement of the Higgs boson mass, and the requirement that the relic density of lightest neutralinos be consistent with observations. Although large regions of the MSSM parameter space can be excluded by these combined bounds, leptophilic versions of the MSSM can survive these constraints. In this paper we consider a scenario in which the requirements of minimal flavor violation, vanishing $CP$-violation, and mass universality are relaxed, specifically focusing on scenarios with light sleptons. We find a large region of parameter space, analogous to the original bulk region, for which the lightest neutralino is a thermal relic with an abundance consistent with that of dark matter. We find that these leptophilic models are constrained by measurements of the magnetic and electric dipole moments of the electron and muon, and that these models have ...

  10. Bulk muscles, loose cables.

    Liyanage, Chamari R D G; Kodali, Venkata

    2014-01-01

    The accessibility and usage of body building supplements is on the rise with stronger internet marketing strategies by the industry. The dangers posed by the ingredients in them are underestimated. A healthy young man came to the emergency room with palpitations and feeling unwell. Initial history and clinical examination were non-contributory to find the cause. ECG showed atrial fibrillation. A detailed history for any over the counter or herbal medicine use confirmed that he was taking supplements to bulk muscle. One of the components in these supplements is yohimbine; the onset of symptoms coincided with the ingestion of this product and the patient is symptom free after stopping it. This report highlights the dangers to the public of consuming over the counter products with unknown ingredients and the consequential detrimental impact on health. PMID:25326558

  11. Early survival factor deprivation in the olfactory epithelium enhances activity-dependent survival

    Adrien eFrançois

    2013-12-01

    Full Text Available The neuronal olfactory epithelium undergoes permanent renewal because of environmental aggression. This renewal is partly regulated by factors modulating the level of neuronal apoptosis. Among them, we had previously characterized endothelin as neuroprotective. In this study, we explored the effect of cell survival factor deprivation in the olfactory epithelium by intranasal delivery of endothelin receptors antagonists to rat pups. This treatment induced an overall increase of apoptosis in the olfactory epithelium. The responses to odorants recorded by electroolfactogram were decreased in treated animal, a result consistent with a loss of olfactory sensory neurons (OSNs. However, the treated animal performed better in an olfactory orientation test based on maternal odor compared to non-treated littermates. This improved performance could be due to activity-dependent neuronal survival of OSNs in the context of increased apoptosis level. In order to demonstrate it, we odorized pups with octanal, a known ligand for the rI7 olfactory receptor (Olr226. We quantified the number of OSN expressing rI7 by RT-qPCR and whole mount in situ hybridization. While this number was reduced by the survival factor removal treatment, this reduction was abolished by the presence of its ligand. This improved survival was optimal for low concentration of odorant and was specific for rI7-expressing OSNs. Meanwhile, the number of rI7-expressing OSNs was not affected by the odorization in non-treated littermates; showing that the activity-dependant survival of OSNs did not affect the OSN population during the 10 days of odorization in control conditions. Overall, our study shows that when apoptosis is promoted in the olfactory mucosa, the activity-dependent neuronal plasticity allows faster tuning of the olfactory sensory neuron population towards detection of environmental odorants.

  12. Inflation from bulk viscosity

    Bamba, Kazuharu

    2015-01-01

    We explore the perfect fluid description of the inflationary universe. In particular, we investigate a fluid model with the bulk-viscosity term. We find that the three observables of inflationary cosmology: the spectral index of the curvature perturbations, the tensor-to-scalar ratio of the density perturbations, and the running of the spectral index, can be consistent with the recent Planck results. We also reconstruct the explicit equation of state (EoS) of the viscous fluid from the spectral index of the curvature perturbations compatible with the Planck analysis. In the reconstructed models of the viscous fluid, the tensor-to-scalar ratio of the density perturbations can satisfy the constraints obtained from the Planck satellite. The running of the spectral index can explain the Planck data. In addition, it is demonstrated that in the reconstructed models of the viscous fluid, the graceful exit from inflation can be realized. Furthermore, we show that the singular inflation can occur in the viscous fluid ...

  13. Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

    Qi He

    Full Text Available The endocytosis of transferrin receptor (TfR has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

  14. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    Coffey, Sam; Costacou, Tina; Orchard, Trevor; Erkan, Elif

    2015-01-01

    Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage. PMID:26465605

  15. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    Sam Coffey

    Full Text Available Diabetes mellitus (DM has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160 and cytoplasmic tail of megalin. Mice with type 1 DM (T1D displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN at an earlier stage.

  16. Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

    de la Vega Michelle

    2011-12-01

    Full Text Available Abstract Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

  17. Bulk-Fill Resin Composites

    Benetti, Ana Raquel; Havndrup-Pedersen, Cæcilie; Honoré, Daniel;

    2015-01-01

    restorative procedure. The aim of this study, therefore, was to compare the depth of cure, polymerization contraction, and gap formation in bulk-fill resin composites with those of a conventional resin composite. To achieve this, the depth of cure was assessed in accordance with the International Organization...... for Standardization 4049 standard, and the polymerization contraction was determined using the bonded-disc method. The gap formation was measured at the dentin margin of Class II cavities. Five bulk-fill resin composites were investigated: two high-viscosity (Tetric EvoCeram Bulk Fill, SonicFill) and...... three low-viscosity (x-tra base, Venus Bulk Fill, SDR) materials. Compared with the conventional resin composite, the high-viscosity bulk-fill materials exhibited only a small increase (but significant for Tetric EvoCeram Bulk Fill) in depth of cure and polymerization contraction, whereas the low...

  18. Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization

    1992-01-01

    Macrophages and B-lymphocytes express two major isoforms of Fc receptor (FcRII-B2 and FcRII-B1) that exhibit distinct capacities for endocytosis. This difference in function reflects the presence of an in- frame insertion of 47 amino acids in the cytoplasmic domain of the lymphocyte isoform (FcRII-B1) due to alternative mRNA splicing. By expressing wild type and mutant FcRII cDNAs in fibroblasts, we have now examined the mechanism by which the insertion acts to prevent coated pit localization...

  19. Role of coated vesicles, microfilaments, and calmodulin in receptor- mediated endocytosis by cultured B lymphoblastoid cells

    1980-01-01

    Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) bindi...

  20. Role of endocytosis and cathepsin-mediated activation in Nipah virus entry

    The recent discovery that the Nipah virus (NiV) fusion protein (F) is activated by endosomal cathepsin L raised the question if NiV utilize pH- and protease-dependent mechanisms of entry. We show here that the NiV receptor ephrin B2, virus-like particles and infectious NiV are internalized from the cell surface. However, endocytosis, acidic pH and cathepsin-mediated cleavage are not necessary for the initiation of infection of new host cells. Our data clearly demonstrate that proteolytic activation of the NiV F protein is required before incorporation into budding virions but not after virus entry

  1. Uptake of fluorescent nano beads into BY2-cells involves clathrin-dependent and clathrin-independent endocytosis.

    Bandmann, Vera; Müller, Jasmin Daniela; Köhler, Tim; Homann, Ulrike

    2012-10-19

    To follow endocytosis in BY-2 cells we made use of fluorescent nano beads. Beads with 20nm in diameter were internalised rapidly and accumulated partially in compartments also labelled by the endocytic marker FM4-64. Studies in BY-2 cells and protoplasts revealed that larger beads (100nm) were excluded from uptake into turgescent and plasmolysed cells while protoplasts were able to internalise beads with a diameter of up to 1000nm. Endocytosis of beads was only partially inhibited by the clathrin-specific inhibitor Ikarugamycin and strongly blocked by wortmannin. These results imply that uptake of beads involves clathrin-dependent and clathrin-independent endocytic mechanisms and supports the hypothesis that clathrin-independent endocytosis plays a general role in plants. PMID:23046971

  2. Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis

    Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kindey cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments

  3. Size-dependent endocytosis of gold nanoparticles studied by three-dimensional mapping of plasmonic scattering images

    Lee Chia-Wei

    2010-12-01

    Full Text Available Abstract Background Understanding the endocytosis process of gold nanoparticles (AuNPs is important for the drug delivery and photodynamic therapy applications. The endocytosis in living cells is usually studied by fluorescent microscopy. The fluorescent labeling suffers from photobleaching. Besides, quantitative estimation of the cellular uptake is not easy. In this paper, the size-dependent endocytosis of AuNPs was investigated by using plasmonic scattering images without any labeling. Results The scattering images of AuNPs and the vesicles were mapped by using an optical sectioning microscopy with dark-field illumination. AuNPs have large optical scatterings at 550-600 nm wavelengths due to localized surface plasmon resonances. Using an enhanced contrast between yellow and blue CCD images, AuNPs can be well distinguished from cellular organelles. The tracking of AuNPs coated with aptamers for surface mucin glycoprotein shows that AuNPs attached to extracellular matrix and moved towards center of the cell. Most 75-nm-AuNPs moved to the top of cells, while many 45-nm-AuNPs entered cells through endocytosis and accumulated in endocytic vesicles. The amounts of cellular uptake decreased with the increase of particle size. Conclusions We quantitatively studied the endocytosis of AuNPs with different sizes in various cancer cells. The plasmonic scattering images confirm the size-dependent endocytosis of AuNPs. The 45-nm-AuNP is better for drug delivery due to its higher uptake rate. On the other hand, large AuNPs are immobilized on the cell membrane. They can be used to reconstruct the cell morphology.

  4. The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis

    Chang Henry C

    2008-05-01

    Full Text Available Abstract Background Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux, a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood. Results To understand the role of auxilin in Notch ligand endocytosis, we have analyzed several point mutations affecting specific domains of dAux. In agreement with previous work, analysis using these stronger dAux alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in dAux deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing dAux phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in dAux mutant cells, Delta accumulates at the cell surface. In dAux mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures. Conclusion Our data suggest that dAux mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in dAux mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, our observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization.

  5. Activity-dependent regulation of calcium and ribosomes in the chick cochlear nucleus.

    Call, C L; Hyson, R L

    2016-03-01

    Cochlea removal results in the death of 20-30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). Two potentially cytotoxic events, a dramatic rise in intracellular calcium concentration ([Ca(2+)]i) and a decline in the integrity of ribosomes are observed within 1h of deafferentation. Glutamatergic input from the auditory nerve has been shown to preserve NM neuron health by activating metabotropic glutamate receptors (mGluRs), maintaining both normal [Ca(2+)]i and ribosomal integrity. One interpretation of these results is that a common mGluR-activated signaling cascade is required for the maintenance of both [Ca(2+)]i and ribosomal integrity. This could happen if both responses are influenced directly by a common messenger, or if the loss of mGluR activation causes changes in one component that secondarily causes changes in the other. The present studies tested this common-mediator hypothesis in slice preparations by examining activity-dependent regulation of [Ca(2+)]i and ribosomes in the same tissue after selectively blocking group I mGluRs (1-Aminoindan-1,5-dicarboxylic acid (AIDA)) or group II mGluRs (LY 341495) during unilateral auditory nerve stimulation. Changes in [Ca(2+)]i of NM neurons were measured using fura-2 ratiometric calcium imaging and the tissue was subsequently processed for Y10B immunoreactivity (Y10B-ir), an antibody that recognizes a ribosomal epitope. The group I mGluR antagonist blocked the activity-dependent regulation of both [Ca(2+)]i and Y10B-ir, but the group II antagonist blocked only the activity-dependent regulation of Y10B-ir. That is, even when group II receptors were blocked, stimulation continued to maintain low [Ca(2+)]i, but it did not maintain Y10B-ir. These results suggest a dissociation in how calcium and ribosomes are regulated in NM neurons and that ribosomes can be regulated through a mechanism that is independent of calcium regulation. PMID:26739326

  6. Diacylglycerol Guides the Hopping of Clathrin-Coated Pits along Microtubules for Exo-Endocytosis Coupling.

    Yuan, Tianyi; Liu, Lin; Zhang, Yongdeng; Wei, Lisi; Zhao, Shiqun; Zheng, Xiaolu; Huang, Xiaoshuai; Boulanger, Jerome; Gueudry, Charles; Lu, Jingze; Xie, Lihan; Du, Wen; Zong, Weijian; Yang, Lu; Salamero, Jean; Liu, Yanmei; Chen, Liangyi

    2015-10-12

    Many receptor-mediated endocytic processes are mediated by constitutive budding of clathrin-coated pits (CCPs) at spatially randomized sites before slowly pinching off from the plasma membrane (60-100 s). In contrast, clathrin-mediated endocytosis (CME) coupled with regulated exocytosis in excitable cells occurs at peri-exocytic sites shortly after vesicle fusion (∼10 s). The molecular mechanism underlying this spatiotemporal coupling remains elusive. We show that coupled endocytosis makes use of pre-formed CCPs, which hop to nascent fusion sites nearby following vesicle exocytosis. A dynamic cortical microtubular network, anchored at the cell surface by the cytoplasmic linker-associated protein on microtubules and the LL5β/ELKS complex on the plasma membrane, provides the track for CCP hopping. Local diacylglycerol gradients generated upon exocytosis guide the direction of hopping. Overall, the CCP-cytoskeleton-lipid interaction demonstrated here mediates exocytosis-coupled fast recycling of both plasma membrane and vesicular proteins, and it is required for the sustained exocytosis during repetitive stimulations. PMID:26439397

  7. Seamless tube shape is constrained by endocytosis-dependent regulation of active Moesin.

    Schottenfeld-Roames, Jodi; Rosa, Jeffrey B; Ghabrial, Amin S

    2014-08-01

    Most tubes have seams (intercellular or autocellular junctions that seal membranes together into a tube), but "seamless" tubes also exist. In Drosophila, stellate-shaped tracheal terminal cells make seamless tubes, with single branches running through each of dozens of cellular extensions. We find that mutations in braided impair terminal cell branching and cause formation of seamless tube cysts. We show that braided encodes Syntaxin7 and that cysts also form in cells deficient for other genes required either for membrane scission (shibire) or for early endosome formation (Rab5, Vps45, and Rabenosyn-5). These data define a requirement for early endocytosis in shaping seamless tube lumens. Importantly, apical proteins Crumbs and phospho-Moesin accumulate to aberrantly high levels in braided terminal cells. Overexpression of either Crumbs or phosphomimetic Moesin induced lumenal cysts and decreased terminal branching. Conversely, the braided seamless tube cyst phenotype was suppressed by mutations in crumbs or Moesin. Indeed, mutations in Moesin dominantly suppressed seamless tube cyst formation and restored terminal branching. We propose that early endocytosis maintains normal steady-state levels of Crumbs, which recruits apical phosphorylated (active) Moe, which in turn regulates seamless tube shape through modulation of cortical actin filaments. PMID:25065756

  8. The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

    Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions. Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C; vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content

  9. Kinetics of cellular uptake of viruses and nanoparticles via clathrin-mediated endocytosis

    Banerjee, Anand; Berezhkovskii, Alexander; Nossal, Ralph

    2016-02-01

    Several viruses exploit clathrin-mediated endocytosis to gain entry into host cells. This process is also used extensively in biomedical applications to deliver nanoparticles (NPs) to diseased cells. The internalization of these nano-objects is controlled by the assembly of a clathrin-containing protein coat on the cytoplasmic side of the plasma membrane, which drives the invagination of the membrane and the formation of a cargo-containing endocytic vesicle. Current theoretical models of receptor-mediated endocytosis of viruses and NPs do not explicitly take coat assembly into consideration. In this paper we study cellular uptake of viruses and NPs with a focus on coat assembly. We characterize the internalization process by the mean time between the binding of a particle to the membrane and its entry into the cell. Using a coarse-grained model which maps the stochastic dynamics of coat formation onto a one-dimensional random walk, we derive an analytical formula for this quantity. A study of the dependence of the mean internalization time on NP size shows that there is an upper bound above which this time becomes extremely large, and an optimal size at which it attains a minimum. Our estimates of these sizes compare well with experimental data. We also study the sensitivity of the obtained results on coat parameters to identify factors which significantly affect the internalization kinetics.

  10. Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes

    Hirai Toshiro

    2011-01-01

    Full Text Available Abstract Background Clarifying the physicochemical properties of nanomaterials is crucial for hazard assessment and the safe application of these substances. With this in mind, we analyzed the relationship between particle size and the in vitro effect of amorphous nanosilica (nSP. Specifically, we evaluated the relationship between particle size of nSP and the in vitro biological effects using human keratinocyte cells (HaCaT. Results Our results indicate that exposure to nSP of 70 nm diameter (nSP70 induced an elevated level of reactive oxygen species (ROS, leading to DNA damage. A markedly reduced response was observed using submicron-sized silica particles of 300 and 1000 nm diameter. In addition, cytochalasin D-treatment reduced nSP70-mediated ROS generation and DNA damage, suggesting that endocytosis is involved in nSP70-mediated cellular effects. Conclusions Thus, particle size affects amorphous silica-induced ROS generation and DNA damage of HaCaT cells. We believe clarification of the endocytosis pathway of nSP will provide useful information for hazard assessment as well as the design of safer forms of nSPs.

  11. Effect of endocytosis inhibitors on Coxiella burnetii interaction with host cells

    The obligate intracellular rickettsia Coxiella burnetii has previously been reported to reach the intra-vacuolar compartment of host cells by phagocytosis. With the aim to further examine the mechanisms of C. burnetii internalisation, macrophage monolayers were treated with well characterised inhibitors of endocytosis. The treatment with two general inhibitors, colchicine and methylamine, resulted in a pronounced dose-dependent decrease of radiolabelled phase II rickettsiae retained from the intracellular fraction. A third inhibitor used, amiloride, has been reported to reduce effectively clathrin-independent pinocytic pathways. The internalisation of C. burnetii was shown to be substantially reduced also by amiloride and the effect was dependent on its concentration. The passive role of C. burnetii in the internalisation was verified by using heat-killed C. burnetii. Host cells treated with either of the three inhibitors (amiloride, colchicine and methylamine) showed a similar reduction of intracellular C. burnetii after exposure to killed as weal as live organisms. The data presented indicate that different endocytic mechanisms, pinocytosis as well as phagocytosis, may mediate the uptake of C. burnetii by a host cell. Key words: Coxiella burnetii; internalisation; endocytosis (authors)

  12. New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy.

    Farrell, Kristen B; Grossman, Caitlin; Di Pietro, Santiago M

    2015-11-01

    Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identify previously unknown regulatory factors of the endocytic machinery. We further studied the top scoring protein identified in the screen, Ubx3, a member of the conserved ubiquitin regulatory X (UBX) protein family. In vivo and in vitro approaches demonstrate that Ubx3 is a new coat component. Ubx3-GFP has typical endocytic coat protein dynamics with a patch lifetime of 45 ± 3 sec. Ubx3 contains a W-box that mediates physical interaction with clathrin and Ubx3-GFP patch lifetime depends on clathrin. Deletion of the UBX3 gene caused defects in the uptake of Lucifer Yellow and the methionine transporter Mup1 demonstrating that Ubx3 is needed for efficient endocytosis. Further, the UBX domain is required both for localization and function of Ubx3 at endocytic sites. Mechanistically, Ubx3 regulates dynamics and patch lifetime of the early arriving protein Ede1 but not later arriving coat proteins or actin assembly. Conversely, Ede1 regulates the patch lifetime of Ubx3. Ubx3 likely regulates CME via the AAA-ATPase Cdc48, a ubiquitin-editing complex. Our results uncovered new components of the CME machinery that regulate this fundamental process. PMID:26362318

  13. Osmotic induction of fluid-phase endocytosis in onion epidermal cells.

    Oparka, K J; Prior, D A; Harris, N

    1990-03-01

    A transient plasmolysis/deplasmolysis (plasmolytic cycle) of onion epidermal cells has been shown to induce the formation of fluid-phase endocytic vesicles. Plasmolysis in the presence of the membrane-impermeant fluorescent probes Lucifer Yellow CH (LYCH) and Cascade Blue hydrazide resulted in the uptake of these probes by fluid-phase endocytosis. Following deplasmolysis, many of the dye-containing vesicles left their parietal positions within the cell and underwent vigorous streaming in the cytoplasm. Vesicles were observed to move within transvacuolar strands and their movements were recorded over several hours by video-microscopy. Within 2 h of deplasmolysis several of the larger endocytic vesicles had clustered around the nuclear membrane, apparently lodged in the narrow zone of cytoplams surrounding the nucleus. In further experiments LYCH was endocytically loaded into the cells during the first plasmolytic cycle and Cascade Blue subsequently loaded during a second plasmolytic cycle. This resulted in the introduction of two populations of endocytic vesicles into the cells, each containing a different probe. Both sets of vesicles underwent cytoplasmic streaming. The data are discussed in the light of previous observations of fluid-phase endocytosis in plant cells. PMID:24202101

  14. CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells

    Pritschet, Kathrin; Donhauser, Norbert; Schuster, Philipp; Ries, Moritz; Haupt, Sabrina; Kittan, Nicolai A.; Korn, Klaus [Institute of Clinical and Molecular Virology, National Reference Centre for Retroviruses, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 91054 Erlangen (Germany); Poehlmann, Stefan [Institute of Virology, Hannover Medical School, 30625 Hannover (Germany); Holland, Gudrun; Bannert, Norbert [Robert Koch-Institute, Center for Biological Security 4, 13353 Berlin (Germany); Bogner, Elke [Institute of Virology, Charite University Hospital, 10117 Berlin (Germany); Schmidt, Barbara, E-mail: baschmid@viro.med.uni-erlangen.de [Institute of Clinical and Molecular Virology, National Reference Centre for Retroviruses, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 91054 Erlangen (Germany)

    2012-02-20

    Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p < 0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

  15. CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells

    Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p < 0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

  16. Single wall carbon nanotubes enter cells by endocytosis and not membrane penetration

    Lösche Mathias

    2011-09-01

    Full Text Available Abstract Background Carbon nanotubes are increasingly being tested for use in cellular applications. Determining the mode of entry is essential to control and regulate specific interactions with cells, to understand toxicological effects of nanotubes, and to develop nanotube-based cellular technologies. We investigated cellular uptake of Pluronic copolymer-stabilized, purified ~145 nm long single wall carbon nanotubes (SWCNTs through a series of complementary cellular, cell-mimetic, and in vitro model membrane experiments. Results SWCNTs localized within fluorescently labeled endosomes, and confocal Raman spectroscopy showed a dramatic reduction in SWCNT uptake into cells at 4°C compared with 37°C. These data suggest energy-dependent endocytosis, as shown previously. We also examined the possibility for non-specific physical penetration of SWCNTs through the plasma membrane. Electrochemical impedance spectroscopy and Langmuir monolayer film balance measurements showed that Pluronic-stabilized SWCNTs associated with membranes but did not possess sufficient insertion energy to penetrate through the membrane. SWCNTs associated with vesicles made from plasma membranes but did not rupture the vesicles. Conclusions These measurements, combined, demonstrate that Pluronic-stabilized SWCNTs only enter cells via energy-dependent endocytosis, and association of SWCNTs to membrane likely increases uptake.

  17. Calcium channel types contributing to chromaffin cell excitability, exocytosis and endocytosis.

    Mahapatra, S; Calorio, C; Vandael, D H F; Marcantoni, A; Carabelli, V; Carbone, E

    2012-01-01

    Voltage gated Ca(2+) channels are effective voltage sensors of plasma membrane which convert cell depolarizations into Ca(2+) signaling. The chromaffin cells of the adrenal medulla utilize a large number of Ca(2+) channel types to drive the Ca(2+)-dependent release of catecholamines into blood circulation, during normal or stress-induced conditions. Some of the Ca(2+) channels expressed in chromaffin cells (L, N, P/Q, R and T), however, do not control only vesicle fusion and catecholamine release. They also subserve a variety of key activities which are vital for the physiological and pathological functioning of the cell, like: (i) shaping the action potentials of electrical oscillations driven either spontaneously or by ACh stimulation, (ii) controlling the action potential frequency of tonic or bursts firing, (iii) regulating the compensatory and excess endocytosis following robust exocytosis and (iv) driving the remodeling of Ca(2+) signaling which occurs during stressors stimulation. Here, we will briefly review the well-established properties of voltage-gated Ca(2+) channels accumulated over the past three decades focusing on the most recent discoveries on the role that L- (Cav1.2, Cav1.3) and T-type (Cav3.2) channels play in the control of excitability, exocytosis and endocytosis of chromaffin cells in normal and stress-mimicking conditions. PMID:22317919

  18. Increased expression of endocytosis-Related proteins in rat hippocampus following 10-day electroconvulsive seizure treatment.

    Enomoto, Shingo; Shimizu, Kunio; Nibuya, Masashi; Toda, Hiroyuki; Yoshino, Aihide; Suzuki, Eiji; Kondo, Takashi; Fukuda, Hiroshi

    2016-06-15

    Although electroconvulsive therapy (ECT) is clinically used for severe depression and drug-resistant Parkinson's disease, its exact biological background and mechanism have not yet been fully elucidated. Two potential explanations have been presented so far to explain the increased neuroplastic and resilient profiles of multiple ECT administrations. One is the alteration of central neurotransmitter receptor densities and the other is the expressional upregulation of brain derived neurotrophic factor in various brain regions with enhanced hippocampal neurogenesis and mossy fiber sprouting. In the present report, western blot analyses revealed significantly upregulated expression of various endocytosis-related proteins following 10-day electroconvulsive seizure (ECS) treatment in rat hippocampal homogenates and hippocampal lipid raft fractions extracted using an ultracentrifugation procedure. Upregulated proteins included endocytosis-related scaffolding proteins (caveolin-1, flotillin-1, and heavy and light chains of clathrin) and small GTPases (Rab5, Rab7, Rab11, and Rab4) specifically expressed on various types of endosomes. Two scaffolding proteins, caveolin-1 and flotillin-1, were also increased in the lipid raft fraction. Together with our previous finding of increased autophagy-related proteins in the hippocampal region, the present results suggest membrane trafficking machinery is enhanced following 10-day ECS treatment. We consider that the membrane trafficking machinery that transports functional proteins in the neuronal cells and from or into the synaptic membranes is one of the new candidates supporting the cellular and behavioral neuroplastic profiles of ECS treatments in animal experiments and ECT administrations in clinical settings. PMID:27177725

  19. Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification

    Dejonghe, Wim; Kuenen, Sabine; Mylle, Evelien; Vasileva, Mina; Keech, Olivier; Viotti, Corrado; Swerts, Jef; Fendrych, Matyáš; Ortiz-Morea, Fausto Andres; Mishev, Kiril; Delang, Simon; Scholl, Stefan; Zarza, Xavier; Heilmann, Mareike; Kourelis, Jiorgos; Kasprowicz, Jaroslaw; Nguyen, Le Son Long; Drozdzecki, Andrzej; Van Houtte, Isabelle; Szatmári, Anna-Mária; Majda, Mateusz; Baisa, Gary; Bednarek, Sebastian York; Robert, Stéphanie; Audenaert, Dominique; Testerink, Christa; Munnik, Teun; Van Damme, Daniël; Heilmann, Ingo; Schumacher, Karin; Winne, Johan; Friml, Jiří; Verstreken, Patrik; Russinova, Eugenia

    2016-01-01

    ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane. PMID:27271794

  20. The ulcerative colitis marker protein WAFL interacts with accessory proteins in endocytosis

    You Fu Pan, Ing-Marie Viklund, Heng Hang Tsai, Sven Pettersson, Ichiro N. Maruyama

    2010-01-01

    Full Text Available Ulcerative colitis (UC is one of the major forms of inflammatory bowel disease with unknown cause. A molecular marker, WAFL, has recently been found to be up-regulated in the inflamed colonic mucosa of UC patients. Towards understanding biological function of WAFL, we analyzed proteins interacting with WAFL in HEK-293 cells by immunoprecipitation and mass spectrometry. Among four proteins found to specifically interact with WAFL, both KIAA0196 and KIAA1033 bind to α-appendage of the adaptor protein complex 2 (AP2, which acts as an interaction hub for accessory proteins in endocytosis mediated by clathrin-coated vesicle (CCV. The specific interaction between WAFL and KIAA0196 was also confirmed in human colorectal carcinoma HCT-116 cells by co-immunoprecipitation with specific antibodies. Meta-analyses of the databases of expressed genes suggest that the three genes are co-expressed in many tissues and cell types, and that their molecular function may be classified in the category of 'membrane traffic protein'. Therefore, these results suggest that WAFL may play an important role in endocytosis and subsequent membrane trafficking by interacting with AP2 through KIAA0196 and KIAA1033.

  1. Mining the bulk positron lifetime

    Aourag, H.; Guittom, A. [Centre de Recherche Nucleaire d' Alger (CRNA), Alger Gare - Algiers (Algeria)

    2009-02-15

    We introduce a new approach to investigate the bulk positron lifetimes of new systems based on data-mining techniques. Through data mining of bulk positron lifetimes, we demonstrate the ability to predict the positron lifetimes of new semiconductors on the basis of available semiconductor data already studied. Informatics techniques have been applied to bulk positron lifetimes for different tetrahedrally bounded semiconductors in order to discover computational design rules. (copyright 2009 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  2. Mining the bulk positron lifetime

    We introduce a new approach to investigate the bulk positron lifetimes of new systems based on data-mining techniques. Through data mining of bulk positron lifetimes, we demonstrate the ability to predict the positron lifetimes of new semiconductors on the basis of available semiconductor data already studied. Informatics techniques have been applied to bulk positron lifetimes for different tetrahedrally bounded semiconductors in order to discover computational design rules. (copyright 2009 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  3. Advances in bulk port development

    Soros, P. (Soros Associates Consulting Engineers, New York, NY (USA))

    1991-03-01

    The article features several recently developed bulk ports which illustrate aspects of new technology or concepts in maritime transport. Low handling capacity bulk terminals at Ponta da Madeira, Brazil and Kooragang Island, Australia and the low-cost bulk port at Port of Corpus Christi, Texas are described. Operations at the ports of Pecket and Tocopilla in Chile, which had special technical problems, are mentioned. Coal terminals at Port Kembla, Australia and St. Johns River in Florid Jacksonville, Florida are featured as examples of terminals which had to be designed to meet high environmental standards. 13 refs., 2 figs., 14 photos.

  4. The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis

    Waldhoer, Maria; Casarosa, Paola; Rosenkilde, Mette M;

    2003-01-01

    US28 is one of four 7 transmembrane (7TM) chemokine receptors encoded by human cytomegalovirus and has been shown to both signal and endocytose in a ligand-independent, constitutively active manner. Here we show that the constitutive activity and constitutive endocytosis properties of US28 are se...... a 7TM receptor can camouflage the agonist properties of a ligand....

  5. Sphingosine 1-phosphate-induced motility and endocytosis of dendritic cells is regulated by SWAP-70 through RhoA.

    Ocaña-Morgner, Carlos; Reichardt, Peter; Chopin, Michaël; Braungart, Sarah; Wahren, Christine; Gunzer, Matthias; Jessberger, Rolf

    2011-05-01

    The phospholipid mediator sphingosine 1-phosphate (S1P) enhances motility and endocytosis of mature dendritic cells (DCs). We show that in vitro migration of Swap-70(-/-) bone marrow-derived DCs (BMDCs) in response to S1P and S1P-induced upregulation of endocytosis are significantly reduced. S1P-stimulated movement of Swap-70(-/-) BMDCs, specifically retraction of their trailing edge, in a collagen three-dimensional environment is impaired. These in vitro observations correlate with delayed entry into lymphatic vessels and migration to lymph nodes of skin DCs in Swap-70(-/-) mice. Expression of S1P receptors (S1P(1-3)) by wild-type and Swap-70(-/-) BMDCs is similar, but Swap-70(-/-) BMDCs fail to activate RhoA and to localize Rac1 and RhoA into areas of actin polymerization after S1P stimulus. The Rho-activating G protein Gα(i) interacts with SWAP-70, which also supports the localization of Gα(13) to membrane rafts in BMDCs. LPS-matured Swap-70(-/-) BMDCs contain significantly more active RhoA than wild-type DCs. Preinhibition of Rho activation restored migration to S1P, S1P-induced upregulation of endocytosis in mature Swap-70(-/-) BMDCs, and localization of Gα(13) to membrane rafts. These data demonstrate SWAP-70 as a novel regulator of S1P signaling necessary for DC motility and endocytosis. PMID:21421853

  6. The polyene antimycotics nystatin and filipin disrupt the plasma membrane, whereas natamycin inhibits endocytosis in germinating conidia of Penicillium discolor

    van Leeuwen, M.R.; Golovina, E.A.; Dijksterhuis, J.

    2009-01-01

    AIMS: To investigate the differences in membrane permeability and the effect on endocytosis of the polyene antimycotics nystatin, filipin and natamycin on germinating fungal conidia. METHODS AND RESULTS: The model system was Penicillium discolor, a food spoilage fungus. Filipin resulted in permeabil

  7. The polyene antimycotics nystatin and filipin disrupt the plasma membrane, whereas natamycin inhibits endocytosis in germinating conidia of Penicillium discolor

    Leeuwen, van M.R.; Golovina, E.A.; Dijksterhuis, J.

    2009-01-01

    To investigate the differences in membrane permeability and the effect on endocytosis of the polyene antimycotics nystatin, filipin and natamycin on germinating fungal conidia. Methods and Results: The model system was Penicillium discolor, a food spoilage fungus. Filipin resulted in permeabilizatio

  8. Light-regulated stapled peptides to inhibit protein-protein interactions involved in clathrin-mediated endocytosis.

    Nevola, Laura; Martín-Quirós, Andrés; Eckelt, Kay; Camarero, Núria; Tosi, Sébastien; Llobet, Artur; Giralt, Ernest; Gorostiza, Pau

    2013-07-22

    Control of membrane traffic: Photoswitchable inhibitors of protein-protein interactions were applied to photoregulate clathrin-mediated endocytosis (CME) in living cells. Traffic light (TL) peptides acting as "stop" and "go" signals for membrane traffic can be used to dissect the role of CME in receptor internalization and in cell growth, division, and differentiation. PMID:23775788

  9. Bulk Nuclear Properties from Reactions

    Danielewicz, P.

    2002-01-01

    Extraction of bulk nuclear properties by comparing reaction observables to results from semiclassical transport-model simulations is discussed. Specific properties include the nuclear viscosity, incompressibility and constraints on the nuclear pressure at supranormal densities.

  10. Bulk charges in eleven dimensions

    Hawking, Stephen William

    1998-01-01

    Eleven dimensional supergravity has electric type currents arising from the Chern-Simon and anomaly terms in the action. However the bulk charge integrates to zero for asymptotically flat solutions with topological trivial spatial sections. We show that by relaxing the boundary conditions to generalisations of the ALE and ALF boundary conditions in four dimensions one can obtain static solutions with a bulk charge preserving between 1/16 and 1/4 of the supersymmetries. One can introduce membranes with the same sign of charge into these backgrounds. This raises the possibility that these generalized membranes might decay quantum mechanically to leave just a bulk distribution of charge. Alternatively and more probably, a bulk distribution of charge can decay into a collection of singlely charged membranes. Dimensional reductions of these solutions lead to novel representations of extreme black holes in four dimensions with up to four charges. We discuss how the eleven-dimensional Kaluza-Klein monopole wrapped a...

  11. MPTP-meditated hippocampal dopamine deprivation modulates synaptic transmission and activity-dependent synaptic plasticity

    Parkinson's disease (PD)-like symptoms including learning deficits are inducible by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Therefore, it is possible that MPTP may disturb hippocampal memory processing by modulation of dopamine (DA)- and activity-dependent synaptic plasticity. We demonstrate here that intraperitoneal (i.p.) MPTP injection reduces the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN) within 7 days. Subsequently, the TH expression level in SN and hippocampus and the amount of DA and its metabolite DOPAC in striatum and hippocampus decrease. DA depletion does not alter basal synaptic transmission and changes pair-pulse facilitation (PPF) of field excitatory postsynaptic potentials (fEPSPs) only at the 30 ms inter-pulse interval. In addition, the induction of long-term potentiation (LTP) is impaired whereas the duration of long-term depression (LTD) becomes prolonged. Since both LTP and LTD depend critically on activation of NMDA and DA receptors, we also tested the effect of DA depletion on NMDA receptor-mediated synaptic transmission. Seven days after MPTP injection, the NMDA receptor-mediated fEPSPs are decreased by about 23%. Blocking the NMDA receptor-mediated fEPSP does not mimic the MPTP-LTP. Only co-application of D1/D5 and NMDA receptor antagonists during tetanization resembled the time course of fEPSP potentiation as observed 7 days after i.p. MPTP injection. Together, our data demonstrate that MPTP-induced degeneration of DA neurons and the subsequent hippocampal DA depletion alter NMDA receptor-mediated synaptic transmission and activity-dependent synaptic plasticity. - Highlights: → I.p. MPTP-injection mediates death of dopaminergic neurons. → I.p. MPTP-injection depletes DA and DOPAC in striatum and hippocampus. → I.p. MPTP-injection does not alter basal synaptic transmission. → Reduction of LTP and enhancement of LTD after i.p. MPTP-injection. → Attenuation of NMDA-receptors mediated

  12. Endocytosis of pro-inflammatory cytokine receptors and its relevance for signal transduction.

    Hermanns, Heike M; Wohlfahrt, Julia; Mais, Christine; Hergovits, Sabine; Jahn, Daniel; Geier, Andreas

    2016-08-01

    The pro-inflammatory cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) are key players of the innate and adaptive immunity. Their activity needs to be tightly controlled to allow the initiation of an appropriate immune response as defense mechanism against pathogens or tissue injury. Excessive or sustained signaling of either of these cytokines leads to severe diseases, including rheumatoid arthritis, inflammatory bowel diseases (Crohn's disease, ulcerative colitis), steatohepatitis, periodic fevers and even cancer. Studies carried out in the last 30 years have emphasized that an elaborate control system for each of these cytokines exists. Here, we summarize what is currently known about the involvement of receptor endocytosis in the regulation of these pro-inflammatory cytokines' signaling cascades. Particularly in the last few years it was shown that this cellular process is far more than a mere feedback mechanism to clear cytokines from the circulation and to shut off their signal transduction. PMID:27071147

  13. Epidermal growth factor receptor and cancer: control of oncogenic signalling by endocytosis

    Grandal, Michael Vibo; Madshus, I.H.

    2008-01-01

    prevents its down-regulation, underscoring the importance of the cellular background for EGFR effects. Signalling from ErbB proteins can either be terminated by dissociation of ligand resulting in dephosphorylation, or blunted by degradation of the receptors. Although proteasomal targeting of ErbB proteins...... has been described, lysosomal degradation upon ligand-induced endocytosis seems to play the major role in EGFR down-regulation. Preclinical and clinical data have demonstrated that EGFR is a central player in cancer, especially in carcinomas, some brain tumours and in non-small cell lung cancer. Such...... studies have further validated EGFR as an important molecular target in cancer treatment. This review focuses on mechanisms involved in ligand-induced EGFR activation and endocytic down-regulation. A better understanding of EGFR biology should allow development of more tumour-selective therapeutic...

  14. Membrane tension is a key determinant of bud morphology in clathrin-mediated endocytosis

    Hassinger, Julian E; Drubin, David G; Rangamani, Padmini

    2016-01-01

    In clathrin-mediated endocytosis (CME), clathrin and various adaptor proteins coat a patch of the plasma membrane, which is reshaped to form a budded vesicle. Experimental studies have demonstrated that elevated membrane tension can inhibit bud formation by a clathrin coat. In this study, we investigate the impact of membrane tension on the mechanics of membrane budding by simulating clathrin coats that either grow in area or progressively induce greater curvature. At low membrane tension, progressively increasing the area of a curvature-generating coat causes the membrane to smoothly evolve from a flat to budded morphology, whereas the membrane remains essentially flat at high membrane tensions. Interestingly, at physiologically relevant, intermediate membrane tensions, the shape evolution of the membrane undergoes a snapthrough instability in which increasing coat area causes the membrane to "snap" from an open, U-shaped bud to a closed, $\\Omega$-shaped bud. This instability is accompanied by a large energy...

  15. Non-26S Proteasome Proteolytic Role of Ubiquitin in Plant Endocytosis and Endosomal Trafficking

    Miaomiao Tian; Qi Xie

    2013-01-01

    The 76 amino acid protein ubiquitin (Ub) is highly conserved in all eukaryotic species.It plays important roles in many cellular processes by covalently attaching to the target proteins.The best known function of Ub is marking substrate proteins for degradation by the 26S proteasome.In fact,other consequences of ubiquitination have been discovered in yeast and mammals,such as membrane trafficking,DNA repair,chromatin modification,and protein kinase activation.The common mechanism underlying these processes is that Ub serves as a signal to sort proteins to the vacuoles or lysosomes for degradation as opposed to 26S proteasome-dependent degradation.To date,several reports have indicated that a similar function of Ub also exists in plants.This review focuses on a summary and analysis of the recent research progress on Ub acting as a signal to mediate endocytosis and endosomal trafficking in plants.

  16. Intestinal alkaline phosphatase: selective endocytosis from the enterocyte brush border during fat absorption

    Hansen, Gert Helge; Niels-Christiansen, Lise-Lotte; Immerdal, Lissi;

    2007-01-01

    explants. By immunofluorescence microscopy, fat absorption caused a translocation of IAP from the enterocyte brush border to the interior of the cell, whereas other brush-border enzymes were unaffected. By electron microscopy, the translocation occurred by a rapid (5 min) induction of endocytosis via...... clathrin-coated pits. By 60 min, IAP was seen in subapical endosomes and along membranes surrounding fat droplets. IAP is a well-known lipid raft-associated protein, and fat absorption was accompanied by a marked change in the density and morphology of the detergent-resistant membranes harboring IAP. A...... IAP and may contribute to the appearance of the enzyme in serum and surfactant-like particles....

  17. Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening.

    Daniel, James A; Malladi, Chandra S; Kettle, Emma; McCluskey, Adam; Robinson, Phillip J

    2012-08-01

    Small molecules modulating synaptic vesicle endocytosis (SVE) may ultimately be useful for diseases where pathological neurotransmission is implicated. Only a small number of specific SVE modulators have been identified to date. Slow progress is due to the laborious nature of traditional approaches to study SVE, in which nerve terminals are identified and studied in cultured neurons, typically yielding data from 10-20 synapses per experiment. We provide a protocol for a quantitative, high-throughput method for studying SVE in thousands of nerve terminals. Rat forebrain synaptosomes are attached to 96-well microplates and depolarized; SVE is then quantified by uptake of the dye FM4-64, which is imaged by high-content screening. Synaptosomes that have been frozen and stored can be used in place of fresh synaptosomes, reducing the experimental time and animal numbers required. With a supply of frozen synaptosomes, the assay can be performed within a day, including data analysis. PMID:22767087

  18. Three-dimensional imaging of single nanotube molecule endocytosis on plasmonic substrates

    Hong, Guosong; Robinson, Joshua T; Wang, Hailiang; Zhang, Bo; Dai, Hongjie

    2012-01-01

    Investigating the cellular internalization pathways of single molecules or single nano-objects is important to understanding cell-matter interactions and to applications in drug delivery and discovery. Imaging and tracking the motion of single molecules on cell plasma membrane require high spatial resolution in three dimensions (3D). Fluorescence imaging along the axial dimension with nanometer resolution has been highly challenging but critical to revealing displacements in trans-membrane events. Here, utilizing a plasmonic ruler based on the sensitive distance dependence of near-infrared fluorescence enhancement (NIR-FE) of carbon nanotubes on a gold plasmonic substrate, we probe ~10 nm scale trans-membrane displacements through changes in nanotube fluorescence intensity, enabling observations of single nanotube endocytosis in 3D. Cellular uptake and trans-membrane displacements show clear dependences to temperature and clathrin assembly on cell membrane, suggesting that the cellular entry mechanism for a n...

  19. CD4-independent human immunodeficiency virus infection involves participation of endocytosis and cathepsin B.

    Hiroaki Yoshii

    Full Text Available During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1 strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 µM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1.

  20. Mechanisms involved in calcium oxalate endocytosis by Madin-Darby canine kidney cells

    A.H. Campos

    2000-01-01

    Full Text Available Calcium oxalate (CaOx crystals adhere to and are internalized by tubular renal cells and it seems that this interaction is related (positively or negatively to the appearance of urinary calculi. The present study analyzes a series of mechanisms possibly involved in CaOx uptake by Madin-Darby canine kidney (MDCK cells. CaOx crystals were added to MDCK cell cultures and endocytosis was evaluated by polarized light microscopy. This process was inhibited by an increase in intracellular calcium by means of ionomycin (100 nM; N = 6; 43.9% inhibition; P<0.001 or thapsigargin (1 µM; N = 6; 33.3% inhibition; P<0.005 administration, and via blockade of cytoskeleton assembly by the addition of colchicine (10 µM; N = 8; 46.1% inhibition; P<0.001 or cytochalasin B (10 µM; N = 8; 34.2% inhibition; P<0.001. Furthermore, CaOx uptake was reduced when the activity of protein kinase C was inhibited by staurosporine (10 nM; N = 6; 44% inhibition; P<0.01, or that of cyclo-oxygenase by indomethacin (3 µM; N = 12; 17.2% inhibition; P<0.05; however, the uptake was unaffected by modulation of potassium channel activity with glibenclamide (3 µM; N = 6, tetraethylammonium (1 mM; N = 6 or cromakalim (1 µM; N = 6. Taken together, these data indicate that the process of CaOx internalization by renal tubular cells is similar to the endocytosis reported for other systems. These findings may be relevant to cellular phenomena involved in early stages of the formation of renal stones.

  1. C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

    FrançoisMarceau

    2013-09-01

    Full Text Available The C-C chemokine receptor-7 (CCR7 is a G protein coupled receptor that has a role in leukocyte homing, but that is also expressed in aggressive tumor cells. Preclinical research supports that CCR7 is a valid target in oncology. In view of the increasing availability of therapeutic monoclonal antibodies that carry cytotoxic cargoes, we studied the feasibility of forcing intact cells to internalize known monoclonal antibodies by exploiting the cycle of endocytosis and recycling triggered by the CCR7 agonist CCL19. Firstly, an anti-CCR7 antibody (CD197; clone 150503 labeled surface recombinant CCR7 expressed in intact HEK 293a cells and the fluorescent antibody was internalized following CCL19 treatment. Secondly, a recombinant myc-tagged CCL19 construction was exploited along the anti-myc monoclonal antibody 4A6. The myc-tagged ligand was produced as a conditioned medium of transfected HEK 293a cells that contained the equivalent of 430 ng/ml of immunoreactive CCL19 (average value, ELISA determination. CCL19-myc, but not authentic CCL19, carried the fluorophore-labeled antibody 4A6 into other recipient cells that expressed recombinant CCR7 (microscopy, cytofluorometry. The immune complexes were apparent in endosomal structures, colocalized well with the small GTPase Rab5 and progressed toward Rab7-positive endosomes. A dominant negative form of Rab5 (GDP-locked inhibited this endocytosis. Further, endosomes in CCL19-myc- or CCL19-stimulated cells were positive for β-arrestin2, but rarely for β-arrestin1. Following treatment with CCL19-myc and the 4A6 antibody, the melanoma cell line A375 that expresses endogenous CCR7 was specifically stained using a secondary peroxidase-conjugated antibody. Agonist-stimulated CCR7 can transport antibody-based cargoes, with possible therapeutic applications in oncology.

  2. Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons.

    Yepes, M; Wu, F; Torre, E; Cuellar-Giraldo, D; Jia, D; Cheng, L

    2016-04-01

    The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca(2+)-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis. PMID:26820595

  3. Developmental and activity-dependent plasticity of filiform hair receptors in the locust

    HaraldWolf

    2013-08-01

    Full Text Available A group of wind sensitive filiform hair receptors on the locust thorax and head makes contact onto a pair of identified interneuron, A4I1. The hair receptors’ central nervous projections exhibit pronounced structural dynamics during nymphal development, for example, by gradually eliminating their ipsilateral dendritic field while maintaining the contralateral one. These changes are dependent not only on hormones controlling development but on neuronal activity as well. The hair-to-interneuron system has remarkably high gain (close to 1 and makes contact to flight steering muscles. During stationary flight in front of a wind tunnel, interneuron A4I1 is active in the wing beat rhythm, and in addition it responds strongly to stimulation of sensory hairs in its receptive field. A role of the hair-to-interneuron in flight steering is thus suggested. This system appears suitable for further study of developmental and activity-dependent plasticity in a sensorimotor context with known connectivity patterns.

  4. Long lasting protein synthesis- and activity-dependent spine shrinkage and elimination after synaptic depression.

    Yazmín Ramiro-Cortés

    Full Text Available Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD mediated by metabotropic glutamate receptors (mGluRs through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation.

  5. Activity-dependent synaptic plasticity modulates the critical phase of brain development.

    Chaudhury, Sraboni; Sharma, Vikram; Kumar, Vivek; Nag, Tapas C; Wadhwa, Shashi

    2016-04-01

    Plasticity or neuronal plasticity is a unique and adaptive feature of nervous system which allows neurons to reorganize their interactions in response to an intrinsic or extrinsic stimulation and shapes the formation and maintenance of a functional neuronal circuit. Synaptic plasticity is the most important form of neural plasticity and plays critical role during the development allowing the formation of precise neural connectivity via the process of pruning. In the sensory systems-auditory and visual, this process is heavily dependent on the external cues perceived during the development. Environmental enrichment paradigms in an activity-dependent manner result in early maturation of the synapses and more efficient trans-synaptic signaling or communication flow. This has been extensively observed in the avian auditory system. On the other hand, stimuli results in negative effect can cause alterations in the synaptic connectivity and strength resulting in various developmental brain disorders including autism, fragile X syndrome and rett syndrome. In this review we discuss the role of different forms of activity (spontaneous or environmental) during the development of the nervous system in modifying synaptic plasticity necessary for shaping the adult brain. Also, we try to explore various factors (molecular, genetic and epigenetic) involved in altering the synaptic plasticity in positive and negative way. PMID:26515724

  6. Cell Biological Mechanisms of Activity-Dependent Synapse to Nucleus Translocation of CRTC1 in Neurons

    Toh Hean eCh'ng

    2015-09-01

    Full Text Available Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1 in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of synaptic glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules. We identify a nuclear localization signal within CRTC1, and characterize three conserved serine residues whose dephosphorylation is required for nuclear import. Domain analysis reveals that the amino-terminal third of CRTC1 contains all of the signals required for regulated nucleocytoplasmic trafficking. We fuse this region to Dendra2 to generate a reporter construct and perform live-cell imaging coupled with local uncaging of glutamate and photoconversion to characterize the dynamics of stimulus-induced retrograde transport and nuclear accumulation.

  7. Activity-dependent BDNF release via endocytic pathways is regulated by synaptotagmin-6 and complexin.

    Wong, Yu-Hui; Lee, Chia-Ming; Xie, Wenjun; Cui, Bianxiao; Poo, Mu-ming

    2015-08-11

    Brain-derived neurotrophic factor (BDNF) is known to modulate synapse development and plasticity, but the source of synaptic BDNF and molecular mechanisms regulating BDNF release remain unclear. Using exogenous BDNF tagged with quantum dots (BDNF-QDs), we found that endocytosed BDNF-QDs were preferentially localized to postsynaptic sites in the dendrite of cultured hippocampal neurons. Repetitive neuronal spiking induced the release of BDNF-QDs at these sites, and this process required activation of glutamate receptors. Down-regulating complexin 1/2 (Cpx1/2) expression eliminated activity-induced BDNF-QD secretion, although the overall activity-independent secretion was elevated. Among eight synaptotagmin (Syt) isoforms examined, down-regulation of only Syt6 impaired activity-induced BDNF-QD secretion. In contrast, activity-induced release of endogenously synthesized BDNF did not depend on Syt6. Thus, neuronal activity could trigger the release of endosomal BDNF from postsynaptic dendrites in a Cpx- and Syt6-dependent manner, and endosomes containing BDNF may serve as a source of BDNF for activity-dependent synaptic modulation. PMID:26216953

  8. Expression of activity-dependent neuroprotective protein in the brain of adult rats.

    Gennet, N; Herden, C; Bubb, V J; Quinn, J P; Kipar, A

    2008-03-01

    Activity-dependent neuroprotective protein (ADNP) is a VIP-regulated gene, which is essential for brain development. A synthetic peptide (NAP) derived from the ADNP sequence is highly neuroprotective, therefore it has been hypothesised that ADNP has a similar role. ADNP contains classical transcription factor motifs and nuclear localisation domains, but it has also been reported to be secreted and to co-localise with microtubules, indicating that ADNP may have multiple functions. We investigated the pattern of ADNP expression by immunohistology in normal rat brain, in order to generate a framework for future studies examining changes in ADNP expression in response to noxious stimuli or in models of disease. We found widespread ADNP-like immunoreactivity in neurons throughout the rat brain, with the highest expression in the cerebellum, and strong expression in the thalamus, mesencephalon, pons and medulla oblongata. ADNP-like immunoreactivity was mainly observed in the cytoplasm of neurons, and fibre tracts were often strongly positive as well. In addition, positive neuronal nuclei were occasionally observed. ADNP-like immunoreactivity was lost in degenerating "dark" neurons, whereas it appeared to locate to the nucleus in some of the morphologically unaltered adjacent cells. Occasional astrocyte and microglial cells were also positive. We suggest that the widespread expression of ADNP may correlate with the wide-ranging protective effects of NAP, and that the cytoplasmic and axonal localisation of ADNP-like immunoreactivity suggests additional, non-transcriptional functions of ADNP. PMID:18072088

  9. Activity-dependent synaptic plasticity of a chalcogenide electronic synapse for neuromorphic systems.

    Li, Yi; Zhong, Yingpeng; Zhang, Jinjian; Xu, Lei; Wang, Qing; Sun, Huajun; Tong, Hao; Cheng, Xiaoming; Miao, Xiangshui

    2014-01-01

    Nanoscale inorganic electronic synapses or synaptic devices, which are capable of emulating the functions of biological synapses of brain neuronal systems, are regarded as the basic building blocks for beyond-Von Neumann computing architecture, combining information storage and processing. Here, we demonstrate a Ag/AgInSbTe/Ag structure for chalcogenide memristor-based electronic synapses. The memristive characteristics with reproducible gradual resistance tuning are utilised to mimic the activity-dependent synaptic plasticity that serves as the basis of memory and learning. Bidirectional long-term Hebbian plasticity modulation is implemented by the coactivity of pre- and postsynaptic spikes, and the sign and degree are affected by assorted factors including the temporal difference, spike rate and voltage. Moreover, synaptic saturation is observed to be an adjustment of Hebbian rules to stabilise the growth of synaptic weights. Our results may contribute to the development of highly functional plastic electronic synapses and the further construction of next-generation parallel neuromorphic computing architecture. PMID:24809396

  10. Activity-dependent neuroprotective protein (ADNP) exhibits striking sexual dichotomy impacting on autistic and Alzheimer's pathologies.

    Malishkevich, A; Amram, N; Hacohen-Kleiman, G; Magen, I; Giladi, E; Gozes, I

    2015-01-01

    Activity-dependent neuroprotective protein (ADNP) is a most frequent autism spectrum disorder (ASD)-associated gene and the only protein significantly decreasing in the serum of Alzheimer's disease (AD) patients. Is ADNP associated with ASD being more prevalent in boys and AD more prevalent in women? Our results revealed sex-related learning/memory differences in mice, reflecting hippocampal expression changes in ADNP and ADNP-controlled AD/ASD risk genes. Hippocampal ADNP transcript content was doubled in male vs female mice, with females showing equal expression to ADNP haploinsufficient (ADNP(+/)(-)) males and no significant genotype-associated reduction. Increased male ADNP expression was replicated in human postmortem hippocampal samples. The hippocampal transcript for apolipoprotein E (the major risk gene for AD) was doubled in female mice compared with males, and further doubled in the ADNP(+/-) females, contrasting a decrease in ADNP(+/-) males. Previously, overexpression of the eukaryotic translation initiation factor 4E (eIF4E) led to ASD-like phenotype in mice. Here, we identified binding sites on ADNP for eIF4E and co-immunoprecipitation. Furthermore, hippocampal eIF4E expression was specifically increased in young ADNP(+/-) male mice. Behaviorally, ADNP(+/-) male mice exhibited deficiencies in object recognition and social memory compared with ADNP(+/+) mice, while ADNP(+/-) females were partially spared. Contrasting males, which preferred novel over familiar mice, ADNP(+/+) females showed no preference to novel mice and ADNP(+/-) females did not prefer mice over object. ADNP expression, positioned as a master regulator of key ASD and AD risk genes, introduces a novel concept of hippocampal gene-regulated sexual dimorphism and an ADNP(+/-) animal model for translational psychiatry. PMID:25646590

  11. Cellular mechanisms of activity-dependent BDNF expression in primary sensory neurons.

    Vermehren-Schmaedick, A; Khanjian, R A; Balkowiec, A

    2015-12-01

    Brain-derived neurotrophic factor (BDNF) is abundantly expressed by both developing and adult rat visceral sensory neurons from the nodose ganglion (NG) in vivo and in vitro. We have previously shown that BDNF is released from neonatal NG neurons by activity and regulates dendritic development in their postsynaptic targets in the brainstem. The current study was carried out to examine the cellular and molecular mechanisms of activity-dependent BDNF expression in neonatal rat NG neurons, using our established in vitro model of neuronal activation by electrical field stimulation with patterns that mimic neuronal activity in vivo. We show that BDNF mRNA (transcript 4) increases over threefold in response to a 4-h tonic or bursting pattern delivered at the frequency of 6 Hz, which corresponds to the normal heart rate of a newborn rat. No significant increase in BDNF expression was observed following stimulation at 1 Hz. The latter effect suggests a frequency-dependent mechanism of regulated BDNF expression. In addition to BDNF transcript 4, which is known to be regulated by activity, transcript 1 also showed significant upregulation. The increases in BDNF mRNA were followed by BDNF protein upregulation of a similar magnitude after 24h of stimulation at 6 Hz. Electrical stimulation-evoked BDNF expression was inhibited by pretreating neurons with the blocker of voltage-gated sodium channels tetrodotoxin and by removing extracellular calcium. Moreover, our data show that repetitive stimulation-evoked BDNF expression requires calcium influx through N-, but not L-type, channels. Together, our study reveals novel mechanisms through which electrical activity stimulates de novo synthesis of BDNF in sensory neurons, and points to the role of N-type calcium channels in regulating BDNF expression in sensory neurons in response to repetitive stimulation. PMID:26459016

  12. Looking for a bulk point

    Maldacena, Juan; Zhiboedov, Alexander

    2015-01-01

    We consider Lorentzian correlators of local operators. In perturbation theory, singularities occur when we can draw a position-space Landau diagram with null lines. In theories with gravity duals, we can also draw Landau diagrams in the bulk. We argue that certain singularities can arise only from bulk diagrams, not from boundary diagrams. As has been previously observed, these singularities are a clear diagnostic of bulk locality. We analyze some properties of these perturbative singularities and discuss their relation to the OPE and the dimensions of double-trace operators. In the exact nonperturbative theory, we expect no singularity at these locations. We prove this statement in 1+1 dimensions by CFT methods.

  13. Gamma probe dry bulk densities

    The gamma density probe is a useful instrument for measuring water content in small volumes of soil. Essentially, the gamma probe measures the density of the soil and water between a source and a detector. To transpose the gamma densities into water content, the dry bulk density of the soil is needed. A nondestructive method for estimating dry bulk densities for use with the gamma probe is proposed. The procedure is based on the assumption that water content values in a field dry condition were more stable than the dry bulk density values and could be transferred from one point to another. The procedure was successfully used on three areas in Reynolds Creek Experimental Watershed in southwest Idaho. (U.S.)

  14. Bulk Viscosity of Interacting Hadrons

    Wiranata, A.; M. Prakash

    2009-01-01

    We show that first approximations to the bulk viscosity $\\eta_v$ are expressible in terms of factors that depend on the sound speed $v_s$, the enthalpy, and the interaction (elastic and inelastic) cross section. The explicit dependence of $\\eta_v$ on the factor $(\\frac 13 - v_s^2)$ is demonstrated in the Chapman-Enskog approximation as well as the variational and relaxation time approaches. The interesting feature of bulk viscosity is that the dominant contributions at a given temperature ari...

  15. Bulk Viscosity of Interacting Hadrons

    Wiranata, A

    2009-01-01

    We show that first approximations to the bulk viscosity $\\eta_v$ are expressible in terms of factors that depend on the sound speed $v_s$, the enthalpy, and the interaction (elastic and inelastic) cross section. The explicit dependence of $\\eta_v$ on the factor $(\\frac 13 - v_s^2)$ is demonstrated in the Chapman-Enskog approximation as well as the variational and relaxation time approaches. The interesting feature of bulk viscosity is that the dominant contributions at a given temperature arise from particles which are neither extremely nonrelativistic nor extremely relativistic. Numerical results for a model binary mixture are reported.

  16. Geomagnetic and solar activity dependence of ionospheric upflowing O+: FAST observations

    Zhao, K.; Jiang, Y.; Chen, K. W.; Huang, L. F.

    2016-09-01

    This paper investigates the dependence of the occurrence frequency of ionospheric upflowing oxygen (O+) ions on the sunspot cycle and geomagnetic activity. We examine the upflows response to the geomagnetic disturbances as well as the influence of the ion energy factor in controlling the magnitude of the occurrence frequency and the net energy flux. We discuss the spatial distribution of the upflow occurrence frequency and construct a regression model as a function of the magnetic latitude. The results show an overall enhancement of the upflow occurrence frequency during magnetically disturbed periods and indicate that the high-occurrence area spreads out from the source regions during magnetically quiet periods. The high-occurrence areas are located at 70° magnetic latitude (mLat) in the dayside auroral oval zone and between 76-80° mLat in the dayside polar cusp region. In the nightside auroral oval zone, these areas are near 60° mLat, penetrating further equatorward to 55° mLat during magnetically disturbed periods. High energy (≥1 keV) upflowing ions are common in the nightside auroral oval zone while low energy (<1 keV) upflowing ions are found escaping from the high latitude dayside cusp region. A Gaussian function is shown to be a good fit to the occurrence frequency over the magnetic latitude. For high energy upflowing O+ ions, the occurrence frequency exhibits a single peak located at about 60° mLat in the nightside auroral oval zone while for low energy upflowing O+ ions, it exhibits two peaks, one near 60° mLat in the auroral oval zone and the other near 78° mLat in the cusp region. We study the solar activity dependence by analyzing the relationship between the upflow occurrence frequency and the sunspot number (RZ). The statistical result shows that the frequency decreases with declining solar activity level, from ˜30 % at solar maximum to ˜5 % at solar minimum. In addition, the correlation coefficient between the occurrence frequency and RZ

  17. Bulk charges in eleven dimensions

    Hawking, S. W.; Taylor-Robinson, M. M.

    1998-07-01

    Eleven dimensional supergravity has electric type currents arising from the Chern-Simon and anomaly terms in the action. However the bulk charge integrates to zero for asymptotically flat solutions with topological trivial spatial sections. We show that by relaxing the boundary conditions to generalisations of the ALE and ALF boundary conditions in four dimensions one can obtain static solutions with a bulk charge. Solutions involving anomaly terms preserve between 1/16 and 1/4 of the supersymmetries but Chern-Simons fluxes generally break all of the remaining supersymmetry. One can introduce membranes with the same sign of charge into these backgrounds. This raises the possibility that these generalized membranes might decay quantum mechanically to leave just a bulk distribution of charge. Alternatively and more probably, a bulk distribution of charge can decay into a collection of singly charged membranes. Dimensional reductions of these solutions lead to novel representations of extreme black holes in four dimensions with up to four charges. We discuss how the eleven-dimensional Kaluza-Klein monopole wrapped around a space with non-zero first Pontryagin class picks up an electric charge proportional to the Pontryagin number.

  18. Bulk viscosity and deflationary universes

    Lima, J A S; Waga, I

    2007-01-01

    We analyze the conditions that make possible the description of entropy generation in the new inflationary model by means of a nearequilibrium process. We show that there are situations in which the bulk viscosity cannot describe particle production during the coherent field oscillations phase.

  19. Longitudinal bulk acoustic mass sensor

    Hales, Jan Harry; Teva, Jordi; Boisen, Anja;

    2009-01-01

    A polycrystalline silicon longitudinal bulk acoustic cantilever is fabricated and operated in air at 51 MHz. A mass sensitivity of 100 Hz/fg (1 fg=10(-15) g) is obtained from the preliminary experiments where a minute mass is deposited on the device by means of focused ion beam. The total noise i...

  20. Activity-dependent increases in local oxygen consumption correlate with postsynaptic currents in the mouse cerebellum in vivo

    Mathiesen, Claus; Caesar, Kirsten; Thomsen, Kirsten Engelund;

    2011-01-01

    metabolism via mitochondrial signaling, but whether this also occurs in the intact brain is unknown. Here we applied a pharmacological approach to dissect the effects of ionic currents and cytosolic Ca(2+) rises of neuronal origin on activity-dependent rises in CMRO(2). We used two-photon microscopy and...

  1. Activity-dependent increases in local oxygen consumption correlate with postsynaptic currents in the mouse cerebellum in vivo

    Mathiesen, Claus; Caesar, Kirsten; Thomsen, Kirsten Engelund;

    2011-01-01

    Evoked neural activity correlates strongly with rises in cerebral metabolic rate of oxygen (CMRO(2)) and cerebral blood flow (CBF). Activity-dependent rises in CMRO(2) fluctuate with ATP turnover due to ion pumping. In vitro studies suggest that increases in cytosolic Ca(2+) stimulate oxidative...

  2. Activity-dependent increases in local oxygen consumption correlate with post-synaptic currents in the mouse cerebellum in vivo

    Mathiesen, Claus; Caesar, Kirsten; Thomsen, Kirsten Joan;

    2011-01-01

    Evoked neural activity correlates strongly with rises in cerebral metabolic rate of oxygen (CMRO2) and cerebral blood flow. Activity-dependent rises in CMRO2 fluctuate with ATP turnover due to ion pumping. In vitro studies suggest that increases in cytosolic Ca2+ stimulate oxidative metabolism via...

  3. Biodistribution and endocytosis of ICAM-1-targeting antibodies versus nanocarriers in the gastrointestinal tract in mice

    Mane V

    2012-08-01

    Full Text Available Viraj Mane,1 Silvia Muro1, 21Institute for Bioscience and Biotechnology Research, 2Fischell Department of Bioengineering, University of Maryland, College Park, MD, USAAbstract: Drug delivery to the gastrointestinal (GI tract is key for improving treatment of GI maladies, developing oral vaccines, and facilitating drug transport into circulation. However, delivery of formulations to the GI tract is hindered by pH changes, degradative enzymes, mucus, and peristalsis, leading to poor GI retention. Targeting may prolong residence of therapeutics in the GI tract and enhance their interaction with this tissue, improving such aspects. We evaluated nanocarrier (NC and ligand-mediated targeting in the GI tract following gastric gavage in mice. We compared GI biodistribution, degradation, and endocytosis between control antibodies and antibodies targeting the cell surface determinant intercellular adhesion molecule 1 (ICAM-1, expressed on GI epithelium and other cell types. These antibodies were administered either as free entities or coated onto polymer NCs. Fluorescence and radioisotope tracing showed proximal accumulation, with preferential retention in the stomach, jejunum, and ileum; and minimal presence in the duodenum, cecum, and colon by 1 hour after administration. Upstream (gastric retention was enhanced in NC formulations, with decreased downstream (jejunal accumulation. Of the total dose delivered to the GI tract, ~60% was susceptible to enzymatic (but not pH-mediated degradation, verified both in vitro and in vivo. Attenuation of peristalsis by sedation increased upstream retention (stomach, duodenum, and jejunum. Conversely, alkaline NaHCO3, which enhances GI transit by decreasing mucosal viscosity, favored downstream (ileal passage. This suggests passive transit through the GI tract, governed by mucoadhesion and peristalsis. In contrast, both free anti-ICAM and anti-ICAM NCs demonstrated significantly enhanced upstream (stomach and duodenum

  4. Parameter estimation with bio-inspired meta-heuristic optimization: modeling the dynamics of endocytosis

    Tashkova Katerina

    2011-10-01

    Full Text Available Abstract Background We address the task of parameter estimation in models of the dynamics of biological systems based on ordinary differential equations (ODEs from measured data, where the models are typically non-linear and have many parameters, the measurements are imperfect due to noise, and the studied system can often be only partially observed. A representative task is to estimate the parameters in a model of the dynamics of endocytosis, i.e., endosome maturation, reflected in a cut-out switch transition between the Rab5 and Rab7 domain protein concentrations, from experimental measurements of these concentrations. The general parameter estimation task and the specific instance considered here are challenging optimization problems, calling for the use of advanced meta-heuristic optimization methods, such as evolutionary or swarm-based methods. Results We apply three global-search meta-heuristic algorithms for numerical optimization, i.e., differential ant-stigmergy algorithm (DASA, particle-swarm optimization (PSO, and differential evolution (DE, as well as a local-search derivative-based algorithm 717 (A717 to the task of estimating parameters in ODEs. We evaluate their performance on the considered representative task along a number of metrics, including the quality of reconstructing the system output and the complete dynamics, as well as the speed of convergence, both on real-experimental data and on artificial pseudo-experimental data with varying amounts of noise. We compare the four optimization methods under a range of observation scenarios, where data of different completeness and accuracy of interpretation are given as input. Conclusions Overall, the global meta-heuristic methods (DASA, PSO, and DE clearly and significantly outperform the local derivative-based method (A717. Among the three meta-heuristics, differential evolution (DE performs best in terms of the objective function, i.e., reconstructing the output, and in terms of

  5. Polymerisationseigenschaften von Bulk-Fill Kompositen

    Maier, Eva

    2015-01-01

    Hintergrund und Ziele: Untersuchung der Polymerisationseigenschaften von Bulk-Fill Kompositen bzgl. Konversionsrate (degree of conversion = DC), Vickers-Härte (HV), Polymerisationsschrumpfungsstress (PSS) und Polymerisationsvolumenschrumpfung (PVS) im Vergleich zu konventionellen Kompositen. Material und Methode: Untersucht wurden die Bulk-Fill Komposite Filtek Bulk Fill Flowable (FBF, 3M ESPE, Seefeld), Surefil Smart Dentin Replacement (SDR, Dentsply, Konstanz), Tetric EvoCeram Bulk Fill...

  6. Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

    Fine, Michael; Llaguno, Marc C.; Lariccia, Vincenzo; Lin, Mei-Jung; Yaradanakul, Alp; Hilgemann, Donald W.

    2011-01-01

    The roles that lipids play in endocytosis are the subject of debate. Using electrical and imaging methods, we describe massive endocytosis (MEND) in baby hamster kidney (BHK) and HEK293 cells when the outer plasma membrane monolayer is perturbed by the nonionic detergents, Triton X-100 (TX100) and NP-40. Some alkane detergents, the amphipathic drugs, edelfosine and tamoxifen, and the phospholipase inhibitor, U73122, are also effective. Uptake of the membrane tracer, FM 4–64, into vesicles and...

  7. Preferred sites of exocytosis and endocytosis colocalize during high but not lower frequency stimulation in mouse motor nerve terminals

    Gaffield, Michael A.; Tabares, Lucia; Betz, William J.

    2009-01-01

    The spatial relationship of exocytosis and endocytosis in motor nerve terminals has been explored, with varied results, mostly in fixed preparations and without direct information on the utilization of each exocytic site. We sought to determine these spatial properties in real time using synaptopHluorin (spH) and FM 4-64. Earlier we showed that nerve stimulation elicits the appearance of spH fluorescence hot spots, which mark preferred sites of exocytosis. Here we show that nerve stimulation ...

  8. Membrane glycoprotein M6A promotes μ-opioid receptor endocytosis and facilitates receptor sorting into the recycling pathway

    Ying-Jian Liang; Dai-Fei Wu; Ralf Stumm; Volker H(o)llt; Thomas Koch

    2008-01-01

    The interaction of μ-opioid receptor (MOPr) with the neuronal membrane glycoprotein M6a is known to facilitate MOPr endocytosis in human embryonic kidney 293 (HEK293) cells. To further study the role of M6a in the post-endocytotic sorting of MOPr, we investigated the agonist-induced co-internalization of MOPr and M6a and protein targeting after internalization in HEK293 cells that co-expressed HA-tagged MOPr and Myc-tagged M6a. We found that M6a, MOPr, and Rab 11, a marker for recycling endosomes, co-localized in endocytotic vesicles, indicating that MOPr and M6a are primarily targeted to recycling endosomes after endocytosis. Furthermore, co-expression of M6a augmented the post-endocytotic sorting of δ-opioid receptors into the recycling pathway, indicating that M6a might have a more general role in opioid receptor post-ndocytotic sorting. The enhanced post-endocytotic sorting of MOPr into the recycling pathway was accompanied by a decrease in agonist-induced receptor down-regulation of M6a in co-expressing cells. We tested the physiological relevance of these findings in primary cultures of cortical neurons and found that co-expression of M6a markedly increased the translocation of MOPrs from the plasma membrane to intracellular vesicles at steady state and significantly enhanced both constitutive and agonist-induced receptor endocytosis. In conclusion, our results strongly indicate that M6a modulates MOPr endocytosis and post-endocytotic sorting and has an important role in receptor regulation.

  9. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of β-glucuronidase

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human β-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3% of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of β-glucuronidase. At pH 7.5, the rate of endocytosis was only 14% the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized β-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized β-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor

  10. Influenza A virus H5N1 entry into host cells is through clathrin-dependent endocytosis

    2009-01-01

    Influenza A virus H5N1 presents a major threat to human health. The entry of influenza virus into host cells is believed to be mediated by hemagglutinin (HA), a virus surface glycoprotein that can bind terminal sialic acid residues on host cell glycoproteins and glycolipids. In this study, we elucidated the pathways through which H5N1 enters human lung carcinoma cell line A549. We first proved that H5N1 can enter A549 cells via endocytosis, as lysosomotropic agents, such as bafilomycin A1 and chloroquine, can rescue H5N1-induced A549 cell death. By using specific inhibitors, and siRNAs that target the clathrin pathway, we further found that H5N1 could enter A549 cells via clathrin-mediated endocytosis, while inhibitors targeting caveolae-mediated endocytosis could not inhibit H5N1 cell entry. These findings expand our understanding of H5N1 pathogenesis and provide new information for anti-viral drug research.

  11. Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles

    Chiang, Hsueh-Cheng; Shin, Wonchul; Zhao, Wei-Dong; Hamid, Edaeni; Sheng, Jiansong; Baydyuk, Maryna; Wen, Peter J.; Jin, Albert; Momboisse, Fanny; Wu, Ling-Gang

    2014-02-01

    Vesicle fusion with the plasma membrane generates an Ω-shaped membrane profile. Its pore is thought to dilate until flattening (full-collapse), followed by classical endocytosis to retrieve vesicles. Alternatively, the pore may close (kiss-and-run), but the triggering mechanisms and its endocytic roles remain poorly understood. Here, using confocal and stimulated emission depletion microscopy imaging of dense-core vesicles, we find that fusion-generated Ω-profiles may enlarge or shrink while maintaining vesicular membrane proteins. Closure of fusion-generated Ω-profiles, which produces various sizes of vesicles, is the dominant mechanism mediating rapid and slow endocytosis within ~1-30 s. Strong calcium influx triggers dynamin-mediated closure. Weak calcium influx does not promote closure, but facilitates the merging of Ω-profiles with the plasma membrane via shrinking rather than full-collapse. These results establish a model, termed Ω-exo-endocytosis, in which the fusion-generated Ω-profile may shrink to merge with the plasma membrane, change in size or change in size then close in response to calcium, which is the main mechanism to retrieve dense-core vesicles.

  12. Influenza A virus H5N1 entry into host cells is through clathrin-dependent endocytosis

    WANG HongLiang; JIANG ChengYu

    2009-01-01

    Influenza A virus H5N1 presents a major threat to human health. The entry of influenza virus into host cells is believed to be mediated by hemagglutinin (HA), a virus surface glycoprotein that can bind ter-minal sialic acid residues on host cell glycoproteins and glycolipids. In this study, we elucidated the pathways through which H5N1 enters human lung carcinoma cell line A549. We first proved that H5N1 can enter A549 cells via endocytosis, as lysosomotropic agents, such as bafilomycin A1 and chloro. quine, can rescue H5Nl-induced A549 cell death. By using specific inhibitors, and siRNAs that target the clathrin pathway, we further found that H5N1 could enter A549 cells via clathrin-mediated endocy-tosis, while inhibitors targeting caveolae-mediated endocytosis could not inhibit H5N1 cell entry. These findings expand our understanding of H5N1 pathogenesis and provide new information for anti-viral drug research.

  13. UBE3A Regulates Synaptic Plasticity and Learning and Memory by Controlling SK2 Channel Endocytosis.

    Sun, Jiandong; Zhu, Guoqi; Liu, Yan; Standley, Steve; Ji, Angela; Tunuguntla, Rashmi; Wang, Yubin; Claus, Chad; Luo, Yun; Baudry, Michel; Bi, Xiaoning

    2015-07-21

    Gated solely by activity-induced changes in intracellular calcium, small-conductance potassium channels (SKs) are critical for a variety of functions in the CNS, from learning and memory to rhythmic activity and sleep. While there is a wealth of information on SK2 gating, kinetics, and Ca(2+) sensitivity, little is known regarding the regulation of SK2 subcellular localization. We report here that synaptic SK2 levels are regulated by the E3 ubiquitin ligase UBE3A, whose deficiency results in Angelman syndrome and overexpression in increased risk of autistic spectrum disorder. UBE3A directly ubiquitinates SK2 in the C-terminal domain, which facilitates endocytosis. In UBE3A-deficient mice, increased postsynaptic SK2 levels result in decreased NMDA receptor activation, thereby impairing hippocampal long-term synaptic plasticity. Impairments in both synaptic plasticity and fear conditioning memory in UBE3A-deficient mice are significantly ameliorated by blocking SK2. These results elucidate a mechanism by which UBE3A directly influences cognitive function. PMID:26166566

  14. Enhancement of saporin cytotoxicity by Gypsophila saponins--more than stimulation of endocytosis.

    Weng, A; Bachran, C; Fuchs, H; Krause, E; Stephanowitz, H; Melzig, M F

    2009-10-30

    Saporin is a type I ribosome-inactivating protein with N-glycosidase activity. It removes adenine residues from the 28S ribosomal RNA resulting in inhibition of protein synthesis. Recently we have shown that saporin exerts no cytotoxicity on seven human cell lines. However, the combination of saporin with a special mixture of Gypsophila saponins (Soapwort saponins) from Gypsophila paniculata L. (baby's breath) rendered saporin to a potent cytotoxin comparable to viscumin, a highly toxic type II ribosome-inactivating protein. In this study we investigated whether the enhancement of the saporin-cytotoxicity by Gypsophila saponins is mediated by a saponin-triggered modulation of endocytosis, exocytosis or impaired degradation processes of his-tagged saporin ((his)saporin) in ECV-304 cells. For this purpose (his)saporin was labelled with tritium and cytotoxicity of the toxin alone and in combination with Gypsophila saponins was scrutinized. The transport and degradation processes of (his)saporin were not different in Gypsophila saponin-treated and control cells. However, after ultracentrifugation of a post-nuclear supernatant the amount of cytosolic (his)saporin was significantly higher in saponin-treated cells than in cells, which were only incubated with (his)saporin. This indicates a saponin mediated endosomal escape of saporin. PMID:19615984

  15. Catabolism of circulating enzymes: plasma clearance, endocytosis, and breakdown of lactate dehydrogenase-1 in rabbits

    Smit, M.J.; Beekhuis, H.; Duursma, A.M.; Bouma, J.M.; Gruber, M.

    1988-12-01

    Lactate dehydrogenase-1, intravenously injected into rabbits, was cleared with first-order kinetics (half-life 27 min), until at least 80% of the injected activity had disappeared from plasma. Radioactivity from injected SVI-labeled enzyme disappeared at this same rate. Trichloroacetic-acid-soluble breakdown products started to appear in the circulation shortly after injection of the labeled enzyme. Body scans of the rabbits for 80 min after injection of T I-labeled enzyme revealed rapid accumulation of label in the liver, peaking 10-20 min after injection. Subsequently, activity in the liver declined and radioactivity (probably labeled breakdown products of low molecular mass) steadily accumulated in the bladder. Tissue fractionation of liver, 19 min after injection of labeled enzyme, indicated that the radioactivity was present both in endosomes and in lysosomes, suggesting uptake by endocytosis, followed by breakdown in the lysosomes. Measurements of radioactivity in liver and plasma suggest that the liver is responsible for the breakdown of at least 75% of the injected enzyme. Radioautography of tissue sections of liver and spleen showed accumulated radioactivity in sinusoidal liver cells and red pulpa, respectively. These results are very similar to those for lactate dehydrogenase-5, creatine kinase MM, and several other enzymes that we have previously studied in rats.

  16. Catabolism of circulating enzymes: plasma clearance, endocytosis, and breakdown of lactate dehydrogenase-1 in rabbits.

    Smit, M J; Beekhuis, H; Duursma, A M; Bouma, J M; Gruber, M

    1988-12-01

    Lactate dehydrogenase-1 (EC 1.1.1.27), intravenously injected into rabbits, was cleared with first-order kinetics (half-life 27 min), until at least 80% of the injected activity had disappeared from plasma. Radioactivity from injected 125I-labeled enzyme disappeared at this same rate. Trichloroacetic-acid-soluble breakdown products started to appear in the circulation shortly after injection of the labeled enzyme. Body scans of the rabbits for 80 min after injection of 131I-labeled enzyme revealed rapid accumulation of label in the liver, peaking 10-20 min after injection. Subsequently, activity in the liver declined and radioactivity (probably labeled breakdown products of low molecular mass) steadily accumulated in the bladder. Tissue fractionation of liver, 19 min after injection of labeled enzyme, indicated that the radioactivity was present both in endosomes and in lysosomes, suggesting uptake by endocytosis, followed by breakdown in the lysosomes. Measurements of radioactivity in liver and plasma suggest that the liver is responsible for the breakdown of at least 75% of the injected enzyme. Radioautography of tissue sections of liver and spleen showed accumulated radioactivity in sinusoidal liver cells and red pulpa, respectively. These results are very similar to those for lactate dehydrogenase-5, creatine kinase MM, and several other enzymes that we have previously studied in rats. PMID:3197286

  17. Catabolism of circulating enzymes: plasma clearance, endocytosis, and breakdown of lactate dehydrogenase-1 in rabbits

    Lactate dehydrogenase-1, intravenously injected into rabbits, was cleared with first-order kinetics (half-life 27 min), until at least 80% of the injected activity had disappeared from plasma. Radioactivity from injected 125I-labeled enzyme disappeared at this same rate. Trichloroacetic-acid-soluble breakdown products started to appear in the circulation shortly after injection of the labeled enzyme. Body scans of the rabbits for 80 min after injection of 131I-labeled enzyme revealed rapid accumulation of label in the liver, peaking 10-20 min after injection. Subsequently, activity in the liver declined and radioactivity (probably labeled breakdown products of low molecular mass) steadily accumulated in the bladder. Tissue fractionation of liver, 19 min after injection of labeled enzyme, indicated that the radioactivity was present both in endosomes and in lysosomes, suggesting uptake by endocytosis, followed by breakdown in the lysosomes. Measurements of radioactivity in liver and plasma suggest that the liver is responsible for the breakdown of at least 75% of the injected enzyme. Radioautography of tissue sections of liver and spleen showed accumulated radioactivity in sinusoidal liver cells and red pulpa, respectively. These results are very similar to those for lactate dehydrogenase-5, creatine kinase MM, and several other enzymes that we have previously studied in rats

  18. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria

    E.V. Seliverstova

    2015-04-01

    Full Text Available The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes, and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.

  19. Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis.

    Smaczynska-de Rooij, Iwona I; Allwood, Ellen G; Mishra, Ritu; Booth, Wesley I; Aghamohammadzadeh, Soheil; Goldberg, Martin W; Ayscough, Kathryn R

    2012-02-01

    Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin-related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset of invagination and disassemble concomitant with inward vesicle movement. Rvs167-GFP localization is reduced in cells lacking vps1 suggesting that Vps1 influences Rvs167 association with the endocytic complex. Unlike classical dynamins, Vps1 does not have a proline-arginine domain that could interact with SH3 domain-containing proteins. Thus, while Rvs167 has an SH3 domain, it is not clear how an interaction would be mediated. Here, we demonstrate an interaction between Rvs167 SH3 domain and the single type I SH3-binding motif in Vps1. Mutant Vps1 that cannot bind Rvs167 rescues all membrane fusion/fission functions associated with Vps1 except for endocytic function, demonstrating the specificity and mechanistic importance of the interaction. In vitro, an Rvs161/Rvs167 heterodimer can disassemble Vps1 oligomers. Overall, the data support the idea that Vps1 and the amphiphysins function together to mediate scission during endocytosis in yeast. PMID:22082017

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

    Alabi, AbdulRasheed A; Tsien, Richard W

    2013-01-01

    Regulated exocytosis and endocytosis are critical to the function of many intercellular networks, particularly the complex neural circuits underlying mammalian behavior. Kiss-and-run (KR) is an unconventional fusion between secretory vesicles and a target membrane that releases intravesicular content through a transient, nanometer-sized fusion pore. The fusing vesicle retains its gross shape, precluding full integration into the planar membrane, and enough molecular components for rapid retrieval, reacidification, and reuse. KR makes judicious use of finite presynaptic resources, and mounting evidence suggests that it influences synaptic information transfer. Here we detail emerging perspectives on KR and its role in neurotransmission. We additionally formulate a restraining force hypothesis as a plausible mechanistic basis for KR and its physiological modulation in small nerve terminals. Clarification of the mechanism and function of KR has bearing on understanding the kinetic transitions underlying SNARE-mediated fusion, interactions between vesicles and their local environment, and the influence of release dynamics on neural information processing. PMID:23245563

  1. Membrane Tension Inhibits Deformation by Coat Proteins in Clathrin-Mediated Endocytosis

    Hassinger, Julian; Drubin, David; Oster, George; Rangamani, Padmini

    2016-02-01

    In clathrin-mediated endocytosis (CME), clathrin and various adaptor proteins coat a patch of the plasma membrane, which is reshaped to form a budded vesicle. Experimental studies have demonstrated that elevated membrane tension can inhibit bud formation by a clathrin coat. In this study, we investigate the impact of membrane tension on the mechanics of membrane budding by simulating clathrin coats that either grow in area or progressively induce greater curvature. At low membrane tension, progressively increasing the area of a curvature-generating coat causes the membrane to smoothly evolve from a flat to budded morphology, whereas the membrane remains essentially flat at high membrane tensions. Interestingly, at physiologically relevant, intermediate membrane tensions, the shape evolution of the membrane undergoes a snapthrough instability in which increasing coat area causes the membrane to "snap" from an open, U-shaped bud to a closed, $\\Omega$-shaped bud. This instability is accompanied by a large energy barrier, which could cause a developing endocytic pit to stall if the binding energy of additional coat is insufficient to overcome this barrier. Similar results were found for a coat of constant area in which the spontaneous curvature progressively increases. Additionally, a pulling force on the bud, simulating a force from actin polymerization, is sufficient to drive a transition from an open to closed bud, overcoming the energy barrier opposing this transition.

  2. Inactivation of Tor proteins affects the dynamics of endocytic proteins in early stage of endocytosis

    Brandon Tenay; Evin Kimberlin; Michelle Williams; Juliette Denise; Joshua Fakilahyel; Kyoungtae Kim

    2013-06-01

    Tor2 is an activator of the Rom2/Rho1 pathway that regulates -factor internalization. Since the recruitment of endocytic proteins such as actin-binding proteins and the amphiphysins precedes the internalization of -factor, we hypothesized that loss of Tor function leads to an alteration in the dynamics of the endocytic proteins. We report here that endocytic proteins, Abp1 and Rvs167, are less recruited to endocytic sites not only in tor2 but also tor1 mutants. Furthermore, we found that the endocytic proteins Rvs167 and Sjl2 are completely mistargeted to the cytoplasm in tor1tor2ts double mutant cells. We also demonstrate here that the efficiency of endocytic internalization or scission in all tor mutants was drastically decreased. In agreement with the Sjl2 mislocalization, we found that in tor1tor2ts double mutant cells, as well as other tor mutant cells, the overall PIP2 level was dramatically increased. Finally, the cell wall chitin content in tor2ts and tor1tor2ts mutant cells was also significantly increased. Taken together, both functional Tor proteins, Tor1 and Tor2, are essentially required for proper endocytic protein dynamics at the early stage of endocytosis.

  3. Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting.

    Anselmo, Aaron C; Zhang, Mengwen; Kumar, Sunny; Vogus, Douglas R; Menegatti, Stefano; Helgeson, Matthew E; Mitragotri, Samir

    2015-03-24

    The impact of physical and chemical modifications of nanoparticles on their biological function has been systemically investigated and exploited to improve their circulation and targeting. However, the impact of nanoparticles' flexibility (i.e., elastic modulus) on their function has been explored to a far lesser extent, and the potential benefits of tuning nanoparticle elasticity are not clear. Here, we describe a method to synthesize polyethylene glycol (PEG)-based hydrogel nanoparticles of uniform size (200 nm) with elastic moduli ranging from 0.255 to 3000 kPa. These particles are used to investigate the role of particle elasticity on key functions including blood circulation time, biodistribution, antibody-mediated targeting, endocytosis, and phagocytosis. Our results demonstrate that softer nanoparticles (10 kPa) offer enhanced circulation and subsequently enhanced targeting compared to harder nanoparticles (3000 kPa) in vivo. Furthermore, in vitro experiments show that softer nanoparticles exhibit significantly reduced cellular uptake in immune cells (J774 macrophages), endothelial cells (bEnd.3), and cancer cells (4T1). Tuning nanoparticle elasticity potentially offers a method to improve the biological fate of nanoparticles by offering enhanced circulation, reduced immune system uptake, and improved targeting. PMID:25715979

  4. Egg drop syndrome virus enters duck embryonic fibroblast cells via clathrin-mediated endocytosis.

    Huang, Jingjing; Tan, Dan; Wang, Yang; Liu, Caihong; Xu, Jiamin; Wang, Jingyu

    2015-12-01

    Previous studies of egg drop syndrome virus (EDSV) is restricted to serological surveys, disease diagnostics, and complete viral genome analysis. Consequently, the infection characteristics and entry routes of EDSV are poorly understood. Therefore, we aimed to explore the entry pathway of EDSV into duck embryonic fibroblast (DEF) cells as well as the infection characteristics and proliferation of EDSV in primary DEF and primary chicken embryo liver (CEL) cells. Transmission electron microscopy revealed that the virus triggered DEF cell membrane invagination as early as 10 min post-infection and that integrated endocytic vesicles formed at 20 min post-infection. The virus yield in EDSV-infected DEF cells treated with chlorpromazine (CPZ), sucrose, methyl-β-cyclodextrin (MβCD), or NH4Cl was measured by quantitative real-time PCR. Compared with the mock treatment, CPZ and sucrose greatly inhibited the production of viral progeny in a dose-dependent manner, while MβCD treatment did not result in a significant difference. Furthermore, NH4Cl had a strong inhibitory effect on the production of EDSV progeny. In addition, indirect immunofluorescence demonstrated that virus particles clustered on the surface of DEF cells treated with CPZ or sucrose. These results indicate that EDSV enters DEF cells through clathrin-mediated endocytosis followed by a pH-dependent step, which is similar to the mechanism of entry of human adenovirus types 2 and 5. PMID:26200954

  5. Endocytosis of the major yolk proteins of the silkmoth, Hyalophora cecropia: Uptake kinetics and interactions

    The oocytes of Lepidopteran insects take up several yolk proteins in defined proportions even though their relative availability in the hemolymph changes during the several days required to complete yolk formation in all the eggs. There are three hemolymph yolk precursors, vitellogenin, microvitellogenin and lipophorin; one precursor, paravitellogenin is produced in the ovary. The control mechanism for their proportional endocytosis is not known. In this thesis, the author describe the purification of all four proteins and the radiolabeling of the hemolymph precursors. The radiolabeled proteins were tested with an in vitro incubation system to assess the biological activity of the proteins and the reliability of the incubation methods. All of the labeled probes were transferred from the incubation medium to yolk spheres within the oocyte in a saturable, energy-dependent, and stage-specific manner. The rates of uptake were similar to the estimated rates of uptake in situ. The concentration dependence of in vitro uptake was investigated and found to be consistent with in situ concentrations and the composition of yolk in mature eggs. Two precursors, vitellogenin and lipophorin, competed for uptake indicating that they share a common binding site while the third, microvitellin, did not compete with the others. Though vitellogenin and lipophorin competed for uptake, only vitellogenin displayed the unique ability to increase the uptake rate of microvitellin and fluid in vitro

  6. Evolutionary Changes on the Way to Clathrin-Mediated Endocytosis in Animals.

    Dergai, Mykola; Iershov, Anton; Novokhatska, Olga; Pankivskyi, Serhii; Rynditch, Alla

    2016-01-01

    Endocytic pathways constitute an evolutionarily ancient system that significantly contributed to the eukaryotic cell architecture and to the diversity of cell type-specific functions and signaling cascades, in particular of metazoans. Here we used comparative proteomic studies to analyze the universal internalization route in eukaryotes, clathrin-mediated endocytosis (CME), to address the issues of how this system evolved and what are its specific features. Among 35 proteins crucially required for animal CME, we identified a subset of 22 proteins common to major eukaryotic branches and 13 gradually acquired during evolution. Based on exploration of structure-function relationship between conserved homologs in sister, distantly related and early diverged branches, we identified novel features acquired during evolution of endocytic proteins on the way to animals: Elaborated way of cargo recruitment by multiple sorting proteins, structural changes in the core endocytic complex AP2, the emergence of the Fer/Cip4 homology domain-only protein/epidermal growth factor receptor substrate 15/intersectin functional complex as an additional interaction hub and activator of AP2, as well as changes in late endocytic stages due to recruitment of dynamin/sorting nexin 9 complex and involvement of the actin polymerization machinery. The evolutionary reconstruction showed the basis of the CME process and its subsequent step-by-step development. Documented changes imply more precise regulation of the pathway, as well as CME specialization for the uptake of specific cargoes and cell type-specific functions. PMID:26872775

  7. West Nile virus infection causes endocytosis of a specific subset of tight junction membrane proteins.

    Zaikun Xu

    Full Text Available West Nile virus (WNV is a blood-borne pathogen that causes systemic infections and serious neurological disease in human and animals. The most common route of infection is mosquito bites and therefore, the virus must cross a number of polarized cell layers to gain access to organ tissue and the central nervous system. Resistance to trans-cellular movement of macromolecules between epithelial and endothelial cells is mediated by tight junction complexes. While a number of recent studies have documented that WNV infection negatively impacts the barrier function of tight junctions, the intracellular mechanism by which this occurs is poorly understood. In the present study, we report that endocytosis of a subset of tight junction membrane proteins including claudin-1 and JAM-1 occurs in WNV infected epithelial and endothelial cells. This process, which ultimately results in lysosomal degradation of the proteins, is dependent on the GTPase dynamin and microtubule-based transport. Finally, infection of polarized cells with the related flavivirus, Dengue virus-2, did not result in significant loss of tight junction membrane proteins. These results suggest that neurotropic flaviviruses such as WNV modulate the host cell environment differently than hemorrhagic flaviviruses and thus may have implications for understanding the molecular basis for neuroinvasion.

  8. Down syndrome fibroblast model of Alzheimer-related endosome pathology: accelerated endocytosis promotes late endocytic defects.

    Cataldo, Anne M; Mathews, Paul M; Boiteau, Anne Boyer; Hassinger, Linda C; Peterhoff, Corrinne M; Jiang, Ying; Mullaney, Kerry; Neve, Rachael L; Gruenberg, Jean; Nixon, Ralph A

    2008-08-01

    Endocytic dysfunction is an early pathological change in Alzheimer's disease (AD) and Down's syndrome (DS). Using primary fibroblasts from DS individuals, we explored the interactions among endocytic compartments that are altered in AD and assessed their functional consequences in AD pathogenesis. We found that, like neurons in both AD and DS brains, DS fibroblasts exhibit increased endocytic uptake, fusion, and recycling, and trafficking of lysosomal hydrolases to rab5-positive early endosomes. Moreover, late endosomes identified using antibodies to rab7 and lysobisphosphatidic acid increased in number and appeared as enlarged, perinuclear vacuoles, resembling those in neurons of both AD and DS brains. In control fibroblasts, similar enlargement of rab5-, rab7-, and lysobisphosphatidic acid-positive endosomes was induced when endocytosis and endosomal fusion were increased by expression of either a rab5 or an active rab5 mutant, suggesting that persistent endocytic activation results in late endocytic dysfunction. Conversely, expression of a rab5 mutant that inhibits endocytic uptake reversed early and late endosomal abnormalities in DS fibroblasts. Our results indicate that DS fibroblasts recapitulate the neuronal endocytic dysfunction of AD and DS, suggesting that increased trafficking from early endosomes can account, in part, for downstream endocytic perturbations that occur in neurons in both AD and DS brains. PMID:18535180

  9. RAB-5- and DYNAMIN-1-Mediated Endocytosis of EFF-1 Fusogen Controls Cell-Cell Fusion

    Ksenia Smurova

    2016-02-01

    Full Text Available Cell-cell fusion plays essential roles during fertilization and organogenesis. Previous studies in C. elegans led to the identification of the eukaryotic fusion protein (EFF-1 fusogen, which has structural homology to class II viral fusogens. Transcriptional repression of EFF-1 ensures correct fusion fates, and overexpression of EFF-1 results in embryonic lethality. EFF-1 must be expressed on the surface of both fusing cells; however, little is known regarding how cells regulate EFF-1 surface exposure. Here, we report that EFF-1 is actively removed from the plasma membrane of epidermal cells by dynamin- and RAB-5-dependent endocytosis and accumulates in early endosomes. EFF-1 was transiently localized to apical domains of fusion-competent cells. Effective cell-cell fusion occurred only between pairs of cell membranes in which EFF-1 localized. Downregulation of dynamin or RAB-5 caused EFF-1 mislocalization to all apical membrane domains and excessive fusion. Thus, internalization of EFF-1 is a safety mechanism preventing excessive cell fusion.

  10. Coulombic Fluids Bulk and Interfaces

    Freyland, Werner

    2011-01-01

    Ionic liquids have attracted considerable interest in recent years. In this book the bulk and interfacial physico-chemical characteristics of various fluid systems dominated by Coulomb interactions are treated which includes molten salts, ionic liquids as well as metal-molten salt mixtures and expanded fluid metals. Of particular interest is the comparison of the different systems. Topics in the bulk phase concern the microscopic structure, the phase behaviour and critical phenomena, and the metal-nonmetal transition. Interfacial phenomena include wetting transitions, electrowetting, surface freezing, and the electrified ionic liquid/ electrode interface. With regard to the latter 2D and 3D electrochemical phase formation of metals and semi-conductors on the nanometer scale is described for a number of selected examples. The basic concepts and various experimental methods are introduced making the book suitable for both graduate students and researchers interested in Coulombic fluids.

  11. Nitrative DNA damage induced by multi-walled carbon nanotube via endocytosis in human lung epithelial cells

    Guo, Feiye, E-mail: zhizi0269@doc.medic.mie-u.ac.jp [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507 (Japan); Ma, Ning, E-mail: maning@suzuka-u.ac.jp [Faculty of Health Science, Suzuka University of Medical Science, 1001-1 Kishioka-cho, Suzuka, Mie, 510-0293 (Japan); Horibe, Yoshiteru, E-mail: violinteru@yahoo.co.jp [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507 (Japan); Kawanishi, Shosuke, E-mail: kawanisi@suzuka-u.ac.jp [Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Minami-Tamagaki-cho, Suzuka, Mie, 513-8670 (Japan); Murata, Mariko, E-mail: mmurata@doc.medic.mie-u.ac.jp [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507 (Japan); Hiraku, Yusuke, E-mail: y-hiraku@doc.medic.mie-u.ac.jp [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507 (Japan)

    2012-04-15

    Carbon nanotube (CNT) has a promising usage in the field of material science for industrial purposes because of its unique physicochemical property. However, intraperitoneal administration of CNT was reported to cause mesothelioma in experimental animals. Chronic inflammation may contribute to carcinogenesis induced by fibrous materials. 8-Nitroguanine is a mutagenic DNA lesion formed during inflammation and may play a role in CNT-induced carcinogenesis. In this study, we examined 8-nitroguanine formation in A549 human lung alveolar epithelial cells treated with multi-walled CNT (MWCNT) by fluorescent immunocytochemistry. Both MWCNTs with diameter of 20–30 nm (CNT20) and 40–70 nm (CNT40) significantly induced 8-nitroguanine formation at 5 and 10 μg/ml (p < 0.05), which persisted for 24 h, although there was no significant difference in DNA-damaging abilities of these MWCNTs. MWCNTs significantly induced the expression of inducible nitric oxide synthase (iNOS) for 24 h (p < 0.05). MWCNTs also significantly increased the level of nitrite, a hydrolysis product of oxidized NO, in the culture supernatant at 4 and 8 h (p < 0.05). MWCNT-induced 8-nitroguanine formation and iNOS expression were largely suppressed by inhibitors of iNOS (1400 W), nuclear factor-κB (Bay11-7082), actin polymerization (cytochalasin D), caveolae-mediated endocytosis (methyl-β-cyclodextrin, MBCD) and clathrin-mediated endocytosis (monodansylcadaverine, MDC). Electron microscopy revealed that MWCNT was mainly located in vesicular structures in the cytoplasm, and its cellular internalization was reduced by MBCD and MDC. These results suggest that MWCNT is internalized into cells via clathrin- and caveolae-mediated endocytosis, leading to inflammatory reactions including iNOS expression and resulting nitrative DNA damage, which may contribute to carcinogenesis. Highlights: ►Multi-walled carbon nanotube (MWCNT) caused DNA damage in A549 cells. ►MWCNT formed 8-nitroguanine, a DNA lesion

  12. Nitrative DNA damage induced by multi-walled carbon nanotube via endocytosis in human lung epithelial cells

    Carbon nanotube (CNT) has a promising usage in the field of material science for industrial purposes because of its unique physicochemical property. However, intraperitoneal administration of CNT was reported to cause mesothelioma in experimental animals. Chronic inflammation may contribute to carcinogenesis induced by fibrous materials. 8-Nitroguanine is a mutagenic DNA lesion formed during inflammation and may play a role in CNT-induced carcinogenesis. In this study, we examined 8-nitroguanine formation in A549 human lung alveolar epithelial cells treated with multi-walled CNT (MWCNT) by fluorescent immunocytochemistry. Both MWCNTs with diameter of 20–30 nm (CNT20) and 40–70 nm (CNT40) significantly induced 8-nitroguanine formation at 5 and 10 μg/ml (p < 0.05), which persisted for 24 h, although there was no significant difference in DNA-damaging abilities of these MWCNTs. MWCNTs significantly induced the expression of inducible nitric oxide synthase (iNOS) for 24 h (p < 0.05). MWCNTs also significantly increased the level of nitrite, a hydrolysis product of oxidized NO, in the culture supernatant at 4 and 8 h (p < 0.05). MWCNT-induced 8-nitroguanine formation and iNOS expression were largely suppressed by inhibitors of iNOS (1400 W), nuclear factor-κB (Bay11-7082), actin polymerization (cytochalasin D), caveolae-mediated endocytosis (methyl-β-cyclodextrin, MBCD) and clathrin-mediated endocytosis (monodansylcadaverine, MDC). Electron microscopy revealed that MWCNT was mainly located in vesicular structures in the cytoplasm, and its cellular internalization was reduced by MBCD and MDC. These results suggest that MWCNT is internalized into cells via clathrin- and caveolae-mediated endocytosis, leading to inflammatory reactions including iNOS expression and resulting nitrative DNA damage, which may contribute to carcinogenesis. Highlights: ►Multi-walled carbon nanotube (MWCNT) caused DNA damage in A549 cells. ►MWCNT formed 8-nitroguanine, a DNA lesion

  13. Study of uptake and endocytosis of gamma rays-irradiated crotoxin by mice peritoneal macrophages

    The purpose was to investigate the uptake and endocytosis of 2000 Gy 60Co irradiated crotoxin through mouse peritoneal macrophages, correlating with native one and another non related protein, the ovalbumin. Native (CTXN) or 2000 Gy 60 Co γ-rays (dose rate 540 Gy/hour) irradiated crotoxin (CTXI) or ovalbumin processed of same manner (OVAN - OVAI) were offered to mouse peritoneal macrophages and their uptake was evaluated by immunohistochemistry and quantitative in situ ELISA. The involvement of scavenger receptors (ScvR) was evaluated by using blockers drugs (Probuco-PBC or Dextran Sulfate - SD) or with nonspecific blocking using fetal calf serum (FBS). The morphology and viability of macrophages were preserved during the experiments. CTXI showed irradiation-induced aggregates and formation of oxidative changing were observed on this protein after gamma rays treatment. By immunohistochemistry we could observe heavy stained phagocytic vacuole on macrophages incubated with CTXI, as compared with CTXN. Quantitatively by in situ ELISA, the sema pattern was observed, displaying a 2-fold CTXI incorporation. In presence of PBC or SD we could find a significant decrease of CTXI uptake but not of CTXN. However the CTXN uptake was depressed by FBS, not observed with CTXI. OVA, after gamma rays treatment, underwent a high degradation suffering a potent incorporation and metabolism by macrophages, with a major uptake of OVAI in longer incubation (120 minutes). Gamma rays (60 Co) produced oxidative changes on CTX molecule, leading to a uptake by ScvR-mice peritoneal macrophages, suggesting that the relation antigen-presenting cells and gamma rays-modified proteins are responsible for the better immune response presented by irradiated antigens. (author)

  14. Clathrin and LRP-1-independent constitutive endocytosis and recycling of uPAR.

    Katia Cortese

    Full Text Available BACKGROUND: The urokinase receptor (uPAR/CD87 is highly expressed in malignant tumours. uPAR, as a GPI anchored protein, is preferentially located at the cell surface, where it interacts with its ligands urokinase (uPA and the extracellular matrix protein vitronectin, thus promoting plasmin generation, cell-matrix interactions and intracellular signalling events. Interaction with a complex formed by uPA and its inhibitor PAI-1 induces cell surface down regulation and recycling of the receptor via the clathrin-coated pathway, a process dependent on the association to LRP-1. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have found that along with the ligand-induced down-regulation, uPAR also internalizes and recycles constitutively through a second pathway that is independent of LRP-1 and clathrin but shares some properties with macropinocytosis. The ligand-independent route is amiloride-sensitive, does not require uPAR partitioning into lipid rafts, is independent of the activity of small GTPases RhoA, Rac1 and Cdc42, and does not require PI3K activity. Constitutively endocytosed uPAR is found in EEA1 positive early/recycling endosomes but does not reach lysosomes in the absence of ligands. Electron microscopy analysis reveals the presence of uPAR in ruffling domains at the cell surface, in macropinosome-like vesicles and in endosomal compartments. CONCLUSIONS/SIGNIFICANCE: These results indicate that, in addition to the ligand-induced endocytosis of uPAR, efficient surface expression and membrane trafficking might also be driven by an uncommon macropinocytic mechanism coupled with rapid recycling to the cell surface.

  15. Bulk metallic glass tube casting

    Research highlights: → Tubular specimens of Zr55Cu30Al10Ni5 cast in custom arc-melting furnace. → Tilt casting supplemented by suction casting. → Bulk metallic glass formed only with optimized processing parameters. → Fully amorphous tubes with 1.8 mm wall thickness and 25 mm diameter. - Abstract: Tubular bulk metallic glass specimens were produced, using a custom-built combined arc-melting tilt-casting furnace. Zr55Cu30Al10Ni5 tubes with outer diameter of 25 mm and 0.8-3 mm wall thicknesses were cast, with both tilt and suction casting to ensure mold filling. Tilt casting was found to fill one side of the tube mold first, with the rest of the tube circumference filled subsequently by suction casting. Optimized casting parameters were required to fully fill the mold and ensure glass formation. Too small melt mass and too low arc power filled the mold only partially. However, too large melt mass and higher arc power which lead to the best mold filling also lead to partial crystallization. Variations in processing parameters were explored, until a glassy ring with 1.8 mm thickness was produced. Different sections of the as-cast ring were investigated by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and instrumented indentation to ensure amorphous microstructure. Atomic force microscopy (AFM) was used to compare the surface qualities of the first- and last-filled sections. These measurements confirmed the glassy structure of the cast ring, and that, the tilt cast tube section consistently showed better surface quality than the suction cast section. Optimized casting parameters are required to fully realize the potential of directly manufacturing complex shapes out of high-purity bulk metallic glasses by tilt casting.

  16. Bulk Moisture and Salinity Sensor

    Nurge, Mark; Monje, Oscar; Prenger, Jessica; Catechis, John

    2013-01-01

    Measurement and feedback control of nutrient solutions in plant root zones is critical to the development of healthy plants in both terrestrial and reduced-gravity environments. In addition to the water content, the amount of fertilizer in the nutrient solution is important to plant health. This typically requires a separate set of sensors to accomplish. A combination bulk moisture and salinity sensor has been designed, built, and tested with different nutrient solutions in several substrates. The substrates include glass beads, a clay-like substrate, and a nutrient-enriched substrate with the presence of plant roots. By measuring two key parameters, the sensor is able to monitor both the volumetric water content and salinity of the nutrient solution in bulk media. Many commercially available moisture sensors are point sensors, making localized measurements over a small volume at the point of insertion. Consequently, they are more prone to suffer from interferences with air bubbles, contact area of media, and root growth. This makes it difficult to get an accurate representation of true moisture content and distribution in the bulk media. Additionally, a network of point sensors is required, increasing the cabling, data acquisition, and calibration requirements. measure the dielectric properties of a material in the annular space of the vessel. Because the pore water in the media often has high salinity, a method to measure the media moisture content and salinity simultaneously was devised. Characterization of the frequency response for capacitance and conductance across the electrodes was completed for 2-mm glass bead media, 1- to 2-mm Turface (a clay like media), and 1- to 2-mm fertilized Turface with the presence of root mass. These measurements were then used to find empirical relationships among capacitance (C), the dissipation factor (D), the volumetric water content, and the pore water salinity.

  17. Gold based bulk metallic glass

    Schroers, Jan; Lohwongwatana, Boonrat; Johnson, William L.; Peker, Atakan

    2005-01-01

    Gold-based bulk metallic glass alloys based on Au-Cu-Si are introduced. The alloys exhibit a gold content comparable to 18-karat gold. They show very low liquidus temperature, large supercooled liquid region, and good processibility. The maximum casting thickness exceeds 5 mm in the best glassformer. Au49Ag5.5Pd2.3Cu26.9Si16.3 has a liquidus temperature of 644 K, a glass transition temperature of 401 K, and a supercooled liquid region of 58 K. The Vickers hardness of the alloys in this system...

  18. Iron - based bulk amorphous alloys

    R. Babilas

    2010-07-01

    Full Text Available Purpose: The paper presents a structure characterization, thermal and soft magnetic properties analysis of Fe-based bulk amorphous materials in as-cast state and after crystallization process. In addition, the paper gives some brief review about achieving, formation and structure of bulk metallic glasses as a special group of amorphous materials.Design/methodology/approach: The studies were performed on Fe72B20Si4Nb4 metallic glass in form of ribbons and rods. The amorphous structure of tested samples was examined by X-ray diffraction (XRD, transmission electron microscopy (TEM and scanning electron microscopy (SEM methods. The thermal properties of the glassy samples were measured using differential thermal analysis (DTA and differential scanning calorimetry (DSC. The magnetic properties contained initial and maximum magnetic permeability, coercive force and magnetic after-effects measurements were determined by the Maxwell-Wien bridge and VSM methods.Findings: The X-ray diffraction and transmission electron microscopy investigations revealed that the studied as-cast bulk metallic glasses in form of ribbons and rods were amorphous. Two stage crystallization process was observed for studied bulk amorphous alloy. The differences of crystallization temperature between ribbons and rods with chosen thickness are probably caused by different amorphous structures as a result of the different cooling rates in casting process. The SEM images showed that studied fractures could be classified as mixed fractures with indicated two zones contained “river” and “smooth” areas. The changing of chosen soft magnetic properties (μr, Bs, Hc obtained for samples with different thickness is a result of the non-homogenous amorphous structure of tested metallic glasses. The annealing process in temperature range from 373 to 773 K causes structural relaxation of tested amorphous materials, which leads to changes in their physical properties. The qualitative

  19. Bulk dynamics for interfacial growth models

    López, Cristóbal; Santos, Fernando; Garrido, P. L.

    2000-01-01

    We study the influence of the bulk dynamics of a growing cluster of particles on the properties of its interface. First, we define a general bulk growth model by means of a continuum Master equation for the evolution of the bulk density field. This general model just considers an arbitrary addition of particles (though it can be easily generalized to consider subtraction) with no other physical restriction. The corresponding Langevin equation for this bulk density field is derived where the i...

  20. Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes.

    Moscatelli, A; Idilli, A I; Rodighiero, S; Caccianiga, M

    2012-09-01

    Pollen tube growth depends on the integrity of the actin cytoskeleton that regulates cytoplasmic streaming and secretion. To clarify whether actin also plays a role in pollen tube endocytosis, Latrunculin B (LatB) was employed in internalisation experiments with tobacco pollen tubes, using the lipophilic dye FM4-64 and charged nanogold. Time-lapse analysis and dissection of endocytosis allowed us to identify internalisation pathways with different sensitivity to LatB. Co-localisation experiments and ultrastructural observations using positively charged nanogold revealed that LatB significantly inhibited endocytosis in the pollen tube shank, affecting internalisation of the plasma membrane (PM) recycled for secretion, as well as that conveyed to vacuoles. In contrast, endocytosis of negatively charged nanogold in the tip, which is also conveyed to vacuoles, was not influenced. Experiments of fluorescence recovery after photobleaching (FRAP) of the apical and subapical PM revealed domains with different rates of fluorescence recovery and showed that these differences depend on the actin cytoskeleton integrity. These results show the presence of distinct degradation pathways by demonstrating that actin-dependent and actin-indepedent endocytosis both operate in pollen tubes, internalising tracts of PM to be recycled and broken down. Intriguingly, although most studies concentrate on exocytosis and distension in the apex, the present paper shows that uncharacterised, actin-dependent secretory activity occurs in the shank of pollen tubes. PMID:22288466

  1. Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis, and Thyroglobulin Internalization in a Thyroid Cell Line.

    Mascia, Anna; Gentile, Flaviana; Izzo, Antonella; Mollo, Nunzia; De Luca, Maria; Bucci, Cecilia; Nitsch, Lucio; Calì, Gaetano

    2016-08-01

    Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over-expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over-expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over-expressing cells, Rab7 and LC3B-II co-localized and co-localization in large circular structures occurred only in Rab7 over-expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co-localization of Rab7 with cortactin and f-actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over-expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP-stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over-expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695-1708, 2016. © 2015 Wiley Periodicals, Inc. PMID:26599499

  2. Caveolin-1 and CDC42 mediated endocytosis of silica-coated iron oxide nanoparticles in HeLa cells

    Nils Bohmer

    2015-01-01

    Full Text Available Nanomedicine is a rapidly growing field in nanotechnology, which has great potential in the development of new therapies for numerous diseases. For example iron oxide nanoparticles are in clinical use already in the thermotherapy of brain cancer. Although it has been shown, that tumor cells take up these particles in vitro, little is known about the internalization routes. Understanding of the underlying uptake mechanisms would be very useful for faster and precise development of nanoparticles for clinical applications. This study aims at the identification of key proteins, which are crucial for the active uptake of iron oxide nanoparticles by HeLa cells (human cervical cancer as a model cell line. Cells were transfected with specific siRNAs against Caveolin-1, Dynamin 2, Flotillin-1, Clathrin, PIP5Kα and CDC42. Knockdown of Caveolin-1 reduces endocytosis of superparamagnetic iron oxide nanoparticles (SPIONs and silica-coated iron oxide nanoparticles (SCIONs between 23 and 41%, depending on the surface characteristics of the nanoparticles and the experimental design. Knockdown of CDC42 showed a 46% decrease of the internalization of PEGylated SPIONs within 24 h incubation time. Knockdown of Dynamin 2, Flotillin-1, Clathrin and PIP5Kα caused no or only minor effects. Hence endocytosis in HeLa cells of iron oxide nanoparticles, used in this study, is mainly mediated by Caveolin-1 and CDC42. It is shown here for the first time, which proteins of the endocytotic pathway mediate the endocytosis of silica-coated iron oxide nanoparticles in HeLa cells in vitro. In future studies more experiments should be carried out with different cell lines and other well-defined nanoparticle species to elucidate possible general principles.

  3. Functional analysis of Abp1p-interacting proteins involved in endocytosis of the MCC component in Aspergillus oryzae.

    Matsuo, Kento; Higuchi, Yujiro; Kikuma, Takashi; Arioka, Manabu; Kitamoto, Katsuhiko

    2013-07-01

    We have investigated the functions of three endocytosis-related proteins in the filamentous fungus Aspergillus oryzae. Yeast two-hybrid screening using the endocytic marker protein AoAbp1 (A.oryzae homolog of Saccharomyces cerevisiae Abp1p) as a bait identified four interacting proteins named Aip (AoAbp1 interacting proteins). In mature hyphae, EGFP (enhanced green fluorescent protein) fused to Aips colocalized with AoAbp1 at the hyphal tip region and the plasma membrane, suggesting that Aips function in endocytosis. aipA is a putative AAA ATPase and its function has been dissected (Higuchi et al., 2011). aipB, the homolog of A. nidulans myoA, encodes an essential class I myosin and its conditional mutant showed a germination defect. aipC and aipD do not contain any recognizable domains except some proline-rich regions which may interact with two SH3 (Src homology 3) domains of AoAbp1. Neither aipC nor aipD disruptants showed any defects in their growth, but the aipC disruptant formed less conidia compared with the control strain. In addition, the aipC disruptant was resistant to the triazole antifungal drugs that inhibit ergosterol biosynthesis. Although no aip disruptants showed any defects in the uptake of the fluorescent dye FM4-64, the endocytosis of the arginine permease AoCan1, one of the MCC (membrane compartment of Can1p) components, was delayed in both aipC and aipD disruptants. In A. oryzae, AoCan1 localized mainly at the plasma membrane in the basal region of hyphae, suggesting that different endocytic mechanisms exist in apical and basal regions of highly polarized cells. PMID:23597630

  4. Interactions between the Yeast SM22 Homologue Scp1 and Actin Demonstrate the Importance of Actin Bundling in Endocytosis* S⃞

    Gheorghe, Dana M.; Aghamohammadzadeh, Soheil; Rooij, Iwona I. Smaczynska-de; Allwood, Ellen G.; Winder, Steve J.; Ayscough, Kathryn R.

    2008-01-01

    The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that all...

  5. Glutathione-Conjugate Transport by RLIP76 is required for Clathrin-Dependent Endocytosis and Chemical Carcinogenesis**

    SINGHAL, SHARAD S.; Wickramarachchi, Dilki; Yadav, Sushma; Singhal, Jyotsana; Leake, Kathryn; Vatsyayan, Rit; Chaudhary, Pankaj; Lelsani, Poorna; Suzuki, Sumihiro; Yang, Shaohua; Awasthi, Yogesh C.; Awasthi, Sanjay

    2011-01-01

    Targeted depletion of the RALBP1 encoded 76 kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancer without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad-spectrum of RLIP76-targeted therapy is because its glutathione-conjugate (GS-E) transport-activity is required for clathrin-dependent endocytosis (CDE), that regulates all ligand-receptor signaling, and that RLIP76 is required not o...

  6. The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.

    Moreau, V; Galan, J M; Devilliers, G; Haguenauer-Tsapis, R; Winsor, B

    1997-01-01

    The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway. Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protec...

  7. alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis

    1981-01-01

    alpha 2-Macroglobulin (alpha 2 M) was adsorbed to colloidal gold and used as a new tool in the study of receptor-mediated endocytosis. alpha 2 M-gold is easy to prepare and is clearly visualized at the electron microscope level. When cells were incubated with alpha 2 M-gold at 0 degrees C, gold was visualized both diffusely over the cell surface and concentrated in coated pits. After cells to which alpha 2 M-gold had been bound at 0 degrees C were warmed, the gold was rapidly internalized int...

  8. Handling of bulk solids theory and practice

    Shamlou, P A

    1990-01-01

    Handling of Bulk Solids provides a comprehensive discussion of the field of solids flow and handling in the process industries. Presentation of the subject follows classical lines of separate discussions for each topic, so each chapter is self-contained and can be read on its own. Topics discussed include bulk solids flow and handling properties; pressure profiles in bulk solids storage vessels; the design of storage silos for reliable discharge of bulk materials; gravity flow of particulate materials from storage vessels; pneumatic transportation of bulk solids; and the hazards of solid-mater

  9. Gold based bulk metallic glass

    Schroers, Jan; Lohwongwatana, Boonrat; Johnson, William L.; Peker, Atakan

    2005-08-01

    Gold-based bulk metallic glass alloys based on Au-Cu-Si are introduced. The alloys exhibit a gold content comparable to 18-karat gold. They show very low liquidus temperature, large supercooled liquid region, and good processibility. The maximum casting thickness exceeds 5mm in the best glassformer. Au49Ag5.5Pd2.3Cu26.9Si16.3 has a liquidus temperature of 644K, a glass transition temperature of 401K, and a supercooled liquid region of 58K. The Vickers hardness of the alloys in this system is ˜350Hv, twice that of conventional 18-karat crystalline gold alloys. This combination of properties makes the alloys attractive for many applications including electronic, medical, dental, surface coating, and jewelry.

  10. Gold based bulk metallic glass

    Gold-based bulk metallic glass alloys based on Au-Cu-Si are introduced. The alloys exhibit a gold content comparable to 18-karat gold. They show very low liquidus temperature, large supercooled liquid region, and good processibility. The maximum casting thickness exceeds 5 mm in the best glassformer. Au49Ag5.5Pd2.3Cu26.9Si16.3 has a liquidus temperature of 644 K, a glass transition temperature of 401 K, and a supercooled liquid region of 58 K. The Vickers hardness of the alloys in this system is ∼350 Hv, twice that of conventional 18-karat crystalline gold alloys. This combination of properties makes the alloys attractive for many applications including electronic, medical, dental, surface coating, and jewelry

  11. Bulk analysis using nuclear techniques

    Bulk analysis techniques developed for the mining industry are reviewed. Using penetrating neutron and #betta#-radiations, measurements are obtained directly from a large volume of sample (3-30 kg) #betta#-techniques were used to determine the grade of iron ore and to detect shale on conveyor belts. Thermal neutron irradiation was developed for the simultaneous determination of iron and aluminium in iron ore on a conveyor belt. Thermal-neutron activation analysis includes the determination of alumina in bauxite, and manganese and alumina in manganese ore. Fast neutron activation analysis is used to determine silicon in iron ores, and alumina and silica in bauxite. Fast and thermal neutron activation has been used to determine the soil in shredded sugar cane. (U.K.)

  12. Cellular mechanisms regulating activity-dependent release of native brain-derived neurotrophic factor from hippocampal neurons.

    Balkowiec, Agnieszka; Katz, David M

    2002-12-01

    Brain-derived neurotrophic factor (BDNF) plays a critical role in activity-dependent modifications of neuronal connectivity and synaptic strength, including establishment of hippocampal long-term potentiation (LTP). To shed light on mechanisms underlying BDNF-dependent synaptic plasticity, the present study was undertaken to characterize release of native BDNF from newborn rat hippocampal neurons in response to physiologically relevant patterns of electrical field stimulation in culture, including tonic stimulation at 5 Hz, bursting stimulation at 25 and 100 Hz, and theta-burst stimulation (TBS). Release was measured using the ELISA in situ technique, developed in our laboratory to quantify secretion of native BDNF without the need to first overexpress the protein to nonphysiological levels. Each stimulation protocol resulted in a significant increase in BDNF release that was tetrodotoxin sensitive and occurred in the absence of glutamate receptor activation. However, 100 Hz tetanus and TBS, stimulus patterns that are most effective in inducing hippocampal LTP, were significantly more effective in releasing native BDNF than lower-frequency stimulation. For all stimulation protocols tested, removal of extracellular calcium, or blockade of N-type calcium channels, prevented BDNF release. Similarly, depletion of intracellular calcium stores with thapsigargin and treatment with dantrolene, an inhibitor of calcium release from caffeine-ryanodine-sensitive stores, markedly inhibited activity-dependent BDNF release. Our results indicate that BDNF release can encode temporal features of hippocampal neuronal activity. The dual requirement for calcium influx through N-type calcium channels and calcium mobilization from intracellular stores strongly implicates a role for calcium-induced calcium release in activity-dependent BDNF secretion. PMID:12451139

  13. Isotopic signatures by bulk analyses

    Los Alamos National Laboratory has developed a series of measurement techniques for identification of nuclear signatures by analyzing bulk samples. Two specific applications for isotopic fingerprinting to identify the origin of anthropogenic radioactivity in bulk samples are presented. The first example is the analyses of environmental samples collected in the US Arctic to determine the impact of dumping of radionuclides in this polar region. Analyses of sediment and biota samples indicate that for the areas sampled the anthropogenic radionuclide content of sediments was predominantly the result of the deposition of global fallout. The anthropogenic radionuclide concentrations in fish, birds and mammals were very low. It can be surmised that marine food chains are presently not significantly affected. The second example is isotopic fingerprinting of water and sediment samples from the Rocky Flats Facility (RFP). The largest source of anthropogenic radioactivity presently affecting surface-waters at RFP is the sediments that are currently residing in the holding ponds. One gram of sediment from a holding pond contains approximately 50 times more plutonium than 1 liter of water from the pond. Essentially 100% of the uranium in Ponds A-1 and A-2 originated as depleted uranium. The largest source of radioactivity in the terminal Ponds A-4, B-5 and C-2 was naturally occurring uranium and its decay product radium. The uranium concentrations in the waters collected from the terminal ponds contained 0.05% or less of the interim standard calculated derived concentration guide for uranium in waters available to the public. All of the radioactivity observed in soil, sediment and water samples collected at RFP was naturally occurring, the result of processes at RFP or the result of global fallout. No extraneous anthropogenic alpha, beta or gamma activities were detected. The plutonium concentrations in Pond C-2 appear to vary seasonally

  14. Guidelines for Measuring Bulk Density of Soil

    Bulk density is defined as the dry weight of soil per unit volume of undisturbed soil. • Bulk density can be used to give an indication of the porosity and structure of the soil influencing O2 and H2O movement in the soil. • Soils with a bulk density higher than 1.6 g/cm3 may restrict root development. • Bulk density is also a measurement of the degree of compaction of the soil. • Bulk density increases with compaction and tends to increase with soil depth. • Sandy soils tend to have higher bulk density (1.4-1.5 g/cm3) than clay soils (1.2-1/3g/cm3). The measurement of soil bulk density is carried out by collecting undisturbed soil samples through inserting metal rings (with a known volume) into the soil, and determining the weight of the collected soil after drying

  15. Activity-dependent plasticity of electrical synapses: increasing evidence for its presence and functional roles in the mammalian brain.

    Haas, Julie S; Greenwald, Corey M; Pereda, Alberto E

    2016-01-01

    Gap junctions mediate electrical synaptic transmission between neurons. While the actions of neurotransmitter modulators on the conductance of gap junctions have been extensively documented, increasing evidence indicates they can also be influenced by the ongoing activity of neural networks, in most cases via local interactions with nearby glutamatergic synapses. We review here early evidence for the existence of activity-dependent regulatory mechanisms as well recent examples reported in mammalian brain. The ubiquitous distribution of both neuronal connexins and the molecules involved suggest this phenomenon is widespread and represents a property of electrical transmission in general. PMID:27230776

  16. GRP75 upregulates clathrin-independent endocytosis through actin cytoskeleton reorganization mediated by the concurrent activation of Cdc42 and RhoA.

    Chen, Hang; Gao, Zhihui; He, Changzheng; Xiang, Rong; van Kuppevelt, Toin H; Belting, Mattias; Zhang, Sihe

    2016-05-01

    Therapeutic macromolecules are internalized into the cell by either clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). Although some chaperone proteins play an essential role in CME (e.g. Hsc70 in clathrin uncoating), relatively few of these proteins are functionally involved in CIE. We previously revealed a role for the mitochondrial chaperone protein GRP75 in heparan sulfate proteoglycan (HSPG)-mediated, membrane raft-associated macromolecule endocytosis. However, the mechanism underlying this process remains unclear. In this study, using a mitochondrial signal peptide-directed protein trafficking expression strategy, we demonstrate that wild-type GRP75 expression enhanced the uptakes of HSPG and CIE marker cholera toxin B subunit but impaired the uptake of CME marker transferrin. The endocytosis regulation function of GRP75 is largely mediated by its subcellular location in mitochondria and is essentially determined by its ATPase domain. Interestingly, the mitochondrial expression of GRP75 or its ATPase domain significantly stimulates increases in both RhoA and Cdc42 activation, remarkably induces stress fibers and enhances filopodia formation, which collectively results in the promotion of CIE, but the inhibition of CME. Furthermore, silencing of Cdc42 or RhoA impaired the ability of GRP75 overexpression to increase CIE. Therefore, these results suggest that endocytosis vesicle enrichment of GRP75 by mitochondria trafficking upregulates CIE through an actin cytoskeleton reorganization mechanism mediated by the concurrent activation of Cdc42 and RhoA. This finding provides novel insight into organelle-derived chaperone signaling and the regulation of different endocytosis pathways in cells. PMID:27090015

  17. Dynamic bio-adhesion of polymer nanoparticles on MDCK epithelial cells and its impact on bio-membranes, endocytosis and paracytosis

    He, Bing; Yuan, Lan; Dai, Wenbing; Gao, Wei; Zhang, Hua; Wang, Xueqing; Fang, Weigang; Zhang, Qiang

    2016-03-01

    Nowadays, concern about the use of nanotechnology for biomedical application is unprecedentedly increasing. In fact, nanosystems applied for various potential clinical uses always have to cross the primary biological barrier consisting of epithelial cells. However, little is really known currently in terms of the influence of the dynamic bio-adhesion of nanosystems on bio-membranes as well as on endocytosis and transcytosis. This was investigated here using polymer nanoparticles (PNs) and MDCK epithelial cells as the models. Firstly, the adhesion of PNs on cell membranes was found to be time-dependent with a shift of both location and dispersion pattern, from the lateral adhesion of mainly mono-dispersed PNs initially to the apical coverage of the PN aggregate later. Then, it was interesting to observe in this study that the dynamic bio-adhesion of PNs only affected their endocytosis but not their transcytosis. It was important to find that the endocytosis of PNs was not a constant process. A GM1 dependent CDE (caveolae dependent endocytosis) pathway was dominant in the preliminary stage, followed by the co-existence of a CME (clathrin-mediated endocytosis) pathway for the PN aggregate at a later stage, in accordance with the adhesion features of PNs, suggesting the modification of PN adhesion patterns on the endocytosis pathways. Next, the PN adhesion was noticed to affect the structure of cell junctions, via altering the extra- and intra-cellular calcium levels, leading to the enhanced paracellular transport of small molecules, but not favorably enough for the obviously increased passing of PNs themselves. Finally, FRAP and other techniques all demonstrated the obvious impact of PN adhesion on the membrane confirmation, independent of the adhesion location and time, which might lower the threshold for the internalization of PNs, even their aggregates. Generally, these findings confirm that the transport pathway mechanism of PNs through epithelial cells is rather

  18. Targeted, activity-dependent spinal stimulation produces long-lasting motor recovery in chronic cervical spinal cord injury.

    McPherson, Jacob G; Miller, Robert R; Perlmutter, Steve I

    2015-09-29

    Use-dependent movement therapies can lead to partial recovery of motor function after neurological injury. We attempted to improve recovery by developing a neuroprosthetic intervention that enhances movement therapy by directing spike timing-dependent plasticity in spared motor pathways. Using a recurrent neural-computer interface in rats with a cervical contusion of the spinal cord, we synchronized intraspinal microstimulation below the injury with the arrival of functionally related volitional motor commands signaled by muscle activity in the impaired forelimb. Stimulation was delivered during physical retraining of a forelimb behavior and throughout the day for 3 mo. Rats receiving this targeted, activity-dependent spinal stimulation (TADSS) exhibited markedly enhanced recovery compared with animals receiving targeted but open-loop spinal stimulation and rats receiving physical retraining alone. On a forelimb reach and grasp task, TADSS animals recovered 63% of their preinjury ability, more than two times the performance level achieved by the other therapy groups. Therapeutic gains were maintained for 3 additional wk without stimulation. The results suggest that activity-dependent spinal stimulation can induce neural plasticity that improves behavioral recovery after spinal cord injury. PMID:26371306

  19. The DEG/ENaC cation channel protein UNC-8 drives activity-dependent synapse removal in remodeling GABAergic neurons

    Miller-Fleming, Tyne W; Petersen, Sarah C; Manning, Laura; Matthewman, Cristina; Gornet, Megan; Beers, Allison; Hori, Sayaka; Mitani, Shohei; Bianchi, Laura; Richmond, Janet; Miller, David M

    2016-01-01

    Genetic programming and neural activity drive synaptic remodeling in developing neural circuits, but the molecular components that link these pathways are poorly understood. Here we show that the C. elegans Degenerin/Epithelial Sodium Channel (DEG/ENaC) protein, UNC-8, is transcriptionally controlled to function as a trigger in an activity-dependent mechanism that removes synapses in remodeling GABAergic neurons. UNC-8 cation channel activity promotes disassembly of presynaptic domains in DD type GABA neurons, but not in VD class GABA neurons where unc-8 expression is blocked by the COUP/TF transcription factor, UNC-55. We propose that the depolarizing effect of UNC-8-dependent sodium import elevates intracellular calcium in a positive feedback loop involving the voltage-gated calcium channel UNC-2 and the calcium-activated phosphatase TAX-6/calcineurin to initiate a caspase-dependent mechanism that disassembles the presynaptic apparatus. Thus, UNC-8 serves as a link between genetic and activity-dependent pathways that function together to promote the elimination of GABA synapses in remodeling neurons. DOI: http://dx.doi.org/10.7554/eLife.14599.001 PMID:27403890

  20. Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection

    Piccini, Luana E.; Castilla, Viviana; Damonte, Elsa B.

    2015-01-01

    The endocytic uptake and intracellular trafficking for penetration of DENV-3 strain H-87 into Vero cells was analyzed by using several biochemical inhibitors and dominant negative mutants of cellular proteins. The results presented show that the infective entry of DENV-3 into Vero cells occurs through a non-classical endocytosis pathway dependent on low pH and dynamin, but non-mediated by clathrin. After uptake, DENV-3 transits through early endosomes to reach Rab 7-regulated late endosomes, and according with the half-time for ammonium chloride resistance viral nucleocapsid is released into the cytosol approximately at 12 min post-infection. Furthermore, the influence of the clathrin pathway in DENV-3 infective entry in other mammalian cell lines of human origin, such as A549, HepG2 and U937 cells, was evaluated demonstrating that variable entry pathways are employed depending on the host cell. Results show for the first time the simultaneous coexistence of infective and non -infective routes for DENV entry into the host cell, depending on the usage of clathrin-mediated endocytosis. PMID:26469784

  1. Transferrin receptor number, synthesis, and endocytosis during erythropoietin-induced maturation of Friend virus-infected erythroid cells

    Erythropoietin (EP) responsive Friend virus-infected erythroid cells had 200,000 steady-state binding sites for transferrin at 370C when isolated from the spleens of Friend virus-infected mice. Upon culture of these cells with EP, the synthesis of transferrin receptors increased 4- to 7-fold and the number of transferrin-binding sites per cell doubled after 24 h. However, the rate of uptake of 59Fe from transferrin remained constant at approximately 35,000 atoms of 59Fe per minute per cell during this period in culture. The amount of 125I-transferrin internalized during the steady-state binding did not change during this culture period while the transferrin bound to the surface increased 3-fold. At all stages of erythroid maturation, the maximum rate of endocytosis was determined to be 18,000 molecules of transferrin per minute per cell, and the interval that 125I-transferrin remains in the interior of the cell was calculated to be 6.9 min. After 48 h of culture with EP, the number of steady-state transferrin-binding sites was reduced in part due to the sequestration of surface receptors within the cell. The uptake of iron from transferrin was limited by the level of endocytosis of transferrin during the initial phase of culture and the number of transferrin receptors at the cell surface during the latter stages of erythroid maturation of these cells

  2. Entry of human papillomavirus type 16 by actin-dependent, clathrin- and lipid raft-independent endocytosis.

    Mario Schelhaas

    Full Text Available Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16, the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na⁺/H⁺ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH.

  3. Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection.

    Piccini, Luana E; Castilla, Viviana; Damonte, Elsa B

    2015-01-01

    The endocytic uptake and intracellular trafficking for penetration of DENV-3 strain H-87 into Vero cells was analyzed by using several biochemical inhibitors and dominant negative mutants of cellular proteins. The results presented show that the infective entry of DENV-3 into Vero cells occurs through a non-classical endocytosis pathway dependent on low pH and dynamin, but non-mediated by clathrin. After uptake, DENV-3 transits through early endosomes to reach Rab 7-regulated late endosomes, and according with the half-time for ammonium chloride resistance viral nucleocapsid is released into the cytosol approximately at 12 min post-infection. Furthermore, the influence of the clathrin pathway in DENV-3 infective entry in other mammalian cell lines of human origin, such as A549, HepG2 and U937 cells, was evaluated demonstrating that variable entry pathways are employed depending on the host cell. Results show for the first time the simultaneous coexistence of infective and non -infective routes for DENV entry into the host cell, depending on the usage of clathrin-mediated endocytosis. PMID:26469784

  4. Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection.

    Luana E Piccini

    Full Text Available The endocytic uptake and intracellular trafficking for penetration of DENV-3 strain H-87 into Vero cells was analyzed by using several biochemical inhibitors and dominant negative mutants of cellular proteins. The results presented show that the infective entry of DENV-3 into Vero cells occurs through a non-classical endocytosis pathway dependent on low pH and dynamin, but non-mediated by clathrin. After uptake, DENV-3 transits through early endosomes to reach Rab 7-regulated late endosomes, and according with the half-time for ammonium chloride resistance viral nucleocapsid is released into the cytosol approximately at 12 min post-infection. Furthermore, the influence of the clathrin pathway in DENV-3 infective entry in other mammalian cell lines of human origin, such as A549, HepG2 and U937 cells, was evaluated demonstrating that variable entry pathways are employed depending on the host cell. Results show for the first time the simultaneous coexistence of infective and non -infective routes for DENV entry into the host cell, depending on the usage of clathrin-mediated endocytosis.

  5. Relative entropy equals bulk relative entropy

    Jafferis, Daniel L; Maldacena, Juan; Suh, S Josephine

    2015-01-01

    We consider the gravity dual of the modular Hamiltonian associated to a general subregion of a boundary theory. We use it to argue that the relative entropy of nearby states is given by the relative entropy in the bulk, to leading order in the bulk gravitational coupling. We also argue that the boundary modular flow is dual to the bulk modular flow in the entanglement wedge, with implications for entanglement wedge reconstruction.

  6. Coupling brane fields to bulk supergravity

    In this note we present a simple, general prescription for coupling brane localized fields to bulk supergravity. We illustrate the procedure by considering 6D N=2 bulk supergravity on a 2D orbifold, with brane fields localized at the fixed points. The resulting action enjoys the full 6D N=2 symmetries in the bulk, and those of 4D N=1 supergravity at the brane positions. (orig.)

  7. Coupling brane fields to bulk supergravity

    Parameswaran, Susha L. [Uppsala Univ. (Sweden). Theoretical Physics; Schmidt, Jonas [Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany)

    2010-12-15

    In this note we present a simple, general prescription for coupling brane localized fields to bulk supergravity. We illustrate the procedure by considering 6D N=2 bulk supergravity on a 2D orbifold, with brane fields localized at the fixed points. The resulting action enjoys the full 6D N=2 symmetries in the bulk, and those of 4D N=1 supergravity at the brane positions. (orig.)

  8. Bulk scalar field in DGP braneworld cosmology

    Ansari, Rizwan ul Haq

    2007-01-01

    We investigated the effects of bulk scalar field in the braneworld cosmological scenario. The Friedmann equations and acceleration condition in presence of the bulk scalar field for a zero tension brane and cosmological constant are studied. In DGP model the effective Einstein equation on the brane is obtained with bulk scalar field. The rescaled bulk scalar field on the brane in the DGP model behaves as an effective four dimensional field, thus standard type cosmology is recovered. In present study of the DGP model, the late-time accelerating phase of the universe can be explained .

  9. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

    1995-01-01

    We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decre...

  10. The down-stream effects of mannan-induced lectin complement pathway activation depend quantitatively on alternative pathway amplification

    Harboe, Morten; Garred, Peter; Karlstrøm, Ellen;

    2009-01-01

    of AP was not observed even at high mannan concentrations since addition of the inhibiting anti-MBL mAb 3F8 completely abolished generation of the terminal C5b-9 complex (TCC). However, selective blockade of AP by anti-factor D inhibited more than 80% of TCC release into the fluid phase after LP...... activation showing that AP amplification is quantitatively responsible for the final effect of initial specific LP activation. TCC generation on the solid phase was distinctly but less inhibited by anti-fD. C2 bypass of the LP pathway could be demonstrated, and AP amplification was also essential during C2...... bypass in LP as shown by complete inhibition of TCC generation in C2-deficient serum by anti-fD and anti-properdin antibodies. In conclusion, the down-stream effect of LP activation depends strongly on AP amplification in normal human serum and in the C2 bypass pathway....

  11. The solar activity dependence of nonmigrating tides in electron density at low and middle latitudes observed by CHAMP and GRACE

    Zhou, Yun-Liang; Wang, Li; Xiong, Chao; Lühr, Hermann; Ma, Shu-Ying

    2016-04-01

    In this paper we use more than a decade of in situ electron density observations from CHAMP and GRACE satellites to investigate the solar activity dependence of nonmigrating tides at both low and middle latitudes. The results indicate that the longitudinal patterns of F region electron density vary with season and latitude, which are exhibiting a wavenumber 4 (WN4) pattern around September equinox at low latitudes and WN1/WN2 patterns during local summer at the southern/northern middle latitudes. These wave patterns in the F region ionosphere can clearly be seen during both solar maximum and minimum years. At low latitudes the absolute amplitudes of DE3 (contributing to the WN4 pattern) are found to be highly related to the solar activity, showing larger amplitudes during solar maximum years. Similarly a solar activity dependence can also be found for the absolute amplitudes of D0, DW2 and DE1 (contributing to the WN1 and WN2 pattern) at middle latitudes. The relative amplitudes (normalized by the zonal mean) of these nonmigrating tides at both low and middle altitudes show little dependence on solar activity. We further found a clear modulation by the quasi-biennial oscillation (QBO) of the relative DE3 amplitudes in both satellite observations, which is consistent with the QBO dependence as reported for the E region temperatures and zonal wind. It also supports the strong coupling of the low-latitude nonmigrating tidal activity between the E and F regions. However, the QBO dependence cannot be found for the relative amplitudes of the nonmigrating tides at middle latitudes, which implies that these tides are generated in situ at F region altitudes.

  12. 27 CFR 20.191 - Bulk articles.

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Bulk articles. 20.191... Users of Specially Denatured Spirits Operations by Users § 20.191 Bulk articles. Users who convey articles in containers exceeding one gallon may provide the recipient with a photocopy of subpart G of...

  13. Bulk equations of motion from CFT correlators

    Kabat, Daniel

    2015-01-01

    To O(1/N) we derive, purely from CFT data, the bulk equations of motion for interacting scalar fields and for scalars coupled to gauge fields and gravity. We first uplift CFT operators to mimic local AdS fields by imposing bulk microcausality. This requires adding an infinite tower of smeared higher-dimension double-trace operators to the CFT definition of a bulk field, with coefficients that we explicitly compute. By summing the contribution of the higher-dimension operators we derive the equations of motion satisfied by these uplifted CFT operators and show that we precisely recover the expected bulk equations of motion. We exhibit the freedom in the CFT construction which corresponds to bulk field redefinitions.

  14. Investigation of the receptor-mediated endocytosis of transcobalamin/intrinsic factor-vitamin B12 complexes

    Beedholm, Rasmus; Grissom, Charles B.; Fedosov, Sergey N.; Nexø, Ebba; Moestrup, Søren K.

    unknown receptor structure. This receptor is suggested to be regulated by the vitamin B12 level in the cells, which is interesting in relation to cancer growth. The cellular endocytosis of TC- B12 complex by this unknown receptor is being investigated, using confocal microscopy. Fluorescently labeled B12......  The transport of vitamin B12 (B12)/cobalamin in the tissue-fluids is facilitated by three different binding-proteins: Intrinsic factor (IF), transcobalamin (TC) and haptocorrin. Especially the first two are important for the cellular uptake of B12. Intrinsic factor is produced in the ventricle...... and is essential for the B12 uptake in the distal part of ileum by the receptor complex cubilin/amionless. TC is important for the uptake of B12 from plasma. In the kidney, megalin is the receptor for the TC- B12 complex, whereas uptake of TC- B12 in the extrarenal tissue occurs by means of a still...

  15. Machine-Learning-Based Analysis in Genome-Edited Cells Reveals the Efficiency of Clathrin-Mediated Endocytosis

    Sun Hae Hong

    2015-09-01

    Full Text Available Cells internalize various molecules through clathrin-mediated endocytosis (CME. Previous live-cell imaging studies suggested that CME is inefficient, with about half of the events terminated. These CME efficiency estimates may have been confounded by overexpression of fluorescently tagged proteins and inability to filter out false CME sites. Here, we employed genome editing and machine learning to identify and analyze authentic CME sites. We examined CME dynamics in cells that express fluorescent fusions of two defining CME proteins, AP2 and clathrin. Support vector machine classifiers were built to identify and analyze authentic CME sites. From inception until disappearance, authentic CME sites contain both AP2 and clathrin, have the same degree of limited mobility, continue to accumulate AP2 and clathrin over lifetimes >∼20 s, and almost always form vesicles as assessed by dynamin2 recruitment. Sites that contain only clathrin or AP2 show distinct dynamics, suggesting they are not part of the CME pathway.

  16. Constitutive endocytosis and turnover of the neuronal glycine transporter GlyT2 is dependent on ubiquitination of a C-terminal lysine cluster.

    Jaime de Juan-Sanz

    Full Text Available Inhibitory glycinergic neurotransmission is terminated by sodium and chloride-dependent plasma membrane glycine transporters (GlyTs. The mainly glial glycine transporter GlyT1 is primarily responsible for the completion of inhibitory neurotransmission and the neuronal glycine transporter GlyT2 mediates the reuptake of the neurotransmitter that is used to refill synaptic vesicles in the terminal, a fundamental role in the physiology and pathology of glycinergic neurotransmission. Indeed, inhibitory glycinergic neurotransmission is modulated by the exocytosis and endocytosis of GlyT2. We previously reported that constitutive and Protein Kinase C (PKC-regulated endocytosis of GlyT2 is mediated by clathrin and that PKC accelerates GlyT2 endocytosis by increasing its ubiquitination. However, the role of ubiquitination in the constitutive endocytosis and turnover of this protein remains unexplored. Here, we show that ubiquitination of a C-terminus four lysine cluster of GlyT2 is required for constitutive endocytosis, sorting into the slow recycling pathway and turnover of the transporter. Ubiquitination negatively modulates the turnover of GlyT2, such that increased ubiquitination driven by PKC activation accelerates transporter degradation rate shortening its half-life while decreased ubiquitination increases transporter stability. Finally, ubiquitination of GlyT2 in neurons is highly responsive to the free pool of ubiquitin, suggesting that the deubiquitinating enzyme (DUB ubiquitin C-terminal hydrolase-L1 (UCHL1, as the major regulator of neuronal ubiquitin homeostasis, indirectly modulates the turnover of GlyT2. Our results contribute to the elucidation of the mechanisms underlying the dynamic trafficking of this important neuronal protein which has pathological relevance since mutations in the GlyT2 gene (SLC6A5 are the second most common cause of human hyperekplexia.

  17. Interactions between the yeast SM22 homologue Scp1 and actin demonstrate the importance of actin bundling in endocytosis.

    Gheorghe, Dana M; Aghamohammadzadeh, Soheil; Smaczynska-de Rooij, Iwona I; Allwood, Ellen G; Winder, Steve J; Ayscough, Kathryn R

    2008-05-30

    The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185 to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process. PMID:18400761

  18. Dynamic analysis of Arabidopsis AP2 σ subunit reveals a key role in clathrin-mediated endocytosis and plant development.

    Fan, Lusheng; Hao, Huaiqing; Xue, Yiqun; Zhang, Liang; Song, Kai; Ding, Zhaojun; Botella, Miguel A; Wang, Haiyang; Lin, Jinxing

    2013-09-01

    Clathrin-mediated endocytosis, which depends on the AP2 complex, plays an essential role in many cellular and developmental processes in mammalian cells. However, the function of the AP2 complex in plants remains largely unexplored. Here, we show in Arabidopsis that the AP2 σ subunit mutant (ap2 σ) displays various developmental defects that are similar to those of mutants defective in auxin transport and/or signaling, including single, trumpet-shaped and triple cotyledons, impaired vascular pattern, reduced vegetative growth, defective silique development and drastically reduced fertility. We demonstrate that AP2 σ is closely associated and physically interacts with the clathrin light chain (CLC) in vivo using fluorescence cross-correlation spectroscopy (FCCS), protein proximity analyses and co-immunoprecipitation assays. Using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), we show that AP2 σ-mCherry spots colocalize with CLC-EGFP at the plasma membrane, and that AP2 σ-mCherry fluorescence appears and disappears before CLC-EGFP fluorescence. The density and turnover rate of the CLC-EGFP spots are significantly reduced in the ap2 σ mutant. The internalization and recycling of the endocytic tracer FM4-64 and the auxin efflux carrier protein PIN1 are also significantly reduced in the ap2 σ mutant. Further, the polar localization of PIN1-GFP is significantly disrupted during embryogenesis in the ap2 σ mutant. Taken together, our results support an essential role of AP2 σ in the assembly of a functional AP2 complex in plants, which is required for clathrin-mediated endocytosis, polar auxin transport and plant growth regulation. PMID:23924631

  19. Nephrotoxicity of Bence-Jones proteins: correlation with endocytosis by BHK cells and intracellular movement

    Ana Lucia Nicastri

    2001-06-01

    Full Text Available The aim of this investigation was to evaluate the endocytosis of two Bence-Jones proteins by renal cells in order to elucidate the interference of their physical and chemical characteristics on nephrotoxicity. Bence-Jones proteins (AK and GL were purified and isolated from the urine of two patients with multiple myeloma. The isotype of both proteins was characterised as being human monoclonal lambda light chain. The AK protein presented mainly an Ip>7.0, a high content of galactose and a low amount of sialic acid molecules. On the other hand, the GL protein presented a single band with an Ip of 4.3, a higher level of sialic acid and a reduced amount of galactose, in comparison with the AK protein. Baby Hamster Kidney (BHK cells were maintained in culture in bottles at 37ºC, using DMEM culture media supplemented with 10% of calf serum with a pH of 7.4. Once the monolayer was observed to be confluent, the BHK cells were incubated with the two proteins, dissolved in a serum-free medium for 1, 5, 15, 30, 60 minutes and 24 hours. Control cells were established omitting the incubation with Bence-Jones proteins, but maintaining all of the other conditions. After, this the cells were washed, trypsinised, centrifuged and fixed in a solution of 4% paraformaldehyde and 0.5% glutaraldehyde on a 0.1 M, pH 7.4 phosphate buffer. Cells were processed for immunocytochemical reactions by using protein A coupled with colloidal gold and further silver enhancement. Semi-thin sections of the pellets were obtained and submitted to the cytochemical reactions. Detection of labelling was made by using light microscopy. It was observed that GL protein tended to be directed towards a perinuclear position, whereas the AK protein tended to suffer lysosomal deviation, suggesting that there is a direct contribution of physical and chemical characteristics on intracellular direction taken by Bence-Jones proteins.O objetivo deste trabalho foi avaliar a endocitose de duas prote

  20. Bulk Viscosity in Holographic Lifshitz Hydrodynamics

    Hoyos, Carlos; Oz, Yaron

    2013-01-01

    We compute the bulk viscosity in holographic models dual to theories with Lifshitz scaling and/or hyperscaling violation, using a generalization of the bulk viscosity formula derived in arXiv:1103.1657 from the null focusing equation. We find that only a class of models with massive vector fields are truly Lifshitz scale invariant, and have a vanishing bulk viscosity. For other holographic models with scalars and/or massless vector fields we find a universal formula in terms of the dynamical exponent and the hyperscaling violation exponent.

  1. Bulk viscosity in holographic Lifshitz hydrodynamics

    Carlos Hoyos; Bom Soo Kim; Yaron Oz

    2014-01-01

    We compute the bulk viscosity in holographic models dual to theories with Lifshitz scaling and/or hyperscaling violation, using a generalization of the bulk viscosity formula derived in arXiv:1103.1657 from the null focusing equation. We find that only a class of models with massive vector fields are truly Lifshitz scale invariant, and have a vanishing bulk viscosity. For other holographic models with scalars and/or massless vector fields we find a universal formula in terms of the dynamical ...

  2. Bulk viscosity in holographic Lifshitz hydrodynamics

    We compute the bulk viscosity in holographic models dual to theories with Lifshitz scaling and/or hyperscaling violation, using a generalization of the bulk viscosity formula derived in arXiv:1103.1657 from the null focusing equation. We find that only a class of models with massive vector fields are truly Lifshitz scale invariant, and have a vanishing bulk viscosity. For other holographic models with scalars and/or massless vector fields we find a universal formula in terms of the dynamical exponent and the hyperscaling violation exponent

  3. Bulk viscosity of hot and dense hadrons

    The bulk viscosity of hot and dense hadrons has been estimated within the framework of hadronic resonance gas model. We observe that the bulk viscosity to entropy ratio increases faster with temperature for higher μB. The magnitude of ζ is more at high μB. This results will have crucial importance for fire-ball produced at low energy nuclear collisions (FAIR, NICA). We note that the bulk to shear viscosity ratio remains above the bound set by AdS/CFT

  4. Activity-Dependent NPAS4 Expression and the Regulation of Gene Programs Underlying Plasticity in the Central Nervous System

    José Fernando Maya-Vetencourt

    2013-01-01

    Full Text Available The capability of the brain to change functionally in response to sensory experience is most active during early stages of development but it decreases later in life when major alterations of neuronal network structures no longer take place in response to experience. This view has been recently challenged by experimental strategies based on the enhancement of environmental stimulation levels, genetic manipulations, and pharmacological treatments, which all have demonstrated that the adult brain retains a degree of plasticity that allows for a rewiring of neuronal circuitries over the entire life course. A hot spot in the field of neuronal plasticity centres on gene programs that underlie plastic phenomena in adulthood. Here, I discuss the role of the recently discovered neuronal-specific and activity-dependent transcription factor NPAS4 as a critical mediator of plasticity in the nervous system. A better understanding of how modifications in the connectivity of neuronal networks occur may shed light on the treatment of pathological conditions such as brain damage or disease in adult life, some of which were once considered untreatable.

  5. Two-Photon Correlation Spectroscopy in Single Dendritic Spines Reveals Fast Actin Filament Reorganization during Activity-Dependent Growth.

    Jian-Hua Chen

    Full Text Available Two-photon fluorescence correlation spectroscopy (2P-FCS within single dendritic spines of living hippocampal pyramidal neurons was used to resolve various subpopulations of mobile F-actin during activity-dependent structural changes such as potentiation induced spine head growth. Two major classes of mobile F-actin were discovered: very dynamic and about a hundred times less dynamic F-actin. Spine head enlargement upon application of Tetraethylammonium (TEA, a protocol previously used for the chemical induction of long-term potentiation (cLTP strictly correlated to changes in the dynamics and filament numbers in the different actin filament fractions. Our observations suggest that spine enlargement is governed by a mechanism in which longer filaments are first cut into smaller filaments that cooperate with the second, increasingly dynamic shorter actin filament population to quickly reorganize and expand the actin cytoskeleton within the spine head. This process would allow a fast and efficient spine head enlargement using a major fraction of the actin filament population that was already present before spine head growth.

  6. Brain-derived neurotrophic factor in arterial baroreceptor pathways: implications for activity-dependent plasticity at baroafferent synapses.

    Martin, Jessica L; Jenkins, Victoria K; Hsieh, Hui-ya; Balkowiec, Agnieszka

    2009-01-01

    Functional characteristics of the arterial baroreceptor reflex change throughout ontogenesis, including perinatal adjustments of the reflex gain and adult resetting during hypertension. However, the cellular mechanisms that underlie these functional changes are not completely understood. Here, we provide evidence that brain-derived neurotrophic factor (BDNF), a neurotrophin with a well-established role in activity-dependent neuronal plasticity, is abundantly expressed in vivo by a large subset of developing and adult rat baroreceptor afferents. Immunoreactivity to BDNF is present in the cell bodies of baroafferent neurons in the nodose ganglion, their central projections in the solitary tract, and terminal-like structures in the lower brainstem nucleus tractus solitarius. Using ELISA in situ combined with electrical field stimulation, we show that native BDNF is released from cultured newborn nodose ganglion neurons in response to patterns that mimic the in vivo activity of baroreceptor afferents. In particular, high-frequency bursting patterns of baroreceptor firing, which are known to evoke plastic changes at baroreceptor synapses, are significantly more effective at releasing BDNF than tonic patterns of the same average frequency. Together, our study indicates that BDNF expressed by first-order baroreceptor neurons is a likely mediator of both developmental and post-developmental modifications at first-order synapses in arterial baroreceptor pathways. PMID:19054281

  7. Technical specifications for the bulk shielding reactor

    This report provides information concerning the technical specifications for the Bulk Shielding Reactor. Areas covered include: safety limits and limiting safety settings; limiting conditions for operation; surveillance requirements; design features; administrative controls; and monitoring of airborne effluents. 10 refs

  8. The bulk radio expansion of Cassiopeia A

    Comparison, in the visibility plane, or radio observations of Cassiopeia A made at 151 MHz over a 2.3 yr interval indicates that the bulk of the radio emitting material has not been decelerated strongly

  9. Faecal bulking efficacy of Australasian breakfast cereals.

    Monro, John A

    2002-01-01

    Faecal bulk may play an important role in preventing a range of disorders of the large bowel, but as yet there is little information available on the relative faecal bulking capacities of various foods. Breakfast cereals are often promoted as a good source of potential bulk for 'inner health' because they provide dietary fibre, but their relative abilities to provide faecal bulk per se have not been described. The faecal bulking efficacy of 28 representative Australasian breakfast cereals was therefore measured. A rat model developed for the purpose, and shown to give similar responses as humans to cereal fibres, was used to measure faecal bulking efficacy as increases in fully hydrated faecal weight/100 g diet, based on precise measurements of food intake, faecal dry matter output and faecal water-holding capacity (g water held without stress/g faecal dry matter). Compared to a baseline diet containing 50% sucrose, increments in hydrated faecal weight due to 50% breakfast cereal ranged from slightly negative (Cornflakes, -2 g/100 g diet) to about 80 g/100 g diet (San Bran). Most breakfast cereals increased hydrated faecal weight by between 10 and 20 g/100 g diet from a baseline of 21 +/- 1.5 g/100 g diet, but four products containing high levels of wheat bran had an exceptionally large impact on hydrated faecal weight (increment > 20 g/100 g diet), and the changes resulted more from relative changes in dry matter output than in faecal water retention/gram. However, as faecal water retention was about 2.5 g water/g faecal dry matter on average, increases in dry matter represented large increases in faecal water load. Faecal bulking indices (FBI) for most of the breakfast cereals were less than 20 (wheat bran = 100). The content of wheat bran equivalents for faecal bulk (WBE(fb)) in the breakfast cereals was calculated from FBI. Most breakfast cereals contributed, per serve, less than 10% of a theoretical daily reference value for faecal bulk (DRV(fb) = 63 WBE

  10. PHONON ECHOES IN BULK AND POWDERED MATERIALS

    Kajimura, K.

    1981-01-01

    Experimental and theoretical studies of phonon echoes in bulk and powdered materials are reviewed. Phonon echoes have been observed in many materials such as bulk piezoelectric crystals, paramagnets, glasses, doped semiconductors, and piezoelectric, magnetic, and metallic powders, etc. The echoes arise from a time reversal of the phase, like spin echoes, of a primary pulsed acoustic excitation due to a second acoustic or rf pulse. The phase reversal occurs through the nonlinear interactions o...

  11. Orbital magnetization in insulators: Bulk versus surface

    Bianco, Raffaello; Resta, Raffaele

    2016-05-01

    The orbital magnetic moment of a finite piece of matter is expressed in terms of the one-body density matrix as a simple trace. We address a macroscopic system, insulating in the bulk, and we show that its orbital moment is the sum of a bulk term and a surface term, both extensive. The latter only occurs when the transverse conductivity is nonzero and it is due to conducting surface states. Simulations on a model Hamiltonian validate our theory.

  12. An intrinsic mobility ceiling of Si bulk

    Garcia-Castello, Nuria; Prades, Joan Daniel; Cirera, Albert

    2011-01-01

    We compute by Density Functional Theory-Non Equilibrium Green Functions Formalism (DFT-NEGFF) the conductance of bulk Si along different crystallographic directions. We find a ceiling value for the intrinsic mobility of bulk silicon of $8.4\\cdot10^6 cm^2/V\\cdot s$. We suggest that this result is related to the lowest effective mass of the $$ direction.

  13. Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.

    Zhang Deshui

    2012-11-01

    Full Text Available Abstract Background Transferrin (TF plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF, and evaluate its suitability for biopharmaceutical applications. Result Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2 and Caco-2 human colon carcinoma cells (HTB-37, we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240 and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72, for supporting their proliferation, differentiation, and physiological function of antibody production. Conclusion The functional similarity between rice derived rhTF and native hTF in

  14. Multi-timescale modeling of activity-dependent metabolic coupling in the neuron-glia-vasculature ensemble.

    Jolivet, Renaud; Coggan, Jay S; Allaman, Igor; Magistretti, Pierre J

    2015-02-01

    Glucose is the main energy substrate in the adult brain under normal conditions. Accumulating evidence, however, indicates that lactate produced in astrocytes (a type of glial cell) can also fuel neuronal activity. The quantitative aspects of this so-called astrocyte-neuron lactate shuttle (ANLS) are still debated. To address this question, we developed a detailed biophysical model of the brain's metabolic interactions. Our model integrates three modeling approaches, the Buxton-Wang model of vascular dynamics, the Hodgkin-Huxley formulation of neuronal membrane excitability and a biophysical model of metabolic pathways. This approach provides a template for large-scale simulations of the neuron-glia-vasculature (NGV) ensemble, and for the first time integrates the respective timescales at which energy metabolism and neuronal excitability occur. The model is constrained by relative neuronal and astrocytic oxygen and glucose utilization, by the concentration of metabolites at rest and by the temporal dynamics of NADH upon activation. These constraints produced four observations. First, a transfer of lactate from astrocytes to neurons emerged in response to activity. Second, constrained by activity-dependent NADH transients, neuronal oxidative metabolism increased first upon activation with a subsequent delayed astrocytic glycolysis increase. Third, the model correctly predicted the dynamics of extracellular lactate and oxygen as observed in vivo in rats. Fourth, the model correctly predicted the temporal dynamics of tissue lactate, of tissue glucose and oxygen consumption, and of the BOLD signal as reported in human studies. These findings not only support the ANLS hypothesis but also provide a quantitative mathematical description of the metabolic activation in neurons and glial cells, as well as of the macroscopic measurements obtained during brain imaging. PMID:25719367

  15. Activity-dependent endogenous taurine release facilitates excitatory neurotransmission in the neocortical marginal zone of neonatal rats

    Taizhe eQian

    2014-02-01

    Full Text Available In the developing cerebral cortex, the marginal zone (MZ, consisting of early-generated neurons such as Cajal-Retzius cells, plays an important role in cell migration and lamination. There is accumulating evidence of widespread excitatory neurotransmission mediated by γ-aminobutyric acid (GABA in the MZ. Cajal-Retzius cells express not only GABAA receptors but also α2/β subunits of glycine receptors, and exhibit glycine receptor-mediated depolarization due to high [Cl−]i. However, the physiological roles of glycine receptors and their endogenous agonists during neurotransmission in the MZ are yet to be elucidated. To address this question, we performed optical imaging from the MZ using the voltage-sensitive dye JPW1114 on tangential neocortical slices of neonatal rats. A single electrical stimulus evoked an action-potential-dependent optical signal that spread radially over the MZ. The amplitude of the signal was not affected by glutamate receptor blockers, but was suppressed by either GABAA or glycine receptor antagonists. Combined application of both antagonists nearly abolished the signal. Inhibition of Na+, K+-2Cl− cotransporter by 20 µM bumetanide reduced the signal, indicating that this transporter contributes to excitation. Analysis of the interstitial fluid obtained by microdialysis from tangential neocortical slices with high-performance liquid chromatography revealed that GABA and taurine, but not glycine or glutamate, were released in the MZ in response to the electrical stimulation. The ambient release of taurine was reduced by the addition of a voltage-sensitive Na+ channel blocker. Immunohistochemistry and immunoelectron microscopy indicated that taurine was stored both in Cajal-Retzius and non-Cajal-Retzius cells in the MZ, but was not localized in presynaptic structures. Our results suggest that activity-dependent non-synaptic release of endogenous taurine facilitates excitatory neurotransmission through activation of

  16. Activity-dependent release of endogenous brain-derived neurotrophic factor from primary sensory neurons detected by ELISA in situ.

    Balkowiec, A; Katz, D M

    2000-10-01

    To define activity-dependent release of endogenous brain-derived neurotrophic factor (BDNF), we developed an in vitro model using primary sensory neurons and a modified ELISA, termed ELISA in situ. Dissociate cultures of nodose-petrosal ganglion cells from newborn rats were grown in wells precoated with anti-BDNF antibody to capture released BDNF, which was subsequently detected using conventional ELISA. Conventional ELISA alone was unable to detect any increase in BDNF concentration above control values following chronic depolarization with 40 mM KCl for 72 hr. However, ELISA in situ demonstrated a highly significant increase in BDNF release, from 65 pg/ml in control to 228 pg/ml in KCl-treated cultures. The efficacy of the in situ assay appears to be related primarily to rapid capture of released BDNF that prevents BDNF binding to the cultured cells. We therefore used this approach to compare BDNF release from cultures exposed for 30 min to either continuous depolarization with elevated KCl or patterned electrical field stimulation (50 biphasic rectangular pulses of 25 msec, at 20 Hz, every 5 sec). Short-term KCl depolarization was completely ineffective at evoking any detectable release of BDNF, whereas patterned electrical stimulation increased extracellular BDNF levels by 20-fold. In addition, the magnitude of BDNF release was dependent on stimulus pattern, with high-frequency bursts being most effective. These data indicate that the optimal stimulus profile for BDNF release resembles that of other neuroactive peptides. Moreover, our findings demonstrate that BDNF release can encode temporal features of presynaptic neuronal activity. PMID:11007900

  17. Multi-timescale Modeling of Activity-Dependent Metabolic Coupling in the Neuron-Glia-Vasculature Ensemble

    Jolivet, Renaud

    2015-02-26

    Glucose is the main energy substrate in the adult brain under normal conditions. Accumulating evidence, however, indicates that lactate produced in astrocytes (a type of glial cell) can also fuel neuronal activity. The quantitative aspects of this so-called astrocyte-neuron lactate shuttle (ANLS) are still debated. To address this question, we developed a detailed biophysical model of the brain’s metabolic interactions. Our model integrates three modeling approaches, the Buxton-Wang model of vascular dynamics, the Hodgkin-Huxley formulation of neuronal membrane excitability and a biophysical model of metabolic pathways. This approach provides a template for large-scale simulations of the neuron-glia-vasculature (NGV) ensemble, and for the first time integrates the respective timescales at which energy metabolism and neuronal excitability occur. The model is constrained by relative neuronal and astrocytic oxygen and glucose utilization, by the concentration of metabolites at rest and by the temporal dynamics of NADH upon activation. These constraints produced four observations. First, a transfer of lactate from astrocytes to neurons emerged in response to activity. Second, constrained by activity-dependent NADH transients, neuronal oxidative metabolism increased first upon activation with a subsequent delayed astrocytic glycolysis increase. Third, the model correctly predicted the dynamics of extracellular lactate and oxygen as observed in vivo in rats. Fourth, the model correctly predicted the temporal dynamics of tissue lactate, of tissue glucose and oxygen consumption, and of the BOLD signal as reported in human studies. These findings not only support the ANLS hypothesis but also provide a quantitative mathematical description of the metabolic activation in neurons and glial cells, as well as of the macroscopic measurements obtained during brain imaging.

  18. Cytotoxicity mechanism of α-MMC in normal liver cells through LRP1 mediated endocytosis and JNK activation.

    Wang, Ling; Shen, Fubing; Zhang, Min; He, Qianchuan; Zhao, Hui; Yu, Xiaoping; Yang, Shuxia; Liu, Yang; Deng, Nianhua; Zheng, Juecun; Zhu, Lixia; Liu, Xiaolan

    2016-05-16

    Alpha-momorcharin (α-MMC), a type I ribosome-inactivating protein isolated from Momordica charantia, is a potential drug candidate with strong anti-tumor activity. However, α-MMC has a severe hepatotoxicity when applied in vivo, which may greatly hinders its use in clinic in the future. The biological mechanism of hepatotoxicity induced by α-MMC is largely unknown, especially the mechanism by which α-MMC enters the hepatocytes. In this study, we investigated α-MMC-induced cytotoxicity in normal liver L02 cell line as well as the mechanism underlying it. As expected, α-MMC is more toxic in L02 cells than in various normal cells from other organs. The cytotoxic effect of α-MMC on L02 cells is found to be mediated through cell apoptosis as detected by flow cytometry and fluorescence microscopy. Importantly, α-MMC was shown to bind to a specific receptor on cell membrane, as the density of the cell membrane receptor is closely related to both the amount of α-MMC endocytosed and the cytotoxicity in different cell lines. By using LRP1 competitive inhibitor α2-M or siRNA targeting LRP1, we further identified that LRP1 protein served as the membrane receptor for α-MMC. Both α2-M and siRNA targeting LRP1 can significantly inhibit α-MMC's endocytosis as well as its cytotoxicity in L02 cells. In addition, it was found that α-MMC can activate the JNK signalling pathways via LRP1 in L02 cells. As JNK activation often leads to cell apoptosis, the activation of JNK may play an important role in α-MMC-induced cytotoxicity. To our knowledge, this is the first report showing that LRP1 mediates the cytotoxicity of α-MMC through (1) endocytosis and induced apoptosis and (2) the activation of the JNK pathway. Our findings shed light on the fundamental mechanism of hepatotoxicity of α-MMC and offer reference to understand its mechanism of lymphocytotoxicity and neurotoxicity. PMID:27262837

  19. RNASEK Is a V-ATPase-Associated Factor Required for Endocytosis and the Replication of Rhinovirus, Influenza A Virus, and Dengue Virus

    Jill M. Perreira

    2015-08-01

    Full Text Available Human rhinovirus (HRV causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase proton pump. RNASEK is required for endocytosis, and its depletion produces enlarged clathrin-coated pits (CCPs at the cell surface. These enlarged CCPs contain endocytic cargo and are bound by the scissioning GTPase, DNM2. Loss of RNASEK alters the localization of multiple V-ATPase subunits and lowers the levels of the ATP6AP1 subunit. Together, our results show that RNASEK closely associates with the V-ATPase and is required for its function; its loss prevents the early events of endocytosis and the replication of multiple pathogenic viruses.

  20. Module 13: Bulk Packaging Shipments by Highway

    The Hazardous Materials Modular Training Program provides participating United States Department of Energy (DOE) sites with a basic, yet comprehensive, hazardous materials transportation training program for use onsite. This program may be used to assist individual program entities to satisfy the general awareness, safety training, and function specific training requirements addressed in Code of Federal Regulation (CFR), Title 49, Part 172, Subpart H -- ''Training.'' Module 13 -- Bulk Packaging Shipments by Highway is a supplement to the Basic Hazardous Materials Workshop. Module 13 -- Bulk Packaging Shipments by Highway focuses on bulk shipments of hazardous materials by highway mode, which have additional or unique requirements beyond those addressed in the ten module core program. Attendance in this course of instruction should be limited to those individuals with work experience in transporting hazardous materials utilizing bulk packagings and who have completed the Basic Hazardous Materials Workshop or an equivalent. Participants will become familiar with the rules and regulations governing the transportation by highway of hazardous materials in bulk packagings and will demonstrate the application of these requirements through work projects and examination

  1. Development of superconductor bulk for superconductor bearing

    Kim, Chan Joong; Jun, Byung Hyuk; Park, Soon Dong (and others)

    2008-08-15

    Current carrying capacity is one of the most important issues in the consideration of superconductor bulk materials for engineering applications. There are numerous applications of Y-Ba-Cu-O (YBCO) bulk superconductors e.g. magnetic levitation train, flywheel energy storage system, levitation transportation, lunar telescope, centrifugal device, magnetic shielding materials, bulk magnets etc. Accordingly, to obtain YBCO materials in the form of large, single crystals without weak-link problem is necessary. A top seeded melt growth (TSMG) process was used to fabricate single crystal YBCO bulk superconductors. The seeded and infiltration growth (IG) technique was also very promising method for the synthesis of large, single-grain YBCO bulk superconductors with good superconducting properties. 5 wt.% Ag doped Y211 green compacts were sintered at 900 .deg. C {approx} 1200 .deg.C and then a single crystal YBCO was fabricated by an infiltration method. A refinement and uniform distribution of the Y211 particles in the Y123 matrix were achieved by sintering the Ag-doped samples. This enhancement of the critical current density was ascribable to a fine dispersion of the Y211 particles, a low porosity and the presence of Ag particles. In addition, we have designed and manufactured large YBCO single domain with levitation force of 10-13 kg/cm{sup 2} using TSMG processing technique.

  2. Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana

    Paudyal, R; Jamaluddin, A.; Warren, JP; Doyle, SM; Robert, S.; Warriner, SL; Baker, A.

    2014-01-01

    Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins,...

  3. Human epidermal Langerhans cells cointernalize by receptor-mediated endocytosis "nonclassical" major histocompatibility complex class I molecules (T6 antigens) and class II molecules (HLA-DR antigens).

    Hanau, D.; Fabre, M.; Schmitt, D A; Garaud, J C; Pauly, G; Tongio, M M; Mayer, S.; Cazenave, J. P.

    1987-01-01

    HLA-DR and T6 surface antigens are expressed only by Langerhans cells and indeterminate cells in normal human epidermis. We have previously demonstrated that T6 antigens are internalized in Langerhans cells and indeterminate cells by receptor-mediated endocytosis. This process is induced by the binding of BL6, a monoclonal antibody directed against T6 antigens. In the present study, using a monoclonal antibody directed against HLA-DR antigens, on human epidermal cells in suspension, we show t...

  4. RNASEK Is a V-ATPase-Associated Factor Required for Endocytosis and the Replication of Rhinovirus, Influenza A Virus, and Dengue Virus

    Jill M. Perreira; Aaron M. Aker; George Savidis; Christopher R. Chin; William M. McDougall; Jocelyn M. Portmann; Paul Meraner; Miles C. Smith; Motiur Rahman; Richard E. Baker; Annick Gauthier; Michael Franti; Abraham L. Brass

    2015-01-01

    Human rhinovirus (HRV) causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase) proton pump. RNASEK is required for endocytosis, and its de...

  5. Luminescent Ruthenium(II) Complex Bearing Bipyridine and N-Heterocyclic Carbene-based C∧N∧C Pincer Ligand for Live-Cell Imaging of Endocytosis

    Tsui, Wai-Kuen; Chung, Lai-Hon; Wong, Matthew Man-Kin; Tsang, Wai-Him; Lo, Hoi-Shing; Liu, Yaxiang; Leung, Chung-Hang; Ma, Dik-Lung; Chiu, Sung-Kay; Wong, Chun-Yuen

    2015-03-01

    Luminescent ruthenium(II)-cyanide complex with N-heterocyclic carbene pincer ligand C∧N∧C = 2,6-bis(1-butylimidazol-2-ylidene)pyridine and 2,2'-bipyridine (bpy) shows minimal cytotoxicity to both human breast carcinoma cell (MCF-7) and human retinal pigmented epithelium cell (RPE) in a wide range of concentration (0.1-500 μM), and can be used for the luminescent imaging of endocytosis of the complex in these cells.

  6. Reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibirets1

    1983-01-01

    Temperature-induced structural changes in the cortical region of the garland cell, which is considered to be active in endocytosis, were investigated in a temperature-sensitive, single gene mutant of Drosophila melanogaster, shibirets1 (shi) and wild-type (Oregon-R). At 19 degrees C, both shi and wild type showed similar structural features: an irregularly extended network of labyrinthine channels, coated pits and vesicles, tubular elements and alpha vacuoles. Tannic acid (TA) impregnation sh...

  7. Intestinal Cell Tight Junctions Limit Invasion of Candida albicans through Active Penetration and Endocytosis in the Early Stages of the Interaction of the Fungus with the Intestinal Barrier.

    Marianne Goyer

    Full Text Available C. albicans is a commensal yeast of the mucous membranes in healthy humans that can also cause disseminated candidiasis, mainly originating from the digestive tract, in vulnerable patients. It is necessary to understand the cellular and molecular mechanisms of the interaction of C. albicans with enterocytes to better understand the basis of commensalism and pathogenicity of the yeast and to improve the management of disseminated candidiasis. In this study, we investigated the kinetics of tight junction (TJ formation in parallel with the invasion of C. albicans into the Caco-2 intestinal cell line. Using invasiveness assays on Caco-2 cells displaying pharmacologically altered TJ (i.e. differentiated epithelial cells treated with EGTA or patulin, we were able to demonstrate that TJ protect enterocytes against invasion of C. albicans. Moreover, treatment with a pharmacological inhibitor of endocytosis decreased invasion of the fungus into Caco-2 cells displaying altered TJ, suggesting that facilitating access of the yeast to the basolateral side of intestinal cells promotes endocytosis of C. albicans in its hyphal form. These data were supported by SEM observations of differentiated Caco-2 cells displaying altered TJ, which highlighted membrane protrusions engulfing C. albicans hyphae. We furthermore demonstrated that Als3, a hypha-specific C. albicans invasin, facilitates internalization of the fungus by active penetration and induced endocytosis by differentiated Caco-2 cells displaying altered TJ. However, our observations failed to demonstrate binding of Als3 to E-cadherin as the trigger mechanism of endocytosis of C. albicans into differentiated Caco-2 cells displaying altered TJ.

  8. The endocytosis gene END3 is essential for the glucose-induced rapid decline of small vesicles in the extracellular fraction in Saccharomyces cerevisiae

    Chiang, Hui-Ling; Giardina, Bennett J.; Stein, Kathryn

    2014-01-01

    Background: Protein secretion is a fundamental process in all living cells. Gluconeogenic enzymes are secreted when Saccharomyces cerevisiae are grown in media containing low glucose. However, when cells are transferred to media containing high glucose, they are internalized. We investigated whether or not gluconeogenic enzymes were associated with extracellular vesicles in glucose-starved cells. We also examined the role that the endocytosis gene END3 plays in the internalization of extracel...

  9. The endocytosis of epidermal growth factor in A431 cells: A pH of microenvironment and the dynamics of receptor complex dissociation

    The endocytosis and intracellular fate of epidermal growth factor (EGF) were studied in A431 cells. After 15-20 min of internalization at 37 degree C, rhodomaine-labeled [125-I] EGF (EGR-Rh) accumulated into large juxtanuclear compartment consisting of closely related vesicles. This structure was shown to be localized in the para-Golgi region. Fluorescein-labeled transferrin (Tr-FITC) was observed in the same region when added to the cell simultaneously with EGF-Rh. Using microscopy spectrofluorometer, the authors determined that the Tr-FITC-containing para-Golgi structures have a pH of 6.1±0.3 while lysosomes containing dextran-fluorescein have a pH of 5.0±0.2. To study the dynamics of EGF-receptor dissociation during endocytosis a mild detergent treatment of living cells was used for extraction of an intracellular receptor-unbound EGF. These results suggest that EGF remains associated with receptors during endocytosis in A431 cells until it is transferred to lysosomes where the pH of the EGF microenvironment is dropped to 5. A prolonged presence of EGF-receptor complexes in the para-Golgi region might be of importance in mitotic signaling

  10. Fission yeast arrestin-related trafficking adaptor, Arn1/Any1, is ubiquitinated by Pub1 E3 ligase and regulates endocytosis of Cat1 amino acid transporter

    Akio Nakashima

    2014-05-01

    Full Text Available The Tsc1–Tsc2 complex homologous to human tuberous sclerosis complex proteins governs amino acid uptake by regulating the expression and intracellular distribution of amino acid transporters in Schizosaccharomyces pombe. Here, we performed a genetic screening for molecules that are involved in amino acid uptake and found Arn1 (also known as Any1. Arn1 is homologous to ART1, an arrestin-related trafficking adaptor (ART in Saccharomyces cerevisiae, and contains a conserved arrestin motif, a ubiquitination site, and two PY motifs. Overexpression of arn1+ confers canavanine resistance on cells, whereas its disruption causes hypersensitivity to canavanine. We also show that Arn1 regulates endocytosis of the Cat1 amino acid transporter. Furthermore, deletion of arn1+ suppresses a defect of amino acid uptake and the aberrant Cat1 localization in tsc2Δ. Arn1 interacts with and is ubiquitinated by the Pub1 ubiquitin ligase, which is necessary to regulate Cat1 endocytosis. Cat1 undergoes ubiquitinations on lysine residues within the N-terminus, which are mediated, in part, by Arn1 to determine Cat1 localization. Correctively, Arn1 is an ART in S. pombe and contributes to amino acid uptake through regulating Cat1 endocytosis in which Tsc2 is involved.

  11. A diphoton resonance from bulk RS

    Csáki, Csaba; Randall, Lisa

    2016-07-01

    Recent LHC data hinted at a 750 GeV mass resonance that decays into two photons. A significant feature of this resonance is that its decays to any other Standard Model particles would be too low to be detected so far. Such a state has a compelling explanation in terms of a scalar or a pseudoscalar that is strongly coupled to vector states charged under the Standard Model gauge groups. Such a scenario is readily accommodated in bulk RS with a scalar localized in the bulk away from but close to the Higgs. Turning this around, we argue that a good way to find the elusive bulk RS model might be the search for a resonance with prominent couplings to gauge bosons.

  12. Micro benchtop optics by bulk silicon micromachining

    Lee, Abraham P.; Pocha, Michael D.; McConaghy, Charles F.; Deri, Robert J.

    2000-01-01

    Micromachining of bulk silicon utilizing the parallel etching characteristics of bulk silicon and integrating the parallel etch planes of silicon with silicon wafer bonding and impurity doping, enables the fabrication of on-chip optics with in situ aligned etched grooves for optical fibers, micro-lenses, photodiodes, and laser diodes. Other optical components that can be microfabricated and integrated include semi-transparent beam splitters, micro-optical scanners, pinholes, optical gratings, micro-optical filters, etc. Micromachining of bulk silicon utilizing the parallel etching characteristics thereof can be utilized to develop miniaturization of bio-instrumentation such as wavelength monitoring by fluorescence spectrometers, and other miniaturized optical systems such as Fabry-Perot interferometry for filtering of wavelengths, tunable cavity lasers, micro-holography modules, and wavelength splitters for optical communication systems.

  13. Bulk amorphous Mg-based alloys

    Pryds, Nini

    The present paper describes the preparation and properties of bulk amorphous quarternary Mg-based alloys and the influence of additional elements on the ability of the alloy to form bulk amorphous. The main goal is to find a Mg-based alloy system which shows both high strength to weight ratio and a...... low glass transition temperature. The alloys were prepared by using a relatively simple technique, i.e. rapid cooling of the melt in a copper wedge mould. The essential structural changes that are achieved by going from the amorphous to the crystalline state through the supercooled liquid state are...... discussed in this paper. On the basis of these measurements phase diagrams of the different systems were constructed. Finally, it is demonstrated that when pressing the bulk amorphous alloy onto a metallic dies at temperatures within the supercooled liquid region, the alloy faithfully replicates the surface...

  14. Orchestrating Bulk Data Movement in Grid Environments

    Vazhkudai, SS

    2005-01-25

    Data Grids provide a convenient environment for researchers to manage and access massively distributed bulk data by addressing several system and transfer challenges inherent to these environments. This work addresses issues involved in the efficient selection and access of replicated data in Grid environments in the context of the Globus Toolkit{trademark}, building middleware that (1) selects datasets in highly replicated environments, enabling efficient scheduling of data transfer requests; (2) predicts transfer times of bulk wide-area data transfers using extensive statistical analysis; and (3) co-allocates bulk data transfer requests, enabling parallel downloads from mirrored sites. These efforts have demonstrated a decentralized data scheduling architecture, a set of forecasting tools that predict bandwidth availability within 15% error and co-allocation architecture, and heuristics that expedites data downloads by up to 2 times.

  15. Bulk Comptonization by Turbulence in Accretion Disks

    Kaufman, J

    2016-01-01

    Radiation pressure dominated accretion discs around compact objects may have turbulent velocities that greatly exceed the electron thermal velocities within the disc. Bulk Comptonization by the turbulence may therefore dominate over thermal Comptonization in determining the emergent spectrum. Bulk Comptonization by divergenceless turbulence is due to radiation viscous dissipation only. It can be treated as thermal Comptonization by solving the Kompaneets equation with an equivalent "wave" temperature, which is a weighted sum over the power present at each scale in the turbulent cascade. Bulk Comptonization by turbulence with non-zero divergence is due to both pressure work and radiation viscous dissipation. Pressure work has negligible effect on photon spectra in the limit of optically thin turbulence, and in this limit radiation viscous dissipation alone can be treated as thermal Comptonization with a temperature equivalent to the full turbulent power. In the limit of extremely optically thick turbulence, ra...

  16. Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen

    G. Dennis Sprott

    2003-01-01

    Full Text Available Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis of archaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation.

  17. Elucidating the mechanisms of nickel compound uptake: A review of particulate and nano-nickel endocytosis and toxicity

    Nickel (Ni) is a worldwide pollutant and contaminant that humans are exposed to through various avenues resulting in multiple toxic responses — most alarming is its clear carcinogenic nature. A variety of particulate Ni compounds persist in the environment and can be distinguished by characteristics such as solubility, structure, and surface charge. These characteristics influence cellular uptake and toxicity. Some particulate forms of Ni are carcinogenic and are directly and rapidly endocytized by cells. A series of studies conducted in the 1980s observed this process, and we have reanalyzed the results of these studies to help elucidate the molecular mechanism of particulate Ni uptake. Originally the process of uptake observed was described as phagocytosis, however in the context of recent research we hypothesize that the process is macropinocytosis and/or clathrin mediated endocytosis. Primary considerations in determining the route of uptake here include calcium dependence, particle size, and inhibition through temperature and pharmacological approaches. Particle characteristics that influenced uptake include size, charge, surface characteristics, and structure. This discussion is relevant in the context of nanoparticle studies and the emerging interest in nano-nickel (nano-Ni), where toxicity assessments require a clear understanding of the parameters of particulate uptake and where establishment of such parameters is often obscured through inconsistencies across experimental systems. In this regard, this review aims to carefully document one system (particulate nickel compound uptake) and characterize its properties.

  18. Interactions between calcium oxalate monohydrate crystals and Madin-Darby canine kidney cells: endocytosis and cell proliferation.

    Kohjimoto, Y; Ebisuno, S; Tamura, M; Ohkawa, T

    1996-01-01

    The present investigation was designed to study the biological responses in cultures of Madin-Darby canine kidney (MDCK) cells exposed to calcium oxalate monohydrate (COM) crystals, the most common type of urinary crystals. The addition of COM crystals significantly accelerated the multiplication of MDCK cells and significantly activated the cell viability. After exposure of MDCK cells to COM crystals, scanning electron microscopy revealed that some crystals adhered to the plasma membrane and others were endocytosed by the cell. This cellular uptake of crystals was time dependent from 1 to 8 h and showed a specificity according to crystal type. However, the endocytosis of aggregated COM crystals was less marked than that of non-aggregated crystals. Pre-treatment with each of the glycosaminoglycans (sodium pentosan polysulphate, heparin, and chondroitin sulphate C) produced a significant reduction of the cellular uptake of COM crystals, suggesting that these glycosaminoglycans may play some critical roles in preventing the cellular uptake of crystals. Although investigation in further detail is necessary, we speculate that these crystal-cell interactions, that is, the cellular uptake of crystals and cell proliferation, may be among the earliest processes in the formation of kidney stones. PMID:8873377

  19. Polypeptide hormone receptor phosphorylation: is there a role in receptor-mediated endocytosis of human growth hormone

    To determine whether receptor phosphorylation is a critical step in the internalization of polypeptide hormones and their receptors, the authors have studied a model system wherein insulin stimulates phosphorylation of its receptor and is also internalized. Using insulin as a positive control, they found that it stimulated a partially purified plasma membrane preparation of IM-9 lymphocytes to autophosphorylate its receptor and to catalyze the phosphorylation of a tyrosine-containing substrate. The human GH (hGH) receptor of the IM-9 lymphocytes, when coupled to [125I]iodo-hGH, migrated as a 140,000-dalton protein on polyacrylamide gel electrophoresis. This protein, in contrast to the insulin receptor, was not phosphorylated by the addition of hGH, nor did hGH stimulate this preparation to phosphorylate the tyrosine-containing substrate poly-(GluNa,Tyr)4:1, casein, or histone f2b under a variety of conditions. The authors conclude that receptor phosphorylation is not a critical intermediate in the receptor-mediated endocytosis of hGH and probably other polypeptide hormones and growth factors

  20. NECAP 1 regulates AP-2 interactions to control vesicle size, number, and cargo during clathrin-mediated endocytosis.

    Brigitte Ritter

    2013-10-01

    Full Text Available AP-2 is the core-organizing element in clathrin-mediated endocytosis. During the formation of clathrin-coated vesicles, clathrin and endocytic accessory proteins interact with AP-2 in a temporally and spatially controlled manner, yet it remains elusive as to how these interactions are regulated. Here, we demonstrate that the endocytic protein NECAP 1, which binds to the α-ear of AP-2 through a C-terminal WxxF motif, uses an N-terminal PH-like domain to compete with clathrin for access to the AP-2 β2-linker, revealing a means to allow AP-2-mediated coordination of accessory protein recruitment and clathrin polymerization at sites of vesicle formation. Knockdown and functional rescue studies demonstrate that through these interactions, NECAP 1 and AP-2 cooperate to increase the probability of clathrin-coated vesicle formation and to control the number, size, and cargo content of the vesicles. Together, our data demonstrate that NECAP 1 modulates the AP-2 interactome and reveal a new layer of organizational control within the endocytic machinery.

  1. Endocytosis-inducer adhesins produced by enteropathogenic serogroups of Escherichia coli participate on bacterial attachment to infant enterocytes

    João Ramos Costa Andrade

    1987-03-01

    Full Text Available Enteropathogenic E. coli (EPEC infection of Hep-2 cells preoceeds through bacterial attachment to cell surface and internalization of adhered bacteria. EPEC attachment is a prerequisite for cell infection and is mediated by adhesins that recognize carbohydrate-containing receptors on cell membrane. Such endocytosis-inducer adhesins (EIA also promote EPEC binding to infant enterocytes, suggesting that EIA may have an important role on EPEC gastroenteritis.A infecção de células Hep-2 por E. coli enteropatogênicas (ECEP implica na aderência bacteriana e posterior interiorização dos microrganismos aderidos por um mecanismo de endocitose. A aderência das ECEP é pré-requisito para a infecção e é mediada por adesinas que reconhecem receptores inibidos por certas oses na membrana celular. Tais "adesinas indutoras da endocitose" (AIE também promovem a ligação bacteriana a enterócitos obtidos do intestino delgado de lactente, sugerindo que as AIE possam desempenhar algum papel nas diarréias causadas por ECEP.

  2. Elucidating the mechanisms of nickel compound uptake: A review of particulate and nano-nickel endocytosis and toxicity

    Muñoz, Alexandra; Costa, Max, E-mail: Max.Costa@nyumc.org

    2012-04-01

    Nickel (Ni) is a worldwide pollutant and contaminant that humans are exposed to through various avenues resulting in multiple toxic responses — most alarming is its clear carcinogenic nature. A variety of particulate Ni compounds persist in the environment and can be distinguished by characteristics such as solubility, structure, and surface charge. These characteristics influence cellular uptake and toxicity. Some particulate forms of Ni are carcinogenic and are directly and rapidly endocytized by cells. A series of studies conducted in the 1980s observed this process, and we have reanalyzed the results of these studies to help elucidate the molecular mechanism of particulate Ni uptake. Originally the process of uptake observed was described as phagocytosis, however in the context of recent research we hypothesize that the process is macropinocytosis and/or clathrin mediated endocytosis. Primary considerations in determining the route of uptake here include calcium dependence, particle size, and inhibition through temperature and pharmacological approaches. Particle characteristics that influenced uptake include size, charge, surface characteristics, and structure. This discussion is relevant in the context of nanoparticle studies and the emerging interest in nano-nickel (nano-Ni), where toxicity assessments require a clear understanding of the parameters of particulate uptake and where establishment of such parameters is often obscured through inconsistencies across experimental systems. In this regard, this review aims to carefully document one system (particulate nickel compound uptake) and characterize its properties.

  3. Introduction of caveolae structural proteins into the protozoan Toxoplasma results in the formation of heterologous caveolae but not caveolar endocytosis.

    Bao Lige

    Full Text Available Present on the plasma membrane of most metazoans, caveolae are specialized microdomains implicated in several endocytic and trafficking mechanisms. Caveolins and the more recently discovered cavins are the major protein components of caveolae. Previous studies reported that caveolar invaginations can be induced de novo on the surface of caveolae-negative mammalian cells upon heterologous expression of caveolin-1. However, it remains undocumented whether other components in the transfected cells participate in caveolae formation. To address this issue, we have exploited the protozoan Toxoplasma as a heterologous expression system to provide insights into the minimal requirements for caveogenesis and caveolar endocytosis. Upon expression of caveolin-1, Toxoplasma accumulates prototypical exocytic caveolae 'precursors' in the cytoplasm. Toxoplasma expressing caveolin-1 alone, or in conjunction with cavin-1, neither develops surface-located caveolae nor internalizes caveolar ligands. These data suggest that the formation of functional caveolae at the plasma membrane in Toxoplasma and, by inference in all non-mammalian cells, requires effectors other than caveolin-1 and cavin-1. Interestingly, Toxoplasma co-expressing caveolin-1 and cavin-1 displays an impressive spiraled network of membranes containing the two proteins, in the cytoplasm. This suggests a synergistic activity of caveolin-1 and cavin-1 in the morphogenesis and remodeling of membranes, as illustrated for Toxoplasma.

  4. Mannose 6-phosphate receptor-dependent endocytosis of lysosomal enzymes is increased in sulfatide-storing kidney cells.

    Klein, Diana; Yaghootfam, Afshin; Matzner, Ullrich; Koch, Bettina; Braulke, Thomas; Gieselmann, Volkmar

    2009-01-01

    Metachromatic leukodystrophy is a lysosomal disorder caused by the deficiency of arylsulfatase A (ASA). This leads to the storage of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide) in various cell types, such as renal tubular cells. Examination of mannose 6-phosphate receptor (MPR300)-dependent endocytosis revealed that uptake of lysosomal enzymes is more than two-fold increased in sulfatide-storing kidney cells. Expression of MPR300 and its internalization rate is increased in these cells, whereas the recycling rate is decreased. Similar alterations can be found for the transferrin receptor, indicating that sulfatide storage leads to a general alteration of the endocytotic pathway. These data allow calculating that the endosomal pool from which receptors can recycle is 1.4- to 2-fold increased in lipid-storing cells. Immunocytochemistry demonstrates that the MPR300 in lipid-storing cells does not co-localize with accumulated sulfatide, suggesting that the kinetics of internalization and recycling appear to be altered indirectly. PMID:19007310

  5. Fluorescently labeled methyl-beta-cyclodextrin enters intestinal epithelial Caco-2 cells by fluid-phase endocytosis.

    Ferenc Fenyvesi

    Full Text Available Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10(-8 cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

  6. Differential Use of the C-Type Lectins L-SIGN and DC-SIGN for Phlebovirus Endocytosis.

    Léger, Psylvia; Tetard, Marilou; Youness, Berthe; Cordes, Nicole; Rouxel, Ronan N; Flamand, Marie; Lozach, Pierre-Yves

    2016-06-01

    Bunyaviruses represent a growing threat to humans and livestock globally. The receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely unidentified and poorly characterized. DC-SIGN is a C-type lectin highly expressed on dermal dendritic cells that has been found to act as an authentic entry receptor for many phleboviruses (Bunyaviridae), including Rift Valley fever virus (RVFV), Toscana virus (TOSV) and Uukuniemi virus (UUKV). We found that these phleboviruses can exploit another C-type lectin, L-SIGN, for infection. L-SIGN shares 77% sequence homology with DC-SIGN and is expressed on liver sinusoidal endothelial cells. L-SIGN is required for UUKV binding but not for virus internalization. An endocytosis-defective mutant of L-SIGN was still able to mediate virus uptake and infection, indicating that L-SIGN acts as an attachment receptor for phleboviruses rather than an endocytic receptor. Our results point out a fundamental difference in the use of the C-type lectins L-SIGN and DC-SIGN by UUKV to enter cells, although both proteins are closely related in terms of molecular structure and biological function. This study sheds new light on the molecular mechanisms by which phleboviruses target the liver and also highlights the added complexity in virus-receptor interactions beyond attachment. PMID:26990254

  7. CaV1.3 as pacemaker channels in adrenal chromaffin cells: specific role on exo- and endocytosis?

    Comunanza, Valentina; Marcantoni, Andrea; Vandael, David H; Mahapatra, Satyajit; Gavello, Daniela; Carabelli, Valentina; Carbone, Emilio

    2010-01-01

    Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca (2+) -dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (CaV1.2 and CaV1.3), CaV1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using CaV1 .3 (-/-) KO mice and the AP-clamp it has been possible to resolve the time course of CaV1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells CaV1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca (2+) currents and activate BK channels confers to CaV1.3 the unique feature of driving Ca (2+) loading during long interspike intervals and, possibly, to control the Ca (2+) -dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling. PMID:21084859

  8. Tobacco Mosaic Virus-Based 1D Nanorod-Drug Carrier via the Integrin-Mediated Endocytosis Pathway.

    Tian, Ye; Gao, Sijia; Wu, Man; Liu, Xiangxiang; Qiao, Jing; Zhou, Quan; Jiang, Shidong; Niu, Zhongwei

    2016-05-01

    For cancer therapy, viruses have been utilized as excellent delivery vehicles because of their facile transfection efficiency in their host cells. However, their inherent immunogenicity has become the major obstacle for their translation into approved pharmaceuticals. Herein, we utilized rodlike plant virus, tobacco mosaic virus (TMV), which is nontoxic to mammals and mainly infects tobacco species, as anticancer nanorod-drug vector for cancer therapy study. Doxorubicin (DOX) was installed in the inner cavity of TMV by hydrazone bond, which enabled the pH-sensitive drug release property. Conjugation of cyclic Arg-Gly-Asp (cRGD) on the surface of TMV can enhance HeLa cell uptake of the carrier via the integrin-mediated endocytosis pathway. Comparing with free DOX, the cRGD-TMV-hydra-DOX vector had similar cell growth inhibition and much higher apoptosis efficiency on HeLa cells. Moreover, the in vivo assay assumed that cRGD-TMV-hydra-DOX behaved similar antitumor efficiency but much lower side effect on HeLa bearing Balb/c-nu mice. Our work provides novel insights into potentially cancer therapy based on rodlike plant viral nanocarriers. PMID:27062971

  9. Synthesis of Bulk Superconducting Magnesium Diboride

    Margie Olbinado

    2002-06-01

    Full Text Available Bulk polycrystalline superconducting magnesium diboride, MgB2, samples were successfully prepared via a one-step sintering program at 750°C, in pre Argon with a pressure of 1atm. Both electrical resistivity and magnetic susceptibility measurements confirmed the superconductivity of the material at 39K, with a transition width of 5K. The polycrystalline nature, granular morphology, and composition of the sintered bulk material were confirmed using X-ray diffractometry (XRD, scanning electron microscopy (SEM, and energy dispersive X-ray analysis (EDX.

  10. Thermal relics in cosmology with bulk viscosity

    In this paper we discuss some consequences of cosmological models in which the primordial cosmic matter is described by a relativistic imperfect fluid. The latter takes into account the dissipative effects (bulk viscosity) arising from different cooling rates of the fluid components in the expanding Universe. We discuss, in particular, the effects of the bulk viscosity on Big Bang Nucleosynthesis and on the thermal relic abundance of particles, looking at recent results of PAMELA experiment. The latter has determined an anomalous excess of positron events, which cannot be explained by conventional cosmology and particle physics. (orig.)

  11. Remedial investigations for quarry bulk wastes

    The US Department of Energy proposes, as a separate operable unit of the Weldon Spring Site Remedial Action Project, to remove contaminated bulk wastes from the Weldon Spring quarry and transport them approximately four miles to the chemical plant portion of the raffinate pits and chemical plant area. The wastes will be held in temporary storage prior to the record of decision for the overall remedial action. The decision on the ultimate disposal of these bulk wastes will be included as part of the decision for management of the waste materials resulting from remedial action activities at the raffinate pits and chemical plant area. 86 refs., 71 figs., 83 tabs

  12. Radiation-hardened bulk CMOS technology

    The evolutionary development of a radiation-hardened bulk CMOS technology is reviewed. The metal gate hardened CMOS status is summarized, including both radiation and reliability data. The development of a radiation-hardened bulk silicon gate process which was successfully implemented to a commercial microprocessor family and applied to a new, radiation-hardened, LSI standard cell family is also discussed. The cell family is reviewed and preliminary characterization data is presented. Finally, a brief comparison of the various radiation-hardened technologies with regard to performance, reliability, and availability is made

  13. Thermal relics in cosmology with bulk viscosity

    Iorio, A. [Charles University in Prague, Faculty of Mathematics and Physics, Prague (Czech Republic); Lambiase, G. [Universita di Salerno, Dipartimento di Fisica E.R. Caianiello, Fisciano (Italy); INFN, Gruppo Collegato di Salerno, Fisciano (Italy)

    2015-03-01

    In this paper we discuss some consequences of cosmological models in which the primordial cosmic matter is described by a relativistic imperfect fluid. The latter takes into account the dissipative effects (bulk viscosity) arising from different cooling rates of the fluid components in the expanding Universe. We discuss, in particular, the effects of the bulk viscosity on Big Bang Nucleosynthesis and on the thermal relic abundance of particles, looking at recent results of PAMELA experiment. The latter has determined an anomalous excess of positron events, which cannot be explained by conventional cosmology and particle physics. (orig.)

  14. Bulk Entropy in Loop Quantum Gravity

    Livine, Etera R; Terno, Daniel R.

    2007-01-01

    In the framework of loop quantum gravity (LQG), having quantum black holes in mind, we generalize the previous boundary state counting (gr-qc/0508085) to a full bulk state counting. After a suitable gauge fixing we are able to compute the bulk entropy of a bounded region (the "black hole") with fixed boundary. This allows us to study the relationship between the entropy and the boundary area in details and we identify a holographic regime of LQG where the leading order of the entropy scales w...

  15. Bulk Entropy in Loop Quantum Gravity

    Livine, Etera R

    2007-01-01

    In the framework of loop quantum gravity (LQG), having quantum black holes in mind, we generalize the previous boundary state counting (gr-qc/0508085) to a full bulk state counting. After a suitable gauge fixing we are able to compute the bulk entropy of a bounded region (the "black hole") with fixed boundary. This allows us to study the relationship between the entropy and the boundary area in details and we identify a holographic regime of LQG where the leading order of the entropy scales with the area. We show that in this regime we can fine tune the factor between entropy and area without changing the Immirzi parameter.

  16. 网格蛋白介导型内吞作用与广谱抗病毒药%Clathrin-mediated endocytosis and broad-spectrum antivirals

    周丽; 杨晓虹; 徐利保; 肖军海

    2013-01-01

    Viral disease is a serious threat for human health. Alhough plenty of antiviral agents have been used in clinical treatment, many viruses are resistant to them via virus mutation. And novel harmful viruses emerge in endlessly. So research and development of new antiviral drugs, especially the agents that are of broad-spectrum antiviral activity is particularly important. Clathrin-mediated endocytosis is the most common pathway used by viruses and pathogens for entering host cells. The inhibitors of clathrin-me-diated endocytosis may block the entry of viruses and pathogens, thus prevent viral infection. For the inhibitors do not directly act on the virus itself, it is hard to induce virus mutations which produce drug resistance. Clathrin-mediated endocytosis is the potential target of broad-spectrum antiviral agents in recent years. This review focuses on the mechanism of virus entry through clathrin-mediated endocytosis, the recent advances of clathrin-mediated endocytosis inhibitors and their potential applications in broad-spectrum antiviral therapeutics field.%病毒性疾病对人类的健康造成了巨大的威胁,虽然有很多药物用于临床治疗,但由于病毒的易变异性,对现有的抗病毒药物极易产生耐药性,而新发病毒又层出不穷,因此研发新的抗病毒药物尤其是广谱且不易产生耐药的抗病毒药物对于病毒性疾病的治疗就显得尤为重要.网格蛋白介导型内吞是许多病毒和病原体进入宿主细胞的主要途径,抑制此途径可阻断病毒进入宿主细胞,从而抑制病毒感染,由于其功能和机制与病毒自身无关,不易产生耐药,是近年来广谱抗病毒药物的潜在作用靶标.本文结合国内外最新研究报道,简要综述了病毒依赖网格蛋白介导型内吞入胞的机制,网格蛋白介导型内吞抑制剂的研究现状,及其在广谱抗病毒药物研发中的潜在应用前景.

  17. THE OPTIMIZATION OF PLUSH YARNS BULKING PROCESS

    VINEREANU Adam

    2014-05-01

    Full Text Available This paper presents the experiments that were conducted on the installation of continuous bulking and thermofixing “SUPERBA” type TVP-2S for optimization of the plush yarns bulking process. There were considered plush yarns Nm 6.5/2, made of the fibrous blend of 50% indigenous wool sort 41 and 50% PES. In the first stage, it performs a thermal treatment with a turboprevaporizer at a temperature lower than thermofixing temperature, at atmospheric pressure, such that the plush yarns - deposed in a freely state on a belt conveyor - are uniformly bulking and contracting. It was followed the mathematical modeling procedure, working with a factorial program, rotatable central composite type, and two independent variables. After analyzing the parameters that have a direct influence on the bulking degree, there were selected the pre-vaporization temperature (coded x1,oC and the velocity of belt inside pre-vaporizer (coded x 2, m/min. As for the dependent variable, it was chosen the plush yarn diameter (coded y, mm. There were found the coordinates of the optimal point, and then this pair of values was verified in practice. These coordinates are: x1optim= 90oC and x 2optim= 6.5 m/min. The conclusion is that the goal was accomplished: it was obtained a good cover degree f or double-plush carpets by reducing the number of tufts per unit surface.

  18. Determining the bulk strength of strata

    The bulk rock strength of strata is determined by logging a bore-hole with an instrument to obtain a neutron-neutron log of the hole and determining in conjunction with the lithology of the strata the strength by applying an inverse relation formula. (author)

  19. Realistic anomaly mediation with bulk gauge fields

    We present a simple general framework for realistic models of supersymmetry breaking driven by anomaly mediation. We consider a 5-dimensional 'brane universe' where the visible and hidden sectors are localized on different branes, and the standard model gauge bosons propagate in the bulk. In this framework there can be charged scalar messengers that have contact interactions with the hidden sector, either localized in the hidden sector or in the bulk. These scalars obtain soft masses that feed into visible sector scalar masses at two loop order via bulk gauge interactions. This contribution is automatically flavor-blind, and can be naturally positive. If the messengers are in the bulk this contribution is automatically the same order of magnitude as the anomaly mediated contribution, independent of the brane spacing. If the messengers are localized to a brane the two effects are of the same order for relatively small brane spacings. The gaugino masses and A terms are determined completely by anomaly mediation. In order for anomaly mediation to dominate over radion mediation the radion must be is stabilized in a manner that preserves supersymmetry, with supergravity effects included. We show that this occurs in simple models. We also show that the mu problem can be solved by the vacuum expectation value of a singlet in this framework. (author)

  20. Crevice chemistry estimation from bulk water chemistry

    Since the first PWR plant in Japan started commercial operation in 1970, 22 plants are running in Japan as of the end of 1994. The main purpose of secondary water chemistry control is to minimize the corrosion possibility of the secondary system equipment, especially steam generators (SG). To achieve this objective, much effort has been concentrated on improving secondary water chemistry control. As a result of this effort, the recent secondary water chemistry in Japanese plants is well maintained in every stage of operation. However, to ensure and improve the reliability of SG, it is necessary to control crevice environments, which are located at tube/tube support plate intersections and under the sludge pile on the tube sheet. According to recent crevice monitoring examination results, the concentration behavior impurities in SG bulk water at the crevice is different for each species, and SG bulk water and crevice chemical compositions are not always equal. From these results, to control the crevice chemistry, improving bulk water chemistry control methods and a new type of molar ratio control index is needed. This paper introduces a brief summary of a recent crevice chemistry evaluation technique and bulk water chemistry control method, which is employed for crevice chemistry control, based on crevice monitoring examination results

  1. A Stereoscopic Look into the Bulk

    Czech, Bartlomiej; McCandlish, Samuel; Mosk, Benjamin; Sully, James

    2016-01-01

    We present the foundation for a holographic dictionary with depth perception. The dictionary consists of natural CFT operators whose duals are simple, diffeomorphism-invariant bulk operators. The CFT operators of interest are the "OPE blocks," contributions to the OPE from a single conformal family. In holographic theories, we show that the OPE blocks are dual at leading order in 1/N to integrals of effective bulk fields along geodesics or homogeneous minimal surfaces in anti-de Sitter space. One widely studied example of an OPE block is the modular Hamiltonian, which is dual to the fluctuation in the area of a minimal surface. Thus, our operators pave the way for generalizing the Ryu-Takayanagi relation to other bulk fields. Although the OPE blocks are non-local operators in the CFT, they admit a simple geometric description as fields in kinematic space--the space of pairs of CFT points. We develop the tools for constructing local bulk operators in terms of these non-local objects. The OPE blocks also allow ...

  2. Teaching Advanced SQL Skills: Text Bulk Loading

    Olsen, David; Hauser, Karina

    2007-01-01

    Studies show that advanced database skills are important for students to be prepared for today's highly competitive job market. A common task for database administrators is to insert a large amount of data into a database. This paper illustrates how an up-to-date, advanced database topic, namely bulk insert, can be incorporated into a database…

  3. Longitudinal bulk a coustic mass sensor

    Hales, Jan Harry; Teva, Jordi; Boisen, Anja;

    2009-01-01

    Design, fabrication and characterization, in terms of mass sensitivity, is presented for a polycrystalline silicon longitudinal bulk acoustic cantilever. The device is operated in air at 51 MHz, resulting in a mass sensitivity of 100 HZ/fg (1 fg = 10{su−15 g). The initial characterization is cond...

  4. Bulk sulfur (S) deposition in China

    Liu, Lei; Zhang, Xiuying; Wang, Shanqian; Zhang, Wuting; Lu, Xuehe

    2016-06-01

    A systematic dataset of an observation network on a national scale has been organized to investigate the spatial distribution of bulk sulfur (S) deposition (Sdep) throughout China during 2000-2013, representing by far the most detailed data set to track the bulk sulfur deposition throughout China since 2000. Such a dataset is needed for ecosystem studies and for developing emission control policies. Bulk Sdep values showed great variations, ranging from 2.17 to 70.55 kg ha-1 y-1, with an average of 22.99 kg ha-1 y-1. The average rate of bulk Sdep located in East Coastal region (35.97 kg ha-1 y-1), Middle Yangtze region (57.90 kg ha-1 y-1), Middle Yellow River region (23.42 kg ha-1 y-1), North Coastal region (42.19 kg ha-1 y-1), Northeast region (34.28 kg ha-1 y-1), South Coastal region (36.97 kg S ha-1 y-1), Southwest region (33.85 kg ha-1 y-1) was 4.50, 7.24, 2.93, 5.28, 4.29, 4.63 and 4.24 times than that in Northwest region (7.99 kg ha-1 y-1). Bulk Sdep over China was mainly from fossil fuel combustion (76.96%), biomass burning (7.64%), crust (6.22%), aged sea salt (5.48%) and agriculture (3.68%). A systematic observation network on a national scale should be established to conduct a long-term monitoring atmospheric Sdep (including wet and dry deposition), based on exiting ecological stations administrated by different departments in China.

  5. 27 CFR 1.90 - Distilled spirits in bulk.

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Distilled spirits in bulk..., NONINDUSTRIAL USE OF DISTILLED SPIRITS AND WINE, BULK SALES AND BOTTLING OF DISTILLED SPIRITS Bulk Sales and Bottling of Distilled Spirits Warehouse Receipts § 1.90 Distilled spirits in bulk. By the terms of the...

  6. Bulk solitary waves in elastic solids

    Samsonov, A. M.; Dreiden, G. V.; Semenova, I. V.; Shvartz, A. G.

    2015-10-01

    A short and object oriented conspectus of bulk solitary wave theory, numerical simulations and real experiments in condensed matter is given. Upon a brief description of the soliton history and development we focus on bulk solitary waves of strain, also known as waves of density and, sometimes, as elastic and/or acoustic solitons. We consider the problem of nonlinear bulk wave generation and detection in basic structural elements, rods, plates and shells, that are exhaustively studied and widely used in physics and engineering. However, it is mostly valid for linear elasticity, whereas dynamic nonlinear theory of these elements is still far from being completed. In order to show how the nonlinear waves can be used in various applications, we studied the solitary elastic wave propagation along lengthy wave guides, and remarkably small attenuation of elastic solitons was proven in physical experiments. Both theory and generation for strain soliton in a shell, however, remained unsolved problems until recently, and we consider in more details the nonlinear bulk wave propagation in a shell. We studied an axially symmetric deformation of an infinite nonlinearly elastic cylindrical shell without torsion. The problem for bulk longitudinal waves is shown to be reducible to the one equation, if a relation between transversal displacement and the longitudinal strain is found. It is found that both the 1+1D and even the 1+2D problems for long travelling waves in nonlinear solids can be reduced to the Weierstrass equation for elliptic functions, which provide the solitary wave solutions as appropriate limits. We show that the accuracy in the boundary conditions on free lateral surfaces is of crucial importance for solution, derive the only equation for longitudinal nonlinear strain wave and show, that the equation has, amongst others, a bidirectional solitary wave solution, which lead us to successful physical experiments. We observed first the compression solitary wave in the

  7. Live imaging provides new insights on dynamic F-actin filopodia and differential endocytosis during myoblast fusion in Drosophila.

    Shruti Haralalka

    Full Text Available The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs, and cell adhesion molecules Kin-of-IrreC (Kirre and Sticks-and-stones (Sns on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and

  8. Lysosomal and endosomal heterogeneity in the liver: A comparison of the intracellular pathways of endocytosis in rat liver cells

    Air-filled albumin microspheres, asialoorosomucoid and formaldehyde-treated serum albumin are selectively taken up by endocytosis in rat liver Kupffer cells, parenchymal cells and endothelial cells, respectively. Intracellular transport and degradation of endocytosed material were studied by subcellular fractionation in sucrose and Nycodenz gradients after intravenous injection of the ligand. By using ligands labeled with 125I-tyramine-cellobiose, the subcellular distribution of labeled degradation products can be studied because they are trapped at the site of formation. The results show that the kinetics of intracellular transport are different in hepatic parenchymal, endothelial and Kupffer cells. In endothelial cells, the ligand is associated with two types of endosomes during the first minutes after internalization and then is transferred rapidly to the lysosomes. In parenchymal cells, 125I-tyramine-cellobiose-asialoorosomucoid was located in a relatively slowly sedimenting vesicle during the first minute after internalization and subsequently in denser endosomes. Degradation of 125I-tyramine-cellobiose-asialoorosomucoid in parenchymal cells started later than that of 125I-tyramine-cellobiose-formaldehyde-treated serum albumin in endothelial cells. Furthermore, the ligand seemed to be transferred relatively slowly from endosomes to lysosomes, and most of the undegraded ligand was in the endosomes. The rate-limiting step of proteolysis in parenchymal cells is probably the transport from endosomes to lysosomes. In Kupffer cells, most 125I-tyramine-cellobiose-microspheres are found as undegraded material in very dense endosomes up to 3 hr after injection. After 20 hr, most of the ligand is degraded in lysosomes distributed at a lower density than the endosomes in Nycodenz and sucrose gradients

  9. Influence of the surface charge of PLGA nanoparticles on their in vitro genotoxicity, cytotoxicity, ROS production and endocytosis.

    Platel, Anne; Carpentier, Rodolphe; Becart, Elodie; Mordacq, Gwendoline; Betbeder, Didier; Nesslany, Fabrice

    2016-03-01

    With the ongoing commercialization of nanotechnology products, human exposure to nanoparticles (NPs) is set to increase dramatically and an evaluation of their potential adverse effects is essential. Surface charge, among other physico-chemicals parameters, is a key criterion that should be considered when using a definition for nanomaterials in a regulatory context. It has recently been recognized as an important factor in determining the toxicity of NPs; however, a complete understanding of the mechanisms involved is still lacking. In this context, the aim of the present study was to investigate the influence of the surface charge modification of NPs on in vitro toxicity assays. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles bearing different surface charges, positive(+), neutral(n) or negative(-), were synthesized. In vitro genotoxicity assays (micronucleus and comet assays) coupled with an assessment of cytotoxicity, were performed in different cell lines (L5178Y mouse lymphoma cells, TK6 human B-lymphoblastoid cells and 16HBE14o- human bronchial epithelial cells). Reactive oxygen species (ROS) production and endocytosis studies were also performed. Our results showed that PLGA(+) NPs were cytotoxic. They are endocytosed by the clathrin pathway and induced ROS in the three cell lines. They led to chromosomal aberrations without primary DNA damage in 16HBE14o- cells, suggesting that aneuploidy may be considered as an important biomarker when assessing the genotoxic potential of NPs. Moreover, 16HBE14o- cells seem to be more suitable for the in vitro screening of inhaled NPs than the regulatory L5178Y and TK6 cells. PMID:26487569

  10. Angelman Syndrome Protein Ube3a Regulates Synaptic Growth and Endocytosis by Inhibiting BMP Signaling in Drosophila.

    Li, Wenhua; Yao, Aiyu; Zhi, Hui; Kaur, Kuldeep; Zhu, Yong-Chuan; Jia, Mingyue; Zhao, Hui; Wang, Qifu; Jin, Shan; Zhao, Guoli; Xiong, Zhi-Qi; Zhang, Yong Q

    2016-05-01

    Altered expression of the E3 ubiquitin ligase UBE3A, which is involved in protein degradation through the proteasome-mediated pathway, is associated with neurodevelopmental and behavioral defects observed in Angelman syndrome (AS) and autism. However, little is known about the neuronal function of UBE3A and the pathogenesis of UBE3A-associated disorders. To understand the in vivo function of UBE3A in the nervous system, we generated multiple mutations of ube3a, the Drosophila ortholog of UBE3A. We found a significantly increased number of total boutons and satellite boutons in conjunction with compromised endocytosis in the neuromuscular junctions (NMJs) of ube3a mutants compared to the wild type. Genetic and biochemical analysis showed upregulation of bone morphogenetic protein (BMP) signaling in the nervous system of ube3a mutants. An immunochemical study revealed a specific increase in the protein level of Thickveins (Tkv), a type I BMP receptor, but not other BMP receptors Wishful thinking (Wit) and Saxophone (Sax), in ube3a mutants. Ube3a was associated with and specifically ubiquitinated lysine 227 within the cytoplasmic tail of Tkv, and promoted its proteasomal degradation in Schneider 2 cells. Negative regulation of Tkv by Ube3a was conserved in mammalian cells. These results reveal a critical role for Ube3a in regulating NMJ synapse development by repressing BMP signaling. This study sheds new light onto the neuronal functions of UBE3A and provides novel perspectives for understanding the pathogenesis of UBE3A-associated disorders. PMID:27232889

  11. Integration of bulk piezoelectric materials into microsystems

    Aktakka, Ethem Erkan

    Bulk piezoelectric ceramics, compared to deposited piezoelectric thin-films, provide greater electromechanical coupling and charge capacity, which are highly desirable in many MEMS applications. In this thesis, a technology platform is developed for wafer-level integration of bulk piezoelectric substrates on silicon, with a final film thickness of 5-100microm. The characterized processes include reliable low-temperature (200°C) AuIn diffusion bonding and parylene bonding of bulk-PZT on silicon, wafer-level lapping of bulk-PZT with high-uniformity (+/-0.5microm), and low-damage micro-machining of PZT films via dicing-saw patterning, laser ablation, and wet-etching. Preservation of ferroelectric and piezoelectric properties is confirmed with hysteresis and piezo-response measurements. The introduced technology offers higher material quality and unique advantages in fabrication flexibility over existing piezoelectric film deposition methods. In order to confirm the preserved bulk properties in the final film, diaphragm and cantilever beam actuators operating in the transverse-mode are designed, fabricated and tested. The diaphragm structure and electrode shapes/sizes are optimized for maximum deflection through finite-element simulations. During tests of fabricated devices, greater than 12microm PP displacement is obtained by actuation of a 1mm2 diaphragm at 111kHz with properties of bulk-PZT5A are mostly preserved without any necessity of repolarization. Three generations of resonant vibration energy harvesters are designed, simulated and fabricated to demonstrate the competitive performance of the new fabrication process over traditional piezoelectric deposition systems. An unpackaged PZT/Si unimorph harvester with 27mm3 active device volume produces up to 205microW at 1.5g/154Hz. The prototypes have achieved the highest figure-of-merits (normalized-power-density x bandwidth) amongst previously reported inertial energy harvesters. The fabricated energy harvester is

  12. Premelting at Defects Within Bulk Colloidal Crystals

    Alsayed, A. M.; Islam, M. F.; Zhang, J.; Collings, P. J.; Yodh, A. G.

    2005-08-01

    Premelting is the localized loss of crystalline order at surfaces and defects at temperatures below the bulk melting transition. It can be thought of as the nucleation of the melting process. Premelting has been observed at the surfaces of crystals but not within. We report observations of premelting at grain boundaries and dislocations within bulk colloidal crystals using real-time video microscopy. The crystals are equilibrium close-packed, three-dimensional colloidal structures made from thermally responsive microgel spheres. Particle tracking reveals increased disorder in crystalline regions bordering defects, the amount of which depends on the type of defect, distance from the defect, and particle volume fraction. Our observations suggest that interfacial free energy is the crucial parameter for premelting in colloidal and atomic-scale crystals.

  13. Fully antisymmetrised dynamics for bulk fermion systems

    The neutron star's crust and mantel are typical examples of non-uniform bulk systems with spacial localisations. When modelling such systems at low temperatures, as is the case in the crust, one has to work with antisymmetrised many-body states to get the correct fermion behaviour. Fermionic molecular dynamics, which works with an antisymmetrised product of localised wave packets, should be an appropriate choice. Implementing periodic boundary conditions into the fermionic molecular dynamics formalism would allow the study of the neutron star's crust as a bulk quantum system. Unfortunately, the antisymmetrisation is a non-local entanglement which reaches far out of the periodically repeated unit cell. In this proceeding, we give a brief overview how periodic boundary conditions and fermionic molecular dynamics can be combined without truncating the long-range many-body correlation induced by the antisymmetry of the many-body state.

  14. NPP bulk equipment dismantling problems and experience

    NPP bulk equipment dismantling problems and experience are summarized. 'ECOMET-S' JSC is shown as one of the companies which are able to make NPPs industrial sites free from stored bulk equipment with its further utilization. 'ECOMET-S' JSC is the Russian Federation sole specialized metallic LLW (MLLW) treatment and utilization facility. Company's main objectives are waste predisposal volume reduction and treatment for the unrestricted release as a scrap. Leningrad NPP decommissioned main pumps and moisture separators/steam super heaters dismantling results are presented. Prospective fragmentation technologies (diamond and electro-erosive cutting) testing results are described. The electro-erosive cutting machine designed by 'ECOMET-S' JSC is presented. The fragmentation technologies implementation plans for nuclear industry are presented too. (author)

  15. Bulk viscous cosmology with causal transport theory

    We consider cosmological scenarios originating from a single imperfect fluid with bulk viscosity and apply Eckart's and both the full and the truncated Müller-Israel-Stewart's theories as descriptions of the non-equilibrium processes. Our principal objective is to investigate if the dynamical properties of Dark Matter and Dark Energy can be described by a single viscous fluid and how such description changes when a causal theory (Müller-Israel-Stewart's, both in its full and truncated forms) is taken into account instead of Eckart's non-causal one. To this purpose, we find numerical solutions for the gravitational potential and compare its behaviour with the corresponding ΛCDM case. Eckart's and the full causal theory seem to be disfavoured, whereas the truncated theory leads to results similar to those of the ΛCDM model for a bulk viscous speed in the interval 10−11 || cb2 ∼−8

  16. Bulk band gaps in divalent hexaborides

    Denlinger, Jonathan; Clack, Jules A.; Allen, James W.; Gweon, Gey-Hong; Poirier, Derek M.; Olson, Cliff G.; Sarrao, John L.; Bianchi, Andrea D.; Fisk, Zachary

    2002-08-01

    Complementary angle-resolved photoemission and bulk-sensitive k-resolved resonant inelastic x-ray scattering of divalent hexaborides reveal a >1 eV X-point gap between the valence and conduction bands, in contradiction to the band overlap assumed in several models of their novel ferromagnetism. This semiconducting gap implies that carriers detected in transport measurements arise from defects, and the measured location of the bulk Fermi level at the bottom of the conduction band implicates boron vacancies as the origin of the excess electrons. The measured band structure and X-point gap in CaB6 additionally provide a stringent test case for proper inclusion of many-body effects in quasi-particle band calculations.

  17. Induction detection of concealed bulk banknotes

    Bulk cash smuggling is a serious issue that has grown in volume in recent years. By building on the magnetic characteristics of paper currency, induction sensing is found to be capable of quickly detecting large masses of banknotes. The results show that this method is effective in detecting bulk cash through concealing materials such as plastics, cardboards, fabrics and aluminum foil. The significant difference in the observed phase between the received signals caused by conducting materials and ferrite compounds, found in banknotes, provides a good indication that this process can overcome the interference by metal objects in a real sensing application. This identification strategy has the potential to not only detect the presence of banknotes, but also the number, while still eliminating false positives caused by metal objects

  18. Surface-Bulk Vibrational Correlation Spectroscopy.

    Roy, Sandra; Covert, Paul A; Jarisz, Tasha A; Chan, Chantelle; Hore, Dennis K

    2016-05-01

    Homo- and heterospectral correlation analysis are powerful methods for investigating the effects of external influences on the spectra acquired using distinct and complementary techniques. Nonlinear vibrational spectroscopy is a selective and sensitive probe of surface structure changes, as bulk molecules are excluded on the basis of symmetry. However, as a result of this exquisite specificity, it is blind to changes that may be occurring in the solution. We demonstrate that correlation analysis between surface-specific techniques and bulk probes such as infrared absorption or Raman scattering may be used to reveal additional details of the adsorption process. Using the adsorption of water and ethanol binary mixtures as an example, we illustrate that this provides support for a competitive binding model and adds new insight into a dimer-to-bilayer transition proposed from previous experiments and simulations. PMID:27058265

  19. Bulk metamaterials: Design, fabrication and characterization

    Andryieuski, Andrei; Malureanu, Radu; Alabastri, Alessandro;

    2009-01-01

    Bulk metamaterials claim a lot of attention worldwide. We report about our activity and advances in design, fabrication and characterization of metal-dielectric composites with three-dimensional lattices. The nomenclature of designs exhibiting negative index behaviour in the near infrared include......-layers-thick polymer woodpile photonic crystal. Characterization of such samples before and after metal deposition in the 700 nm 1700 nm range exposes some unpredictable features like an enhanced broadband transmission, which still waits to be explained.......Bulk metamaterials claim a lot of attention worldwide. We report about our activity and advances in design, fabrication and characterization of metal-dielectric composites with three-dimensional lattices. The nomenclature of designs exhibiting negative index behaviour in the near infrared includes...

  20. Multilayer Integrated Film Bulk Acoustic Resonators

    Zhang, Yafei

    2013-01-01

    Multilayer Integrated Film Bulk Acoustic Resonators mainly introduces the theory, design, fabrication technology and application of a recently developed new type of device, multilayer integrated film bulk acoustic resonators, at the micro and nano scale involving microelectronic devices, integrated circuits, optical devices, sensors and actuators, acoustic resonators, micro-nano manufacturing, multilayer integration, device theory and design principles, etc. These devices can work at very high frequencies by using the newly developed theory, design, and fabrication technology of nano and micro devices. Readers in fields of IC, electronic devices, sensors, materials, and films etc. will benefit from this book by learning the detailed fundamentals and potential applications of these advanced devices. Prof. Yafei Zhang is the director of the Ministry of Education’s Key Laboratory for Thin Films and Microfabrication Technology, PRC; Dr. Da Chen was a PhD student in Prof. Yafei Zhang’s research group.

  1. Nuclear Matter Bulk Parameter Scales and Correlations

    We study the arising of correlations among some isovector bulk parameters in nonrelativistic and relativistic hadronic mean-field models. For the former, we investigate correlations in the nonrelativistic (NR) limit of relativistic point-coupling models. We provide analytical correlations, for the NR limit model, between the symmetry energy and its derivatives, namely, the symmetry energy slope, curvature, skewness and fourth order derivative, discussing the conditions in which they are linear ones. We also show that some correlations presented in the NR limit model are reproduced for relativistic models presenting cubic and quartic self-interactions in its scalar field. As a direct application of such linear correlations, we remark its association with possible crossing points in the density dependence of the linearly correlated bulk parameter. (author)

  2. Brane plus Bulk Supersymmetry in Ten Dimensions

    Bergshoeff, E A; Ortín, Tomas; Roest, D; Van Proeyen, A

    2001-01-01

    We discuss a generalized form of IIA/IIB supergravity depending on all R-R potentials C^(p) (p=0,1,...,9) as the effective field theory of Type IIA/IIB superstring theory. For the IIA case we explicitly break this R-R democracy to either p=5 which allows us to write a new bulk action that can be coupled to N=1 supersymmetric brane actions. The case of 8-branes is studied in detail using the new bulk & brane action. The supersymmetric negative tension branes without matter excitations can be viewed as orientifolds in the effective action. These D8-branes and O8-planes are fundamental in Type I' string theory. A BPS 8-brane solution is given which satisfies the jump conditions on the wall. As an application of our results we derive a quantization of the mass parameter and the cosmological constant in string units.

  3. Melt Grown ZnO Bulk Crystals

    Schulz, Detlev; Ganschow, Steffen; Klimm, Detlef

    2009-01-01

    Bulk crystals of zinc oxide can be grown from the melt by a Bridgman technique under pressure. This new technology using an iridium crucible shows the potential to yield large single crystals of good crystalline perfection. Crystals with diameters up to 33 mm and a length of up to 50 mm have been demonstrated. The impurity content can be strongly reduced by using the crucibles repeatedly.

  4. Oscillatory Dynamical Switching System of Bulk Ferroelectrics

    M. N.A. Halif; S. Daud; Junaidah Osman

    2005-01-01

    This study gives a detailed account of calculation of the bulk ferroelectric (FE) oscillatory dynamical system switching to first and second-order phase transition, respectively. All the formalism is delineated in the framework of Landau free-energy expansion and Landau-Khalatnikov (LK) equation of motion where the effect of external energy may affray FE atoms similar to the spring damped oscillatory system. Here we scrutinized the switching properties from free-energy expansion and hysteresi...

  5. Effective Pure States for Bulk Quantum Computation

    Knill, Emanuel; Chuang, Isaac; Laflamme, Raymond

    1997-01-01

    In bulk quantum computation one can manipulate a large number of indistinguishable quantum computers by parallel unitary operations and measure expectation values of certain observables with limited sensitivity. The initial state of each computer in the ensemble is known but not pure. Methods for obtaining effective pure input states by a series of manipulations have been described by Gershenfeld and Chuang (logical labeling) and Cory et al. (spatial averaging) for the case of quantum computa...

  6. Superconducting RF cavities film of bulk

    Darriulat, Pierre

    1999-01-01

    The successful operation of LEP2 has demonstrated the feasibility of using on a large scale copper accelerating cavities coated with a thin superconducting niobium film. Yet other existing or planned installations such as CEBAF and TESLA, rely instead on the bulk niobium technology. The reason is a wide spread belief that the film technology would suffer from fundamental limitations preventing high gradients to be reached...

  7. Depleted Bulk Heterojunction Colloidal Quantum Dot Photovoltaics

    Barkhouse, D. Aaron R.

    2011-05-26

    The first solution-processed depleted bulk heterojunction colloidal quantum dot solar cells are presented. The architecture allows for high absorption with full depletion, thereby breaking the photon absorption/carrier extraction compromise inherent in planar devices. A record power conversion of 5.5% under simulated AM 1.5 illumination conditions is reported. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Material profile influences in bulk-heterojunctions

    Roehling, J.D.; Rochester, C.W.; Ro, H.W.; Wang, P.; Majewski, J; Batenburg, Joost; Arslan, I; Delongchamp, D.M.; Moulé, A.J.

    2014-01-01

    The morphology in mixed bulk-heterojunction films are compared using three different quantitative measurement techniques. We compare the vertical composition changes using high-angle annular dark-field scanning transmission electron microscopy with electron tomography and neutron and x-ray reflectometry. The three measurement techniques yield qualitatively comparable vertical concentration measurements. The presence of a metal cathode during thermal annealing is observed to alter the fulleren...

  9. Genome-Wide Analysis of MEF2 Transcriptional Program Reveals Synaptic Target Genes and Neuronal Activity-Dependent Polyadenylation Site Selection

    Flavell, Steven W; Kim, Tae-Kyung; Gray, Jesse M.; Harmin, David A.; Hemberg, Martin; Hong, Elizabeth J.; Markenscoff-Papadimitriou, Eirene; Bear, Daniel M.; Greenberg, Michael E.

    2008-01-01

    Although many transcription factors are known to control important aspects of neural development, the genome-wide programs that are directly regulated by these factors are not known. We have characterized the genetic program that is activated by MEF2, a key regulator of activity-dependent synapse development. These MEF2 target genes have diverse functions at synapses, revealing a broad role for MEF2 in synapse development. Several of the MEF2 targets are mutated in human neurological disorder...

  10. Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection

    Flavell, Steven W; Kim, Tae-Kyung; Gray, Jesse M.; Harmin, David A.; Hemberg, Martin; Hong, Elizabeth J.; Markenscoff-Papadimitriou, Eirene; Bear, Daniel M.; Greenberg, Michael E.

    2008-01-01

    Although many transcription factors are known to control important aspects of neural development, the genome-wide programs that are directly regulated by these factors are not known. We have characterized the genetic program that is activated by MEF2, a key regulator of activity-dependent synapse development. These MEF2 target genes have diverse functions at synapses, revealing a broad role for MEF2 in synapse development. Several of the MEF2 targets are mutated in human neurological disorder...

  11. Statistical Determination of Bulk Flow Motions

    Song, Yong-Seon; Nichol, Robert C; Miller, Christopher J

    2010-01-01

    We present here a new parameterization for the bulk motions of galaxies and clusters (in the linear regime) that can be measured statistically from the shape and amplitude of the two-dimensional two-point correlation function. We further propose the one-dimensional velocity dispersion (v_p) of the bulk flow as a complementary measure of redshift-space distortions, which is model-independent and not dependent on the normalisation method. As a demonstration, we have applied our new methodology to the C4 cluster catalogue constructed from Data Release Three (DR3) of the Sloan Digital Sky Survey. We find v_p=270^{+433}km/s (also consistent with v_p=0) for this cluster sample (at z=0.1), which is in agreement with that predicted for a WMAP5-normalised LCDM model (i.e., v_p(LCDM=203km/s). This measurement does not lend support to recent claims of excessive bulk motions (\\simeq1000 km/s) which appear in conflict with LCDM, although our large statistical error cannot rule them out. From the measured coherent evolutio...

  12. Superconducting State Parameters of Bulk Amorphous Alloys

    Aditya M. Vora

    2012-12-01

    Full Text Available Well recognized empty core (EMC pseudopotential of Ashcroft is used to investigate the superconducting state parameters viz; electron-phonon coupling strength λ, Coulomb pseudopotential μ*, transition temperature TC, isotope effect exponent α and effective interaction strength NOV of some (Ni33Zr671 – xVx (x = 0, 0.05, 0.1, 0.15 bulk amorphous alloys. We have incorporated five different types of local field correction functions, proposed by Hartree (H, Taylor (T, Ichimaru-Utsumi (IU, Farid et al. (F and Sarkar et al. (S to show the effect of exchange and correlation on the aforesaid properties. Very strong influence of the various exchange and correlation functions is concluded from the present study. The TC obtained from Sarkar et al. (S local field correction function are found an excellent agreement with available theoretical data. Quadratic TC equation has been proposed, which provide successfully the TC values of bulk amorphous alloys under consideration. Also, the present results are found in qualitative agreement with other such earlier reported data, which confirms the superconducting phase in the s bulk amorphous alloys.

  13. Bulk viscous cosmology in early Universe

    C P Singh

    2008-07-01

    The effect of bulk viscosity on the early evolution of Universe for a spatially homogeneous and isotropic Robertson-Walker model is considered. Einstein's field equations are solved by using `gamma-law' equation of state = ( - 1)ρ, where the adiabatic parameter gamma () depends on the scale factor of the model. The `gamma' function is defined in such a way that it describes a unified solution of early evolution of the Universe for inflationary and radiation-dominated phases. The fluid has only bulk viscous term and the coefficient of bulk viscosity is taken to be proportional to some power function of the energy density. The complete general solutions have been given through three cases. For flat space, power-law as well as exponential solutions are found. The problem of how the introduction of viscosity affects the appearance of singularity, is briefly discussed in particular solutions. The deceleration parameter has a freedom to vary with the scale factor of the model, which describes the accelerating expansion of the Universe.

  14. DEMONSTRATION BULK VITRIFICATION SYSTEM (DBVS) EXTERNAL REVIEW

    The Hanford mission to retrieve and immobilize 53 million gallons of radioactive waste from 177 underground storage tanks will be accomplished using a combination of processing by the waste treatment plant currently under construction, and a supplemental treatment that would process low-activity waste. Under consideration for this treatment is bulk vitrification, a versatile joule-heated melter technology which could be deployed in the tank farms. The Department proposes to demonstrate this technology under a Research, Development and Demonstration (RD and D) permit issued by the Washington State Department of Ecology using both non-radioactive simulant and blends of actual tank waste. From the demonstration program, data would be obtained on cost and technical performance to enable a decision on the potential use of bulk vitrification as the supplemental treatment technology for Hanford. An independent review by sixteen subject matter experts was conducted to assure that the technical basis of the demonstration facility design would be adequate to meet the objectives of the Demonstration Bulk Vitrification System (DBVS) program. This review explored all aspects of the program, including flowsheet chemistry, project risk, vitrification, equipment design and nuclear safety, and was carried out at a time when issues can be identified and corrected. This paper describes the mission need, review approach, technical recommendations and follow-on activities for the DBVS program

  15. Multiphase composites with extremal bulk modulus

    Gibiansky, L. V.; Sigmund, Ole

    2000-01-01

    This paper is devoted to the analytical and numerical study of isotropic elastic composites made of three or more isotropic phases. The ranges of their effective bulk and shear moduli are restricted by the Hashin-Shtrikman-Walpole (HSW) bounds. For two-phase composites, these bounds are attainabl...... isotropic three-dimensional three-phase composites with cylindrical inclusions of arbitrary cross-sections (plane strain problem) or transversely isotropic thin plates (plane stress or bending of plates problems). (C) 2000 Elsevier Science Ltd. All rights reserved.......This paper is devoted to the analytical and numerical study of isotropic elastic composites made of three or more isotropic phases. The ranges of their effective bulk and shear moduli are restricted by the Hashin-Shtrikman-Walpole (HSW) bounds. For two-phase composites, these bounds are attainable......, that is, there exist composites with extreme bulk and shear moduli. For multiphase composites, they may or may not be attainable depending on phase moduli and volume fractions. Sufficient conditions of attainability of the bounds and various previously known and new types of optimal composites are...

  16. Reluctance motors with bulk HTS material

    In recent years we have successfully designed, built and tested several reluctance motors with YBCO bulk material incorporated into the rotor, working at 77 K. Our last motor type SRE150 was tested up to 200 kW. The aim of our investigations is the construction of motors with extremely high power density and dynamics. In comparison to conventional motor types the advantage of HTS reluctance motors with respect to size and dynamics could be demonstrated. Some fields of possible future applications will be described. These motors show a significant improvement in performance using high quality HTS bulk elements in the rotor. Until now the motor parameters have been limited by the current density which could be obtained in the bulk material at 77 K and by the geometric dimensions of the segments available. Therefore we expect further improvements in the case of these materials. Since the total motor including stator and rotor is working at low temperature we have to optimize the windings and the magnetic circuit to these operation conditions. A new design of a 200 kW motor in order to achieve increased power density and the theoretical results of our calculations will be shown

  17. Bulk Comptonization by turbulence in accretion discs

    Kaufman, J.; Blaes, O. M.

    2016-06-01

    Radiation pressure dominated accretion discs around compact objects may have turbulent velocities that greatly exceed the electron thermal velocities within the disc. Bulk Comptonization by the turbulence may therefore dominate over thermal Comptonization in determining the emergent spectrum. Bulk Comptonization by divergenceless turbulence is due to radiation viscous dissipation only. It can be treated as thermal Comptonization by solving the Kompaneets equation with an equivalent `wave' temperature, which is a weighted sum over the power present at each scale in the turbulent cascade. Bulk Comptonization by turbulence with non-zero divergence is due to both pressure work and radiation viscous dissipation. Pressure work has negligible effect on photon spectra in the limit of optically thin turbulence, and in this limit radiation viscous dissipation alone can be treated as thermal Comptonization with a temperature equivalent to the full turbulent power. In the limit of extremely optically thick turbulence, radiation viscous dissipation is suppressed, and the evolution of local photon spectra can be understood in terms of compression and expansion of the strongly coupled photon and gas fluids. We discuss the consequences of these effects for self-consistently resolving and interpreting turbulent Comptonization in spectral calculations in radiation magnetohydrodynamic simulations of high luminosity accretion flows.

  18. The Etv1 transcription factor activity-dependently downregulates a set of genes controlling cell growth and differentiation in maturing cerebellar granule cells.

    Okazawa, Makoto; Abe, Haruka; Nakanishi, Shigetada

    2016-05-13

    In the early postnatal period, cerebellar granule cells exhibit an activity-dependent downregulation of a set of immaturation genes involved in cell growth and migration and are shifted to establishment of a mature network formation. Through the use of a granule cell culture and both pharmacological and RNA interference (siRNA) analyses, the present investigation revealed that the downregulation of these immaturation genes is controlled by strikingly unified signaling mechanisms that operate sequentially through the stimulation of AMPA and NMDA receptors, tetrodotoxin-sensitive Na(+) channels and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). This signaling cascade induces the Etv1 transcription factor, and knockdown of Etv1 by a siRNA technique prevented this activity-dependent downregulation of immaturation genes. Thus, taken into consideration the mechanism that controls the upregulation of maturation genes involved in synaptic formation, these results indicate that Etv1 orchestrates the activity-dependent regulation of both maturation and immaturation genes in developing granule cells and plays a key role in specifying the identity of mature granule cells in the cerebellum. PMID:27059140

  19. Clathrin light chain directs endocytosis by influencing the binding of the yeast Hip1R homologue, Sla2, to F-actin

    Boettner, Douglas R.; Friesen, Helena; Andrews, Brenda; Lemmon, Sandra K.

    2011-01-01

    The role of clathrin light chain (CLC) in clathrin-mediated endocytosis is not completely understood. Previous studies showed that the CLC N-terminus (CLC-NT) binds the Hip1/Hip1R/Sla2 family of membrane/actin–binding factors and that overexpression of the CLC-NT in yeast suppresses endocytic defects of clathrin heavy-chain mutants. To elucidate the mechanistic basis for this suppression, we performed synthetic genetic array analysis with a clathrin CLC-NT deletion mutation (clc1-Δ19-76). clc...

  20. The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment

    Hansen, Gert Helge; Delmas, B; Besnardeau, L;

    1998-01-01

    Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive...... and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as...... the site of fusion for TGEV....

  1. Detecting autophagy in Arabidopsis roots by membrane-permeable cysteine protease inhibitor E-64d and endocytosis tracer FM4–64

    Oh-ye, Yuumi; Inoue, Yuko; Moriyasu, Yuji

    2011-01-01

    Autophagy is the process by which cells degrade their own components in lysosomes or vacuoles. Autophagy in tobacco BY-2 cells cultured in sucrose-free medium takes place in formed, autolysosomes in the presence of a cysteine protease inhibitor. The autolysosomes in BY-2 cells are located in the endocytotic pathway and thus can be stained with fluorescent endocytosis marker FM4–64. In the present study, in order to detect autophagy in the root cells of Arabidopsis, we incubated root tips from...

  2. Delayed onset of positive feedback activation of Rab5 by Rabex-5 and Rabaptin-5 in endocytosis.

    Huaiping Zhu

    Full Text Available BACKGROUND: Rabex-5 is a guanine nucleotide exchange factor (GEF that specifically activates Rab5, i.e., converting Rab5-GDP to Rab5-GTP, through two distinct pathways to promote endosome fusion and endocytosis. The direct pathway involves a pool of membrane-associated Rabex-5 that targets to the membrane via an early endosomal targeting (EET domain. The indirect pathway, on the other hand, involves a cytosolic pool of Rabex-5/Rabaptin-5 complex. The complex is recruited to the membrane via Rabaptin-5 binding to Rab5-GTP, suggesting a positive feedback mechanism. The relationship of these two pathways for Rab5 activation in the cell is unclear. METHODOLOGY/PRINCIPAL FINDINGS: We dissect the relative contribution of each pathway to Rab5 activation via mathematical modeling and kinetic analysis in the cell. These studies show that the indirect pathway constitutes a positive feedback loop for converting Rab5-GDP to Rab5-GTP on the endosomal membrane and allows sensitive regulation of endosome fusion activity by the levels of Rab5 and Rabex-5 in the cell. The onset of this positive feedback effect, however, contains a threshold, which requires above endogenous levels of Rab5 or Rabex-5 in the cell. We term this novel phenomenon "delayed response". The presence of the direct pathway reduces the delay by increasing the basal level of Rab5-GTP, thus facilitates the function of the Rabex-5/Rabaptin-5-mediated positive feedback loop. CONCLUSION: Our data support the mathematical model. With the model's guidance, the data reveal the affinity of Rabex-5/Rabaptin-5/Rab5-GTP interaction in the cell, which is quantitatively related to the Rabex-5 concentration for the onset of the indirect positive feedback pathway. The presence of the direct pathway and increased Rab5 concentration can reduce the Rabex-5 concentration required for the onset of the positive feedback loop. Thus the direct and indirect pathways cooperate in the regulation of early endosome

  3. Evolution of bulk damage initiation in DKDP

    Carr, Christopher W.; McMillian, T. H.; Staggs, Mike C.; Radousky, Harry B.; Demos, Stavros G.

    2003-05-01

    We investigate the evolution of laser-induced damage initiated in the bulk of DKDP crystals using in-situ microscopy. Experimental results indicate that at peek fluences greater than 10 J/cm2, damage sites are formed with increasing number as a function of the laser fluence. Following plasma formation, cracks are observed which grow in size for tens of seconds after the termination of the laser pulse. Subsequent irradiation leads to modest increase in size only during the initial 2-5 pulses. Experimental results suggest that there is also relaxation of the stresses adjacent to a damage site for several hours after initial damage.

  4. Calculated Bulk Properties of the Actinide Metals

    Skriver, Hans Lomholt; Andersen, O. K.; Johansson, B.

    1978-01-01

    Self-consistent relativistic calculations of the electronic properties for seven actinides (Ac-Am) have been performed using the linear muffin-tin orbitals method within the atomic-sphere approximation. Exchange and correlation were included in the local spin-density scheme. The theory explains t...... the variation of the atomic volume and the bulk modulus through the 5f series in terms of an increasing 5f binding up to plutonium followed by a sudden localisation (through complete spin polarisation) in americium...

  5. Neutron moisture gage for bulk material

    Desing and operation of neutron moisture gage of bulk materials intended for the determination of moisture of coke, agglomerated charge, and iron ore concentrate in black metallurgy is described. The moisture gage operates both under ''measurement'' and ''calibration'' conditions, contains a fast neutron source, and two groups of slow neutron detectors. Technical and economic efficiency of the moisture gage utilization consists in the improved accuracy of moisture detection at the expense of more accurate calibration, optimum arrangement of the carriage in a hopper, and stabilization of detector temperature. The device service is also simplified

  6. Oscillatory Dynamical Switching System of Bulk Ferroelectrics

    M. N.A. Halif

    2005-01-01

    Full Text Available This study gives a detailed account of calculation of the bulk ferroelectric (FE oscillatory dynamical system switching to first and second-order phase transition, respectively. All the formalism is delineated in the framework of Landau free-energy expansion and Landau-Khalatnikov (LK equation of motion where the effect of external energy may affray FE atoms similar to the spring damped oscillatory system. Here we scrutinized the switching properties from free-energy expansion and hysteresis loop. The polarization and current switching appropriate to the estimated complete switching time, changing of temperature, electric field and damping are scrutinized and discussed.

  7. Active neutron multiplicity counting of bulk uranium

    This paper describes a new nondestructive assay technique being developed to assay bulk uranium containing kilogram quantities of 235U. The new technique uses neutron multiplicity analysis of data collected with a coincidence counter outfitted with AmLi neutron sources. We have calculated the expected neutron multiplicity count rate and assay precision for this technique and will report on its expected performance as a function of detector design characteristics, 235U sample mass, AmLi source strength, and source-to-sample coupling. 11 refs., 2 figs., 2 tabs

  8. The bulk composition of exo-planets

    Gaensicke, Boris; Dufour, Patrick; Farihi, Jay; Jura, Michael; Kilic, Mukremin; Melis, Carl; Veras, Dimitri; Xu, Siyi; Zuckerman, Ben

    2015-01-01

    Priorities in exo-planet research are rapidly moving from finding planets to characterizing their physical properties. Of key importance is their chemical composition, which feeds back into our understanding of planet formation. For the foreseeable future, far-ultraviolet spectroscopy of white dwarfs accreting planetary debris remains the only way to directly and accurately measure the bulk abundances of exo-planetary bodies. The exploitation of this method is limited by the sensitivity of HST, and significant progress will require a large-aperture space telescope with a high-throughput ultraviolet spectrograph.

  9. Heavy Quark Energy Loss and Bulk Physics

    We show that the low-pT data and high-pT data from RHIC and LHC give a contradictory picture of the relevant dynamics of a quark-gluon plasma. While the bulk observables imply a strongly coupled fluid best described by AdS/CFT the rare probes imply a weakly coupled gas best described using thermal pQCD. These conclusions are based on leading order theoretical calculations, and we speculate that more sophisticated calculations that include higher order effects might provide a resolution to the current puzzle

  10. New Class of Plastic Bulk Metallic Glass

    An intrinsic plastic Cu45Zr46Al7Ti2 bulk metallic glass (BMG) with high strength and superior compressive plastic strain of up to 32.5% was successfully fabricated by copper mold casting. The superior compressive plastic strain was attributed to a large amount of randomly distributed free volume induced by Ti minor alloying, which results in extensive shear band formation, branching, interaction and self-healing of minor cracks. The mechanism of plasticity presented here suggests that the creation of a large amount of free volume in BMGs by minor alloying or other methods might be a promising new way to enhance the plasticity of BMGs

  11. Improving the bulk data transfer experience

    Guok, Chin; Guok, Chin; Lee, Jason R.; Berket, Karlo

    2008-05-07

    Scientific computations and collaborations increasingly rely on the network to provide high-speed data transfer, dissemination of results, access to instruments, support for computational steering, etc. The Energy Sciences Network is establishing a science data network to provide user driven bandwidth allocation. In a shared network environment, some reservations may not be granted due to the lack of available bandwidth on any single path. In many cases, the available bandwidth across multiple paths would be sufficient to grant the reservation. In this paper we investigate how to utilize the available bandwidth across multiple paths in the case of bulk data transfer.

  12. Bulk hydrogen analysis by neutron scattering

    The efforts of the UCT-based group now working on bulk analysis using neutrons are directed mainly towards developing methods for detecting concealed explosive or other hidden contraband, such as drugs. The technique which we are developing, called fast neutron scattering analysis (FNSA), is based on the detection and measurement of hydrogen and other elements, particularly carbon, nitrogen and oxygen, by means of fast neutron scattering measurements. This report describes progress achieved over the past year in the work on fast neutron scattering analysis and in the development of detection techniques for plastic land mines

  13. Forming of bulk metallic glass microcomponents

    Wert, John A.; Thomsen, Christian; Jensen, Rune Debel;

    2009-01-01

    The present article considers forward extrusion, closed-die forging and backward extrusion processes for fabrication of individual microcomponents from two bulk metallic glass (BMG) compositions: Mg60Cu30Y10 and Zr44Cu40Ag8Al8. Two types of tooling were used in the present work: relatively massive...... die sets characteristic of cold forming operations for crystalline metals and lightweight die sets adapted to the special characteristics of BMGs. In addition to demonstrating that microcomponents of several geometries can be readily fabricated from BMGs, rheological properties are combined with...

  14. Characterization of the dominating bulk recombination in bulk-heterojunction blends using photoinduced absorption

    Sandén, S.; Wilson, N. M.; Sandberg, O. J.; Ã-sterbacka, R.

    2016-05-01

    In this work we show how to clarify the dominating bulk recombination in organic solar cells by using photoinduced absorption. We show how to use the intensity and frequency dependence of the in-phase and quadrature signals to obtain the effective reaction order. For trap-assisted recombination, we can show using a multiple trapping and retrapping model with an exponential tail-state distribution that a temperature dependent reaction order is obtained which allows for determination of the characteristic energy of the exponential distribution of trap-states. In the model system pBTTT:PC60BM, we show that trap-assisted recombination is the dominating bulk recombination in 1:1 blends with a characteristic energy of the exponential trap distribution E c h = 44 ± 5 meV. The 1:4 blend, on the other hand, shows temperature independent behavior in good agreement with a dominating 2D Langevin bulk recombination.

  15. A Batch Feeder for Inhomogeneous Bulk Materials

    Vislov, I. S.; Kladiev, S. N.; Slobodyan, S. M.; Bogdan, A. M.

    2016-04-01

    The work includes the mechanical analysis of mechanical feeders and batchers that find application in various technological processes and industrial fields. Feeders are usually classified according to their design features into two groups: conveyor-type feeders and non-conveyor feeders. Batchers are used to batch solid bulk materials. Less frequently, they are used for liquids. In terms of a batching method, they are divided into volumetric and weighting batchers. Weighting batchers do not provide for sufficient batching accuracy. Automatic weighting batchers include a mass controlling sensor and systems for automatic material feed and automatic mass discharge control. In terms of operating principle, batchers are divided into gravitational batchers and batchers with forced feed of material using conveyors and pumps. Improved consumption of raw materials, decreased loss of materials, ease of use in automatic control systems of industrial facilities allows increasing the quality of technological processes and improve labor conditions. The batch feeder suggested by the authors is a volumetric batcher that has no comparable counterparts among conveyor-type feeders and allows solving the problem of targeted feeding of bulk material batches increasing reliability and hermeticity of the device.

  16. Effective pure states for bulk quantum computation

    In bulk quantum computation one can manipulate a large number of indistinguishable quantum computers by parallel unitary operations and measure expectation values of certain observables with limited sensitivity. The initial state of each computer in the ensemble is known but not pure. Methods for obtaining effective pure input states by a series of manipulations have been described by Gershenfeld and Chuang (logical labeling) [Science 275, 350 (1997)] and Cory et al. (spatial averaging) [Proc. Natl. Acad. Sci. USA 94, 1634 (1997)] for the case of quantum computation with nuclear magnetic resonance. We give a different technique called temporal averaging. This method is based on classical randomization, requires no ancilla quantum bits, and can be implemented in nuclear magnetic resonance without using gradient fields. We introduce several temporal averaging algorithms suitable for both high-temperature and low-temperature bulk quantum computing and analyze the signal-to-noise behavior of each. Most of these algorithms require only a constant multiple of the number of experiments needed by the other methods for creating effective pure states. copyright 1998 The American Physical Society

  17. Ideal bulk pressure of active Brownian particles

    Speck, Thomas; Jack, Robert L.

    2016-06-01

    The extent to which active matter might be described by effective equilibrium concepts like temperature and pressure is currently being discussed intensely. Here, we study the simplest model, an ideal gas of noninteracting active Brownian particles. While the mechanical pressure exerted onto confining walls has been linked to correlations between particles' positions and their orientations, we show that these correlations are entirely controlled by boundary effects. We also consider a definition of local pressure, which describes interparticle forces in terms of momentum exchange between different regions of the system. We present three pieces of analytical evidence which indicate that such a local pressure exists, and we show that its bulk value differs from the mechanical pressure exerted on the walls of the system. We attribute this difference to the fact that the local pressure in the bulk does not depend on boundary effects, contrary to the mechanical pressure. We carefully examine these boundary effects using a channel geometry, and we show a virial formula for the pressure correctly predicts the mechanical pressure even in finite channels. However, this result no longer holds in more complex geometries, as exemplified for a channel that includes circular obstacles.

  18. Enhancing bulk superconductivity by engineering granular materials

    Mayoh, James; García García, Antonio

    2014-03-01

    The quest for higher critical temperatures is one of the main driving forces in the field of superconductivity. Recent theoretical and experimental results indicate that quantum size effects in isolated nano-grains can boost superconductivity with respect to the bulk limit. Here we explore the optimal range of parameters that lead to an enhancement of the critical temperature in a large three dimensional array of these superconducting nano-grains by combining mean-field, semiclassical and percolation techniques. We identify a broad range of parameters for which the array critical temperature, TcArray, can be up to a few times greater than the non-granular bulk limit, Tc 0. This prediction, valid only for conventional superconductors, takes into account an experimentally realistic distribution of grain sizes in the array, charging effects, dissipation by quasiparticles and limitations related to the proliferation of thermal fluctuations for sufficiently small grains. For small resistances we find the transition is percolation driven. Whereas at larger resistances the transition occurs above the percolation threshold due to phase fluctuations. JM acknowledes support from an EPSRC Ph.D studentship, AMG acknowledges support from EPSRC, grant No. EP/I004637/1, FCT, grant PTDC/FIS/111348/2009 and a Marie Curie International Reintegration Grant PIRG07-GA-2010-268172.

  19. Substantial bulk photovoltaic effect enhancement via nanolayering.

    Wang, Fenggong; Young, Steve M; Zheng, Fan; Grinberg, Ilya; Rappe, Andrew M

    2016-01-01

    Spontaneous polarization and inversion symmetry breaking in ferroelectric materials lead to their use as photovoltaic devices. However, further advancement of their applications are hindered by the paucity of ways of reducing bandgaps and enhancing photocurrent. By unravelling the correlation between ferroelectric materials' responses to solar irradiation and their local structure and electric polarization landscapes, here we show from first principles that substantial bulk photovoltaic effect enhancement can be achieved by nanolayering PbTiO3 with nickel ions and oxygen vacancies ((PbNiO2)x(PbTiO3)(1-x)). The enhancement of the total photocurrent for different spacings between the Ni-containing layers can be as high as 43 times due to a smaller bandgap and photocurrent direction alignment for all absorption energies. This is due to the electrostatic effect that arises from nanolayering. This opens up the possibility for control of the bulk photovoltaic effect in ferroelectric materials by nanoscale engineering of their structure and composition. PMID:26791545

  20. Perovskite oxides: Oxygen electrocatalysis and bulk structure

    Carbonio, R. E.; Fierro, C.; Tryk, D.; Scherson, D.; Yeager, Ernest

    1987-01-01

    Perovskite type oxides were considered for use as oxygen reduction and generation electrocatalysts in alkaline electrolytes. Perovskite stability and electrocatalytic activity are studied along with possible relationships of the latter with the bulk solid state properties. A series of compounds of the type LaFe(x)Ni1(-x)O3 was used as a model system to gain information on the possible relationships between surface catalytic activity and bulk structure. Hydrogen peroxide decomposition rate constants were measured for these compounds. Ex situ Mossbauer effect spectroscopy (MES), and magnetic susceptibility measurements were used to study the solid state properties. X ray photoelectron spectroscopy (XPS) was used to examine the surface. MES has indicated the presence of a paramagnetic to magnetically ordered phase transition for values of x between 0.4 and 0.5. A correlation was found between the values of the MES isomer shift and the catalytic activity for peroxide decomposition. Thus, the catalytic activity can be correlated to the d-electron density for the transition metal cations.

  1. 19 CFR 151.24 - Unlading facilities for bulk sugar.

    2010-04-01

    ... 19 Customs Duties 2 2010-04-01 2010-04-01 false Unlading facilities for bulk sugar. 151.24 Section... OF THE TREASURY (CONTINUED) EXAMINATION, SAMPLING, AND TESTING OF MERCHANDISE Sugars, Sirups, and Molasses § 151.24 Unlading facilities for bulk sugar. When dutiable sugar is to be imported in bulk, a...

  2. 7 CFR 58.313 - Print and bulk packaging rooms.

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Print and bulk packaging rooms. 58.313 Section 58.313 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards....313 Print and bulk packaging rooms. Rooms used for packaging print or bulk butter and related...

  3. Bulk forming of industrial micro components in conventional metals and bulk metallic glasses

    Arentoft, Mogens; Paldan, Nikolas Aulin; Eriksen, Rasmus Solmer;

    2007-01-01

    For production of micro components in large numbers, forging is an interesting and challenging process. The conventional metals like silver, steel and aluminum often require multi-step processes, but high productivity and increased strength justify the investment. As an alternative, bulk metallic...

  4. Organic hybrid planar-nanocrystalline bulk heterojunctions

    Forrest, Stephen R.; Yang, Fan

    2013-04-09

    A photosensitive optoelectronic device having an improved hybrid planar bulk heterojunction includes a plurality of photoconductive materials disposed between the anode and the cathode. The photoconductive materials include a first continuous layer of donor material and a second continuous layer of acceptor material. A first network of donor material or materials extends from the first continuous layer toward the second continuous layer, providing continuous pathways for conduction of holes to the first continuous layer. A second network of acceptor material or materials extends from the second continuous layer toward the first continuous layer, providing continuous pathways for conduction of electrons to the second continuous layer. The first network and the second network are interlaced with each other. At least one other photoconductive material is interspersed between the interlaced networks. This other photoconductive material or materials has an absorption spectra different from the donor and acceptor materials.

  5. Contact characteristics for YBCO bulk superconductors

    Yamamoto, Naoki; Sakai, Tomokazu; Sawa, Koichiro; Tomita, Masaru; Murakami, Masato

    2003-10-01

    We have studied the contact characteristics of two resin-impregnated YBCO (a composite of YBa 2Cu 3O y and Y 2BaCuO 5) bulk superconductors in mechanical contact. A switching phenomenon could be observed at a threshold current or a transfer current value in the V- I curves of the YBCO contact. The transfer current exceeded the previous value of 13.5 A at 77 K in the contact when the sample surfaces were carefully polished. The present results suggest that a pair of YBCO blocks might be applicable to the mechanical persistent current switch for superconducting magnetic energy storage and other superconducting systems run in a persistent current mode.

  6. Crack evolution in bulk metallic glasses

    In the present study, the mechanisms underlying plastic deformation of a Ni-based bulk metallic glass (BMG) are explored. Based on the microstructural investigations, a model is proposed how fracture emerges in BMGs. After deformation, the glass is macroscopically more fragile indicating a decrease in the viscosity within the shear bands due to shear softening. These fluctuations of viscosity and therefore Poisson ratio between the deformed and undeformed regions appear to be the initiation sites for nanometer-scale cracks, which are aligned parallel to the applied force. Coalescence of voids is believed to form these small cracks, which eventually interconnect along the interface between the sheared and unsheared regions to form a detrimental defect resulting in fracture.

  7. Bulk nanoscale materials in steel products

    Although a number of nanoscale metallic materials exhibit interesting mechanical properties the fabrication paths are often complex and difficult to apply to bulk structural materials. However a number of steels which exhibit combinations of plasticity and phase transitions can be deformed to produce ultra high strength levels in the range 1 to 3 GPa. The resultant high stored energy and complex microstructures allow new nanoscale structures to be produced by combinations of recovery and recrystallisation. The resultant structures exhibit totally new combinations of strength and ductility to be achieved. In specific cases this also enables both the nature of the grain boundary structure and the spatial variation in structure to be controlled. In this presentation both the detailed microstructural features and their relation to the strength, work-hardening capacity and ductility will be discussed for a number of martensitic and austenitic steels.

  8. Bulk disk resonator based ultrasensitive mass sensor

    Cagliani, Alberto; Davis, Zachary James

    2009-01-01

    In the framework of developing an innovative label-free sensor for multiarrayed biodetection applications, we present a novel bulk resonator based mass sensor. The sensor is a polysilicon disk which shows a Q-factor of 6400 in air at 68.8 MHz, resulting in mass resolutions down in the femtogram...... range. The sensor has been characterized in terms of sensitivity both for distributed mass detection, performing six consecutive depositions of e-beam evaporated Au, and localized mass detection, depositing approximately 7.5 pg of Pt/Ga/C three times consecutively with a Focused Ion Beam system....... The sensor has an extremely high distributed mass to frequency shift sensitivity of 60104 Hzcm2/¿g and shows a localized mass to frequency sensitivity up to 4405 Hz/pg with a localized mass resolution down to 15 fg. The device has been fabricated with a new microfabrication process that uses only two...

  9. Holographic bulk viscosity: GPR vs EO

    Buchel, Alex; Kiritsis, Elias

    2011-01-01

    Recently Eling and Oz (EO) proposed a formula for the holographic bulk viscosity, in arXiv:1103.1657, derived from the null horizon focusing equation. This formula seems different from that obtained earlier by Gubser, Pufu and Rocha (GPR) in arXiv:0806.0407 calculated from the IR limit of the two-point function of the trace of the stress tensor. The two were shown to agree only for some simple scaling cases. We point out that the two formulae agree in two non-trivial holographic theories describing RG flows. The first is the strongly coupled N=2* gauge theory plasma. The second is the semi-phenomenological model of Improved Holographic QCD.

  10. A route to transparent bulk metals

    Schwingenschlögl, Udo

    2012-07-23

    Hypothetical compounds based on a sapphire host are investigated with respect to their structural as well as electronic features. The results are obtained by electronic structure calculations within density functional theory and the generalized gradient approximation. A quarter of the Al atoms in Al 2O 3 is replaced by a 4d transition metal M ion, with d 0 to d 9 electronic configuration. We perform structure optimizations for all the compounds and analyze the electronic states. Due to the sizeable band gap of the Al 2O 3 host, we can identify promising candidates for transparent bulk metals. We explain the mechanisms leading to this combination of materials properties. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. DEPLOYMENT OF THE BULK TRITIUM SHIPPING PACKAGE

    Blanton, P.

    2013-10-10

    A new Bulk Tritium Shipping Package (BTSP) was designed by the Savannah River National Laboratory to be a replacement for a package that has been used to ship tritium in a variety of content configurations and forms since the early 1970s. The BTSP was certified by the National Nuclear Safety Administration in 2011 for shipments of up to 150 grams of Tritium. Thirty packages were procured and are being delivered to various DOE sites for operational use. This paper summarizes the design features of the BTSP, as well as associated engineered material improvements. Fabrication challenges encountered during production are discussed as well as fielding requirements. Current approved tritium content forms (gas and tritium hydrides), are reviewed, as well as, a new content, tritium contaminated water on molecular sieves. Issues associated with gas generation will also be discussed.

  12. Tuneable film bulk acoustic wave resonators

    Gevorgian, Spartak Sh; Vorobiev, Andrei K

    2013-01-01

    To handle many standards and ever increasing bandwidth requirements, large number of filters and switches are used in transceivers of modern wireless communications systems. It makes the cost, performance, form factor, and power consumption of these systems, including cellular phones, critical issues. At present, the fixed frequency filter banks based on Film Bulk Acoustic Resonators (FBAR) are regarded as one of the most promising technologies to address performance -form factor-cost issues. Even though the FBARs improve the overall performances the complexity of these systems remains high.  Attempts are being made to exclude some of the filters by bringing the digital signal processing (including channel selection) as close to the antennas as possible. However handling the increased interference levels is unrealistic for low-cost battery operated radios. Replacing fixed frequency filter banks by one tuneable filter is the most desired and widely considered scenario. As an example, development of the softwa...

  13. Universe Models with Negative Bulk Viscosity

    Brevik, Iver

    2013-01-01

    The concept of negative temperatures has occasionally been used in connection with quantum systems. A recent example of this sort is reported in the paper of S. Braun et al. [Science 339,52 (2013)], where an attractively interacting ensemble of ultracold atoms is investigated experimentally and found to correspond to a negative-temperature system since the entropy decreases with increasing energy at the high end of the energy spectrum. As the authors suggest, it would be of interest to investigate whether a suitable generalization of standard cosmological theory could be helpful, in order to elucidate the observed accelerated expansion of the universe usually explained in terms of a positive tensile stress (negative pressure). In the present note we take up this basic idea and investigate a generalization of the standard viscous cosmological theory, not by admitting negative temperatures but instead by letting the bulk viscosity take negative values. Evidently, such an approach breaks standard thermodynamics,...

  14. Combustion of bulk titanium in oxygen

    Clark, A. F.; Moulder, J. C.; Runyan, C. C.

    1975-01-01

    The combustion of bulk titanium in one atmosphere oxygen is studied using laser ignition and several analytical techniques. These were high-speed color cinematography, time and space resolved spectra in the visible region, metallography (including SEM) of specimens quenched in argon gas, X-ray and chemical product analyses, and a new optical technique, the Hilbert transform method. The cinematographic application of this technique for visualizing phase objects in the combustion zone is described. The results indicate an initial vapor phase reaction immediately adjacent to the molten surface but as the oxygen uptake progresses the evaporation approaches the point of congruency and a much reduced evaporation rate. This and the accumulation of the various soluble oxides soon drive the reaction zone below the surface where gas formation causes boiling and ejection of particles. The buildup of rutile cuts off the oxygen supply and the reaction ceases.

  15. Bulk semiconducting scintillator device for radiation detection

    Stowe, Ashley C.; Burger, Arnold; Groza, Michael

    2016-08-30

    A bulk semiconducting scintillator device, including: a Li-containing semiconductor compound of general composition Li-III-VI.sub.2, wherein III is a Group III element and VI is a Group VI element; wherein the Li-containing semiconductor compound is used in one or more of a first mode and a second mode, wherein: in the first mode, the Li-containing semiconductor compound is coupled to an electrical circuit under bias operable for measuring electron-hole pairs in the Li-containing semiconductor compound in the presence of neutrons and the Li-containing semiconductor compound is also coupled to current detection electronics operable for detecting a corresponding current in the Li-containing semiconductor compound; and, in the second mode, the Li-containing semiconductor compound is coupled to a photodetector operable for detecting photons generated in the Li-containing semiconductor compound in the presence of the neutrons.

  16. Vortices in superconducting bulk, films and SQUIDs

    Ernst Helmut Brandt

    2006-01-01

    The properties of the ideal periodic vortex lattice in bulk superconductors and in films of any thickness can be calculated from Ginzburg-Landau theory by an iteration method using Fourier series. The London theory yields general analytic expressions for the magnetic field and energy of arbitrary arrangements of straight or curved vortex lines. The elasticity of the vortex lattice is highly nonlocal. The magnetic response of superconductors of realistic shapes like thin and thick strips and disks or thin rectangular plates or films, containing pinned vortices, can be computed within continuum theory by solving an integral equation. A useful example is a thin square with a central hole and a radial slit, used as superconducting quantum interference device (SQUID).

  17. Assessment of bioburden encapsulated in bulk materials

    Schubert, Wayne W.; Newlin, Laura; Chung, Shirley Y.; Ellyin, Raymond

    2016-05-01

    The National Aeronautics and Space Administration (NASA) imposes bioburden limitations on all spacecraft destined for solar system bodies that might harbor evidence of extant or extinct life. The subset of microorganisms trapped within solid materials during manufacture and assembly is referred to as encapsulated bioburden. In the absence of spacecraft-specific data, NASA relies on specification values to estimate total spacecraft encapsulated bioburden, typically 30 endospores/cm3 or 300 viable cells/cm3 in non-electronic materials. Specification values for endospores have been established conservatively, and represent no less than an order of magnitude greater abundance than that derived from empirical assessments of actual spacecraft materials. The goal of this study was to generate data germane to determining whether revised bulk encapsulated material values (lower than those estimated by historical specifications) tailored specifically to the materials designated in modern-day spacecraft design could be used, on a case-by-case basis, to comply with planetary protection requirements. Organic materials having distinctly different chemical properties and configurations were selected. This required more than one experimental and analytical approach. Filtration was employed for liquid electrolytes, lubricants were suspended in an aqueous solution and solids (wire and epoxy sealant) were cryogenically milled. The final data characteristic for all bioburden estimates was microbial colony formation in rich agar growth medium. To assess survival potential, three non-spore-forming bacterial cell lines were systematically encapsulated in an epoxy matrix, liberated via cryogenic grinding, and cultured. Results suggest that bulk solid materials harbor significantly fewer encapsulated microorganisms than are estimated by specification values. Lithium-ion battery electrolyte reagents housed fewer than 1 CFU/cm3. Results also demonstrated that non-spore-forming microorganisms

  18. Performance and applications of quench melt-growth bulk magnets

    Nariki, S.; Teshima, H.; Morita, M.

    2016-03-01

    This paper describes the progress in quench melt-growth (QMG) bulk magnets, developed by the Nippon Steel & Sumitomo Metal Corporation, which consist of single crystalline RE123 phase and finely dispersed RE211 particles. QMG bulks can trap high magnetic fields. The field-trapping ability of QMG bulks is largely increased with an improvement in its J c and size, promising the realization of various applications such as flywheel energy-storage systems, ship motors, NMR/MRI spectrometers, wind-power generators and so on. Intensive research has revealed that the optimal RE element is different depending on application requirements. Gd-QMG bulk is the most promising material for several high-field engineering applications. The trapped magnetic field of Gd-QMG bulk 60 mm in diameter at 77 K is twice as large as that of Y-QMG bulk with a similar size due to its excellent J c properties. The large Gd-based QMG bulks up to 150 mm in diameter are fabricated by incorporating the RE compositional gradient method. Compact NMR/MRI spectrometers are one of the promising applications of bulk superconductors. Eu-QMG bulks are suitable for NMR magnets. NMR applications require extremely homogeneous magnetic fields. In the Eu-system, the small paramagnetic moment of a Eu ion compared to a Gd ion improves the field homogeneity in the bulk. For the application of current leads, Dy-based QMG is available by utilizing a low thermal conductivity.

  19. Phosphorylation of threonine 156 of the mu2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo.

    Olusanya, O; Andrews, P D; Swedlow, J R; Smythe, E

    2001-06-01

    The clathrin-coated pit is the major port of entry for many receptors and pathogens and is the paradigm for membrane-based sorting events in higher cells [1]. Recently, it has been possible to reconstitute in vitro the events leading to assembly, invagination, and budding off of clathrin-coated vesicles, allowing dissection of the machinery required for sequestration of receptors into these structures [2-6]. The AP2 adaptor complex is a key element of this machinery linking receptors to the coat lattice, and it has previously been reported that AP2 can be phosphorylated both in vitro and in vivo [7-10]. However, the physiological significance of this has never been established. Here, we show that phosphorylation of a single threonine residue (Thr156) of the mu2 subunit of the AP2 complex is essential for efficient endocytosis of transferrin both in an in vitro coated-pit budding assay and in living cells. PMID:11516654

  20. On methods of estimating cosmological bulk flows

    Nusser, Adi

    2015-01-01

    We explore similarities and differences between several estimators of the cosmological bulk flow, $\\bf B$, from the observed radial peculiar velocities of galaxies. A distinction is made between two theoretical definitions of $\\bf B$ as a dipole moment of the velocity field weighted by a radial window function. One definition involves the three dimensional (3D) peculiar velocity, while the other is based on its radial component alone. Different methods attempt at inferring $\\bf B$ for either of these definitions which coincide only for a constant velocity field. We focus on the Wiener Filtering (WF, Hoffman et al. 2015) and the Constrained Minimum Variance (CMV,Feldman et al. 2010) methodologies. Both methodologies require a prior expressed in terms of the radial velocity correlation function. Hoffman et al. compute $\\bf B$ in Top-Hat windows from a WF realization of the 3D peculiar velocity field. Feldman et al. infer $\\bf B$ directly from the observed velocities for the second definition of $\\bf B$. The WF ...

  1. On methods of estimating cosmological bulk flows

    Nusser, Adi

    2016-01-01

    We explore similarities and differences between several estimators of the cosmological bulk flow, B, from the observed radial peculiar velocities of galaxies. A distinction is made between two theoretical definitions of B as a dipole moment of the velocity field weighted by a radial window function. One definition involves the three-dimensional (3D) peculiar velocity, while the other is based on its radial component alone. Different methods attempt at inferring B for either of these definitions which coincide only for the case of a velocity field which is constant in space. We focus on the Wiener Filtering (WF) and the Constrained Minimum Variance (CMV) methodologies. Both methodologies require a prior expressed in terms of the radial velocity correlation function. Hoffman et al. compute B in Top-Hat windows from a WF realization of the 3D peculiar velocity field. Feldman et al. infer B directly from the observed velocities for the second definition of B. The WF methodology could easily be adapted to the second definition, in which case it will be equivalent to the CMV with the exception of the imposed constraint. For a prior with vanishing correlations or very noisy data, CMV reproduces the standard Maximum Likelihood estimation for B of the entire sample independent of the radial weighting function. Therefore, this estimator is likely more susceptible to observational biases that could be present in measurements of distant galaxies. Finally, two additional estimators are proposed.

  2. Studies of bulk heterojunction solar cells

    Cossel, Raquel; McIntyre, Max; Tzolov, Marian

    We are studying bulk heterojunction solar cells that were fabricated using a mixture of PCPDTBT and PCBM­C60. The impedance data of the cells in dark responded like a simple RC circuit. The value of the dielectric constant derived from these results is consistent with the values reported in the literature for these materials. We are showing that the parallel resistance in the equivalent circuit of linear lump elements can be interpreted using the DC current­voltage measurements. The impedance spectra under light illumination indicated the existence of additional polarization. This extra feature can be described by a model that includes a series RC circuit in parallel with the equivalent circuit for a device in dark. The physical interpretation of the additional polarization is based on photo­generated charges getting trapped in wells, which have a characteristic relaxation time corresponding to the observed break frequency in the impedance spectra. We have studied the influence of the anode and cathode interface on this phenomena, either by using different interface materials, or by depositing the metal electrode while the substate is heated.

  3. Leachables evaluation for bulk drug substance.

    Tsui, Victor; Somma, Maria S; Zitzner, Linda A

    2009-01-01

    This study describes a comprehensive analytical approach for evaluating potential leachables from product-contacting material surfaces in the manufacture of bulk drug substance (BDS) for use in parenteral products. A qualitative step-by-step evaluation of the process stream for the production, purification, and storage of the BDS was performed. The product-contact equipment surfaces were then grouped based on their materials of construction and prioritized according to the potential risk of contributing leachables to the BDS. Based on this evaluation, 13 potential leachable species were identified and classified either as volatile organic compounds (VOCs), semi-volatile organic compounds (SVOCs), anions, cations, or trace metals. The BDS was first screened for the presence of VOCs, SVOCs, anions, cations, and trace metals using analytical methods that were qualified for their application with the BDS. Thirteen potential leachables were then spiked in the BDS and in the water for injection for spike-recovery studies. The analysis of the BDS for potential leachables showed that the potential impurities were not present in the BDS except for a trace amount of silicon, and that the residual solvent concentrations were lower than the recommended limits established by the International Committee on Harmonisation. The spike-recovery studies confirmed that the analytical methods could effectively determine the leachable compounds in the BDS. Moreover, the 13 contaminants did not form a complex with the protein in the BDS and did not represent a potential risk to the BDS's safety and stability. PMID:19634355

  4. Casimir effect in dielectrics: Bulk energy contribution

    In a recent series of papers, Schwinger discussed a process that he called the dynamical Casimir effect. The key essence of this effect is the change in zero-point energy associated with any change in a dielectric medium. (In particular, if the change in the dielectric medium is taken to be the growth or collapse of a bubble, this effect may have relevance to sonoluminescence.) The kernel of Schwinger close-quote s result is that the change in Casimir energy is proportional to the change in the volume of the dielectric, plus finite-volume corrections. Other papers have called into question this result, claiming that the volume term should actually be discarded, and that the dominant term remaining is proportional to the surface area of the dielectric. In this paper, which is an expansion of an earlier Letter on the same topic, we present a careful and critical review of the relevant analyses. We find that the Casimir energy, defined as the change in zero-point energy due to a change in the medium, has at leading order a bulk volume dependence. This is in full agreement with Schwinger close-quote s result, once the correct physical question is asked. We have nothing new to say about sonoluminescence itself. copyright 1997 The American Physical Society

  5. Bulk viscous cosmology: statefinder and entropy

    He, X

    2006-01-01

    The statefinder diagnostic pair is adopted to differentiate viscous cosmology models and it is found that the trajectories of these viscous cosmology models on the statefinder pair $s-r$ plane are quite different from those of the corresponding non-viscous cases. Particularly for the quiessence model, the singular properties of state parameter $w=-1$ are obviously demonstrated on the statefinder diagnostic pair planes. We then discuss the entropy of the viscous / dissipative cosmology system which may be more practical to describe the present cosmic observations as the perfect fluid is just a global approximation to the complicated cosmic media in current universe evolution. When the bulk viscosity takes the form of $\\zeta=\\zeta_{1}\\dot{a}/a$($\\zeta_{1}$ is constant), the relationship between the entropy $S$ and the redshift $z$ is explicitly given out. We find that the entropy of the viscous cosmology is always increasing and consistent with the thermodynamics arrow of time for the universe evolution. With t...

  6. Photoelectron spectroscopy bulk and surface electronic structures

    Suga, Shigemasa

    2014-01-01

    Photoelectron spectroscopy is now becoming more and more required to investigate electronic structures of various solid materials in the bulk, on surfaces as well as at buried interfaces. The energy resolution was much improved in the last decade down to 1 meV in the low photon energy region. Now this technique is available from a few eV up to 10 keV by use of lasers, electron cyclotron resonance lamps in addition to synchrotron radiation and X-ray tubes. High resolution angle resolved photoelectron spectroscopy (ARPES) is now widely applied to band mapping of materials. It attracts a wide attention from both fundamental science and material engineering. Studies of the dynamics of excited states are feasible by time of flight spectroscopy with fully utilizing the pulse structures of synchrotron radiation as well as lasers including the free electron lasers (FEL). Spin resolved studies also made dramatic progress by using higher efficiency spin detectors and two dimensional spin detectors. Polarization depend...

  7. Thermodynamic properties of bulk and confined water

    Mallamace, Francesco, E-mail: francesco.mallamace@unime.it [Dipartimento di Fisica e Scienza della Terra Università di Messina and CNISM, I-98168 Messina (Italy); Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States); Center for Polymer Studies and Department of Physics, Boston University, Boston, Massachusetts 02215 (United States); Corsaro, Carmelo [Dipartimento di Fisica e Scienza della Terra Università di Messina and CNISM, I-98168 Messina (Italy); Mallamace, Domenico [Dipartimento di Scienze dell' Ambiente, della Sicurezza, del Territorio, degli Alimenti e della Salute, Università di Messina, I-98166 Messina (Italy); Vasi, Sebastiano; Vasi, Cirino [IPCF-CNR, I-98166 Messina (Italy); Stanley, H. Eugene [Center for Polymer Studies and Department of Physics, Boston University, Boston, Massachusetts 02215 (United States)

    2014-11-14

    The thermodynamic response functions of water display anomalous behaviors. We study these anomalous behaviors in bulk and confined water. We use nuclear magnetic resonance (NMR) to examine the configurational specific heat and the transport parameters in both the thermal stable and the metastable supercooled phases. The data we obtain suggest that there is a behavior common to both phases: that the dynamics of water exhibit two singular temperatures belonging to the supercooled and the stable phase, respectively. One is the dynamic fragile-to-strong crossover temperature (T{sub L} ≃ 225 K). The second, T{sup *} ∼ 315 ± 5 K, is a special locus of the isothermal compressibility K{sub T}(T, P) and the thermal expansion coefficient α{sub P}(T, P) in the P–T plane. In the case of water confined inside a protein, we observe that these two temperatures mark, respectively, the onset of protein flexibility from its low temperature glass state (T{sub L}) and the onset of the unfolding process (T{sup *})

  8. Interactions between the Yeast SM22 Homologue Scp1 and Actin Demonstrate the Importance of Actin Bundling in Endocytosis*S⃞

    Gheorghe, Dana M.; Aghamohammadzadeh, Soheil; Rooij, Iwona I. Smaczynska-de; Allwood, Ellen G.; Winder, Steve J.; Ayscough, Kathryn R.

    2008-01-01

    The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185 to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process. PMID:18400761

  9. The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

    Chiara Sandri; Guido Serini; Francesca Caccavari; Donatella Valdembri; Chiara Camillo; Stefan Veltel; Martina Santambrogio; Letizia Lanzetti; Fedenco Bussolino; Johanna Ivaska

    2012-01-01

    During developmental and tumor angiogenesis,semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases.R-Ras is mainly expressed in vascular cells,where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms.We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and-angiogenic activity of R-Ras.Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia.Upon binding,GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF)to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes.Here,the R-Ras/RIN2/Rab5 signaling module activates Racl-dependent cell adhesion via TIAM1,a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate.In conclusion,the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Racl.

  10. The timing of cortical granule fusion, content dispersal, and endocytosis during fertilization of the hamster egg: an electrophysiological and histochemical study.

    Kline, D; Stewart-Savage, J

    1994-03-01

    To determine the temporal relationship between cortical granule exocytosis and the repetitive calcium transients, which are characteristic of mammalian fertilization, we monitored membrane addition from exocytosis during fertilization of hamster eggs. Continuous measurement of membrane capacitance by applying a 3.1-nA alternating current at 375 Hz showed addition of cortical granule membrane. Simultaneous measurement of membrane potential revealed each calcium transient by the appearance of transient hyperpolarizing responses due to calcium-activated potassium channels in the egg. The initial membrane capacitance of the eggs averaged 736 +/- 44 pF (mean +/- SD; n = 7) and an increase in capacitance of 61 +/- 19 pF occurred within 4 sec of the start of the first hyperpolarizing response (HR) after fertilization. Immediately after the first increase in capacitance there was a gradual decline in membrane capacitance in all eggs and in five/seven eggs the capacitance returned to the unfertilized level in 7.8 +/- 4.4 min. The gradual decline in capacitance after the first increase indicated endocytosis, which was confirmed by the internalization of fluorescently labeled dextran. Superimposed on the gradual decline in membrane capacitance were smaller increases in capacitance that occurred with the second and later HRs. The total increase in capacitance from the first three events averaged 72 +/- 19 pF, representing an average increase in capacitance of about 10% of the capacitance of the unfertilized egg. By labeling eggs before and after permeabilization with two different fluorochromes attached to Lens culinaris agglutinin, we demonstrate that the dispersal of the cortical granules contents does not occur immediately after exocytosis. Our results demonstrate that cortical granule exocytosis in hamster eggs is closely coupled to the periodic increases in calcium, that the contents of the cortical granules are slow to disperse, and that after exocytosis, the surface

  11. Endocytosis of indium-tin-oxide nanoparticles by macrophages provokes pyroptosis requiring NLRP3-ASC-Caspase1 axis that can be prevented by mesenchymal stem cells.

    Naji, Abderrahim; Muzembo, Basilua André; Yagyu, Ken-Ichi; Baba, Nobuyasu; Deschaseaux, Frédéric; Sensebé, Luc; Suganuma, Narufumi

    2016-01-01

    The biological effects of indium-tin-oxide (ITO) are of considerable importance because workers exposed to indium compounds have been diagnosed with interstitial lung disease or pulmonary alveolar proteinosis; however, the pathophysiology of these diseases is undefined. Here, mice intraperitoneally inoculated with ITO-nanoparticles (ITO-NPs) resulted in peritonitis dependent in NLRP3 inflammasome, with neutrophils recruitment and interleukin-1β (IL-1β) production. Withal peritoneal macrophages exposed ex vivo to ITO-NPs caused IL-1β secretion and cytolysis. Further, alveolar macrophages exposed to ITO-NPs in vitro showed ITO-NP endocytosis and production of tumor necrosis factor-α (TNF-α) and IL-1β, ensued cell death by cytolysis. This cell death was RIPK1-independent but caspase1-dependent, and thus identified as pyroptosis. Endocytosis of ITO-NPs by activated THP-1 cells induced pyroptosis with IL-1β/TNF-α production and cytolysis, but not in activated THP-1 cells with knockdown of NLRP3, ASC, or caspase1. However, exposing activated THP-1 cells with NLRP3 or ASC knockdown to ITO-NPs resulted in cell death but without cytolysis, with deficiency in IL-1β/TNF-α, and revealing features of apoptosis. While, mesenchymal stem cells (MSCs) co-cultured with macrophages impaired both inflammation and cell death induced by ITO-NPs. Together, our findings provide crucial insights to the pathophysiology of respiratory diseases caused by ITO particles, and identify MSCs as a potent therapeutic. PMID:27194621

  12. Immunomodulatory effects of human umbilical cord Wharton's jelly-derived mesenchymal stem cells on differentiation, maturation and endocytosis of monocyte-derived dendritic cells.

    Saeidi, Mohsen; Masoud, Ahmad; Shakiba, Yadollah; Hadjati, Jamshid; Mohyeddin Bonab, Mandana; Nicknam, Mohammad Hossein; Latifpour, Mostafa; Nikbin, Behrooz

    2013-03-01

    The Wharton's jelly of the umbilical cord is believed to be a source of mesenchymal stem cells (MSCs) which can be therapeutically applied in degenerative diseases.In this study, we investigated the immunomodulatory effect of umbilical cord derived-mesenchymal stem cells (UC-MSCs) and bone marrow-derived-mesenchymal stem cells (BM-MSCs) on differentiation, maturation, and endocytosis of monocyte-derived dendritic cells in a transwell culture system under laboratory conditions. Monocytes were differentiated into immature dendritic cells (iDCs) in the presence of GM-CSF and IL-4 for 6 days and then differentiated into mature dendritic cells (mDCs) in the presence of TNF-α for 2 days. In every stage of differentiation, immature and mature dendritic cells were separately co-cultured with UC-MSCs and BM-MSCs. The findings showed that UC-MSCs and BM-MSCs inhibited strongly differentiation and maturation of dendritic cells at higher dilution ratios (1:1). The BM-MSCs and UC-MSCs showed more inhibitory effect on CD1a, CD83, CD86 expression, and dendritic cell endocytic activity, respectively. On the other hand, these cells severely up-regulated CD14 marker expression. We concluded that UC-MSCs and BM-MSCs could inhibit differentiation, maturation and endocytosis in monocyte-derived DCs through the secreted factors and free of any cell-cell contacts under laboratory conditions. As DCs are believed to be the main antigen presenting cells for naïve T cells in triggering immune responses, it would be logical that their inhibitory effect on differentiation, maturation and function can decrease or modulate immune and inflammatory responses. PMID:23454777

  13. Immunomodulatory effects of human umbilical cord Wharton's jelly-derived mesenchymal stem cells on differentiation, maturation and endocytosis of monocyte-derived dendritic cells.

    Mohsen Saeidi

    2013-03-01

    Full Text Available The Wharton’s jelly of the umbilical cord is believed to be a source of mesenchymal stem cells (MSCs which can be therapeutically applied in degenerative diseases.In this study, we investigated the immunomodulatory effect of umbilical cord derived- mesenchymal stem  cells (UC-MSCs and  bone  marrow-derived-mesenchymal stem  cells (BM-MSCs on differentiation, maturation, and endocytosis of monocyte-derived dendritic cells in a transwell culture system under laboratory conditions. Monocytes were differentiated into immature dendritic cells (iDCs in the presence of GM-CSF and IL-4 for 6 days and then differentiated into mature dendritic cells (mDCs in the presence of TNF-α for 2 days. In every stage of differentiation, immature and mature dendritic cells were separately co- cultured with UC-MSCs and BM-MSCs.The findings showed that UC-MSCs and BM-MSCs inhibited strongly differentiation and maturation of dendritic cells at higher dilution ratios (1:1. The BM-MSCs and UC-MSCs showed more inhibitory effect on CD1a, CD83, CD86 expression, and dendritic cell endocytic activity, respectively. On the other hand, these cells severely up-regulated CD14 marker expression. We concluded that UC-MSCs and BM-MSCs could inhibit differentiation, maturation and endocytosis in monocyte-derived DCs through the secreted factors and free of any cell- cell contacts  under  laboratory conditions. As DCs  are believed to  be the  main antigen presenting cells for naïve T cells in triggering immune responses, it would be logical that their inhibitory effect on differentiation, maturation and function can decrease or modulate immune and inflammatory responses.

  14. Macroscopic and direct light propulsion of bulk graphene material

    Zhang, Tengfei; Wu, Yingpeng; Xiao, Peishuang; Yi, Ningbo; Lu, Yanhong; Ma, Yanfeng; Huang, Yi; Zhao, Kai; Yan, Xiao-Qing; Liu, Zhi-Bo; Tian, Jian-Guo; Chen, Yongsheng

    2015-01-01

    It has been a great challenge to achieve the direct light manipulation of matter on a bulk scale. In this work, the direct light propulsion of matter was observed on a macroscopic scale for the first time using a bulk graphene based material. The unique structure and properties of graphene and the morphology of the bulk graphene material make it capable of not only absorbing light at various wavelengths but also emitting energetic electrons efficiently enough to drive the bulk material following Newtonian mechanics. Thus, the unique photonic and electronic properties of individual graphene sheets are manifested in the response of the bulk state. These results offer an exciting opportunity to bring about bulk scale light manipulation with the potential to realize long-sought proposals in areas such as the solar sail and space transportation driven directly by sunlight.

  15. A CFT Perspective on Gravitational Dressing and Bulk Locality

    Lewkowycz, Aitor; Verlinde, Herman

    2016-01-01

    We revisit the construction of local bulk operators in AdS/CFT with special focus on gravitational dressing and its consequences for bulk locality. Specializing to 2+1-dimensions, we investigate these issues via the proposed identification between bulk operators and cross-cap boundary states. We obtain explicit expressions for correlation functions of bulk fields with boundary stress tensor insertions, and find that they are free of non-local branch cuts but do have non-local poles. We recover the HKLL recipe for restoring bulk locality for interacting fields as the outcome of a natural CFT crossing condition. We show that, in a suitable gauge, the cross-cap states solve the bulk wave equation for general background geometries, and satisfy a conformal Ward identity analogous to a soft graviton theorem, Virasoro symmetry, the large N conformal bootstrap and the uniformization theorem all play a key role in our derivations.

  16. Gravitational potential wells and the cosmic bulk flow

    Kumar, Abhinav; Feldman, Hume A; Watkins, Richard

    2015-01-01

    The bulk flow is a volume average of the peculiar velocities and a useful probe of the mass distribution on large scales. The gravitational instability model views the bulk flow as a potential flow that obeys a Maxwellian Distribution. We use two N-body simulations, the LasDamas Carmen and the Horizon Run, to calculate the bulk flows of various sized volumes in the simulation boxes. Once we have the bulk flow velocities as a function of scale, we investigate the mass and gravitational potential distribution around the volume. We found that matter densities can be asymmetrical and difficult to detect in real surveys, however, the gravitational potential and its gradient may provide better tools to investigate the underlying matter distribution. This study shows that bulk flows are indeed potential flows and thus provides information on the flow sources. We also show that bulk flow magnitudes follow a Maxwellian distribution on scales $>10\\ h^{-1}$Mpc.

  17. Gravitational potential wells and the cosmic bulk flow

    Wang, Yuyu; Kumar, Abhinav; Feldman, Hume; Watkins, Richard

    2016-03-01

    The bulk flow is a volume average of the peculiar velocities and a useful probe of the mass distribution on large scales. The gravitational instability model views the bulk flow as a potential flow that obeys a Maxwellian Distribution. We use two N-body simulations, the LasDamas Carmen and the Horizon Run, to calculate the bulk flows of various sized volumes in the simulation boxes. Once we have the bulk flow velocities as a function of scale, we investigate the mass and gravitational potential distribution around the volume. We found that matter densities can be asymmetrical and difficult to detect in real surveys, however, the gravitational potential and its gradient may provide better tools to investigate the underlying matter distribution. This study shows that bulk flows are indeed potential flows and thus provides information on the flow sources. We also show that bulk flow magnitudes follow a Maxwellian distribution on scales > 10h-1 Mpc.

  18. Bulk temperature measurement in thermally striped pipe flows

    The hot leg flows in some Pressurized Water Reactor (PWR) designs have a temperature distribution across the pipe cross-section. This condition is often referred to as a thermally striped flow. Here, the bulk temperature measurement of pipe flows with thermal striping is explored. An experiment is conducted to examine the feasibility of using temperature measurements on the external surface of the pipe to estimate the bulk temperature of the flow. Simple mixing models are used to characterize the development of the temperature profile in the flow. Simple averaging techniques and Backward Propagating Neural Net are used to predict bulk temperature from the external temperature measurements. Accurate bulk temperatures can be predicted. However, some temperature distributions in the flow effectively mask the bulk temperature from the wall and cause significant error in the bulk temperature predicted using this technique

  19. The functionalized amino acid (S-Lacosamide subverts CRMP2-mediated tubulin polymerization to prevent constitutive and activity-dependent increase in neurite outgrowth

    Sarah M Wilson

    2014-07-01

    Full Text Available Activity-dependent neurite outgrowth is a highly complex, regulated process with important implications for neuronal circuit remodeling in development as well as in seizure-induced sprouting in epilepsy. Recent work has linked outgrowth to collapsin response mediator protein 2 (CRMP2, an intracellular phosphoprotein originally identified as axon guidance and growth cone collapse protein. The neurite outgrowth promoting function of CRMP2 is regulated by its phosphorylation state. In this study, depolarization (potassium chloride-driven activity increased the level of active CRMP2 by decreasing its phosphorylation by GSK3β via a reduction in priming by Cdk5. To determine the contribution of CRMP2 in activity-driven neurite outgrowth, we screened a limited set of compounds for their ability to reduce neurite outgrowth but not modify voltage-gated sodium channel (VGSC biophysical properties. This led to the identification of (S-lacosamide ((S-LCM, a stereoisomer of the clinically used antiepileptic drug (R-LCM (Vimpat®, as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Whereas (S-LCM was ineffective in targeting VGSCs, the presumptive pharmacological targets of (R-LCM, (S-LCM was more efficient than (R-LCM in subverting neurite outgrowth. Biomolecular interaction analyses revealed that (S-LCM bound to wildtype CRMP2 with low micromolar affinity, similar to (R-LCM. Through the use of this novel tool, the activity-dependent increase in neurite outgrowth observed following depolarization was characterized to be reliant on CRMP2 function. Knockdown of CRMP2 by siRNA in cortical neurons resulted in reduced CRMP2-dependent neurite outgrowth; incubation with (S-LCM phenocopied this effect. Other CRMP2-mediated processes were unaffected. (S-LCM subverted neurite outgrowth not by affecting the canonical CRMP2-tubulin association but rather by impairing the ability of CRMP2 to promote tubulin polymerization, events that are

  20. LOGISTICAL CRITERIONS AND STRATEGIES FOR STORAGE OF BULK MATERIALS

    Péter Telek

    2009-01-01

    The aim of my paper is to analyze the applicability of criterions used for selection of storage methods of individual units and general logistic strategies for storage of bulk materials. To reach this aim paper gives an overview about the advanced storage methods of bulk materials and their application possibilities. The second part shows the main criterions and logistic strategies in deep and describes their usability for bulk materials.

  1. Nanocomposite RE-Ba-Cu-O bulk superconductors

    Iida, Kazumasa

    2016-01-01

    Nanocomposite oxide high-temperature bulk superconductors can be used as quasi-magnets. Thanks to the recent progress of material processing, quasi-magnet with 26 mm diameter can generate a large field of 17.6 T at 26 K. These results are highly attractive for applications, involving levitation of permanent magnets on the bulk superconductors. Indeed, several other applications such as motors and magnetic resonance microscope using bulk superconductors have been proposed and demonstrated. In ...

  2. Extracting the bulk viscosity of the quark–gluon plasma

    We investigate the implications of a nonzero bulk viscosity coefficient on the azimuthal momentum anisotropy of ultracentral relativistic heavy ion collisions at the Large Hadron Collider. We find that, with IP-Glasma initial conditions, a finite bulk viscosity coefficient leads to a better description of the flow harmonics in ultracentral collisions. We then extract optimal values of bulk and shear viscosity coefficients that provide the best agreement with flow harmonic coefficients data in this centrality class

  3. Can local bulk effects explain the galactic dark matter?

    Heydari-Fard, Malihe; Sepangi, Hamid R, E-mail: m.heydarifard@mail.sbu.ac.ir, E-mail: hr-sepangi@sbu.ac.ir [Department of Physics, Shahid Beheshti University, Evin, Tehran 19839 (Iran, Islamic Republic of)

    2008-08-15

    We obtain the virial theorem within the context of a brane-world model without mirror symmetry or any form of junction condition. Taking a constant curvature bulk (neglecting non-local bulk effects), the local bulk effects generate a geometrical mass, contributing to the gravitational energy which may be used to explain the virial mass discrepancy in clusters of galaxies. We fix the parameters of this model in agreement with observational data.

  4. Can local bulk effects explain the galactic dark matter?

    Heydari-Fard, Malihe; Sepangi, Hamid R.

    2008-01-01

    We obtain the virial theorem within the context of a brane-world model without mirror symmetry or any form of junction condition. Taking a constant curvature bulk (neglecting non-local bulk effects), the local bulk effects generate a geometrical mass, contributing to the gravitational energy which may be used to explain the virial mass discrepancy in clusters of galaxies. We fix the parameter of this model in agreement with observational data.

  5. Bulk flow scaling for turbulent channel and pipe flows

    Chen, Xi; She, Zhen-Su

    2016-01-01

    We report a theory deriving bulk flow scaling for canonical wall-bounded flows. The theory accounts for the symmetries of boundary geometry (flat plate channel versus circular pipe) by a variational calculation for a large-scale energy length, which characterizes its bulk flow scaling by a simple exponent, i.e. $m=4$ for channel and 5 for pipe. The predicted mean velocity shows excellent agreement with several dozen sets of quality empirical data for a wide range of the Reynolds number (Re), with a universal bulk flow constant $\\kappa\\approx0.45$. Predictions for dissipation and turbulent transport in the bulk flow are also given, awaiting data verification.

  6. On Eling-Oz formula for the holographic bulk viscosity

    Buchel, Alex

    2011-01-01

    Recently Eling and Oz [1] proposed a simple formula for the bulk viscosity of holographic plasma. They argued that the formula is valid in the high temperature (near-conformal) regime, but is expected to break down at low temperatures. We point out that the formula is in perfect agreement with the previous computations of the bulk viscosity of the cascading plasma [2,3], as well as with the previous computations of the bulk viscosity of N=2^* plasma [4,5]. In the latter case it correctly reproduces the critical behaviour of the bulk viscosity in the vicinity of the critical point with the vanishing speed of sound.

  7. Locality, bulk equations of motion and the conformal bootstrap

    Kabat, Daniel

    2016-01-01

    We develop an approach to construct local bulk operators in a CFT to order 1/N^2. Since 4-point functions are not fixed by conformal invariance we use the OPE to categorize possible forms for a bulk operator. Using previous results on 3-point functions we construct a local bulk operator in each OPE channel. We then impose the condition that the bulk operators constructed in different channels agree, and hence give rise to a well-defined bulk operator. We refer to this condition as the "bulk bootstrap." We argue and explicitly show in some examples that the bulk bootstrap leads to some of the same results as the regular conformal bootstrap. In fact the bulk bootstrap provides an easier way to determine some CFT data, since it does not require knowing the form of the conformal blocks. This analysis clarifies previous results on the relation between bulk locality and the bootstrap for theories with a 1/N expansion, and it identifies a simple and direct way in which OPE coefficients and anomalous dimensions deter...

  8. Mechanical properties of Ag added Dy123 low porosity bulks

    Mechanical properties of Ag added DyBa2Cu3Ox (Dy123) low porosity bulks were evaluated by bending tests of specimens cut from the bulks. The bending strength of the Ag added low porosity bulks was higher than that of a Dy123 low porosity bulk without Ag addition tested in the previous study. No significant difference in the area fraction of Ag particles was observed among the bending test specimens. On the other hand, an increase of the bending strength with decrease of the average Ag particle size was observed. It is deduced that small Ag particles effectively suppress the crack propagation.

  9. Xerophilic mycopopulations of teas in bulk

    Škrinjar Marija M.

    2011-01-01

    Full Text Available d.o.o., Novi Sad AU Krunić Vesna J. AF EKOLd.o.o., Novi Sad KW teas % mould contamination % thermal treatment KR nema Other the water, tea is the most popular beverage in the world today. They are used for ages, in the beginning as refreshing drinks, and later more for their healing properties. Teas have been demonstrated to show antioxidative, anti-carcinogenic, and anti-microbial properties. Considering that the teas, during the production, are not treated with any temperature, there is high risk for contamination with different type of microorganisms, especially with moulds. Moulds are ubiquitously distributed in nature and their spores can be found in the atmosphere even at high altitudes and under favorable conditions of temperature and humidity, moulds grow on many commodities including cereals, oil seeds, nuts, herbs and spices. Most of them are potential producers of mycotoxins which present a real hazard to human health. The aim of this work was to investigate total mould count and to identify moulds isolated from teas in bulk, than from teas treated with hot, sterile, distilled water and from the tea filtrates. Tested teas were peppermint, sage, yarrow, black tea, bearberry, lemon balm, mixture of teas from Zlatibor. In teas in balk was observed high contamination with different kinds of moulds (1.84-4.55 cfu/g, such as Aspergillus awamori, A. lovaniensis, A niger, A. phoenicus, A. repens, A. restrictus, A. sydowii, A. versicolor, Eurotium amstelodami, E. chevalieri, E. herbariorum, Penicillium chrysogenum, and Scopulariopsis brevicaulis. The most frequent were species from Aspergillus and Eurotium genera. Thermal treatment with hot, sterile, distilled water reduced the number of fungal colonies. Aspergillus awamori was the most resistant and appeared in six samples of filtrates of tea, Aspergillus niger in one sample and Penicillium chrysogenum in one sample.

  10. Cavitation instability in bulk metallic glasses

    Dai L.H.

    2015-01-01

    Full Text Available Recent experiments have shown that fracture surfaces of bulk metallic glasses (BMGs usually exhibit an intriguing nanoscale corrugation like fractographic feature mediated by nanoscale void formation. We attribute the onset of this nanoscale corrugation to TTZs (tension transformation zones mediated cavitation. In our recent study, the spall experiments of Zr-based BMG using a single-stage light gas gun were performed. To uncover the mechanisms of the spallation damage nucleation and evolution, the samples were designed to be subjected to dynamic tensile loadings of identical amplitude but with different durations by making use of the multi-stress pulse and the double-flyer techniques. It is clearly revealed that the macroscopic spall fracture in BMGs originates from the nucleation, growth and coalescence of micro-voids. Then, a microvoid nucleation model of BMGs based on free volume theory is proposed, which indicates that the nucleation of microvoids at the early stage of spallation in BMGs is resulted from diffusion and coalescence of free volume. Furthermore, a theoretical model of void growth in BMGs undergoing remote dynamic hydrostatic tension is developed. The critical condition of cavitation instability is obtained. It is found that dynamic void growth in BMGs can be well controlled by a dimensionless inertial number characterizing the competition between intrinsic and extrinsic time scales. To unveil the atomic-level mechanism of cavitation, a systematic molecular dynamics (MD simulation of spallation behaviour of a binary metallic glass with different impact velocities was performed. It is found that micro-void nucleation is determined TTZs while the growth is controlled by shear transformation zones (STZs at atomic scale.

  11. Cavitation instability in bulk metallic glasses

    Dai, L. H.; Huang, X.; Ling, Z.

    2015-09-01

    Recent experiments have shown that fracture surfaces of bulk metallic glasses (BMGs) usually exhibit an intriguing nanoscale corrugation like fractographic feature mediated by nanoscale void formation. We attribute the onset of this nanoscale corrugation to TTZs (tension transformation zones) mediated cavitation. In our recent study, the spall experiments of Zr-based BMG using a single-stage light gas gun were performed. To uncover the mechanisms of the spallation damage nucleation and evolution, the samples were designed to be subjected to dynamic tensile loadings of identical amplitude but with different durations by making use of the multi-stress pulse and the double-flyer techniques. It is clearly revealed that the macroscopic spall fracture in BMGs originates from the nucleation, growth and coalescence of micro-voids. Then, a microvoid nucleation model of BMGs based on free volume theory is proposed, which indicates that the nucleation of microvoids at the early stage of spallation in BMGs is resulted from diffusion and coalescence of free volume. Furthermore, a theoretical model of void growth in BMGs undergoing remote dynamic hydrostatic tension is developed. The critical condition of cavitation instability is obtained. It is found that dynamic void growth in BMGs can be well controlled by a dimensionless inertial number characterizing the competition between intrinsic and extrinsic time scales. To unveil the atomic-level mechanism of cavitation, a systematic molecular dynamics (MD) simulation of spallation behaviour of a binary metallic glass with different impact velocities was performed. It is found that micro-void nucleation is determined TTZs while the growth is controlled by shear transformation zones (STZs) at atomic scale.

  12. Silicon bulk micromachined hybrid dimensional artifact.

    Claudet, Andre A.; Tran, Hy D.; Bauer, Todd Marks; Shilling, Katherine Meghan; Oliver, Andrew David

    2010-03-01

    A mesoscale dimensional artifact based on silicon bulk micromachining fabrication has been developed and manufactured with the intention of evaluating the artifact both on a high precision coordinate measuring machine (CMM) and video-probe based measuring systems. This hybrid artifact has features that can be located by both a touch probe and a video probe system with a k=2 uncertainty of 0.4 {micro}m, more than twice as good as a glass reference artifact. We also present evidence that this uncertainty could be lowered to as little as 50 nm (k=2). While video-probe based systems are commonly used to inspect mesoscale mechanical components, a video-probe system's certified accuracy is generally much worse than its repeatability. To solve this problem, an artifact has been developed which can be calibrated using a commercially available high-accuracy tactile system and then be used to calibrate typical production vision-based measurement systems. This allows for error mapping to a higher degree of accuracy than is possible with a glass reference artifact. Details of the designed features and manufacturing process of the hybrid dimensional artifact are given and a comparison of the designed features to the measured features of the manufactured artifact is presented and discussed. Measurement results from vision and touch probe systems are compared and evaluated to determine the capability of the manufactured artifact to serve as a calibration tool for video-probe systems. An uncertainty analysis for calibration of the artifact using a CMM is presented.

  13. Bulk forming of industrial micro components in conventional metals and bulk metallic glasses

    Arentoft, Mogens; Paldan, Nikolas Aulin; Eriksen, Rasmus Solmer; Gastaldi, T.; Wert, John A.; Eldrup, Morten Mostgaard

    2007-01-01

    glasses will at elevated temperatures behave like a highly viscous liquid, which can easily form even complicated geometries in 1 step. The strengths and limitations of forming the 2 materials are analyzed for a micro 3D component in a silver alloy and an Mg-Cu-Y BMG. ©2007 American Institute of Physics......For production of micro components in large numbers, forging is an interesting and challenging process. The conventional metals like silver, steel and aluminum often require multi-step processes, but high productivity and increased strength justify the investment. As an alternative, bulk metallic...

  14. Calculation of structurally related properties of bulk and surface Si

    The self-consistent pseudopotential method is applied to study the bulk and surface structurally related properties of Si. Equilibrium configurations are determined by minimizing the total energy of the system; the calculated bulk properties and the surface relaxation of Si are found to be in good agreement with experiment. The surface energy and the surface reconstruction of Si are briefly discussed

  15. Advanced and new developments in bulk metal forming

    Bay, Niels; Wanheim, Tarras; Ravn, Bjarne Gottlieb; Arentoft, Mogens

    Increasing demands to manufacturing industry of faster, better and cheaper production has intensified the research and development of bulk metal forming. The present paper gives examples on European industrial research on secondary bulk metal forming processes. The R&D follows three lines of appr...

  16. Synthesizing Bulk Density for Soils with Abundant Rock Fragments

    Vincent, K. R.; Chadwick, O. A.

    1994-01-01

    Bulk density is a fundamental soil property that is difficult to determine for gravelly to extremely gravelly soils because results vary significantly with sample volume. For such coarse soils, the representative volume (for whole-soil bulk density) should be large, but guidelines for selecting an appropriate sample volume do not exist.

  17. T-Duality Simplifies Bulk-Boundary Correspondence

    Mathai, Varghese; Thiang, Guo Chuan

    2016-07-01

    Recently, we introduced T-duality in the study of topological insulators. In this paper, we study the bulk-boundary correspondence for three phenomena in condensed matter physics, namely, the quantum Hall effect, the Chern insulator, and time reversal invariant topological insulators. In all of these cases, we show that T-duality trivializes the bulk-boundary correspondence.

  18. 77 FR 12293 - PCBs Bulk Product v. Remediation Waste

    2012-02-29

    ... AGENCY PCBs Bulk Product v. Remediation Waste AGENCY: Environmental Protection Agency (EPA). ACTION... remediation waste. The proposed reinterpretation is ] in response to questions EPA received about the... regarding PCB bulk product and PCB remediation waste under regulations promulgated at 40 CFR part 761....

  19. 7 CFR 58.211 - Packaging room for bulk products.

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Packaging room for bulk products. 58.211 Section 58... Service 1 Rooms and Compartments § 58.211 Packaging room for bulk products. A separate room or area shall... dust within the packaging room and where needed, a dust collector shall be provided and...

  20. 27 CFR 24.301 - Bulk still wine record.

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Bulk still wine record. 24..., DEPARTMENT OF THE TREASURY LIQUORS WINE Records and Reports § 24.301 Bulk still wine record. A proprietor who produces or receives still wine in bond, (including wine intended for use as distilling material or...

  1. Stability of bulk metallic glass structure

    Jain, H.; Williams, D.B.

    2003-06-18

    The fundamental origins of the stability of the (Pd-Ni){sub 80}P{sub 20} bulk metallic glasses (BMGs), a prototype for a whole class of BMG formers, were explored. While much of the properties of their BMGs have been characterized, their glass-stability have not been explained in terms of the atomic and electronic structure. The local structure around all three constituent atoms was obtained, in a complementary way, using extended X-ray absorption fine structure (EXAFS), to probe the nearest neighbor environment of the metals, and extended energy loss fine structure (EXELFS), to investigate the environment around P. The occupied electronic structure was investigated using X-ray photoelectron spectroscopy (XPS). The (Pd-Ni){sub 80}P{sub 20} BMGs receive their stability from cumulative, and interrelated, effects of both atomic and electronic origin. The stability of the (Pd-Ni){sub 80}P{sub 20} BMGs can be explained in terms of the stability of Pd{sub 60}Ni{sub 20}P{sub 20} and Pd{sub 30}Ni{sub 50}P{sub 20}, glasses at the end of BMG formation. The atomic structure in these alloys is very similar to those of the binary phosphide crystals near x=0 and x=80, which are trigonal prisms of Pd or Ni atoms surrounding P atoms. Such structures are known to exist in dense, randomly-packed systems. The structure of the best glass former in this series, Pd{sub 40}Ni{sub 40}P{sub 20} is further described by a weighted average of those of Pd{sub 30}Ni{sub 50}P{sub 20} and Pd{sub 60}Ni{sub 20}P{sub 20}. Bonding states present only in the ternary alloys were found and point to a further stabilization of the system through a negative heat of mixing between Pd and Ni atoms. The Nagel and Tauc criterion, correlating a decrease in the density of states at the Fermi level with an increase in the glass stability, was consistent with greater stability of the Pd{sub x}Ni{sub (80-x)}P{sub 20} glasses with respect to the binary alloys of P. A valence electron concentration of 1.8 e/a, which

  2. Renormalization group approach to causal bulk viscous cosmological models

    Belinchon, J A [Grupo Inter-Universitario de Analisis Dimensional, Dept. Fisica ETS Arquitectura UPM, Av. Juan de Herrera 4, Madrid (Spain); Harko, T [Department of Physics, University of Hong Kong, Pokfulam Road, Hong Kong (China); Mak, M K [Department of Physics, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong (China)

    2002-06-07

    The renormalization group method is applied to the study of homogeneous and flat Friedmann-Robertson-Walker type universes, filled with a causal bulk viscous cosmological fluid. The starting point of the study is the consideration of the scaling properties of the gravitational field equations, the causal evolution equation of the bulk viscous pressure and the equations of state. The requirement of scale invariance imposes strong constraints on the temporal evolution of the bulk viscosity coefficient, temperature and relaxation time, thus leading to the possibility of obtaining the bulk viscosity coefficient-energy density dependence. For a cosmological model with bulk viscosity coefficient proportional to the Hubble parameter, we perform the analysis of the renormalization group flow around the scale-invariant fixed point, thereby obtaining the long-time behaviour of the scale factor.

  3. Renormalization group approach to causal bulk viscous cosmological models

    The renormalization group method is applied to the study of homogeneous and flat Friedmann-Robertson-Walker type universes, filled with a causal bulk viscous cosmological fluid. The starting point of the study is the consideration of the scaling properties of the gravitational field equations, the causal evolution equation of the bulk viscous pressure and the equations of state. The requirement of scale invariance imposes strong constraints on the temporal evolution of the bulk viscosity coefficient, temperature and relaxation time, thus leading to the possibility of obtaining the bulk viscosity coefficient-energy density dependence. For a cosmological model with bulk viscosity coefficient proportional to the Hubble parameter, we perform the analysis of the renormalization group flow around the scale-invariant fixed point, thereby obtaining the long-time behaviour of the scale factor

  4. Carbon nanotubes grown on bulk materials and methods for fabrication

    Menchhofer, Paul A.; Montgomery, Frederick C.; Baker, Frederick S.

    2011-11-08

    Disclosed are structures formed as bulk support media having carbon nanotubes formed therewith. The bulk support media may comprise fibers or particles and the fibers or particles may be formed from such materials as quartz, carbon, or activated carbon. Metal catalyst species are formed adjacent the surfaces of the bulk support material, and carbon nanotubes are grown adjacent the surfaces of the metal catalyst species. Methods employ metal salt solutions that may comprise iron salts such as iron chloride, aluminum salts such as aluminum chloride, or nickel salts such as nickel chloride. Carbon nanotubes may be separated from the carbon-based bulk support media and the metal catalyst species by using concentrated acids to oxidize the carbon-based bulk support media and the metal catalyst species.

  5. HP1 recruits activity-dependent neuroprotective protein to H3K9me3 marked pericentromeric heterochromatin for silencing of major satellite repeats.

    Kerstin Mosch

    Full Text Available H3 lysine 9 trimethylation (H3K9me3 is a histone posttranslational modification (PTM that has emerged as hallmark of pericentromeric heterochromatin. This constitutive chromatin domain is composed of repetitive DNA elements, whose transcription is differentially regulated. Mammalian cells contain three HP1 proteins, HP1α, HP1β and HP1γ These have been shown to bind to H3K9me3 and are thought to mediate the effects of this histone PTM. However, the mechanisms of HP1 chromatin regulation and the exact functional role at pericentromeric heterochromatin are still unclear. Here, we identify activity-dependent neuroprotective protein (ADNP as an H3K9me3 associated factor. We show that ADNP does not bind H3K9me3 directly, but that interaction is mediated by all three HP1 isoforms in vitro. However, in cells ADNP localization to areas of pericentromeric heterochromatin is only dependent on HP1α and HP1β. Besides a PGVLL sequence patch we uncovered an ARKS motif within the ADNP homeodomain involved in HP1 dependent H3K9me3 association and localization to pericentromeric heterochromatin. While knockdown of ADNP had no effect on HP1 distribution and heterochromatic histone and DNA modifications, we found ADNP silencing major satellite repeats. Our results identify a novel factor in the translation of H3K9me3 at pericentromeric heterochromatin that regulates transcription.

  6. Ephedrae herba stimulates hepatocyte growth factor-induced MET endocytosis and downregulation via early/late endocytic pathways in gefitinib-resistant human lung cancer cells.

    Nishimura, Yukio; Hyuga, Sumiko; Takiguchi, Soichi; Hyuga, Masashi; Itoh, Kazuyuki; Hanawa, Toshihiko

    2016-05-01

    The MET tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), are known to be overexpressed in a variety of malignant tumor cells, and are implicated in the development of gefitinib-resistance in human non-small cell lung cancer (NSCLC) cells. Ephedrae herba was previously reported to prevent HGF-induced cancer cell motility by directly suppressing HGF/MET signaling through the inhibition of MET tyrosine kinase, and treatment with its extract also considerably reduced MET protein levels. To further investigate the mechanism underlying the Ephedrae herba-induced inhibition of MET phosphorylation as well as its degradation and subsequent disappearance, we examined the effect of Ephedrae herba on HGF-stimulated MET endocytosis and downregulation via early/late endocytic pathways in an NSCLC cell line. Using immunofluorescence microscopy, we found that pretreatment of cells with Ephedrae herba extract dramatically changed the intracellular distribution of plasma membrane-associated MET, and that the resultant MET staining was distributed throughout the cytoplasm. Pretreatment of the cells with Ephedrae herba extract also led to the rapid loss of MET and phosphorylated (p)-MET in HGF-stimulated cells. In contrast, inefficient endocytic delivery of MET and p-MET from early to late endosomes was observed in the absence of Ephedrae herba extract, since considerable amounts of the internalized MET accumulated in the early endosomes and were not delivered to lysosomes up to 1 h after HGF-stimulation. Furthermore, large amounts of MET and p-MET that had accumulated in late endosomes of Ephedrae herba-pretreated cells after HGF stimulation were observed along with bafilomycin A1. Therefore, we inferred that degradation of MET occurred in the late endosome/lysosome pathway. Moreover, western blot analysis revealed the accelerated degradation of MET and p-MET proceeds in cells pretreated with Ephedrae herba extract. Collectively, our results suggest that

  7. Engineering nanostructural routes for enhancing thermoelectric performance: bulk to nanoscale

    Rajeshkumar eMohanraman

    2015-11-01

    Full Text Available Thermoelectricity is a very important physical property, especially its significance in heat-electricity conversion. If thermoelectric devices can be effectively applied to the recovery of the renewable energies, such as waste heat and solar energy, the energy shortage and global warming issues may be greatly relieved. This review focusses recent developments on the thermoelectric performance of a low-dimensional material, bulk nanostructured materials, conventional bulk materials etc. Particular emphasis is given on, how the nanostructure in nanostructured composites, confinement effects in one-dimensional nanowires and doping effects in conventional bulk composites plays an important role in ZT enhancement.

  8. Engineering Nanostructural Routes for Enhancing Thermoelectric Performance: Bulk to Nanoscale

    Mohanraman, Rajeshkumar; Lan, Tian-Wey; Hsiung, Te-Chih; Amada, Dedi; Lee, Ping-Chung; Ou, Min-Nan; Chen, Yang-Yuan

    2015-01-01

    Thermoelectricity is a very important phenomenon, especially its significance in heat-electricity conversion. If thermoelectric devices can be effectively applied to the recovery of the renewable energies, such as waste heat and solar energy, the energy shortage, and global warming issues may be greatly relieved. This review focusses recent developments on the thermoelectric performance of a low-dimensional material, bulk nanostructured materials, conventional bulk materials etc. Particular emphasis is given on, how the nanostructure in nanostructured composites, confinement effects in one-dimensional nanowires and doping effects in conventional bulk composites plays an important role in ZT enhancement. PMID:26913280

  9. Theoretical analysis of ac losses in high temperature superconducting bulks

    Ac loss behavior in HTS bulk materials is studied by developing an analytic method. The self-heating effect on the distributions of temperature in HTS bulks has been calculated. A critical frequency which sets the upper limit for driving frequency of the external field in sustaining a superconducting state is obtained. Our model provides useful information of understanding the experimental phenomena, including an anomalous ac loss behavior, a retardation of the inner field respect to the external ac field, etc., in HTS bulk materials

  10. Eco Issues in Bulk Materials Handling Technologies in Ports

    Nenad Zrnić

    2011-09-01

    Full Text Available This paper deals with eco issues in bulk materials handling in ports. Solid, free-flowing materials are said to be in bulk. Bulk materials handling is very difficult, because it incorporates all the features of liquids, gasses and mass solids. Energy efficiency, dust emissions in nearby environment, dust explosions, jamming, noise, handling of hazardous materials and protection of materials from contamination are issues that will be considered in this paper. Here are also presented possible solutions for some of these issues

  11. Bulk Local States and Crosscaps in Holographic CFT

    Nakayama, Yu

    2016-01-01

    In a weakly coupled gravity theory in the anti-de Sitter space, local states in the bulk are linear superpositions of Ishibashi states for a crosscap in the dual conformal field theory. The superposition structure can be constrained either by the microscopic causality in the bulk gravity or the bootstrap condition in the boundary conformal field theory. We show, contrary to some expectation, that these two conditions are not compatible to each other in the weak gravity regime. We also present an evidence to show that bulk local states in three dimensions are not organized by the Virasoro symmetry.

  12. Theoretical evaluation of bulk viscosity: Expression for relaxation time

    Hossein Mohammad Zaheri, Ali; Srivastava, Sunita; Tankeshwar, K.

    2007-10-01

    A theoretical calculation of bulk viscosity has been carried out by deriving an expression for the relaxation time which appears in the formula for bulk viscosity derived by Okumura and Yonezawa. The expression involved a pair distribution function and interaction potential. Numerical results have been obtained over a wide range of densities and temperatures for Lennard-Jones fluids. It is found that our results provide a good description of bulk viscosity as has been judged by comparing the results with nonequilibrium molecular dynamics results. In addition, our results demonstrate the importance of the multiparticle correlation function.

  13. Unipolar memristive switching in bulk positive temperature coefficient ceramic thermistor

    Wu, Hongya; Wang, Caihui; Fu, Hua; Zhou, Ji; Zheng, Shuzhi

    2016-01-01

    A memristive switching phenomena was investigated in macroscale bulk positive temperature coefficient (PTC) thermosensitive ceramics. (BaxSr1-x)TiO3, which is a well-known PTC thermistor, was taken as an example to analyze the memristive behavior of those macroscale bulk ceramics. Hysteretic current-voltage (I-V) characteristics, which are the features of memristor were obtained. The origin of the effect is attributed to the PTC thermosensitive characteristic of the bulk ceramics, and a switching mechanism driven by competing field-driven heat generation and heat dissipation was proposed.

  14. Bulk sound velocity of porous materials at high pressures

    耿华运; 吴强; 谭华; 蔡灵仓; 经福谦

    2002-01-01

    A correction of Walsh's method for bulk sound velocity calculation for shocked porous materials is accomplishedbased on the Wu-Jing thermodynamic equation of state. The corrected bulk velocities for solid and porous sampleswith low porosities are in good agreement with the corresponding experimental data published previously. On the basisof this corrected equation, the influence of thermoelectrons on the bulk velocity of shocked materials is discussed indetail at pressures of 50, 70 and 200 GPa. Some interesting phenomena are revealed, which seem to be the uniquefeatures of a dynamic-pressure-loading process and could not be found in static experiments.

  15. Spontaneous localization of bulk fields: the six-dimensional case

    We study N=2 supersymmetric gauge theories with d=6 bulk and d=4 brane fields charged under a U(1) gauge symmetry. Radiatively induced Fayet-Iliopoulos terms lead to an instability of the bulk fields. We compute the profile of the bulk zero modes and observe the phenomenon of spontaneous localization towards the position of the branes. While this mechanism is quite similar to the d=5 case, the mass spectrum of the excited Kaluza-Klein modes shows a crucial difference

  16. Evaluation of All Nonsynonymous Single-Nucleotide Polymorphisms in the Gene Encoding Human Deoxyribonuclease I-Like 1, Possibly Implicated in the Blocking of Endocytosis-Mediated Foreign Gene Transfer

    Ueki, Misuzu; Kimura-Kataoka, Kaori; Fujihara, Junko; Takeshita, Haruo; Iida, Reiko; Yasuda, Toshihiro

    2014-01-01

    Many nonsynonymous single-nucleotide polymorphisms (SNPs) in the human deoxyribonuclease I-like 1 (DNase 1L1) gene, possibly implicated in the blocking of endocytosis-mediated foreign gene transfer, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all 21 nonsynonymous human DNase 1L1 SNPs was performed in 16 different populations representing three ethnic groups using the PCR-restriction fragment...

  17. Practical technique of pulsed field magnetization for bulk HTS application

    We studied a pulsed field magnetization (PFM) of bulk HTS assembled into a synchronous motor as a field-pole. The PFM is essential to apply bulk HTS inside the machine as a practical technique. In the present study, we developed a PFM technique that is a usage of Controlled Magnetic density Distribution Coil (CMDC). The coil is composed of inner vortex coil and outer solenoid. We successfully obtained the trapped flux density with 1.3 T by the step-wise cooling method with CMDC at 38 K in the motor. The bulk was cooled by a condensed neon. In addition, we studied the PFM for Gd-bulk of 140 mm diameter. By using the CMDC, we obtained the trapped flux density distribution with regular shape. In this paper we report these advanced PFM techniques for a practical machinery applications

  18. Bulk metallic glasses: A new class of engineering materials

    Joysurya Basu; S Ranganathan

    2003-06-01

    Bulk glass-forming alloys have emerged over the past fifteen years with attractive properties and technological promise. A number of alloy systems based on lanthanum, magnesium, zirconium, palladium, iron, cobalt and nickel have been discovered. Glass-forming ability depends on various factors like enthalpy of mixing, atomic size and multicomponent alloying. A number of processes is available to synthesise bulk metallic glasses. The crystallisation behaviour and mechanical properties of these alloys pose interesting scientific questions. Upon crystallisation many of these glasses transform to bulk nanocrystals and nanoquasicrystals. A detailed study of the structure and the crystallisation behaviour of glasses has enabled the elucidation of the possible atomic configuration in liquid alloys. Their crystallisation behaviour can be exploited to synthesise novel nanocomposite microstructures and their mechanical properties can be enhanced. A broad overview of the present status of the science and technology of bulk metallic glasses and their potential technological uses is presented.

  19. Control over magnetic properties in bulk hybrid materials

    Urban, Christian; Quesada, Adrian; Saerbeck, Thomas; Rubia, Miguel Angel De La; Garcia, Miguel Angel; Fernandez, Jose Francisco; Schuller, Ivan K.; UCSD Collaboration; Instituto de Ceramica, Madrid Collaboration; Institut Laue-Langevin, Grenoble Collaboration

    We present control of coercivity and remanent magnetization of a bulk ferromagnetic material embedded in bulk vanadium sesquioxide (V2O3) by using a standard bulk synthesis procedure. The method generalizes the use of structural phase transitions of one material to control structural and magnetic properties of another. A structural phase transition (SPT) in the V2O3 host material causes magnetic properties of Ni to change as function of temperature. The remanent magnetization and the coercivity are reversibly controlled by the SPT without additional external magnetic fields. The reversible tuning shown here opens the pathway for controlling the properties of a vast variety of magnetic hybrid bulk systems. This Work is supported by the Office of Basic Energy Science, U.S. Department of Energy, BES-DMS funded by the Department of Energy's Office of Basic Energy Science, DMR under grant DE FG02 87ER-45332.

  20. Fluid-fluid transitions at bulk supercritical conditions.

    Xie, Fei; Woodward, Clifford E; Forsman, Jan

    2013-02-26

    We use three different polymer solvent mixture models to theoretically determine the existence of capillary-induced phase separation in simple pores under supercritical bulk conditions. These models undergo bulk demixing, due to quite different mechanisms, yet readily display supercritical transitions without the use of esoteric interactions in the capillary. The theoretical method used to analyze these systems is density functional theory. We find that capillary demixing is not reliant on the presence of a pure surface transition but may occur in the absence of the latter. This is shown by considering cases where the surface enhancement factor is too weak to cause demixing at a single surface or else the bulk conditions are supercritical to both bulk and surface transitions. This phenomenon may prove useful in applications involving adsorption from mixtures into porous particles. PMID:23360176