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Sample records for activates junctional endocytosis

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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...

  8. 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...

  9. Actin Depolymerization Disrupts Tight Junctions via Caveolae-mediated EndocytosisV⃞

    Shen, Le; Turner, Jerrold R.

    2005-01-01

    The tight junction (TJ) determines epithelial barrier function. Actin depolymerization disrupts TJ structure and barrier function, but the mechanisms of this effect remain poorly understood. The goal of this study was to define these mechanisms. Madin-Darby canine kidney (MDCK) cells expressing enhanced green fluorescent protein-, enhanced yellow fluorescent protein-, or monomeric red fluorescent protein 1-fusion proteins of β-actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and cav...

  10. 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...

  11. 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

  12. 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

  13. 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

  14. 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...

  15. Activated Microglia do not form Functional Gap Junctions in vivo

    Wasseff, Sameh K.; Scherer, Steven S.

    2014-01-01

    We investigated whether microglia form gap junctions with themselves, or with astrocytes, oligodendrocytes, or neurons in vivo in normal mouse brains, and in pathological conditions that induce microglial activation - brain injury, a model of Alzheimer’s disease. Although microglia are in close physical proximity to glia and neurons, they do not form functional gap junctions under these pathological conditions.

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

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

    2014-01-01

    Most tubes have “seams” – intercellular or autocellular junctions that seal membranes together into a tube – but “seamless” tubes also exist [1-3]. 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 ...

  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. 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

  20. 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 ...

  1. Junction temperature estimation for an advanced active power cycling test

    Choi, Uimin; Blaabjerg, Frede; Jørgensen, S.

    estimation method using on-state VCE for an advanced active power cycling test is proposed. The concept of the advanced power cycling test is explained first. Afterwards the junction temperature estimation method using on-state VCE and current is presented. Further, the method to improve the accuracy of the...

  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. Thermally activated magnetization reversal in magnetic tunnel junctions

    Zhou Guang-Hong; Wang Yin-Gang; Qi Xian-Jin; Li Zi-Quan; Chen Jian-Kang

    2009-01-01

    In this paper, the magnetization reversal of the ferromagnetic layers in the lrMn/CoFe/AlOx/CoFe magnetic tunnel junction has been investigated using bulk magnetometry. The films exhibit very complex magnetization processes and reversal mechanism. Thermal activation phenomena such as the training effect, the asymmetry of reversal, the loop broadening and the decrease of exchange field while holding the film at negative saturation have been observed on the hysteresis loops of the pinned ferromagnetic layer while not on those of the free ferromagnetic layer. The thermal activation phenomena observed can be explained by the model of two energy barrier distributions with different time constants.

  4. Active zone stability:insights from fly neuromuscular junction

    Xiaolin Tian; Chunlai Wu

    2015-01-01

    The presynaptic active zone is a dynamic structure that orchestrates regulated release of neurotrans-mitters. Developmental and aging processes, and changes in neuronal network activity can all modulate the number, size and composition of active zone and thereby synaptic efifcacy. However, very little is known about the mechanism that controls the structural stability of active zone. By study-ing a model synapse, theDrosophila neuromuscular junction, our recent work shed light on how two scaffolding proteins at the active zone regulate active zone stability by promoting a localized dephos-phorylation event at the nerve terminal. Here we discuss the major insights from our ifndings and their implications for future research.

  5. 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

  6. 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.

  7. 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.

  8. 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...

  9. Enhanced BBB permeability of osmotically active poly(mannitol-co-PEI) modified with rabies virus glycoprotein via selective stimulation of caveolar endocytosis for RNAi therapeutics in Alzheimer's disease.

    Park, Tae-Eun; Singh, Bijay; Li, Huishan; Lee, Jun-Yeong; Kang, Sang-Kee; Choi, Yun-Jaie; Cho, Chong-Su

    2015-01-01

    RNA interference (RNAi) holds one of the promising tools for Alzheimer's disease (AD) treatment by directly arresting the causative genes. For successful RNAi therapeutics for AD, limited access of therapeutic genes to the brain needs to be overcome by developing siRNA delivery system that could cross the blood-brain barrier (BBB). Here, we report a non-viral vector, rabies virus glycoprotein (RVG)-modified poly(mannitol-co-PEI) gene transporter (PMT), R-PEG-PMT. The RVG ligand directed the PMT/siRNA complexes toward the brain through binding to nicotinic acetylcholine receptors expressed on BBB. In mechanistic study using in vitro BBB model, we observed that osmotically-active PMT enhanced the receptor-mediated transcytosis by stimulating the caveolar endocytosis. The potential of RNAi therapeutics for AD using R-PEG-PMT/siBACE1 complexes was demonstrated in vitro and in vivo. Our results suggest that R-PEG-PMT is a powerful gene carrier system for brain targeted RNAi therapeutics with synergistic effect of RVG ligand and PMT on well-modulated receptor-mediated transcytosis through BBB. PMID:25457984

  10. 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 ...

  11. E-cadherin junction formation involves an active kinetic nucleation process.

    Biswas, Kabir H; Hartman, Kevin L; Yu, Cheng-han; Harrison, Oliver J; Song, Hang; Smith, Adam W; Huang, William Y C; Lin, Wan-Chen; Guo, Zhenhuan; Padmanabhan, Anup; Troyanovsky, Sergey M; Dustin, Michael L; Shapiro, Lawrence; Honig, Barry; Zaidel-Bar, Ronen; Groves, Jay T

    2015-09-01

    Epithelial (E)-cadherin-mediated cell-cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role. PMID:26290581

  12. ApoER2 expression increases Aβ production while decreasing Amyloid Precursor Protein (APP endocytosis: Possible role in the partitioning of APP into lipid rafts and in the regulation of γ-secretase activity

    Bu Guojun

    2007-07-01

    Full Text Available Abstract Background The generation of the amyloid-β peptide (Aβ through the proteolytic processing of the amyloid precursor protein (APP is a central event in the pathogenesis of Alzheimer's disease (AD. Recent studies highlight APP endocytosis and localization to lipid rafts as important events favoring amyloidogenic processing. However, the precise mechanisms underlying these events are poorly understood. ApoER2 is a member of the low density lipoprotein receptor (LDL-R family exhibiting slow endocytosis rate and a significant association with lipid rafts. Despite the important neurophysiological roles described for ApoER2, little is known regarding how ApoER2 regulates APP trafficking and processing. Results Here, we demonstrate that ApoER2 physically interacts and co-localizes with APP. Remarkably, we found that ApoER2 increases cell surface APP levels and APP association with lipid rafts. The increase of cell surface APP requires the presence of ApoER2 cytoplasmic domain and is a result of decreased APP internalization rate. Unexpectedly, ApoER2 expression correlated with a significant increase in Aβ production and reduced levels of APP-CTFs. The increased Aβ production was dependent on the integrity of the NPxY endocytosis motif of ApoER2. We also found that expression of ApoER2 increased APP association with lipid rafts and increased γ-secretase activity, both of which might contribute to increased Aβ production. Conclusion These findings show that ApoER2 negatively affects APP internalization. However, ApoER2 expression stimulates Aβ production by shifting the proportion of APP from the non-rafts to the raft membrane domains, thereby promoting β-secretase and γ-secretase mediated amyloidogenic processing and also by incrementing the activity of γ-secretase.

  13. 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 ...

  14. The Effects of Exercise-induced Fatigue on Acetylcholinesterase Expression and Activity at Rat Neuromuscular Junctions

    Wen, Guo; Hui, Wang; Dan, Chen; Xiao-Qiong, Wu; Jian-Bin, Tong; Chang-Qi, Li; De-Liang, Lei; Wei-Jun, Cai; Zhi-Yuan, Li; Xue-Gang, Luo

    2009-01-01

    Acetylcholinesterase is the enzyme that terminates neurotransmission by hydrolyzing the acetylcholine released by the motoneurons at the neuromuscular junctions. Although acetylcholinesterase has been studied for almost a century, the underlying relationship between exercise-induced fatigue and acetylcholinesterase activity at the synaptic cleft is not clear. The purpose of this study was to assess the effects of exercise-induced fatigue on the expression and activity of acetylcholinesterase ...

  15. 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

  16. 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.

  17. Mathematical modeling of gap junction coupling and electrical activity in human β-cells

    Loppini, Alessandro; Braun, Matthias; Filippi, Simonetta; Gram Pedersen, Morten

    2015-12-01

    Coordinated insulin secretion is controlled by electrical coupling of pancreatic β-cells due to connexin-36 gap junctions. Gap junction coupling not only synchronizes the heterogeneous β-cell population, but can also modify the electrical behavior of the cells. These phenomena have been widely studied with mathematical models based on data from mouse β-cells. However, it is now known that human β-cell electrophysiology shows important differences to its rodent counterpart, and although human pancreatic islets express connexin-36 and show evidence of β-cell coupling, these aspects have been little investigated in human β-cells. Here we investigate theoretically, the gap junction coupling strength required for synchronizing electrical activity in a small cluster of cells simulated with a recent mathematical model of human β-cell electrophysiology. We find a lower limit for the coupling strength of approximately 20 pS (i.e., normalized to cell size, ˜2 pS pF-1) below which spiking electrical activity is asynchronous. To confront this theoretical lower bound with data, we use our model to estimate from an experimental patch clamp recording that the coupling strength is approximately 100-200 pS (10-20 pS pF-1), similar to previous estimates in mouse β-cells. We then investigate the role of gap junction coupling in synchronizing and modifying other forms of electrical activity in human β-cell clusters. We find that electrical coupling can prolong the period of rapid bursting electrical activity, and synchronize metabolically driven slow bursting, in particular when the metabolic oscillators are in phase. Our results show that realistic coupling conductances are sufficient to promote synchrony in small clusters of human β-cells as observed experimentally, and provide motivation for further detailed studies of electrical coupling in human pancreatic islets.

  18. An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor.

    Staerk, Judith; Lacout, Catherine; Sato, Takeshi; Smith, Steven O.; Vainchenker, William; Constantinescu, Stefan

    2006-01-01

    Ligand binding to the thrombopoietin receptor (TpoR) is thought to impose a dimeric receptor conformation(s) leading to hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. Unlike other cytokine receptors, such as the erythropoietin receptor, TpoR contains an amphipathic KWQFP motif at the junction between the transmembrane (TM) and cytoplasmic domains. We show here that a mutant TpoR (delta5TpoR), where this sequence was deleted, is constitutively active. I...

  19. Triplet supercurrent due to spin-active zones in a Josephson junction

    Linder, Jacob; Sudbø, Asle

    2010-07-01

    Motivated by a recent experiment evidencing triplet superconductivity in a ferromagnetic Josephson junction with a Cu2MnAl -Heusler barrier, we construct a theoretical model accounting for this observation. The key ingredients in our model which generate the triplet supercurrent are spin-active zones, characterized by an effective canted interface magnetic moment. Using a numerical solution of the quasiclassical equations of superconductivity with spin-active boundary conditions, we find qualitatively very good agreement with the experimentally observed supercurrent. Further experimental implications of the spin-active zones are discussed.

  20. Activation energy of fractional vortices and spectroscopy of a vortex molecule in long Josephson junction

    This thesis is divided into two parts, the measurement of the activation energy of a fractional vortex and the spectroscopy of a vortex-molecule. Fractional vortices can be studied in long 0-κ Josephson junctions, where a jump of the Josephson phase is created artificially with a pair of tiny current injectors. To compensate for this phase discontinuity, a ρ vortex is formed. Here, ρ describes the vortex's so called topological charge. The ρ vortices are pinned at the discontinuity and they carry the fraction (ρ/2).Φ0 of magnetic flux, with the magnetic flux quantum Φ0 2.07.10-15. Two stable vortex configurations are possible, a direct Vortex and a complementary one. ρ depends on the injector current. When the bias current of the junction exceeds a characteristic threshold, which dependents on ρ, the Lorentz force is bigger than the pinning force of the vortex and a fluxon is pulled away. In this case a complementary (ρ-2π) vortex is left behind. This switching of the ρ vortex and the resulting emission of a fluxon can be described as a Kramers like escape of a particle out of a tilted washboard potential. The washboard potential is tilted to the point where the barrier is small enough, so that the particle can escape via thermal or quantum fluctuations. In the case of thermal fluctuations the barrier height is called activation energy. The activation energy can be determined by measuring the junction's switching current statistics. In this thesis, the activation energy, necessary for the vortex escape, was measured as a function of ρ and a homogenous external magnetic field perpendicular to the junction. The main focus was the investigation of 0-π junctions. The temperature dependence of the activation energy was investigated, too. It turns out, that the transition-state-theory is convenient to describe the switching probability of the standard Nb-AlOx-Nb junctions at 4.2 K. For the measurements at 0.5 K a model of low to intermediate damping results

  1. 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...

  2. 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

  3. Neural progenitor cells isolated from the subventricular zone present hemichannel activity and form functional gap junctions with glial cells

    Talaverón, Rocío; Fernández, Paola; Escamilla, Rosalba; Pastor, Angel M.; Matarredona, Esperanza R.; Sáez, Juan C.

    2015-01-01

    The postnatal subventricular zone (SVZ) lining the walls of the lateral ventricles contains neural progenitor cells (NPCs) that generate new olfactory bulb interneurons. Communication via gap junctions between cells in the SVZ is involved in NPC proliferation and in neuroblast migration towards the olfactory bulb. SVZ NPCs can be expanded in vitro in the form of neurospheres that can be used for transplantation purposes after brain injury. We have previously reported that neurosphere-derived NPCs form heterocellular gap junctions with host glial cells when they are implanted after mechanical injury. To analyze functionality of NPC-glial cell gap junctions we performed dye coupling experiments in co-cultures of SVZ NPCs with astrocytes or microglia. Neurosphere-derived cells expressed mRNA for at least the hemichannel/gap junction channel proteins connexin 26 (Cx26), Cx43, Cx45 and pannexin 1 (Panx1). Dye coupling experiments revealed that gap junctional communication occurred among neurosphere cells (incidence of coupling: 100%). Moreover, hemichannel activity was also detected in neurosphere cells as evaluated in time-lapse measurements of ethidium bromide uptake. Heterocellular coupling between NPCs and glial cells was evidenced in co-cultures of neurospheres with astrocytes (incidence of coupling: 91.0 ± 4.7%) or with microglia (incidence of coupling: 71.9 ± 6.7%). Dye coupling in neurospheres and in co-cultures was inhibited by octanol, a gap junction blocker. Altogether, these results suggest the existence of functional hemichannels and gap junction channels in postnatal SVZ neurospheres. In addition, they demonstrate that SVZ-derived NPCs can establish functional gap junctions with astrocytes or microglia. Therefore, cell-cell communication via gap junctions and hemichannels with host glial cells might subserve a role in the functional integration of NPCs after implantation in the damaged brain. PMID:26528139

  4. 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 ...

  5. [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

  6. 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.

  7. Block of gap junctions eliminates aberrant activity and restores light responses during retinal degeneration.

    Toychiev, Abduqodir H; Ivanova, Elena; Yee, Christopher W; Sagdullaev, Botir T

    2013-08-28

    Retinal degeneration leads to progressive photoreceptor cell death, resulting in vision loss. Subsequently, inner retinal neurons develop aberrant synaptic activity, compounding visual impairment. In retinal ganglion cells, light responses driven by surviving photoreceptors are obscured by elevated levels of aberrant spiking activity. Here, we demonstrate in rd10 mice that targeting disruptive neuronal circuitry with a gap junction antagonist can significantly reduce excessive spiking. This treatment increases the sensitivity of the degenerated retina to light stimuli driven by residual photoreceptors. Additionally, this enhances signal transmission from inner retinal neurons to ganglion cells, potentially allowing the retinal network to preserve the fidelity of signals either from prosthetic electronic devices, or from cells optogenetically modified to transduce light. Thus, targeting maladaptive changes to the retina allows for treatments to use existing neuronal tissue to restore light sensitivity, and to augment existing strategies to replace lost photoreceptors. PMID:23986234

  8. 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

  9. 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-...

  10. Metabolic stabilization of acetylcholine receptors in vertebrate neuromuscular junction by muscle activity

    The effects of muscle activity on the growth of synaptic acetylcholine receptor (AChR) accumulations and on the metabolic AChR stability were investigated in rat skeletal muscle. Ectopic end plates induced surgically in adult soleus muscle were denervated early during development when junctional AChR number and stability were still low and, subsequently, muscles were either left inactive or they were kept active by chronic exogenous stimulation. AChR numbers per ectopic AChR cluster and AChR stabilities were estimated from the radioactivity and its decay with time, respectively, of end plate sites whose AChRs had been labeled with 125I-alpha-bungarotoxin (alpha-butx). The results show that the metabolic stability of the AChRs in ectopic clusters is reversibly increased by muscle activity even when innervation is eliminated very early in development. 1 d of stimulation is sufficient to stabilize the AChRs in ectopic AChR clusters. Muscle stimulation also produced an increase in the number of AChRs at early denervated end plates. Activity-induced cluster growth occurs mainly by an increase in area rather than in AChR density, and for at least 10 d after denervation is comparable to that in normally developing ectopic end plates. The possible involvement of AChR stabilization in end plate growth is discussed

  11. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  12. Hydrogenated Amorphous Silicon Germanium Active Layer for Top Cell of a Multi Junction Cell Structure.

    Cho, Jaehyun; Iftiquar, S M; Kim, Minbum; Park, Jinjoo; Jung, Junhee; Kim, Jiwoong; Yi, Junsin

    2016-05-01

    Intrinsic hydrogenated amorphous silicon-germanium (a-SiGe:H) alloy is generally used in the bottom cell because of its low band gap. The a-SiGe:H has a higher photo conductivity in comparison to the a-Si:H; thus, it is expected that the a-SiGe:H can show better short circuit current density than that of the a-Si:H based solar cell. Therefore, we optimized a-SiGe:H active layer that can be a suitable choice for the front cell of a multi junction.solar cell. Furthermore, we carried out a comparative study of the solar cells that have a-SiGe:H and a-Si:H as respective active layers. The a-SiGe:H based solar cells show higher short circuit current density, while the a-Si:H based cells show higheropen circuit voltage. The current-voltage characteristics of these cells are as follows: (a) V(oc) = 770 mV, J(sc) = 15.0 mA/cm2, FF = 64.5%, and η = 7.47% for a-SiGe:H based cell; and (b) V(oc) = 826 mV, J(sc) = 13.63 mA/cm2, FF = 72.0%, and η = 8.1% for a-Si:H based cell. PMID:27483837

  13. 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...

  14. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne;

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in...... extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx......43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium....

  15. 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

    misbalance between endocytosis and exocytosis could be involved in the anticonvulsant activity of the ketogenic diet. PMID:26748385

  16. Prostaglandin E2 Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Activity and Airway Surface Liquid Volume Requires Gap Junctional Communication

    Scheckenbach, K E Ludwig; Losa, Davide; Dudez, Tecla; Bacchetta, Marc; O'Grady, Scott; Crespin, Sophie; Chanson, Marc

    2010-01-01

    Stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by protease-activated receptors (PARs) at the basolateral membranes and by adenosine receptors (ADO-Rs) at the apical membrane maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Both pathways involve the release of prostaglandin E2 (PGE2) and the stimulation of their basolateral receptors (EP-Rs). We sought to determine whether gap junctions contribute to the coord...

  17. Prostaglandin E₂regulation of cystic fibrosis transmembrane conductance regulator activity and airway surface liquid volume requires gap junctional communication

    Scheckenbach, K E Ludwig; Losa, Davide; Dudez, Tecla; Bacchetta, Marc; O'Grady, Scott; Crespin, Sophie; Chanson, Marc

    2011-01-01

    Stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by protease-activated receptors (PARs) at the basolateral membranes and by adenosine receptors (ADO-Rs) at the apical membrane maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Both pathways involve the release of prostaglandin E₂ (PGE₂) and the stimulation of their basolateral receptors (EP-Rs). We sought to determine whether gap junctions contribute to the coord...

  18. 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.

  19. Conductance of redox-active single molecular junctions: an electrochemical approach

    The conductance of molecular junctions formed of N,N'-bis(n-thioalkyl)-4,4'-bipyridinium bromides or alkanedithiols between a gold (Au) scanning tunnelling microscope tip and a Au(111)-(1 x 1) electrode has been studied at electrified solid/liquid interfaces. A statistical analysis based on large sets of individual current-distance traces was applied to obtain the electrical conductance of single junctions. The one-electron reduction of the viologen moiety from the dication V2+ to the radical cation state V+. gives rise to a 50% increase of the junction conductance. Increasing the length of the alkyl spacer units leads to a tunnelling decay constant βCH2 = 5.9-6.1 nm-1. This value is significantly lower than βCH2 = 8.2 nm-1 estimated for molecular junctions of alkanedithiols. The difference is attributed to conformational changes within the two junctions. The contact conductance was estimated to 10 μS

  20. Musical molecules: the molecular junction as an active component in audio distortion circuits

    Molecular junctions that have a non-linear current–voltage characteristic consistent with quantum mechanical tunneling are demonstrated as analog audio clipping elements in overdrive circuits widely used in electronic music, particularly with electric guitars. The performance of large-area molecular junctions fabricated at the wafer level is compared to currently standard semiconductor diode clippers, showing a difference in the sound character. The harmonic distributions resulting from the use of traditional and molecular clipping elements are reported and discussed, and differences in performance are noted that result from the underlying physics that controls the electronic properties of each clipping component. In addition, the ability to tune the sound using the molecular junction is demonstrated. Finally, the hybrid circuit is compared to an overdriven tube amplifier, which has been the standard reference electric guitar clipped tone for over 60 years. In order to investigate the feasibility of manufacturing molecular junctions for use in commercial applications, devices are fabricated using a low-density format at the wafer level, where 38 dies per wafer, each containing two molecular junctions, are made with exceptional non-shorted yield (99.4%, representing 718 out of 722 tested devices) without requiring clean room facilities. (paper)

  1. Musical molecules: the molecular junction as an active component in audio distortion circuits

    Bergren, Adam Johan; Zeer-Wanklyn, Lucas; Semple, Mitchell; Pekas, Nikola; Szeto, Bryan; McCreery, Richard L.

    2016-03-01

    Molecular junctions that have a non-linear current-voltage characteristic consistent with quantum mechanical tunneling are demonstrated as analog audio clipping elements in overdrive circuits widely used in electronic music, particularly with electric guitars. The performance of large-area molecular junctions fabricated at the wafer level is compared to currently standard semiconductor diode clippers, showing a difference in the sound character. The harmonic distributions resulting from the use of traditional and molecular clipping elements are reported and discussed, and differences in performance are noted that result from the underlying physics that controls the electronic properties of each clipping component. In addition, the ability to tune the sound using the molecular junction is demonstrated. Finally, the hybrid circuit is compared to an overdriven tube amplifier, which has been the standard reference electric guitar clipped tone for over 60 years. In order to investigate the feasibility of manufacturing molecular junctions for use in commercial applications, devices are fabricated using a low-density format at the wafer level, where 38 dies per wafer, each containing two molecular junctions, are made with exceptional non-shorted yield (99.4%, representing 718 out of 722 tested devices) without requiring clean room facilities.

  2. 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

  3. Gap Junctions

    Goodenough, Daniel A.; Paul, David L.

    2009-01-01

    Gap junctions are aggregates of intercellular channels that permit direct cell–cell transfer of ions and small molecules. Initially described as low-resistance ion pathways joining excitable cells (nerve and muscle), gap junctions are found joining virtually all cells in solid tissues. Their long evolutionary history has permitted adaptation of gap-junctional intercellular communication to a variety of functions, with multiple regulatory mechanisms. Gap-junctional channels are composed of hex...

  4. Gap Junctions

    Nielsen, Morten Schak; Axelsen, Lene Nygaard; Sorgen, Paul L.; Verma, Vandana; Delmar, Mario; Holstein-Rathlou, Niels-Henrik

    2012-01-01

    Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of ...

  5. Temporo-Parietal Junction Activity in Theory-of-Mind Tasks: Falseness, Beliefs, or Attention

    Aichhorn, Markus; Perner, Josef; Weiss, Benjamin; Kronbichler, Martin; Staffen, Wolfgang; Ladurner, Gunther

    2009-01-01

    By combining the false belief (FB) and photo (PH) vignettes to identify theory-of-mind areas with the false sign (FS) vignettes, we re-establish the functional asymmetry between the left and right temporo-parietal junction (TPJ). The right TPJ (TPJ-R) is specially sensitive to processing belief information, whereas the left TPJ (TPJ-L) is equally…

  6. 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

  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. Functional chromaffin cell plasticity in response to stress: focus on nicotinic, gap junction, and voltage-gated Ca2+ channels.

    Guérineau, Nathalie C; Desarménien, Michel G; Carabelli, Valentina; Carbone, Emilio

    2012-10-01

    An increase in circulating catecholamines constitutes one of the mechanisms whereby human body responds to stress. In response to chronic stressful situations, the adrenal medullary tissue exhibits crucial morphological and functional changes that are consistent with an improvement of chromaffin cell stimulus-secretion coupling efficiency. Stimulus-secretion coupling encompasses multiple intracellular (chromaffin cell excitability, Ca(2+) signaling, exocytosis, endocytosis) and intercellular pathways (splanchnic nerve-mediated synaptic transmission, paracrine and endocrine communication, gap junctional coupling), each of them being potentially subjected to functional remodeling upon stress. This review focuses on three chromaffin cell incontrovertible actors, the cholinergic nicotinic receptors and the voltage-dependent T-type Ca(2+) channels that are directly involved in Ca(2+)-dependent events controlling catecholamine secretion and electrical activity, and the gap junctional communication involved in the modulation of catecholamine secretion. We show here that these three actors react differently to various stressors, sometimes independently, sometimes in concert or in opposition. PMID:22252244

  10. Neuroinflammation leads to region-dependent alterations in astrocyte gap junction communication and hemichannel activity

    Karpuk, Nikolay; Burkovetskaya, Maria; Fritz, Teresa; Angle, Amanda; Kielian, Tammy

    2011-01-01

    Inflammation attenuates gap junction (GJ) communication in cultured astrocytes. Here we utilized a well-characterized model of experimental brain abscess as a tool to query effects of the CNS inflammatory milieu on astrocyte GJ communication and electrophysiological properties. Whole-cell patch-clamp recordings were performed on GFP-positive astrocytes in acute brain slices from GFAP-GFP mice at 3 or 7 days following S. aureus infection in the striatum. Astrocyte GJ communication was signific...

  11. 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

  12. 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

  13. Synthesis and enhanced photoelectrocatalytic activity of p–n junction Co3O4/TiO2 nanotube arrays

    Highlights: ► Co3O4/TiO2 nanotube arrays (NTs) were prepared by an impregnating–deposition–decompostion method treatment. ► Co3O4/TiO2 NTs exhibit high photoelectrocatalytic (PEC) activity. ► The high PEC activity was attribute to the formation of p–n junction between Co3O4 and TiO2. - Abstract: Co3O4/TiO2 nanotube arrays (NTs) were prepared by depositing Co3O4 nanoparticles (NPs) on the tube wall of the self-organized TiO2 NTs using an impregnating–deposition–decompostion method. The prepared samples were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and UV–vis absorption spectroscopy. The photoelectrocatalytic (PEC) activity is evaluated by degradation of methyl orange (MO) aqueous solution. The prepared Co3O4/TiO2 NTs exhibit much higher PEC activity than TiO2 NTs due to the p–n junction formed between Co3O4 and TiO2.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. Fabrication of BiOBr nanosheets@TiO2 nanobelts p-n junction photocatalysts for enhanced visible-light activity

    Zhao, Yang; Huang, Xiang; Tan, Xin; Yu, Tao; Li, Xiangli; Yang, Libin; Wang, Shucong

    2016-03-01

    The construction of p-n junction structure is a smart strategy for improving the photocatalytic activity, since p-n junctions can inhibit the recombination of photo-induced charges. Herein, BiOBr nanosheets@TiO2 nanobelts p-n junction photocatalysts were prepared by assembling BiOBr nanosheets on the surface of TiO2 nanobelts via a hydrothermal route followed by a co-precipitation process. BiOBr@TiO2 p-n junction photocatalysts exhibited enhanced photocatalytic activity in photocatalytic H2 production over water splitting and photodegradation of Rhodamine B (RhB) under visible light irradiation. Mott-Schottky plots confirmed the formation of p-n junctions in the interface of BiOBr and TiO2. The enhanced photocatalytic performance can be ascribed to the 1D nanostructure and the formation of p-n junctions. This work shows a potential application of low cost BiOBr as a substitute for noble metals in photocatalytic H2 production under visible light irradiation.

  19. The nature of single-ion activity coefficients calculated from potentiometric measurements on cells with liquid junctions

    Highlights: → Problem of ionic activity coefficients, determined by potentiometry, is reconsidered. → They are found to be functions of mean activity coefficients and transport numbers of ions. → The finding is verified by calculations and comparing the results with reported data. → Calculations are performed for systems with single electrolytes and binary mixtures. - Abstract: Potentiometric measurements on cells with liquid junctions are sometimes used for calculations of single-ion activity coefficients in electrolyte solutions, the incidence of this being increased recently. As surmised by Guggenheim in the 1930s, such coefficients (of ions i), γi, are actually complicated functions of mean ionic activity coefficients, γ±, and transport numbers of ions, ti. In the present paper specific functions γi(γ±, ti) are derived for a number of cell types with an arbitrary mixture of strong electrolytes in a one-component solvent in the liquid-junction system. The cell types include cells with (i) identical electrodes, (ii) dissimilar electrodes reversible to the same ions, (iii) dissimilar electrodes reversible to ions of opposite charge signs, (iv) dissimilar electrodes reversible to different ions of the same charge sign, and (v) identical reference electrodes and an ion-selective membrane permeable to ions of only one type. Pairs of functions for oppositely charged ions are found to be consistent with the mean ionic activity coefficients as would be expected for pairs of the proper γi quantities by definition of γ±. The functions are tested numerically on some of the reported γi datasets that are the more tractable. A generally good agreement is found with data reported for cells with single electrolytes HCl and KCl in solutions, and with binary mixtures in the liquid-junction systems of KCl from the reference solutions and NaCl and HCl from the test solutions. It is found that values of γi(γ±, ti) functions, in general, do depend on the

  20. 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.

  1. 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.

  2. 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...

  3. Ultra-Shallow P+/N Junction Formation in Si Using Low Temperature Solid Phase Epitaxy Assisted with Laser Activation

    A combination of Ge pre-amorphization implantation (Ge-PAI), low-energy B implantation and laser annealing is a promising method to form highly-activated, abrupt and ultra-shallow junctions (USJ). In our previous report of IIT 2006, we succeeded in forming pn junctions less than 10 nm using non-melt double-pulsed green laser. However, a large leakage current under reverse bias was observed consequently due to residual defects in the implanted layer. In this study, a method to form USJ is proposed: a combination of low-temperature solid phase epitaxy and non-melt laser irradiation for B activation. Ge pre-amorphization implantation was performed at energy of 6 keV with a dose of 3x1014/cm2. Then B implantation was performed at energy of 0.2 keV with a dose of 1.2x1015/cm2. Samples were annealed at 400 deg. C for 10 h in nitrogen atmosphere. Subsequently, non-melt laser irradiation was performed at energy of 690 mJ/cm2 and pulse duration of 100 ns with intervals of 300 ns. As a result, USJ around 10 nm with better crystallinity was successfully formed. And the leakage current of pn diodes was reduced significantly. Moreover, it is proven from secondary ion mass spectroscopy (SIMS) analysis that transient enhanced diffusion (TED) of B is specifically suppressed.

  4. Electrochemical gate-controlled electron transport of redox-active single perylene bisimide molecular junctions

    We report a scanning tunneling microscopy (STM) experiment in an electrochemical environment which studies a prototype molecular switch. The target molecules were perylene tetracarboxylic acid bisimides modified with pyridine (P-PBI) and methylthiol (T-PBI) linker groups and with bulky tert-butyl-phenoxy substituents in the bay area. At a fixed bias voltage, we can control the transport current through a symmetric molecular wire Au|P-PBI(T-PBI)|Au by variation of the electrochemical 'gate' potential. The current increases by up to two orders of magnitude. The conductances of the P-PBI junctions are typically a factor 3 larger than those of T-PBI. A theoretical analysis explains this effect as a consequence of shifting the lowest unoccupied perylene level (LUMO) in or out of the bias window when tuning the electrochemical gate potential VG. The difference in on/off ratios reflects the variation of hybridization of the LUMO with the electrode states with the anchor groups. IT-ES(T) curves of asymmetric molecular junctions formed between a bare Au STM tip and a T-PBI (P-PBI) modified Au(111) electrode in an aqueous electrolyte exhibit a pronounced maximum in the tunneling current at -0.740, which is close to the formal potential of the surface-confined molecules. The experimental data were explained by a sequential two-step electron transfer process

  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. Electric field control of spin dynamics in a magnetically active tunnel junction

    The dynamics of a single spin embedded in a tunnelling junction is studied. Within a nonequilibrium Keldysh Green's function technique, we derive a quantum Langevin equation describing the spin dynamics. In the high temperature limit, it reduces to a Bloch equation, for which the spin relaxation rate, as determined by the temporal fluctuation, is linearly proportional to the temperature. In the opposite limit, the relaxation rate depends on the applied voltage, in contrast to the case of a spin in an equilibrium environment. We also show that spin-flip transition processes during electron tunnelling convert the applied electric field (i.e. voltage bias) into an effective magnetic field. Consequently, the dynamics of the spin, otherwise precessing along the static magnetic field, will have either a frequency shift proportional to the dc bias or a magnetic resonance driven indirectly by an ac electric field at the Larmor frequency ωL. An experiment to measure this effect is also proposed

  8. Ultra shallow junction formation and dopant activation study of Ga implanted Si

    Gwilliam, R. [Advanced Technology Institute, University of Surrey, Guildford, Surrey GU2 7XH (United Kingdom)]. E-mail: r.gwilliam@surrey.ac.uk; Gennaro, S. [ITC-irst Istituto Trentino di Cultura, Centro per la Ricerca Scientifica e Tecnologica, Via Sommarive 18, 38050 Povo (Trento) (Italy); Claudio, G. [Advanced Technology Institute, University of Surrey, Guildford, Surrey GU2 7XH (United Kingdom); Sealy, B.J. [Advanced Technology Institute, University of Surrey, Guildford, Surrey GU2 7XH (United Kingdom); Mulcahy, C. [Cascade Scientific Ltd., ETC Building, Brunel Science Park Uxbridge, Middlesex UB8 3PH (United Kingdom); Biswas, S. [Cascade Scientific Ltd., ETC Building, Brunel Science Park Uxbridge, Middlesex UB8 3PH (United Kingdom)

    2005-08-01

    The trend for decreasing geometries within CMOS architecture is driving the need for ever shallower, highly doped, low resistivity layers in silicon. The conventional dopant of choice, boron, as a result of its light mass requires that implant energies be ever reduced to meet the demands of these shallow junctions, with the inevitable effect on throughput due to implanter beam current limitations. In this paper we investigate using secondary ion mass spectrometry (SIMS), spreading resistance profiling (SRP) and Hall effect measurements, the alternate p-type dopant species of Ga and its behaviour in the energy range 2-5 keV, implanted into both single crystal Si and pre-amorphised material.

  9. 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

  10. 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

  11. 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....

  12. 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....

  13. 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....

  14. 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.

  15. 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

  16. 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...

  17. Inhibiting Invasion into Human Bladder Carcinoma 5637 Cells with Diallyl Trisulfide by Inhibiting Matrix Metalloproteinase Activities and Tightening Tight Junctions

    Yung Hyun Choi

    2013-10-01

    Full Text Available Diallyl trisulfide (DATS, an organosulfur compound in garlic, possesses pronounced anti-cancer potential. However, the anti-invasive mechanism of this compound in human bladder carcinoma is not fully understood. In this study, we evaluated the anti-invasive effects of DATS on a human bladder carcinoma (5637 cell line and investigated the underlying mechanism. The results indicated that DATS suppressed migration and invasion of 5637 cells by reducing the activities and expression of matrix metalloproteinase (MMP-2 and MMP-9 at both the protein and mRNA levels. DATS treatment up-regulated expression of tissue inhibitor of metalloproteinase (TIMP-1 and TIMP-2 in 5637 cells. The inhibitory effects of DATS on invasiveness were associated with an increase in transepithelial electrical resistance and repression of the levels of claudin family members. Although further studies are needed, our data demonstrate that DATS exhibits anti-invasive effects in 5637 cells by down-regulating the activity of tight junctions and MMPs. DATS may have future utility in clinical applications for treating bladder cancer.

  18. 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

  19. Tryptophan at the transmembrane–cytosolic junction modulates thrombopoietin receptor dimerization and activation

    Defour, Jean-Philippe; Itaya, Miki; Gryshkova, Vitalina; Brett, Ian C.; Pecquet, Christian; Sato, Takeshi; Smith, Steven O.; Stefan N. Constantinescu

    2013-01-01

    Dimerization of single-pass membrane receptors is essential for activation. In the human thrombopoietin receptor (TpoR), a unique amphipathic RWQFP motif separates the transmembrane (TM) and intracellular domains. Using a combination of mutagenesis, spectroscopy, and biochemical assays, we show that W515 of this motif impairs dimerization of the upstream TpoR TM helix. TpoR is unusual in that a specific residue is required for this inhibitory function, which prevents receptor self-activation....

  20. 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

  1. 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.

  2. OSCILLATING FLUID FLOW ACTIVATION OF GAP JUNCTION HEMICHANNELS INDUCES ATP RELEASE FROM MLO-Y4 OSTEOCYTES

    Genetos, Damian C.; Kephart, Curtis J.; Zhang, Yue; Yellowley, Clare E.; Donahue, Henry J.

    2007-01-01

    Mechanical loads are required for optimal bone mass. One mechanism whereby mechanical loads are transduced into localized cellular signals is strain-induced fluid flow through lacunae and canaliculi of bone. Gap junctions (GJ) between osteocytes and osteoblasts provides a mechanism whereby flow-induced signals are detected by osteocytes and transduced to osteoblasts. We have demonstrated the importance of GJ and gap junctional intercellular communication (GJIC) in intracellular calcium and pr...

  3. 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.

  4. 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

  5. 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.

  6. Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase

    McSherry, Elaine A

    2011-03-23

    Abstract Introduction The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. Methods MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. Results JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6

  7. Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase.

    McSherry, Elaine A

    2011-03-23

    ABSTRACT: INTRODUCTION: The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS: MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS: JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF

  8. Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase.

    McSherry, Elaine A

    2012-02-01

    INTRODUCTION: The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS: MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and beta1-integrin, we examined activation of the beta1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and beta1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS: JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the beta1-integrin substrate fibronectin. This was accompanied by reduced protein expression of beta1-integrin and its binding partners alphaV- and alpha5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and beta1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between

  9. Attenuated expression of the tight junction proteins is involved in clopidogrel-induced gastric injury through p38 MAPK activation

    Highlights: ► Clopidogrel suppressed GES-1 cell viability in a concentration- and time-dependent manner. ► Clopidogrel significantly increased dextran permeability, reduced occludin and ZO-1 expression, and induced cell apoptosis. ► Clopidogrel activated p38 MAPK signaling pathway. ► Activation of p38 activity was involved in clopidogrel-induced increase in gastric epithelial cells permeability and disruption of TJ. -- Abstract: Bleeding complications and delayed healing of gastric ulcer associated with use of clopidogrel is a common clinical concern; however, the underlying mechanisms remain to be determined. This study aimed to clarify whether clopidogrel could cause the damage of the human gastric epithelial cells and to further elucidate the mechanisms involved. After human gastric epithelial cell line GES-1 had been treated with clopidogrel (0.5–2.5 mM), the cell proliferation was examined by MTT assay, apoptosis was measured with DAPI staining and flow cytometry analysis, and the barrier function of the tight junctions (TJ) was evaluated by permeability measurement and transmission electron microscopy. Moreover, expression of the TJ proteins occludin and ZO-1 and the phosphorylation of the mitogen-activated protein kinases (MAPK) p38, ERK, and JNK were examined by western blot. In addition, three MAPK inhibitors specific to p38, ERK and JNK were used, respectively, to verify the signaling pathways responsible for regulating the expression of the TJ proteins being tested. Results showed that clopidogrel significantly increased dextran permeability, induced apoptosis, suppressed GES-1 cell viability, and reduced the expression of the TJ proteins (occludin and ZO-1), acting through p38 MAPK phosphorylation. Furthermore, these observed effects were partially abolished by SB-203580 (a p38 MAPK inhibitor), rather than by either U-0126 (an ERK inhibitor) or SP-600125 (a JNK inhibitor), suggesting that clopidogrel-induced disruption in the gastric

  10. 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

  11. Abnormal Activation of RhoA/ROCK-I Signaling in Junctional Zone Smooth Muscle Cells of Patients With Adenomyosis.

    Wang, S; Duan, H; Zhang, Y; Sun, F Q

    2016-03-01

    Adenomyosis (ADS) is a common estrogen-dependent gynecological disease with unknown etiology. The RhoA/Rho-kinase (ROCK) signaling pathway is involved in various cellular functions, including migration, proliferation, and smooth muscle contraction. Here we examined the potential role of this pathway in junctional zone (JZ) contraction in women with and without ADS. We demonstrated that in the normal JZ, RhoA and ROCK-I messenger RNA (mRNA) and protein expression was significantly higher in the proliferative phase of the menstrual cycle than in the secretory phase. Expression of RhoA and ROCK-I in the JZ from women with ADS was significantly higher than in the control women and showed no significant differences across the menstrual cycle. Treatment of JZ smooth muscle cells (JZSMCs) with estrogen at 0, 1, 10, or 100 nmol/L for 24 hours resulted in increased expression of RhoA, ROCK-I, and myosin light-chain (MLC) phosphorylation (p-MLC) in a dose-dependent manner. In parallel to its effects on p-MLC, estrogen-mediated, dose-dependent contraction responses in JZSMCs. Estrogen-mediated contraction in the ADS group was significantly higher than in the controls and also showed no significant differences across the menstrual cycle. These effects were suppressed in the presence of ICI 182780 or Y27632, supporting an estrogen receptor-dependent and RhoA activation-dependent mechanism. Our results indicate that the level of RhoA and ROCK-I increases in patients with ADS and the cyclic change is lost. Estrogen may affect uterine JZ contraction of ADS by enhancing RhoA/ ROCK-I signaling. PMID:26335177

  12. Impact of multiple sub-melt laser scans on the activation and diffusion of shallow Boron junctions

    Rosseel, E.; Vandervorst, W.; Clarysse, T.;

    2008-01-01

    Sub-melt laser annealing is a promising technique to achieve the required sheet resistance and junction depth specifications for the 32 nm technology node and beyond. In order to obtain a production worthy process with minimal sheet resistance variation at a macroscopic and microscopic level, car...... concentration dependent diffusion component....

  13. Similar oxysterols may lead to opposite effects on synaptic transmission: Olesoxime versus 5α-cholestan-3-one at the frog neuromuscular junction.

    Kasimov, M R; Zakyrjanova, G F; Giniatullin, A R; Zefirov, A L; Petrov, A M

    2016-07-01

    Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle. PMID:27102612

  14. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Xie, Yuchao [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Vinken, Mathieu [Department of Toxicology, Center for Pharmaceutical Sciences, Vrije Universiteit Brussels, 1090 Brussels (Belgium); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2013-12-15

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB

  15. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB

  16. 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...

  17. 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.

  18. 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.

  19. Activation energy of fractional vortices and spectroscopy of a vortex molecule in long Josephson junction; Aktivierungsenergie fraktionaler Flusswirbel und Spektroskopie an Vortex-Molekuelen in langen Josephsonkontakten

    Buckenmaier, Kai

    2010-06-09

    This thesis is divided into two parts, the measurement of the activation energy of a fractional vortex and the spectroscopy of a vortex-molecule. Fractional vortices can be studied in long 0-{kappa} Josephson junctions, where a jump of the Josephson phase is created artificially with a pair of tiny current injectors. To compensate for this phase discontinuity, a {rho} vortex is formed. Here, {rho} describes the vortex's so called topological charge. The {rho} vortices are pinned at the discontinuity and they carry the fraction ({rho}/2).{phi}{sub 0} of magnetic flux, with the magnetic flux quantum {phi}{sub 0} 2.07.10{sup -15}. Two stable vortex configurations are possible, a direct Vortex and a complementary one. {rho} depends on the injector current. When the bias current of the junction exceeds a characteristic threshold, which dependents on {rho}, the Lorentz force is bigger than the pinning force of the vortex and a fluxon is pulled away. In this case a complementary ({rho}-2{pi}) vortex is left behind. This switching of the {rho} vortex and the resulting emission of a fluxon can be described as a Kramers like escape of a particle out of a tilted washboard potential. The washboard potential is tilted to the point where the barrier is small enough, so that the particle can escape via thermal or quantum fluctuations. In the case of thermal fluctuations the barrier height is called activation energy. The activation energy can be determined by measuring the junction's switching current statistics. In this thesis, the activation energy, necessary for the vortex escape, was measured as a function of {rho} and a homogenous external magnetic field perpendicular to the junction. The main focus was the investigation of 0-{pi} junctions. The temperature dependence of the activation energy was investigated, too. It turns out, that the transition-state-theory is convenient to describe the switching probability of the standard Nb-AlO{sub x}-Nb junctions at 4.2 K

  20. Modulation of neural activity in the temporoparietal junction with transcranial direct current stimulation changes the role of beliefs in moral judgment

    Hang eYe

    2015-12-01

    Full Text Available Judgments about whether an action is morally right or wrong typically depend on our capacity to infer the actor’s beliefs and the outcomes of the action. Prior neuroimaging studies have found that mental state (e.g., beliefs, intentions attribution for moral judgment involves a complex neural network that includes the temporoparietal junction (TPJ. However, neuroimaging studies cannot demonstrate a direct causal relationship between the activity of this brain region and mental state attribution for moral judgment. In the current study, we used transcranial direct current stimulation (tDCS to transiently alter neural activity in the TPJ. The participants were randomly assigned to one of three stimulation treatments (right anodal/left cathodal tDCS, left anodal/right cathodal tDCS, or sham stimulation. Each participant was required to complete two similar tasks of moral judgment before receiving tDCS and after receiving tDCS. We studied whether tDCS to the TPJ altered mental state attribution for moral judgment. The results indicated that restraining the activity of the right temporoparietal junction (RTPJ or the left the temporoparietal junction (LTPJ decreased the role of beliefs in moral judgments and led to an increase in the dependence of the participants’ moral judgments on the action’s consequences. We also found that the participants exhibited reduced reaction times both in the cases of intentional harms and attempted harms after receiving right cathodal/left anodal tDCS to the TPJ. These findings inform and extend the current neural models of moral judgment and moral development in typically developing people and in individuals with neurodevelopmental disorders such as autism.

  1. Effects of junction geometry in crossover temperature to macroscopic quantum tunneling regime of intrinsic Josephson junctions

    We investigated the phase dynamics of Bi-2212 intrinsic Josephson junctions with two types of junction geometry. We found that a crossover temperature to the macroscopic quantum tunneling regime was quite different between the two types of junction geometry. The observed behavior is discussed in terms of an edge effect in long Josephson junctions dependent on the junction geometry. We investigated the phase dynamics of long intrinsic Josephson junctions, which were fabricated on a narrow bridge structure of Bi2Sr2CaCu2Oy (Bi-2212) single crystals by using a focused ion-beam etching. We measured the probability distribution of the switching events from the zero-voltage state for two types of junction geometry. One is a junction where the bridge width (L1) is larger than the Josephson penetration depth, λJ, and the distance between two slits (L2) is comparable to λJ, while the other is a junction where L1 is comparable to λJ and L2 is larger than λJ. We found that a crossover temperature from the thermally activated regime to the macroscopic quantum tunneling regime was quite different between the two types of junction geometry. We discuss the observed behavior in terms of an edge effect in long Josephson junctions dependent on the junction geometry.

  2. 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

  3. The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5β1 Integrin at the Macrophage-Amebae Intercellular Junction.

    Leanne Mortimer

    2015-05-01

    Full Text Available Entamoeba histolytica (Eh is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α5β1 integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α5β1 integrin and NLRP3 into the intercellular junction, where α5β1 integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α5β1 integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X7 receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α5β1 integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α5β1 integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α5β1 integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection.

  4. 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

  5. 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.

  6. Calcium-mediated transductive systems and functionally active gap junctions in astrocyte-like GL15 cells

    Steimberg Nathalie

    2001-05-01

    Full Text Available Abstract Background It has been proposed that GL15, a human cell line derived from glioblastoma multiforme, is a possible astroglial-like cell model, based on the presence of cytoplasmic glial fibrillary acidic protein. Results The aim of this work was to delineate the functional characteristics of GL15 cells using various experimental approaches, including the study of morphology, mechanism of induction of intracellular Ca2+ increase by different physiological agonists, and the presence and permeability of the gap-junction system during cell differentiation. Immunostaining experiments showed the presence and localization of specific glial markers, such as glial fibrillary acidic protein and S100B, and the lack of the neuronal marker S100A. Notably, all the Ca2+ pathways present in astrocytes were detected in GL15 cells. In particular, oscillations in intracellular Ca2+ levels were recorded either spontaneously, or in the presence of ATP or glutamate (but not KCl. Immunolabelling assays and confocal microscopy, substantiated by Western blot analyses, revealed the presence of connexin43, a subunit of astrocyte gap-junction channels. The protein is organised in characteristic spots on the plasma membrane at cell-cell contact regions, and its presence and distribution depends on the differentiative status of the cell. Finally, a microinjection/dye-transfer assay, employed to determine gap-junction functionality, clearly demonstrated that the cells were functionally coupled, albeit to varying degrees, in differentiated and undifferentiated phenotypes. Conclusions In conclusion, results from this study support the use of the GL15 cell line as a suitable in vitro astrocyte model, which provides a valuable guide for studying glial physiological features at various differentiation phases.

  7. 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...

  8. 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.

  9. 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.

  10. 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

  11. Macroscopic quantum tunneling in Josephson tunnel junctions and Coulomb blockade in single small tunnel junctions

    Experiments investigating the process of macroscopic quantum tunneling in a moderately-damped, resistively shunted, Josephson junction are described, followed by a discussion of experiments performed on very small capacitance normal-metal tunnel junctions. The experiments on the resistively-shunted Josephson junction were designed to investigate a quantum process, that of the tunneling of the Josephson phase variable under a potential barrier, in a system in which dissipation plays a major role in the dynamics of motion. All the parameters of the junction were measured using the classical phenomena of thermal activation and resonant activation. Theoretical predictions are compared with the experimental results, showing good agreement with no adjustable parameters; the tunneling rate in the moderately damped (Q ∼ 1) junction is seen to be reduced by a factor of 300 from that predicted for an undamped junction. The phase is seen to be a good quantum-mechanical variable. The experiments on small capacitance tunnel junctions extend the measurements on the larger-area Josephson junctions from the region in which the phase variable has a fairly well-defined value, i.e. its wavefunction has a narrow width, to the region where its value is almost completely unknown. The charge on the junction becomes well-defined and is predicted to quantize the current through the junction, giving rise to the Coulomb blockade at low bias. I present the first clear observation of the Coulomb blockade in single junctions. The electrical environment of the tunnel junction, however, strongly affects the behavior of the junction: higher resistance leads are observed to greatly sharpen the Coulomb blockade over that seen with lower resistance leads. I present theoretical descriptions of how the environment influences the junctions; comparisons with the experimental results are in reasonable agreement

  12. Density functional theory based direct comparison of coherent tunneling and electron hopping in redox-active single-molecule junctions

    Kastlunger, Georg; Stadler, Robert

    2015-03-01

    To define the conductance of single-molecule junctions with a redox functionality in an electrochemical cell, two conceptually different electron transport mechanisms, namely, coherent tunneling and vibrationally induced hopping, compete with each other, where implicit parameters of the setup such as the length of the molecule and the applied gate voltage decide which mechanism is the dominant one. Although coherent tunneling is most efficiently described within Landauer theory and the common theoretical treatment of electron hopping is based on Marcus theory, both theories are adequate for the processes they describe without introducing accuracy-limiting approximations. For a direct comparison, however, it has to be ensured that the crucial quantities obtained from electronic structure calculations, i.e., the transmission function T (E ) in Landauer theory and the transfer integral V , the reorganization energy λ , and the driving force Δ G0 in Marcus theory, are derived from similar grounds, as pointed out by Nitzan and coworkers in a series of publications. In this paper our framework is a single-particle picture, for which we perform density functional theory calculations for the conductance corresponding to both transport mechanisms for junctions with the central molecule containing one, two, or three Ruthenium centers, from which we extrapolate our results in order to define the critical length of the transition point of the two regimes which we identify at 5.76nm for this type of molecular wire. We also discuss trends in the dependence on an electrochemically induced gate potential.

  13. Morphological transformation and effect on gap junction intercellular communication in Syrian hamster embryo cells as screening tests for carcinogens devoid of mutagenic activity.

    Rivedal, E; Mikalsen, S O; Sanner, T

    2000-04-01

    A large fraction of chemicals observed to cause cancer in experimental animals is devoid of mutagenic activity. It is therefore of importance to develop methods that can be used to detect and study environmental carcinogenic agents that do not interact directly with DNA. Previous studies have indicated that induction of in vitro cell transformation and inhibition of gap junction intercellular communication are endpoints that could be useful for the detection of non-genotoxic carcinogens. In the present work, 13 compounds [chlordane, Arochlor 1260, di(2-ethylhexyl)phthalate, 1,1,1-trichloro-2, 2-bis(4-chlorophenyl)ethane, limonene, sodium fluoride, ethionine, o-anisidine, benzoyl peroxide, o-vanadate, phenobarbital, 12-O-tetradecanoylphorbol 13-acetate and clofibrate] have been tested for their ability to induce morphological transformation and affect intercellular communication in Syrian hamster embryo cells. The substances were selected on the basis of being proven or suspected non-genotoxic carcinogens, and thus difficult to detect in short-term tests. The data show that nine of the 13 compounds induced morphological transformation, and seven of the 13 inhibited intercellular communication in hamster embryo cells. Taken together, 12 of the 13 substances either induced transformation or caused inhibition of communication. The data suggest that the combined use of morphological transformation and gap junction intercellular communication in Syrian hamster embryo cells may be beneficial when screening for non-genotoxic carcinogens. PMID:10793297

  14. 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

  15. 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

  16. 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

  17. Modulatory effects of cAMP and PKC activation on gap junctional intercellular communication among thymic epithelial cells

    Neves-dos-Santos Sandra

    2010-01-01

    Full Text Available Abstract Background We investigated the effects of the signaling molecules, cyclic AMP (cAMP and protein-kinase C (PKC, on gap junctional intercellular communication (GJIC between thymic epithelial cells (TEC. Results Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC. Conclusions Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.

  18. Solitons in Josephson junctions

    Ustinov, A. V.

    1998-11-01

    Magnetic flux quanta in Josephson junctions, often called fluxons, in many cases behave as solitons. A review of recent experiments and modelling of fluxon dynamics in Josephson circuits is presented. Classic quasi-one-dimensional junctions, stacked junctions (Josephson superlattices), and discrete Josephson transmission lines (JTLs) are discussed. Applications of fluxon devices as high-frequency oscillators and digital circuits are also addressed.

  19. 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...

  20. 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...

  1. Specific deletion of AMP-activated protein kinase (α1AMPK in murine oocytes alters junctional protein expression and mitochondrial physiology.

    Michael J Bertoldo

    Full Text Available Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK, an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues. Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.

  2. 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.

  3. Current distributions of thermal switching in extremely underdamped Josephson junctions

    The first measurements of the switching current distribution of an extremely underdamped Josephson junction are presented at various temperatures. Careful fitting of the data provides an experimental verification of the thermal activation theory in the very low damping limit. Moreover, the fitting allows us to obtain the ''effective'' resistance of a Josephson tunnel junction, thus providing an important indication as to the proper junction resistance to be used in the resistively shunted junction model. These values of junction resistance show the temperature dependence of a subgap resistance, i.e., exp(Δ/k/sub B/T), due to activation of quasiparticles over the superconductor energy gap Δ

  4. 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

  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 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 ...

  6. 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

  7. 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.

  8. Supercurrent decay in extremely underdamped Josephson junctions

    We present an experimental study of the effective dissipation relevant in the thermally activated supercurrent decay of extremely underdamped Josephson junctions. Data referring to the supercurrent decay of Nb/AlOx/Nb Josephson junctions are compared with the Kramers theory. Our measurements allow us to obtain the open-quotes effectiveclose quotes resistance to be used in the resistively shunted junction model that results to be the subgap resistance due to the presence of thermally activated quasiparticles. The extremely low dissipation level obtained at low temperatures renders our result quite interesting in view of experiments in the quantum limit. copyright 1998 The American Physical Society

  9. Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

    Ma, W.H.; Liu, Y.J.; Wang, W.; Zhang, Y.Z. [The Third Hospital of Hebei Medical University, The Provincial Key Laboratory for Orthopedic Biomechanics of Hebei, Shijiazhuang, Hebei Province (China)

    2015-02-13

    Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation.

  10. Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

    Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation

  11. Spinal Gap Junction Channels in Neuropathic Pain

    Jeon, Young Hoon; Youn, Dong Ho

    2015-01-01

    Damage to peripheral nerves or the spinal cord is often accompanied by neuropathic pain, which is a complex, chronic pain state. Increasing evidence indicates that alterations in the expression and activity of gap junction channels in the spinal cord are involved in the development of neuropathic pain. Thus, this review briefly summarizes evidence that regulation of the expression, coupling, and activity of spinal gap junction channels modulates pain signals in neuropathic pain states induced...

  12. 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

  13. Homotypic gap junctional communication associated with metastasis increases suppression increases with PKA kinase activity and is unaffected by P13K inhibition

    Loss of gap junctional intercellular communication (GJIC) between cancer cells is a common characteristic of malignant transformation. This communication is mediated by connexin proteins that make up the functional units of gap junctions. Connexins are highly regulated at the protein level and phosp...

  14. 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...

  15. 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.

  16. 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

  17. 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...

  18. 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.

  19. The Masticatory Contractile Load Induced Expression and Activation of Akt1/PKBα in Muscle Fibers at the Myotendinous Junction within Muscle-Tendon-Bone Unit

    Yüksel Korkmaz

    2010-01-01

    Full Text Available The cell specific detection of enzyme activation in response to the physiological contractile load within muscle-tendon-bone unit is essential for understanding of the mechanical forces transmission from muscle cells via tendon to the bone. The hypothesis that the physiological mechanical loading regulates activation of Akt1/PKBα at Thr308 and at Ser473 in muscle fibers within muscle-tendon-bone unit was tested using quantitative immunohistochemistry, confocal double fluorescence analysis, and immunoblot analysis. In comparison to the staining intensities in peripheral regions of the muscle fibers, Akt1/PKBα was detected with a higher staining intensity in muscle fibers at the myotendinous junction (MTJ areas. In muscle fibers at the MTJ areas, Akt1/PKBα is dually phosphorylated at Thr308 and Ser473. The immunohistochemical results were confirmed by immunoblot analysis. We conclude that contractile load generated by masticatory muscles induces local domain-dependent expression of Akt1/PKBα as well as activation by dually phosphorylation at Thr308 and Ser473 in muscle fibers at the MTJ areas within muscle-tendon-bone unit.

  20. Molecular electronic junction transport

    Solomon, Gemma C.; Herrmann, Carmen; Ratner, Mark

    2012-01-01

    Whenasinglemolecule,oracollectionofmolecules,isplacedbetween two electrodes and voltage is applied, one has a molecular transport junction. We discuss such junctions, their properties, their description, and some of their applications. The discussion is qualitative rather than quantitative......, and focuses on mechanism, structure/function relations, regimes and mechanisms of transport, some molecular regularities, and some substantial challenges facing the field. Because there are many regimes and mechanisms in transport junctions, we will discuss time scales, geometries, and inelastic scattering...

  1. Zipper and freeway shear zone junctions

    Passchier, Cees; Platt, John

    2016-04-01

    Ductile shear zones are usually presented as isolated planar high-strain domains in a less deformed wall rock, characterised by shear sense indicators such as characteristic deflected foliation traces. Many shear zones, however, form branched systems and if movement on such branches is contemporaneous, the resulting geometry can be complicated and lead to unusual fabric geometries in the wall rock. For Y-shaped shear zone junctions with three simultaneously operating branches, and with slip directions at a high angle to the branch line, eight basic types of shear zone triple junctions are possible, divided into three groups. The simplest type, called freeway junctions, have similar shear sense on all three branches. If shear sense is different on the three branches, this can lead to space problems. Some of these junctions have shear zone branches that join to form a single branch, named zipper junctions, or a single shear zone which splits to form two, known as wedge junctions. Closing zipper junctions are most unusual, since they form a non-active high-strain zone with opposite deflection of foliations. Shear zipper and shear wedge junctions have two shear zones with similar shear sense, and one with the opposite sense. All categories of shear zone junctions show characteristic flow patterns in the shear zone and its wall rock. Shear zone junctions with slip directions normal to the branch line can easily be studied, since ideal sections of shear sense indicators lie in the plane normal to the shear zone branches and the branch line. Expanding the model to allow slip oblique and parallel to the branch line in a full 3D setting gives rise to a large number of geometries in three main groups. Slip directions can be parallel on all branches but oblique to the branch line: two slip directions can be parallel and a third oblique, or all three branches can have slip in different directions. Such more complex shear zone junctions cannot be studied to advantage in a

  2. 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.

  3. 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

  4. 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.

  5. Magnetic tunnel junctions (MTJs)

    2001-01-01

    We review the giant tunnel magnetoresistance (TMR) in ferromagnetic-insulator-ferromagnetic junctions discovered in recent years, which is the magnetoresistance (MR) associated with the spin-dependent tunneling between two ferromagnetic metal films separated by an insulating thin tunnel barrier. The theoretical and experimental results including junction conductance, magnetoresistance and their temperature and bias dependences are described.

  6. Stacked Josephson Junctions

    Madsen, Søren Find; Pedersen, Niels Falsig; Christiansen, Peter Leth

    2010-01-01

    Long Josephson junctions have for some time been considered as a source of THz radiation. Solitons moving coherently in the junctions is a possible source for this radiation. Analytical computations of the bunched state and bunching-inducing methods are reviewed. Experiments showing THz radiation...

  7. 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

  8. 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

  9. 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

  10. 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...

  11. 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...

  12. Significance and prognostic value of lysosomal enzyme activities measured in surgically operated adenocarcinomas of the gastroesophageal junction and squamous cell carcinomas of the lower third of esophagus

    Aron Altorjay; Balazs Paal; Nicolette Sohar; Janos Kiss; Imre Szanto; Istvan Sohar

    2005-01-01

    AIM: To establish whether there are fundamental differences in the biochemistries of adenocarcinomas of the gastroesophageal junction (GEJ) and the squamous cell carcinomas of the lower third of the esophagus (LTE).METHODS: Between February 1, 1997 and February 1,2000, we obtained tissue samples at the moment of resection from 54 patients for biochemical analysis. The full set of data could be comprehensively analyzed in 47 of 54 patients' samples (81%). Of these, 29 were adenocarcinomas of the GEJ Siewert type Ⅰ (n = 8), type Ⅱ (n = 12), type Ⅲ (n = 9), and 18 presented as squamous cell carcinomas of the LTE. We evaluated the mean values of 11-lysosomal enzyme and 1-cytosol protease activities of the tumorous and surrounding mucosae as well as their relative activities, measured as the ratio of activity in tumor and normal tissues from the same patient.These data were further analyzed to establish the correlation with tumor localization, TNM stage (lymph-node involvement), histological type (papillary, signet-ring cell,tubular), state of differentiation (good, moderate, poor),and survival (≤24 or ≥24 mo).RESULTS: In adenocarcinomas, the activity of α-mannosidase (AMAN), cathepsin B (CB) and dipeptidyl-peptidase Ⅰ (DPP Ⅰ) increased significantly as compared to the normal gastric mucosa. In squamous cell carcinomas of the esophagus, we also found a significant difference in the activity of cathepsin L and tripeptidyl-peptidase I in addition to these three. There was a statistical correlation of AMAN,CB, and DPP Ⅰ activity between the level of differentiation of adenocarcinomas of the GEJ and lymph node involvement,because tumors with no lymph node metastases histologically confirmed as well-differentiated, showed a significantly lower activity. The differences in CB and DPP Ⅰ activity correlated well with the differences in survival rates, since the CB and DPP Ⅰ values of those who died within 24 mo following surgical intervention were

  13. 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

  14. 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.

  15. Bronchoalveolar Lavage Fluid Utilized Ex Vivo to Validate In Vivo Findings: Inhibition of Gap Junction Activity in Lung Tumor Promotion is Toll-Like Receptor 4-Dependent

    Hill, Thomas; Osgood, Ross S; Velmurugan, Kalpana; Alexander, Carla-Maria; Upham, Brad L; Bauer, Alison K

    2013-01-01

    TLR4 protects against lung tumor promotion and pulmonary inflammation in mice. Connexin 43 (Cx43), a gap junction gene, was increased in Tlr4 wildtype compared to Tlr4-mutant mice in response to promotion, which suggests gap junctional intercellular communication (GJIC) may be compromised. We hypothesized that the early tumor microenvironment, represented by Bronchoalveolar Lavage Fluid (BALF) from Butylated hydroxytoluene (BHT; promoter)-treated mice, would produce TLR4-dependent changes in ...

  16. Macroscopic quantum tunneling and thermal activation in a small mesa structured Bi2Sr2CaCu2Oy intrinsic Josephson junctions

    Kitano, H.; Ota, K.; Hamada, K.; Takemura, R.; Ohmaki, M.; Maeda, A.; Suzuki, M.

    2009-03-01

    A nanometer-thick small mesa consiting of only two or three Bi2Sr2CaCu2Oy intrinsic Josephson junctions (IJJs) is studied through the switching current distribution measurements down to 0.4 K. Experimental results clearly show that the first switching events from the zero-voltage state for 1 K IJJs with several tens of junctions, in contrast to the recent result on a similar mesa-structured surface IJJ.

  17. 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

  18. 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

  19. Structural properties of the active layer of discotic hexabenzocoronene/perylene diimide bulk hetero junction photovoltaic devices: The role of alkyl side chain length

    We investigate thin blend films of phenyl-substituted hexa-peri-hexabenzocoronenes (HBC) with various alkyl side chain lengths ((CH2)n, n = 6, 8, 12 and 16)/perylenediimide (PDI). These blends constitute the active layers in bulk-hetero junction organic solar cells we studied recently [1]. Their structural properties are studied by both scanning electron microscopy and X-ray diffraction measurements. The results support the evidence for the formation of HBC donor-PDI acceptor complexes in all blends regardless of the side chain length of the HBC molecule. These complexes are packed into a layered structure parallel to the substrate for short side chain HBC molecules (n = 6 and 8). The layered structure is disrupted by increasing the side chain length of the HBC molecule and eventually a disordered structure is formed for long side chains (n > 12). We attribute this behavior to the size difference between the aromatic parts of the HBC and PDI molecules. For short side chains, the size difference results in a room for the side chains of the two molecules to fill in the space around the aromatic cores. For long side chains (n > 12), the empty space will not be enough to accommodate this increase, leading to the disruption of the layered structure and a rather disordered structure is formed. Our results highlight the importance of the donor-acceptor interaction in a bulk heterojunction active layer as well as the geometry of the two molecules and their role in determining the structure of the active layer and thus their photovoltaic performance.

  20. FSH modulates PKAI and GPR3 activities in mouse oocyte of COC in a gap junctional communication (GJC-dependent manner to initiate meiotic resumption.

    Junxia Li

    Full Text Available Many studies have shown that cyclic adenosine-5'-monophosphate (cAMP-dependent protein kinase A (PKA and G-protein-coupled receptor 3 (GPR3 are crucial for controlling meiotic arrest in oocytes. However, it is unclear how gonadotropins modulate these factors to regulate oocyte maturation, especially by gap junctional communication (GJC. Using an in vitro meiosis-arrested mouse cumulus-oocyte complex (COC culture model, we showed that there is a close relationship between follicle-stimulating hormone (FSH and the PKA type I (PKAI and GPR3. The effect of FSH on oocyte maturation was biphasic, initially inhibitory and then stimulatory. During FSH-induced maturation, rapid cAMP surges were observed in both cumulus cells and oocyte. Most GJC between cumulus cells and oocyte ceased immediately after FSH stimulation and recommenced after the cAMP surge. FSH-induced maturation was blocked by PKAI activator 8-AHA-cAMP. Levels of PKAI regulatory subunits and GPR3 decreased and increased, respectively, after FSH stimulation. In the presence of the GJC inhibitor carbenoxolone (CBX, FSH failed to induce the meiotic resumption and the changes in PKAI, GPR3 and cAMP surge in oocyte were no longer detected. Furthermore, GPR3 was upregulated by high cAMP levels, but not by PKAI activation. When applied after FSH stimulation, the specific phosphodiesterase 3A (PDE3A inhibitor cilostamide immediately blocked meiotic induction, regardless of when it was administered. PKAI activation inhibited mitogen-activated protein kinase (MAPK phosphorylation in the oocytes of COCs, which participated in the initiation of FSH-induced meiotic maturation in vitro. Just before FSH-induced meiotic maturation, cAMP, PKAI, and GPR3 returned to basal levels, and PDE3A activity and MAPK phosphorylation increased markedly. These experiments show that FSH induces a transient increase in cAMP levels and regulates GJC to control PKAI and GPR3 activities, thereby creating an inhibitory

  1. Promoting and avoiding recombination: contrasting activities of the Escherichia coli RuvABC Holliday junction resolvase and RecG DNA translocase.

    Zhang, Jing; Mahdi, Akeel A; Briggs, Geoffrey S; Lloyd, Robert G

    2010-05-01

    RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalyzing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam, or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb potentially pathological replication initiated via PriA protein at sites remote from oriC. PMID:20157002

  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. A Needle-Type p-i-n Junction Semiconductor Detector for In-Vivo Measurement of Beta Tracer Activity

    A miniature detector probe has been developed for in-vivo detection of beta tracer activity. A lithium-drifted p-i-n detector shaped as a cylinder 0.9 mm in diameter and 3 mm long acts as the sensing element. The detector is encased in a stainless steel tube 50 mm long, fastened to a holder fitted with a miniature coaxial contact. The free end of the tube has a syringe-like, entirely tight tip. The steel tube has an outer diameter of 1.4 mm except for 10 mm at the free end where the outer diameter is 1.1 mm corresponding to a wall thickness of 005 mm. The detector is placed in the 1.1 mm part of the tube. The construction and the properties of the probe are described

  4. Mechanically activated switching of Si-based single-molecule junction as imaged with three-dimensional dynamic probe

    Nakamura, Miki; Yoshida, Shoji; Katayama, Tomoki; Taninaka, Atsushi; Mera, Yutaka; Okada, Susumu; Takeuchi, Osamu; Shigekawa, Hidemi

    2015-10-01

    Understanding and extracting the full functions of single-molecule characteristics are key factors in the development of future device technologies, as well as in basic research on molecular electronics. Here we report a new methodology for realizing a three-dimensional (3D) dynamic probe of single-molecule conductance, which enables the elaborate 3D analysis of the conformational effect on molecular electronics, by the formation of a Si/single molecule/Si structure using scanning tunnelling microscopy (STM). The formation of robust covalent bonds between a molecule and Si electrodes, together with STM-related techniques, enables the stable and repeated control of the conformational modulation of the molecule. By 3D imaging of the conformational effect on a 1,4-diethynylbenzene molecule, a binary change in conductance with hysteresis is observed for the first time, which is considered to originate from a mechanically activated conformational change.

  5. 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...

  6. 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

  7. 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.

  8. Estimation of active faulting in a slow deformation area: Culoz fault as a case study (Jura-Western Alps junction).

    de La Taille, Camille; Jouanne, Francois; Crouzet, Christian; Jomard, Hervé; Beck, Christian; de Rycker, Koen; van Daele, Maarten; Lebourg, Thomas

    2014-05-01

    The north-western Alps foreland is considered as still experiencing distal effects of Alpine collision, resulting in both horizontal and vertical relative displacements. Based on seismological and geodetic surveys, detailed patterns of active faulting (including subsurface décollements, blind ramps and deeper crustal thrusts have been proposed (Thouvenot et al., 1998), underlining the importance of NW-SE left-lateral strike-slip offsets as along the Vuache and Culoz faults (cf. the 1996 Epagny event: M=5.4; Thouvenot et al., 1998 and the 1822 Culoz event I=VII-VIII; Vogt, 1979). In parallel to this tectonic evolution, the last glaciation-deglaciation cycles contributed to develop large and over-deepened lacustrine basins, such as Lake Le Bourget (Perrier, 1980). The fine grain, post LGM (ie post 18 ky), sedimentary infill gives a good opportunity to evidence late quaternary tectonic deformations. This study focuses on the Culoz fault, extending from the Jura to the West, to the Chautagne swamp and through the Lake Le Bourget to the East. Historical earthquakes are known nearby this fault as ie the 1822 Culoz event. The precise location and geometry of the main fault is illustrated but its Eastern termination still needs to be determined. High resolution seismic sections and side-scan sonar images performed in the 90's (Chapron et al., 1996) showed that the Col du Chat and Culoz faults have locally deformed the quaternary sedimentary infill of the lake. These studies, mainly devoted to paleo-climate analysis were not able to determine neither the geometry of the fault, or to quantify the observed deformations. A new campaign devoted to highlight the fault geometry and associated deformation, has been performed in October 2013. Very tight profiles were performed during this high resolution seismic survey using seistec boomer and sparker sources. In several places the rupture reaches the most recent seismic reflectors underlying that these faults were active during

  9. 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

  10. 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...

  11. 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...

  12. 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...

  13. 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...

  14. Quantum Junction Solar Cells

    Tang, Jiang

    2012-09-12

    Colloidal quantum dot solids combine convenient solution-processing with quantum size effect tuning, offering avenues to high-efficiency multijunction cells based on a single materials synthesis and processing platform. The highest-performing colloidal quantum dot rectifying devices reported to date have relied on a junction between a quantum-tuned absorber and a bulk material (e.g., TiO 2); however, quantum tuning of the absorber then requires complete redesign of the bulk acceptor, compromising the benefits of facile quantum tuning. Here we report rectifying junctions constructed entirely using inherently band-aligned quantum-tuned materials. Realizing these quantum junction diodes relied upon the creation of an n-type quantum dot solid having a clean bandgap. We combine stable, chemically compatible, high-performance n-type and p-type materials to create the first quantum junction solar cells. We present a family of photovoltaic devices having widely tuned bandgaps of 0.6-1.6 eV that excel where conventional quantum-to-bulk devices fail to perform. Devices having optimal single-junction bandgaps exhibit certified AM1.5 solar power conversion efficiencies of 5.4%. Control over doping in quantum solids, and the successful integration of these materials to form stable quantum junctions, offers a powerful new degree of freedom to colloidal quantum dot optoelectronics. © 2012 American Chemical Society.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. Pattern analysis of gap junction plaques with open and closed pores

    Müller, A.; Blanz, Wolf-Ekkehard; Laub, Gerhard; Hülser, Dieter F.

    1985-01-01

    The structure of freeze-fractured gap junctions was studied by electron microscopy and subsequent pattern analysis using a computer controlled image processing system. Rat mammary tumor cells (BICR/WIR-k) which are permanently coupled via gap junctions when cultered as monolayers were used under different fixed and unfixed conditions. Active (coupling competent) gap junctions seem to be characterized by loosely packed connexons, whereas non-active (permanently closed) gap junctions may consis...

  5. Spin currents in TFT-Josephson junction

    The spin of the Cooper pair in a triplet superconductor provides a new degree of freedom in Josephson junction physics. This can be accessed by using a magnetically-active tunneling barrier, leading to a rich variety of unconventional Josephson effects. Because of the triplet state of the pairing wavefunction, triplet superconductor junctions in general also display a Josephson spin current, which can flow even when the equilibrium charge current is vanishing. Using the quasiclassical Green's function theory, we have examined the more general situation of a magnetically-active barrier which does not conserve the spin of a tunneling Cooper-pair. We demonstrate that the Josephson spin currents on either side of the barrier need not be identical, with the magnitude, sign and orientation all allowed to differ. Not only do our calculations enhance the physical understanding of transport through triplet superconductor junctions, but they also open the possibility of novel spintronic Josephson devices.

  6. 75 FR 76294 - Radio Broadcasting Services: Pacific Junction, IA

    2010-12-08

    ... Junction, in overcoming objections raised by the FAA to the activation of this allotment. See 75 FR 30756... From the Federal Register Online via the Government Publishing Office FEDERAL COMMUNICATIONS COMMISSION 47 CFR Part 73 Radio Broadcasting Services: Pacific Junction, IA AGENCY: Federal...

  7. Junction-FET dosimeter

    The performance of a new junction-FET dosimeter and its application to the beam profile measurement are presented. One of the two junction FET's making up an astable multivibrator is used as a small-size (approx.0.4x0.4 mm) high-level dose detector. The irradiated dose can be estimated by the amount of the decrease of the oscillator period of the multivibrator. The distinct advantages in its small size and superior resistive property to radiation effect enable us to measure the cross-sectional profile of the electron beam from a linac with high spatial resolution of about 0.4 mm

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. Regulation of gap junctions by protein phosphorylation

    Sáez J.C.

    1998-01-01

    Full Text Available Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions.

  16. 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

  17. Endocytic activity of Sertoli cells grown in bicameral culture chambers

    Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding 125I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived

  18. Reduced Order Projective and Hybrid Projective Combination-Combination Synchronization of Four Chaotic Josephson Junctions

    K. S. Ojo; Njah, A. N.; O. I. Olusola; Omeike, M. O.

    2014-01-01

    This paper investigates the reduced order projective and hybrid projective combination-combination synchronization of four chaotic Josephson junctions consisting of two third order Josephson junctions as the drives and two second order chaotic Josephson junctions as the response systems via active backstepping technique. The investigation confirms the achievement of reduced order projective and hybrid projective combination-combination synchronization among four chaotic Josephson junctions vi...

  19. Proteomic and bioinformatic analysis of epithelial tight junction reveals an unexpected cluster of synaptic molecules

    Tang Vivian W

    2006-12-01

    Full Text Available Abstract Background Zonula occludens, also known as the tight junction, is a specialized cell-cell interaction characterized by membrane "kisses" between epithelial cells. A cytoplasmic plaque of ~100 nm corresponding to a meshwork of densely packed proteins underlies the tight junction membrane domain. Due to its enormous size and difficulties in obtaining a biochemically pure fraction, the molecular composition of the tight junction remains largely unknown. Results A novel biochemical purification protocol has been developed to isolate tight junction protein complexes from cultured human epithelial cells. After identification of proteins by mass spectroscopy and fingerprint analysis, candidate proteins are scored and assessed individually. A simple algorithm has been devised to incorporate transmembrane domains and protein modification sites for scoring membrane proteins. Using this new scoring system, a total of 912 proteins have been identified. These 912 hits are analyzed using a bioinformatics approach to bin the hits in 4 categories: configuration, molecular function, cellular function, and specialized process. Prominent clusters of proteins related to the cytoskeleton, cell adhesion, and vesicular traffic have been identified. Weaker clusters of proteins associated with cell growth, cell migration, translation, and transcription are also found. However, the strongest clusters belong to synaptic proteins and signaling molecules. Localization studies of key components of synaptic transmission have confirmed the presence of both presynaptic and postsynaptic proteins at the tight junction domain. To correlate proteomics data with structure, the tight junction has been examined using electron microscopy. This has revealed many novel structures including end-on cytoskeletal attachments, vesicles fusing/budding at the tight junction membrane domain, secreted substances encased between the tight junction kisses, endocytosis of tight junction

  20. 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)

  1. Model building to facilitate understanding of holliday junction and heteroduplex formation, and holliday junction resolution.

    Selvarajah, Geeta; Selvarajah, Susila

    2016-07-01

    Students frequently expressed difficulty in understanding the molecular mechanisms involved in chromosomal recombination. Therefore, we explored alternative methods for presenting the two concepts of the double-strand break model: Holliday junction and heteroduplex formation, and Holliday junction resolution. In addition to a lecture and computer-animated video, we included a model building activity using pipe cleaners. Biotechnology undergraduates (n = 108) used the model to simulate Holliday junction and heteroduplex formation, and Holliday junction resolution. Based on student perception, an average of 12.85 and 78.35% students claimed that they completely and partially understood the two concepts, respectively. A test conducted to ascertain their understanding about the two concepts showed that 66.1% of the students provided the correct response to the three multiple choice questions. A majority of the 108 students attributed the inclusion of model building to their better understanding of Holliday junction and heteroduplex formation, and Holliday junction resolution. This underlines the importance of incorporating model building, particularly in concepts that require spatial visualization. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(4):381-390, 2016. PMID:26899144

  2. 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.

  3. 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.

  4. 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

  5. Electron transport through molecular junctions

    At present, metal–molecular tunnel junctions are recognized as important active elements in molecular electronics. This gives a strong motivation to explore physical mechanisms controlling electron transport through molecules. In the last two decades, an unceasing progress in both experimental and theoretical studies of molecular conductance has been demonstrated. In the present work we give an overview of theoretical methods used to analyze the transport properties of metal–molecular junctions as well as some relevant experiments and applications. After a brief general description of the electron transport through molecules we introduce a Hamiltonian which can be used to analyze electron–electron, electron–phonon and spin–orbit interactions. Then we turn to description of the commonly used transport theory formalisms including the nonequilibrium Green’s functions based approach and the approach based on the “master” equations. We discuss the most important effects which could be manifested through molecules in electron transport phenomena such as Coulomb, spin and Frank–Condon blockades, Kondo peak in the molecular conductance, negative differential resistance and some others. Bearing in mind that first principles electronic structure calculations are recognized as the indispensable basis of the theory of electron transport through molecules, we briefly discuss the main equations and some relevant applications of the density functional theory which presently is often used to analyze important characteristics of molecules and molecular clusters. Finally, we discuss some kinds of nanoelectronic devices built using molecules and similar systems such as carbon nanotubes, various nanowires and quantum dots.

  6. Bronchoalveolar Lavage Fluid Utilized Ex Vivo to Validate In Vivo Findings: Inhibition of Gap Junction Activity in Lung Tumor Promotion is Toll-Like Receptor 4-Dependent.

    Hill, Thomas; Osgood, Ross S; Velmurugan, Kalpana; Alexander, Carla-Maria; Upham, Brad L; Bauer, Alison K

    2013-12-27

    TLR4 protects against lung tumor promotion and pulmonary inflammation in mice. Connexin 43 (Cx43), a gap junction gene, was increased in Tlr4 wildtype compared to Tlr4-mutant mice in response to promotion, which suggests gap junctional intercellular communication (GJIC) may be compromised. We hypothesized that the early tumor microenvironment, represented by Bronchoalveolar Lavage Fluid (BALF) from Butylated hydroxytoluene (BHT; promoter)-treated mice, would produce TLR4-dependent changes in pulmonary epithelium, including dysregulation of GJIC in the Tlr4-mutant (BALB (Lps-d) ) compared to the Tlr4-sufficient (BALB; wildtype) mice. BHT (4 weekly doses) was injected ip followed by BALF collection at 24 h. BALF total protein and total macrophages were significantly elevated in BHT-treated BALB (Lps-d) over BALB mice, similar to previous findings. BALF was then utilized in an ex vivo manner to treat C10 cells, a murine alveolar type II cell line, followed by the scrape-load dye transfer assay (GJIC), Cx43 immunostaining, and quantitative RT-PCR (Mcp-1, monocyte chemotactic protein 1). GJIC was markedly reduced in C10 cells treated with BHT-treated BALB (Lps-d) BALF for 4 and 24 h compared to BALB and control BALF from the respective mice (p < 0.05). Mcp-1, a chemokine, was also significantly increased in the BHT-treated BALB (Lps-d) BALF compared to the BALB mice, and Cx43 protein expression in the cell membrane altered. These novel findings suggest signaling from the BALF milieu is involved in GJIC dysregulation associated with promotion and links gap junctions to pulmonary TLR4 protection in a novel ex vivo model that could assist in future potential tumor promoter screening. PMID:25035812

  7. 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.

  8. Radiological audit of remedial action activities at the processing site, transfer site, and Cheney disposal site Grand Junction, Colorado: Audit date, August 9--11, 1993. Final report

    1993-08-01

    The Uranium Mill Tailing Remedial Action (UMTRA) Project`s Technical Assistance Contractor (TAC) performed a radiological audit of the Remedial Action Contractor (RAC), MK-Ferguson and CWM Federal Environmental Services, Inc., at the processing site, transfer site, and Cheney disposal site in Grand Junction, Colorado. Jim Hylko and Bill James of the TAC conducted this audit August 9 through 11, 1993. Bob Cornish and Frank Bosiljevec represented the US Department of Energy (DOE). This report presents one programmatic finding, eleven site-specific observations, one good practice, and four programmatic observations.

  9. Role of autophagy in the regulation of epithelial cell junctions.

    Nighot, Prashant; Ma, Thomas

    2016-01-01

    Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions. PMID:27583189

  10. Disordered graphene Josephson junctions

    Munoz, W. A.; Covaci, L.; Peeters, F. M.

    2014-01-01

    A tight-binding approach based on the Chebyshev-Bogoliubov-de Gennes method is used to describe disordered single-layer graphene Josephson junctions. Scattering by vacancies, ripples or charged impurities is included. We compute the Josephson current and investigate the nature of multiple Andreev reflections, which induce bound states appearing as peaks in the density of states for energies below the superconducting gap. In the presence of single atom vacancies, we observe a strong suppressio...

  11. 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

  12. 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

  13. RhoA-JNK Regulates the E-Cadherin Junctions of Human Gingival Epithelial Cells.

    Lee, G; Kim, H J; Kim, H-M

    2016-03-01

    The junctional epithelium (JE) is unique with regard to its wide intercellular spaces and sparsely developed intercellular junctions. Thus, knowledge of the molecular mechanisms that regulate the formation of the intercellular junctions of the junctional epithelium may be essential to understand the pathophysiology of the JE. HOK-16B cells, a normal human gingival epithelial cell line, were used to identify the molecules involved in the regulation of the formation of intercellular E-cadherin junctions between human gingival epithelial cells. Activation of c-Jun N-terminal kinase (JNK) disrupted the intercellular junctions through the dissociation of E-cadherin. The role of JNK in the formation of these E-cadherin junctions was further confirmed by demonstrating that JNK inhibition induced the formation of intercellular E-cadherin junctions. The upstream signaling of JNK was also examined. Activation of the small GTPase RhoA disrupted the formation of E-cadherin junctions between HOK-16B cells, which was accompanied by JNK activation. Disruption of these intercellular junctions upon RhoA activation was prevented when JNK activity was inhibited. In contrast, RhoA inactivation led to HOK-16B cell aggregation and the formation of intercellular junctions, even under conditions in which the cellular junctions were naturally disrupted by growth on a strongly adhesive surface. Furthermore, the JE of mouse molars had high JNK activity associated with low E-cadherin expression, which was reversed in the other gingival epithelia, including the sulcular epithelium. Interestingly, JNK activity was increased in cells grown on a solid surface, where cells showed higher RhoA activity than those grown on soft surfaces. Together, these results indicate that the decreased formation of intercellular E-cadherin junctions within the JE may be coupled to high JNK activity, which is activated by the upregulation of RhoA on solid tooth surfaces. PMID:26635280

  14. Josephson junctions with ferromagnetic alloy interlayer

    Josephson junctions are used as active devices in superconducting electronics and quantum information technology. Outstanding properties are their distinct non-linear electrical characteristics and a usually sinusoidal relation between the current and the superconducting phase difference across the junction. In general the insertion of ferromagnetic material in the barrier of a Josephson junction is associated with a suppression of superconducting correlations. But also new phenomena can arise which may allow new circuit layouts and enhance the performance of applications. This thesis presents a systematic investigation for two concepts to fabricate Josephson junctions with a rather uncommon negative critical current. Such devices exhibit an intrinsic phase slip of π between the electrodes, so they are also known as π junctions. Both studies go well beyond existing experiments and in one system a π junction is shown for the first time. All the thin film junctions are based on superconducting Nb electrodes. In a first approach, barriers made from Si and Fe were investigated with respect to the realisation of π junctions by spin-flip processes. The distribution of Fe in the Si matrix was varied from pure layers to disperse compounds. The systematic fabrication of alloy barriers was facilitated by the development of a novel timing-based combinatorial sputtering technique for planetary deposition systems. An orthogonal gradient approach allowed to create binary layer libraries with independent variations of thickness and composition. Second, Nb vertical stroke AlOx vertical stroke Nb vertical stroke Ni60Cu40 vertical stroke Nb (SIsFS) double barrier junctions were experimentally studied for the occurrence of proximity effect induced order parameter oscillations. Detailed dependencies of the critical current density on the thickness of s-layer and F-layer were acquired and show a remarkable agreement to existing theoretical predictions. Especially a variation of jc

  15. Josephson junctions with ferromagnetic alloy interlayer

    Himmel, Nico

    2015-07-23

    Josephson junctions are used as active devices in superconducting electronics and quantum information technology. Outstanding properties are their distinct non-linear electrical characteristics and a usually sinusoidal relation between the current and the superconducting phase difference across the junction. In general the insertion of ferromagnetic material in the barrier of a Josephson junction is associated with a suppression of superconducting correlations. But also new phenomena can arise which may allow new circuit layouts and enhance the performance of applications. This thesis presents a systematic investigation for two concepts to fabricate Josephson junctions with a rather uncommon negative critical current. Such devices exhibit an intrinsic phase slip of π between the electrodes, so they are also known as π junctions. Both studies go well beyond existing experiments and in one system a π junction is shown for the first time. All the thin film junctions are based on superconducting Nb electrodes. In a first approach, barriers made from Si and Fe were investigated with respect to the realisation of π junctions by spin-flip processes. The distribution of Fe in the Si matrix was varied from pure layers to disperse compounds. The systematic fabrication of alloy barriers was facilitated by the development of a novel timing-based combinatorial sputtering technique for planetary deposition systems. An orthogonal gradient approach allowed to create binary layer libraries with independent variations of thickness and composition. Second, Nb vertical stroke AlO{sub x} vertical stroke Nb vertical stroke Ni{sub 60}Cu{sub 40} vertical stroke Nb (SIsFS) double barrier junctions were experimentally studied for the occurrence of proximity effect induced order parameter oscillations. Detailed dependencies of the critical current density on the thickness of s-layer and F-layer were acquired and show a remarkable agreement to existing theoretical predictions. Especially

  16. 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

  17. 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

  18. 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

  19. 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

  20. Josephson junction simulation of neurons

    Crotty, Patrick; Schult, Daniel; Segall, Ken

    2010-01-01

    With the goal of understanding the intricate behavior and dynamics of collections of neurons, we present superconducting circuits containing Josephson junctions that model biologically realistic neurons. These "Josephson junction neurons" reproduce many characteristic behaviors of biological neurons such as action potentials, refractory periods, and firing thresholds. They can be coupled together in ways that mimic electrical and chemical synapses. Using existing fabrication technologies, lar...

  1. Fluid Flow at Branching Junctions

    Sochi, Taha

    2013-01-01

    The flow of fluids at branching junctions plays important kinematic and dynamic roles in most biological and industrial flow systems. The present paper highlights some key issues related to the flow of fluids at these junctions with special emphasis on the biological flow networks particularly blood transportation vasculature.

  2. Reduced gap junctional coupling leads to uncorrelated motor neuron firing and precocious neuromuscular synapse elimination

    Personius, Kirkwood E.; Chang, Qiang; Mentis, George Z.; O'Donovan, Michael J.; Rita J Balice-Gordon

    2007-01-01

    During late embryonic and early postnatal life, neuromuscular junctions undergo synapse elimination that is modulated by patterns of motor neuron activity. Here, we test the hypothesis that reduced spinal neuron gap junctional coupling decreases temporally correlated motor neuron activity that, in turn, modulates neuromuscular synapse elimination, by using mutant mice lacking connexin 40 (Cx40), a developmentally regulated gap junction protein expressed in motor and other spinal neurons. In C...

  3. Indomethacin induces increase in gastric epithelial tight junction permeability via redistribution of occludin and activation of p38 MAPK in MKN-28 Cells.

    Thakre-Nighot, Meghali; Blikslager, Anthony T

    2016-01-01

    Tight Junctions (TJ) create a paracellular barrier that is compromised when nonsteriodal anti-inflammatory drugs (NSAIDs) injure the gastric epithelium, leading to increased permeability. However, the mechanism of NSAID-induced gastric injury is unclear. Here, we examined the effect of indomethacin on barrier function and TJ in gastric MKN-28 cells. In concentration response studies, 500 µm indomethacin induced a significant decrease in transepithelial resistance (TER; 380 vs. 220 Ω·cm(2) for control and indomethacin-treated cells respectively, p permeability by 0.2 vs 1.2 g/l (p permeability. Pretreatment with the p38 MAPK inhibitor significantly attenuated the disruption of barrier function, but JNK and MEK/ERK inhibition had no effect. Western blot analysis on gastric mucosa reveled loss of TJ protein occludin by indomethacin, which was prevented by inhibition of p38 MAPK. This data suggests that indomethacin compromises the gastric epithelial barrier via p38 MAPK inducing occludin alterations in the TJs. PMID:27583191

  4. Herlitz junctional epidermolysis bullosa.

    Laimer, Martin; Lanschuetzer, Christoph M; Diem, Anja; Bauer, Johann W

    2010-01-01

    Junctional epidermolysis bullosa type Herlitz (JEB-H) is the autosomal recessively inherited, more severe variant of "lucidolytic" JEB. Characterized by generalized, extensive mucocutaneous blistering at birth and early lethality, this devastating condition is most often caused by homozygous null mutations in the genes LAMA3, LAMB3, or LAMC2, each encoding for 1 of the 3 chains of the heterotrimer laminin-332. The JEB-H subtype usually presents as a severe and clinically diverse variant of the EB group of mechanobullous genodermatoses. This article outlines the epidemiology, presentation, and diagnosis of JEB-H. Morbidity and mortality are high, necessitating optimized protocols for early (including prenatal) diagnosis and palliative care. Gene therapy remains the most promising perspective. PMID:19945616

  5. The human myotendinous junction

    Knudsen, A B; Larsen, M; Mackey, Abigail;

    2015-01-01

    The myotendinous junction (MTJ) is a specialized structure in the musculotendinous system, where force is transmitted from muscle to tendon. Animal models have shown that the MTJ takes form of tendon finger-like processes merging with muscle tissue. The human MTJ is largely unknown and has never...... from all 14 patients. TEM images displayed similarities to observations in animals: Sarcolemmal evaginations observed as finger-like processes from the tendon and endomysium surrounding the muscle fibers, with myofilaments extending from the final Z-line of the muscle fiber merging with the tendon...... been described in three dimensions (3D). The aim of this study was to describe the ultrastructure of the human MTJ and render 3D reconstructions. Fourteen subjects (age 25 ± 3 years) with isolated injury of the anterior cruciate ligament (ACL), scheduled for reconstruction with a semitendinosus...

  6. Disordered graphene Josephson junctions

    Muñoz, W. A.; Covaci, L.; Peeters, F. M.

    2015-02-01

    A tight-binding approach based on the Chebyshev-Bogoliubov-de Gennes method is used to describe disordered single-layer graphene Josephson junctions. Scattering by vacancies, ripples, or charged impurities is included. We compute the Josephson current and investigate the nature of multiple Andreev reflections, which induce bound states appearing as peaks in the density of states for energies below the superconducting gap. In the presence of single-atom vacancies, we observe a strong suppression of the supercurrent, which is a consequence of strong intervalley scattering. Although lattice deformations should not induce intervalley scattering, we find that the supercurrent is still suppressed, which is due to the presence of pseudomagnetic barriers. For charged impurities, we consider two cases depending on whether the average doping is zero, i.e., existence of electron-hole puddles, or finite. In both cases, short-range impurities strongly affect the supercurrent, similar to the vacancies scenario.

  7. t-SNARE Phosphorylation Regulates Endocytosis in Yeast

    Gurunathan, Sangiliyandi; Marash, Michael; Weinberger, Adina; Gerst, Jeffrey E.

    2002-01-01

    Earlier we demonstrated that activation of a ceramide-activated protein phosphatase (CAPP) conferred normal growth and secretion to yeast lacking their complement of exocytic v-SNAREs (Snc1,2) or bearing a temperature-sensitive mutation in an exocytic t-SNARE (Sso2). CAPP activation led to Sso dephosphorylation and enhanced the assembly of t-SNAREs into functional complexes. Thus, exocytosis in yeast is modulated by t-SNARE phosphorylation. Here, we show that endocytic defects in cells lackin...

  8. 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.

  9. Endocytosis against the high turgor of guard cells

    Meckel, Tobias

    2004-01-01

    Stomata are found in the epidermis of photosynthetic active plant organs. They are formed by pairs of guard cells which create a pore to facilitate CO2 and water exchange with the environment. In order to control this gas exchange, guard cells actively change their volume and, consequently, surface area to alter the aperture of the stomatal pore. These changes are achieved by an uptake or release of K+ through K+-selective channels followed by the respective osmotic water fluxes. The quantifi...

  10. 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...

  11. 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...

  12. 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 ...

  13. 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...

  14. 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

  15. 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

  16. 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 ...

  17. 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 ...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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 ...

  6. 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.

  7. 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.

  8. Electronic properties of nanotube junctions

    Lambin, Ph.; Meunier, V.

    1998-08-01

    The possibility of realizing junctions between two different nanotubes has recently attracted a great interest, even though much remains to be done for putting this idea in concrete form. Pentagon-heptagon pair defects in the otherwise perfect graphitic network make such connections possible, with virtually infinite varieties. In this paper, the literature devoted to nanotube junctions is briefly reviewed. A special emphasize is put on the electronic properties of C nanotube junctions, together with an indication on how their current-voltage characteristics may look like.

  9. TRANSITIONAL FLOW IN CHANNEL JUNCTIONS

    NI Han-gen; LIU Ya-kun

    2004-01-01

    On the basis of energy and continuity equations a simple one-dimensional formulation was proposed to predict the transitional flow at an open-channel junction. An empilical relation between the junction losses, the junction angle, and the discharge ratio was suggested which agrees well with the experimental results. The results calculated by the present formulation for the depth ratio were compared with the results of earlier one-dimensional formulations and experiments. It is found that the present results coincide better with experiments than those of others.

  10. Papain Degrades Tight Junction Proteins of Human Keratinocytes In Vitro and Sensitizes C57BL/6 Mice via the Skin Independent of its Enzymatic Activity or TLR4 Activation

    Stremnitzer, Caroline; Manzano-Szalai, Krisztina; Willensdorfer, Anna; Starkl, Philipp(*); Pieper, Mario; König, Peter; Mildner, Michael; Tschachler, Erwin; Reichart, Ursula; Jensen-Jarolim, Erika

    2015-01-01

    Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes....

  11. ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

    Jorgensen, N R; Geist, S T; Civitelli, R;

    1997-01-01

    stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied...... connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U...... receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP...

  12. SIS junction reactance complete compensation

    SIS junction geometrical capacitance together with out of phase current Ikk impedance component forms sufficient junction reactance XSIS = (ωC + BQ)-1. This paper suggests the way to resonate out both ωC and BQ by using additional identical SIS junction connected to the first through a long line impedance inverter and RF + DC biased symmetrically to the first. Pumped IV curves without quantum reactance and frequency impedance patterns of the system were calculated. Calculations demonstrated the presence of high and even negative induced dynamic resistance regions at high order quasiparticle steps for the case of SIS junction reactance complete compensation. The suggested method may be used in SIS mixers and detectors for a better RF matching

  13. Thermal conductance of superlattice junctions

    Lu, Simon; McGaughey, Alan J. H., E-mail: mcgaughey@cmu.edu [Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States)

    2015-05-15

    We use molecular dynamics simulations and the lattice-based scattering boundary method to compute the thermal conductance of finite-length Lennard-Jones superlattice junctions confined by bulk crystalline leads. The superlattice junction thermal conductance depends on the properties of the leads. For junctions with a superlattice period of four atomic monolayers at temperatures between 5 and 20 K, those with mass-mismatched leads have a greater thermal conductance than those with mass-matched leads. We attribute this lead effect to interference between and the ballistic transport of emergent junction vibrational modes. The lead effect diminishes when the temperature is increased, when the superlattice period is increased, and when interfacial disorder is introduced, but is reversed in the harmonic limit.

  14. Thermal conductance of superlattice junctions

    Simon Lu

    2015-05-01

    Full Text Available We use molecular dynamics simulations and the lattice-based scattering boundary method to compute the thermal conductance of finite-length Lennard-Jones superlattice junctions confined by bulk crystalline leads. The superlattice junction thermal conductance depends on the properties of the leads. For junctions with a superlattice period of four atomic monolayers at temperatures between 5 and 20 K, those with mass-mismatched leads have a greater thermal conductance than those with mass-matched leads. We attribute this lead effect to interference between and the ballistic transport of emergent junction vibrational modes. The lead effect diminishes when the temperature is increased, when the superlattice period is increased, and when interfacial disorder is introduced, but is reversed in the harmonic limit.

  15. 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.

  16. 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

  17. Electronic thermometry in tunable tunnel junction

    Maksymovych, Petro

    2016-03-15

    A tunable tunnel junction thermometry circuit includes a variable width tunnel junction between a test object and a probe. The junction width is varied and a change in thermovoltage across the junction with respect to the change in distance across the junction is determined. Also, a change in biased current with respect to a change in distance across the junction is determined. A temperature gradient across the junction is determined based on a mathematical relationship between the temperature gradient, the change in thermovoltage with respect to distance and the change in biased current with respect to distance. Thermovoltage may be measured by nullifying a thermoelectric tunneling current with an applied voltage supply level. A piezoelectric actuator may modulate the probe, and thus the junction width, to vary thermovoltage and biased current across the junction. Lock-in amplifiers measure the derivatives of the thermovoltage and biased current modulated by varying junction width.

  18. Adherens junction distribution mechanisms during cell-cell contact elongation in Drosophila.

    Gabrielle Goldenberg

    Full Text Available During Drosophila gastrulation, amnioserosa (AS cells flatten and spread as an epithelial sheet. We used AS morphogenesis as a model to investigate how adherens junctions (AJs distribute along elongating cell-cell contacts in vivo. As the contacts elongated, total AJ protein levels increased along their length. However, genetically blocking this AJ addition indicated that it was not essential for maintaining AJ continuity. Implicating other remodeling mechanisms, AJ photobleaching revealed non-directional lateral mobility of AJs along the elongating contacts, as well as local AJ removal from the membranes. Actin stabilization with jasplakinolide reduced AJ redistribution, and live imaging of myosin II along elongating contacts revealed fragmented, expanding and contracting actomyosin networks, suggesting a mechanism for lateral AJ mobility. Actin stabilization also increased total AJ levels, suggesting an inhibition of AJ removal. Implicating AJ removal by endocytosis, clathrin endocytic machinery accumulated at AJs. However, dynamin disruption had no apparent effect on AJs, suggesting the involvement of redundant or dynamin-independent mechanisms. Overall, we propose that new synthesis, lateral diffusion, and endocytosis play overlapping roles to populate elongating cell-cell contacts with evenly distributed AJs in this in vivo system.

  19. Josephson junctions with ferromagnetic interlayer

    We report on the fabrication of superconductor/insulator/ferromagnetic metal/superconductor (Nb/AlOx/Pd0.82Ni0.18/Nb) Josephson junctions (SIFS JJs) with high critical current densities, large normal resistance times area products, and high quality factors. For these junctions, a transition from 0- to π-coupling is observed for a thickness dF=6 nm of the ferromagnetic Pd0.82Ni0.18 interlayer. The magnetic field dependence of the critical current of the junctions demonstrates good spatial homogeneity of the tunneling barrier and ferromagnetic interlayer. Magnetic characterization shows that the Pd0.82Ni0.18 has an out-of-plane anisotropy and large saturation magnetization indicating negligible dead layers at the interfaces. A careful analysis of Fiske modes up to about 400 GHz provides valuable information on the junction quality factor and the relevant damping mechanisms. Whereas losses due to quasiparticle tunneling dominate at low frequencies, at high frequencies the damping is explained by the finite surface resistance of the junction electrodes. High quality factors of up to 30 around 200 GHz have been achieved. They allow to study the junction dynamics, in particular the switching probability from the zero-voltage into the voltage state with and without microwave irradiation. The experiments with microwave irradiation are well explained within semi-classical models and numerical simulations. In contrast, at mK temperature the switching dynamics without applied microwaves clearly shows secondary quantum effects. Here, we could observe for the first time macroscopic quantum tunneling in Josephson junctions with a ferromagnetic interlayer. This observation excludes fluctuations of the critical current as a consequence of an unstable magnetic domain structure of the ferromagnetic interlayer and affirms the suitability of SIFS Josephson junctions for quantum information processing.

  20. Josephson junctions with ferromagnetic interlayer

    Wild, Georg Hermann

    2012-03-04

    We report on the fabrication of superconductor/insulator/ferromagnetic metal/superconductor (Nb/AlO{sub x}/Pd{sub 0.82}Ni{sub 0.18}/Nb) Josephson junctions (SIFS JJs) with high critical current densities, large normal resistance times area products, and high quality factors. For these junctions, a transition from 0- to {pi}-coupling is observed for a thickness d{sub F}=6 nm of the ferromagnetic Pd{sub 0.82}Ni{sub 0.18} interlayer. The magnetic field dependence of the critical current of the junctions demonstrates good spatial homogeneity of the tunneling barrier and ferromagnetic interlayer. Magnetic characterization shows that the Pd{sub 0.82}Ni{sub 0.18} has an out-of-plane anisotropy and large saturation magnetization indicating negligible dead layers at the interfaces. A careful analysis of Fiske modes up to about 400 GHz provides valuable information on the junction quality factor and the relevant damping mechanisms. Whereas losses due to quasiparticle tunneling dominate at low frequencies, at high frequencies the damping is explained by the finite surface resistance of the junction electrodes. High quality factors of up to 30 around 200 GHz have been achieved. They allow to study the junction dynamics, in particular the switching probability from the zero-voltage into the voltage state with and without microwave irradiation. The experiments with microwave irradiation are well explained within semi-classical models and numerical simulations. In contrast, at mK temperature the switching dynamics without applied microwaves clearly shows secondary quantum effects. Here, we could observe for the first time macroscopic quantum tunneling in Josephson junctions with a ferromagnetic interlayer. This observation excludes fluctuations of the critical current as a consequence of an unstable magnetic domain structure of the ferromagnetic interlayer and affirms the suitability of SIFS Josephson junctions for quantum information processing.

  1. Neuromuscular junctional disorders.

    Girija, A S; Ashraf, V V

    2008-07-01

    Neuromuscular junctional disorders (NMJ) in children are distinct entity. They may be acquired or hereditary. They pose problem in diagnosis because of the higher occurrence of sero negative Myasthenia Gravis (MG) cases in children. The identity of MusK antibody positivity in a good percentage of sero negative cases further adds to problems in diagnosis. The Congenital Myasthenic Syndrome (CMS) which are rare disorders of hereditary neuromuscular transmission (NMT) has to be differentiated because immunotherapy has no benefit in this group. Molecular genetic studies of these diseases helps to identify specific type of CMS which is important as other drugs like Fluoxetine, Quinidine are found to be effective in some. In infancy, all can manifest as floppy infant syndrome. The important key to diagnosis is by detailed electrophysiological studies including repetitive nerve stimulation at slow and high rates and its response to anticholinesterases and estimation of Acetyl choline receptor antibodies. Other causes of neuromuscular transmission defects viz. snake venom poisoning and that due to drugs are discussed. PMID:18716738

  2. Gap Junction Enhancer Increases Efficacy of Cisplatin to Attenuate Mammary Tumor Growth

    Shishido, Stephanie N.; Nguyen, Thu A.

    2012-01-01

    Cisplatin treatment has an overall 19% response rate in animal models with malignant tumors. Increasing gap junction activity in tumor cells provides the targets to enhance antineoplastic therapies. Previously, a new class of substituted quinolines (PQs) acts as gap junction enhancer, ability to increase the gap junctional intercellular communication, in breast cancer cells. We examined the effect of combinational treatment of PQs and antineoplastic drugs in an animal model, showing an increa...

  3. Confocal Annular Josephson Tunnel Junctions

    Monaco, Roberto

    2016-04-01

    The physics of Josephson tunnel junctions drastically depends on their geometrical configurations and here we show that also tiny geometrical details play a determinant role. More specifically, we develop the theory of short and long annular Josephson tunnel junctions delimited by two confocal ellipses. The behavior of a circular annular Josephson tunnel junction is then seen to be simply a special case of the above result. For junctions having a normalized perimeter less than one, the threshold curves in the presence of an in-plane magnetic field of arbitrary orientations are derived and computed even in the case with trapped Josephson vortices. For longer junctions, a numerical analysis is carried out after the derivation of the appropriate motion equation for the Josephson phase. We found that the system is modeled by a modified and perturbed sine-Gordon equation with a space-dependent effective Josephson penetration length inversely proportional to the local junction width. Both the fluxon statics and dynamics are deeply affected by the non-uniform annulus width. Static zero-field multiple-fluxon solutions exist even in the presence of a large bias current. The tangential velocity of a traveling fluxon is not determined by the balance between the driving and drag forces due to the dissipative losses. Furthermore, the fluxon motion is characterized by a strong radial inward acceleration which causes electromagnetic radiation concentrated at the ellipse equatorial points.

  4. Confocal Annular Josephson Tunnel Junctions

    Monaco, Roberto

    2016-09-01

    The physics of Josephson tunnel junctions drastically depends on their geometrical configurations and here we show that also tiny geometrical details play a determinant role. More specifically, we develop the theory of short and long annular Josephson tunnel junctions delimited by two confocal ellipses. The behavior of a circular annular Josephson tunnel junction is then seen to be simply a special case of the above result. For junctions having a normalized perimeter less than one, the threshold curves in the presence of an in-plane magnetic field of arbitrary orientations are derived and computed even in the case with trapped Josephson vortices. For longer junctions, a numerical analysis is carried out after the derivation of the appropriate motion equation for the Josephson phase. We found that the system is modeled by a modified and perturbed sine-Gordon equation with a space-dependent effective Josephson penetration length inversely proportional to the local junction width. Both the fluxon statics and dynamics are deeply affected by the non-uniform annulus width. Static zero-field multiple-fluxon solutions exist even in the presence of a large bias current. The tangential velocity of a traveling fluxon is not determined by the balance between the driving and drag forces due to the dissipative losses. Furthermore, the fluxon motion is characterized by a strong radial inward acceleration which causes electromagnetic radiation concentrated at the ellipse equatorial points.

  5. The conversion of PN-junction influencing the piezoelectric output of a CuO/ZnO nanoarray nanogenerator and its application as a room-temperature self-powered active H2S sensor

    Room-temperature, high H2S sensing has been realized from a CuO/ZnO nanoarray self-powered, active gas sensor. The piezoelectric output of CuO/ZnO nanoarrays can act not only as the power source of the device, but also as the H2S sensing signal at room temperature. Upon exposure to 800 ppm H2S at room temperature, the piezoelectric output of the device greatly decreased from 0.738 V (in air) to 0.101 V. The sensitivity increased to 629.8, much higher than bare ZnO nanoarrays. As the device was exposed to H2S, a CuO/ZnO PN-junction was converted into a CuS/ZnO Ohmic contact, which greatly increased the electron density in the nanowire and enhanced the screen effect on the piezoelectric output. Our results can stimulate a research trend on designing new composite piezoelectric material for high-performance self-powered active gas sensors. (papers)

  6. Complement upregulation and activation on motor neurons and neuromuscular junction in the SOD1 G93A mouse model of familial amyotrophic lateral sclerosis

    B. Heurich; N.B. El Idrissi; R.M. Donev; S. Petri; P. Claus; J. Neal; B.P. Morgan; V. Ramaglia

    2011-01-01

    Complement activation products are elevated in cerebrospinal fluid, spinal cord and motor cortex of patients with amyotrophic lateral sclerosis (ALS) but are untested in models. We determined complement expression and activation in the SOD1 G93A mouse model of familial ALS (fALS). At 126 days, C3 mR

  7. 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

  8. 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.

  9. The Masticatory Contractile Load Induced Expression and Activation of Akt1/PKBα in Muscle Fibers at the Myotendinous Junction within Muscle-Tendon-Bone Unit

    Yüksel Korkmaz; Franz J. Klinz; Mehrnoush Moghbeli; Klaus Addicks; Wolfgang H.-M. Raab; Wilhelm Bloch

    2010-01-01

    The cell specific detection of enzyme activation in response to the physiological contractile load within muscle-tendon-bone unit is essential for understanding of the mechanical forces transmission from muscle cells via tendon to the bone. The hypothesis that the physiological mechanical loading regulates activation of Akt1/PKBα at Thr308 and at Ser473 in muscle fibers within muscle-tendon-bone unit was tested using quantitative immunohistochemistry, confocal double fluorescence analy...

  10. Quantitative mapping of fast voltage pulses in tunnel junctions by plasmonic luminescence

    Grosse, Christoph; Etzkorn, Markus; Kuhnke, Klaus; Loth, Sebastian; Kern, Klaus

    2013-10-01

    An optical read-out technique is demonstrated that enables mapping the time-dependent electrostatic potential in the tunnel junction of a scanning tunneling microscope with millivolt and nanosecond accuracy. We measure the time-dependent intensity of plasmonic light emitted from the tunnel junction upon excitation with a nanosecond voltage pulse. The light intensity is found to be a quantitative measure of the voltage between tip and sample. This permits non-invasive mapping of fast voltage transients directly at the tunnel junction. Knowledge of the pulse profile reaching the tunnel junction is applied to optimize the experiment's time response by actively shaping the incident pulses.

  11. Modelling of Dual-Junction Solar Cells including Tunnel Junction

    Abdelaziz Amine

    2013-01-01

    Full Text Available Monolithically stacked multijunction solar cells based on III–V semiconductors materials are the state-of-art of approach for high efficiency photovoltaic energy conversion, in particular for space applications. The individual subcells of the multi-junction structure are interconnected via tunnel diodes which must be optically transparent and connect the component cells with a minimum electrical resistance. The quality of these diodes determines the output performance of the solar cell. The purpose of this work is to contribute to the investigation of the tunnel electrical resistance of such a multi-junction cell through the analysis of the current-voltage (J-V characteristics under illumination. Our approach is based on an equivalent circuit model of a diode for each subcell. We examine the effect of tunnel resistance on the performance of a multi-junction cell using minimization of the least squares technique.

  12. 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.

  13. 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.

  14. 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...

  15. 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...

  16. Toxicity of hydroxylated and quinoid PCB metabolites: Inhibition of gap junctional intercellular communication and activation of aryl hydrocarbon and estrogen receptors in hepatic and mammary cells

    Machala, M.; Bláha, L.; Lehmler, H.-J.; Plíšková, M.; Májková, Z.; Kapplová, P.; Sovadinová, I.; Vondráček, Jan; Malmberg, T.; Robertson, L. W.

    2004-01-01

    Roč. 17, č. 3 (2004), s. 340-347. ISSN 0893-228X R&D Projects: GA MZe QC0194; GA AV ČR IPP1050128 Institutional research plan: CEZ:AV0Z5004920 Keywords : hydroxylated polychlorinated biphenyls * AhR-mediated activity * inhibition of GJIC Subject RIV: BO - Biophysics Impact factor: 2.797, year: 2004

  17. The effect of multi active junctions on broadband emission from InAs/InGaAlAs quantum-dash structure

    Tan, Cheeloon

    2009-10-01

    We demonstrate the importance of multi active layer stacking in realizing a semiconductor broadband quantum-dash-in-well laser. The photoluminescence measurements show the negligible factor of largely localized inhomogeneous quantum-dash ensembles in producing ultra-wide envelope of emission. © 2009 IEEE.

  18. Transport properties of molecular junctions

    Zimbovskaya, Natalya A

    2013-01-01

    A comprehensive overview of the physical mechanisms that control electron transport and the characteristics of metal-molecule-metal (MMM) junctions is presented. As far as possible, methods and formalisms presented elsewhere to analyze electron transport through molecules are avoided. This title introduces basic concepts—a description of the electron transport through molecular junctions—and briefly describes relevant experimental methods. Theoretical methods commonly used to analyze the electron transport through molecules are presented. Various effects that manifest in the electron transport through MMMs, as well as the basics of density-functional theory and its applications to electronic structure calculations in molecules are presented. Nanoelectronic applications of molecular junctions and similar systems are discussed as well. Molecular electronics is a diverse and rapidly growing field. Transport Properties of Molecular Junctions presents an up-to-date survey of the field suitable for researchers ...

  19. Gap Junctions in C. elegans

    ChristianC.Naus

    2014-02-01

    Full Text Available As in other multicellular organisms, the nematode Caenorhabditis elegans uses gap junctions to provide direct cell-to-cell contact. The nematode gap junctions are formed by innexins (invertebrate analogs of the connexins; a family of proteins that surprisingly share no primary sequence homology, but do share structural and functional similarity with connexins. The model organism C. elegans contains 25 innexin genes and innexins are found in virtually all cell types and tissues. Additionally, many innexins have dynamic expression patterns during development, and several innexins are essential genes in the nematode. C. elegans is a popular invertebrate model due to several features including a simple anatomy, a complete cell lineage, sequenced genome and an array of genetic resources. Thus the worm has potential to offer valuable insights into the various functions of gap junction mediated intercellular communication.

  20. NbN tunnel junctions

    All-niobium nitride Josephon junctions have been prepared successfully using a new processing called SNOP: Selective Niobium (nitride) Overlap Process. Such a process involves the ''trilayer'' deposition on the whole wafer before selective patterning of the electrodes by optically controlled dry reactive ion etching. Only two photomask levels are need to define an ''overlap'' or a ''cross-type'' junction with a good accuracy. The properties of the niobium nitride films deposited by DC-magnetron sputtering and the surface oxide growth are analysed. The most critical point to obtain high quality and high gap value junctions resides in the early stage of the NbN counterelectrode growth. Some possibilities to overcome such a handicap exist even if the fabrication needs substrate temperatures below 2500C

  1. The nanostructure of myoendothelial junctions contributes to signal rectification between endothelial and vascular smooth muscle cells

    Brasen, Jens Christian; Jacobsen, Jens Christian Brings; von Holstein-Rathlou, Niels-Henrik

    2012-01-01

    can easily drive a concentration change in the head of the myoendothelial protrusion. Subsequently the signal can be amplified in the head, and activate the entire cell. In contrast, a signal in the cell from which the myoendothelial junction originates will be attenuated and delayed in the neck...... region as it travels into the head of the myoendothelial junction and the neighboring cell....

  2. 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

  3. 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

  4. Gap junctions in olfactory neurons modulate olfactory sensitivity

    Zhang Chunbo

    2010-08-01

    Full Text Available Abstract Background One of the fundamental questions in olfaction is whether olfactory receptor neurons (ORNs behave as independent entities within the olfactory epithelium. On the basis that mature ORNs express multiple connexins, I postulated that gap junctional communication modulates olfactory responses in the periphery and that disruption of gap junctions in ORNs reduces olfactory sensitivity. The data collected from characterizing connexin 43 (Cx43 dominant negative transgenic mice OlfDNCX, and from calcium imaging of wild type mice (WT support my hypothesis. Results I generated OlfDNCX mice that express a dominant negative Cx43 protein, Cx43/β-gal, in mature ORNs to inactivate gap junctions and hemichannels composed of Cx43 or other structurally related connexins. Characterization of OlfDNCX revealed that Cx43/β-gal was exclusively expressed in areas where mature ORNs resided. Real time quantitative PCR indicated that cellular machineries of OlfDNCX were normal in comparison to WT. Electroolfactogram recordings showed decreased olfactory responses to octaldehyde, heptaldehyde and acetyl acetate in OlfDNCX compared to WT. Octaldehyde-elicited glomerular activity in the olfactory bulb, measured according to odor-elicited c-fos mRNA upregulation in juxtaglomerular cells, was confined to smaller areas of the glomerular layer in OlfDNCX compared to WT. In WT mice, octaldehyde sensitive neurons exhibited reduced response magnitudes after application of gap junction uncoupling reagents and the effects were specific to subsets of neurons. Conclusions My study has demonstrated that altered assembly of Cx43 or structurally related connexins in ORNs modulates olfactory responses and changes olfactory activation maps in the olfactory bulb. Furthermore, pharmacologically uncoupling of gap junctions reduces olfactory activity in subsets of ORNs. These data suggest that gap junctional communication or hemichannel activity plays a critical role in

  5. Environmental Audit of the Grand Junction Projects Office

    The Grand Junction Projects Office (GJPO) is located in Mesa County, Colorado, immediately south and west of the Grand Junction city limits. The US Atomic Energy Commission (AEC) established the Colorado Raw Materials Office at the present-day Grand Junction Projects Office in 1947, to aid in the development of a viable domestic uranium industry. Activities at the site included sampling uranium concentrate; pilot-plant milling research, including testing and processing of uranium ores; and operation of a uranium mill pilot plant from 1954 to 1958. The last shipment of uranium concentrate was sent from GJPO in January, 1975. Since that time the site has been utilized to support various DOE programs, such as the former National Uranium Resource Evaluation (NURE) Program, the Uranium Mill Tailings Remedial Action Project (UMTRAP), the Surplus Facilities Management Program (SFMP), and the Technical Measurements Center (TMC). All known contamination at GJPO is believed to be the result of the past uranium milling, analyses, and storage activities. Hazards associated with the wastes impounded at GJPO include surface and ground-water contamination and potential radon and gamma-radiation exposure. This report documents the results of the Baseline Environmental Audit conducted at Grand Junction Projects Office (GJPO) located in Grand Junction, Colorado. The Grand Junction Baseline Environmental Audit was conducted from May 28 to June 12, 1991, by the Office of Environmental Audit (EH-24). This Audit evaluated environmental programs and activities at GJPO, as well as GJPO activities at the State-Owned Temporary Repository. 4 figs., 12 tabs

  6. 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

  7. Charge transport in nanoscale junctions.

    Albrecht, Tim; Kornyshev, Alexei; Bjørnholm, Thomas

    2008-09-01

    Understanding the fundamentals of nanoscale charge transfer is pivotal for designing future nano-electronic devices. Such devices could be based on individual or groups of molecular bridges, nanotubes, nanoparticles, biomolecules and other 'active' components, mimicking wire, diode and transistor functions. These have operated in various environments including vacuum, air and condensed matter, in two- or three-electrode configurations, at ultra-low and room temperatures. Interest in charge transport in ultra-small device components has a long history and can be dated back to Aviram and Ratner's letter in 1974 (Chem. Phys. Lett. 29 277-83). So why is there a necessity for a special issue on this subject? The area has reached some degree of maturity, and even subtle geometric effects in the nanojunction and noise features can now be resolved and rationalized based on existing theoretical concepts. One purpose of this special issue is thus to showcase various aspects of nanoscale and single-molecule charge transport from experimental and theoretical perspectives. The main principles have 'crystallized' in our minds, but there is still a long way to go before true single-molecule electronics can be implemented. Major obstacles include the stability of electronic nanojunctions, reliable operation at room temperature, speed of operation and, last but not least, integration into large networks. A gradual transition from traditional silicon-based electronics to devices involving a single (or a few) molecule(s) therefore appears to be more viable from technologic and economic perspectives than a 'quantum leap'. As research in this area progresses, new applications emerge, e.g. with a view to characterizing interfacial charge transfer at the single-molecule level in general. For example, electrochemical experiments with individual enzyme molecules demonstrate that catalytic processes can be studied with nanometre resolution, offering a route towards optimizing biosensors at

  8. The sequence of moderate-size earthquakes at the junction of the Ligurian basin and the Corsica margin (western Mediterranean): The initiation of an active deformation zone revealed?

    Larroque, Christophe; Delouis, Bertrand; Sage, Françoise; Régnier, Marc; Béthoux, Nicole; Courboulex, Françoise; Deschamps, Anne

    2016-04-01

    A new seismically active zone is found in the southern part of the Ligurian basin, 80-km west of Corsica (western Mediterranean). The activity began in February 2011 with a foreshock (ML 4) and a mainshock (ML 5.3) 5 days later, followed by numerous aftershocks. We first analyze the fore- and mainshock in detail. We compare the results obtained using classical methods (linear location in a 1D medium and focal mechanisms from P and S polarities) and new approaches (non-linear location in a 3D medium and waveform modeling for determining the seismic moment and the focal mechanism). Both methods provided similar results for location, depth (in the range of 6-13 km) and focal mechanisms, which reveal reverse faulting with nodal planes oriented N-S and NE-SW. We then locate 27 of the aftershocks in the 3D model and find a 10-km-long NE-SW alignment with a depth between 7 and 16 km. In 2012 and 2013, three other moderate-size events (ML 3.8, 4 and 4.5) occurred and confirm that this zone is still active. The epicentral area is located in the oceanic domain of the Ligurian Basin. From analysis of the bathymetry and high-resolution multi-channel seismic profiles, no morphologic anomaly at surface and no inherited fault in the shallow ~ 4 km depth were imaged, which suggest that no significant deformation occurred in the area since 5 Ma. Thus, the structure(s) activated during the 2011-2013 sequence remain unknown. In light of these results, we point out a notable difference on both sides of the Ligurian Basin: the northern margin, close to the alpine chain, suffered strong earthquakes and large cumulated deformation since 5 Ma, while the southern margin, close to the Corsica-Sardinia continental block, is poorly deformed since 5 Ma.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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)

  14. 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

  15. 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

  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. 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

  18. 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.

  19. Josephson junctions based on pnictide superconductors

    Josephson junctions are a powerful tool for understanding more about the physical behaviour of pnictide superconductors. We built different kinds of Josephson junctions based on pnictide thin films. Planar junctions, edge type junctions, and junctions on bicrystalline substrates were prepared. We present manufacturing techniques and also the electronical properties of the different junctions and compare them. The measurement of I-V-characteristics show a strong excess current. We have to mind this when calculating the IcRN product. The effective IcRN values are 6.5 μV for the grain boundary junction, 7.9 μV for the planar structure, and 7.5 μV for the edge junction.

  20. Investigation of multi-junction solar cells using electrostatic force microscopy methods

    Moczała, M., E-mail: magdalena.moczala@pwr.wroc.pl [Wrocław University of Technology, Faculty of Microsystem Electronics and Photonics, Division of Metrology of Micro- and Nanostructures, ul. Z. Janiszewskiego 11/17, 50-372 Wrocław (Poland); Sosa, N.; Topol, A. [IBM Thomas J. Watson Research Center, P.O. Box 218, Yorktown Heights, NY 10598 (United States); Gotszalk, T. [Wrocław University of Technology, Faculty of Microsystem Electronics and Photonics, Division of Metrology of Micro- and Nanostructures, ul. Z. Janiszewskiego 11/17, 50-372 Wrocław (Poland)

    2014-06-01

    Multi-junction III–V solar cells are designed to have a much broader absorption of the solar spectrum than Si-based or single junctions, thus yield the highest conversion. The conversion efficiency can be further scaled with sun concentration. The ability of high conversion efficiencies makes multi-junction prime candidates for fine-tuning explorations aimed at getting closer to the theoretical efficiencies. In this paper, we report on electrostatic force microscopy (EFM) measurements of the built-in potential of multi-junction III–V semiconductor-based solar cells. Kelvin probe force microscopy (KPFM) was employed to qualitatively study the width and electrical properties of individual junctions, i.e., built-in potential, activity, and thickness of the p–n junctions. In addition, the voltage drops across individual solar cell p–n junctions were measured using Kelvin probe microscopy under various operation conditions: dark; illuminated; short-circuit; and biased. We present a method which enables the measurement of a working structure, while focusing on the electrical characteristics of an individual junction by virtue of selecting the spectral range of the illumination used. We show that these pragmatic studies can provide a feedback to improve photovoltaic device design, particularly of operation under a current mismatched situation. This new analysis technique offers additional insights into behavior of the multi-junction solar cell and shows promise for further progress in this field. - Highlights: • We explore the electronic structure of III–V based high efficiency solar cells. • Qualitative study of the solar cell operation characteristics is presented. • Quantitative study of the electrostatic landscape of operational high efficiency devices is presented. • Precise identification of the epitaxially grown p–n and tunnel junctions in the multi-junction solar cell. • Influence of illumination conditions and cell biasing on each p

  1. Investigation of multi-junction solar cells using electrostatic force microscopy methods

    Multi-junction III–V solar cells are designed to have a much broader absorption of the solar spectrum than Si-based or single junctions, thus yield the highest conversion. The conversion efficiency can be further scaled with sun concentration. The ability of high conversion efficiencies makes multi-junction prime candidates for fine-tuning explorations aimed at getting closer to the theoretical efficiencies. In this paper, we report on electrostatic force microscopy (EFM) measurements of the built-in potential of multi-junction III–V semiconductor-based solar cells. Kelvin probe force microscopy (KPFM) was employed to qualitatively study the width and electrical properties of individual junctions, i.e., built-in potential, activity, and thickness of the p–n junctions. In addition, the voltage drops across individual solar cell p–n junctions were measured using Kelvin probe microscopy under various operation conditions: dark; illuminated; short-circuit; and biased. We present a method which enables the measurement of a working structure, while focusing on the electrical characteristics of an individual junction by virtue of selecting the spectral range of the illumination used. We show that these pragmatic studies can provide a feedback to improve photovoltaic device design, particularly of operation under a current mismatched situation. This new analysis technique offers additional insights into behavior of the multi-junction solar cell and shows promise for further progress in this field. - Highlights: • We explore the electronic structure of III–V based high efficiency solar cells. • Qualitative study of the solar cell operation characteristics is presented. • Quantitative study of the electrostatic landscape of operational high efficiency devices is presented. • Precise identification of the epitaxially grown p–n and tunnel junctions in the multi-junction solar cell. • Influence of illumination conditions and cell biasing on each p

  2. Nano-Molecular Junctions on STM Tips

    Chun Huang∗; Jianshu Yang

    2011-01-01

    We present a technique for building metal-organic-metal junctions, which contain ten or fewer conjugated molecules between each of such junction, and the investigations of the I-V response of these junctions. The junctions are made by self assembling thiolated molecules onto gold coated tips for use in scanning tunneling microscopy. We show that this easy technique probes the qualitative properties of the molecules. Current-voltage characteristics of a Tour wire and a new molecular rectifier are presented.

  3. Interfacial thermal transport in atomic junctions

    Zhang, Lifa; Keblinski, Pawel; Wang, Jian-Sheng; Li, Baowen

    2011-01-01

    We study ballistic interfacial thermal transport across atomic junctions. Exact expressions for phonon transmission coefficients are derived for thermal transport in one-junction and two-junction chains, and verified by numerical calculation based on a nonequilibrium Green's function method. For a single-junction case, we find that the phonon transmission coefficient typically decreases monotonically with increasing freqency. However, in the range between equal frequency spectrum and equal ac...

  4. Dynamics of pi-junction interferometer circuits

    Kornkev, V.K.; Mozhaev, P.B.; Borisenko, I.V.; Ovsyannikov, G.A.; Pedersen, Niels Falsig

    The pi-junction superconducting circuit dynamics was studied by means of numerical simulation technique. Parallel arrays consisting of Josephson junctions of both 0- and pi-type were studied as a model of high-T-c grain-boundary Josephson junction. The array dynamics and the critical current...... dependence on magnetic field are discussed. Experimental results for dc interferometers with 0 and pi high-T-c bi-crystal Josephson junctions are reported and discussed in comparison with numerical simulation....

  5. Josephson tunnel junction microwave attenuator

    Koshelets, V. P.; Shitov, S. V.; Shchukin, A. V.;

    1993-01-01

    A new element for superconducting electronic circuitry-a variable attenuator-has been proposed, designed, and successfully tested. The principle of operation is based on the change in the microwave impedance of a superconductor-insulator-superconductor (SIS) Josephson tunnel junction when dc biased...

  6. Dynamics of pi-junction interferometer circuits

    Kornkev, V.K.; Mozhaev, P.B.; Borisenko, I.V.;

    2002-01-01

    The pi-junction superconducting circuit dynamics was studied by means of numerical simulation technique. Parallel arrays consisting of Josephson junctions of both 0- and pi-type were studied as a model of high-T-c grain-boundary Josephson junction. The array dynamics and the critical current...

  7. delta-biased Josephson tunnel junctions

    Monaco, R.; Mygind, Jesper; Koshelet, V.;

    2010-01-01

    Abstract: The behavior of a long Josephson tunnel junction drastically depends on the distribution of the dc bias current. We investigate the case in which the bias current is fed in the central point of a one-dimensional junction. Such junction configuration has been recently used to detect the...

  8. Soliton excitations in Josephson tunnel junctions

    Lomdahl, P. S.; Sørensen, O. H.; Christiansen, Peter Leth

    1982-01-01

    A detailed numerical study of a sine-Gordon model of the Josephson tunnel junction is compared with experimental measurements on junctions with different L / λJ ratios. The soliton picture is found to apply well on both relatively long (L / λJ=6) and intermediate (L / λJ=2) junctions. We find good...

  9. Soliton bunching in annular Josephson junctions

    Vernik, I.V; Lazarides, Nickos; Sørensen, Mads Peter;

    1996-01-01

    By studying soliton (fluxon) motion in long annular Josephson junctions it is possible to avoid the influence of the boundaries and soliton-soliton collisions present in linear junctions. A new experimental design consisting of a niobium coil placed on top of an annular junction has been used to...

  10. GAP junctional communication in brain secondary organizers.

    Bosone, Camilla; Andreu, Abraham; Echevarria, Diego

    2016-06-01

    Gap junctions (GJs) are integral membrane proteins that enable the direct cytoplasmic exchange of ions and low molecular weight metabolites between adjacent cells. They are formed by the apposition of two connexons belonging to adjacent cells. Each connexon is formed by six proteins, named connexins (Cxs). Current evidence suggests that gap junctions play an important part in ensuring normal embryo development. Mutations in connexin genes have been linked to a variety of human diseases, although the precise role and the cell biological mechanisms of their action remain almost unknown. Among the big family of Cxs, several are expressed in nervous tissue but just a few are expressed in the anterior neural tube of vertebrates. Many efforts have been made to elucidate the molecular bases of Cxs cell biology and how they influence the morphogenetic signal activity produced by brain signaling centers. These centers, orchestrated by transcription factors and morphogenes determine the axial patterning of the mammalian brain during its specification and regionalization. The present review revisits the findings of GJ composed by Cx43 and Cx36 in neural tube patterning and discuss Cx43 putative enrollment in the control of Fgf8 signal activity coming from the well known secondary organizer, the isthmic organizer. PMID:27273333

  11. Physical understanding of cryogenic implant benefits for electrical junction stability

    Adeni Khaja, Fareen; Colombeau, Benjamin; Thanigaivelan, Thirumal; Ramappa, Deepak; Henry, Todd [Applied Materials-Varian Semiconductor Equipment, 35 Dory Road, Gloucester, Massachusetts 01930 (United States)

    2012-03-12

    We investigate the effect of cryogenic temperature implants on electrical junction stability for ultra shallow junction applications for sub-32 nm technology nodes and beyond. A comprehensive study was conducted to gain physical understanding of the impact of cryogenic temperature implants on dopant-defect interactions. Carborane (C{sub 2}B{sub 10}H{sub 12}) molecule, a potential alternative to monomer boron was implanted in carbon preamorphized silicon substrates at cryogenic implant temperatures. Results indicate implants at cryogenic temperatures increase dopant activation with reduced diffusion, resulting in lower sheet resistance for a lower junction depth. Further, this study emphasizes the benefits of co-implants performed at cryogenic temperatures as alternative to traditional preamorphizing implants.

  12. Molecular transport junctions: vibrational effects

    Transport of electrons in a single molecule junction is the simplest problem in the general subject area of molecular electronics. In the past few years, this area has been extended to probe beyond the simple tunnelling associated with large energy gaps between electrode Fermi level and molecular levels, to deal with smaller gaps, with near-resonance tunnelling and, particularly, with effects due to interaction of electronic and vibrational degrees of freedom. This overview is devoted to the theoretical and computational approaches that have been taken to understanding transport in molecular junctions when these vibronic interactions are involved. After a short experimental overview, and discussion of different test beds and measurements, we define a particular microscopic model Hamiltonian. That overall Hamiltonian can be used to discuss all of the phenomena dealt with subsequently. These include transition from coherent to incoherent transport as electron/vibration interaction increases in strength, inelastic electron tunnelling spectroscopy and its interpretation and measurement, affects of interelectronic repulsion treated at the Hubbard level, noise in molecular transport junctions, non-linear conductance phenomena, heating and heat conduction in molecular transport junctions and current-induced chemical reactions. In each of these areas, we use the same simple model Hamiltonian to analyse energetics and dynamics. While this overview does not attempt survey the literature exhaustively, it does provide appropriate references to the current literature (both experimental and theoretical). We also attempt to point out directions in which further research is required to answer cardinal questions concerning the behaviour and understanding of vibrational effects in molecular transport junctions. (topical review)

  13. Fabrication of high quality ferromagnetic Josephson junctions

    Weides, M. [Institute for Solid State Research, Research Centre Juelich, D-52425 Juelich (Germany) and CNI-Center of Nanoelectronic Systems for Information Technology, Research Centre Juelich, D-52425 Juelich (Germany)]. E-mail: m.weides@fz-juelich.de; Tillmann, K. [Institute for Solid State Research, Research Centre Juelich, D-52425 Juelich (Germany); Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Research Centre Juelich, D-52425 Juelich (Germany); Kohlstedt, H. [Institute for Solid State Research, Research Centre Juelich, D-52425 Juelich (Germany); CNI-Center of Nanoelectronic Systems for Information Technology, Research Centre Juelich, D-52425 Juelich (Germany); Department of Material Science and Engineering and Department of Physics, University of Berkeley, CA 94720 (United States)

    2006-05-15

    We present ferromagnetic Nb/Al{sub 2}O{sub 3}/Ni{sub 60}Cu{sub 40}/Nb Josephson junctions (SIFS) with an ultrathin Al{sub 2}O{sub 3} tunnel barrier. The junction fabrication was optimized regarding junction insulation and homogeneity of current transport. Using ion-beam-etching and anodic oxidation we defined and insulated the junction mesas. The additional 2 nm thin Cu-layer below the ferromagnetic NiCu (SINFS) lowered interface roughness and ensured very homogeneous current transport. A high yield of junctional devices with j {sub c} spreads less than 2% was obtained.

  14. Fabrication of high quality ferromagnetic Josephson junctions

    We present ferromagnetic Nb/Al2O3/Ni60Cu40/Nb Josephson junctions (SIFS) with an ultrathin Al2O3 tunnel barrier. The junction fabrication was optimized regarding junction insulation and homogeneity of current transport. Using ion-beam-etching and anodic oxidation we defined and insulated the junction mesas. The additional 2 nm thin Cu-layer below the ferromagnetic NiCu (SINFS) lowered interface roughness and ensured very homogeneous current transport. A high yield of junctional devices with j c spreads less than 2% was obtained

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. Modelling of Dual-Junction Solar Cells including Tunnel Junction

    Abdelaziz Amine; Yamina Mir; Mimoun Zazoui

    2013-01-01

    Monolithically stacked multijunction solar cells based on III–V semiconductors materials are the state-of-art of approach for high efficiency photovoltaic energy conversion, in particular for space applications. The individual subcells of the multi-junction structure are interconnected via tunnel diodes which must be optically transparent and connect the component cells with a minimum electrical resistance. The quality of these diodes determines the output performance of the solar cell. The p...

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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...

  6. 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....

  7. 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

  8. 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...

  9. 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.

  10. Seebeck effect in molecular junctions

    Zimbovskaya, Natalya A.

    2016-05-01

    Advances in the fabrication and characterization of nanoscale systems presently allow for a better understanding of their thermoelectric properties. As is known, the building blocks of thermoelectricity are the Peltier and Seebeck effects. In the present work we review results of theoretical studies of the Seebeck effect in single-molecule junctions and similar systems. The behavior of thermovoltage and thermopower in these systems is controlled by several factors including the geometry of molecular bridges, the characteristics of contacts between the bridge and the electrodes, the strength of the Coulomb interactions between electrons on the bridge, and of electron–phonon interactions. We describe the impact of these factors on the thermopower. Also, we discuss a nonlinear Seebeck effect in molecular junctions.

  11. Seebeck effect in molecular junctions

    Advances in the fabrication and characterization of nanoscale systems presently allow for a better understanding of their thermoelectric properties. As is known, the building blocks of thermoelectricity are the Peltier and Seebeck effects. In the present work we review results of theoretical studies of the Seebeck effect in single-molecule junctions and similar systems. The behavior of thermovoltage and thermopower in these systems is controlled by several factors including the geometry of molecular bridges, the characteristics of contacts between the bridge and the electrodes, the strength of the Coulomb interactions between electrons on the bridge, and of electron–phonon interactions. We describe the impact of these factors on the thermopower. Also, we discuss a nonlinear Seebeck effect in molecular junctions. (topical review)

  12. How coherent are Josephson junctions?

    Paik, Hanhee; Bishop, Lev S; Kirchmair, G; Catelani, G; Sears, A P; Johnson, B R; Reagor, M J; Frunzio, L; Glazman, L; Schoelkopf, R J

    2011-01-01

    Attaining sufficient coherence is a requirement for realizing a large-scale quantum computer. We present a new implementation of a superconducting transmon qubit that is strongly coupled to a three-dimensional superconducting cavity. We observe a reproducible increase in the coherence times of qubit (both $T_1$ and $T_2$ > 10 microseconds) and cavity ($T_{cav}$ ~ 50 microseconds) by more than an order of magnitude compared to the current state-of-art superconducting qubits. This enables the study of the stability and quality of Josephson junctions at precisions exceeding one part per million. Surprisingly, we see no evidence for $1/f$ critical current noise. At elevated temperatures, we observe the dissipation due to a small density (< 1 - 10 ppm) of thermally-excited quasiparticles. The results suggest that the overall quality of Josephson junctions will allow error rates of a few $10^{-4}$, approaching the error correction threshold.

  13. Electron transport in molecular junctions

    Jin, Chengjun

    charge position are in quantitative agreement with the experiments, while pure DFT is not. This is the consequence of the accurate energy level alignment, where the DFT+∑ method corrects the self-interaction error in the standard DFT functional and uses a static image charge model to include the image......This thesis addresses the electron transport in molecular junctions, focusing on the energy level alignment and correlation effects. Various levels of theory have been applied to study the structural and electronic effects in different molecular junctions, starting from the single particle density...... the lowest unoccupied molecular level (LUMO) of the 44BP molecule hybridizes strongly with Ni 3d orbitals, the gating is auxiliary by the so-called spinterface. Finally, the correlation effect of the image charge beyond the energy level renormalization has been studied. It is shown that the finite response...

  14. Presynaptic active zone density during development and synaptic plasticity.

    Gwenaëlle L Clarke

    2012-02-01

    Full Text Available Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs, the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS, active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and β-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated.

  15. Counting Statistics in Nanoscale Junctions

    Liu, Yu-Shen; Chen, Yu-Chang

    2010-01-01

    We present first-principles calculations for moments of the current up to the third order in atomic-scale junctions. The quantum correlations of the current are calculated using the current operator in terms of the wave functions obtained self-consistently within the static density-functional theory. We investigate the relationships of the conductance, the second, and the third moment of the current for carbon atom chains of various lengths bridging two metal electrodes in the linear and nonl...

  16. Imaging of cervicothoracic junction trauma

    Kaewlai, Rathachai

    2013-01-01

    Sirote Wongwaisayawan,1 Ruedeekorn Suwannanon,2 Rathachai Kaewlai11Department of Radiology, Ramathibodi Hospital and Mahidol University, Bangkok, Thailand; 2Department of Radiology, Faculty of Medicine, Prince of Songkla University, Hat Yai, ThailandAbstract: Cervicothoracic junction trauma is an important cause of morbidity and mortality in trauma patients. Imaging has played an important role in identifying injuries and guiding appropriate, timely therapy. Computed tomography is currently a...

  17. Roles of gap junctions, connexins and pannexins in epilepsy

    Shanthini eMylvaganam

    2014-05-01

    Full Text Available Enhanced gap junctional communication (GJC between neurons is considered a major factor underlying the neuronal synchrony driving seizure activity. In addition, the hippocampal sharp wave ripple complexes, associated with learning and seizures, are diminished by GJC blocking agents. Although gap junctional blocking drugs inhibit experimental seizures, they all have other nonspecific actions. Besides interneuronal GJC between dendrites, inter-axonal and inter-glial GJC is also considered important for seizure generation. Interestingly, in most studies of cerebral tissue from animal seizure models and from human patients with epilepsy, there is up-regulation of glial, but not neuronal gap junctional mRNA and protein. Significant changes in the expression and post-translational modification of the astrocytic connexin Cx43, and Panx1 were observed in an in vitro Co++ seizure model, further supporting a role for glia in seizure-genesis, although the reasons for this remain unclear. Further suggesting an involvement of astrocytic GJC in epilepsy, is the fact that the expression of astrocytic Cx mRNAs (Cxs 30 and 43 is several fold higher than that of neuronal Cx mRNAs (Cxs 36 and 45, and the number of glial cells outnumber neuronal cells in mammalian hippocampal and cortical tissue. Pannexin expression is also increased in both animal and human epileptic tissues. Specific Cx43 mimetic peptides, Gap 27 and SLS, inhibit the docking of astrocytic connexin Cx43 proteins from forming intercellular gap junctions, diminishing spontaneous seizures. Besides GJs, Cx membrane hemichannels in glia and Panx membrane channels in neurons and glia are also inhibited by gap junctional pharmacological blockers. Although there is no doubt that connexin-based gap junctions and hemichannels, and pannexin-based membrane channels are related to epilepsy, the specific details of how they are involved and how we can modulate their function for therapeutic purposes remain to

  18. Thermoelectric efficiency of molecular junctions.

    Perroni, C A; Ninno, D; Cataudella, V

    2016-09-21

    Focus of the review is on experimental set-ups and theoretical proposals aimed to enhance thermoelectric performances of molecular junctions. In addition to charge conductance, the thermoelectric parameter commonly measured in these systems is the thermopower, which is typically rather low. We review recent experimental outcomes relative to several junction configurations used to optimize the thermopower. On the other hand, theoretical calculations provide estimations of all the thermoelectric parameters in the linear and non-linear regime, in particular of the thermoelectric figure of merit and efficiency, completing our knowledge of molecular thermoelectricity. For this reason, the review will mainly focus on theoretical studies analyzing the role of not only electronic, but also of the vibrational degrees of freedom. Theoretical results about thermoelectric phenomena in the coherent regime are reviewed focusing on interference effects which play a significant role in enhancing the figure of merit. Moreover, we review theoretical studies including the effects of molecular many-body interactions, such as electron-vibration couplings, which typically tend to reduce the efficiency. Since a fine tuning of many parameters and coupling strengths is required to optimize the thermoelectric conversion in molecular junctions, new theoretically proposed set-ups are discussed in the conclusions. PMID:27420149

  19. Environmental assessment of facility operations at the U.S. Department of Energy Grand Junction Projects Office, Grand Junction, Colorado

    The US Department of Energy (DOE) has prepared a sitewide environmental assessment (EA) of the proposed action to continue and expand present-day activities on the DOE Grand Junction Projects Office (GJPO) facility in Grand Junction, Colorado. Because DOE-GJPO regularly proposes and conducts many different on-site activities, DOE decided to evaluate these activities in one sitewide EA rather than in multiple, activity-specific documents. On the basis of the information and analyses presented in the EA, DOE has determined that the proposed action does not constitute a major Federal action significantly affecting the quality of the human environment, as defined by the National Environmental Policy Act (NEPA) of 1969. Therefore, preparation of an environmental impact statement is not required for facility operations, and DOE is issuing this Finding of No Significant Impact (FONSI)

  20. Environmental assessment of facility operations at the U.S. Department of Energy Grand Junction Projects Office, Grand Junction, Colorado

    NONE

    1996-06-01

    The US Department of Energy (DOE) has prepared a sitewide environmental assessment (EA) of the proposed action to continue and expand present-day activities on the DOE Grand Junction Projects Office (GJPO) facility in Grand Junction, Colorado. Because DOE-GJPO regularly proposes and conducts many different on-site activities, DOE decided to evaluate these activities in one sitewide EA rather than in multiple, activity-specific documents. On the basis of the information and analyses presented in the EA, DOE has determined that the proposed action does not constitute a major Federal action significantly affecting the quality of the human environment, as defined by the National Environmental Policy Act (NEPA) of 1969. Therefore, preparation of an environmental impact statement is not required for facility operations, and DOE is issuing this Finding of No Significant Impact (FONSI).

  1. Algorithms for Junctions in Directed Acyclic Graphs

    Ferreira, Carlos Eduardo

    2012-01-01

    Given a pair of distinct vertices u, v in a graph G, we say that s is a junction of u, v if there are in G internally vertex disjoint directed paths from s to u and from s to v. We show how to characterize junctions in directed acyclic graphs. We also consider the two problems in the following and derive efficient algorithms to solve them. Given a directed acyclic graph G and a vertex s in G, how can we find all pairs of vertices of G such that s is a junction of them? And given a directed acyclic graph G and k pairs of vertices of G, how can we preprocess G such that all junctions of k given pairs of vertices could be listed quickly? All junctions of k pairs problem arises in an application in Anthropology and we apply our algorithm to find such junctions on kinship networks of some brazilian indian ethnic groups.

  2. Palladium Electrodes for Molecular Tunnel Junctions

    Chang, Shuai; Sen, Suman; Zhang, Peiming; Gyarfas, Brett; Ashcroft, Brian; Lefkowitz, Steven; Peng, Hongbo; Lindsay, Stuart

    2012-01-01

    Gold has been the metal of choice for research on molecular tunneling junctions, but it is incompatible with CMOS fabrication because it forms deep level traps in silicon. Palladium electrodes do not contaminate silicon, and also give higher tunnel current signals in the molecular tunnel junctions we have studied. The result is cleaner signals in a recognition-tunneling junction that recognizes the four natural DNA bases as well as 5-methyl cytosine, with no spurious background signals. More ...

  3. Electron optics with ballistic graphene junctions

    Chen, Shaowen; Han, Zheng; Elahi, Mirza M.; Habib, K. M. Masum; Wang, Lei; Wen, Bo; Gao, Yuanda; Taniguchi, Takashi; Watanabe, Kenji; Hone, James; Ghosh, Avik W.; Dean, Cory R.

    2016-01-01

    Electrons transmitted across a ballistic semiconductor junction undergo refraction, analogous to light rays across an optical boundary. A pn junction theoretically provides the equivalent of a negative index medium, enabling novel electron optics such as negative refraction and perfect (Veselago) lensing. In graphene, the linear dispersion and zero-gap bandstructure admit highly transparent pn junctions by simple electrostatic gating, which cannot be achieved in conventional semiconductors. M...

  4. Hysteresis development in superconducting Josephson junctions

    The resistively and capacitive shunted junction model is used to investigate hysteresis development in superconducting Josephson junctions. Two empirical formulas that relate the hysteresis width and the quasi-particle diffusion length in terms of the junctions electrical parameters, temperature and frequency are obtained. The obtained formulas provide a simple tool to investigate the full potentials of the hysteresis phenomena. (author). 9 refs, 3 figs

  5. The Fluxion in a Curved Josephson Junction

    Dobrowolski, Tomasz

    2014-01-01

    The curved Josephson junction is described. In the framework of the Maxwell equations the equation that describes the influence of the curvature on the fluxion motion was obtained. The method of geometrical reduction of the sine-Gordon model from three to lower dimensional manifold was applied to the long Josephson junction. It was argued that the geometrical reduction describes the junctions with slowly varying curvatures.

  6. Gap Junctions Couple Astrocytes and Oligodendrocytes

    Orthmann-Murphy, Jennifer L.; ABRAMS, CHARLES K.; Scherer, Steven S.

    2008-01-01

    In vertebrates, a family of related proteins called connexins form gap junctions (GJs), which are intercellular channels. In the central nervous system (CNS), GJs couple oligodendrocytes and astrocytes (O/A junctions) and adjacent astrocytes (A/A junctions), but not adjacent oligodendrocytes, forming a “glial syncytium.” Oligodendrocytes and astrocytes each express different connexins. Mutations of these connexin genes demonstrate that the proper functioning of myelin and oligodendrocytes req...

  7. String junction as a baryonic constituent

    Kalashnikova, Yu S

    1995-01-01

    We extend the model for QCD string with quarks to consider the Mercedes Benz string configuration describing the three-quark baryon. Under the assumption of adiabatic separation of quark and string junction motion we formulate and solve the classical equation of motion for the junction.We dare to quantize the motion of the junction, and discuss the impact of these modes on the baryon spectra.

  8. Analysis of vertebrate gap junction protein.

    Finbow, M E; Shuttleworth, J.; Hamilton, A.E.; Pitts, J D

    1983-01-01

    A new method for the purification of gap junctions is described which depends on the extraction of cell monolayers or tissue homogenates with Triton X-100. The major band on SDS-polyacrylamide gel electrophoresis (PAGE) of junctional preparations from a variety of vertebrate sources has an apparent mol. wt. of 16,000 (16 K). Further evidence for the junctional origin of the 16 K protein is provided by the results of four different experimental approaches. (i) The junctions form a sharp band i...

  9. Microwave photonics with Josephson junction arrays

    Zueco, David; Solano, Enrique; García-Ripoll, Juan José

    2011-01-01

    We introduce an architecture for a photonic crystal in the microwave regime based on superconducting transmission lines interrupted by Josephson junctions. A study of the scattering properties of a single junction in the line shows that the junction behaves as a perfect mirror when the photon frequency matches the Josephson plasma frequency. We generalize our calculations to periodic arrangements of junctions, demonstrating that they can be used for tunable band engineering, forming what we call a quantum circuit crystal. As a relevant application, we discuss the creation of stationary entanglement between two superconducting qubits interacting through a disordered media.

  10. Thermodynamics of two-dimensional Josephson junctions

    We derive the effective free energy of a two-dimensional Josephson junction in the presence of an external current and predict that the junction has a phase transition at a temperature TJ below the bulk transition temperature Tc. In the range TJ c is reduced by thermal fluctuations; for a junction of size L, Ic ∝ Lb(T) where b(T) J c vanishes at L → ∞) while 0 J. Our results may account for the absence of an observable supercurrent at temperatures below Tc in YBa2Cu3Ox-and Bi2Sr2CaCu2O8-based junctions. (orig.)

  11. Black Diamonds at Brane Junctions

    Chamblin, Andrew; Csaki, Csaba; Erlich, Joshua; Timothy J. Hollowood

    2000-01-01

    We discuss the properties of black holes in brane-world scenarios where our universe is viewed as a four-dimensional sub-manifold of some higher-dimensional spacetime. We consider in detail such a model where four-dimensional spacetime lies at the junction of several domain walls in a higher dimensional anti-de Sitter spacetime. In this model there may be any number p of infinitely large extra dimensions transverse to the brane-world. We present an exact solution describing a black p-brane wh...

  12. 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

  13. 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

  14. 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...

  15. 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 ...

  16. 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.

  17. Liquid phase epitaxy of abrupt junctions in InAs and studies of injection radiative tunneling processes

    The p-n junction in a InAs crystal, by liquid phase epitaxy is obtained. The processes of injection and tunneling radiative recombination by emitted radiation from active region of p-n junction for low injection current are studied. (M.C.K.)

  18. Grand Junction Remedial Action Program

    The Grand Junction Remedial Action Program (hereinafter referred to as the Program) originated in 1972 due to a recognized need to reduce the levels of radiation found in some of the structures identified in Grand Junction, Colorado that were constructed in part with uranium mill tailings. Out of over 640 locations eventually identified as qualifying for corrective action, the Program performed remedial construction on 594 of them. The owners of over 45 unremediated structures either did not wish to participate in the voluntary Program, or the structures were torn down, burned down, or were abandoned before the Program could take action on them. Because this was the first remedial action program of its type, and because its task was to reduce the radiation levels as soon as practical, there was no time for lengthly research and development of remedial methods or techniques. Trial and error combined with basic engineering and health physics produced a Program that learned as it progressed. At a cost of $22.7 million over a 15-year period, a substantial portion of the community had radiation exposure reduced because many public buildings such as schools, churches, and businesses, as well as private residences were remediated. 21 refs., 10 figs., 6 tabs

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. Rapid recycling of ClC-2 chloride channels between plasma membrane and endosomes: role of a tyrosine endocytosis motif in surface retrieval.

    Cornejo, Isabel; Niemeyer, María Isabel; Zúñiga, Leandro; Yusef, Yamil R; Sepúlveda, Francisco V; Cid, L Pablo

    2009-12-01

    ClC-2 chloride channel is present in the brain and some transporting epithelia where its function is poorly understood. We have now demonstrated that the surface channels are rapidly internalised and approximately the 70% of the surface membrane protein recycles after 4- to 8-min internalisation. Endocytosis of ClC-2 was dependent upon tyrosine 179 located within an endocytic motif. Rapid recycling accompanied by an even faster internalisation could account for the abundant presence of ClC-2 in intracellular membranous structures. At least a proportion of ClC-2 resides in lipid rafts. Use of beta-cyclodextrin led to an increase in cell surface channel, but, surprisingly, a decrease in functionally active channels. We suggest that ClC-2 requires residing in beta-cyclodextrin sensitive clusters with other molecules in order to remain active. Regulation of ClC-2 trafficking to and within the membrane could be a means of modulating its activity. PMID:19711355

  4. Linker-dependent Junction Formation Probability in Single-Molecule Junctions

    Yoo, Pil Sun; Kim, Taekyeong [HankukUniversity of Foreign Studies, Yongin (Korea, Republic of)

    2015-01-15

    We compare the junction formation probabilities of single-molecule junctions with different linker molecules by using a scanning tunneling microscope-based break-junction technique. We found that the junction formation probability varies as SH > SMe > NH2 for the benzene backbone molecule with different types of anchoring groups, through quantitative statistical analysis. These results are attributed to different bonding forces according to the linker groups formed with Au atoms in the electrodes, which is consistent with previous works. Our work allows a better understanding of the contact chemistry in the metal.molecule junction for future molecular electronic devices.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. Shot noise in YBCO bicrystal Josephson junctions

    Constantinian, K.Y.; Ovsyannikov, G.A.; Borisenko, I.V.; Mygind, Jesper; Pedersen, Niels Falsig

    2003-01-01

    We measured spectral noise density in YBCO symmetric bicrystal Josephson junctions on sapphire substrates at bias voltages up to 100 mV and T 4.2 K. Normal state resistance of the Josephson junctions, R-N = 20-90 Omega and ICRN up to 2.2 mV have been observed in the experimental samples. Noise me...

  12. Genetics Home Reference: junctional epidermolysis bullosa

    ... Junctional epidermolysis bullosa results from mutations in the LAMA3 , LAMB3 , LAMC2 , and COL17A1 genes. Mutations in each ... of all cases of junctional epidermolysis bullosa . The LAMA3 , LAMB3 , and LAMC2 genes each provide instructions for ...

  13. Gap junctions and connexin-interacting proteins

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the best-studied

  14. Electrophysiological study in neuromuscular junction disorders

    Cherian, Ajith; Baheti, Neeraj N.; Iype, Thomas

    2013-01-01

    This review is on ultrastructure and subcellular physiology at normal and abnormal neuromuscular junctions. The clinical and electrophysiological findings in myasthenia gravis, Lambert-Eaton myasthenic syndrome (LEMS), congenital myasthenic syndromes, and botulinum intoxication are discussed. Single fiber electromyography (SFEMG) helps to explain the basis of testing neuromuscular junction function by repetitive nerve stimulation (RNS). SFEMG requires skill and patience and its availability i...

  15. Hall effect in NS and SNS junctions

    Zhou, F.; Spivak, B.

    1997-01-01

    Hall effect in SN and SNS junctions is considered. It is shown that at small temperature the Hall voltage is significantly suppressed as compared to its normal metal value. The time dependence of the Hall voltage in SNS junctions has a form of narrow pulses with the Josephson frequency.

  16. Suppression of timing errors in short overdamped Josephson junctions

    Pankratov, Andrey L.; Spagnolo, Bernardo

    2004-01-01

    The influence of fluctuations and periodical driving on temporal characteristics of short overdamped Josephson junction is analyzed. We obtain the standard deviation of the switching time in the presence of a dichotomous driving force for arbitrary noise intensity and in the frequency range of practical interest. For sinusoidal driving the resonant activation effect has been observed. The mean switching time and its standard deviation have a minimum as a function of driving frequency. As a co...

  17. Shear-induced reorganization of renal proximal tubule cell actin cytoskeleton and apical junctional complexes.

    Duan, Yi; Gotoh, Nanami; Yan, Qingshang; Du, Zhaopeng; Weinstein, Alan M; Wang, Tong; Weinbaum, Sheldon

    2008-08-12

    In this study, we demonstrate that fluid shear stress (FSS)-induced actin cytoskeletal reorganization and junctional formation in renal epithelial cells are nearly completely opposite the corresponding changes in vascular endothelial cells (ECs) [Thi MM et al. (2004) Proc Natl Acad Sci USA 101:16483-16488]. Mouse proximal tubule cells (PTCs) were subjected to 5 h of FSS (1 dyn/cm(2)) to investigate the dynamic responses of the cytoskeletal distribution of filamentous actin (F-actin), ZO-1, E-cadherin, vinculin, and paxillin to FSS. Immunofluorescence analysis revealed that FSS caused basal stress fiber disruption, more densely distributed peripheral actin bands (DPABs), and the formation of both tight junctions (TJs) and adherens junctions (AJs). A dramatic reinforcement of vinculin staining was found at the cell borders as well as the cell interior. These responses were abrogated by the actin-disrupting drug, cytochalasin D. To interpret these results, we propose a "junctional buttressing" model for PTCs in which FSS enables the DPABs, TJs, and AJs to become more tightly connected. In contrast, in the "bumper-car" model for ECs, all junctional connections were severely disrupted by FSS. This "junctional buttressing" model explains why a FSS of only 1/10 of that used in the EC study can cause a similarly dramatic, cytoskeletal response in these tall, cuboidal epithelial cells; and why junctional buttressing between adjacent cells may benefit renal epithelium in maximizing flow-activated, brush border-dependent, transcellular salt and water reabsorption. PMID:18685100

  18. Electrostatic Control over Temperature-Dependent Tunneling across a Single Molecule Junction

    Alvar R. Garrigues; Wang, Lejia; del Barco, Enrique; Nijhuis, Christian A.

    2016-01-01

    Understanding how the mechanism of charge transport through molecular tunnel junctions depends on temperature is crucial to control electronic function in molecular electronic devices. With just a few systems investigated as a function of bias and temperature so far, thermal effects in molecular tunnel junctions remain poorly understood. Here we report a detailed charge transport study of an individual redox-active ferrocene-based molecule over a wide range of temperatures and applied potenti...

  19. Involvement of Holliday Junction Resolvase in Fluoroquinolone-Mediated Killing of Mycobacterium smegmatis

    Long, Quanxin; Du, Qinglin; Fu, Tiwei; Drlica, Karl; Zhao, Xilin; Xie, Jianping

    2014-01-01

    The absence of the Holliday-junction Ruv resolvase of Mycobacterium smegmatis increased the bacteriostatic and bactericidal activities of the fluoroquinolone moxifloxacin, an important antituberculosis agent. The treatment of ruvAB-deficient cells with thiourea and 2,2′-bipyridyl lowered moxifloxacin lethality to wild-type levels, indicating that the absence of ruvAB stimulates a lethal pathway involving reactive oxygen species. A hexapeptide that traps the Holliday junction substrate of RuvA...

  20. Measuring quantum systems with tunnel junctions

    Full text: We present a formalism that allows to describe a quantum system modulating the transmission of a tunnel junction. The tunnel junction acts as an environment for the quantum system. Contrary to the conventional approach to open quantum systems we retain a degree of freedom of the environment, the charge passed through the junction, after averaging over the bath degrees of freedom, employing a projection operator technique. The resulting object characterizing the joint dynamics of the system and the charge is the charge specific density matrix. We derive a master equation describing the time evolution of the charge specific density matrix. We consider two examples of quantum systems coupled to the junction: a spin and a harmonic oscillator. In the spin case we are able to analyze a quantum measurement process in detail. For the oscillator we investigate the noise in the tunnel junction induced by the coupling. (author)

  1. Spin accumulation in triplet Josephson junction

    We employ a Hamiltonian method to study the equal-spin pairing triplet Josephson junction with different orbital symmetries of pair potentials. Both the spin/charge supercurrent and possible spin accumulation at the interface of the junction are analyzed by means of the Keldysh Green's function. It is found that a spontaneous angle-resolved spin accumulation can form at the junction's interface when the orbital symmetries of Cooper pairs in two triplet superconductors are different, the physical origin is the combined effect of the different orbital symmetries and different spin states of Cooper pairs due to the misalignment of two d vectors in triplet leads. An abrupt current reversal effect induced by misalignment of d vectors is observed and can survive in a strong interface barrier scattering because the zero-energy state appears at the interface of the junction. These properties of the p-wave Josephson junction may be helpful for identifying the order parameter symmetry.

  2. 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

  3. 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

  4. Chaos in junctions and devices

    The plan of the paper is as follows. Section 2 is an introduction into chaos in dissipative systems with an emphasis on period doubling and intermittency. The logistic map and the circle map are discussed and their significance as describing systems of continuous dynamics is emphasized. Section 3 is subdivided into two parts after the introduction of the RSJ equations. The first is on the ac driven Josephson junction without a dc bias and the second on the same with a dc current. Each of these subdivisions includes a discussion of experiments as well. There is also a section on investigations that do not fit into either of the above categories. Section 4 is devoted to the dc-SQUID, in the first part as a magnetic flux gauge and in the second as a four dimensional dynamical system, which can be simulated with great accuracy and compared with one dimensional models. (orig./BUD)

  5. Cylindrical Josephson junctions in magnetic fields

    The radial Josephson current I/sub J/ between co-axial cylinders was measured as a function of axial and azimuthal magnetic fields. The junctions were of two types: 0.25 mm diameter Nb-oxide-Sn single junctions and 0.25 mm film diameter Nb-oxide-Sn film double junctions. The Sn film of the single junctions was 160 nm or 200 nm. The Sn films of the double junctions were both either 155 nm or 230 nm. For a pair of cylinders I/sub J/ is zero except when both members are in the same fluxoid quantum state. When I/sub J/not equal to O, the relative phase is independent of aximuthal angle theta. In all measurements the cylinders were in fluxoid state zero. There was a critical value of axial field B/sub s/ which destroyed the Josephson coupling for each junction. This critical field is smallest for the outer tin junction of the double junction. It depends upon geometry and film thickness but is independent of the value of I/sub J/. The calculated value of the Gibbs function per unit volume of the tin films is, however, nearly the same for all junctions at their respective critical fields. Th Josephson current for the 160 nm Sn film single cylindrical junction was measured as a function of axial field B/sub z/ and azimuthal field B/sub theta/. When the axial field was zero the Josephson current as a function of azimuthal field showed the Fraunhofer like pattern of a flat junction in a magnetic field. As the axial field was increased, the central lobe of the Fraunhofer pattern decreased and disappeared at the critical field leaving the side lobes broadened. It is well known that a Josephson junction may switch to the voltage state at any current less than the maximum Josephson current. For some cylindrical junctions the switching currents are not continuously distributed but discrete with certain values occurring repeatedly. This observation is not understood

  6. Calcium-Ask1-MKK7-JNK2-c-Src Signaling Cascade Mediates Disruption of Intestinal Epithelial Tight Junctions by Dextran Sulfate Sodium

    Samak, Geetha; Chaudhry, Kamaljit K.; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H.; Rao, RadhaKrishna

    2015-01-01

    Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with the symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca2+ concentration, and depletion of intracellular Ca2+ by BAPTA or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of Ask1 or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased Tyr-phosphorylation of occludin, ZO-1, E-cadherin and β-catenin. SP600125 abrogated DSS-induced Tyr-phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto phosphorylation of c-Src. This study demonstrates that Ca2+-Ask1-MKK7-JNK2-cSrc signaling cascade mediates DSS-induced tight junction disruption and barrier dysfunction. PMID:25377781

  7. Shallow trench isolation dimensions effects on leakage current and doping concentration of advanced p-n junction diodes

    Poyai, A. [TMEC, 51/4 Moo 1, Wang-Takien District, Amphur Muang, Chachoengsao 24000 (Thailand)]. E-mail: amporn.poyai@nectec.or.th; Rittaporn, I. [TMEC, 51/4 Moo 1, Wang-Takien District, Amphur Muang, Chachoengsao 24000 (Thailand); Simoen, E. [IMEC, Kapeldreef 75, B-3001 Leuven (Belgium); Claeys, C. [IMEC, Kapeldreef 75, B-3001 Leuven (Belgium); E.E. Department, KU Leuven, Kasteelpark Arenberg 10, B-3001 Leuven (Belgium); Rooyackers, R. [IMEC, Kapeldreef 75, B-3001 Leuven (Belgium)

    2004-12-15

    This paper describes the impact of active area and shallow trench isolation (STI) width on the junction leakage current and doping concentration. A higher junction leakage current is found for a narrower active area and STI width. This is mainly due to a higher compressive stress. This compressive stress also affects the doping concentration near the junction. A higher compressive stress when reduce active area width causes a higher doping concentration while a higher compressive stress when reduce STI width results in a lower doping concentration.

  8. Electric field breakdown in single molecule junctions.

    Li, Haixing; Su, Timothy A; Zhang, Vivian; Steigerwald, Michael L; Nuckolls, Colin; Venkataraman, Latha

    2015-04-22

    Here we study the stability and rupture of molecular junctions under high voltage bias at the single molecule/single bond level using the scanning tunneling microscope-based break-junction technique. We synthesize carbon-, silicon-, and germanium-based molecular wires terminated by aurophilic linker groups and study how the molecular backbone and linker group affect the probability of voltage-induced junction rupture. First, we find that junctions formed with covalent S-Au bonds are robust under high voltage and their rupture does not demonstrate bias dependence within our bias range. In contrast, junctions formed through donor-acceptor bonds rupture more frequently, and their rupture probability demonstrates a strong bias dependence. Moreover, we find that the junction rupture probability increases significantly above ∼1 V in junctions formed from methylthiol-terminated disilanes and digermanes, indicating a voltage-induced rupture of individual Si-Si and Ge-Ge bonds. Finally, we compare the rupture probabilities of the thiol-terminated silane derivatives containing Si-Si, Si-C, and Si-O bonds and find that Si-C backbones have higher probabilities of sustaining the highest voltage. These results establish a new method for studying electric field breakdown phenomena at the single molecule level. PMID:25675085

  9. Photoresponse of a Single Y-Junction Carbon Nanotube.

    Samanta, Sudeshna; Saini, Deepika; Singha, Achintya; Das, Kaustuv; Bandaru, Prabhakar R; Rao, Apparao M; Raychaudhuri, Arup Kumar

    2016-07-27

    We report investigation of optical response in a single strand of a branched carbon nanotube (CNT), a Y-junction CNT composed of multiwalled CNTs. The experiment was performed by connecting a pair of branches while grounding the remaining one. Of the three branch combinations, only one combination is optically active which also shows a nonlinear semiconductor-like I-V curve, while the other two branch combinations are optically inactive and show linear ohmic I-V curves. The photoresponse includes a zero-bias photocurrent from the active branch combination. Responsivity of ≈1.6 mA/W has been observed from a single Y-CNT at a moderate bias of 150 mV with an illumination of wavelength 488 nm. The photoresponse experiment allows us to understand the nature of internal connections in the Y-CNT. Analysis of data locates the region of photoactivity at the junction of only two branches and only the combination of these two branches (and not individual branches) exhibits photoresponse upon illumination. A model calculation based on back-to-back Schottky-type junctions at the branch connection explains the I-V data in the dark and shows that under illumination the barriers at the contacts become lowered due to the presence of photogenerated carriers. PMID:27379988

  10. MQT observation in Bi2212 intrinsic Josephson junctions

    Kashiwaya, Satoshi; Matsumoto, Tetsuro; Kashiwaya, Hiromi; Shibata, Hajime; Eisaki, Hiroshi; Yoshida, Yoshiyuki; Kawabata, Shiro; Tanaka, Yukio

    2007-09-01

    The quantum dynamics of Bi 2Sr 2CaCu 2O 8+ δ intrinsic Josephson junctions (IJJ’s) is studied based on the escape rate measurements. The saturations observed in the escape temperature and the width of the switching current below 0.45 K (= T∗) indicate the transition of the switching mechanism from the thermal activation to the macroscopic quantum tunneling at T∗. It is shown that most of the switching properties are consistently explained in terms of the underdamped Josephson junction with quality factor of about 70 in spite of possible damping due to d-wave superconductivity. The present result gives the upper limit of the dissipation of IJJ’s.

  11. Characterization of escape times of Josephson Junctions for signal detection

    Addesso, Paolo; Pierro, Vincenzo

    2011-01-01

    The measurement of the escape time of a Josephson junction might be used to detect the presence of a sinusoidal signal embedded in noise when standard signal processing tools can be prohibitive. We show that the prescriptions for the experimental set-up and some physical behaviors depend on the detection strategy. More specifically, by exploiting the sample mean of escape times to perform detection, two resonant regions are identified. At low frequencies there is a stochastic resonance/activation phenomenon, while near the plasma frequency a geometric resonance appears. The naive sample mean detector is outperformed, in terms of error probability, by the optimal likelihood ratio test. The latter exhibits only geometric resonance, showing monotonically increasing performance as the bias current approaches the junction critical current. In this regime the escape times are vanishingly small and therefore performance are essentially limited by measurement electronics. The behavior of the likelihood ratio and samp...

  12. Osmotic forces and gap junctions in spreading depression: a computational model

    Shapiro, B. E.

    2001-01-01

    In a computational model of spreading depression (SD), ionic movement through a neuronal syncytium of cells connected by gap junctions is described electrodiffusively. Simulations predict that SD will not occur unless cells are allowed to expand in response to osmotic pressure gradients and K+ is allowed to move through gap junctions. SD waves of [K+]out approximately 25 to approximately 60 mM moving at approximately 2 to approximately 18 mm/min are predicted over the range of parametric values reported in gray matter, with extracellular space decreasing up to approximately 50%. Predicted waveform shape is qualitatively similar to laboratory reports. The delayed-rectifier, NMDA, BK, and Na+ currents are predicted to facilitate SD, while SK and A-type K+ currents and glial activity impede SD. These predictions are consonant with recent findings that gap junction poisons block SD and support the theories that cytosolic diffusion via gap junctions and osmotic forces are important mechanisms underlying SD.

  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. Contour junctions underlie neural representations of scene categories in high-level human visual cortex.

    Choo, Heeyoung; Walther, Dirk B

    2016-07-15

    Humans efficiently grasp complex visual environments, making highly consistent judgments of entry-level category despite their high variability in visual appearance. How does the human brain arrive at the invariant neural representations underlying categorization of real-world environments? We here show that the neural representation of visual environments in scene-selective human visual cortex relies on statistics of contour junctions, which provide cues for the three-dimensional arrangement of surfaces in a scene. We manipulated line drawings of real-world environments such that statistics of contour orientations or junctions were disrupted. Manipulated and intact line drawings were presented to participants in an fMRI experiment. Scene categories were decoded from neural activity patterns in the parahippocampal place area (PPA), the occipital place area (OPA) and other visual brain regions. Disruption of junctions but not orientations led to a drastic decrease in decoding accuracy in the PPA and OPA, indicating the reliance of these areas on intact junction statistics. Accuracy of decoding from early visual cortex, on the other hand, was unaffected by either image manipulation. We further show that the correlation of error patterns between decoding from the scene-selective brain areas and behavioral experiments is contingent on intact contour junctions. Finally, a searchlight analysis exposes the reliance of visually active brain regions on different sets of contour properties. Statistics of contour length and curvature dominate neural representations of scene categories in early visual areas and contour junctions in high-level scene-selective brain regions. PMID:27118087

  15. Graded junction termination extensions for electronic devices

    Merrett, J. Neil (Inventor); Isaacs-Smith, Tamara (Inventor); Sheridan, David C. (Inventor); Williams, John R. (Inventor)

    2007-01-01

    A graded junction termination extension in a silicon carbide (SiC) semiconductor device and method of its fabrication using ion implementation techniques is provided for high power devices. The properties of silicon carbide (SiC) make this wide band gap semiconductor a promising material for high power devices. This potential is demonstrated in various devices such as p-n diodes, Schottky diodes, bipolar junction transistors, thyristors, etc. These devices require adequate and affordable termination techniques to reduce leakage current and increase breakdown voltage in order to maximize power handling capabilities. The graded junction termination extension disclosed is effective, self-aligned, and simplifies the implementation process.

  16. Palladium electrodes for molecular tunnel junctions.

    Chang, Shuai; Sen, Suman; Zhang, Peiming; Gyarfas, Brett; Ashcroft, Brian; Lefkowitz, Steven; Peng, Hongbo; Lindsay, Stuart

    2012-10-26

    Gold has been the metal of choice for research on molecular tunneling junctions, but it is incompatible with complementary metal-oxide-semiconductor fabrication because it forms deep level traps in silicon. Palladium electrodes do not contaminate silicon, and also give higher tunnel current signals in the molecular tunnel junctions that we have studied. The result is cleaner signals in a recognition-tunneling junction that recognizes the four natural DNA bases as well as 5-methyl cytosine, with no spurious background signals. More than 75% of all the recorded signal peaks indicate the base correctly. PMID:23037952

  17. Palladium electrodes for molecular tunnel junctions

    Gold has been the metal of choice for research on molecular tunneling junctions, but it is incompatible with complementary metal–oxide–semiconductor fabrication because it forms deep level traps in silicon. Palladium electrodes do not contaminate silicon, and also give higher tunnel current signals in the molecular tunnel junctions that we have studied. The result is cleaner signals in a recognition-tunneling junction that recognizes the four natural DNA bases as well as 5-methyl cytosine, with no spurious background signals. More than 75% of all the recorded signal peaks indicate the base correctly. (paper)

  18. δ-biased Josephson tunnel junctions

    The behavior of a long Josephson tunnel junction drastically depends on the distribution of the dc bias current. We investigate the case in which the bias current is fed in the central point of a one-dimensional junction. Such junction configuration has been recently used to detect the persistent currents circulating in a superconducting loop. Analytical and numerical results indicate that the presence of fractional vortices leads to remarkable differences from the conventional case of uniformly distributed dc bias current. The theoretical findings are supported by detailed measurements on a number of δ-biased samples having different electrical and geometrical parameters.

  19. Plasticity of single-atom Pb junctions

    Müller, M.; Salgado, C.; Néel, N.; Palacios, J. J.; Kröger, J.

    2016-06-01

    A low-temperature scanning tunneling microscope was used to fabricate atomic contacts on Pb(111). Conductance characteristics of the junctions were simultaneously recorded with forming and subsequent breaking of the contacts. A pronounced hysteresis effect in conductance traces was observed from junctions comprising the clean Pb(111) surface. The hysteretic behavior was less profound in contacts to single Pb atoms adsorbed to Pb(111). Density-functional calculations reproduced the experimental results by performing a full ab initio modeling of plastic junction deformations. A comprehensive description of the experimental findings was achieved by considering different atomic tip apex geometries.

  20. Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin.

    Fontes, Joseph D; Ramsey, Jon; Polk, Jeremy M; Koop, Andre; Denisova, Janna V; Belousov, Andrei B

    2015-01-01

    Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed. PMID:26017008

  1. The Intracellular Na+/H+ Exchanger NHE7 Effects a Na+-Coupled, but Not K+-Coupled Proton-Loading Mechanism in Endocytosis

    Nina Milosavljevic

    2014-05-01

    Full Text Available Vesicular H+-ATPases and ClC-chloride transporters are described to acidify intracellular compartments, which also express the highly conserved Na+/H+ exchangers NHE6, NHE7, and NHE9. Mutations of these exchangers cause autism-spectrum disorders and neurodegeneration. NHE6, NHE7, and NHE9 are hypothesized to exchange cytosolic K+ for H+ and alkalinize vesicles, but this notion has remained untested in K+ because their intracellular localization prevents functional measurements. Using proton-killing techniques, we selected a cell line that expresses wild-type NHE7 at the plasma membrane, enabling measurement of the exchanger’s transport parameters. We found that NHE7 transports Li+ and Na+, but not K+, is nonreversible in physiological conditions and is constitutively activated by cytosolic H+. Therefore, NHE7 acts as a proton-loading transporter rather than a proton leak. NHE7 mediates an acidification of intracellular vesicles that is additive to that of V-ATPases and that accelerates endocytosis. This study reveals an unexpected function for vesicular Na+/H+ exchangers and provides clues for understanding NHE-linked neurological disorders.

  2. 2,8-Diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine potent CCR4 antagonists capable of inducing receptor endocytosis.

    Shukla, Lena; Ajram, Laura A; Begg, Malcolm; Evans, Brian; Graves, Rebecca H; Hodgson, Simon T; Lynn, Sean M; Miah, Afjal H; Percy, Jonathan M; Procopiou, Panayiotis A; Richards, Stephen A; Slack, Robert J

    2016-06-10

    A number of potent 2,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine CCR4 antagonists binding to the extracellular allosteric site were synthesised. (R)-N-(2,4-Dichlorobenzyl)-2-(2-(pyrrolidin-2-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine (R)-(18a) has high affinity in both the [(125)I]-TARC binding assay with a pKi of 8.8, and the [(35)S]-GTPγS functional assay with a pIC50 of 8.1, and high activity in the human whole blood actin polymerisation assay (pA2 = 6.7). The most potent antagonists were also investigated for their ability to induce endocytosis of CCR4 and were found to internalise about 60% of the cell surface receptors, a property which is not commonly shared by small molecule antagonists of chemokine receptors. PMID:26991939

  3. Motorway junction design with emphasis on traffic performance and safety assesment - case study junction Ljubljana Rudnik

    Mlaker, Pavel

    2013-01-01

    Thesis encompasses reconstruction predesign of the motorway junction Ljubljana Rudnik into motorway interchange. In this area is intended to be the junction of main arterial road with highway network, while today serves only as a minor junction of Rudnik and Ig area on the motorway. The purpose of reconstruction is to enable free traffic flow on most congested directions of the interchange, but also preserve the present function, in which Ig and Rudnik area are connected with the motorway. Bu...

  4. UMTRA Project water sampling and analysis plan, Grand Junction, Colorado. Revision 1, Version 6

    This water sampling and analysis plan describes the planned, routine ground water sampling activities at the Grand Junction US DOE Uranium Mill Tailings Remedial Action (UMTRA) Project site (GRJ-01) in Grand Junction, Colorado, and at the Cheney Disposal Site (GRJ-03) near Grand Junction. The plan identifies and justifies the sampling locations, analytical parameters, detection limits, and sampling frequencies for the routine monitoring stations at the sites. Regulatory basis is in the US EPA regulations in 40 CFR Part 192 (1994) and EPA ground water quality standards of 1995 (60 FR 2854). This plan summarizes results of past water sampling activities, details water sampling activities planned for the next 2 years, and projects sampling activities for the next 5 years

  5. Molecular junctions: Single-molecule contacts exposed

    Nichols, Richard J.; Higgins, Simon J.

    2015-05-01

    Using a scanning tunnelling microscopy-based method it is now possible to get an atomistic-level description of the most probable binding and contact configuration for single-molecule electrical junctions.

  6. Current trends in salivary gland tight junctions.

    Baker, Olga J

    2016-01-01

    Tight junctions form a continuous intercellular barrier between epithelial cells that is required to separate tissue spaces and regulate selective movement of solutes across the epithelium. They are composed of strands containing integral membrane proteins (e.g., claudins, occludin and tricellulin, junctional adhesion molecules and the coxsackie adenovirus receptor). These proteins are anchored to the cytoskeleton via scaffolding proteins such as ZO-1 and ZO-2. In salivary glands, tight junctions are involved in polarized saliva secretion and barrier maintenance between the extracellular environment and the glandular lumen. This review seeks to provide an overview of what is currently known, as well as the major questions and future research directions, regarding tight junction expression, organization and function within salivary glands. PMID:27583188

  7. Josephson tunnel junctions in niobium films

    A method of fabricating stable Josephson tunnel junctions with reproducible characteristics is described. The junctions have a sandwich structure consisting of a vacuum evaporated niobium film, a niobium oxide layer produced by the glow discharge method and a lead film deposited by vacuum evaporation. Difficulties in producing thin-film Josephson junctions are discussed. Experimental results suggest that the lower critical field of the niobium film is the most essential parameter when evaluating the quality of these junctions. The dependence of the lower critical field on the film thickness and on the Ginzburg-Landau parameter of the film is studied analytically. Comparison with the properties of the evaporated films and with the previous calculations for bulk specimens shows that the presented model is applicable for most of the prepared samples. (author)

  8. Superconducting switch made of graphene nanoribbon junctions

    Liang, Qifeng; Dong, Jinming

    2008-09-01

    The transmission of superconductor-graphene nanoribbon-superconductor junctions (SGS) has been studied by the non-equilibrium Green's function method. It is found that the on-site potential U in the center zigzag graphene nanoribbon (ZGNR) of the SGS junction plays an important role in the magnitude of the supercurrent Ic. As the effective Fermi energy μeff (μeff = μF-U) goes from negative to positive, the SGS junction would suddenly transform from an 'OFF' state to an 'ON' state. And, as μeff increases further, the Ic will continue to increase. This switching behavior of the SGS junction shares the same origin with the zigzag GNR valley-isospin valve (Rycerz et al 2007 Nat. Phys. 3 172). Besides the valley-isospin, the density of states will also have an effect on the suppression of Ic.

  9. Chirality effect in disordered graphene ribbon junctions

    We investigate the influence of edge chirality on the electronic transport in clean or disordered graphene ribbon junctions. By using the tight-binding model and the Landauer-Büttiker formalism, the junction conductance is obtained. In the clean sample, the zero-magnetic-field junction conductance is strongly chirality-dependent in both unipolar and bipolar ribbons, whereas the high-magnetic-field conductance is either chirality-independent in the unipolar or chirality-dependent in the bipolar ribbon. Furthermore, we study the disordered sample in the presence of magnetic field and find that the junction conductance is always chirality-insensitive for both unipolar and bipolar ribbons with adequate disorders. In addition, the disorder-induced conductance plateaus can exist in all chiral bipolar ribbons provided the disorder strength is moderate. These results suggest that we can neglect the effect of edge chirality in fabricating electronic devices based on the magnetotransport in a disordered graphene ribbon. (paper)

  10. 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

  11. Heat dissipation in atomic-scale junctions

    Lee, Woochul; Kim, Kyeongtae; Jeong, Wonho; Zotti, Linda Angela; Pauly, Fabian; Cuevas, Juan Carlos; Reddy, Pramod

    2013-01-01

    Atomic and single-molecule junctions represent the ultimate limit to the miniaturization of electrical circuits. They are also ideal platforms for testing quantum transport theories that are required to describe charge and energy transfer in novel functional nanometre-scale devices. Recent work has successfully probed electric and thermoelectric phenomena in atomic-scale junctions. However, heat dissipation and transport in atomic-scale devices remain poorly characterized owing to experimenta...

  12. Controllable spin transport in ferromagnetic graphene junctions

    Yokoyama, Takehito

    2008-01-01

    We study spin transport in normal/ferromagnetic/normal graphene junctions where a gate electrode is attached to the ferromagnetic graphene. We find that due to the exchange field of the ferromagnetic graphene, spin current through the junctions has an oscillatory behavior with respect to the chemical potential in the ferromagnetic graphene, which can be tuned by the gate voltage. Especially, we obtain a controllable spin current reversal by the gate voltage. Our prediction of high controllabi...

  13. Degradation of connexins and gap junctions

    Falk, Matthias M.; Kells, Rachael M.; Berthoud, Viviana M.

    2014-01-01

    Connexin proteins are short-lived within the cell, whether present in the secretory pathway or in gap junction plaques. Their levels can be modulated by their rate of degradation. Connexins, at different stages of assembly, are degraded through the proteasomal, endo-/lysosomal, and phago-/lysosomal pathways. In this review, we summarize the current knowledge about connexin and gap junction degradation including the signals and protein-protein interactions that participate in their targeting f...

  14. Supercurrent Switch in Graphene π Junctions

    Linder, Jacob; Yokoyama, Takehito; Huertas-Hernando, Daniel; Sudbø, Asle

    2008-05-01

    We study the supercurrent in a superconductor/ferromagnet/superconductor graphene junction. In contrast to its metallic counterpart, the oscillating critical current in our setup decays only weakly upon increasing the exchange field and junction width. We find an unusually large residual value of the supercurrent at the oscillatory cusps due to a strong deviation from a sinusoidal current-phase relationship. Our findings suggest a very efficient device for dissipationless supercurrent switching.

  15. Supercurrent switch in graphene pi junctions.

    Linder, Jacob; Yokoyama, Takehito; Huertas-Hernando, Daniel; Sudbø, Asle

    2008-05-01

    We study the supercurrent in a superconductor/ferromagnet/superconductor graphene junction. In contrast to its metallic counterpart, the oscillating critical current in our setup decays only weakly upon increasing the exchange field and junction width. We find an unusually large residual value of the supercurrent at the oscillatory cusps due to a strong deviation from a sinusoidal current-phase relationship. Our findings suggest a very efficient device for dissipationless supercurrent switching. PMID:18518411

  16. Exotic hadron and string junction model

    Hadron structure is investigated adopting string junction model as a realization of confinement. Besides exotic hadrons (M4, B5 etc.), unconventional hadrons appear. A mass formula for these hadrons is proposed. New selection rule is introduced which requires the covalence of constituent line at hadron vertex. New duality appears due to the freedom of junction, especially in anti BB→anti BB reaction. A possible assignment of exotic and unconventional hadrons to recently observed narrow meson states is presented. (auth.)

  17. 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...

  18. 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...

  19. Black diamonds at brane junctions

    Chamblin, Andrew; Csáki, Csaba; Erlich, Joshua; Hollowood, Timothy J.

    2000-08-01

    We discuss the properties of black holes in brane-world scenarios where our Universe is viewed as a four-dimensional sub-manifold of some higher-dimensional spacetime. We consider in detail such a model where four-dimensional spacetime lies at the junction of several domain walls in a higher dimensional anti-de Sitter spacetime. In this model there may be any number p of infinitely large extra dimensions transverse to the brane-world. We present an exact solution describing a black p-brane which will induce on the brane-world the Schwarzschild solution. This exact solution is unstable to the Gregory-Laflamme instability, whereby long-wavelength perturbations cause the extended horizon to fragment. We therefore argue that at late times a non-rotating uncharged black hole in the brane-world is described by a deformed event horizon in p+4 dimensions which will induce, to good approximation, the Schwarzschild solution in the four-dimensional brane world. When p=2, this deformed horizon resembles a black diamond and more generally for p>2, a polyhedron.

  20. Black Diamonds at Brane Junctions

    Chamblin, A; Erlich, J; Hollowood, Timothy J; Chamblin, Andrew; Csaki, Csaba; Erlich, Joshua; Hollowood, Timothy J.

    2000-01-01

    We discuss the properties of black holes in brane-world scenarios where ouruniverse is viewed as a four-dimensional sub-manifold of somehigher-dimensional spacetime. We consider in detail such a model wherefour-dimensional spacetime lies at the junction of several domain walls in ahigher dimensional anti-de Sitter spacetime. In this model there may be anynumber p of infinitely large extra dimensions transverse to the brane-world. Wepresent an exact solution describing a black p-brane which will induce on thebrane-world the Schwarzschild solution. This exact solution is unstable to theGregory-Laflamme instability, whereby long-wavelength perturbations cause theextended horizon to fragment. We therefore argue that at late times anon-rotating uncharged black hole in the brane-world is described by a deformedevent horizon in p+4 dimensions which will induce, to good approximation, theSchwarzschild solution in the four-dimensional brane world. When p=2, thisdeformed horizon resembles a black diamond and more gener...

  1. Black diamonds at brane junctions

    We discuss the properties of black holes in brane-world scenarios where our Universe is viewed as a four-dimensional sub-manifold of some higher-dimensional spacetime. We consider in detail such a model where four-dimensional spacetime lies at the junction of several domain walls in a higher dimensional anti-de Sitter spacetime. In this model there may be any number p of infinitely large extra dimensions transverse to the brane-world. We present an exact solution describing a black p-brane which will induce on the brane-world the Schwarzschild solution. This exact solution is unstable to the Gregory-Laflamme instability, whereby long-wavelength perturbations cause the extended horizon to fragment. We therefore argue that at late times a non-rotating uncharged black hole in the brane-world is described by a deformed event horizon in p+4 dimensions which will induce, to good approximation, the Schwarzschild solution in the four-dimensional brane world. When p=2, this deformed horizon resembles a black diamond and more generally for p>2, a polyhedron. (c) 2000 The American Physical Society

  2. Spin-crossover molecule based thermoelectric junction

    Using ab-initio numerical methods, we explore the spin-dependent transport and thermoelectric properties of a spin-crossover molecule (i.e., iron complex of 2-(1H-pyrazol-1-yl)-6-(1H-tetrazole-5-yl)pyridine) based nano-junction. We demonstrate a large magnetoresistance, efficient conductance-switching, and spin-filter activity in this molecule-based two-terminal device. The spin-crossover process also modulates the thermoelectric entities. It can efficiently switch the magnitude as well as spin-polarization of the thermocurrent. We find that thermocurrent is changed by ∼4 orders of magnitude upon spin-crossover. Moreover, it also substantially affects the thermopower and consequently, the device shows extremely efficient spin-crossover magnetothermopower generation. Furthermore, by tuning the chemical potential of electrodes into a certain range, a pure spin-thermopower can be achieved for the high-spin state. Finally, the reasonably large values of figure-of-merit in the presence and absence of phonon demonstrate a large heat-to-voltage conversion efficiency of the device. We believe that our study will pave an alternative way of tuning the transport and thermoelectric properties through the spin-crossover process and can have potential applications in generation of spin-dependent current, information storage, and processing

  3. Spin-crossover molecule based thermoelectric junction

    Ghosh, Dibyajyoti [Chemistry and Physics of Materials Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064 (India); Parida, Prakash [Institute for Theoretical Physics, University of Regensburg, D-93040 Regensburg (Germany); Pati, Swapan K. [Theoretical Sciences Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064 (India)

    2015-05-11

    Using ab-initio numerical methods, we explore the spin-dependent transport and thermoelectric properties of a spin-crossover molecule (i.e., iron complex of 2-(1H-pyrazol-1-yl)-6-(1H-tetrazole-5-yl)pyridine) based nano-junction. We demonstrate a large magnetoresistance, efficient conductance-switching, and spin-filter activity in this molecule-based two-terminal device. The spin-crossover process also modulates the thermoelectric entities. It can efficiently switch the magnitude as well as spin-polarization of the thermocurrent. We find that thermocurrent is changed by ∼4 orders of magnitude upon spin-crossover. Moreover, it also substantially affects the thermopower and consequently, the device shows extremely efficient spin-crossover magnetothermopower generation. Furthermore, by tuning the chemical potential of electrodes into a certain range, a pure spin-thermopower can be achieved for the high-spin state. Finally, the reasonably large values of figure-of-merit in the presence and absence of phonon demonstrate a large heat-to-voltage conversion efficiency of the device. We believe that our study will pave an alternative way of tuning the transport and thermoelectric properties through the spin-crossover process and can have potential applications in generation of spin-dependent current, information storage, and processing.

  4. Gap junction- and hemichannel-independent actions of connexins

    Jiang, Jean X.; Gu, Sumin

    2004-01-01

    Connexins have been known to be the protein building blocks of gap junctions and mediate cell–cell communication. In contrast to the conventional dogma, recent evidence suggests that in addition to forming gap junction channels, connexins possess gap junction-independent functions. One important gap junction-independent function for connexins is to serve as the major functional component for hemichannels, the un-apposed halves of gap junctions. Hemichannels, as independent functional units, p...

  5. Plasmon induced electric current in a molecular junction

    Pal, Partha; Jiang, Nan; Sonntag, Matthew; Chiang, Naihao; Foley, Edward; van Duyne, Richard; Seideman, Tamar

    2015-03-01

    We report light-triggered, plasmon-enhanced charge transport in a tip-molecule-surface molecular junction. Experimentally, enhancement of tunneling current is recorded when a chopped laser beam illuminates the junction. The enhancement is quenched when the sample is devoid of molecules and its amplitude increases steeply when the focus of the beam moves closer to the space between the tip and the mono layered sample. Finite difference time domain calculations indicate that maximum electromagnetic field enhancements due to plasmonic activity, occurs in the space between the tip and the sample which is also the region where the tunneling current perturbation peaks. The perturbation in the transport characteristics at the tip-sample junction is theoretically estimated utilizing a recent formulation for describing the transient electronic distribution due to plasmon decoherences. We find the enhancement in the electronic current to be directly proportional to the plasmon excitations only in the presence of a molecular linker which is in excellent agreement with the experimental results. Further analysis reveals that the nascent distribution allows injection of electrons through additional molecular resonances which were previously inaccessible, thus leading to an increased current.

  6. Junction characteristics of C60/polycarbonate blend on Si substrate

    We report a study of the interface between fullerene (C60) doped polycarbonate (PC) blends and n-type Si substrate. C60 is usually an electron acceptor in interpenetrated networks and an electron transport in photovoltaic cells. We have studied that the guest-host approach to prepare C60 doped polycarbonate blend. In this article, we report the I-V characteristics of C60 doped polycarbonate/n-type Si junction and the annealing effect on these characteristics. In this junction, a nanocomposite of organic semiconductor fullerene (C60), used as the active medium, with an inert polycarbonate matrix was spin coated on n-type Si substrate. We found that the C60 shows the junction characteristics with n-type Si substrate. The knee voltage and dynamic resistance varies with concentration of C60 as well as temperature. Ellipsometry studies showed the annealing effect on the refractive index and thickness of C60 doped polycarbonate blend on n-type Si substrate. The optical micrographs show that fullerene (C60) is spherical molecule and it is blend in the form of crystallites having size of micron order.

  7. Phase dynamics of low critical current density YBCO Josephson junctions

    Highlights: • We study the phase dynamics of YBaCuO Josephson junctions using various tools. • We derive information on the dissipation in a wide range of transport parameters. • Dissipation in such devices can be described by a frequency dependent damping model. • The use of different substrates allows us to tune the shell circuit. - Abstract: High critical temperature superconductors (HTS) based devices can have impact in the study of the phase dynamics of Josephson junctions (JJs) thanks to the wide range of junction parameters they offer and to their unconventional properties. Measurements of current–voltage characteristics and of switching current distributions constitute a direct way to classify different regimes of the phase dynamics and of the transport, also in nontrivial case of the moderately damped regime (MDR). MDR is going to be more and more common in JJs with advances in nanopatterning superconductors and synthesizing novel hybrid systems. Distinctive signatures of macroscopic quantum tunneling and of thermal activation in presence of different tunable levels of dissipation have been detected in YBCO grain boundary JJs. Experimental data are supported by Monte Carlo simulations of the phase dynamics, in a wide range of temperatures and dissipation levels. This allows us to quantify dissipation in the MDR and partially reconstruct a phase diagram as guideline for a wide range of moderately damped systems

  8. Berberine potentizes apoptosis induced by X-rays irradiation probably through modulation of gap junctions

    LIU Bing; WANG Qin; YUAN Dong-dong; HONG Xiao-ting; TAO Liang

    2011-01-01

    Background Clinical combination of some traditional Chinese medical herbs, including berberine, with irradiation is demonstrated to improve efficacy of tumor radiotherapy, yet the mechanisms for such effect remain largely unknown. The present study investigated the effect of berberine on apoptosis induced by X-rays irradiation and the relation between this effect and gap junction intercellular communication (GJIC).Methods The role of gap junctions in the modulation of X-rays irradiation-induced apoptosis was explored by manipulation of connexin (Cx) expression, and gap junction function, using oleamide, a GJIC inhibitor, and berberine.Results In transfected HeLa cells, Cx32 expression increased apoptosis induced by X-rays irradiation, while inhibition of gap junction by oleamide reduced the irradiation responses, indicating the dependence of X-rays irradiation-induced apoptosis on GJIC. Berberine, at the concentrations without cytotoxicity, enhanced apoptosis induced by irradiation only in the presence of functional gap junctions.Conclusions These results suggest that berberine potentizes cell apoptosis induced by X-rays irradiation, probably through enhancement of gap junction activity.

  9. Microwave Annealing for NiSiGe Schottky Junction on SiGe P-Channel

    Yu-Hsien Lin

    2015-11-01

    Full Text Available In this paper, we demonstrated the shallow NiSiGe Schottky junction on the SiGe P-channel by using low-temperature microwave annealing. The NiSiGe/n-Si Schottky junction was formed for the Si-capped/SiGe multi-layer structure on an n-Si substrate (Si/Si0.57Ge0.43/Si through microwave annealing (MWA ranging from 200 to 470 °C for 150 s in N2 ambient. MWA has the advantage of being diffusion-less during activation, having a low-temperature process, have a lower junction leakage current, and having low sheet resistance (Rs and contact resistivity. In our study, a 20 nm NiSiGe Schottky junction was formed by TEM and XRD analysis at MWA 390 °C. The NiSiGe/n-Si Schottky junction exhibits the highest forward/reverse current (ION/IOFF ratio of ~3 × 105. The low temperature MWA is a very promising thermal process technology for NiSiGe Schottky junction manufacturing.

  10. Intracellular spermine prevents acid-induced uncoupling of Cx43 gap junction channels.

    Skatchkov, Serguei N; Bukauskas, Feliksas F; Benedikt, Jan; Inyushin, Mikhail; Kucheryavykh, Yuriy V

    2015-06-17

    Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell-cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions. PMID:26011388

  11. Inhibition of connexin43 gap junction channels by the endocrine disruptor ioxynil

    Gap junctions are intercellular plasma membrane domains containing channels that mediate transport of ions, metabolites and small signaling molecules between adjacent cells. Gap junctions play important roles in a variety of cellular processes, including regulation of cell growth and differentiation, maintenance of tissue homeostasis and embryogenesis. The constituents of gap junction channels are a family of trans-membrane proteins called connexins, of which the best-studied is connexin43. Connexin43 functions as a tumor suppressor protein in various tissue types and is frequently dysregulated in human cancers. The pesticide ioxynil has previously been shown to act as an endocrine disrupting chemical and has multiple effects on the thyroid axis. Furthermore, both ioxynil and its derivative ioxynil octanoate have been reported to induce tumors in animal bioassays. However, the molecular mechanisms underlying the possible tumorigenic effects of these compounds are unknown. In the present study we show that ioxynil and ioxynil octanoate are strong inhibitors of connexin43 gap junction channels. Both compounds induced rapid loss of connexin43 gap junctions at the plasma membrane and increased connexin43 degradation. Ioxynil octanoate, but not ioxynil, was found to be a strong activator of ERK1/2. The compounds also had different effects on the phosphorylation status of connexin43. Taken together, the data show that ioxynil and ioxynil octanoate are potent inhibitors of intercellular communication via gap junctions.

  12. Stretching of BDT-gold molecular junctions: Thiol or thiolate termination?

    Souza, Amaury De Melo

    2014-01-01

    It is often assumed that the hydrogen atoms in the thiol groups of a benzene-1,4-dithiol dissociate when Au-benzene-1,4-dithiol-Au junctions are formed. We demonstrate, by stability and transport property calculations, that this assumption cannot be made. We show that the dissociative adsorption of methanethiol and benzene-1,4-dithiol molecules on a flat Au(111) surface is energetically unfavorable and that the activation barrier for this reaction is as high as 1 eV. For the molecule in the junction, our results show, for all electrode geometries studied, that the thiol junctions are energetically more stable than their thiolate counterparts. Due to the fact that density functional theory (DFT) within the local density approximation (LDA) underestimates the energy difference between the lowest unoccupied molecular orbital and the highest occupied molecular orbital by several electron-volts, and that it does not capture the renormalization of the energy levels due to the image charge effect, the conductance of the Au-benzene-1,4-dithiol-Au junctions is overestimated. After taking into account corrections due to image charge effects by means of constrained-DFT calculations and electrostatic classical models, we apply a scissor operator to correct the DFT energy level positions, and calculate the transport properties of the thiol and thiolate molecular junctions as a function of the electrode separation. For the thiol junctions, we show that the conductance decreases as the electrode separation increases, whereas the opposite trend is found for the thiolate junctions. Both behaviors have been observed in experiments, therefore pointing to the possible coexistence of both thiol and thiolate junctions. Moreover, the corrected conductance values, for both thiol and thiolate, are up to two orders of magnitude smaller than those calculated with DFT-LDA. This brings the theoretical results in quantitatively good agreement with experimental data.

  13. The junctional complex in the intestine of Sagitta setosa (Chaetognatha): the paired septate junction.

    Duvert, M; Gros, D; Salat, C

    1980-04-01

    The junctional complex of the intestine of Sagitta setosa has been studied in tissues stained with uranyl acetate or after lanthanum impregnation, and by freeze-cleavage. All types of junctions have been characterized in both perpendicular and tangential planes. From the apex to the base of the cell the following junctions occur in this order: a zonula adhaerens; a septate junction where the septa occur in pairs; a pleated sheet septate junction; and numerous gap junctions of the A-type. From the upper part of the cells inwards to the septate junction, the membranes follow a relatively straight path. In the lower part of the cells the membranes are deeply interdigitating. At the intersection between 3 cells a very different junction is to be observed where small units, periodically disposed, bind the membranes of the 3 adjoining cells. Each unit is composed of 3 short segments which bind the cell membranes to a central ring 16.6 +/- 2.3 nm in outer diameter. The paired septate junction constitutes a new type. Its main features are that the septa are paired and occur in 2 formations, one the 'loose formation', with elements between the septa of each pair, and the other, a 'tight formation'. After lanthanum impregnation, the thickness of each septum is seen to be about 3 nm and the undulation period 12.6 +/- 1.6 nm. On freeze-fractures 10-nm particles are found on crests on the PF face and in furrows on the EF face. The possible significance of this type of junction is discussed. The junctional complex described is analogous to those found in various invertebrate epithelia. PMID:6105159

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Gap junctions between CA3 pyramidal cells contribute to network synchronization in neonatal hippocampus.

    Molchanova, Svetlana M; Huupponen, Johanna; Lauri, Sari E; Taira, Tomi

    2016-08-01

    Direct electrical coupling between neurons through gap junctions is prominent during development, when synaptic connectivity is scarce, providing the additional intercellular connectivity. However, functional studies of gap junctions are hampered by the unspecificity of pharmacological tools available. Here we have investigated gap-junctional coupling between CA3 pyramidal cells in neonatal hippocampus and its contribution to early network activity. Four different gap junction inhibitors, including the general blocker carbenoxolone, decreased the frequency of network activity bursts in CA3 area of hippocampus of P3-6 rats, suggesting the involvement of electrical connections in the generation of spontaneous network activity. In CA3 pyramidal cells, spikelets evoked by local stimulation of stratum oriens, were inhibited by carbenoxolone, but not by inhibitors of glutamatergic and GABAergic synaptic transmission, signifying the presence of electrical connectivity through axo-axonic gap junctions. Carbenoxolone also decreased the success rate of firing antidromic action potentials in response to stimulation, and changed the pattern of spontaneous action potential firing of CA3 pyramidal cells. Altogether, these data suggest that electrical coupling of CA3 pyramidal cells contribute to the generation of the early network events in neonatal hippocampus by modulating their firing pattern and synchronization. PMID:26926429

  19. Josephson tunnel junctions with ferromagnetic interlayer

    Superconductivity and ferromagnetism are well-known physical properties of solid states that have been widely studied and long thought about as antagonistic phenomena due to difference in spin ordering. It turns out that the combination of both superconductor and ferromagnet leads to a very rich and interesting physics. One particular example, the phase oscillations of the superconducting order parameter inside the ferromagnet, will play a major role for the devices discussed in this work. In this thesis, I present Josephson junctions with a thin Al2O3 tunnel barrier and a ferromagnetic interlayer, i.e. superconductor-insulator-ferromagnet-superconductor (SIFS) stacks. The fabrication of junctions was optimized regarding the insulation of electrodes and the homogeneity of the current transport. The junctions were either in the 0 or π coupled ground state, depending on the thickness of the ferromagnetic layer and on temperature. The influence of ferromagnetic layer thickness on the transport properties and the coupling (0, π) of SIFS tunnel junctions was studied. Furthermore, using a stepped ferromagnetic layer with well-chosen thicknesses, I obtained the so-called 0-π Josephson junction. At a certain temperature this 0-π junction can be made perfectly symmetric. In this case the ground state corresponds to a vortex of supercurrent creating a magnetic flux which is a fraction of the magnetic flux quantum Φ0. Such structures allow to study the physics of fractional vortices and to build various electronic circuits based on them. The SIFS junctions presented here have an exponentially vanishing damping at T → 0. The SIFS technology developed within the framework of this work may be used to construct classical and quantum devices such as oscillators, memory cells and qubits. (orig.)

  20. Josephson tunnel junctions with ferromagnetic interlayer

    Weides, M.P.

    2006-07-01

    Superconductivity and ferromagnetism are well-known physical properties of solid states that have been widely studied and long thought about as antagonistic phenomena due to difference in spin ordering. It turns out that the combination of both superconductor and ferromagnet leads to a very rich and interesting physics. One particular example, the phase oscillations of the superconducting order parameter inside the ferromagnet, will play a major role for the devices discussed in this work. In this thesis, I present Josephson junctions with a thin Al{sub 2}O{sub 3} tunnel barrier and a ferromagnetic interlayer, i.e. superconductor-insulator-ferromagnet-superconductor (SIFS) stacks. The fabrication of junctions was optimized regarding the insulation of electrodes and the homogeneity of the current transport. The junctions were either in the 0 or {pi} coupled ground state, depending on the thickness of the ferromagnetic layer and on temperature. The influence of ferromagnetic layer thickness on the transport properties and the coupling (0, {pi}) of SIFS tunnel junctions was studied. Furthermore, using a stepped ferromagnetic layer with well-chosen thicknesses, I obtained the so-called 0-{pi} Josephson junction. At a certain temperature this 0-{pi} junction can be made perfectly symmetric. In this case the ground state corresponds to a vortex of supercurrent creating a magnetic flux which is a fraction of the magnetic flux quantum {phi}{sub 0}. Such structures allow to study the physics of fractional vortices and to build various electronic circuits based on them. The SIFS junctions presented here have an exponentially vanishing damping at T {yields} 0. The SIFS technology developed within the framework of this work may be used to construct classical and quantum devices such as oscillators, memory cells and qubits. (orig.)

  1. Increasing gap junctional coupling: a tool for dissecting the role of gap junctions

    Axelsen, Lene Nygaard; Haugan, Ketil; Stahlhut, Martin;

    2007-01-01

    Much of our current knowledge about the physiological and pathophysiological role of gap junctions is based on experiments where coupling has been reduced by either chemical agents or genetic modification. This has brought evidence that gap junctions are important in many physiological processes....... In a number of cases, gap junctions have been implicated in the initiation and progress of disease, and experimental uncoupling has been used to investigate the exact role of coupling. The inverse approach, i.e., to increase coupling, has become possible in recent years and represents a new way of testing...... the role of gap junctions. The aim of this review is to summarize the current knowledge obtained with agents that selectively increase gap junctional intercellular coupling. Two approaches will be reviewed: increasing coupling by the use of antiarrhythmic peptide and its synthetic analogs...

  2. Clathrin and Cx43 gap junction plaque endoexocytosis

    In earlier transmission electron microscopic studies, we have described pentilaminar gap junctional membrane invaginations and annular gap junction vesicles coated with short, electron-dense bristles. The similarity between these electron-dense bristles and the material surrounding clathrin-coated pits led us to suggest that the dense bristles associated with gap junction structures might be clathrin. To confirm that clathrin is indeed associated with annular gap junction vesicles and gap junction plaques, quantum dot immuno-electron microscopic techniques were used. We report here that clathrin associates with both connexin 43 (Cx43) gap junction plaques and pentilaminar gap junction vesicles. An important finding was the preferential localization of clathrin to the cytoplasmic surface of the annular or of the gap junction plaque membrane of one of the two contacting cells. This is consistent with the possibility that the direction of gap junction plaque internalization into one of two contacting cells is regulated by clathrin

  3. Gap Junctions Contribute to Ictal/Interictal Genesis in Human Hypothalamic Hamartomas

    Jie Wu

    2016-06-01

    Full Text Available Human hypothalamic hamartoma (HH is a rare subcortical lesion associated with treatment-resistant epilepsy. Cellular mechanisms responsible for epileptogenesis are unknown. We hypothesized that neuronal gap junctions contribute to epileptogenesis through synchronous activity within the neuron networks in HH tissue. We studied surgically resected HH tissue with Western-blot analysis, immunohistochemistry, electron microscopy, biocytin microinjection of recorded HH neurons, and microelectrode patch clamp recordings with and without pharmacological blockade of gap junctions. Normal human hypothalamus tissue was used as a control. Western blots showed increased expression of both connexin-36 (Cx36 and connexin-43 (Cx43 in HH tissue compared with normal human mammillary body tissue. Immunohistochemistry demonstrated that Cx36 and Cx43 are expressed in HH tissue, but Cx36 was mainly expressed within neuron clusters while Cx43 was mainly expressed outside of neuron clusters. Gap-junction profiles were observed between small HH neurons with electron microscopy. Biocytin injection into single recorded small HH neurons showed labeling of adjacent neurons, which was not observed in the presence of a neuronal gap-junction blocker, mefloquine. Microelectrode field recordings from freshly resected HH slices demonstrated spontaneous ictal/interictal-like discharges in most slices. Bath-application of gap-junction blockers significantly reduced ictal/interictal-like discharges in a concentration-dependent manner, while not affecting the action-potential firing of small gamma-aminobutyric acid (GABA neurons observed with whole-cell patch-clamp recordings from the same patient's HH tissue. These results suggest that neuronal gap junctions between small GABAergic HH neurons participate in the genesis of epileptic-like discharges. Blockade of gap junctions may be a new therapeutic strategy for controlling seizure activity in HH patients.

  4. Josephson tunnel junctions with ferromagnetic barrier layer

    We have fabricated Nb/Al2O3/Ni0.6Cu0.4/Nb Josephson tunnel junctions. Depending on the thickness of the ferromagnetic Ni0.6Cu0.4 layer and on the ambient temperature, the junctions were in the 0 or π coupled ground state. The Al2O3 tunnel barrier allows to achieve rather low damping. The critical current density in the π state was up to 5 A/cm2 at T=2.1 K, resulting in a Josephson penetration depth λJ as low as 160 μm. Experimentally determined junction parameters are well described by theory taking into account spin-flip scattering in the Ni0.6Cu0.4 layer and different interface transparencies. Using a ferromagnetic layer with a step-like thickness we obtain a 0-π junction with equal lengths and critical currents of 0 and π parts. The Ic(H) pattern shows a clear minimum in the vicinity of zero field. The ground state of our 330 μm (1.3λJ) long junction corresponds to a spontaneous vortex of supercurrent pinned at the 0-π phase boundary, carrying ∝ 6.7% of the magnetic flux quantum Φ0. (orig.)

  5. Auger voltage imaging for junction delineation

    A new method for the two-dimensional characterization of dopant profiles in semiconductors, called 'Auger Voltage Contrast' (AVC), is introduced, which investigates the effect of the dopant on the electronic properties of the device, e.g. the change of the Fermi level across a semiconductor surface. This change can be detected by extracting the shift of the Si-LVV Auger peak with respect to a reference spectrum. AVC linescans across pn-junctions have been modeled using the MINIMOS-NT device simulator, finding the energy shift across a pn-junction is not directly representative for the dopant distribution itself, but that the turning point of the AVC energy shift coincides with the position of the junction, making AVC an applicable tool for junction delineation. Furthermore, contamination experiments showed that small amounts of oxide on the semiconductor surface do not influence the contrast in an AVC image. For processing such an energy shift map, a software tool has been developed, which is able to obtain a map that assigns four regions to the semiconductor: regions that are p-type, regions that are n-type, regions that cannot be assigned to either type due to contamination and regions that act as the 'error bar' between p and n. Experimental data obtained from two-dimensional test structures have been processed with this tool. The resulting images clearly show the n- and p-type regions, and the width of the region corresponding to the junction are clearly below 50 nm. (author)

  6. Soliton excitations in Josephson tunnel junctions

    A detailed numerical study of a sine-Gordon model of the Josephson tunnel junction is compared with experimental measurements on junctions with different L/lambda/sub J/ ratios. The soliton picture is found to apply well on both relatively (L/lambda/sub J/ = 6) and intermediate (L/lambda/sub J/ = 2) junctions. We find good agreement for the current-voltage characteristics, power output, and for the shape and height of the zero-field steps (ZFS). Two distinct modes of solition oscillations are observed: (i) a bunched or congealed mode giving rise to the fundamental frequency ∫1 on all ZFS's and (ii) a ''symmetric'' mode which on the Nth ZFS yields the frequency N∫1. Coexistence of two adjacent frequencies is found on the third ZFS of the longer junction (L/lambda/sub J/ = 6) in a narrow range of bias current as also found in the experiments. Small asymmetries in the experimental environment, a weak magnetic field, e.g., is introduced via the boundary conditions of our numerical model. This gives a junction response to variations in the applied bias current close to that observed experimentally

  7. Inhomogeneous parallel arrays of Josephson junctions

    Highlights: → New long wave model of an inhomogeneous parallel array of Josephson junctions. → Adapted spectral problem giving resonances in the current-voltage characteristic. → At resonances solution is described by two ordinary differential equations. → Good agreement with the characteristic curve of a real five junction array. - Abstract: We model new inhomogeneous parallel arrays of small Josephson junctions by taking into account the time and space variations of the field in the cavity and the capacity miss-match at the junctions. The model consists in a wave equation with Dirac delta function sine nonlinearities. We introduce an adapted spectral problem whose spectrum gives the resonances in the current-voltage characteristic curve of any array. It is shown that at the resonances the solution is described by two simple ordinary differential equations. The resonances obtained by this approach are in good agreement with the characteristic curve of a real five junction array. This flexible approach is a first step towards building a device tailored for given purposes.

  8. Particle detection with superconducting tunnel junctions

    At the Institute of Experimental Nuclear Physics of the University of Karlsruhe (TH) and at the Institute for Nuclear Physics of the Kernforschungszentrum Karlsruhe we started to produce superconducting tunnel junctions and to investigate them for their suitability as particle detectors. The required facilities for the production of tunnel junctions and the experimental equipments to carry out experiments with them were erected. Experiments are presented in which radiations of different kinds of particles could successfully be measured with the tunnel junctions produced. At first we succeeded in detectioning light pulses of a laser. In experiments with alpha-particles of an energy of 4,6 MeV the alpha-particles were detected with an energy resolution of 1,1%, and it was shown in specific experiments that the phonons originating from the deposition of energy by an alpha-particle in the substrate can be detected with superconducting tunnel junctions at the surface. On that occasion it turned out that the signals could be separated with respect to their point of origin (tunnel junction, contact leads, substrate). Finally X-rays with an energy of 6 keV were detected with an energy resolution of 8% in a test arrangement that makes use of the so-called trapping effect to read out a larger absorber volume. (orig.)

  9. An ultra-small capacitance Josephson junction

    We consider a voltage biased ultra-small capacitance Josephson junction, with the coupling to the external source containing both resistive and inductive elements. In addition we include a phenomenological coupling to an external heat bath. Our goal is to extend and generalize previous studies of current biased ultra-small junctions. Charging effects, due to the presence of discrete charge carriers in the junction, play a crucial role. In particular we find an infinite resistance branch in the I-V characteristic for a d.c. bias, and resistive steps in the I-V curve when the external bias contains an additional a.c. component. These effects are reminiscent of the 'Coulomb blockade' and the inverse Shapiro steps, respectively, predicted earlier in the context of current biased circuits. As a response to an a.c. voltage bias we also predict spikes of the voltage across the junction and a noisy background, when this voltage is plotted as a function of either the external d.c. biasing voltage or the external frequency. Our analysis shows that various circuitry components may qualitatively affect the response of the junction to an external bias. (authors)

  10. Sympathetic innervation controls homeostasis of neuromuscular junctions in health and disease.

    Khan, Muzamil Majid; Lustrino, Danilo; Silveira, Willian A; Wild, Franziska; Straka, Tatjana; Issop, Yasmin; O'Connor, Emily; Cox, Dan; Reischl, Markus; Marquardt, Till; Labeit, Dittmar; Labeit, Siegfried; Benoit, Evelyne; Molgó, Jordi; Lochmüller, Hanns; Witzemann, Veit; Kettelhut, Isis C; Navegantes, Luiz C C; Pozzan, Tullio; Rudolf, Rüdiger

    2016-01-19

    The distribution and function of sympathetic innervation in skeletal muscle have largely remained elusive. Here we demonstrate that sympathetic neurons make close contact with neuromuscular junctions and form a network in skeletal muscle that may functionally couple different targets including blood vessels, motor neurons, and muscle fibers. Direct stimulation of sympathetic neurons led to activation of muscle postsynaptic β2-adrenoreceptor (ADRB2), cAMP production, and import of the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PPARGC1A) into myonuclei. Electrophysiological and morphological deficits of neuromuscular junctions upon sympathectomy and in myasthenic mice were rescued by sympathicomimetic treatment. In conclusion, this study identifies the neuromuscular junction as a target of the sympathetic nervous system and shows that sympathetic input is crucial for synapse maintenance and function. PMID:26733679

  11. Characterization of magnetic tunnel junction test pads

    Østerberg, Frederik Westergaard; Kjær, Daniel; Nielsen, Peter Folmer;

    2015-01-01

    We show experimentally as well as theoretically that patterned magnetic tunnel junctions can be characterized using the current-in-plane tunneling (CIPT) method, and the key parameters, the resistance-area product (RA) and the tunnel magnetoresistance (TMR), can be determined. The CIPT method...... square tunnel junction pads with varying sizes and analyze the measured data using both the original and the modified CIPT model. Thus, we determine in which sample size range the modified CIPT model is needed to ensure validity of the extracted sample parameters, RA and TMR. In addition, measurements as...... a function of position on a square tunnel junction pad are used to investigate the sensitivity of the measurement results to probe misalignment....

  12. Holographic Josephson junction from massive gravity

    Hu, Ya-Peng; Li, Huai-Fan; Zeng, Hua-Bi; Zhang, Hai-Qing

    2016-05-01

    We study the holographic superconductor-normal metal-superconductor (SNS) Josephson junction in de Rham-Gabadadze-Tolley massive gravity. If the boundary theory is independent of spatial directions, i.e., if the chemical potential is homogeneous in spatial directions, we find that the graviton mass parameter will make it more difficult for the normal metal-superconductor phase transition to take place. In the holographic model of the Josephson junction, it is found that the maximal tunneling current will decrease according to the graviton mass parameter. Besides, the coherence length of the junction decreases as well with respect to the graviton mass parameter. If one interprets the graviton mass parameter as the effect of momentum dissipation in the boundary field theory, this indicates that the stronger the momentum dissipation is, the smaller the coherence length is.

  13. Holographic Josephson Junction from Massive Gravity

    Hu, Ya-Peng; Zeng, Hua-Bi; Zhang, Hai-Qing

    2015-01-01

    We study the holographic superconductor-normal metal-superconductor (SNS) Josephon junction in the massive gravity. In the homogeneous case of the chemical potential, we find that the graviton mass will make the normal metal-superconductor phase transition harder to take place. In the holographic model of Josephson junction, it is found that the maximal tunneling current will decrease according to the graviton mass. Besides, the coherence length of the junction decreases as well with respect to the graviton mass. If one interprets the graviton mass as the effect of momentum dissipation in the boundary field theory, it indicates that the stronger the momentum dissipation is, the smaller the coherence length is.

  14. Numerical Investigation of Josephson Junction Structures

    Multilayered long Josephson Junction Structures form an interesting physical system where both nonlinearity and interaction between subsystems play an important role. Such systems allow to study physical effects that do not occur in single Josephson junction.The Sakai-Bodin-Pedersen model--a system of perturbed sine-Gordon equations--is used to study the dynamic states of stacks of inductively coupled long Josephson Junctions (LJJs). The corresponding static problem is numerically investigated as well. In order to study the stability of possible static solutions a Sturm-Liouville problem is generated and solved.The transitions from static to dynamic state and the scenario of these transitions are analyzed depending on the model parameters. Different physical characteristics--current-voltage characteristics, individual instant voltages and internal magnetic fields, are calculated and interpreted.

  15. Electron and Phonon Transport in Molecular Junctions

    Li, Qian

    transmission at the Fermi energy. We propose and analyze a way of using π   stacking to design molecular junctions to control heat transport. We develop a simple model system to identify optimal parameter regimes and then use density functional theory (DFT) to extract model parameters for a number of specific......Molecular electronics provide the possibility to investigate electron and phonon transport at the smallest imaginable scale, where quantum effects can be investigated and exploited directly in the design. In this thesis, we study both electron transport and phonon transport in molecular junctions...... DFT method. It is found that the thermal conductance of π-stacked systems can be reduced by 95%, compared with that in a single-molecule junction. Phonon transmission of π-stacked systems is reduced dramatically in the whole frequency range and the left transmission mainly remains below 5 THz....

  16. Phonon spectroscopy with superconducting tunnel junctions

    Superconducting tunnel junctions can be used as generators and detectors of monochromatic phonons of frequency larger than 80 GHz, as was first devised by Eisenmenger and Dayem (1967) and Kinder (1972a, 1973). In this report, we intend to give a general outline of this type of spectroscopy and to present the results obtained so far. The basic physics underlying phonon generation and detection are described in chapter I, a wider approach being given in the references therein. In chapter II, the different types of junctions are considered with respect to their use. Chapter III deals with the evaporation technique for the superconducting junctions. The last part of this report is devoted to the results that we have obtained on γ-irradiated LiF, pure Si and Phosphorous implanted Si. In these chapters, the limitations of the spectrometer are brought out and suggestions for further work are given

  17. Josephson junction microcalorimeter with a superconductor loop

    Yoshihara, F; Shinada, K

    2003-01-01

    We propose a new microcalorimeter in which the critical current of a Josephson junction can be varied by an electron temperature in the normal metal barrier of the superconductor-normal metal-superconductor (SNS) or superconductor-normal metal-insulator-superconductor (SNIS) junctions. In this detector, a Josephson junction with a radiation absorber is included in a superconductor loop and the change of its critical current is converted into a change of magnetic flux in the loop. We estimated the energy resolution of this detector by calculating a noise equivalent power (NEP) of the detector. The estimated energy resolution and dynamic range are 4.2 eV/5.8 eV and 3.1 keV/6.2 keV, respectively with an Ag absorber of 500 x 500 x 2 mu m sup 3 at 100 mK.

  18. Electronic Veselago lensing in graphene PN junctions

    Dean, Cory

    Ballistic electrons in a uniform 2D electron gas (2DEG) behave in close analogy to light propagating through an optical medium. In the absence of impurity scattering, electrons follow straight-line trajectories, while the associated de Broglie wavelength can give rise to interference and diffraction. Here we present measurements of ballistic graphene devices in which a graphite gate is used to realize an atomically-smooth junction. We demonstrate unambiguous signatures of negative refraction across a PN junction, paving the way for electron optics inspired by Veselago lensing. Comparison with theoretical simulations reveals the importance of the junction profile towards this effort. Opportunities for future device designs that may take advantage of these effects will be discussed.

  19. Magnesium gating of cardiac gap junction channels.

    Matsuda, Hiroyuki; Kurata, Yasutaka; Oka, Chiaki; Matsuoka, Satoshi; Noma, Akinori

    2010-09-01

    We aimed to study kinetics of modulation by intracellular Mg(2+) of cardiac gap junction (Mg(2+) gate). Paired myocytes of guinea-pig ventricle were superfused with solutions containing various concentrations of Mg(2+). In order to rapidly apply Mg(2+) to one aspect of the gap junction, the non-junctional membrane of one of the pair was perforated at nearly the connecting site by pulses of nitrogen laser beam. The gap junction conductance (G(j)) was measured by clamping the membrane potential of the other cell using two-electrode voltage clamp method. The laser perforation immediately increased G(j), followed by slow G(j) change with time constant of 3.5 s at 10 mM Mg(2+). Mg(2+) more than 1.0 mM attenuated dose-dependently the gap junction conductance and lower Mg(2+) (0.6 mM) increased G(j) with a Hill coefficient of 3.4 and a half-maximum effective concentration of 0.6 mM. The time course of G(j) changes was fitted by single exponential function, and the relationship between the reciprocal of time constant and Mg(2+) concentration was almost linear. Based on the experimental data, a mathematical model of Mg(2+) gate with one open state and three closed states well reproduced experimental results. One-dimensional cable model of thirty ventricular myocytes connected to the Mg(2+) gate model suggested a pivotal role of the Mg(2+) gate of gap junction under pathological conditions. PMID:20553744

  20. Transport theory of carbon nanotube Y junctions

    We describe a generalization of Landauer-Buettiker theory for networks of interacting metallic carbon nanotubes. We start with symmetric starlike junctions and then extend our approach to asymmetric systems. While the symmetric case is solved in closed form, the asymmetric situation is treated by a mixture of perturbative and non-perturbative methods. For N > 2 repulsively interacting nanotubes, the only stable fixed point of the symmetric system corresponds to an isolated node. Detailed results for both symmetric and asymmetric systems are shown for N = 3, corresponding to carbon nanotube Y junctions

  1. Tunnel magnetoresistance of an organic molecule junction

    Coherent spin-dependent electronic transport is investigated in a molecular junction based on oligophenylene attached to two the semi-infinite ferromagnetic (FM) electrodes with finite cross sections. The work is based on the tight-binding Hamiltonian model and within the framework of a non-equilibrium Green's function (NEGF) technique. It is shown that tunnel magnetoresistance (TMR) of molecular junction can be large (over 60 %) by adjusting the related parameters, and depends on: (i) the applied voltages and (ii) the length of oligophenylele molecule.

  2. Gap junctions-guards of excitability

    Stroemlund, Line Waring; Jensen, Christa Funch; Qvortrup, Klaus;

    2015-01-01

    Cardiomyocytes are connected by mechanical and electrical junctions located at the intercalated discs (IDs). Although these structures have long been known, it is becoming increasingly clear that their components interact. This review describes the involvement of the ID in electrical disturbances...... of the heart and focuses on the role of the gap junctional protein connexin 43 (Cx43). Current evidence shows that Cx43 plays a crucial role in organizing microtubules at the intercalated disc and thereby regulating the trafficking of the cardiac sodium channel NaV1.5 to the membrane....

  3. Supercurrent in long ballistic graphene Josephson junctions

    Borzenets, I. V.; Amet, F.; Ke, C. T.; Watanabe, K.; Taniguchi, T; Yamamoto, M.; Tarucha, S.; Finkelstein, G

    2016-01-01

    We investigate the critical current $I_C$ in Josephson junctions made of encapsulated graphene/boron-nitride heterostructures. $I_C$ is found to scale with temperature $T$ as $\\propto exp(-k_bT/\\delta E)$, which is consistent with the conventional model for ballistic Josephson junctions that are long compared to the thermal length. The extracted energy $\\delta E$ is independent of the carrier density and consistent with the level spacing of the ballistic cavity, as determined from Fabry-Perot...

  4. Bursting behaviour in coupled Josephson junctions.

    Hongray, Thotreithem; Balakrishnan, J; Dana, Syamal K

    2015-12-01

    We report an interesting bow-tie shaped bursting behaviour in a certain parameter regime of two resistive-capacitative shunted Josephson junctions, one in the oscillatory and the other in the excitable mode and coupled together resistively. The burst emerges in both the junctions and they show near-complete synchronization for strong enough couplings. We discuss a possible bifurcation scenario to explain the origin of the burst. An exhaustive study on the parameter space of the system is performed, demarcating the regions of bursting from other solutions. PMID:26723143

  5. Rectangular-to-circular groove waveguide junction

    CUI; Licheng; (崔立成); YANG; Hongsheng; (杨鸿生)

    2003-01-01

    Mode matching method is used to analyze the scattering characteristics of the rectangular-to-circular groove waveguide junction. Firstly, the scattering matrix equation is obtained by matching the electromagnetic fields at the boundary of the junction. The scattering coefficients can be obtained from the equation. Secondly the scattering characteristics of the iris with rectangular window positioned in circular groove waveguide are briefly analyzed. Thirdly, the convergent problem is discussed and the numerical results are given. At last experiment is made and good agreement is found between the calculated results and the measured results.

  6. Preface: Charge transport in nanoscale junctions

    Albrecht, Tim; Kornyshev, Alexei; Bjørnholm, Thomas

    2008-09-01

    Understanding the fundamentals of nanoscale charge transfer is pivotal for designing future nano-electronic devices. Such devices could be based on individual or groups of molecular bridges, nanotubes, nanoparticles, biomolecules and other 'active' components, mimicking wire, diode and transistor functions. These have operated in various environments including vacuum, air and condensed matter, in two- or three-electrode configurations, at ultra-low and room temperatures. Interest in charge transport in ultra-small device components has a long history and can be dated back to Aviram and Ratner's letter in 1974 (Chem. Phys. Lett. 29 277-83). So why is there a necessity for a special issue on this subject? The area has reached some degree of maturity, and even subtle geometric effects in the nanojunction and noise features can now be resolved and rationalized based on existing theoretical concepts. One purpose of this special issue is thus to showcase various aspects of nanoscale and single-molecule charge transport from experimental and theoretical perspectives. The main principles have 'crystallized' in our minds, but there is still a long way to go before true single-molecule electronics can be implemented. Major obstacles include the stability of electronic nanojunctions, reliable operation at room temperature, speed of operation and, last but not least, integration into large networks. A gradual transition from traditional silicon-based electronics to devices involving a single (or a few) molecule(s) therefore appears to be more viable from technologic and economic perspectives than a 'quantum leap'. As research in this area progresses, new applications emerge, e.g. with a view to characterizing interfacial charge transfer at the single-molecule level in general. For example, electrochemical experiments with individual enzyme molecules demonstrate that catalytic processes can be studied with nanometre resolution, offering a route towards optimizing biosensors at

  7. Improved electrical properties of n-n and p-n Si/SiC junctions with thermal annealing treatment

    Liang, J.; Nishida, S.; Arai, M.; Shigekawa, N.

    2016-07-01

    The effects of annealing process on the electrical properties of n+-Si/n-SiC and p+-Si/n-SiC junctions fabricated by using surface-activated bonding are investigated. It is found by measuring the current-voltage (I-V) characteristics of n+-Si/n-SiC junctions that the reverse-bias current and the ideality factor decreased to 2.0 × 10-5 mA/cm2 and 1.10, respectively, after the junctions annealing at 700 °C. The flat band voltages of n+-Si/n-SiC and p+-Si/n-SiC junctions obtained from capacitance-voltage (C-V) measurements decreased with increasing annealing temperature. Furthermore, their flat band voltages are very close to each other irrespective of the annealing temperature change, which suggests that the Fermi level is still pinned at the bonding interface even for the junctions annealing at high temperature and the interface state density causing Fermi level pinning varies with the junctions annealing. The reverse characteristics of n+-Si/n-SiC junctions are in good agreement with the calculations based on thermionic field emission. In addition, the calculated donor concentration of 4H-SiC epi-layers and flat band voltage is consistent with the values obtained from C-V measurements.

  8. Gap junctions and hemichannels composed of connexins: potential therapeutic targets for neurodegenerative diseases

    Hideyuki Takeuchi

    2014-09-01

    Full Text Available Microglia are macrophage-like resident immune cells that contribute to the maintenance of homeostasis in the central nervous system (CNS. Abnormal activation of microglia can cause damage in the CNS, and accumulation of activated microglia is a characteristic pathological observation in neurologic conditions such as trauma, stroke, inflammation, epilepsy, and neurodegenerative diseases. Activated microglia secrete high levels of glutamate, which damages CNS cells and has been implicated as a major cause of neurodegeneration in these conditions. Glutamate-receptor blockers and microglia inhibitors (e.g. minocycline have been examined as therapeutic candidates for several neurodegenerative diseases; however, these compounds exerted little therapeutic benefit because they either perturbed physiological glutamate signals or suppressed the actions of protective microglia. The ideal therapeutic approach would hamper the deleterious roles of activated microglia without diminishing their protective effects. We recently found that abnormally activated microglia secrete glutamate via gap-junction hemichannels on the cell surface. Moreover, administration of gap-junction inhibitors significantly suppressed excessive microglial glutamate release and improved disease symptoms in animal models of neurologic conditions such as stroke, multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer’s disease. Recent evidence also suggests that neuronal and glial communication via gap junctions amplifies neuroinflammation and neurodegeneration. Elucidation of the precise pathologic roles of gap junctions and hemichannels may lead to a novel therapeutic strategies that can slow and halt the progression of neurodegenerative diseases.

  9. High quality Nb-based junctions for superconductive detectors

    Nb-based superconducting tunnel junctions have been proposed as detectors in nuclear physics. A discussion in terms of the achieved junction quality concerning the energy resolution and the limit performances will be presented. (orig.)

  10. 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...

  11. 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.

  12. Spin and valley transports in junctions of Dirac fermions

    Yokoyama, Takehito

    2014-01-01

    We study spin and valley transports in junctions composed of silicene and topological crystalline insulators. We consider normal/magnetic/normal Dirac metal junctions where a gate electrode is attached to the magnetic region. In normal/antiferromagnetic/normal silicene junction, we show that the current through this junction is valley and spin polarized due to the coupling between valley and spin degrees of freedom, and the valley and spin polarizations can be tuned by local application of a ...

  13. Low-Tc, ramp-type Josephson junctions for SQUIDS

    Podt, M.; Rolink, B.G.A.; Flokstra, J.; Rogalla, H.

    2002-01-01

    The Josephson tunnel junction is the basic element of a superconducting quantum interference device (SQUID). Amongst other parameters, the junction capacitance determines the characteristics of a (digital) SQUID. In a conventional dc SQUID, reducing the junction capacitance decreases the flux noise of the sensor, whereas in digital SQUIDs, the operating frequency can be increased when reducing the junction capacitance. For digital SQUIDs, this means that not only the flux noise decreases, but...

  14. Structure-Property Relationships in Atomic-Scale Junctions: Histograms and Beyond.

    Hybertsen, Mark S; Venkataraman, Latha

    2016-03-15

    Over the past 10 years, there has been tremendous progress in the measurement, modeling and understanding of structure-function relationships in single molecule junctions. Numerous research groups have addressed significant scientific questions, directed both to conductance phenomena at the single molecule level and to the fundamental chemistry that controls junction functionality. Many different functionalities have been demonstrated, including single-molecule diodes, optically and mechanically activated switches, and, significantly, physical phenomena with no classical analogues, such as those based on quantum interference effects. Experimental techniques for reliable and reproducible single molecule junction formation and characterization have led to this progress. In particular, the scanning tunneling microscope based break-junction (STM-BJ) technique has enabled rapid, sequential measurement of large numbers of nanoscale junctions allowing a statistical analysis to readily distinguish reproducible characteristics. Harnessing fundamental link chemistry has provided the necessary chemical control over junction formation, enabling measurements that revealed clear relationships between molecular structure and conductance characteristics. Such link groups (amines, methylsuflides, pyridines, etc.) maintain a stable lone pair configuration that selectively bonds to specific, undercoordinated transition metal atoms available following rupture of a metal point contact in the STM-BJ experiments. This basic chemical principle rationalizes the observation of highly reproducible conductance signatures. Subsequently, the method has been extended to probe a variety of physical phenomena ranging from basic I-V characteristics to more complex properties such as thermopower and electrochemical response. By adapting the technique to a conducting cantilever atomic force microscope (AFM-BJ), simultaneous measurement of the mechanical characteristics of nanoscale junctions as they

  15. 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

  16. Microscopic tunneling theory of long Josephson junctions

    Grønbech-Jensen, N.; Hattel, Søren A.; Samuelsen, Mogens Rugholm

    1992-01-01

    We present a numerical scheme for solving a nonlinear partial integro-differential equation with nonlocal time dependence. The equation describes the dynamics in a long Josephson junction modeled by use of the microscopic theory for tunneling between superconductors. We demonstrate that the...

  17. Axial p–n-junctions in nanowires

    The charge distribution and potential profile of p–n-junctions in thin semiconductor nanowires (NWs) were analyzed. The characteristics of screening in one-dimensional systems result in a specific profile with large electric field at the boundary between the n- and p- regions, and long tails with a logarithmic drop in the potential and charge density. As a result of these tails, the junction properties depend sensitively on the geometry of external contacts and its capacity has an anomalously large value and frequency dispersion. In the presence of an external voltage, electrons and holes in the NWs can not be described by constant quasi-Fermi levels, due to small values of the average electric field, mobility, and lifetime of carriers. Thus, instead of the classical Sah–Noice–Shockley theory, the junction current–voltage characteristic was described by an alternative theory suitable for fast generation–recombination and slow diffusion–drift processes. For the non-uniform electric field in the junction, this theory predicts the forward branch of the characteristic to have a non-ideality factor η several times larger than the values 1<η<2 from classical theory. Such values of η have been experimentally observed by a number of researchers, as well as in the present work. (paper)

  18. Fluxon density waves in long Josephson junctions

    Olsen, O. H.; Ustinov, A. V.; Pedersen, Niels Falsig

    1993-01-01

    Numerical simulations of the multiple fluxon dynamics stimulated by an external oscillating force applied at a boundary of a long Josephson junction are presented. The calculated IV characteristics agree well with a recent experimental observation of rf-induced satellite flux-flow steps. The volt...... density waves....

  19. Graphene-based magnetic tunnel junctions

    Cobas, Enrique

    2013-03-01

    Graphene's in-plane transport has been widely researched and has yielded extraordinary carrier mobilities of 105 cm2/Vs and spin diffusion lengths of exceeding 100 μm. These properties bode well for graphene in future electronics and spintronics technologies. Its out-of-plane transport has been far less studied, although its parent material, graphite, shows a large conductance anisotropy. Recent calculations show graphene's interaction with close-packed ferromagnetic metal surfaces should produce highly spin-polarized transport out-of-plane, an enabling breakthrough for spintronics technology. In this work, we fabricate and measure FM/graphene/FM magnetic tunnel junctions using CVD-grown single-layer graphene. The resulting juctions show non-linear current-voltage characteristics and a very weak temperature dependence consistent with charge tunneling transport. Furthermore, we study spin transport across the junction as a function of bias voltage and temperature. The tunneling magnetoresistance (TMR) peaks at two percent for single-layer graphene junctions and exhibits the expected bias asymmetry and a temperature dependence that fits well with established spin-polarized tunneling models. Results of mutli-layer graphene tunnel junctions will also be discussed.

  20. Multisoliton excitations in long Josephson junctions

    Dueholm, B.; Levring, O. A.; Mygind, Jesper; Pedersen, Niels Falsig; Sørensen, O. H.; Cirillo, M.

    1981-01-01

    The microwave emission from long Josephson tunnel junctions dc-current biased on zero-field and Fiske steps has been measured. The frequency and power variation on all steps of the narrow-linewidth radiation near the fundamental cavity-mode frequency and the observed transitions between different...